PHYSIOLOGICAL REVIEWS
Vol. 79, No. 4, October 1999
Printed in U.S.A.
Ischemic Cell Death in Brain Neurons
PETER LIPTON
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin
0031-9333/99 $15.00 Copyright © 1999 the American Physiological Society
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I. Introduction
A. Defining cell death
B. Major features of ischemic cell death
C. Overview of ischemic cell death
D. Organization and methodology of the review
II. Systems Used to Study Ischemia
A. Insult variables that determine damage
B. Differential vulnerabilities of cell populations
C. In vivo models: introduction
D. Global ischemia
E. Focal ischemia: characteristics of different models
F. Focal ischemia: core and penumbra
G. Focal ischemia: development and measurement of damage
H. Role of intraischemic depolarizations in focal ischemic damage
I. Role of leukocytes in focal ischemic damage
J. Hypoxia/ischemia
K. Postischemic blood flow and damage
L. Effects of ischemia on the vasculature
M. Effects of temperature on damage
N. In vitro Models: brain slice
O. In vitro Models: cell cultures
P. Summary
III. Morphologies and Biochemistries of Ischemic Cell Death
A. Morphological characteristics of necrotic cell death
B. Apoptotic cell change
C. Autophagocytotic cell death
D. Molecular changes underlying morphological end stages
E. Relationships between different forms of cell death
F. Summary
IV. Critical Functional and Structural Changes
A. Altered membrane transport properties
B. Mitochondrial damage
C. Global inhibition of protein synthesis
D. Damage to the cytoskeleton
E. Summary
V. Perpetrators of Functional or Structural Damage
A. Free radical and peroxynitrite actions
B. Ca21-dependent proteases
C. Phospholipid metabolism
D. Long-term and short-term changes in protein kinases and phosphatases
E. Summary
VI. Activators of Processes Causing Functional Changes
A. Ca21
B. Zinc
C. Glutamate
D. pH
VII. Initiators of Ischemic Damage
A. Decreased ATP
B. Anoxic depolarization
C. Increased cell Na1
D. Events in focal ischemia
E. Summary
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VIII. Gene Activation and Tolerance
A. Immediate early genes
B. Heat shock proteins
C. Neurotrophins and neurotrophic factors
D. Bcl-2/Bax system
E. Gene products that enhance damage
F. Ischemic tolerance
G. Summary
IX. Final Conclusions
A. Mechanisms of ischemic damage
B. A final viewpoint: attractors, cellular instability, and cell death
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I. INTRODUCTION
A. Defining Cell Death
1. Cell death is an active process
This review is geared toward those working in the field
of ischemic tissue damage and toward students and others
who want to develop a critical overview of the area. The text
carefully examines the evidence that certain chemical and
ionic changes, protein and enzyme changes, and changes in
key functions are involved in ischemic cell death. The long-
term goal of this review is to make as cogent a model of
ischemic cell death as is possible at present, which is done
in the final section, and to highlight what is not known to
excite rigorous, causally directed, experimental work. The
latter is done throughout the text.
The ischemic cell death that is clinically relevant, and
that is largely considered in this review, is different from
the cell death that would eventually ensue, as a consequence of the second law of thermodynamics, from complete and permanent inhibition of energy metabolism.
That would occur because of the inability to couple oxidative metabolism to endergonic processes and the re-
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Lipton, Peter. Ischemic Cell Death in Brain Neurons. Physiol. Rev. 79: 1431–1568, 1999.—This review is directed
at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are
the two principal rodent models for human disease. Cell death occurs by a necrotic pathway characterized by either
ischemic/homogenizing cell change or edematous cell change. Death also occurs via an apoptotic-like pathway that
is characterized, minimally, by DNA laddering and a dependence on caspase activity and, optimally, by those
properties, additional characteristic protein and phospholipid changes, and morphological attributes of apotosis.
Death may also occur by autophagocytosis. The cell death process has four major stages. The first, the induction
stage, includes several changes initiated by ischemia and reperfusion that are very likely to play major roles in cell
death. These include inhibition (and subsequent reactivation) of electron transport, decreased ATP, decreased pH,
increased cell Ca21, release of glutamate, increased arachidonic acid, and also gene activation leading to cytokine
synthesis, synthesis of enzymes involved in free radical production, and accumulation of leukocytes. These changes
lead to the activation of five damaging events, termed perpetrators. These are the damaging actions of free radicals
and their product peroxynitrite, the actions of the Ca21-dependent protease calpain, the activity of phospholipases,
the activity of poly-ADPribose polymerase (PARP), and the activation of the apoptotic pathway. The second stage
of cell death involves the long-term changes in macromolecules or key metabolites that are caused by the
perpetrators. The third stage of cell death involves long-term damaging effects of these macromolecular and
metabolite changes, and of some of the induction processes, on critical cell functions and structures that lead to the
defined end stages of cell damage. These targeted functions and structures include the plasmalemma, the mitochondria, the cytoskeleton, protein synthesis, and kinase activities. The fourth stage is the progression to the
morphological and biochemical end stages of cell death. Of these four stages, the last two are the least well
understood. Quite little is known of how the perpetrators affect the structures and functions and whether and how
each of these changes contribute to cell death. According to this description, the key step in ischemic cell death is
adequate activation of the perpetrators, and thus a major unifying thread of the review is a consideration of how the
changes occurring during and after ischemia, including gene activation and synthesis of new proteins, conspire to
produce damaging levels of free radicals and peroxynitrite, to activate calpain and other Ca21-driven processes that
are damaging, and to initiate the apoptotic process. Although it is not fully established for all cases, the major driving
force for the necrotic cell death process, and very possibly the other processes, appears to be the generation of free
radicals and peroxynitrite. Effects of a large number of damaging changes can be explained on the basis of their
ability to generate free radicals in early or late stages of damage. Several important issues are defined for future
study. These include determining the triggers for apoptosis and autophagocytosis and establishing greater confidence in most of the cellular changes that are hypothesized to be involved in cell death. A very important outstanding
issue is identifying the critical functional and structural changes caused by the perpetrators of cell death. These
changes are responsible for cell death, and their identity and mechanisms of action are almost completely unknown.
October 1999
CEREBRAL ISCHEMIA ON NEURONS
sulting inability to reduce the entropy of the cell at the
expense of exergonic reactions. Cell entropy would increase; ion gradients would dissipate, macromolecular
synthesis would cease, and cell structure would be lost.
This would be considered a “dissipative” cell death. The
phenomenon considered in this review, and by the vast
majority of studies, ensues from relatively short and/or
relatively mild insults which, themselves, do not lead to
dissipative cell death; rather, they cause acute ionic and
chemical changes that initiate sets of highly interactive
ionic and biochemical changes that cause cell death.
2. A working definition of cell death
B. Major Features of Ischemic Cell Death
1. Three major modes of cell death
There appear to be at least three distinct modes of
cell death that participate in ischemic cell death (149,
205, 706). Two of these, apoptosis and autophagocytotic cell death, clearly involve ordered physiological
processes in that new structures are formed that are
integral to the process, and the progress toward cell
elimination is stereotyped. Furthermore, they are a part
of normal cell function, during development and other
times (205). There is some conflict as to whether autophagocytosis can be profound enough to cause cell
death (149, 205, 835), but there are quite persuasive
arguments by Clarke (205) that it is. These include the
high percentage of neuronal area that is covered by
autophagic vacuoles during some developmental cell
death (205), as well as the ability of 3-methyladenine, a
phosphatidylinositol kinase blocker (101) which prevents autophagocytosis, to completely prevent cell
death in at least one case (149).
A third category of cell death, which has been termed
necrosis or necrotic cell death, is fundamentally different
from apoptosis and autophagocytotic cell death in that it
only occurs after an exogenous insult; furthermore, no
new structures are elaborated as part of the process. Thus
it does not appear to be a programmed, or a normal,
physiological process. Because the process is not yet well
characterized, it is best defined now as cell death that is
neither apoptotic nor autophagocytotic.
There are at least two quite different forms of this
cell death; by far the most common is ischemic/homogenizing cell change, where the cell first shrinks dramatically and becomes very electron dense. Less common is
edematous cell change, where the cell swells greatly and
organelles lose their form. There is no satisfactory term
for these modes of cell death. It is widely termed necrosis
or necrotic cell death, but necrosis is really the “decay”
process after the point of cell death and occurs in all
forms of cell death (1137). However, for the sake of
continuity, necrotic cell death and necrosis will be used in
this review. At this stage, it is not completely known why
a particular mode of death occurs in a given cell as a
result of a particular ischemic insult. However, it is apparent that one insult can spawn more than one mode of
death in the same population of cells and also that in
many cases one cell will manifest signs of more than one
mode of cell death (205, 716). Thus neurons have the
potential for exhibiting all modes of death in response to
an ischemic insult. The pathway taken must be a function
of the nature of the insult, the cell type, age, and very
probably the state of the cell at the time of the insult
(716). This issue is considered in section III.
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To study what causes cell death, cell death has to be
recognized. However, there is no well-accepted definition
of the point at which a cell dies (706, 1137). The most
satisfactory definition of cell death, and the one that
would be the most valuable to know therapeutically, is
the point at which the cell becomes unable to recover its
normal morphology and function even if all processes
leading to dissoution are stopped pharmacologically (the
point of no return). This would be analogous to cessation
of breathing or cardiac function for death of the organism.
At this stage, however, we do not know enough about the
ischemic death process of a cell to be able to identify
what the point is. Thus the only unequivocal definition of
cell death is a morphological one, the elimination of the
cell, which can arise either by cell disintegration or
phagocytosis. These are both very clear end points, and
they are often used in studying mechanisms of cell death.
Before this there are certain metastable morphological
states toward which dying cells tend that are almost
certainly precursors of disintegration or phagocytosis
(121, 814) and that are also commonly used as end points.
In this review these are termed the end stages of the cell
death process and are generally considered to represent
dead cells, undergoing necrosis, autophagocytosis, or the
end stages of apoptosis (Fig. 1, column 5). As defined in
this way, cell death is an anatomical event that represents
a state from which the cell cannot recover to even near
normalcy. The state reflects major macromolecular
change, and the study of cell death, at this stage, is the
study of what leads to these drastic macromolecular
changes.
Ultimately, it will be important to identify the point of
cell death, defined above as the point of no return, because some agents might block the major morphological
change but not block the irreversible damage to the cell.
It seems reasonable that prolonged damage to the plasma
membrane, mitochondria, cytoskeleton, or protein synthesis could each be a point of cell death defined as
above, and section IV is devoted to evaluating their roles in
ischemic cell death.
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FIG. 1. Pathways of ischemic cell death. This is an overview of processes involved in ischemic cell death that is consistent
with organization of review. It illustrates major events that are hypothesized to contribute to cell death and also the extremely
complex interactions between these events. This figure is not meant to be complete, but does include processes known or
thought to be important at this stage. Column 5 lists 5 principal morphological forms taken by dying or dead cells after an
ischemic insult. Determining how these end stages are reached is the ultimate goal of research on ischemic cell death. Column
4 lists 6 long-term functional or structural changes, all of which, except changes in membrane permeability, are known to
occur as a result of ischemia. It is hypothesized that one or more of these account for one or more forms of ischemic cell
death. As indicated by lack of 1:1 connections in figure, no direct causal sequence has been established between any of
functional changes in this column and particular end stages shown in column 5. Potential relationships are discussed
extensively in text (see sects. III and IV). Column 3 lists actions that are likely to cause long-term functional changes described
in column 4. These are termed “perpetrators” because they are considered to be key damaging events in ischemic cell death.
No direct effects of perpetrators on critical functional changes are insinuated in figure because none has been completely
established. Many such effects are, however, reasonably well established and are discussed in text. Columns 2 and 1 show
changes in many variables, initiated by original inhibition of electron transport, whose most important end result is considered
to be activation of perpetrators, but which may also have more direct effects on cell damage, that are not shown. Major
outputs of these changes, lumped together as initiators and activators, are changes shown in column 2, which activate
perpetrators. Direct causal interactions between specific changes are shown for these earlier events because far more is
known than about interactions at later stages of the process. However, all these interactions are not established with same
degree of assurance, as discussed in text. These causal interactions are indicated by including changes within same toned
horizontal band, as shown for columns 2 and 3. For example, both increased nitric oxide and free radicals lead to damaging
actions of free radicals and peroxynitrite. They are also indicated by including causal changes within a box whose outline
color is same as that of variable they are changing. For example, both increased calcium and gene activation contribute to
formation of increased nitric oxide. This way of describing events indicates the very high level of interaction between different
changes caused by ischemia, involving many positive-feedback loops. Interpreting the figure: as indicated above, colored
boxes encompass different changes that cause change in variable represented by that color. For example, increased nitric
oxide (peach color) results from increased calcium and gene activation. Colored horizontal arrowheads represent all events
within box of that color. For example, red arrowhead (for ATP) represents loss of oxygen and increase in sodium, which are
enclosed within red box associated with ATP (near bottom of figure). Using this notation allows the very large connectivity
of system to be represented in a manageable way. For example, changes putatively contributing to increase in sodium,
enclosed within green box at top of figure, are extremely numerous when “contents” of each arrowhead are considered. In
addition, changes contributing to increase in cytosolic calcium, and hence calpain activity, are all those included in boxes and
arrowheads in top gray band. Large number of positive-feedback cycles is readily seen. Depol, depolarization; pHi, intracellular
pH; Nai, intracellular Na1; Cai, intracellular Ca21; FFA, free fatty acids; PAF, platelet-activating factor; e2 Transport, electron
transport.
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
2. Ischemic cell death is usually delayed
C. Overview of Ischemic Cell Death
1. Stages of ischemic cell death
Ischemic cell death is initiated by changes that result
directly from inhibition of oxidative phosphorylation and
create an early maelstrom of activity. These changes include decreased pH, decreased ATP, initiation of free
radical production by the mitochondrial chain, increased
cell Na1 and membrane depolarization as a result of the
loss of ATP substrate for the Na1-K1 pump. These lead to
secondary changes in ion and chemical concentrations
which, in concert with the initiating changes, result in
activation of damaging processes. These damaging processes are termed “perpetrators” in this review and are
characterized by their abilities to produce long-term
changes in macromolecules. Activation of one or more of
the perpetrators is considered the key step in necrotic
aspects of ischemic cell death. The perpetrators include
proteases, phospholipases, and free radical actions. The
macromolecular changes they cause, and the ensuing
functional changes, are considered to be the proximal
causes of cell death. At present, the way that ischemic
apoptosis is activated is not well enough understood to
include the same perpetrators in that process with any
certainty, although caspases certainly are involved. Thus
the process of cell death has at least three major stages:
an early development of ionic and chemical changes, a
resulting activation of perpetrators, and a subsequent
change in critical functions and structures that denote or
lead to cell death by one of three different routes.
The overall process is extremely complex due to the
large number of interactions between the variables, illustrated by Figure 1. Although incomplete, Figure 1 provides a good working description of the probable flow of
damage and the very complex interactions between different changes.
2. Limitations in current knowledge
Two very important limitations in our understanding
are not apparent from Figure 1.
The first is the degree of certainty that the different
changes shown are actually involved in ischemic cell
death. It is fairly certain that most, or all, of the activators
and perpetrators in Figure 1 are actually involved in ischemic cell death, although, as will be seen, experimental
evidence is often surprisingly weak. Much less is known
about how these changes lead to cell death (Fig. 1, columns 4 and 5). As will be seen, the identities of the
critical functional changes, how they actually might lead
to cell death, and the end stages are little known for any
of the three modes of cell death.
The temporal aspect of cell death is also missing
from Figure 1. End stages or cell disintegration develop
anywhere from 6 h to several days after the ischemic
insult. The basis for these time delays is not yet known.
Gene activation and subsequent synthesis of damaging
proteins such as cytokines, inducible cyclooxygenase-2
(COX-2), inducible nitric oxide (NO) synthase (iNOS) may
be important. However, it is also likely that there are
long-term low-level activations of perpetrators when the
insult is relatively mild so that major changes in cell
function occur only after prolonged periods. The factors
that determine the temporal aspects of ischemic death are
poorly understood at the moment.
D. Organization and Methodology of the Review
Figure 1 provides a framework for the organization of
the review. After the different model systems used for
studying ischemic damage are considered (see sect. II),
the main body of the review begins from the right-hand
side of Figure 1 and essentially walks backwards; for
example, section III describes the end stages of ischemic
cell death. Knowing the downstream events makes it
easier to evaluate the role of a particular change. For
example, understanding that mitochondrial dysfunction
may be a mediator of cell death (see sect. IV) provides a
focus for examining the targets of free radical action.
Despite the connectivity of the different major parts, each
section is designed to be usefully read independently of
its order of appearance and to fully examine the possible
contribution of a different variable to damage.
The achievable goal of the review is to critically
evaluate the involvement of different variables in ischemic cell death. This entails describing the changes in the
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In addition to its multimodal nature, ischemic cell
death is also characterized by a long delay between the
insult and manifestation of major cell damage. This delay
varies greatly, depending on the nature of the insult and
the brain region being affected. In some cases it is as long
as several days or even weeks (276, 582), whereas in
others it is a few hours or less (742). It is clear that, all
other factors being equal, the greater the energy deprivation, the less time it takes for damage to develop (579).
The very prolonged delay is quite an extraordinary phenomenon; it shows that very short-lived, albeit profound,
derangements of metabolic patterns can initiate the development of damage and cell death that takes place,
between a day and 3 wk later. Remarkably, normal homeostatic mechanisms are unable to prevent this process.
Thus lethal ischemic insults are those that move cells far
enough from their steady state to eliminate the possibility
of reattaining that steady state; sublethal insults produce
early damage, but this is able to be recouped by the cell.
One very important and unresolved issue is what the basis
is for this “destabilization” of the cell. It is discussed
theoretically in section IX.
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TABLE
1. Changes in ATP during ischemia in different systems
Volume 79
Duration of Ischemia, min
System
228, 294, 682, 1182
731, 1254
295, 763, 961
962, 1210
3, 783
1168
0.5
1
2
70
92
45/14
40
8
75
3
5
10–15
12
11
6
10
15
50
5*
328, 329, 1201
1084
6
1
6
56
30
70
26
15
35
20
981
1213
686
6, 530, 532
638
341,‡ 134§
791
97
50†
80
60
50
25‡
50
15§
Values are given as percent of control. 2-VO, 2-vessel occlusion; 4-VO, 4-vessel occlusion. * 1.5 Min. † 2.5 min.
variable that occur during and after ischemia, determining
how they are likely to occur, critically assessing pharmacological and gene manipulation studies done to show
that the change contributes to cell death, and then trying
to understand the mechanism by which the change is
contributing to cell death.
In section IX, a still speculative attempt is made to
synthesize the different sections and describe probable
sets of events that lead to cell death in the different
experimental models. It is hoped that this will provide
useful directions for future studies.
focus on what has been learned from rodent models. In
vitro models that include exposure of neuronal, neuronal/
glial or organotypic slice cultures, or the freshly isolated
hippocampal brain slice, to anoxia or to anoxia in the
absence of glucose (in vitro ischemia) have provided
important information and are discussed when they provide insight into events in vivo. Other single-cell or synaptosomal models have been used very effectively (307,
340) but will not be considered because the metabolic
states of these preparations may be quite different from in
vivo (307).
II. SYSTEMS USED TO STUDY ISCHEMIA
A. Insult Variables That Determine Damage
Many experimental models are used to study ischemic damage. Mechanisms of cell damage are determined by testing effects of different manipulations on
the extent of cell death in a model. To meaningfully
compare results between models, the essential differences between the ischemic insults and the way damage is assessed in these models needs to be appreciated. The factors influencing development of damage in
the different models need to be known, and problems in
interpreting measurements of cell death have to be
recognized.
The three main classes of in vivo rodent models are
global ischemia, focal ischemia, and hypoxia/ischemia. In
the latter, vessel occlusion is combined with breathing an
hypoxic mixture; this modified Levine preparation is now
almost exclusively used in young animals. Larger mammals have been used to study ischemia (534), as have
nonmammalian vertebrates (453), but this review will
The degree to which blood flow is reduced directly
determines the changes in three variables that are almost
undoubtedly the genesis of all future changes. Two of
these, inhibition of electron transport and decreased ATP,
appear to be directly related to the extent of flow reduction (see Table 1). The third variable, free radical production, is likely to be a more complex function of flow rate
because it is activated both by electron transport inhibition and also by oxygen so that it will be maximal at blood
flow values greater than zero. (The precise relationship
between flow rate and free radical production has not
been determined.) Other damaging changes such as decreased pH and leukocyte accumulation are more severe
in the presence of residual blood flow. Thus the amount of
damage need not be a direct function of the decrease in
blood flow. Elevated blood glucose enhances damage in
many cases despite maintaining ATP levels so that the fall
in ATP is not a reliable indicator of the efficacy of an
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Global ischemia
Rat complete ischemia
Mouse decapitation
Rat 2-VO
Rat 4-VO
Global 2-VO
Rat cardiac arrest
Focal ischemia
Penumbra
Core
Ischemia/hypoxia
Carotid/3% O2
Carotid/8% O2
Hippocampal slice (CA1 region/36–37°C)
Ischemia
Anoxia
Cultures
Anoxia
Ischemia
Chemical ischemia
Reference No.
October 1999
CEREBRAL ISCHEMIA ON NEURONS
insult either. As a further complication, the nature of
damage (e.g., extents of apoptosis vs. necrosis) may be a
complex function of the amount of the ATP/electron
transport decrease and free radical increase and thus may
be a complex function of the flow rate during ischemia
(194, 870). Thus, except for increased duration of an
insult, which always enhances and accelerates damage, it
is difficult to estimate, a priori, the potential impact of an
insult based simply on the degree of ischemia and fall in
ATP.
B. Differential Vulnerabilities of Cell Populations
C. In Vivo Models: Introduction
The Levine model, in which hypoxia is combined
with unilateral carotid occlusion (644), adapted in the
mid-1960s in landmark studies by Brown, Brierly, and
co-workers (121, 129, 742), established the basis for systematic study of brain cell damage in rodents. Most in
vivo models now rely on vessel occlusions that predominantly affect the forebrain, developed between the late
1970s and early 1980s (331, 366, 461). They are generally
divided into two categories, global and focal ischemia, but
a modified Levine preparation (hypoxia/ischemia) has
some properties of both models.
D. Global Ischemia
Global ischemic insults are most commonly produced
by vessel occlusions, and less commonly by complete brain
circulatory arrest. Although the former are not actually
global, a large portion of the forebrain is quite uniformly
affected.
1. Anatomy: effects on flow and electrophysiology
The three most widely used global ischemia models
are four-vessel occlusion (4-VO) and two-vessel occlusion
(2-VO) combined with hypotension in the rat, and twovessel occlusion in the gerbil. The importance of transgenic mice has spawned the use of the 2-VO in the mouse
also, and the latter is now being systematically studied
(588, 1074). Results to date are similar to those with rat
and gerbil (588).
A) 4-VO IN RAT. The 4-VO in rat involves permanent
coagulation of the vertebral arteries (which has no deleterious effects) and temporary ligation of the two common carotids. Ligation of temporal muscles reduces flow
more reliably (926, 928). In Wistar rats, which show loss
of righting reflex within 15 s, blood flow is reliably ,3% of
control values in hippocampus, striatum, and neocortex
(931). The electroencephalogram (EEG) generally becomes isoelectric within 30 – 40 s (935), and spontaneous
cortical activity is abolished within 1 min when skull
temperature is maintained at 37°C (1233). No anesthetic is
required during ischemia, and usually none is used.
B) 2-VO IN RAT. This involves ligation of the common
carotid arteries only, along with a blood pressure reduction to ;50 mmHg, and has slightly more profound effects
than 4-VO. Blood flows fall to ;1% in hippocampus, neocortex, and striatum (1055, 1056), and the EEG generally
becomes isoelectric within 15–25 s. However, in the hands
of one group, blood flow is only reduced to 15% of control
levels (1089). This less-intense insult is more easily protected against, for example, by N-methyl-D-aspartate
(NMDA) receptor blockade, and this has led to some
confusion in the literature that will be dealt with in section VIC. Anesthetic is used during ischemia.
C) 2-VO IN GERBIL. This is induced by temporarily ligating the carotid arteries, with no reduction in blood pressure. Because there are no posterior communicating arteries in gerbils, this produces profound forebrain
ischemia (583). Changes are similar to those in the rat
models; blood flow in cortex is ,1% and in hippocampus
is ;4% of control values (548), whereas EEG failure occurs within 20 s (1088). Damage has been successfully
dissociated from the occurrence of postischemic seizures
that also characterize the insult in gerbils (493). Anesthetic is not used during ischemia.
D) COMPLETE VERSUS INCOMPLETE GLOBAL ISCHEMIA. Finally,
there is complete global ischemia, generally achieved by
neck-cuff (259, 297, 682), cardiac arrest (228, 294), or by
ligating or compressing all arteries stemming from the
heart (560, 915). Blood flow to the whole brain is zero or
,1% in these models (297).
Vessel occlusion is often termed “incomplete isch-
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In vivo, distinct neuronal populations have very different vulnerabilities to ischemia, and these have been
reviewed relatively recently (169). For example, 5-min
global ischemia caused delayed death in almost all CA1
pyramidal cell neurons with no effects on other populations, whereas 20 min caused cell death in CA3 neurons
but had almost no effect on dentate granule cells in the
hippocampus (579, 583) or on interneurons in CA1 (322).
The basis for differential vulnerability has not been determined, although, as will be seen, there are many differences between vulnerable and less vulnerable cells that
might well account for it, as described in Table 5. In
addition to these, differences in presynaptic events may
be responsible for apparent selective vulnerabilities of
postsynaptic neurons as will be seen in section VIB. The
large range of selective vulnerabilities is quite an extraordinary phenomenon. It indicates large differences in metabolism between different cell types that presumably
reflect their different physiological functions and lead,
serendipitously, to differential vulnerabilities to ischemia.
The phenomenon has been used to provide insight into
mechanisms of damage and is discussed in that context
throughout the review.
1437
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PETER LIPTON
2. Changes in energy metabolism
Global insults are generally short, between 3 and
30 min, with 5–20 min being the usual exposure times.
Longer insults become lethal, and these shorter insults
are quite good models for reversible cardiac arrest
pathology. Levels of ATP fall quite rapidly during this
period (Table 1). By 2 min, levels are 15% or lower in
gerbil, and also in rat during complete ischemia. Unfortunately, there are no good kinetic studies for the rat
vessel occlusion models; by 10 min, ATP is 10% or less
of normal values (Table 1), but the rate of decay, which
may be important in determining extent of damage, is
not known.
Levels of ATP in vulnerable cell regions recover quite
well after global ischemia so that delayed damage develops in the face of ATP levels that are at least 70 – 80% of
normal for most of the reperfusion period (162, 929).
Tissue metabolic rate is generally depressed by 50% or
more, for at least 6 h after the ischemia (291, 306, 606,
760). The basis for this is not known, and its consequences have not been determined, but they are likely to
be important.
3. Development and measurement of damage
A) DEVELOPMENT OF DAMAGE. A cardinal feature of global
ischemic insults is a substantial delay between the end of
the short insult and cell death. This is generally between
12 h and several days. The delay depends on the cell
population and the duration of the insult. This “delayed
neuronal death” was first noted and documented in rat by
Pulsinelli and Brierly (926, 927) and in gerbil by Kirino
(580). It is most widely studied in the CA1 region of the
hippocampus, but the striatum, parts of the CA4 region of
hippocampus, and layers 2 and 5 in cerebral cortex also
show the phenomenon in response to short insults.
Overall, gerbil hippocampus is slightly more sensitive
than rat. With body temperature at 36°C, but brain temperature falling normally, 5 min of ischemia caused massive cell death in the hilus of the dentate gyrus (CA4) and
the striatum within 24 h and complete cell loss in the CA1
region of the gerbil hippocampus within 3– 4 days (223,
580). Ischemia for 3.5 min caused 75% cell death in CA1
after 7 days (847), whereas 2 min caused no measurable
damage in that region (543). It did cause extensive damage in the somatostatin-containing cells in the hilar region
(735).
When rat brain was maintained at 36 –37°C, 6 –10 min
of 4-VO caused near-complete CA1 pyramidal cell death in
3 days (647, 681), and 10 min of 2-VO caused almost
complete cell death in 7 days (210, 519, 819, 921). Ischemia for 5 min caused 50% loss (519) compared with
complete cell loss in gerbil, and 2-min ischemia caused a
(very minor) neuronal loss in CA1 and CA4 (1055). As
with the gerbil, hilar and striatal neurons die much more
rapidly than CA1 pyramidal cells (896, 997), although,
interestingly, a greater insult duration was necessary to
initiate the cell death in striatal neurons than in hippocampal neurons (997). This shows a dissociation between factors that initiate the damage and those involved
in its development.
Damage from successive insults can be cumulative.
When spaced 1 h apart, three insults that normally gave
no damage individually caused major damage (543).
B) MORPHOLOGICAL CHANGES DURING DELAYED NEURONAL
DEATH. There are four major sets of changes during development of the delayed death in gerbil. Most notable is a
massive proliferation of stacks of endoplasmic reticulum
(ER) that peaks after ;2 days; ER fragments accumulate,
with lysosomes, around the nucleus (579, 581). After ;3
days, there is a regression of the proliferated ER and the
formation of a large number of autophagosomes with a
very large increase in the lysosomal proteases, cathepsins
B, H, and L (834). The autophagic vacuoles may arise from
the expanded ER as the two have been associated during
developmental cell death (205). Thus a long-term program
for autophagocytosis may have been activated by 5 min of
ischemia.
There is also a gradual increase in the numbers of
small dense bodies ;1 mm or less in diameter in both
somata and dendrites, which largely localize under the
plasmalemma. Throughout the 3 days, the dendrites show
progressive swelling, microvacuolization, and disappearance of microtubules, beginning immediately after ischemia (1236). After early swelling, mitochondria remain
normal until ischemic cell change (ICC) or edematous cell
change (ECC) become apparent.
The delayed death in the rat appears very different,
although it develops at about the same rate. The extent of
ER proliferation is far less (251, 581, 897) and, perhaps
relatedly, no autophagasomes have been explicitly identified. The small dense bodies are somewhat more prevalent than in the gerbil and may be undegraded proteins
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emia” because of the residual blood flow, and there is
controversy as to whether complete or incomplete ischemia is more damaging, with the thought being that the
larger fall in pH or increase in free radicals during incomplete ischemia might enhance damage. This does not
seem to be the case. Complete ischemia for 10 min in rats,
even without maintenance of temperature, causes complete destruction of cells in the CA1 pyramidal cell layer
after 7 days (226). This damage is certainly as severe as
the incomplete global ischemia models (see sect. IID3).
Overall, gerbil vessel occlusion is the most studied
model because the operation is simpler than the rat.
However, there are many differences between the two
species, both in terms of metabolic and genetic changes
during and after ischemia, and the abilities of different
agents to protect against damage. Thus the processes of
cell death may be different; results from one species
cannot necessarily be generalized.
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4. Summary
There are profound reductions in ATP levels for very
short periods during ischemia, possibly for as little as 1–2
min. Nevertheless, there is massive cell death in vulnerable regions. There is a significant delay between the ischemia and obvious morphological signs of cell death. This
delay can be as much as 4 days and as little as 12 h
depending on the length of the insult and the cell population; for any one region, the longer the insult, the
shorter the delay. Understanding why this delayed cell
death arises from such short insults is a major goal of
research in the field.
E. Focal Ischemia: Characteristics
of Different Models
Almost all focal ischemic models, whether larger
mammal such as cat (1077) or nonhuman primate (1092)
or rodent, primarily involve occlusion of one middle cerebral artery (366). In some cases, the carotid artery is
also ligated. The insult is thus differentiated from global
ischemia in two very important ways. First, even at the
core of the lesion, the blood flow is almost always higher
than during global ischemia so that longer insults are
required to get damage. Secondly, there is a significant
gradation of ischemia from the core of the lesion to its
outermost boundary, and hence there are different metabolic conditions within the affected site. Because of its
duration, and heterogeneity, the insult is much more complex than global ischemia, but it is an invaluable model for
stroke and is thus widely studied.
In permanent focal ischemia, the arterial blockage is
maintained throughout an experiment, usually for 1 to
several days, whereas in temporary focal models, vessels
are blocked for up to 3 h, followed by prolonged reperfusion. Despite the big difference in insults, maximal lesion
sizes are comparable in the two cases, and the progression of damage, in terms of the numbers of damaged
neurons and the extent of that damage over 6 –72 h, is
remarkably similar (1283). Nevertheless, there are clear
differences illustrated by effects of different protectants
so that the mechanisms of cell death may well be different. As with global ischemia, mouse models have now
been introduced (434, 588) but will not be discussed
explicitly.
There are variations within each of the two basic
classes of insult: temporary and permanent ischemia. Several different rat strains are used (one is a spontaneously
hypertensive strain that has a less extensive collateral
circulation during ischemia and is more easily damaged;
Refs. 137, 222, 391). Different strains show minor quantitative differences in lesion locations and sizes but qualitative differences in responses to different protectants.
Also, different sites and techniques of artery occlusion are used, to try and obtain reproducibility of flow
reduction and lesion size (462, 860). There are two principal occlusion sites. In proximal occlusion, the middle
cerebral artery (MCA) is occluded close to its branching
from the internal carotid, before the origin of the lenticulostriate arteries (860, 1104, 1105). Appropriate care can
produce lesion sizes with coefficients of variation that are
,20% (860). A newer and now widely used approach to
proximal MCA occlusion is the insertion of a nylon suture
into the carotid artery past the point at which the MCA
branches so that the latter is occluded at its origin (79,
598, 655, 798). Used optimally, the coefficient of variation
in lesion size is ;8% and of blood flow is 35% (79). A
similarly small coefficient of variation in lesion size, along
with a large lesion of ;250 mm3 at 24 h, was obtained
with the silicone-coated suture in Fischer 344 rats (44).
There are some potentially important artifacts with this
widely used method. Flow following temporary ischemia
is somewhat compromised by the partial occlusion of the
carotid arteries with the filament (79). Also, even a temporary occlusion leads to a long-term increase in core
temperature at ;1.5°C due to decreased circulation and
damage to the hypothalamus, at least in Wistar rats (1241,
1297). Finally, there is quite extensive damage to small
arteries in the ischemic field, with loss of endothelium
and muscle damage (832). This occurs after normal insults (e.g., 6-h ischemia or 2-h ischemia and several hours
of reperfusion). It has been suggested that this damage
may affect subsequent neuronal cell death, for example,
by exacerbating the leukocyte response in the period after
ischemia (435). This is not at all proven but needs to be
considered.
In distal MCA occlusion, flow to the basal ganglia is
not blocked so that damage is only cortical. The occlusion
can be made surgically, in which case the lesion is readily
reversible (137) or, less invasively, by creating a (permanent) thrombus by laser irradiation when the artery is
perfused with Rose Bengal (714). Distal MCA occlusion
has to be combined with occlusion of the ipsilateral carotid artery to get adequate flow reduction (122, 189).
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that participate in damage (251, 582), although there is no
explicit evidence.
As the duration of the insult increases, and so presumably the extent of the biochemical changes, so does
the rate of cell death increase (579, 897). The nature of the
process may ultimately change; ER proliferation, for example, is absent in gerbils after a 30-min insult that causes
cell death in 12 h (897).
At a certain point, these rather subtle changes are
transformed into quite abrupt major changes in cell structure associated with the end stages of cell death. These
are discussed in section III. The mechanisms of this dramatic change are not known; presumably accumulated
changes become great enough to cause what is essentially
a “phase change,” as discussed by Gallyas et al. (350).
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PETER LIPTON
F. Focal Ischemia: Core and Penumbra
1. Blood flows
2. Ionic and metabolic changes in the core
and penumbra
As indicated, there are major differences in physiology and biochemistry between the core and penumbra
and, indeed, these represent another way in which the
regions are differentiated. Furthermore, differences in
metabolism and in susceptibilities to protectants strongly
suggest that different mechanisms cause the cell death in
the two regions.
The core undergoes rapid anoxic depolarization
within 1–3 min with a concomitant rise in extracellular K1
to ;70 mM, which may show some short intermittent
returns to baseline (359, 818). There is a large decrease in
extracellular Ca21 within 1–2 min at flow levels that are
present in the core (428, 429), signifying entry into the
tissue. During the reperfusion period, after 2 h temporary
focal ischemia, extracellular K1 returns to control levels
for 6 h before rising slightly (to 5 mM) for the remainder
of a 24-h recirculation period. Despite this return to near
normalcy, the insult produces severe damage with a large
infarct including all of the core (359).
Events in the penumbra are less drastic, although
they still lead to infarct. There is EEG silence (1077) and
very much reduced evoked transmission (977), but there
is no permanent anoxic depolarization with concomitant
ion changes during the ischemia. Presumably ATP levels,
which are maintained at 50 –70% of normal, do not fall
enough to allow the anoxic depolarization. However,
there are sporadic transient depolarizations (369, 759)
that are here termed “intraischemic depolarizations” (462,
818). They arise differently from anoxic depolarizations.
Anoxic depolarizations are independent of NMDA-type
glutamate receptors (394, 611, 635, 934, 1231) and only
very slightly (1231) or not at all (635) dependent on
non-NMDA glutamate receptors. In contrast, the frequency of intraischemic depolarizations is markedly dependent on both classes of ionotropic glutamate receptors (47, 188, 483, 759). The depolarizations may actually
originate from glutamate release in the infarct core. Nitric
oxide synthase knockout mice show reduced frequencies
along with less release of glutamate in the core of the
lesion (1031). The depolarizations occur very abruptly,
last between 3 and 5 min, and have the characteristic form
of spreading depressions. They do not occur during reperfusion (818). As discussed in section IIH, they may well
enhance damage.
3. Energy metabolism in core and penumbra
Levels of ATP fall to ;25% of basal values in the core
and remain there between 5 min and 4 h of ischemia (328,
329, 1084, 1201) (Table 1). Later measurements have not
been reported. After damaging durations of temporary
ischemia, core ATP levels return to about two-thirds normal, for at least 4 h (329), although damage is as great as
in permanent ischemia. Oxygen levels return to normal
(801), showing that compromised flow is not the basis for
the damage that occurs during reoxygenation.
Levels of ATP in the penumbra average ;50 –70% of
normal during ischemia (Table 1). Glucose utilization is
actually elevated in the penumbral region (48, 816, 1252)
at early times but falls to ;50% normal, equal to the core,
by 3.5 h (1252), suggesting significant compromise of cell
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In both proximal and distal MCA occlusions, there
are core ischemic regions, served primarily by the occluded MCA, where blood flow is reduced to ,15%, and
there are penumbral regions where flow is ,40%. There
are also extrapenumbral cortical regions in which flow is
.40%. These approximate flow-based definitions of core
(284, 816, 977, 1104) and of penumbra (48, 369, 462) as
well as the extrapenumbral (or peri-infarct) region are
generally agreed upon, although there is still much discussion. Using flow is a simple and physiologically meaningful way to differentiate the regions, which suffer fundamentally different insults as indicated by efficacies of
protectants, and major morphological changes. Given a
long enough temporary (or permanent) insult, both core
and penumbra become infarcted (1299), whereas the extra penumbral zone only shows death of isolated neurons.
The density of cell death is not great enough to cause
infarct.
In proximal MCA occlusion, the core is in the lateral
portion of the caudate putamen and overlying parietal/
somatosensory cortex (106, 751, 798, 1104). Most other
regions of neocortex and entorhinal cortex, as well as the
medial caudate-putamen, constitute the penumbra (79,
106, 751, 798). In the distal MCA occlusion/carotid artery
occlusion, the striatum is spared, and core and penumbra
span the parietal cortex, with penumbra occupying the
more inferior regions. There is significant variation in the
extent of the different regions among Sprague-Dawley
rats, spontaneously hypertensive rats, and Wistar rats (48,
122, 137, 714).
With development of on-line measurements of flow
using positive emission tomography (PET) scans, and the
use of more detailed time course studies using iodoantipyrene, there is emergent evidence that blood flow varies throughout the penumbral region and also decays with
time, although the extent of decay varies widely in different models (79, 438, 798, 1297). The implications of this
decay are currently being considered. Clearly, it intensifies the insult in that region over time, and so may be
important in the development of penumbral damage
(325). It is considered in greater depth in the abovereferenced papers dedicated to flow measurements and
analysis.
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CEREBRAL ISCHEMIA ON NEURONS
4. Free radical changes in core and penumbra
In contrast to ion and metabolite changes, a recent
study demonstrated very little free radical change in the
core as compared with penumbra (1060). Free radicals, as
measured by hydroxylation of salicylate in microdialysates, increased early during ischemia in the penumbra,
remained elevated throughout 3 h, and then became further elevated with the onset of reperfusion. In marked
contrast, there was no increase in the core during 3 h of
ischemia. There was an increase in the core after ;4 h of
reperfusion. Thus the exposure of the core to free radicals
during the main period of damage development must be
considered minimal. The opposite is true of the penumbra.
G. Focal Ischemia: Development and Measurement
of Damage
Unlike global ischemia, which leads to neuronal cell
death in isolated regions, focal ischemia produces a contiguous mass of damaged brain tissue termed the infarct.
Rather than measuring damage by counting dead cells, as
in global ischemia, damage is generally expressed as the
volume of the infarct, for which there are standard measurement techniques (e.g., Ref. 860). This is a rough measurement in that the appearance of infarct is predicated
on a large fraction of the cells in the region being damaged; otherwise, individual cell damage is noted.
1. Development of the infarct over time
The gold standard of infarct delineation is a pallid
region when tissue is stained with hematoxylin and eosin
or pure Nissl stains (860). The pallor results from vacuolization of the neuropil, including swelling of glia, presynaptic elements and dendritic elements, and the loss of
Nissl staining in the neuropil (355). So identified, it signifies cell damage but not necessarily cell death. This is
exemplified by a delayed reduction in infarct size in animals in which iNOS is blocked (799).
In permanent ischemia, the infarct is first seen after
3–12 h (76, 137, 276, 355, 359, 814, 860). The wide time
range appears to reflect strain differences, with the early
infarct delineation seen in Sprague-Dawley rats (860) and
the later delineation seen in Wistar rats (355), possibly
because the lesion is less uniform in the latter (351, 355).
The infarct begins in the core but reaches close to its
maximal size, which includes core and penumbra, 6 –24 h
after the onset of ischemia. The infarct continues to grow
after 1 day, although at much slower rate, so that between
24 and 72 h it grows by ;30% in Wistar rats (355). Thus
damage continues to increase in penumbral regions.
Different durations of temporary ischemia lead to
graded involvement of different regions in the infarct as
measured after 1–2 days reperfusion (752). Ten to twenty
minutes produces scattered dead neurons in the core
(653, 752), whereas 1 h leads to infarct in the core (354,
526, 752). Infarct in the penumbra develops fully when the
temporary occlusion is 2–3 h (354, 526, 752). In this case,
the size of the infarct is the same as it is in permanent
ischemia (e.g., Refs. 752, 1283), although it develops
slightly more slowly (1283). After ;7 days, there is almost
complete loss of cellular elements in the infarcted region;
this is termed pan-necrosis. As little as 30 min of temporary ischemia also leads to an infarct in the core region,
but this only first appears after 3 days and takes 2–3 wk to
mature to its full size (276).
In summary, development of the infarct is quite rapid,
but it matures over several days, indicating a significant
delayed component of damage in tissue where the insult
intensity is relatively small. As indicated above, expansion of the infarct indicates expansion of the region in
which the large preponderance of cells are damaged. The
relationship of infarct to cell death is considered in section IIG.
2. Specific cell changes during development
of the infarct
Other changes associated with the infarct provide
more specific insight into the nature of the progression of
damage and provide other ways of delineating the lesion.
Diffusion weighted magnetic resonance imaging
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function at this point. Unfortunately, there are no reports
of oxygen utilization during the insult, which would allow
a more complete assessment of the rate of energy metabolism. Although penumbral blood flow declines during the
insult, it remains far higher than in the core; infarct develops despite this flow difference.
Levels of ATP during the reperfusion period after
temporary ischemia do not seem to recover much from
their ischemic values (489, 590), although further measurements are needed to confirm this conclusion. Glucose
utilization in some penumbral tissue is reduced to well
below normal during early reperfusion, and this is considered a reliable indicator of the tissue that will later
become part of the infarction (79, 1299). Presumably, the
low glucose metabolism indicates severe cell damage of
some kind at this early stage.
Thus, as with global ischemia, the core of the focal
lesion undergoes massive ion and metabolite changes that
recover quite strongly after a temporary insult. Despite
this recovery, it is almost impossible to prevent damage
by any intervention during the reperfusion period. It has
also proven extremely difficult to prevent core damage by
any single intervention, even before the onset of ischemia,
when ischemia lasts for 2–3 h. The penumbra can be
rescued by many interventions. These differences suggest
different mechanisms of damage in the two regions. As
with global ischemia, postischemic energy metabolism
appears to be compromised in both core and penumbra.
1441
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PETER LIPTON
3. Individual cell death within the infarct
It is critical to relate the infarct to cell death. Individual cell damage within the infarct develops slowly, but
more rapidly than delayed cell death in global ischemia,
suggesting that the prolonged focal insults have more
impact than the shorter, more intense global insults. The
best descriptions of this cell damage have come from
Garcia and co-workers (351, 355, 1283). Cell death, defined as eosinophilic shrunken neurons (advanced ischemic cell change) or neurons showing autophagocytotic
morphology (178), and also neurons showing apoptotic
morphology (178, 652) (see Fig. 1), first becomes significant at ;6 –12 h in the core of the lesion, with a major
increase between these two time points. At 12 and 24 h,
;80% of the neurons have suffered cell death. A similar
although less extensively described time course is seen in
spontaneously hypertensive rats after 80 min of ligation
(202). Although development of cell death in the penumbra appears to lag development in the core, it is hard to be
sure, from the data provided. A less-detailed time course
study found that 2 days after 2-h focal ischemia, 55% of
the cells in the core and penumbral infarcts were dead,
whereas 30% were damaged but did not show signs of
death; 15% appeared healthy. These may represent different cell types, but that was not discussed (656).
At least for the first 72 h, the rate and extent of
developments of cell death caused by 2-h temporary ischemia are the same as they are for permanent ischemia
(1283). Furthermore, the number of apoptotic cells in the
penumbra is the same in the two models throughout this
72-h period (785).
4. Evaluation of protective treatments
in focal ischemia
Inferred mechanisms of focal ischemic damage are
almost always based on the assumption that when a protective treatment reduces infarct size it is blocking the
process of ischemic cell death in neurons.
For the early infarct (before pan-necrosis), on which
most measurements are made, the implicit assumption is
thus that the uniform pallor and other indicators within
the infarct largely result from neurons so severely damaged that they will die or have already died. Overall, this
is justified in that a large percentage of neurons within the
infarct die after 24 –72 h, at least in Wistar rats. Thus, if a
treatment reduces the radius of the early infarct, it is very
likely to be slowing down cell death in penumbral neurons. On the other hand, in at least one case, infarct size
was reduced after 48 h when delayed protective treatments were applied (1278), indicating that the majority of
cells at the borders of the infarct are not yet dead after
48 h (1278). Overall, it is most satisfactory to measure
infarct size after 72 h, but this is often dificult to do.
In general, there may not be a precise 1:1 correspondence between infarct size and the amount of cell death.
Infarct requires a certain percentage of the cells to die (as
witnessed by the peri-infarct area that has cell death but
no infarct), indicating that the infarct in a certain region
may be abolished but that significant cell death may well
remain. It would be much more useful for determining
cell death mechanisms if cell counting as well as infarct
size were used to assess cell death whenever possible.
This being said, the infarct size provides a reliable semiquantitative measure of the extent of neuronal death and
is far easier to measure than individual cell death.
5. Full development of the infarct into pan-necrosis
The final stage of infarct development in focal ischemia is pan-necrosis, in which the neuronal death is accompanied by glial and vascular cell death and loss of
cellular elements. This does not occur in most global
models that lead to widespread neuronal death, although
it does occur in extremely insulting conditions, such as
low pH (522) or a prolonged very intense insult (259). It
occurs about 1 wk after most focal insults. The basis for
pan-necrosis is not clear, but for some reason, both the
endothelial and glial cells become major targets, suffering
early changes and eventual death (914). In hyperglycemic
global ischemia, which leads to infarct, glial pH falls to 4.3
(607), a level that can kill these cells (608). No measurements were made of endothelial cell pH. It would be very
useful to measure focal ischemic effects on glial (or endothelial cell) pH to see whether drastic lowerings might
explain development of the infarcts in those conditions.
During prolonged hypoxia/ischemia, which also leads
to infarct, there was an early activation of pinocytotic
transport across the endothelial layer, which was quite
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(DWMRI) delineates the core of the (future) infarct as
early as 1 h after the onset of ischemia, and at later times,
DWMRI quite faithfully defines the developing infarct
(151, 362). It is very likely that the altered signal intensity
represents regions of tissue in which there is intracellular
edema (nicely discussed in Ref. 362).
Staining frozen sections with 2,3,5-tetraphenyltetrazolium chloride (TTC) (359), which is colored red by
electron transport in active mitochondria, defines a region
in which mitochondrial function is severely compromised. Damage is not necessarily irreversible. The region
is slightly smaller than the infarct size defined by hematoxylin and eosin (H and E) at early times but overlaps the
H and E staining at 24 h (955).
Finally, the loss of mitogen-activated protein (MAP) 2
immunostaining, which is almost certainly a reflection of
increased cytosolic Ca21 and activation of calpain (Y.
Zhang and P. Lipton, unpublished data) quite faithfully
follows the development of the infarct as defined by H and
E staining (233). Thus several markers of cell dysfunction
parallel the expansion of the infarct as measured by loss
of Nissl stain, indicating that the cellular elements that are
poorly stained are metabolically compromised.
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CEREBRAL ISCHEMIA ON NEURONS
specific to conditions in which infarct was developing
(895, 898). This might be important, but it is not known
whether it occurs in focal ischemia. Direct exposure of
brain to free radical-generating systems leads to infarction-like changes (174), suggesting that prolonged production of free radicals may be leading to pan-necrosis, but
there is no explicit evidence. Thus there are reasonable
lines of investigation of the pan-necrotic mechanism. It
does not seem to relate to neuronal death because it
occurs so late, but it might.
H. Role of Intraischemic Depolarizations in Focal
Ischemic Damage
very useful to measure the effects of the artifactually
increased frequencies of intraischemic depolarizations on
ATP levels to see if there were a correlation between the
latter and the increased damage.
I. Role of Leukocytes in Focal Ischemic Damage
One of the most intriguing lines of study to develop
over the past several years has been three lines of evidence that white blood cells contribute to focal ischemic
damage after temporary insults (41, 204, 248, 431, 592).
These are 1) the appearance of neutrophils in ischemic
tissue at an appropriate time; 2) the protection afforded
against damage by agents that should prevent the accumulation or activation of neutrophils and 3) known damaging actions of these cells when they accumulate in
tissue (1200). Effects of various manipulations suggest
that the neutrophils are important in temporary, but not
permanent, focal ischemia (203, 775, 1285). The evidence
is generally strong and is bolstered by the probable involvement of neutrophils in ischemic injury in other tissues (399, 933). However, there are important conflicting
reports and alternate interpretations of data that need to
be considered. Furthermore, it remains to be determined
whether damage results from effects on blood flow or
from direct toxic effects on the vasculature or the neurons.
1. Accumulation of neutrophils in ischemic tissue
There is an accumulation of neutrophils in infarcted
tissue 24 h after permanent or temporary ischemia (63,
120, 542, 1244). At this time, neutrophils are present in the
vasculature and have, in most cases, infiltrated the parenchyma (63, 353). A very notable exception to the latter is
human stroke where neutrophils accumulate early in severe cases (9) but, in the one relevant study, were almost
exclusively confined to the vessels even more than a day
after the stroke (665).
If it helps cause neuronal damage, neutrophil accumulation should be present well before 24 h after ischemia, and this does seem to be the case. Polymorphonuclear leukocytes accumulate in blood vessels within 4 h of
the onset of permanent (353, 592), or temporary (563)
ischemia (the latter measurements made in baboons).
Perhaps the most striking evidence correlating neutrophil
accumulation with damage emerges from studies using
PET scans on humans. Accumulation begins within 6 h of
the stroke, and there is a striking positive correlation
between the amount and duration of leukocyte accumulation and both the size of the infarct and the severity of
neurological outcome (9).
An important issue is whether the neutrophils act
after infiltration through the blood vessels. Significant
infiltration into the parenchyma only begins 12 h after the
onset of permanent occlusion, by which time there is
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The bases of the intraischemic depolarizations or
spreading depression-like depolarizations (818) discussed
previously are not completely known; they may well be
the spread of increased extracellular K1 and glutamate
from the core of the lesion (463, 818). Their frequencies
are halved by either NMDA or DL-a-amino-3-hydroxy-5methylisoxazole-propionic acid (AMPA) antagonists, indicating that glutamate-mediated depolarization contributes markedly. The evidence linking the development of
damage in the penumbra to these depolarizations is extensive but is largely correlative (47, 151, 462, 463, 818).
For example, factors that reduce the frequency of the
intraischemic depolarizations, such as glutamate antagonists and hypothermia (462, 818), as well as blockade of
iNOS (1031), reduce the size of the infarct that develops in
the penumbral region. The most persuasive direct evidence that they are damaging is that artifactually increasing the numbers of depolarizations during ischemia, by
about twofold, using either KCl (151) or electrical stimulation (47), increased the size of infarct, and peri-infarct
damage, by 30 –100%, respectively. In one case, this was
measured 24 h after distal MCA occlusion and in the other
2 h after the onset of proximal MCA occlusion, using
DWMRI (255). Thus the artifactual depolarizations do
appear to be damaging in ischemic conditions, and this
suggests that the endogenous depolarizations are also
damaging. Unfortunately, it is very difficult to specifically
target the latter with a drug, to more directly establish
their importance.
The basis for the putative damaging effect of the
depolarizations is not known; similar magnitude and duration depolarizations are not damaging in nonischemic
tissues (818), although in those cases there is an increased blood flow that may help to maintain energy
levels. One possibility is that they lower ATP levels because blood flow is reduced and that this is damaging.
There is evidence, from an abstract published several
years ago (1101), that the depolarizations are responsible
for the reduction in ATP in the penumbra. However, ATP
only falls to ;70%, and it is not clear at all that this would
be damaging during the 5-min depolarizations. It would be
1443
1444
PETER LIPTON
2. Mechanism of neutrophil accumulation
Neutrophil accumulation in vessels requires interactions between several adhesion molecules. These include
intracellular adhesion molecule (ICAM)-1 and E- and
P-selectins on the endothelial cells, fibronectin and laminin
in the extracellular matrix, and integrins and L-selectins on
the white blood cells. There are excellent short reviews of
the process (204, 964, 1216) and, indeed, antibodies and
peptides that bind to these molecules profoundly inhibit the
accumulation of neutrophils after transient or permanent
ischemia (197, 1243, 1245, 1285, 1287).
Ischemia activates this adhesion system. On endo-
thelial cells, ICAM is increased 2 h after 2-h MCA
occlusion (542, 1286) and also after human stroke
(665). E-selectin is upregulated some time between 4
and 24 h (427, 1287). On the neutrophils, the integrin
complex CD11/CD18 may also be upregulated, although
this is less certain (63).
There is reasonable but very incomplete evidence that
one or both cytokines, interleukin (IL)-1b and tumor necrosis factor-a (TNF-a), which do upregulate ICAM (965), are
mediators of this response. It seems likely that there is a
cascade involving free radical generation, increased TNF-a
and IL-1b, and the activation of the nuclear transcription
factor NFkB. (1186). The NFkB upregulates more proximally than the cytokines and is probably a critical intermediary step. Other factors are also probably necessary (56).
There may be important positive feedback between the cytokines and NFkB and free radicals as they all are able to
activate each other (1186), and it is not clear which of these
is the initiating step.
Tumor necrosis factor-a is increased within 30 min of
the onset of focal ischemia in the circulation and in brain
tissue (154, 636), as is IL-1b (1295). The only study directly assessing the importance of the cytokines showed
that treatment with the IL-1 receptor antagonist reduced
accumulation of neutrophils during permanent MCA occlusion (352). Less directly, but still significant, blockade
of TNF-a action with a recombinant receptor greatly enhanced the number of perfused microvessels at the end of
4-h permanent MCA occlusion (412).
The postulated interaction is supported by studies in
cultured endothelial cells (1035). Hypoxia induced the
formation of IL-1, the subsequent expression of ICAM and
E-selectin, and increased adherence of leukocytes. Antagonizing IL-1 action attenuated leukocyte adherence by
;60% (unfortunately adhesion molecule changes were
not explicitly measured). A similar effect was observed in
microvascular cells from human brain (1068). Further
studies on these cells show that activation of the transcription factor NFkB is very probably critical to the
expression of ICAM. Hypoxia and reoxygenation caused
activation of NFkB and, later, ICAM (464). Both these
responses were blunted by the proteasome inhibitor ntosyl-Phe-chloromethylketone (464), which blocks the activation of NFkB. The ICAM upregulation was also
blocked by the free radical scavenger pyrrolidine dithiocarbamate, and cerebral endothelial cells in culture produce abundant free radicals under these conditions
(1226).
Although evidence for protective effects of blocking
cytokine action is now abundant (see sect. VIIIE), this
cannot be taken to show that leukocyte accumulation is
damaging; blockade of cytokines reduces tissue damage
even in permanent ischemia (352, 946), against which
adhesion antagonists are ineffective (203, 775, 1285).
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major cell necrosis (353, 1283), but it is more rapid following temporary ischemia, the insult for which there is
evidence that neutrophils are damaging. There was significant accumulation 6 h after 1- to 2-h occlusion in two
different studies (202, 542, 1283), which increased by 50%
at 12 h (1283). This time course is consistent with parenchymal neutrophils causing cell damage. However, in one
of the studies, the location of the early accumulation was
not described (202) and in the other the accumulation did
not reach significance in cortex at 6 or 12 h (1283); cortex
is the region in which damage is prevented by antileukocyte treatment. Thus there is no well-documented evidence that accumulation in the parenchymal tissue is
likely to be causing damage.
In contrast to the large number of results showing
timely accumulation of neutrophils in at least the vasculature, Bartus and co-workers (435) found that there was
no significant neutrophil accumulation until 21 h after 2-h
distal MCA occlusion occlusion despite the occurrence of
infarct by 8 h. There is other evidence that neutrophil
accumulation does not always accompany cell damage.
Two very careful sets of studies on spontaneously hypertensive rats, using essentially identical methodologies,
showed either a large neutrophil accumulation 6 –12 h
after 80 min of MCA occlusion in the penumbral tissue
(63, 202) or no neutrophil accumulation except in severely damaged tissue after 60 min of MCA occlusion
(642). In the latter, there was a major activation of microglia/macrophages ;6 h after the occlusion. Thus as yet
unidentified factors may determine the nature of the inflammatory response to an insult.
It has been suggested that artifactual mechanical
effects of the insult may actually cause the neutrophil
accumulation (for example, filament occlusion or laser
irradiation) (435), and this has not been ruled out by
careful studies. However, neutrophils do accumulate in
the vasculature after human stroke (9, 665), where
there is clearly no mechanical artifact. At present, there
is no simple explanation for the conflicting data on
neutrophil accumulation in rodents. Certainly they suggest that these white blood cells may not always be
important in damage.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
1445
4. Mechanisms of damage
A very large number of studies show that conditions
that reduce or completely block leukocyte accumulation
greatly reduce infarct size. Infarct volume 24 h after 45- to
60-min ischemia was reduced by about two-thirds when
circulating neutrophils in mice (215) or in rats (729) were
completely depleted with an antineutrophil antibody. Inhibiting the activity of the endothelial adhesion molecule
ICAM, either by creating null mice (215, 1062), or using
monoclonal antibodies in rats (197, 730, 1285), strongly
reduced neutrophil accumulation and reduced infarction
size by 50% (rats) to 75% (mice). Slightly smaller reductions, ;33%, were produced in rats by antibodies to the
neutrophil integrins CD10/CD18 (197, 730) and by peptides that interfere with fibronectin binding (1243, 1244)
or laminin binding (1245). A peptide that binds to selectin
(775) decreased infarct volume by .50% in spontaneously
hypertensive rats. Many of these studies involved 45-min
to 1-h exposures to ischemia, but in some, ischemia was
2 or 3 h so the protective effects are not restricted to short
insults. These effects are quite dramatic; there is essentially no further development of infarct once reperfusion
begins.
Importantly, leukocytes do not appear to be involved
in damage during permanent focal ischemia, despite their
accumulation in the tissue vasculature (120, 542). Several
interventions against this insult, which are protective
against temporary ischemic damage, were ineffective in
preventing damage (203, 775, 1285).
Although these studies taken together are impressive
indictments of neutrophils in ischemic damage, there is at
least one exception, which has not been explained away.
In the study where no neutrophil accumulation was measured, neutropenia had no protective effect (435). Because of this, it is important to consider alternate explanations for the effects of the antileukocyte accumulation
studies.
Neutrophil depletion may be protective as a result of
lowering blood levels of damaging cytokines, TNF-a and
IL-1a, that can be produced by these cells (280). Measurements of the effects of neutropenia, or other protective
treatments, on cytokine levels during ischemia would
help interpret results. The adhesion molecules and basal
laminar proteins may also allow critical damage to vasculature or neurons in ways other than via leukocyte
accumulation. Indeed, there is a marked depletion of
lamin and fibronectin that is thought to contribute to
microvascular damage (416). Perhaps the laminin and
fibronectin peptides that attenuate damage (1243, 1245)
are acting by maintaining vascular integrity. Although
these explanations are not particularly likely, it is important to ensure that they, or others, are not correct. This
has not been done.
Leukocytes might induce damage by causing local
vascular occlusion, or they might initiate toxic reactions,
including free radical production by NADH oxidase, production of hypochlorous acid, or protease activation (504,
728, 1200). The latter two require prior free radical production.
A) CHEMICAL TOXICITY. Efforts to determine whether free
radical production is important have not been conclusive.
The one study in which effect of neutrophils on free
radicals was actually measured, carried out by Kogure
and co-workers (728), showed that neutropenia completely abolished the generation of free radicals in cortex
after 60-min MCA occlusion. This very dramatic effect
was seen by measuring formation of ascorbyl free radicals
(728), a reliable semiquantitative method (143). Unfortunately, this is the only such report, and the only study in
which this method has been used to measure free radical
formation. Further experimental support for the conclusion is very necessary as it seems, a priori, unlikely that
neurons and glia themselves do not produce free radicals
during and after ischemia. If it is correct, it ascribes a very
important role to neutrophils in damage, as free radical
attenuation produces major reductions in infarct size.
However, at this stage, it cannot be considered likely.
Neutrophils produce free radicals via activation of
NADPH oxidase. Walder et al. (1185) studied transgenic
mice lacking NADPH oxidase. Infarct size was reduced by
50%, suggesting that leukocyte NADPH oxidase mediated
the damaging effect of the neutrophils. However, Walder
et al. (1185) then found that selective elimination of either
leukocyte or parenchymal NADPH oxidase did not reduce
damage; both had to be eliminated. Thus elimination of
neutrophil NADPH oxidase does not account for the protective effects of eliminating neutrophil accumulation; if it
did, then eliminating the enzyme selectively in neutrophils
would have prevented damage.
An hypothesis that is consistent with both sets of
studies is that neutrophils are necessary for the generation of toxic levels of free radicals but themselves are not
the major source of the free radicals. Thus large-scale free
radical production would be dependent on leukocytes, as
noted by Kogure and co-workers (728), but could be
initiated by NADH oxidase in parenchymal tissue or in the
leukocytes. The way in which leukocytes might lead to
free radical generation is unknown. It might be by producing cytokines (280, 349) which then activate free radical and NO production by parenchymal tissue or by
leukocytes themselves (177, 671). Certainly, the data of
Walder et al. (1185) suggest a central role for NADH
oxidase. Unfortunately this has not been explored further.
B) INDUCIBLE NOS. The inducible form of the enzyme,
iNOS, begins to appear in neutrophils that have invaded
the ischemic tissue between 1 and 2 days after the
onset of permanent focal ischemia (480). Any role for
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3. Effects of preventing leukocyte accumulation
1446
PETER LIPTON
5. Role of leukocytes and microglia in global
ischemic damage
There is no evidence that leukocytes enhance damage after normal short global ischemic episodes. There
was a delayed accumulation of leukocytes 3 h after a
global insult, but only if ischemia was extended to 40 min
or longer, far longer than the 10 min necessary to get
major damage (20). When neutropenic rats were challenged with 15-min global ischemia, there was elevation
of blood flow in the postischemic period relative to control rats, but there was no amelioration of damage to CA1
or CA3 neurons (1007).
On the other hand, there is proliferation of microglia
in damaged regions within a day after short-term global
ischemia, which is prolonged in regions that will go on to
suffer damage (512). Intringuingly, when proliferation of
the microglia is prevented by tetracycline derivatives,
minocycline, or doxycycline, damage after 5-min ischemia
in gerbil is attenuated by 75% (1265). Although this is
consistent with an important role for microglia in damage,
it does not prove it. Nevertheless, the fact that microglia
are major sources of caspase-1 (ICE) by 2 days after
ischemia (94) and so may contribute to IL-1 production
and iNOS production (1265), indicate that these cells may
play an important role in damage (512).
6. Summary
The large number of cases in which agents that prevent leukocyte accumulation also attenuate damage in the
penumbra is strong evidence that this accumulation
somehow makes a major contribution to damage after
temporary focal ischemia. The activation of accumulation
is probably initiated by ischemia-induced cytokine synthesis in endothelial cells or glia, although the evidence
here is not extensive. The trigger for the very early cytokine synthesis is not known.
There are two caveats to the conclusion. The first is
the existence of one very well-documented case and one
somewhat less well-documented case in which there is no
neutrophil accumulation, and in which neutropenia had
no protective effect. This indicates that accumulation
does not always occur and that it may depend on the
nature of the insult in ways that have not yet been determined. A systematic study of neutrophil accumulation
and damage size following different methods of occlusion
would be very helpful. Although artifact may contribute to
accumulation, the accumulation in (nonartifactual) human stroke certainly shows that the phenomenon is a real
one in important conditions.
The second uncertainty stems from the difficulty in
pinpointing a mechanism of damage. This results firstly
from the lack of a definitive answer as to whether there is
a timely invasion of the parenchyma so that it is not
known where the leukocytes are acting. Second, it has not
been determined whether free radical (or other biochemical) action, or effects on flow are the damaging principles, or whether blood vessels or neurons are the targets
of any biochemical action.
A simple model that reconciles some difficulties is
that leukocyte accumulation acts as a “catalyst.” In this
model, activated leukocytes initiate damaging processes
in parenchymal cells or blood vessels by early production
of cytokines or other agents, rather than by producing
free radicals or by blocking vessels. In this case, actions
on flow and on free radical production might mediate
damage. Microglia/macrophages may play the damaging
role that neutrophils play in some focal models and also in
global ischemic damage, but there is no direct evidence
for this.
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this iNOS is very questionable because blocking neutrophil accumulation has no effect on permanent focal
ischemic damage (1285). Thus the fact that aminoguanidine (AG), which inhibits iNOS, attenuated the increase in infarct size that occurred between 24 and 72 h
(481) may well be due to inhibition of the iNOS induced
in endothelial cells (479, 836). Alternatively, AG may be
protective because it is inhibiting another enzyme, for
example, polyamine oxidase, which is very damaging
(208, 498). Although protection was eliminated by the
NO precursor arginine (481), this does not necessarily
show that inhibition of NOS by the AG was giving the
protection. The excess NO production due to arginine
might have overwhelmed the effects of inhibiting another damaging reaction.
Alternatives to free radical-mediated damage are
damage mediated by hypochlorous acid produced by myeloperoxidase, or protease activation; they have not been
explicitly tested.
C) BLOOD FLOW REDUCTION. The alternative to chemically
mediated damage is that leukocytes block blood flow (no
reflow). White cell accumulation does appear to attenuate
blood flow in the early postischemic period (63, 775).
However, evidence that this is damaging is not at all
compelling because, as discussed in section IIK, there is
no evidence that postischemic penumbral blood flow is
limiting. One hour after 3-h ischemia, 40% of the small (,6
mm) capillaries appear to be plugged by leukocytes (247)
but at many later time points after 2-h ischemia there was
much less vessel plugging (;10%) in core or penumbral
regions (1283). Indeed, plugging was far less than during
permanent ischemia, where it was ;50% (353, 1283).
Thus, if vessel plugging is important, it is surprising that
antileukocyte adhesion paradigms do not reduce infarct
size in permanent ischemia.
Hemispheric blood flow 24 h after ischemia was improved by antileukocyte treatment (e.g., Ref. 215). However, this may well reflect the attenuation of infarct size
and hence more actively metabolizing tissue in the protected animals.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
J. Hypoxia/Ischemia
K. Postischemic Blood Flow and Damage
1. Global ischemia
There are major changes in brain blood flow after
global ischemia. It is important to know if they exacerbate
damage because drugs used to study damage mechanisms
often affect blood flow also.
There is a rapid hyperemia following vessel occlusion or complete ischemia, which lasts ;15 min, where
blood flow may be as much as a two- to threefold higher
than control levels (228, 515, 931). There is then almost
(1180) always a marked hypoperfusion that lasts between
6 and 24 h, with blood flow between 30 and 50% of normal
(228, 291, 297, 515, 632, 760, 931, 1070).
The literature strongly suggests that this postischemic hypoperfusion does not cause damage; rather, it
appears to reflect a reduced metabolic rate in the postischemic tissue. The latter may reflect mitochondrial damage or reductions in ATP-utilizing processes. Whatever its
basis, there is an excellent correlation between the lowered glucose utilization and the hypoperfusion after
global ischemia in dog (291, 756) and rat (606, 931). pH,
ATP, and lactate levels are about normal (228). Neutrophil
depletion had no effect on the marked hypoperfusion in
hippocampus and striatum, two very vulnerable structures (396), further indicating that the hypoperfusion in
these vulnerable regions is metabolically driven. Neutro-
phil depletion did increase flow somewhat in nonvulnerable structures (396).
In contrast to the above studies, when ischemia is
prolonged beyond the time required to cause major neuronal death there is a larger reduction in blood flow than
in glucose metabolism or high-energy phosphate utilization, in tissues which will become damaged (367, 645,
646). In one of these cases, the region that suffered damage, the striatum, was the region with the largest dissociation between flow and metabolic rate. Although the mismatch may contribute to damage in extreme cases,
shorter ischemic exposures are lethal in the absence of
any mismatch between flow and metabolism.
2. Focal ischemia and hypoxia/ischemia
There is no compelling evidence that reduced postischemic flow following temporary focal ischemia contributes to development of the infarct. Core regions have
normal blood flow levels (137, 798), whereas flow in the
penumbra is generally ;40 –50% of normal during the 12 h
after temporary focal ischemia, when damage is developing (184, 798). However, as with global ischemia, there is
no evidence for a mismatch between flow and metabolism. In penumbral regions destined for infarct, glucose
utilization is decreased by 50% or more in the first hour
after ischemia, and recent detailed studies show a strong
correlation between the decrease in blood flow and in
glucose metabolism in the postischemic period (79, 1299).
In the few measurements that have been made, ATP levels
during this time are between 70 and 100% of their preischemic values, only a small reduction. One study showed a
reasonable correlation between increased postischemic
blood flow and reduction of early infarct size when prostaglandin synthesis was inhibited (184). However, there
are many other potentially beneficial effects of blocking
prostaglandin metabolism aside from vasodilation, for example, preventing free radical production. Thus the experiments do not establish the importance of postischemic blood flow, and the general preservation of
metabolism-to-flow ratios argues against reduced flow as
a cause of damage. This was also discussed in section III.
3. Summary
At this stage, there is no evidence that damage is
increased by reduced blood flow, after either global or
focal ischemia. The evidence is quite consistent with reduced postischemic metabolism driving the decreased
blood flow. Thus increasing blood flow in the postischemic period should not, in and of itself, be protective.
This conclusion should not obscure the potentially
critical sensitivity of damage to blood flow during the
insult, particularly in focal ischemia where the penumbra
has flow rates that range from very damaging to barely
damaging. Effects of drugs on flow rates during ischemia
may be critical to damage.
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Unilateral carotid occlusion is combined with hypoxia. In adults, the oxygen is lowered to ;3% and leads
to eventual infarction in the MCA territory. It is essentially a focal insult that is now used infrequently (895)
because adult animals do not survive the prolonged
hypoxia reliably enough (948). However, the technique
has been very successfully adapted to neonates that
can reliably survive for days after insults of up to 3.5 h,
and it is considered a very good model for major forms
of neonatal metabolic brain damage (1164). Oxygen is
generally lowered to 8% (for review, see Refs. 948, 959,
1141). This is almost a pure hypoxic insult, because the
hypoxic vasodilation brings blood flow up to near control levels on the ligated side (982), although lateral
cortex and caudoputamen still show blood flow reductions (368).
Fifteen to thirty minutes of hypoxemia (3%) causes
early ischemic cell change and delayed neuronal death in
CA1-CA3, striatum, and layer 5 of cortex in adults, much
like global ischemia (980). In young rats, exposures of 60
min or longer to 8% hypoxia cause delayed development
of infarct providing ATP levels fall to between 50 and 70%
of control values (1213), much like their values in focal
ischemia.
1447
1448
PETER LIPTON
L. Effects of Ischemia on the Vasculature
The role of vascular change, and resulting edema,
will not be considered at any length in this review because
it really is its own topic. Nevertheless, it is useful to have
a general idea of what contribution vascular changes may
make to damage. Certainly capillary endothelial cells are
quite vulnerable to short periods of anoxia and reperfusion. Cultured brain endothelial cells are damaged in a
free radical-dependent manner after 20 min of anoxia, as
assessed by a large increase in lactate dehydrogenase
(LDH) leakage (1226).
The blood-brain barrier becomes severalfold more
permeable to poorly permeant small solutes such as inulin
and sucrose 6 h after global ischemia but then returns to
normal by ;24 h (922). There is little evidence of major
uptake of protein into the brain during much of the postischemic period, except immediately after the insult.
However, starting ;12–24 h before delayed cell death,
there is a quite massive uptake of horseradish peroxidase
or protein-bound Evans blue into the parenchyma (370).
This may well result from activation of pinocytosis in the
endothelial cells because there is no major extracellular
edema and there are no reports of major cell damage to
the endothelium. There is no indication as to whether the
increased blood-brain barrier transport contributes to
damage.
2. Focal ischemia
Focal ischemia is quite different. There is a major
extracellular edema that develops early during the focal
ischemic insult, largely due to activation of Na1 transport
across the blood-brain barrier (91). Reperfusion and very
prolonged permanent ischemia both cause much more
dramatic changes. There is damage to the endothelial cell
layer, along with extravasation of protein and proteinbound Evans blue, as early as 30 min after recirculation
following 60-min ischemia. This does not occur during at
least 6 h of permanent ischemia (527, 800, 1247). The
dependence of this vascular damage on reperfusion is
striking; it may result from enhanced free radical production (849), but this has not been proven. This endothelial
transport change very probably contributes to edema also
(1247).
The relationship of the vascular damage and extracellular edema to cell death has not been well studied.
Some evidence indicates that at least early neuronal death
occurs in the absence of vascular changes. In a study
specifically directed at this question, there was significant
ischemic cell change 6 h after 1-h focal ischemia in caudate putamen, but there was no evidence for extravasation of albumin, or leakage of the smaller molecule, ami-
nobutyric acid from the circulation into the parenchyma
of the caudate-putamen at that time (10).
On the other hand, recent studies suggest that movement of molecules from blood to brain across the “damaged” endothelium are important in the general development of neuronal damage. A matrix metalloproteinase,
MMP-9, was upregulated in endothelial cells during the
first 24 h of permanent ischemia, probably by cytokines,
and when an antibody to this enzyme was administered
systemically, it reduced infarct size after 24 h, by 30%
(958). Both MMP-9 and other matrix metalloproteins act
on the extracellular matrix associated with the vascular
basement membrane, and it is reasonable to assume that
protection by the antibody results from inhibition of basement membrane breakdown and maintenance of vascular
integrity. Further studies are needed to show that vascular integrity is indeed maintained when the enzyme is
inhibited. It would be of much interest to make a similar
study in temporary ischemia, where vascular integrity
seems to be most compromised.
Although there is no established causal relationship
between edema and neuronal damage, there is a very
strong correlation between infarct size and the amount of
hemispheric edema when protective, or damaging, agents
are used to alter cell death (e.g., Ref. 527). Indeed, edema
is often used as a measure of damage. Effects of edema on
blood flow might be important; for example, it may partially account for the progressive decrease in blood flow
in the penumbra during occlusion, discussed above. However, at this stage there is no evidence that edema contributes directly to cell damage. Increased edema may
simply reflect local blood-brain barrier damage caused by
the damaged neurons, glia, and endothelial cells.
M. Effects of Temperature on Damage
1. Artifactual maintenance of temperature
There is little spontaneous change in brain or core
temperature during focal ischemia except in the filament
occlusion model discussed above, where temperature
rises.
However, global ischemia is very different. Brain
temperature in gerbils and rats drops spontaneously by
3–5°C (152, 762, 1241). Core temperature drops by about
the same amount in rats, but actually rises somewhat in
gerbils (211, 584, 628). Temperatures recover within ;1
min of reperfusion (152, 762), although in gerbils there is
a short period of hyperthermia beginning ;15 min after
ischemia and lasting ;45 min. It is 1.5°C in anesthetized
and 2.5°C in unanesthetized animals (768). Because of its
strong effect on damage, brain temperature should be
known or regulated during and for at least 24 h after an
insult (see below). This means maintaining the temperature of the brain at a known level during the insult, with
a lamp or other device, and it means maintaining or
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1. Global ischemia
Volume 79
October 1999
1449
CEREBRAL ISCHEMIA ON NEURONS
TABLE
2. Effects of temperature on damage
Damage in CA1, %
Protocol
37°C
35°C
33°C
15 min of 2-VO in rat (763)
20 min of 4-VO in rat (152)
5 min of 2-VO in Gerbil (228, 1241)
After 20 min of 2-VO in rat (180, 210)*
95
93
80
95
70
20
10
0
45
60
Unless noted otherwise, temperatures are intracranial, during ischemia.
* In this study core temperature was reduced, between 12 and
17 h after ischemic episode. Damage was measured as loss of CA1
pyramidal cells 3–7 days after ischemia. Reference numbers are given in
parentheses.
N. In Vitro Models: Brain Slice
1. Nature of insult
2. Effects of brain temperature on damage
Table 2 shows the dramatic effects of different brain
temperatures during or after global ischemia on the degree of postischemic damage.
Not shown in Table 2, damage is actually exacerbated when brain temperature is maintained at 39°C
(258).
Effects of temperature are manifested for long periods after an insult. Hypothermia is protective when it
occurs up to 12 h after 2-VO, and if temperature is elevated to 39°C even 24 h after mild (5–7 min) 2-VO, global
ischemia damage is strongly enhanced (49). Thus even the
delayed processes involved in cell death are remarkably
temperature sensitive. Temperature differences during
the insult do not just influence the rate of appearance of
damage; they affect the final outcome. For example, hypothermic protection lasts for at least 1 mo in adults and
6 mo in young gerbils (219). Temperature thus strongly
determines the damaging potential of insults.
Temperature is also important in focal ischemic damage. Mild hypothermia reduced infarct size by 35% during
12-h permanent MCA occlusion (53). Reducing temperature to 30°C for 1 h after 2-h temporary focal ischemia
reduced infarct size, measured after a week, by 50%
(1284). Hypothermia (34°C) was very protective against
3-h hypoxia/ischemia in young rats (1235).
These results show that at least one of the major
processes involved in ischemic damage is extremely
sensitive to temperature. Furthermore, the very longlasting sensitivity discussed above indicates that a process occurring at least 24 h after the insult is also
temperature sensitive. Unfortunately, as is discussed in
several of the ensuing sections, a great many potential
contributors to ischemic damage are very sensitive to
temperature so that no particular mechanistic information is provided by the dependence (see Ref. 62 for a
good discussion of this).
Brain slices, and particularly the hippocampal slice,
have become widely used models for studying anoxic or
ischemic damage (531, 673, 686, 695, 1008). In this insult the
bathing solution is rapidly changed from O2/CO2 equilibrated
to N2/CO2 equilibrated. When glucose is maintained in the
anoxic buffer, the insult is termed anoxia (or hypoxia), and
when glucose is omitted, the insult is termed in vitro ischemia or oxygen/glucose deprivation. Adenosine 59-triphosphate falls less completely during in vitro ischemia than it
does during global ischemia and falls more slowly in the
presence of glucose (Table 1). Lowering temperature reduces the degree of damage (949).
2. Nature of damage: comparison with damage in vivo
Generally 5–7 min of in vitro ischemia at 36 –37°C, or
a period of ischemia extending 2–3 min beyond the anoxic
depolarization, leads to rapid damage to various properties of CA1 pyramidal cells which lasts for the 8- to 14-h
life of the slice. Somewhat longer exposures are necessary for damage to dentate granule cells so that slices do
show the same ranking of selective vulnerability as in vivo
tissue. However, differences are not nearly as dramatic
because acute damage, rather than prolonged damage, is
being measured (532), and acute damages are very similar
in different hippocampal regions in vivo (348).
Properties that are damaged include synaptic transmission (531, 1008), protein synthesis (164, 936), maintenance
of ATP levels (916), cytoskeletal integrity (1291), and neuronal morphology (936). Damage to all these occur within
the first 30 min and persist throughout the reperfusion period (286, 348, 916). Changes in protein synthesis are similar
to those observed in vivo, but the profound loss in (evoked)
synaptic transmission and the very intense morphological
damage and disruption of the cytoskeleton are generally not
observed as early or as strongly in vivo, indicating that slices
are more sensitive to ischemic damage than are cells in situ.
Nevertheless, slices fulfill a very important role by facilitat-
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knowing body temperature after the insult. These precautions eliminate the possibility that treatments will affect
brain temperature during the insult via changes in flow or
other variables and eliminate the possibility that they will
affect core temperature, and hence brain temperature,
after the insult. Many agents do the latter when the animal
is not regulated. Brain temperature is most faithfully monitored in the temporalis muscle (152). However, when it is
warmed by a lamp, the skull faithfully remains ;1–2°C
below brain temperature (211, 762) and is easier to monitor. Although brain temperature is now generally well
regulated during an insult (152, 211, 762), there is quite a
lot of variability in the duration for which core temperature is maintained after an insult, and this seriously compromises some studies. These cases are pointed out in the
review.
1450
PETER LIPTON
ing analysis of mechanisms of early changes. Although it is
always possible that these are different from in vivo, rates of
change of ions, metabolites, and protein synthesis that have
been measured so far are quite similar to those measured in
vivo, suggesting that mechanisms are the same.
Shorter insults, which are stopped just when the
anoxic depolarization occurs, lead to a more slowly developing damage, requiring ;12 h to be manifested. This
is closer to the delayed neuronal death seen in vivo. To
date, only damage to synaptic transmission has been studied in this way (1188), but the approach holds much
promise for more sophisticated comparisons between
slice and in vivo conditions.
1. Different models
Primary neuronal/glial cultures from cortex (237,
376), hippocampus (704, 1199), cerebellum, and hypothalamus (791) of embryo or perinatal rats and mice have
been used extensively to study anoxic or ischemic damage since 1983 (966). Organotypic hippocampal slice cultures from perinatal rats were first used in about 1995 and
are being used increasingly (1072, 1073). They promise to
become more valuable models, particularly as they show
delayed death (923, 1072, 1073). However, although they
do show some selective vulnerability of CA1 versus CA3
versus dentate gyrus if the in vitro ischemia is kept relatively short (30 min) (65, 1072), it is not very dramatic.
There is twice as much cell death in CA1 as CA3. Longer
durations (60 min) produce larger and equal amounts of
cell death in all hippocampal regions (947, 1072). Like the
cultured cells, this system also requires prolonged in vitro
ischemia to produce cell damage (1072).
In most studies the insult is in vitro ischemia, although there are several studies of “chemical” anoxia or
“chemical” ischemia in which oxygen is maintained but
mitochondrial inhibitors are used (1199). The latter is a
poor model, and its results will not be considered here.
Free radical generation is almost undoubtedly much
higher than during anoxic conditions. Indeed, in liver
cells, such damage is often markedly decreased by concurrent anoxia (115, 234).
A major difference from damage in vivo is the very
long duration of oxygen and glucose deprivation that is
generally required to induce cell death. This is true for
both primary cultures and organotypic cultures (376,
1072). Although 30- to 60-min deprivation is not long for
focal insults, the degree of oxygen and glucose deprivation that cultures are exposed to is similar to that in global
ischemia, where 5–10 min at 36°C produces profound
delayed damage. There are several possible reasons for
the difference. In the widely used model developed by
Goldberg and Choi (376), ATP does not fall nearly as
much as it does in vivo (Table 1) and, probably relatedly,
the release of glutamate is very delayed (376). The small
fall in ATP would certainly explain the requirement for
the prolonged insult, because it approximates that in the
focal ischemic penumbra (Table 1). Why the energy disturbance is less is not apparent, but an intriguing possibility is that the cells in culture may adopt protective
mechanisms that reduce ATP turnover during hypoxia as
has been nicely discussed for the general case by
Hochachka et al. (453). This possibility is given added
credibility by the fact that the ATP fall during chemical
hypoxia is far greater than during actual anoxia (Table 1).
Another significant difference between cell cultures
and in vivo tissue is that damage in primary cultures
following the 30 – 45 min exposures is completely dependent on activation of NMDA receptors (376). Longer insults (90 min) produce an NMDA-independent form of
damage that appears largely apoptotic (405). In organotypic cultures, NMDA antagonists provide only partial
protection (5), and combined glutamate receptor antagonists are required to protect against 60-min in vitro ischemia (1073). In that sense, the organotypic cultures seem
better models for in vivo events and at this stage seem a
better route to take.
Although damage in cultures is easily studied, it is, of
course, essential to verify any inferences by studies of in
vivo tissue. Neuronal gene expression is likely to alter
very significantly in culture; the anatomical relationships
between cells are very different, and there are no vascular
cells. The latter two objections do not pertain for organotypic cultures.
P. Summary
2. Nature of insult and nature of damage
1. In vitro systems
Damage usually develops 8 –24 h after 30- to 60-min
in vitro ischemia and is generally monitored as a gross
increase in membrane permeability to dyes or protein, in
particular leak of LDH (791) or nonexclusion of trypan
blue or propidium iodide (1073). Sometimes frank disappearance or disintegration of the cells is measured (376),
and more recently apoptotic and necrotic changes are
being monitored by standard methods of nuclear staining
(405).
The goal of research on ischemia is to understand
what occurs in vivo. In vitro systems are able to provide
clues, but for reasons discussed above, and others, conclusions cannot be simply extended to in vivo situations.
The absence of blood flow as a variable, and the absence
of blood vessels themselves in cultures, facilitates interpretations. However, by the same token, the absences
eliminate what may be important components of the damage process. It must be borne in mind that all tissue types
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O. In Vitro Models: Cell Cultures
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2. In vivo systems
Global and focal ischemias are two very different
insults. Global ischemia involves a short (usually 15 min
or less) very intense insult in which ATP is severely
lowered. It is characterized by a slow development of cell
death during reperfusion, which shows great selectivity.
There is no intimation of involvement of the vasculature,
although there is a real possibility that microglia/macrophages play a role in the process. Overall, the insult is
straightforward and quite uniform, but in the hands of one
group, residual blood flow is much higher than the usual
1–3% (1089).
Focal ischemia is a far more complex insult, with
several unresolved issues. There are many variations of
the model including whether or not there is reperfusion
(temporary vs. permanent), where the lesion is, the strain
of rat, and the degree of temperature control. These lead
to a very complex literature. One goal of this section has
been to describe these variations well enough so that
results obtained in different systems can be related to
each other.
A) CORE AND PENUMBRA. Ionic and metabolic changes in
the core and penumbra are dramatically different, and
these probably account for the great disparity in how well
the two regions can be protected by pharmacological
interventions. The fall in ATP in the penumbra is quite
small (levels do not fall much below 70% of control during
development of damage in permanent ischemia), and it
really is not clear that this fall per se is adequate to cause
damage. For example, when damage was initiated by
mitochondrial blockers, ATP fell to 25% for several hours,
and the lesion developed over 1–2 days (736). A real
possibility is that effects in the core add to the insult in
the penumbra and are essential for damage. Examples are
diffusion of K1 and glutamate into the region. The intraischemic depolarizations that may be important in development of damage in the penumbra may well be driven by
ions and glutamate generated in the core. Core damage is
terribly severe when ischemia is maintained for .1 h.
B) TEMPORARY VERSUS PERMANENT FOCAL ISCHEMIA. Development of damage after 2- to 3-h temporary ischemia and
during permanent ischemia are very similar in many detailed respects. One explanation for this is that all the
important events occur in the first 2–3 h so that reperfusion does not have important effects; there is no doubt
that this is true to some extent. As an important example,
ATP levels in the penumbra after temporary ischemia are
about the same as during permanent ischemia; they do
not appear to recover very well, indicating permanently
compromised metabolism. This might help maintain the
similarity between the two insults. However, there are
important differences; free radical and NO generation are
both enhanced during the first 40 – 60 min of reperfusion
(622, 893). Vascular damage is much greater after temporary ischemia. The most glaring difference is the apparent
importance of leukocytes in damage following temporary
ischemia. In some way, these cells appear necessary to
allow damage to continue to develop in the reperfusion
period. They are not necessary for development of damage in permanent ischemia.
C) GLIAL AND VASCULAR RESPONSES. The astroglial responses to focal and global ischemia are very different
from each other. Global ischemia is characterized by quite
rapid glial hypertrophy, increased protein synthesis, and
hyperplasia (642), whereas the more prolonged focal ischemia is characterized by quite rapid glial swelling and,
eventually, cell death during formation of the infarct
(355). Glial cell death in the core, and possibly penumbra,
of the infarct might well result from combination of lowered pH and ischemia; combining pH 6.2 with respiratory
chain inhibition kills glia cells in culture within several
hours (1090). It is important to consider that glial damage
may, in some way, be enhancing neuronal damage.
Roles for vascular changes and extracellular edema
in development of damage in focal ischemia are not yet
compelling, although evidence that a metalloprotease that
attacks the basement membrane is likely to be important
in damage raises the issue of the role of the vasculature.
There is currently no resolution.
3. Some outstanding issues
There are important technical issues in evaluating
results from the models. The most important technical
issue in producing reliable data on protective effects of
drugs is careful and prolonged temperature control; control for at least 24 – 48 h after an insult now seems necessary. This is difficult to do and so is usually not done.
Another key issue is determining whether artifactual vascular damage occurs in some focal ischemic insults and
alters the sequence of events. Finally, the relationship of
infarct size in to neuronal death in focal ischemia needs to
be clarified as long as it is the former that is generally
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suffer ischemic damage, so to show that both neuronal
cultures and in vivo brain tissue suffer delayed ischemic
damage does not mean that the mechanisms are the same.
The composition of brain slices is closer to that of in
vivo tissue, and there is little time for major genetic
change. However, slices are in a compromised metabolic
state, with low ATP values and elevated aerobic glycolysis
(677), and are hypersensitive to ischemic insults. They
show different morphologies from in vivo tissue, and the
preparation of the slice itself induces transcription of
some stress-related mRNA and immediate early genes, as
well as accumulation of Fos and Jun immunoreactivities
(1303). The consequences of this are not known but may
alter ischemic sensitivity. Culture and slice work can be
used as bases for later in vivo studies but the latter,
eventually, must be done.
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PETER LIPTON
III. MORPHOLOGIES AND BIOCHEMISTRIES
OF ISCHEMIC CELL DEATH
As discussed in section I, there are at least three
recognizable pathways of ischemic cell death: necrotic
cell death, apoptotic cell death, and, very probably, autophagocytotic cell death.
The purpose of this section is to describe the end
stages of these pathways and to understand why different
pathways are taken in different cases. Another elusive
goal is to understand the molecular changes that constitute these end stages.
A. Morphological Characteristics of Necrotic
Cell Death
Most of the morphological changes associated with
ischemic cell death were defined by Spielmeyer in the
early part of the 20th century (1063) and led to work in the
1960s and 1970s by Brown and Brierly (129, 130, 355) that
quite rigorously defined different stages of cell damage
including forms of cell death. The earlier work, as well as
that of Brown and Brierly, has been nicely reviewed (121,
128, 392). There are three categories of necrotic cell
death.
1. Edematous or pale cell change (ECC)
A) DESCRIPTION. The cytoplasm is very swollen. Some
mitochondria are swollen with disrupted cristae, but others are rounded and slightly shrunken. Endoplasmic reticulum, the Golgi apparatus, and polysomes are no longer
apparent as complete structures but remnants, often with
attached ribosomes that exist and often accumulate close
to the nucleus. Microtubules and other filamentous structures are absent, and the cytoplasm is almost clear. The
plasma membrane is irregular and sometimes shows
frank breaks, but this is very rare. The nucleus is normal
except for irregular clumping of chromatin (520, 521,
942). These changes have some characteristics of what is
termed peripheral chromatolysis (392, 897).
B) OCCURRENCE. This end stage is not seen very often.
It is predominant after 3- to 4-h hypobaric/ischemia in
young animals (490), in whom it is also produced by
glutamate exposure (490, 852, 920). Agonists of NMDA
induce it in striatum of adult brain (919). It occurs 90 min
after 30- or 10-min global vessel-occlusion ischemia during hyperglycemic conditions (494, 522) and occurs in the
final stages of delayed death in gerbil and rat global
ischemia, where the existence of some “swollen cells” is
described, along with other end-stage morphologies (579,
582, 897, 942) and where, in one case, electron micrographs show this morphology (897).
2. Ischemic cell change (ICC)
A) DESCRIPTION. This end stage contrasts markedly with
ECC and is far more common. In its more exaggerated
phase, it is termed ICC with incrustations (128). There is
a major darkening and shrinkage of the nucleus and cytoplasm (128, 130, 494, 521, 896, 897), and the nucleolus
often assumes a honeycomb appearance (319). The
plasma membrane and the nuclear membrane are both
very irregular, and the cell often assumes a triangular
shape (in 2 dimensions). The cytoplasm generally contains many large (1–2 mm diameter) vacuoles, some of
which are greatly swollen mitochondria with disrupted
cristae (130), but most of which are swollen Golgi cisternae (896), ER (130), or unidentified. Many of the latter
may well be autophagic vacuoles (128, 834). Normal ER
and Golgi organization into stacks is disrupted. There are
few, if any, polysomes, but there is a plethora of ribosomes. At least some microtubules are present. This end
stage and its incidence are very elegantly described in a
recent review (716).
Viewed in the light microscope, these cells are intensely acidophilic (eosinophilic), which is a defining
characteristic of this morphological stage; they are also
intensely argyrophilic (223, 437).
B) OCCURRENCE. This end stage is prominent in many
ischemic models. The cells coexist with the edematous
cells in CA1 at the end of delayed degeneration in rat and
gerbil global ischemic models (579, 582, 897), where frank
eosinophilia is very apparent (899). They are the dominant form in the quite rapid (within hours) death that
occurs in CA4 after global ischemia in gerbil (579) and rat
(896). During or after focal ischemia, cells showing this
change emerge and remain for several days in the periinfarct cortex (814). They are abundant in the penumbra
for 1–2 days and in the core of the lesion for a relatively
short time before the cells disintegrate. They are widespread in adult rats after 40 min sporadic hypoxia/ischemia (121) where they take ;30 min to 1 h to appear after
40-min hypoxia and where they persist for ;24 h. Thus
they constitute a relatively stable state toward which cells
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measured. To what extent does reduction in infarct size
after 24 and 48 h reflect protection against neuronal
death?
Of paramount importance is the question of how well
these animal models replicate events during and after
human stroke. This issue is outside the scope of this
review. However, the difficulty to date in successfully
applying principles learned from these models to human
therapy trials suggests that there are added complexities
in the human disease. These may include greater sensitivities to detrimental side effects of some drugs, such as
MK-801 and its analogs (135, 299), or more complex processes of cell death that cannot be halted by only one
pharmacological intervention.
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CEREBRAL ISCHEMIA ON NEURONS
tend following ischemia. Ischemic cell change may well
require reperfusion to form, because it is not seen immediately after even prolonged complete ischemia (521).
This has not been rigorously tested. If so, there are many
possible reasons such as the requirement for free radicals
or near-normal levels of ATP.
3. Homogenizing cell change/ghost cells
4. Summary
Although these end stages are considered to represent dead cells, this has not been rigorously established
except for homogenizing cell change (HCC) which, in the
electron microscope is clearly associated with badly fragmented cells. Metabolism and function have not been
carefully measured in cells showing ICC or ECC, and
although the morphology is very distorted, the possibility
of recovery cannot be ruled out. However, after focal
ischemia, the disappearance of neurons showing ICC
semiquantitatively parallels the decrease in neuronal density in the peri-infarct region of focal lesions (814). Furthermore, although silver-stained ICC neurons were visualized in the infarct after 24 h, several days later only
silver-stained terminals remained (484). These results certainly suggest degeneration of the ICC neurons.
B. Apoptotic Cell Change
1. General considerations
Apoptosis, in its purest form, is best considered as
one of the processes by which cells die in response to
normal physiological stimuli (205, 565, 1227). In this
sense, it is a “programmed” physiological process. The
question is the extent to which this program of changes
has been coopted by ischemic cell death.
This would be quite simple to answer if the morphological changes and biochemical events of apoptosis were
unambiguously defined. However, there is no overwhelming consensus on this at present. In the next section, a set
of criteria that allow this to be done as well as possible
are described and discussed.
2. Minimal criteria for apoptosis
A large number of reviews detail changes associated
with apoptosis in different cell types and conditions (32,
1004). The following are reasonable minimal criteria for
identifying apoptosis in ischemia. A key is differentiating
it from ischemic cell change that has somewhat similar
characteristics.
A) MORPHOLOGICAL CHANGES. The most unequivocal morphological markers are the formation of smoothly contoured spherical-, or lunar crescent-shaped masses of
chromatin within the nucleus and the subsequent formation of apoptotic bodies that are cell membrane-bound
structures containing cytoplasm and dark chromatin
masses (32, 564, 652). This contrasts with ICC where the
chromatin is irregularly clumped. Before formation of the
apoptotic bodies, the cytoplasm (in electron microscopy)
becomes darkened and pynknotic, as in ICC, but neither it
nor the nucleus becomes nearly as dark as in ICC, so this
is a reasonable way to identify apoptosis (716). The organelles are relatively normal (as distinct from the very
swollen mitochondria and cisternae of ICC) (32, 564, 716),
but there is often some vacuolization (564, 1006), so the
presence of vacuoles cannot be used to identify ICC.
Mitochondria usually only become visibly swollen and
malformed in the end stages of apoptotic change (541,
919), and even then, changes are often very minor (564)
compared with necrotic cell death (541, 564, 919). However, they do swell so that, as with vacuolization, swollen
mitochondria cannot unequivocally be used to identify
necrosis. However, cell shrinkage and darkening, in the
absence of mitochondrial swelling and vacuoles, can be
used to identify apoptosis. This, however, is not seen after
ischemia, so the issue is not simple. Importantly, in contrast to ICC, cytoplasm does not become eosinophilic.
This is a very useful way to distinguish the two processes.
B) BIOCHEMICAL CHANGES. Double-stranded breakdown
of DNA into nucleosomal segments is a very strong criterion (111, 859) and is manifested as DNA laddering, with
fragments being multiples of ;200 bp. This is not unambiguous identification of apoptosis because it is observed
in populations that almost certainly have undergone necrotic cell death (1147). Glutamate toxicity in cultures
(404) and in vivo (918) leads to prominent nucleosomal
fragments of DNA at the same time as the cell population
shows a distinctly necrotic morphology. In situ end label-
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A) DESCRIPTION. The nucleus is somewhat shrunken
and slightly darker than normal, often with a fragmented
membrane. There is irregular chromatin clumping. The
cytoplasm is slightly shrunken and appears fragmented
with small vesicles and dense bodies (128, 351, 355), but
still retains its delimited structure. Although there are
some shrunken mitochondria, there are no other recognizable organelles. The cell membrane appears disrupted.
Cells are much paler than normal cells in the electron
microscope and are weakly eosinophilic, giving the cells
their “ghostlike” appearance. Nuclear “integrity” is maintained longer than cytoplasmic integrity (128, 355). This is
the most disrupted of the three morphologies.
B) OCCURRENCE. This stage is generally assumed to
follow from ICC, and this seems likely based on location
and timing (128, 351). It arises ;24 h after anoxia/ischemia (128) and ;6 –12 h into permanent focal ischemia in
rat, in the core of the lesion (351), and sometime before
48 h after 2-h focal ischemia in the penumbra (652). It has
not been described after global ischemic insults, although
reported analyses have not been very rigorous (579, 896,
897).
1453
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PETER LIPTON
caspases, and/or inhibition of cell death by caspase inhibitors, extracellular binding of annexin V, and release of
cytochrome c into the cytoplasm are all strong indications
that the apoptosis process has been activated and is making a major contribution to ischemic cell death.
With the use of these criteria as best as can be done
at this time, there is now reasonable evidence that apoptosis contributes significantly to cell death in focal ischemia and anoxia/ischemia. Evidence for its role in global
ischemia is much weaker, although aspects of the process
almost undoubtedly contribute to cell death there also.
3. Extent to which ischemic cell death is via apoptosis
A) GLOBAL ISCHEMIA. I) Chromosomal and morphological changes. There are now many reports of both nucleosomal DNA laddering and TUNEL labeling in extracted
tissue and sections of the CA1 region of hippocampus,
following short durations of global ischemia in rats (443,
698) and in gerbils (568, 848, 951, 1016). These occur
during the period that neuronal death is developing. When
observed in detail, TUNEL staining occurs only over cells
undergoing severe cell shrinkage and not in adjacent,
healthy, cells (568, 834). However, as discussed, these
chromosomal changes are not unambiguous identifications of apoptosis (899).
Morphological evidence for apoptosis is weak, despite the title of one publication claiming that delayed
neuronal death is apoptosis (834). In that paper, light
microscope staining was intense with TUNEL when the
cells were dying, but there was no light microscope evidence for chromatin condensation or apoptotic bodies.
Some (it is not stated how many) nuclei showed condensed chromatin, but it was not at all smooth edged
when viewed in electron microscopy, and the shrunken
dark cells showing the condensed chromatin were highly
vacuolated and contained autophagosomes. In fact, the
process seems far more akin to autophagocytotic cell
death than apoptosis. Two studies of rat hippocampus,
after quite mild 2-VO (251) and 4-VO (899) insults, failed
to note any morphological signs of apoptosis. Only ischemic cell change was noted. This is also true in cat
hippocampus after 10-min global ischemia and in Purkinje
cells (716).
Apoptotic bodies probably have a short lifetime (;3
h), and it has been suggested that this might explain their
absence (150). This seems unlikely because apoptotic
cells are readily seen in focal ischemia, hypoxia/ischemia
(see sect. IIIB), and also in a global ischemic-like insult to
spinal cord (541).
II) Biochemical studies. There are no reported studies of cytochrome c localization changes. There is a series
of studies on caspases and their inhibitors which suggest
that these enzymes are involved in cell death and thus
suggest a contribution from apoptotic processes.
CA1 neurons in the gerbil were almost completely
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ing of DNA using terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling (TUNEL), a histochemical approach, is even less selective for apoptosis
because it labels both nucleosomal and nonnucleosomal
fragments (918); the latter are formed during necrotic cell
death. Although not at all selective for nucleosomal cleavage, TUNEL is relatively selective for double-stranded
breaks as distinct from singe-stranded nicks (82), so is
somewhat discriminatory.
Although the biochemistry of apoptotic death is not
completely understood, there are extremely useful identifying characteristics. The activation of caspases, particularly caspase-3 (719, 886), and inhibition of cell death by
inhibition of these proteases (886, 1172, 1301) is the strongest criterion, although the ever-emerging complexity of
the caspase system makes the situation somewhat uncertain (739). Caspases do not appear to be activated in
necrotic death (e.g., Refs. 33, 387). There are several
manifestations of caspase-3 activation that can be measured quite readily including cleavage of poly(ADP-ribose) polymerase (PARP) (318), cleavage of caspase itself
(953), and characteristic cleavage of spectrin (811). Another important criterion is the release of cytochrome c
from mitochondria (941), which activates caspase-3 by
binding to the ced4 analog APAF3 and cleaving the procaspase (441). This now appears to be an almost universal
step in apoptosis (591, 941); in some cases, another mitochondrial protein, apoptotic initiation factor, may play a
role analogous to cytochrome c (1275). Neither of these
changes has been noted in necrotic cell change to date.
Another unique feature of apoptosis is the transfer of
phosphatidylserine to the outer leaflet of the plasma membrane as part of the process by which the cell is targeted
for phagocytosis. This can be monitored by binding of
annexin V, which preferentially binds to this phospholipid
in the presence of Ca21 (1189).
Another frequently occurring feature of apoptotic
death is an increase in the ratio of Bax or other proapoptotic members of the Bcl family to the antiapoptotic
members of this family, Bcl-2 or Bcl-xL, (940). Thus there
are a large number of unique characteristics of apoptosis
that can be measured or observed.
Blockade of cell death by inhibition of protein synthesis is sometimes used as a criterion for apoptosis
(194), but it is not satisfactory. Not only is apoptosis often
immune to inhibition of protein synthesis (61, 718, 1125),
but more importantly there is strong evidence for postischemic synthesis of two very damaging enzymes, COX-2
and iNOS (477, 479, 837). Indeed, in a study of effects of
mild focal ischemia, cycloheximide was very protective
but abolished both DNA laddering and DNA smearing,
indicating strong effects on necrotic death as well as
apoptotic (304).
Thus, at this stage, uniformly condensed chromatin
and apoptotic bodies identified in the light microscope, a
shrunken cytoplasm that is not eosinophilic, activation of
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CEREBRAL ISCHEMIA ON NEURONS
cells after global ischemia before and during the time of
maximal cell death.
III) Summary. Evidence is conflicting. Chemical evidence suggests a contribution of apoptotic processes to
cell death after global ischemia insofar as there is DNA
laddering, caspase activation that is reasonably timely,
and some attenuation of damage by caspase inhibitors.
BAX and BCl-Xs expression are also consistent with an
apoptotic pathway. Certainly the attenuation of cell death
by caspase blockers is not nearly as strong as it is when
death is clearly via apoptosis, for example, in nerve
growth factor (NGF) or serum withdrawal (739, 1106), but
it is substantial.
In ischemia, the caspase inhibitor was protective
when added 2 h after the insult (183), and it had to be
injected within 12 h of the insult (451). This is early in the
cell death process, which lasts ;3– 4 days. Caspase activation is a very late step in many cases of apoptosis (963,
1106, 1302), generally being activated by mitochondrial
release of cytochrome c (441), but this apparently is not
the case in global ischemia, or if it is, then early caspase
activation is also important. The implications of this are
considered in section IVC.
Although biochemical evidence strongly indicates
that apoptotic processes occur, morphological evidence
does not support a standard apoptotic process. Most importantly, there is no evidence of spherical nuclear chromatin clumping/apoptotic bodies at the light microscope
level, and these are a hallmark of classical apoptosis.
Perhaps the caspase-activated endonuclease (301) is not
able to act after ischemia. At the electron microscopic
level, the cytoplasms and nuclei of cells appear too dark
and too disorganized (e.g., Ref. 716). Furthermore, almost
all cells are eosinophilic.
The most reasonable overall assessment of the situation is that caspases are very clearly activated and that
their proteolytic actions contribute to the cell death. However, other important processes occur that bias the final
morphology away from classical apoptosis.
B) FOCAL ISCHEMIA AND HYPOXIA/ISCHEMIA. I) Morphology.
There is DNA laddering in the penumbra of permanent
lesions after one to several hours (669, 670, 1124), 24 h
after 2-h temporary occlusion (652), and several days
after very short (30 min) temporary lesions (276, 304).
Unlike in global ischemia, there is also quite strong morphological evidence for apoptotic changes in both focal
ischemia and hypoxia/ischemia.
Two groups showed similar effects of temporary
ischemia (178, 652). Apoptotic nuclei, showing condensed
chromatin /apoptotic bodies, first appeared in the infarct
core ;15–30 min after beginning ischemia (653) and were
very apparent there within 30 min after 1- or 2-h temporary MCA occlusion (178, 652). After this time, they were
continually present at high density in the periphery of the
infarct (178, 652) for at least 2 days (652). They also
appeared in regions of individual cell necrosis after milder
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protected against cell death by a broad-spectrum caspase
inhibitor (451), with no effect on temperature. The protection persisted, albeit at a lower level, when the hippocampus was injected with IL-1b at levels that were
adequate to enhance ischemic damage, indicating that
protection did not result from inhibition of ICE
(caspase-1) and subsequent reduction of IL-1. However,
these studies did not specify the caspase that is involved.
Less, but significant, protection was afforded in rat by the
more specific caspase-3 inhibitor ZVAD-CHO; 4.5 mg injected into the ventricle saved ;40% of the CA1 neurons
after 3 days and 20% of the neurons after 7 days (183). The
specific ICE inhibitor YVAD had no effect. These protective effects are not terribly strong but do show an involvement of caspases.
Less direct, but important, evidence for caspase involvement is the upregulation of the enzyme. This occurs
either by cleaving the existing procaspases (699) or by
synthesizing a new enzyme that is then cleaved. Caspase3-like mRNA is upregulated between 8 and 24 h after 15to 30-min ischemia in rat, predominantly in CA1 pyramidal cells, and total caspase-3 protein is increased by 8 h
(183, 826). Cleavage of the procaspase increased by 4 h
after ischemia, indicating an early activation of the enzyme, although measurements of enzyme activity indicated a somewhat later activation. Caspase-3-like activity
measured on artificial substrate was elevated ;10-fold in
hippocampus between 8 and 24 h after ischemia, and
PARP breakdown began sometime between 24 and 72 h
after the insult (183). This timing is consistent with the
appearance of active caspase-3 fragments at 24 h after
12-min global ischemia (1232). In gerbils, there was also
cleavage of procaspase-3, but the timing was not ascertained (451).
Overall, these studies indicate that activation of
caspase-3 occurs fairly early in development of damage,
which normally takes 3– 4 days. There are some timing
details that are difficult to explain such as the fact that
total caspase-3 was elevated slightly before the elevation
in mRNA and that breakdown of a synthetic substrate by
caspase was far earlier than the action on a natural substrate, PARP. These may reflect subtleties of the apoptotic
process that are not yet apparent; the work is quite recent,
and small discrepancies are likely to become resolved
with time.
Although caspase-3 appears to be important in the
rat, caspase 2 (Nedd-2) may be more important in gerbil.
Nedd-2 mRNA was upregulated severalfold in CA1 and
CA3 pyramidal neurons 3– 6 h after 5-min ischemia in
gerbil. By 12 h, it was down to baseline (572). There was
no change in caspase-3 mRNA, nor was caspase-2 upregulated in rat (826). Thus there may be a real species difference here. No protein measurements have been reported.
There are further chemical changes that are suggestive of apoptosis; the proapoptotic proteins BAX (185,
420) and Bcl-Xs (264) are expressed in vulnerable CA1
1455
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PETER LIPTON
than core of lesion, following shorter rather than longer
insults, Ref. 178), but the apoptotic changes certainly
exist in the lesion core.
II) Biochemical studies. Mutant mice in which ICE is
knocked out (993) or rendered ineffective (421) have
infarcts that are 50% the volume of controls after transient
focal ischemia. However, the mutants have reduced buildup of IL-1b after ischemia (421), which could account for
protection. The relatively specific caspase-3 inhibitor ZDEVD.FMK reduced infarct size by 27% in a mouse model
of 2-h ischemia followed by 18-h reperfusion (422). It was
only effective if added within 1 h of the end of the ischemia, not when added at 2 h (694), again indicating that
caspases play a role early in the cell death process.
Caspase-3-like activity was more important to overall
cell death in a less severe model (30-min ischemia). ZDEVD.FMK reduced infarct size by ;60% when added
anytime before 6 h after the insult. It was ineffective
added 18 h after the insult but partially protected when
added at 12 h (304). An elegant more detailed time course
study showed that caspase inhibition was effective only
up to to 9 h in this model, and that corresponded to the
point at which caspase-3 was activated (324). As with the
effects of caspases in global ischemia, this is still early
relative to the final stages of cell death, which do not
occur until 3 days or more after ischemia in this model.
There are many caspases (11 at present), and the full
contribution of caspase activation, as distinct from its
role in IL synthesis, will require improved inhibitor specificity. Also, two more universal inhibitors, V-ICEinh and
BAF, which prevent neuronal apoptosis that is unaffected
by the inhibitors of caspase-3 and caspase-1 (877), will be
of help in showing the extent of caspase involvement in
ischemic cell death. It may be greater than indicated by
the ;25% inhibition produced by caspase-3 inhibition
with Z-DEVD.
Several studies have been carried out on caspase
changes after one or more hours of focal ischemia. As in
global ischemia, the increase in de novo synthesis of
caspases appears rather leisurely, but there is an earlier
activation of the procaspase-3 by cleavage. Upregulation
of caspase-3 mRNA in MCA territory is delayed until
16 –24 h after the onset of permanent focal ischemia,
whereas caspase-2 mRNA is upregulated by 4 – 8 h, and is
down to baseline by 16 h (42). Two hours of temporary
focal ischemia in mouse (807) led to an increase in
caspase-3 protein levels in the penumbra after 24-h recovery; there was no change in the focus. [There is a recent
report in abstract form showing a far more rapid increase
in caspase-3 mRNA in layers 2 and 5 cortical neurons in
spontaneously hypertensive rats, beginning within 15 min
of occlusion, with no change in caspase-2 mRNA (825).]
There was an early increase in the 20-kDa caspase cleavage product, peaking between 1 and 4 h after the 2-h insult
but actually beginning during the ischemia. This was accompanied by a manyfold increase in caspase-3-like ac-
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insults (653). Thus apoptotic neurons appear to arise
quite soon after a focal insult and to predominate either
early or in regions where the insult is less severe. The
ratio of apoptotic cells to necrotic (ghost) cells was 9:1 in
the penumbra versus 1:1 in the core 4 h after 2-h temporary ischemia (178). One study compared apoptosis in
temporary and permanent ischemia (785). The time
course of development and the number of apoptotic cells
were identical in the penumbras in the two insults; however, there were about one-half the number of apoptotic
cells in the core area of the permanent lesion, probably
reflecting the continuing profound energy deprivation
(785).
Cells with spherical condensed chromatin structures
are present in great numbers 24 h after permanent or 1-h
temporary ischemia in mouse. In the penumbra, they
constitute ;5–10% of the total number of cells (601, 786),
indicating a major apoptotic component to cell death.
TUNEL staining was almost exclusively over neurons as
opposed to glia (304); no more than 10% of the TUNEL
cells were colabeled with glial fibrillary acidic protein
(652). This confirms the neuronal nature of the apoptotic
cells.
Apoptotic death was convincingly demonstrated after 60 min of ischemia/hypoxia in newborn piglets (745).
There was coincident strong basophilia of the nuclei,
TUNEL labeling, and presence of apoptotic bodies and
condensed cells in the cingulate gyrus 2 days after the
mild insult, which reduced ATP levels to ;50%. A similar
insult, applied to young rats, yielded cells with greatly
increased labeling by annexin V 48 h after the ischemia
(1189), also suggesting apoptotic change.
High-resolution study of the cell changes in the various models indicate that the morphology is often not that
of classical apoptosis. The cells showing apoptotic nuclear morphologies are mostly shrunken, but some of the
apoptotic nuclei are in distinctly swollen cells, with no
possible apoptotic morphology (178). At the level of electron microscopy, the condensed chromatin in nuclei of
cells that very probably had stained positive with TUNEL
did not actually have the smooth appearance of chromatin
in apoptotic cells and furthermore was associated with
cells that were highly vacuolated and were often undergoing quite severe homogenizing-like cell change (155).
This is a secondary necrosis, following major apoptotic
changes.
Overall, these morphological studies strongly indicate nuclear changes associated with apoptosis. However, the cytoplasm often shows much more degeneration
than is expected in classical apoptosis, including vacuolization and disintegration. More morphological studies
would be very beneficial, along the lines of the elegant
studies of Portera-Cailleau and co-workers on glutamate
toxicity and global ischemia (716, 919).
In most studies, apoptosis tends to be favored in
regions where insult intensity is less (penumbra rather
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
4. Basis for initiating apoptosis
A) OVERVIEW. Apoptosis can be induced by a large
number of different stimuli, and many of the conditions
that prevail during ischemia are capable of inducing apo-
ptosis in one or more cell types. These conditions include
free radical generation (380, 502, 938, 1207), NO production (830), reduced mitochondrial activity and membrane
potential (503, 894, 1220, 1275), microtubule disaggregation (110), increased Ca21 (535, 831), activation of calpain
(811, 1065), increased levels of ceramide (619), and increased expression of a mutant P53 (654, 978). The broadspectrum protein kinase inhibitor staurosporine causes
apoptosis in many cell types, including neurons (700), and
both protein kinase C (PKC) and Ca21/calmodulin-dependent protein kinase II (CaMKII) are severely downregulated for many hours after global and (for CaMKII) focal
ischemia; PKC has not been measured in the latter (35,
418, 780).
B) BAX, TNF, CD-95L (FAS LIGAND), AND CYCLINS. More specific inducers of apoptosis are also changed appropriately
by ischemia. This includes a decreased ratio of Bcl-2 to
BAX (365), upregulation of cJun (415), and changes in the
“cell death domain” receptor ligands. Tumor necrosis factor-a is elevated after global ischemia (975, 1155) and has
been shown to be damaging in focal ischemia (636, 746),
where it is also elevated within 20 min (636). Importantly,
reported in abstract form, the Fas ligand, or CD-95 ligand,
was upregulated in the ischemic region for 24 h after 2- to
3-h temporary focal ischemia and, as with TNF-a, this
appeared to be very damaging. Lesion size at 3 days was
much reduced by injected antibody to CD-95 and also in
mice in which the Fas ligand was knocked out (722).
Although TNF-a has many effects in addition to initiating
apoptosis, this is not generally true for the CD-95 system.
Thus these data suggest it may play a critical role in the
ischemic apoptosis. Such a role is consistent with the
widespread upregulation of CD-95 in postmortem brains
showing many different neurodegenerative diseases
(246). It would be of interest to measure ischemic damage
in FADD knock-out mice; if lesion size was reduced, it
would strongly support the role of the CD-95 system (43).
The cyclins are thought to be instrumental in activating apoptosis in some systems, including NGF withdrawal
from sympathetic neurons. Cyclin D1 levels increase after
withdrawal, and apoptosis is effectively prevented by inhibition of the cyclin-dependent kinases (876). Cyclin D1
mRNA is increased selectively in CA1 and subiculum
48 –72 h after 20-min global ischemia in the rat, and cyclin
D1 was expressed in individual CA1 pyramidal neurons by
72 h, before their undergoing ischemic cell change or
apoptosis (1117). Neither the mechanism nor significance
of this recent finding has yet been determined, but it is
reasonable to think the activation of the protein plays a
part in apoptotic cell death.
C) DIRECT EFFECTS ON MITOCHONDRIA. Another mechanism
for cytochrome c release (and hence activation of apoptosis) is suggested by the recent observation that the combination of mitochondrial membrane depolarization and
elevated perimitochondrial Ca21, conditions which occur
during ischemia, cause release of cytochrome c from
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tivity, measured with an artificial substrate, which was
maximal immediately after ischemia and decayed within
;6 h. This is consistent with the requirement for very
early inhibition of caspase-3 to attenuate damage in this
mouse model (422, 694). The very delayed synthesis of
caspase-3, and even the less-delayed synthesis of
caspase-2, is perhaps too late to be of great functional
significance in focal ischemic damage, which is strongly
manifested between 6 and 12 h, but this needs to be tested
by future studies.
Caspase activation via movement of cytochrome c
from the mitochondria into the cytosol is a cardinal feature of most apoptotic systems. There is good evidence
that this occurs in a large number of cells during development of focal ischemic damage (342). Substantial intracellular redistribution of cytochrome c from mitochondria
to cytosol was measured both by immunocytochemistry
and by Western blots within 4 h of reperfusion after
90-min ischemia, and this increased over the next 24 h.
This was selective in that cytochrome oxidase did not
show this change. This time course is in keeping with the
measured activations of caspase.
Hypoxia/ischemia in young rats produces large numbers of apoptotic-like cells. Caspase activity increases and
is half-maximal by ;12 h (191). Neuronal death and brain
volume loss were markedly attenuated by the nonspecific
caspase inhibitor BAF when administered within 3 h of
the insult (191), at levels which did not affect core temperature for at least 2 h.
Taken together, these studies suggest that apoptosis
contributes to cell death during focal ischemia and hypoxia/ischemia and also that caspases are important.
Somewhat surprisingly, neither caspase antagonists nor
knockouts have been tested in permanent focal ischemia,
although the presence of apoptotic profiles suggests that
apoptosis is important. The relative importance of the
apoptosis in temporary focal damage, as evaluated by the
efficacy of the caspase inhibitor, decreased as the duration of the insult increased, and this is in line with the
general notion that less severe insults favor apoptosis
over necrotic cell death (111). The role of caspase synthesis, as distinct from its early activation by proteolysis,
is not clear. The former occurs quite late; it may play a
role in ongoing cell death, perhaps that which is activated
by induced enzymes such as iNOS and/or inducible
COX-2.
It is, of course, possible that caspases aside from
caspase-3-like forms can perpetrate apoptosis so that the
effect of z-DEVD.CHO may represent a lower limit on the
amount of apoptosis that occurs.
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PETER LIPTON
5. Importance and role of protein synthesis
In some cases apoptosis requires protein synthesis,
whereas in others it does not. Whether or not it does so in
ischemic death provides insight into the mechanism of its
activation. Protein synthesis inhibition does ameliorate
ischemic damage in many cases; however, damaging proteins such as iNOS and COX-2 are synthesized and may be
the target of this protective action (as may temperature
reduction; the studies are not generally well controlled for
this). Thus the challenge is to demonstrate that inhibition
of synthesis protects because it specifically prevents
apoptosis. The most persuasive pertinent studies on in
vivo ischemia are those in which mild transient focal
ischemia (30-min distal MCA occlusion) was the insult
and where artifactual inhibition of protein synthesis,
which probably lasted for 12 h, almost completely protected against the damage (276, 304). The evidence that
apoptosis was occurring was not strong in one of the
studies, comprising only TUNEL labeling and DNA laddering, but it was stronger in the other where there was
chromatin condensation also. There is a caveat. Apoptotic
cell death following ischemia is very sensitive to small
decreases in temperature, being abolished by a 2°C fall
(292). Unfortunately, there were no controls for possible
reduction of temperature by cycloheximide at the levels
that were used in the studies (1 and 10 mg/kg), beyond 1 h
after ischemia. The studies thus suggest, but do not prove,
that protein synthesis is required for ischemia-induced
apoptosis. A similar conclusion emerged from work on
cell cultures where non-NMDA-mediated cell death after
ischemia showed TUNEL labeling and was blocked by
cycloheximide (405); here again, independent evidence
for apoptosis was not terribly strong, although the cell
death was inhibited by the general caspase inhibitor ZVAD FMK (387). There is no possibility of a temperature
artifact in culture. Although more careful studies would
be very useful, testing a correlation between protein synthesis inhibition and identified apoptotic-like death with
good temperature control, the current evidence indicates
that ischemia-induced apoptotic death requires protein
synthesis.
If protein synthesis is required, it might explain the
absence of apoptosis following global ischemia, where
protein synthesis is severely inhibited throughout the
postischemic period (see sect. IVC).
If synthesis is required, then the question is what
proteins are likely to be synthesized, and what is the
trigger? The answers are not known. Caspase-3 expression increases in global and focal ischemia, but the timing
of the increase is quite late for it to be playing a major role
in apoptosis, as discussed above. Quite extensive work on
other systems, and particularly on thymocytes, strongly
suggests that the newly synthesized proteins are required
to enable one or more steps that cause the release of
cytochrome c from mitochondria and ultimate activation
of caspases (710, 909). For example, thymocyte apoptosis
initiated “physiologically,” by cortisone requires protein
synthesis. However, thymocyte apoptosis initiated by directly opening the mitochondrial transition pore does not
(710). Clearly, there may be differences in different cell
types. However, it is possible that one effect of ischemia
is to trigger synthesis of proteins that allow eventual
release of cytochrome c by acting on the mitochondrial
membranes. A challenge is to find such proteins.
6. Conclusion
The extent of apoptosis is more problematic than
ECC or ICC, because it is more difficult to define. The
former are straightforward morphological classifications.
Presently, there is no good morphological evidence that
apoptosis occurs after global ischemia, but there is such
evidence, in the nucleus, after focal ischemia and hypoxia/ischemia. TUNEL staining, which has been used to
imply apoptosis in global ischemia, is not a valid measure.
There is DNA laddering in all the ischemic models,
although the laddering of DNA may proceed by a somewhat different mechanism than in normal apoptosis. The
DNA fragments produced by either focal or global ischemia in adult rats, or by hypoxia-ischemia in young rats,
have staggered ends, as distinct from blunt ends seen in
classical apoptosis. The 39-end is recessed ;8 –10 bp (J.
MacManus, personal communication). One possibility is
that an additional endonuclease may be activated in cerebral ischemia, which acts after the classical apoptotic
nuclease(s) (301). There is strong evidence for caspase
activation and some attenuation of cell death by caspase
inhibition in global and focal insults, which seems to be
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isolated mitochondria in the absence of the mitochondrial
pore transition (24).
D) TIMING OF CASPASE ACTIVATION. The timing of caspase
activation with respect to cell death remains something of
an enigma. In both global and focal ischemia, apparent
activation of caspase-3 occurs many hours and in fact
days before signs of cell death are manifested. This is true
when evaluated by appearance of fragments, by appearance of proteolytic activity, and by the time at which
caspase-3 inhibitor has to be added to prevent apoptotic
cell death. There are several systems where caspase activation is very early, including the death-domain ligand/
receptor interactions and staurosporine (618). However,
this is generally an activation of a “precursor” caspase
such as caspase-1 (618) or caspase-8 (1004, 1186, 1219). It
is hard to envision how caspase-3, with its multiplicity of
downstream critical targets, would take days to kill cells
(this can be compared with NGF withdrawal where nuclear condensation and cell death follows caspase-3 activation within 1 h or less). Understanding this time course
should give important insight into the control of ischemic
apoptosis.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
C. Autophagocytotic Cell Death
1. Basic properties of autophagocytotic cell death
A third distinct form of cell death that may pertain
during ischemia is autophagocytotic cell death. This was
classified by Clarke as type II (apoptotic death is type I)
cell death and has been noted in several cases during
development of the nervous system (205). The possibility
that this occurs has been suggested in some discussions
(178), but it has not been studied at all rigorously. In fact,
though, it may be quite prevalent.
Autophagosomes are fusion products of lysosomes
with mono- or multilayered lipid membrane vesicles enclosing regions of cytoplasm (637), which often include
organelles (371). Normal protein turnover by this mechanism occurs at ;1%/h in most cells, and it is thought to be
the principal mechanism of steady-state protein breakdown (637, 1015). Its involvement as a principal player in
death implies that it is strongly activated, and there are
now a number of studies implicating autophagocytosis in
cell death, indicating that it is indeed capable of killing
cells when activated well beyond its normal rate (205).
The characteristic morphology of autophagocytotic cell
death includes a condensed cytoplasm containing many
large vacuoles, most of which are autophagosomes or proliferating lysosomes, and a nucleus in which chromatin is
irregularly clumped (205). This is not very different from
ischemic cell change, or from cytoplasmic morphologies
described for some cases of apoptosis. The nucleus is generally less pynknotic and less dense than in ICC.
2. Evidence for autophagocytosis after ischemia
The rate of autophagocytosis is greatly increased in
some cells that have undergone injury (371) and is approximately doubled in liver 24 h after 60-min ischemia
(724). There is only one direct identification of autophagocytosis in brain ischemia, but it is quite persuasive
(834). Structures that appear to be autophagosomes proliferate ;3 days after 5-min ischemia in gerbil CA1 pyramidal cells. These structures are bordered by multiple
membranes, contain organelles or parts of organelles, and
in addition contain cathepsin. They thus appear to be true
autophagosomes (371). This is the only study in which
autophagosomes were looked for specifically, and it is
possible that they are peculiar to gerbil global ischemia,
because that insult is characterized by a huge proliferation of ER-like membrane, which could be the precursor
to the structures. On the other hand, based on appearance
of cells, it has been suggested as the principal mode of
cell death in rat global ischemia and as very important in
focal ischemia (178), and there is a large increase in
immunoreactivity of the cathepsin B precursor protein
within 24 h of 12- to 15-min global ischemia in rat (448).
Further work in which lysosomal enzymes are localized
and subjected to inhibition and where autophagosomes
are identified would be needed to establish that the process is important in ischemic cell death. There are now
relatively specific inhibitors of cathepsins, such as
3-methyladenine, that appear to work in vivo (149).
There is almost no pertinent information on what
ischemic change might trigger autophagy. Basal autophagy in hepatocytes is strongly inhibited by depleting ER
stores of Ca21 (383), indicating that ischemia-induced
changes in ER could be important. Alternatively, because
phosphorylation of the ribosomal protein S6 inhibits autophagy in liver (100), it is possible that dephosphorylation of this protein occurs in the postischemic period and
so activates autophagocytosis.
D. Molecular Changes Underlying Morphological
End Stages
The first three parts of this section describe the morphological and biochemical aspects of the three major
pathways of cell death. These lead to the key questions,
which are 1) what are the molecular changes underlying
these end stages and their development, and 2) what are
the relationships between the different pathways of cell
death? The first of these is considered in the current
section. The answer is simple, albeit disheartening. There
really is no published work that describes the molecular
changes that form the basis for the end stages, or that
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greater for milder insults, a relatively common property of
the apoptosis/necrosis dichotomy. Further work on the
very rapidly developing field of caspase biology will,
hopefully, clarify the roles of these enzymes.
The nuclear morphology of apoptosis is apparent in
focal ischemia. What is not yet clear is how completely
processes in the cytoplasm, which are almost undoubtedly the keys to cell death, mimic classical apoptosis and
also what percentage of the population is showing the
apoptotic events (699). At present, it is clear that the
cytoplasm in neurons showing apoptotic changes is generally highly vacuolated compared with normal apoptosis,
although the latter do often show vacuolation. It is also
clear that there are a large number of eosinophilic or
ghost neurons and that some of these show apoptotic
nuclei, indicating a very mixed cell death pathway. Future
studies in which characteristics of apoptotic cell death,
and ischemic cell death, are defined more clearly will be
of great help in determining what is occurring. Part of this
should include characterizing the presence of specifically
apoptotic protein changes such as spectrin and F-actin
(gelsolin) cleavages, as well as phosphatidylserine redistribution in the plasmalemma. A promising line of work
that is developing is the careful comparison between
properties of apoptotic death in neurons, induced for
example by staurosporine, and ischemic cell death (700).
This issue is considered in section IIIE.
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PETER LIPTON
describes metabolism in these end stages. All that can be
done here is to briefly speculate.
1. Edematous cell change
The key alterations in edematous cell change are the
nearly empty and structureless cytoplasm and the swelling of the cells. The nucleus shows relatively mild chromatin clumping. The clearing of the cytoplasm implies a
loss of protein and of the cytoskeletal structure. The
swelling is most likely to result from inhibition of the
Na1-K1-ATPase or from plasma membrane leakiness.
The cytoplasmic features of ischemic cell change are
very striking. They include severe shrinkage and condensation to a quite triangular shape (in 2 dimensions), increased electron density of the cytoplasm, and darkening/
pynknosis of the nucleus. There is strong eosinophilia of
the cytoplasm when stained by H and E and argyrophilia
when it is silver stained.
Steady-state regulation of cell volume is not well
understood. Shrinkage of sympathetic neurons after NGF
withdrawal is strongly correlated with loss of protein
(289, 333) so that the shrinkage could be explained by
massive proteolysis. There are now several very specific
cell-permeant inhibitors of proteasomal activity (749,
1033), and their effects on ICC would be of interest.
Indeed, a proteasome inhibitor, PS-519, does strongly decrease the size of the striatal core lesion after temporary
focal ischemia (902), although this might not be related to
prevention of massive protein breakdown but, rather, to
prevention of activation of the transcription factor NFkB
(56). Individual cells were not studied in this report. Cell
volume might also be decreased by activation of K1 and
Cl2 channels as occurs in regulatory volume decrease
(1116). The latter is triggered by increased cytosolic Ca21,
and it is possible that it could be activated by this or other
mechanisms.
Argyrophilia might also reflect proteolysis, since it is
greatly enhanced by puromycin, which produces small peptide fragments (1091). On the other hand, Gallyas et al. (350)
emphasize the all-or-none quality of argyrophilia (350) and
speculate that there is a cooperative macromolecular-skeletal change that exposes protein side chains, causing the
argyrophilia and the increased electron density.
Eosinophilia is partly due a loss of hematoxylin or
Nissl staining, whose basis is not known, perhaps loss of
ribosomes or of polysomes. There may also be an increase in eosin staining. This could be due to major
changes in pKa and/or structure of proteins (1137).
Each of these speculations could be addressed experimentally to help determine the macromolecular basis for
ICC.
3. Apoptotic cell change
Cell shrinkage and increased electron density are
also features of apoptosis, although eosinophilia and argyrophilia do not occur. As discussed above, a net decrease in protein synthesis likely accounts for cell shrinkage in at least one model of apoptosis; it has not been
studied in others. Specific cell changes in apoptosis are
not very well known. However, the central role played by
caspases, and by calpain (909), has revealed a wealth of
potential protein changes. In cytoplasm, fodrin/spectrin is
a caspase-3-like target (811), as are proteins involved in
actin stabilization such as gelsolin (605). Thus a fairly
massive attack on the cytoskeleton is suggested, which
may well account for the apoptotic death. Caspase-3 also
cleaves the endogenous regulator of protein phosphatase
2A, activating that enzyme (987). This could strongly affect interactions between cytoskeletal proteins. Of these
changes, only caspase-mediated spectrin degradation has
been shown during ischemia, in cultures (810). Actin
breakdown has not been noted (183).
4. Summary
It is clear that much work is required if the molecular
changes underlying the morphological end stages are to
be determined. Almost nothing is known. Measurements
of metabolism in different end stages would also be very
valuable. Knowing these would show the targets of the
processes that might be occurring during development of
damage.
E. Relationships Between Different Forms
of Cell Death
1. ECC and ICC
Edematous cell change and ICC change appear to be
independent pathways of cell death. Only ECC is seen in
hyperglycemic global ischemia and prolonged hypoxia/
ischemia in young animals, whereas ECC has not been
noted in focal ischemia. However, the two morphologies
are seen together in some cases, in particular at the end
stage of global ischemia and after NMDA injection in
striatum. In the latter case, a detailed study indicated that
they developed along parallel paths for 12 h and that by
24 h both types of damage showed major disintegration
(919), suggesting no crossover. It seems most likely that
when cell damage reaches a certain point it can lead to
one or the other pathways; the basis for the very dramatic
bifurcation is not known.
2. ICC and apoptotic cell change
Apoptotic changes and ICC are seen at the same time
and in the same populations during focal ischemia, hypoxia/ischemia (292), and after glutamate injection into
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2. Ischemic cell change
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
sorting of cultured cortical neurons into necrotic or
apoptotic-like pathways determined by whether or not
there is permanent damage to the mitochondrial membrane potential when cells are exposed to NMDA (25).
The reason that different insult intensities do predispose to one or other pathways is not known. A simple
idea is that when ATP levels, or other parameters such as
Ca21, change drastically they disrupt apoptotic pathways,
thus biasing the process toward necrosis. As an example,
if protein synthesis, or phosphorylation, is required for
apoptosis and it is drastically inhibited, then apoptosis
may not be allowed. There are many other such possibilities and, indeed, in lymphocytes (452) artifactual opening
of the mitochondrial transition pore (MTP) causes apoptosis in a cell that is set up to carry out the process but
causes necrosis if, for example, appropriate caspases are
inhibited. Thus necrosis may be a fallback pathway if
apoptosis is somehow not available!
The final stages of apoptosis after ischemia or excitotoxicity are very similar to those of necrotic cell death
(699). Unlike the controlled phagocytosis in physiological
apoptosis, there is a “secondary necrosis” at the final
stages of apoptosis so that cell dissolution occurs, as
shown in a careful study of excitotoxicity (919). This
further complicates identification of apoptosis.
3. Autophagocytotic cell change and ICC
The evidence for autophagocytosis is clearly sparse
because it has been studied very little. At this stage, it must
be considered that it may coexist with either ICC or apoptotic cell change; that is, the vacuoles associated with both
these end stages may, at least in part, be autophagocytotic.
The critical factor is whether this is true and, if it is, the
extent to which the autophagocytosis is responsible for cell
death. Alternatively, ICC and apoptotic cell change may be
completely independent of autophagosomes, and autophagocytosis may occur in a separate population of cells.
There really are no data that bear on this yet.
F. Summary
There is strong evidence for at least three different
pathways of ischemic cell death: ECC, which occurs least
frequently; ICC (leading to homogenizing cell change),
which is most prevalent; and apoptotic cell change. There
is preliminary evidence for a fourth programmed pathway, autophagocytotic cell death, that may be very important in global ischemia, at least in the gerbil.
There is strong morphological and biochemical evidence that populations of cells showing apoptosis and ICC
coexist during focal ischemia and also during hypoxia/ischemia in neonates. In the delayed death following global
ischemia, there is biochemical evidence for apoptosis but no
morphological evidence, and at present, it is concluded that
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striatum (919). However, evidence suggests they are quite
distinct modes of death.
In piglet hypoxia/ischemia, reducing temperature by
2°C eliminated apoptotic death but caused no apparent
change in the numbers of necrotic neurons (292). This is
very difficult to reconcile with a sequential process. In
spinal cord ischemia, different populations of cells, segregated by lamina, show almost entirely necrotic cell
death, or apoptotic cell death (541). Thus different populations subjected to the same insult appear to take one or
the other pathways. When two different glutamate agonists were injected into striatum of the adult rat, the
NMDA analog quinolinic acid caused exclusively necrotic
cell death while kainic acid caused almost exclusively
apoptotic-like death (919), again indicating two distinct
pathways. The distinctiveness of the two pathways is
further suggested by the major morphological differences,
particularly the eosinophilia/argyrophilia of the necrotic
pathway as well as the very different nuclear morphology
(although a transition from condensed spherical chromatin to uniformly condensed chromatin does not, at least
naively, seem to be prohibitive). A recent study in cultured neurons exposed to either staurosporine (apoptosis) or high levels of glutamate (considered to be necrosis) emphasized the difference in nuclear morphology; the
very darkened pynknotic nucleus in necrosis verses the
less pynknotic-punctate chromatin nucleus in apoptosis
(700).
If the two pathways are, indeed, independent, then
the critical question becomes why different cells within
the same apparent population proceed by different pathways during certain insults. One possibility is that the
cells are actually from different populations, which have
not yet been recognized. Other possibilities include subtle
differences such as densities of NMDA receptors because
these have a tendency to force cells into a necrotic rather
than an apoptotic mode in some systems. This was mentioned above and is seen in some culture systems where
NMDA-mediated damage is necrotic and non-NMDA-mediated damage is apoptotic (405, 870).
A quite large number of studies show that increased
insult intensity appears to favor necrosis as indicated by
the fraction of apoptotic-like cells following severe and
mild focal insults (compare Refs. 422 and 304), by the
relative abundances of apoptotic and necrotic cells in the
core and penumbra of focal lesions (178, 785), and by the
fraction of cultured cells taking the two pathways as
NMDA or NO levels are increased (111). A nice study in
kidney tubule cells shows a very strong relationship between the percentage decrement in ATP by metabolic
inhibition and the cell death pathway taken. Apoptosis
proceeded when ATP levels were .25% and necrosis
when levels were 15% (658). Thus certain cells, because of
their state at the time of the insult, may tend to one or the
other pathway because the insult’s effective intensity is
different. This is suggested by the apparently random
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PETER LIPTON
IV. CRITICAL FUNCTIONAL AND
STRUCTURAL CHANGES
The basis for this section is the hypothesis that development of cell death and of the end stages results from
long-term damage to key cell functions or structures. Cell
functions and structures whose changes seem most likely
to be the proximal cause of necrotic or apoptotic cell
change (membrane damage, mitochondrial damage, inhibition of protein synthesis, and cytoskeletal damage) are
examined to see if they play a critical role in ischemic cell
death.
The hypothesis may be wrong. Cell death may result
from continued activation of damaging biochemical processes (perpetrators) set in motion by the ischemic insult,
with ultimate breakdown of the cell as a unit. Cell death
may not be perpetrated by an intermediary functional
defect in one or more key processes. Nevertheless, this
section describes ischemic changes in major intracellular
systems because they are deemed likely to be involved in
cell death.
A. Altered Membrane Transport Properties
1. Membrane permeability
The question is whether there are large increases in
membrane permeability to ions or metabolites that might
lead to cell death. There is a paucity of data on this very
important subject.
There are a great number of studies of ischemia in
culture showing a correlation between the number of
disintegrated or necrotic cells and membrane breakdown,
measured as LDH release or uptake of vital stains including trypan blue, propidium iodide, and ethidium bromide
(376, 791, 1072, 1156). However, there are almost no
studies aimed at determining whether the membrane
changes precede large-scale cellular changes. Propidium
iodide does enter neurons in culture that look normal but
are in the process of dying by necrosis, indicating that
membrane leakiness may be a proximal cause of damage
in cell culture (697). Unfortunately, this work in culture is
the only study addressing the issue.
The mobility of a spin probe (5-NS) within the membrane bilayer of synaptosomes increased during the first
hour after 10-min ischemia and then increased again transiently ;8 h later (411). This may have reflected increased
disorder in the phospholipid bilayer. Unfortunately, the
study has not been followed up.
Breaks were noted in the postsynaptic dendritic
membrane during the first hour after 10-min complete
compression ischemia in rat, and there was also a marked
increase in neuronal permeability to horseradish peroxidase (256). Unfortunately, this finding was not pursued so
that neither its reproducibility nor its significance are
known.
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autophagocytosis or ICC, and some ECC, account for main
features of delayed cell death in that model.
Some insight is now being gained into how the
apoptotic end stage is reached. The broad outlines of
the apoptotic pathway are identified in focal ischemia
and hypoxia/ischemia, and although the details including the triggers for apoptosis, the caspases involved,
the molecular changes involved, whether mitochondrial
release of cytochrome c is involved, are still unresolved, there is some evidence that the Fas (CD-95)
ligand system, and possibly TNF-a along with protein
kinases, may well be involved in initiating the process,
and there is quite good evidence for the timely release
of cytochrome c from mitochondria. There is almost no
insight into how other end stages are reached, although
the strong protective effect of proteasome inhibition
indicates that large-scale proteolysis may be important
in ICC.
The marked difference in nuclear morphology between focal ischemia and delayed neuronal death from
global ischemia is currently a puzzle. Given that caspases
play a part in death from both insults, and possibly BAX
also, why do the nuclei not show the condensed spherical
chromatin bodies after global ischemia? Possibly the
caspase activation, or some other process, during global
ischemia is not strong enough to cause this change, thus
leading to some of the biochemistry of apoptosis but not
the morphology.
There is also very little understanding at present of
the molecular changes, or metabolic changes, underlying
the gross structural changes of ICC, or ECC, and these
seem particularly important to determine.
Although there do appear to be separate, predominantly apoptotic or necrotic pathways, the former may
be unique to ischemic cell death, different from apoptotic pathways taken in the absence of a metabolic or
physical insult. Apoptotic nuclei appear associated
with very highly vacuolated cells, or even cells showing
changes similar to ECC and homogenizing cell change.
This certainly suggests that elements of both pathways
may coexist in conditions where nuclei show clear
evidence of apoptotic change and where caspases are
involved in the cell death process. DNA cleavage sites
are also unique.
In conclusion, the ionic and biochemical changes
to be discussed in ensuing parts of the review lead to
the cells adopting one of several possible pathways to
cell death. Some of those changes, along with the nature of the cells affected, are the determinants of the
pathway that is selected, but at this stage, the basis for
selecting a particular pathway is not known. Relatedly,
the critical changes in molecular structures that are
associated with the end stages of the different pathways are not known.
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
2. Na1-K1 pump or other transporters
One to two days of ;50% inhibition of the Na1 pump
with ouabain does cause an apoptotic-like death in cultures (713) so that if there is submaximal Na1 pump
inhibition after ischemia it might well contribute to apoptosis. Also, profound pump inhibition might lead to cell
swelling and ECC.
Reasonably short exposures to several different free
radical species cause severe irreversible damage to the
Na1 pump (187, 473, 1022) and also make it more susceptible to proteolytic attack (473). There is some evidence
that free radical generation following various ischemic
insults does inhibit the pump (382, 701, 866). Sixty minutes of 2-VO in gerbil followed by 15- to 30-min recirculation caused a 70% reduction in ATPase of frontal cortex
and hippocampus, which was blocked by pretreatment
with agents that should reduce free radicals (866). However, there was no pump inhibition after 15-min ischemia
(866), a duration that invariably leads to delayed neuronal
death. The latter suggests that pump inhibition does not
play a role in delayed neuronal damage from global ischemic damage. However, it may well be important in focal
damage, based on the effects of 60-min ischemia. Studies
of this possibility have not been reported and would be of
great interest.
Currently, there are no reports of long-term effects of
ischemia on other plasma membrane transporters or
channels that might cause significant ion or volume imbalance. Such studies would be very valuable.
3. Conclusion
Overall, there is little hard evidence implicating membrane changes in ischemic cell death. This is very surprising given the lability of membrane structure, the many
biochemical changes associated with ischemia, and the
huge potential for cell damage if membrane function is
disrupted. The problem may be the lack of studies that
have addressed the question.
B. Mitochondrial Damage
1. General background
Mitochondria are assuming an increasingly important
role in hypotheses about both apoptotic and necrotic cell
death (591, 617, 1250), but definitive demonstrations that
they play roles in ischemic damage are still lacking. There
are three general ways in which mitochondrial damage or
change might make an important contribution to ischemic
cell death.
1) Inhibition of mitochondrial oxidative phosphorylation or significant uncoupling will lower ATP levels,
increase mitochondrial free radical production, and remove the Ca21 buffering ability of the organelle. These
effects are capable of causing grave damage. This could
occur as a result of direct effects on the tricarboxylic acid
cycle enzymes, on the mitochondrial electron transport/
oxidative phosphorylation system or the lipid composition of the mitochondrial membrane, or as a result of
long-term opening of the MTP.
2) In addition, opening of the MTP may produce
damaging effects by releasing intramitochondrial molecules and ions, for example, a factor able to increase
L-channel Ca21 permeability as recently shown by
Nowicky and Duchen (841).
3) Finally, release of cytochrome c and or at least one
other protein from the intermembrane space is now recognized as a critical step in apoptosis in all systems in
which it has been studied. Such release can be effected by
opening of the MTP but is very frequently due to more
direct effects on the integrity of the outer mitochondrial
membrane, probably mediated by a member of the BCl2/Bax protein family, such as Bar (309, 941).
Alterations in mitochondria therefore have enormous
potential for causing severe cell damage. Despite this, and
despite well-informed speculation (625), there is still a
lack of evidence that convincingly implicates mitochondrial changes in either necrotic or apoptotic ischemic cell
death. There is, though, a body of work that bears
strongly on the issue.
In assessing this work, it is notable that isolating
mitochondria from ischemic or damaged tissue may actually lead to mitochondrial damage (34). This is not
usually considered.
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Although not related to a specific function, the fact
that the phosphatidylcholine precursor cytidine diphosphate choline (cyticholine or CDP-choline) is protective
in ischemia indicates that membrane damage may be
critical (516, 855). When administered intraperitoneally,
CDP-choline very significantly ameliorates the neurological deficit days after 20- to 30-min 4-VO (516) and survival
after focal ischemia (337), and it decreases infarct size
either in combination with NMDA blockade (855) or when
used after short insults (36). In temporary ischemia, the
molecule has several effects (516); however, it does very
clearly act as a precursor to phosphatidylcholine incorporation into cell membranes (516), and it dramatically
decreases the net liberation of free fatty acids (FFA)
during ischemia (268, 1133), presumably by activating
resynthesis of phospholipid. The interpretation of the protective studies is far from unequivocal at this stage; however, they may indicate that loss of membrane integrity is
contributing to the neuronal death.
In conclusion, despite a widespread assumption that
gross plasma membrane changes are important in ischemic cell death, there is almost no evidence for this,
particularly in vivo.
1463
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PETER LIPTON
2. Damage early after ischemia
preferentially inhibit complex I (475), which may explain
the apparent involvement of this complex in inhibition.
Several electron microscope analyses show an accumulation of Ca21 in mitochondria very soon after global
ischemia, which persists for several hours (285, 596, 1051,
1165, 1181, 1272) (there are no equivalent studies in focal
ischemia), observations that are at least consistent with
Lee’s hypothesis.
21
C) ROLE FOR NO? The basis for the prolonged Ca
accumulation is not known; however, there is interesting
evidence implicating NO. Accumulation in vivo was
strongly blocked when an inhibitor of NOS, NG-nitro-Larginine, was added intraventricularly before 5-min ischemia in the gerbil (596). Intracellularly generated NO, at
levels that arise during and shortly after ischemia,
strongly depolarizes mitochondria, inhibiting ATP production (127). Although not tested, it is possible that this
results from large-scale Ca21 accumulation and so accounts for the observed effect of blocking NOS. Both the
above effects could be due to NO or to peroxynitrite (see
sect. VA). In the latter case, they would also depend on
free radical production.
If this is the case, and if Ca21 accumulation is tied to
damage, then free radical and NOS blockade should prevent the early mitochondrial damage. Although studies
are not very extensive, some data are at least compatible
with this. Blockade of FFA production with the plateletactivating factor (PAF) antagonist BN50739 prevented
;50% of the postischemic mitochondrial inhibition and
several free radical scavengers attenuated the early decrease in ATP after global ischemia in the dog (360).
Furthermore, swelling of mitochondria 1 h after 5-min
ischemia in hippocampal slices was prevented by calmidazolium (316), which, among other things, prevents activation of NOS. Thus data are consistent with peroxynitrite-mediated uptake of Ca21 by mitochondria and
subsequent inhibition of function. A mechanism whereby
NO or peroxynitrite leads to Ca21 accumulation is not yet
known.
2. Delayed mitochondrial damage
There are several reports indicating delayed damage
in isolated mitochondria, but again results are quite variable, and timing does not always clearly show that the
mitochondrial damage precedes cell death (e.g., Ref. 786).
Several hours after 11-min anoxia in cats there was a
reduced tissue oxygen consumption that was strongly
associated with inhibited isolated mitochondria (1180).
State 3 respiration measured on isolated rat mitochondria
was reinhibited by ;50% 5 h after 30-min 7-VO in the rat
(1012). In the very sensitive striatum, mitochondria became permanently inhibited 30 –90 min after 2-VO ischemia and 6 h after 4-VO (449, 944, 1054), and they were
inhibited 48 h after the ischemia in CA1 (1054), but only
by 20%. There was a delayed loss of the respiratory con-
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A) EVIDENCE FOR DAMAGE. Mitochondria almost always
undergo a transient swelling for a few hours after any
form of ischemia (130, 742, 896, 897, 1002, 1236). The
origin has not been studied but may be the reversible
opening of the MTP due to free radical generation and
Ca21 accumulation (278, 410). This swelling has not been
directly correlated with function, but it certainly appears
that mitochondria are inhibited during this period.
Tissue metabolic rate, as measured by glucose utilization, is generally temporarily depressed by 50% or more,
for at least 6 h after transient global ischemia (291, 606,
760, 931), and is depressed in the penumbra during and
after focal insults (79, 1252, 1299). The depression after
global ischemia does not occur in all regions but always
occurs in vulnerable cells; in gerbil hippocampus, only
CA1 is affected (760). Along with the decreased metabolic
rate, ATP levels are reduced by 20 –30% for several hours
after 30-min 4-VO and 2-VO in the rat and 10-min complete
ischemia in the dog (1070) and cat (1179). Furthermore,
cytochrome complex aa3 is hyperoxygenated in intact
tissue after global ischemia, consistent with failure of
adequate substrate supply to the respiratory chain (961).
These data are consistent with, but do not establish,
mitochondrial dysfunction. They could equally be explained by a failure of glucose metabolism.
Unfortunately, the totality of studies on isolated mitochondria show a great deal of variability, some of which
may well result from difficulties in their isolation. However, overall they do indicate inhibition in the postischemic period. State 3 respiration of isolated mitochondria
is generally severely depressed (by 50% or more) immediately after global ischemia (449, 450, 668, 1012, 1054)
and, in most measurements, for at least 1 h afterward
(449, 944, 1080), although in one study it recovered within
an hour (1054). Respiration of penumbral (and focal)
tissue mitochondria is inhibited by 40% for at least 4 h
after transient focal ischemia of between 1 and 2 h (624,
625). The damage almost always affects the NADH-linked
portion of the respiratory chain (complex I) (743).
B) MECHANISM OF DAMAGE. Lee and co-workers (1012)
suggest that the basis for the early mitochondrial inhibition is accumulation of mitochondrial Ca21 during, and
possibly shortly after, the ischemia. The conclusion was
based on its very rapid reversal when EGTA or ruthenium
red (RuRed) were added to the isolated mitochondria
(1012). This is consistent with the site of action being the
MTP, which is known to be rapidly activated by increased
cystosolic Ca21 (278), due to accumulation in the matrix
(410), and which rapidly closes when isolated mitochondria are exposed to a chelator (475). This being said, it is
very probable that other processes are involved; for example, the preferential blockade of complex I and hyperoxidation of cytochrome aa3 are not obvious consequences of the MPT. Calcium accumulation appears to
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
4. Possible bases for delayed
mitochondrial dysfunction
Mitochondria are known to be susceptible to free
radicals and Ca21 accumulation, both via the MPT and
other mechanisms. However, the nature of transition from
reversible to irreversible mitochondrial damage is still not
well understood
A) FREE RADICAL AND PEROXYNITRITE ACTIONS. I) In vivo.
Free radicals seem to be at least partially responsible for
the putative delayed mitochondrial damage. As described
in section VA, free radicals are elevated after both global
and focal ischemia.
Pharmacological studies suggest the involvement of
free radicals. The delayed loss of energy charge, and ATP,
following temporary focal ischemia was protected by the
spin trap N-tert-butyl-a-phenylnitrone (BPN) (329), and
the delayed mitochondrial dysfunction measured after
7-VO in the rat was largely prevented by ascorbate (1011),
as was the mitochondrial inhibition several hours after
transient focal ischemia (801). The latter was also protected by the calcineurin inhibitor FK-506 (801), possibly
because it prevents activation of constitutive NOS (cNOS)
and subsequent accumulation of peroxynitrite, although
this has not been shown.
Somewhat less directly, Mn-superoxide dismutase
knockout mice generated more free radicals during permanent focal ischemia, showed a larger lesion, and
showed a much larger number of cells with a loss of
mitochondrial membrane potential after 24 h (786), demonstrating that excess free radical production enhances
mitochondrial damage. However, this result does not explicitly show that normal free radical production causes
the damage. All these studies are suggestive; however,
they do not rule out the possibility that mitochondrial
damage is an indirect result of free radical damage to
another process (e.g., Ca21 homeostasis).
II) In vitro. Exogenously generated free radicals severely damage mitochondrial state 3 respiration, particularly in the presence of elevated Ca21 (119), and these
effects may be the basis for the in vivo effects described
above. Very short (5 min) exposures of cultured neurons
to peroxynitrite cause ;50% inhibition of cytochrome-c
oxidase and succinate-cytochrome c reductase that matures over 24 h (107). This is a very profound effect that
has the possibility of occurring in vivo and being very
important.
Nitric oxide donors (e.g., S-nitroso glutathione) applied to isolated mitochondria, and to thymocytes, open
the MTP, leading to membrane depolarization and subsequent increased superoxide generation by the mitochondria (458). Generation of peroxynitrite from superoxide
and NO at rates calculated to be similar to those encountered in pathological conditions, causes Ca21 release and
depolarization of liver mitochondrial membranes, with
opening of the transition pore (863).
21
B) EFFECTS OF CA . In at least one study, the timing of
21
Ca accumulation is consistent with its causing delayed
inhibition; it increased in mitochondria of the striatum 6 h
after 4-VO, shortly before this region began to degenerate
(1272). There was a large increase in total cell Ca21
beginning 24 h after global ischemia in the gerbil that may
well reflect large scale Ca21 accumulation by the mitochondria. There was a concomitant fall in ATP (432).
Unfortunately, there are no other good studies of delayed
changes in mitochondrial Ca21.
There is quite a large body of evidence that Ca21
accumulation by mitochondria is a major mechanism of
mitochondrial damage and subsequent cell toxicity in
response to glutamate, raising the possibility of a similar
effect after ischemia. Glutamate-induced necrosis in several culture systems (25) is associated with large-scale
accumulation of Ca21 by mitochondria, subsequent mitochondrial depolarization (26, 994, 1206), and lowering
of ATP/ADP, down to 30% of normal levels (142). The
inhibitor of the mitochondrial Ca21/2Na1 exchanger CGP37157, which enhances mitochondrial Ca21 accumulation
and decreases cytosolic Ca21 accumulation, enhances depolarization and damage, establishing quite nicely that the
mitochondrial Ca21 accumulation is causing the depolarization and damage (1206). There is other strong evidence
implicating mitochondrial Ca21 accumulation as a key
factor in this necrotic event (829, 1071).
The basis for the Ca21 accumulation in those cases is
the glutamate-induced cytosolic Ca21 accumulation,
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trol ratio 2– 4 h after temporary MCA occlusion, in mitochondria isolated from both the core and the penumbra
(625) as well as a decrease in state 3 respiration (801).
There was a large decrease in mitochondrial membrane
potential, measured in situ, after 24 h of focal ischemia
(786). All of these results, except the last, strongly indicate mitochondrial damage in reasonably late stages of
damage development. The last result could reflect
changes in dead or dying cells because there is a lot of cell
damage by 24 h.
There are decreases in tissue ATP levels before or
around the time of cell death in global ischemia (760, 929)
and in the penumbra in focal ischemia (329). Levels after
global ischemia fall by 25 and 40% in rat CA1 hippocampus and striatum, respectively (929), and probably further
in gerbil at 24 h (432). It is not known, though, that they
result from mitochondrial damage. For example, in one
study, the falls in ATP after focal ischemia could be
explained by the falls in total adenylates (329). Decreased
levels could also result from inhibited glucose metabolism.
Overall, these studies suggest late, functionally important, mitochondrial damage. They are not strong
enough to establish this unequivocally, and further careful
studies are required. Studies of the status of the MTP and
also of cytochrome c leakage would be very valuable.
1465
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PETER LIPTON
This has not been measured in ischemia but does occur
when the MTP is opened and mitochondria generate free
radicals (452). Cardiolipin changes may well produce defects in the electron transport chain (872).
The mitochondrial pore transition represents a reasonable target for damage; it could be mediated by increased Ca21 or by free radicals, and by both acting
synergistically (410). It produces profound changes in cell
function (89, 410). Although early evidence concerning
the pore opening was conflicting, it now appears that it
does play a role in, at least, focal ischemic damage. Cyclosporin A, which inhibits the MPT, was very protective
in a global ischemic model (1145), but the drug also
inhibits calcineurin, and FK-506, the calcineurin blocker,
is also protective after global ischemia (272). FK0506 is
also protective after temporary focal ischemia (627), but
in this insult, a derivative of cyclosporin, methyl-valine
cyclosporin A, which does not inhibit calcineurin, reduces
the infarct size by .50% (528). This result implicates
opening of the transition pore in, at least, focal ischemic
damage, although it does not show whether it is early or
late opening that is damaging.
5. Damaging actions of mitochondrial dysfunction
There are four apparent mechanisms by which mitochondrial dysfunction in the postischemic period could
lead to cell death. These include maintaining low ATP
levels, overproduction of free radicals, initiation of apoptosis or other damage by leakage of macromolecules,
and decreased ability to buffer Ca21 loads. There may be
other, unknown effects of mitochondrial damage.
A) LOWERED ATP LEVELS. The issue is whether and how
maintaining small (20 –30%) reductions in ATP for many
hours, as occurs in the postischemic phase, will lead to
cell death. Elegant and important in vivo studies by Beale,
Greenemayre, and co-workers show that brain-injected
mitochondrial inhibitors cause a delayed cell death. However, the long transient fall in ATP, of ;50 – 60% at 3 h,
more closely parallels conditions during focal ischemia,
so the studies are not necessarily germane (1001). More
pertinent studies have been done in culture where
chronic treatment with mitochondrial inhibitors leads to
cell death within 8 –24 h (870, 1276, 1277). In one of these,
ATP levels were maintained at 70 – 80% of normal. Cell
death developed in 4 – 8 h (870) and was preventable by
glutamate receptor blockade (870, 1277). Death showed
selective vulnerability (1277), and there were mixed populations of apoptotic and necrotic neurons at 48 h (870).
Thus low-level mitochondrial inhibition produces both
apoptotic and necrotic populations in a timely fashion,
but in this case in culture. Although not tested, the assumption is that MTP did not open and that there was not
an enhanced production of mitochondrial free radicals so
that this represents a “pure” effect of lowered ATP. Even
if this is not so the results indicate that mitochondrial
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which reaches several micromolar (476). If this mechanism of mitochondrial damage is to occur after ischemia,
some factor must elevate mitochondrial Ca21 uptake.
This could be either increased cytosolic Ca21 or, perhaps,
an action on a mitochondrial Ca21 transport system. Although NO or peroxynitrite generation seems capable of
such action (see above), and there is delayed production
of iNOS that might contribute to damage in this way, there
is no evidence for such a mechanism at present.
C) MITOCHONDRIAL TRANSCRIPTION. Mitochondrial transcription of cytochrome oxidase mRNA is damaged
within ;3 h of 3.5-min global ischemia in the gerbil. There
was a progressive loss in enzyme activity and mRNA, with
both falling to 20% of normal after 2 days, well before
major manifestations of cell death. This was confined to
the CA1 region (2). This is consistent with the fairly
dramatic delayed lowering of ATP that was measured
qualitatively in CA1 of the gerbil (432). This phenomenon
has not been well-studied in other models but clearly may
be important. The mechanism by which transcription is
damaged is not known.
D) OTHER POSSIBILITIES. Ceramide, whose concentration
rises within 6 h of starting focal ischemia (619), has been
shown to reversibly inhibit isolated mitochondria (398).
The elevated ceramide concentration is maintained
throughout the ischemia, so it may inhibit in situ though it
seems unlikely it would account for inhibition of the
isolated mitochondria, since it is readily reversed on
washing (398).
Another possible basis for damage is that a large
release of cytochrome c, the probable activator of
caspases, compromises the respiratory chain.
E) SUMMARY. There are several reasonable mechanisms
for delayed mitochondrial damage following ischemia.
Evidence for free radicals is strong in that several free
radical scavengers, or agents which should reduce their
generation such as PAF inhibitors, attenuate mitochondrial damage, and also protect against ischemic damage
(1025). Furthermore, free radicals and peroxynitrite severely damage mitochondria in vitro. Further studies with
NOS inhibitors are essential to determine the role of NO
and/or ONOO2 in vivo.
Mitochondrial Ca21 accumulation also remains a viable possibility as a damaging agent and may certainly act
along with free radical production. More evidence concerning mitochondrial Ca21 changes that presage damage
would be very useful. Inhibition of cytochrome oxidase
synthesis and/or loss of cytochrome c are also very reasonable mechanisms of damage.
If Ca21 or free radicals are the active agents, then the
molecular target needs to be determined. Delayed damage
is not caused by irreversible damage to any of the dehydrogenase complexes (862). Free radicals might be the
agents acting on mitochondrial DNA to block transcription of cytochrome oxidase mRNA. Free radicals may
oxidize cardiolipins, the principal mitochondrial lipid.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
6. Conclusions
There are many strong heuristic arguments for the
involvement of mitochondrial change or damage in ischemic cell death. These include measured effects of conditions prevailing during ischemia, such as peroxynitrite
generation, on isolated mitochondria and also effects of
mitochondrial damage on variables such as free radical
generation, protein leakage, and indeed on cell viability in
several model systems. However, at this stage, there is
very little positive evidence that these changes play a role
in ischemic cell death. In fact, these are confined to
protective effects of methyl-valine cyclosporin A in focal
ischemia (global ischemia has not yet been tested) and
the movement of cytochrome c from mitochondria to
cytosol also seen after that insult.
There are many reports of damage to isolated mitochondria after global and focal ischemia, but these are
somewhat unsettling because no consistent basis for the
dysfunction has been pinpointed; damage to respiratory
chain complexes, blockade of synthesis of, or leakage of,
respiratory chain components, opening of MTP, and accumulation of Ca21 have all been noted or suggested, but
no coherent account has arisen to date. Furthermore,
there is not a strong consensus among different laboratories on times at which mitochondria are inhibited. Much
of the uncertainy probably arises from difficulties in preparation from damaged tissue, tissue heterogeneity, and
difficulties inherent in making measurements on isolated
mitochondria, such as appropriate choice of buffer. Thus,
at this stage, it is difficult to be definitive about the timing
and severity of damage to isolated mitochondria.
Damage in situ has not been well established at all
except in one case where measurements were made 24
and 48 h after focal ischemia, when cell damage was
widespread. Perhaps the best evidence is the widespread
pallor observed within the developing infarct when tissue
is exposed to the mitochondrial function dye TTC. This
certainly indicates compromised mitochondria, although
decreased metabolic rate or glucose metabolism would
lead to the same result. Certainly mitochondria appear to
be greatly swollen and disorganized in cells showing ICC,
which is consistent with damage, but this does not necessarily translate to functional damage.
If mitochondrial function is indeed compromised after ischemia, then evidence from cell cultures indicates
that even the low-level inhibition that appears to exist at
that time, evaluated by ATP concentrations, could cause
apoptotic and necrotic cell death. Unfortunately, there
are no pertinent studies in vivo, in which effects of prolonged mild inhibition of mitochondrial function have
been tested. The studies of glutamate toxicity indicate
that profound mitochondrial Ca21 accumulation is a major factor leading to cell death, although the mechanisms
are not well understood (829). There is, though, no particular reason to think this occurs before ischemic cell
death.
Thus, although mitochondrial lesions seem to be
strong candidates for sites of cell death, or severe damage
leading to death (1044), there is no compelling evidence at
the present time except the very recent data on the effects
of methyl-valine cyclosporin A, presently in abstract form.
Whether or not lesions in this organelle are important in
cell death, this section has pointed out that there is much
to be done in determining the nature of mitochondrial
damage associated with ischemia.
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inhibition, to a level which approximates the change in
vivo, can lead to cell death in a timely fashion.
B) FREE RADICAL GENERATION. Although in vitro studies of
mitochondria strongly indicate that their production of
free radicals should be increased by ischemia (see sect.
VA), the actual evidence for this is really limited to only
one paper. It was shown, using the salicylate trap to
measure OH production, that free radical generation during the 1 h after global ischemia was completely prevented by blocking the mitochondrial respiratory chain
with rotenone (908). No effects on subsequent damage
were measured, and further work is clearly required.
C) INDUCTION OF APOPTOSIS. It is now clear that mitochondria play a central role in most (if not all) cases of
apoptosis (1275) by releasing cytochrome c (591, 941,
1250), and possibly other proteins (1275), from the intermembrane space, and it is clear that adequate cytochrome
c can induce apoptotic changes, at least in part by activating caspases (617, 941). There are several pathways by
which cytochrome c could be released including the MPT
(452, 617, 894, 1274). A possible mechanism for release
after ischemia is by direct interaction of BAX with the
outer mitochondrial membrane (941, 1161). Overexpression of Bax induces cytochrome c release from mitochondria in several systems (220, 882, 963), and recombinant
Bax causes release of cytochrome c from isolated mitochondria (514). Bax is indeed upregulated in vulnerable
cell populations after ischemia (185, 420, 1306), and such
a pathway would explain the apparent requirement for
protein synthesis in ischemic apoptosis.
D) OTHER MECHANISMS . A compromised mitochondrial
membrane potential due to inhibition of oxidative phosphorylation will compromise the Ca21 uptake pathway
in mitochondria and hence reduce the Ca21 buffering
that occurs during normal high-frequency neural activity (1202). This would lead, on average, to chronically
elevated cytosolic Ca21 levels and could thus be damaging.
Another possibility is that other heretofore unidentified proteins may be released from mitochondria once
the transition pore is opened, and lead to necrotic
changes, e.g., proteins which enhance Ca21 channel
opening (841).
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Volume 79
3. Basis for prolonged inhibition of protein synthesis
Prolonged inhibition of protein synthesis, if uncompensated by a decrease in breakdown of protein, would
clearly lead to massive changes in cell properties and cell
death. Protein synthesis is a complex process that is
critically dependent on energy charge (936), intracellular
K1/Na1 (672), and the integrity and phosphorylation level
of a large number of proteins and RNA species (1230). It
is strongly and permanently inhibited by ischemia in vulnerable cells, and the question is whether or not this is
responsible for any forms of ischemic cell death. The
question has to be considered in light of the marked
activation of synthesis of certain damaging proteins in
those same areas in which global synthesis is inhibited,
making it a complex but very intriguing issue.
Protein synthesis is extremely sensitive to cell energy
charge and ion contents (672, 936). However, the persistent postischemic inhibition of synthesis occurs despite
return of energy charge to normal values in slices (936)
and of ATP to normal levels in vivo (12). Ribosomes from
ischemic tissue seem competent (243) so are unlikely to
be the site of damage.
A) INITIATION FACTORS. At this stage, the only changes
that have been positively implicated in the decreased
synthesis are changes in the two initiation factors, eIF-2
and eIF-4, consistent with the major loss in polyribosomes after ischemia (104).
eIF-4G is a substrate for calpain, and there is a 30 –
50% loss in its immunoreactivity after 10- and 20-min
decapitiation ischemia in rabbit (821). This is very likely
due to calpain-mediated proteolysis because the 70% loss
of immunoreactivity at the end of 20-min cardiac arrest is
completely prevented by MDL-28170, the quite selective
calpain antagonist (820). There is a prolonged inhibition
of protein synthesis in vascular endothelial cells as a
result of free radical attack on eIF-4 family-like proteins
(513), showing that eIF-4 is also inactivated for a long
time by free radicals. Thus it is a very likely target.
There is also evidence that eIF-2 is involved in the
inhibition. Ternary initiation complex formation in tissue
homogenates is decreased throughout the cerebrum 30 min
after ischemia and is decreased in vulnerable regions only
(striatum and CA1 of hippocampus) 6 h after 15-min 2-VO
ischemia (146, 466). There is conflict as to the basis for this
inhibition. A priori, phosphorylation of EIF-2a or dephosphorylation of guanine nucleotide exchange factor (GEF)
are the most likely inhibitory points (466, 1230). One set of
studies concluded that there was no increase in EIF-2a
phosphorylation but demonstrated a decrease in GEF activity that appeared to be due to dephosphorylation of a site
that was normally phosphorylated by a tyrosine kinase. The
decreased activity lasted at least 6 h in striatum and CA1 of
hippocampus (466, 470). There was a correlation between
decreased phosphorylation of MAP kinase and cell damage/
protein synthesis inhibition (468), and it was speculated that
inhibition of synthesis resulted from the lack of neurotrophin upregulation in vulnerable populations and resulting
dephosphorylation of GEF. Certainly neurotrophin withdrawal dramatically and rapidly lowers protein synthesis in
dependent cultures (239).
With the use of somewhat more sensitive methodology than used previously (466), inhibition of synthesis at
30-min postischemia was actually associated with a 20 –
30% increase in phosphorylation of eIF-2 (146). Furthermore, there is a threefold increase in the eIF-2 kinase
activity of neocortical brain homogenates 30 min after
30-min ischemia in the rat, suggesting activation of protein kinase R (PKR) and providing a basis for the phosphorylation (147). More recent work, in which the anti-
1. Problems of measurement
Glial proliferation occurs after most global ischemic
episodes, and the increased protein synthesis (which is
seen as soon as 3 h after ischemia in the hippocampal
slice, Ref. 936) can more than compensate for any decrease in neuronal synthesis (332). Thus autoradiographic
measurements are necessary to properly assess changes
in neuronal protein synthesis.
2. Inhibition of synthesis by ischemia
There is a profound brain-wide depression of protein
synthesis in the period during and immediately after
global ischemia that persists in most brain regions of
many different species for 1–24 h depending on the model
(218, 257, 348, 790, 1113, 1209). Over 12– 48 h there is
complete, or near complete, recovery in the regions
where cells will recover from the insult. In contrast, regions in which the cells will die never regain normal levels
of synthesis or in some cases regain and lose them again
(102, 257, 348, 790, 1113). In gerbils, CA1 pyramidal layer
synthesis fell to unmeasurably low values 12 h after 5-min
ischemia and recovered no further; polysomes remained
dissociated (581, 790, 1113). In another study of gerbils,
synthesis in the CA1 region was ;30% of basal values
after 24 h (348). In the monkey, synthesis in hippocampus
showed a transient recovery 6 h after complete compression ischemia, but became strongly inhibited between 12
and 24 h afterward. Synthesis was depressed in core and
penumbra during at least 12 h of focal ischemia (757, 758);
there are no reports of protein synthesis after temporary
focal ischemia. Protein synthesis in CA1 pyramidal cells is
reduced to ;40% 3 h after ischemia in rat hippocampal
slices (936).
Thus, overall, neuronal protein synthesis remains
profoundly depressed in vulnerable areas after global
ischemia and during permanent focal ischemia. It would
be useful to know what occurs after temporary focal
ischemia.
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C. Global Inhibition of Protein Synthesis
CEREBRAL ISCHEMIA ON NEURONS
October 1999
4. Ischemic changes that might lead to inhibition
of synthesis
21
A) INCREASED CYTOSOLIC CA
. There is evidence from
different sources that increased cytosolic Ca21 is involved in inhibition of synthesis.
N-methyl-D-aspartate antagonism, which blocks
much of the increase in cell Ca21 in focal ischemia, protected against inhibition of synthesis in that model (758).
Inhibition of synthesis in the CA1 pyramidal cell layer of
hippocampal slices does not occur if the rise in cytosolic
Ca21 is prevented by incubating slices in 0-Ca21 and 200
mM ketamine during ischemia (936), a combination which
almost completely suppresses the increase in cytosolic
Ca21 (1288). Combined NMDA and AMPA/kainate receptor blockade also attenuated the inhibition (164, 1171),
and this combination largely prevents the increase in
cytosolic Ca21 during ischemia (1288). It has been suggested that Ca21 is not involved in inhibition because in
one study protein synthesis in brain slices was inhibited
by ischemia even in the absence of extracellular Ca21
(265). However, there is a large rise in cytosolic Ca21 in
that condition that was not explicitly recognized by the
authors (769, 1288).
Although increased cytosolic Ca21 certainly should
activate calpain-mediated breakdown of eIF-4, it might
also act on eIF-2. A-23187 leads to a prolonged 70% increase in EIF-2a phosphorylation in cultured neurons (11)
as does glutamate (712). The Ca21 might activate PKR,
which phosphorylates eIF-2. The protein is regulated in a
complex way (507) and might be activated by Ca21-mediated effects.
B) FREE RADICAL GENERATION. The free radical-mediated
inhibition of protein synthesis in vascular endothelial cells
described above (513) indicates that the abundant free radical production in ischemia might cause the damage.
21
C) DEPLETION OF ER CA . It has been suggested (244)
and previously theorized (880) that depletion of ER Ca21
might inhibit synthesis after ischemia. This depletion is
known to inhibit protein synthesis, by activating PKR and
hence phosphorylating and inhibiting eIF-2a (925). It is
not known whether this ER depletion occurs during ischemia, but there are reasons to think it might. Arachidonic
acid, at levels that are present after ischemia (30 mM),
causes depletion of Ca21 from the ER and inhibition of
protein synthesis in cells (244, 969). Also, Ca21 uptake
into isolated ER microsomes is inhibited after ischemia,
although the mechanism is not known. If this occurs in
vivo, it would certainly lower ER Ca21 (878). Presently,
though, there is no definitive evidence for a change in ER
Ca21 at all so the mechanism is purely speculative (879).
5. Evidence linking inhibition of protein synthesis
and cell death
This is most crucial. There are strong correlations
between inhibition of synthesis and eventual cell death
(460). Although 2-min ischemia does not cause cell death,
3-min ischemia leads to persistent inhibition of protein
synthesis in CA1 and to cell death in the gerbil (31).
Ischemic tolerance in gerbil (see sect. VIIIF) is associated
with restoration of protein synthesis in CA1 (348, 544);
hypothermia protects against histological damage and
also prevents delayed inhibition of protein synthesis in
the 4-VO model in rat (1209) as do barbiturates (114).
Unfortunately, such correlations are by no means unique
to global protein synthesis and so cannot be thought to
implicate the process in cell death.
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body for this phosphorylated form of the initiation factor
was used, amplified this considerably (244). There was a
20-fold increase in the phosphorylated form of eIF-2a that
was confined to vulnerable CA1 cells, and some CA3 cells,
10 min to 1 h after 10-min cardiac arrest in rats. At 4 h, the
antibody to this form was still present in the vulnerable
cells, and although a great deal had shifted to the nucleus
(244), a significant amount remained in the cytoplasm
(1078). Importantly, when insulin, which dephosphorylated eIF-2, was injected several hours after ischemia,
protein synthesis was restored to its normal level,
strongly suggesting that the eIF-2 phosphorylation was
the basis for the inhibition of synthesis (1078).
There is at least one caveat, however. The absence of
the phosphorylated form in dentate granule cells after 10min reperfusion (244) is inconsistent with the very profound
inhibition of synthesis that occurs even in nonvulnerable cell
populations for the first 6–24 h (348). Furthermore, it is
essential to explain the very prolonged inhibition in CA1
(days). It is not clear that phosphorylation of eIF-2 would be
maintained that long. For example, glutamate activates eIF-2
phosphorylation and inhibits protein synthesis. However,
the phosphorylation is transient; the eIF-2 dephosphorylates
within 1–2 h of removing glutamate (712). Prolonged maintenance of GEF dephosphorylation, which may result from
inhibited tyrosine phosphorylation, could be accounted for
by chronically lowered brain-derived neutrotrophic factor
(BDNF) (599), as occurs in vulnerable populations. Clearly,
proteolysis of eIF-4 is likely to be an enduring effect. However, it is not established that this factor is adequately rate
limiting in brain to account for the inhibition of synthesis.
This could be tested fairly readily by measuring the effects
of the cell-permeant calpain inhibitors on long-term inhibition of protein synthesis.
B) RNA LEVELS. Unlike protein synthesis, global RNA
synthesis seems to be remarkably stable, showing no
decrease in one study up to at least 4 h after 5-min
ischemia in the gerbil (103). However, there is a decrease
in total mRNA in CA1 3 h after 5-min global ischemia in
the same species (723). Thus there may be activation of
mRNA breakdown, which could account for loss of
mRNA and inhibition of synthesis. This needs to be tested
further.
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PETER LIPTON
Volume 79
There is a paucity of good evidence linking synthesis
inhibition with damage. For example, there are no reports
on effects of agents that prevent cell death, such as calpain
inhibitors or free radical scavengers on protein synthesis
after ischemia. It is important to know if they prevent the
inhibition of protein synthesis before preventing cell death.
Also, it would be very useful to have good pharmacological
studies using inhibitors of protein synthesis to see whether
partial inhibition would eventually cause cell death in normoxia. In this context, the existing data argue against a role
for protein synthesis inhibition in the cell death. In particular, 2-min ischemia in the gerbil does not cause any delayed
morphological damage in spite of strongly inhibiting protein
synthesis in CA1 for between 5 and 24 h (348). The best test
of the role of protein synthesis would be an injection of
insulin soon after the global ischemia. As described above,
this dephosphorylates eIF-2 and restores protein synthesis,
without causing significant hypoglycemia. If it is very protective it would suggest protein synthesis inhibition is an
important factor.
Although the studies of inhibition of global synthesis
in normoxic or only mildly insulted brain tend to argue
against a crucial role for overall inhibition of synthesis in
cell death, studies of the role of protein synthesis in
ischemia, described in section VIIIF, suggest otherwise.
The inability to synthesize protective proteins after ischemia, shown by vulnerable cells, may well be the basis for
their ultimate demise. Furthermore, several key proteins
are inactivated by ischemic events (e.g., protein kinases),
and the inability to resynthesize these proteins could be
very detrimental to the cell. These effects of inhibited
protein synthesis would only be manifested following
very damaging insults and so would not be observed in
the mild insult of 2-min ischemia.
focal ischemia. Indeed, the protein content within the
core of a focal lesion is severely reduced. However, another possible explanation is that proteasome activity is
damaging because it allows activation of NFkB (56). Protein degradation after both global and focal ischemia
needs to be measured to resolve this issue.
6. Protein degradation
Protein synthesis is profoundly and permanently inhibited in vulnerable cells after ischemia. The mechanism
is not known, but there is reasonable evidence suggesting
that changes in initiation factor complexes (eIF2/GEF or
eIF4) are responsible and one study indicating that mRNA
breakdown may be responsible. The latter would probably reflect activated RNase activity. Evidence from brain
slice studies suggests that increased Ca21 is involved in
causing the inhibition of synthesis. Another suggestion is
that it results from depletion of Ca21 from the ER and
another that it results from free radical action. The last
two have not been demonstrated in brain.
The localization and time course of bulk protein synthesis inhibition is completely consistent with its involvement in cell death, as is the correlation between inhibition
of synthesis and cell death in several artifactual paradigms. However, it is difficult to positively implicate inhibition of synthesis in the ischemic death, as has been
suggested (460, 757), particularly for focal ischemia.
The bulk turnover times of proteins are so long that
even profound inhibition of protein synthesis should not
There are studies that seem to directly contradict
the possibility that protein synthesis inhibition causes
damage, in that damage is prevented by inhibitors of
synthesis.
Two studies showed that fairly well-maintained inhibition of synthesis for 2 days was remarkably protective
against global ischemic damage in rat (386) and gerbil
(1027). Unfortunately, however, temperature regulation
was very transient and, as acknowledged by the authors,
the protection may well have resulted from hypothermia.
Inhibition of protein synthesis for ;12 h after the
insult also attenuates damage after mild (30-min ischemia) (276, 304) or relatively mild (90-min ischemia) (275)
transient focal ischemia. This is consistent with the large
component of apoptotic cell death in these paradigms. In
one of these studies (275), temperature was actually monitored for 2 days after the insult and was unaffected by the
cycloheximide treatment, so temperature is almost undoubtedly not an artifact. While important for these mild
insults, these results do not show that profound inhibition
of synthesis for one or more days, as occurs after global
ischemia, is not damaging. Synthesis was inhibited for
only several hours after the insult.
8. Conclusions
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There is very little known about rates of protein
degradation after ischemia. The average turnover times of
brain proteins are 3–5 days (390, 1020). If normal protein
degradation is blocked after ischemia, it would reduce the
impact of synthesis inhibition. Such a close coupling has
been seen in cultured sympathetic neurons where a 50%
inhibition of synthesis is matched by an ;50% inhibition
of degradation rates, although this matching requires the
presence of growth factors (333).
There is a marked decrease in ubiquitin immunoreactivity in the CA1 region after global ischemia (703),
suggesting that a major degradation pathway is blocked,
and this might well lessen the impact of protein synthesis
inhibition. However, degradation rates really need to be
measured.
During focal ischemia, blocking proteasome activity
using PS-519 was extremely protective to the core of the
lesion (902). One explanation for this is that protein degradation makes a major contribution to core damage, in
7. Protective effects of inhibitors of protein synthesis
during an ischemic insult
October 1999
CEREBRAL ISCHEMIA ON NEURONS
D. Damage to the Cytoskeleton
Much of cell structure, protein localization, and almost all transport into and from dendrites and axons is
mediated by cytoskeletal molecules. Their gross disruption would be expected to cause major losses in ordering
and in transport within the cell and hence cell death by
necrosis or by triggering apoptotic changes.
1. Microtubule changes during ischemia
It is clear that microtubule dissolution occurs in ECC
(495, 521, 522) and may play a major role in allowing this
form of damage. This has not been tested with microtubule stabilizing drugs such as taxol, and little is known
about the importance of microtubule dissolution (1122,
1123, 1236, 1237).
In the gerbil, microtubules in the distal dendrites of
the very vulnerable CA1/subiculum region disassemble
immediately after 5-min ischemia; this disassembly
reaches the proximal dendrites after 10 min (1237). Loss
of MAP2 and tubulin antibody staining accompanies the
microtubule breakdown (1237, 1242). The cells within
CA1 that are somewhat less vulnerable require longer
durations of ischemia to show this early dissolution. However, between 6 and 24 h after 5-min ischemia there is a
major microtubule disassembly in proximal (and distal)
apical dendrites of all CA1 pyramidal cells (348, 1236).
Semiquantitative estimates show a 50% loss in microtubules at this time (348). In general, these changes are
irreversible in cells that are destined to die. They also
occur in nonvulnerable cells where they recover after
12–24 h (348). There is complete loss of MAP2 antibody
staining in apical dendrites of the CA1 pyramidal cells at
this time also (726).
Kinesin and cytoplasmic dynein immunoreactivities
are greatly decreased in CA1 at 3 and 8 h after 5-min
ischemia (29), before the major microtubule disruption,
suggesting an early blockade of dendritic transport of
most proteins (29). This could be very important, and the
result will, hopefully, be pursued.
Microtubule changes following global ischemia in the
rat are much less dramatic than in gerbil, even up to 24 h
after 2-VO (251, 357, 581, 1127). However, microtubules
do become disorganized between 24 and 48 h, well before
cell death. It is not at all clear why the gerbil and rat
behave so differently; there may be more stabilizing elements such as calpastatin in rat, but this is not known.
There are no measurements to see whether motor proteins might be compromised, as they are in gerbil.
There are no reports concerning early microtubule
changes in focal ischemia.
2. Mechanisms of microtubule changes
Known mechanisms of microtubule dissociation include MAP2 phosphorylation or proteolysis, dissociation
of the putative microtubule-stabilizing protein STOP decreased GTP/GDP, or proteolysis of tubulin (708, 770). All
of these are activated by Ca21 with the probable exception of the decrease in GTP/GDP (273). MAP2 phosphorylation and dissociation from microtubules is also activated by PKC and other kinases that may be activated
very early during ischemia (132, 459, 491). Thus there are
many potential mechanisms for microtubule dissolution.
Recent studies from our laboratory (316, 1291) on rat
hippocampal slices, where there was significant breakdown of microtubules in CA1 dendrites during 6-min ischemia, indicate that the breakdown is almost certainly
mediated by Ca21. It was greatly attenuated when slices
were incubated in 0 Ca21 buffer and ketamine, a combination that prevents 80% of the normal rise in cytosolic
Ca21 (1288), or lidocaine and 6-cyano-7-nitroquinoxaline2,3-dione (CNQX), which similarly blocked the cytosolic
Ca21 rise. It was also blocked by inclusion of 25 mM
calmidazolium, the calmodulin antagonist (316) (which
did not attenuate the cytosolic Ca21 increase), and by the
calpain inhibitor MDL-27,180 (1291).
The mechanism of breakdown is not known, but it is
not mediated by MAP2 proteolysis or by proteolysis of
microtubule only stabilizing protein, STOP or tubulin
(Zhang and Lipton, unpublished data). The involvement of
calmodulin might be because it causes dissociation of
STOP from the microtubules, which it does in vitro (912),
but this has not been shown for ischemia. These considerations suggest that prolonged, or delayed, elevation of
Ca21 might be responsible for the profound microtubule
dissociation in gerbil and the milder disorganization in
rat, but there is no current evidence.
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alter composition enough to cause cell death in the 12–24
h after focal ischemia. This is supported by the absence of
any ill effects after 2-min ischemia in gerbil despite a
profoundly depressed protein synthesis for between 5 and
24 h after the ischemia. Damage would be still less likely
if turnover of protein was diminished by the ischemic
insult.
These considerations are not germane to short global
ischemic episodes, where death often requires 3– 4 days.
If synthesis is depressed for that long it might well affect
function severely. Effects of prolonged pharmacological
inhibition of protein synthesis would be very informative,
along with measurements of protein turnover after ischemia.
Independently of the effects of long-term loss of protein, the global inhibition of synthesis after ischemia may
be extremely important in death of vulnerable cell populations by preventing synthesis of protective proteins at
critical times and preventing resynthesis of proteins that
are inactivated by the immediate sequelae of ischemia.
1471
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PETER LIPTON
3. Evidence linking microtubule breakdown
to ischemic cell death
creases in band intensities of native spectrin on Western
blots (13, 455, 641, 952, 973), even in the core of the lesion
after 3-h focal ischemia (455). Thus the percentage reduction is small. It may, however, be important.
Caspase-activated spectrin breakdown, which may
well be an important component of apoptosis, has not
been investigated in vivo, but it is prominent during in
vitro ischemia in rat cortical cell cultures (810).
Ankyrin plays a major role in linking membrane proteins, such as Na1-K1-ATPase and Na1 channels to the
cytoskeleton. Two major isoforms, ankyrin B and ankyrin
R, are localized to membrane fractions including the
crude synaptosomal fraction (426). Thirty minutes of
global ischemia in rat, followed by 60-min reperfusion,
caused a 20% loss of immunoreactivity of ankyrin B and
breakdown of ankyrin R, possibly mediated by calpain.
The breakdown was largely in the postischemic period,
suggesting that reperfusion sensitizes the system. These
changes could certainly lead to malfunction of the Na1
pump or other membrane proteins and so contribute to
cell death. This may account for the observed inhibition
of the Na1 pump after 60-min ischemia (866). Effects of
shorter, but still lethal, durations of global ischemia need
to be demonstrated, as do effects of focal ischemia.
B) OTHER CYTOSKELETAL PROTEINS. There is a 30% loss of
immunoreactivity of a 200-kDa neurofilament subunit 1
day after either 5- or 8-min 2-VO in the rat (519); the role
of the protein is not known.
4. Other cytoskeletal proteins
5. Conclusions
A) SPECTRIN AND ANKYRIN. Spectrin provides major linkages between the cell membrane and membrane-associated proteins, which in turn are involved in maintaining
integrity and localization of integral membrane proteins
(80, 81, 471). It is very susceptible to calpain (471) and to
caspase-3-like proteases (810), which cleave it at different
sites (810).
After 5-min ischemia in the gerbil there is a rapid
(within 30 min) breakdown of spectrin by calpain (641,
952, 973) in many brain regions but oddly, perhaps, not in
CA1 pyramidal cells of hippocampus (952). However,
there is a delayed breakdown that is measurable in dendrites of CA1 pyramidal cells after 1 day, peaking at 2
days, and persisting until cell degeneration (952, 973,
1258). This suggests a delayed activation of calpain in CA1
that is probably very important and is discussed in section
VB. There is breakdown at the core of a focal ischemic
lesion after 3 h (455), but no substantial studies have been
reported for focal ischemia.
These results are certainly consistent with a role for
spectrin breakdown in the delayed cell death. However,
arguing against this somewhat is that only a very small
percentage of total spectrin in a region is broken down.
Although several studies clearly show the appearance of
spectrin breakdown product, there are no notable de-
The data and the literature support the possibility
that microtubule dissolution contributes significantly to
apoptotic or necrotic cell death. However, there are major
uncertainties. There are no studies of microtubule
changes in focal ischemia, and changes in the rat during
global ischemia are much later, and less profound, than in
gerbil. Mechanisms of cell death in the gerbil and rat may
differ, or changes in rat may be more subtle but still
important. Certainly, though, in the gerbil, changes are
very rapid and profound and highly correlated with eventual cell death. It is difficult to think that these do not
strongly influence cell death given the profound effects of
microtubule dissolution on cell viability. Unfortunately,
though, there are no data showing a causal relationship
between microtubule breakdown and ischemic cell death,
and these are essential. If microtubule changes are important, then a key question becomes how their dissolution is
maintained over time. Early effects of Ca21 and phosphorylation might well be expected to be reversed. Indeed, it
is possible that an insult causes irreversible damage and
death when it is intense enough to cause a loss of the
ability of microtubules to reassemble. This could be via
proteolysis of key components.
The decrease in motor proteins seen in the gerbil is
potentially very important, since it would stop distribu-
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The microtubule-dissociating drug colchicine causes
cell death in cultured cerebellar granule cells (110), as
well as other cell types (659, 1140, 1193). Death appears to
be apoptotic. Furthermore, photoactivated disruption of
microtubules leads to loss of intracellular organelle motility and quite rapid cell death in cultured kidney epithelial cells (639). Thus there is reason to think that the
ischemic breakdown of microtubules would contribute to
the cell death. Unfortunately, there are no data that support (or contradict) the possibility.
There are data showing correlations between microtubule dissolution and ischemic cell death. Ischemic tolerance in gerbils is associated with the reestablishment of
the microtubules in the CA1 pyramidal cells (348), and
MAP2 breakdown is affected by temperature in the same
way as cell death. Reducing temperature to 33°C during
ischemia markedly decreased loss of MAP2 immunoreactivity, whereas increasing temperature to 39.7°C markedly
enhanced it (293).
The most exciting study to see would be the effect of
the microtubule-stabilizing drug taxol on development of
ischemic cell death. Unfortunately, there are no such
reports. Taxol does attenuate ischemic microtubule
breakdown in the slice (Zhang and Lipton, unpublished
data), so it may well provide useful information if used in
vivo.
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CEREBRAL ISCHEMIA ON NEURONS
evidence for the importance of purely degradative processes. There is equivocal evidence that large-scale proteolysis mediated by proteasomes may be important in
focal ischemia and that autophagocytosis may be important in global ischemia.
V. PERPETRATORS OF FUNCTIONAL
OR STRUCTURAL DAMAGE
This section examines biochemical processes that
are likely to play a part in cell death by causing long-term
changes in macromolecules. These will lead to the functional changes discussed in section IV or other effects
related to cell death. They are considered perpetrators of
damage.
A. Free Radical and Peroxynitrite Actions
E. Summary
1. Free radical, NO, and peroxynitrite changes during
and after ischemia
Four functional changes have been examined which,
a priori, seemed most likely to account for necrotic cell
death. Two of these changes, in mitochondrial function
and microtubular/cytoskeletal structure, could also be
important in apoptotic death. Unfortunately, there is no
compelling evidence that any of these functional changes
cause the cell death, although it is difficult to think that
they are not, among them, largely responsible.
Both plasmalemma and mitochondrial damage could
clearly cause cell death but, so far, there are no convincing demonstrations that either occurs to the extent that
would do this. Evidence that appropriate mitochondrial
changes occur is better than for plasmalemma changes,
including leakage of cytochrome c and protective effects
of methyl-valine cyclosporin A in focal ischemia but is not
yet compelling. Global protein synthesis is clearly drastically inhibited, but in this case, the difficulty is in showing
that such inhibition will actually cause cell death; judicious use of insulin may be very helpful in this regard.
Microtubule dissolution in gerbil is dramatic, and independent studies suggest this should lead to cell death.
However, no pharmacological interventions have been
tried to see if the changes are important in this case. Also
of concern is the great disparity between the extent of
microtubule changes in the gerbil and rat. Changes in the
latter are much less, at the ultrastructural level. They may
still be important, but this would need to be shown.
As noted at the beginning of section IV, cell death
might not result from a functional defect in one or more
of the key processes examined here; rather, it may result
from continued activation of perpetrators (see sect. V) set
in motion by the ischemic insult, with ultimate breakdown of the cell as a unit. In this regard, there is some
The possible roles of free radicals, NO, and peroxynitrite in ischemic cell damage have been extensively reviewed (170, 985, 1043, 1046) since the early suggestions
of Siesjo (1041). As discussed in section VA2, there are
many ways in which free radicals (including NO) and
peroxynitrite may be generated during ischemia. There is
massive evidence from many systems that these species
can cause cell damage. The question addressed in this
section is the extent to which they are actually involved in
ischemic cell death and the ways in which they are involved.
A) GLOBAL ISCHEMIA. I) Free radicals. Several studies
show increased free radical formation during vessel occlusion. The electron-spin resonance signal increases
over 10-fold during 15 min of 4-VO, measured on extracellular fluid collected in a cortical cup (906). There is a
similar increase during this period in the striatum (1305)
and in hippocampus (908) as measured with microprobes
containing a spin trap or salicylate, respectively. There is
also an increase in PBN spin adducts after 20-min 2-VO
(976). There are some contradictory results. In one study
there was no evidence of increased free radicals at the
end of 10-min ischemia in gerbil (851), and no change in
reduced glutathione was measured after 30 min of 4-VO in
the rat (217). However, overall the evidence showing
increases is persuasive.
The increase in free radicals becomes larger and
unambiguous during the early reperfusion period (263,
851, 906, 908, 976, 1260, 1305). The persistence of the
increase depends on the duration of the ischemia. After
10-min ischemia in rat, lucigen chemiluminescence levels
fell from two times baseline to baseline after 40 min.
However, after 15-min ischemia, the levels of 2,3-DHBA, a
measure of free radical or peroxynitrite attack on micro-
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tion of substances by microtubule-based flow processes.
This could be very damaging. In light of the large difference in microtubule dissolution between rats and gerbils,
it would be very interesting if rats also showed the loss of
motor proteins. It would add weight to the conclusion
that there is a cytoskeletal component to cell death.
Other cytoskeletal proteins, such as spectrin and
ankyrin, also undergo breakdown, and many more as yet
unidentified probably do also, since they are prime targets
for calpain, which seems to play a major role in apoptotic
and necrotic cell death. Many important cytoskeletal
changes are also effected by caspases.
There are many reasons to think cytoskeletal changes
would play a major role in cell death. Cytoskeleton plays
essential roles in distribution of protein and lipid and has a
major role in maintaining cell structure, which is clearly
dramatically altered in ischemic cell death. To date, though,
there is no strong positive evidence that the cytoskeletal
changes are important in the ischemic damage.
1473
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PETER LIPTON
show marked increases in free radical formation in the
peri-infarct penumbra (where flow levels are ;50%) during and after focal ischemia. During the ischemia, there
were about twofold increases in indicator signal that occurred within 15 min of onset in one study (312) and
within 100-min of onset in another (773). These persisted
throughout 2–3 h of ischemia, indicating continual generation. Reperfusion after 1 or 2 h gave a large burst that
lasted ;40 min and persisted throughout the remaining
2 h of the study at a steady level about twofold over
baseline (312, 773).
Free radical production appears largely restricted to
the penumbral region. When localized salicylate microprobes were used, continued generation was measured in
the penumbra during 3 h of ischemia and for 6 h after
ischemia. There was no production in the core through
3-h ischemia and until after 3-h reperfusion (1060). There
is major damage by this time. This is an important study,
suggesting that damage in the core of the lesion is unlikely
to result from free radicals, although, as seen below, NO
and peroxynitrite do appear to rise in the core. The absence of free radical generation in the core is somewhat
surprising given the apparent generation of free radicals
during global ischemia, where O2 levels are lower than in
the ischemic core, and confirming studies will be important.
Actual rates of superoxide generation within the
ischemic region, measured by the cytochrome c electrode, were ;25 mM/min (312). This is undoubtedly much
lower than actual values, because some free radicals
would have been scavenged before measurement.
I) NO and peroxynitrite. Nitric oxide appears to rise
during focal ischemia. There are three reports of a transient (20 min) increase (622, 707, 1293) to ;2 mM (707).
There is a continual generation in the postischemic period
(622), although it is somewhat smaller than the early
increase during ischemia (;0.6 mM) (707, 1293). It lasts
for at least 60 min.
The increase in NO was enhanced by superoxide dismutase (SOD) infusion, which competes for superoxide radicals (74), indicating that the NO is normally reacting with
O2
2 to produce peroxynitrite (622). Measurements of 3-nitrosotyrosine confirmed a substantial generation of peroxynitrite (347). There was about equal formation in core and
penumbra at the end of 2 h of focal ischemia and approximate doubling of this in the penumbral region during 3 h of
reperfusion. This was all blocked when NOS was inhibited
by N G-monomethyl-L-arginine (347). Levels of 3-nitrosotyrosine rose to ;1% of all tyrosine residues, far higher than in
basal conditions when there is no measurable nitrosotyrosine, and 5 times the levels seen in Huntington’s disease.
This is a major increase.
The rise of nitrosotyrosine in the core appears to
conflict with the lack of free radical generation in the core
(1060). More studies are required to resolve this, but it is
quite possible that the enhanced NO production com-
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probe salicylate, was still rising 60 min after the end of
ischemia (908), and after 20-min ischemia, chemiluminescence levels remained at 150% baseline for at least 2 h.
Thus durations of global ischemia that lead to profound
delayed neuronal death cause free radical increases that
are probably maintained for at least 2 h. Most interestingly, 24 h after 10-min global ischemia, there is a large
elevation in superoxide that is restricted to the vulnerable
CA1 pyramidal layer in the hippocampus (173). This was
measured using hydroethidine fluorescence. The increase
is approximately coincident with an upregulation of the
enzyme COX-2 in CA1 pyramidal neurons. This occurs
between 8 and 24 h after the ischemia (806) and is a very
likely source of free radicals.
II) NO and peroxynitrite. There was an increase in
NO, to ;11 mM, at the end of 15-min 2-VO global ischemia
(1126), as measured with an NO-sensing electrode, and
there was also an increase after 7-min 2-VO in hippocampus (850). No on-line measurements have been made after
the end of global ischemia, so it is not known if the radical
is continually generated. Peroxynitrite, which is generated from NO and superoxide, is specifically monitored by
measuring formation of nitrosotyrosine. The latter is elevated 4 h after 30-min global ischemia in rats (330), but
this is the only reported study. Further quite strong evidence for peroxynitrite formation, from the same study, is
that blocking NOS with NG-nitro-L-arginine (L-NNA) increased the net production of superoxide (330), suggesting the latter is normally metabolized to peroxynitrite.
Thus insofar as measured, there is evidence for NO and
peroxynitrite accumulation in global ischemia. At present,
there is no detailed knowledge about the time course of
peroxynitrate except that it occurs at some time during
the first 4 h.
There may well be delayed production. Inducible
NOS is greatly increased in the astroglia that border the
CA1 pyramidal layer after 10-min 4-VO global ischemia
(302, 303). The increase is first seen 1 day after the insult
and becomes prominent 2 days later. The increase is
confined to the CA1 region and is adjacent to pyramidal
cells, which die at about this time. No measurements of
nitrosotyrosine have been reported.
B) FOCAL ISCHEMIA. Early indirect measurements of
thiobutyric acid reactants (TBAR) accumulation (777), of
loss of scavengers such as a-tocopherol, and of reduced
forms of ascorbate and ubiquinones (577) strongly indicated that free radicals were produced within 30 min of
initiating focal ischemia. These early studies were very
important in establishing that there was production of
free radicals. Moreover, the study in which depletion of
scavengers was measured indicated that there was continual production of free radicals for at least 6 –12 h of
ischemia (577).
More sophisticated on-line measurements, using a
cytochrome c electrode (312), or measuring lucigenen
enhanced chemiluminescence in a cranial window (893),
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CEREBRAL ISCHEMIA ON NEURONS
2. Mechanisms of net free radical production
during ischemia
Normally the rates of free radical production and
elimination are equal, leading to a steady state that is
presumably tolerated by the cell. Ischemia creates several
conditions that could account for the increased net production of free radicals (336, 413, 488).
A) XANTHINE/HYPOXANTHINE OXIDATION. The breakdown of
adenine nucleotides during ischemia leads to accumulation of hypoxanthine within 10 min (the earliest published
measurement, Ref. 407), which is then metabolized by
xanthine oxidase (XO) or xanthine dehydrogenate (XDH).
Xanthine oxidase produces free radicals, but XDH does
not. The basal level of XO in brain tissue is similar to that
in other organs such as liver and intestine (92).
The reaction for generating superoxide is as follows
Hypoxanthine 1 O 2 3 O 2
2 z 1 H 2 O 2 1 urate
Accumulation of urate also results from the action of
xanthine dehydrogenase (XD), and allopurinol, which inhibits the XO, also scavenges free radicals. Thus unequivocally demonstrating this mode of free radical production
in the tissue is difficult.
At the moment, the evidence favors its importance in
global ischemia (578) but not in focal ischemia (92). This
is reasonable because changes in ATP and, probably,
Ca21, are larger during the global ischemic insult. The
activity of XO, produced by proteolytic cleavage of XDH,
was increased about fivefold 30 min after 15-min global
ischemia in rat, and there was concomitant urate production (578). This is reasonable evidence that free radicals
are being produced by XO. Although there is a large
production of urate (tens of mM) during focal ischemia
(92, 577, 1152), there is no particular evidence that it
reflects XO activity rather than xanthine dehydrogenase
activity. There is no protease-mediated conversion of XD
into XO during focal ischemia, measured at 24 h (92), and,
more importantly, allopurinol does not prevent edema at
doses that almost completely block the urate production
(92). Other free radical scavengers do protect against
edema in the same condition. The results thus suggest
that free radical formation does not result from this reaction in permanent focal ischemia. There are no data for
temporary focal ischemia.
B) ACCUMULATION OF EICOSANOIDS. Oxidative metabolism
of accumulated arachidonic acid (549, 1111) via the cyclooxygenase pathway (602, 620, 623) or via the lipoxygenase pathway (488, 623) leads to superoxide or OHz
production. Phospholipid breakdown into arachidonic
acid is then further activated by free radicals (45, 175).
There is a manyfold increase of FFA, including arachidonic acid, during global and focal ischemia (804, 1111,
1292), and arachidonic acid metabolites are still 7–30
times basal 30 min after global ischemia (1111). No measurements were made right after focal ischemia, but by
4 h, there was no elevation (1292). The metabolism of
arachidonic acid must thus be considered a likely source
of the free radicals during and soon after both global and
focal ischemia. However, there are no pharmacological
studies directly establishing this in brain. There are such
studies in retina, where inhibition of arachidonic acid
metabolism reduced lipid peroxide formation and retinal
damage (179).
A major mechanism for eicosanoid accumulation is
the ischemia-induced synthesis of COX-2 that occurs 6 –24
h after both global (806) and focal (837) ischemia. Inhibitors of this enzyme, such as SC-58125 or NS-398, dramatically reduce the delayed accumulation of prostaglandin
E2 in both insults (806, 836). This COX-2-induced eico-
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bined with a normal steady-state superoxide production
will allow formation of peroxynitrite (74).
C) INDUCED EXPRESSION OF NOS AND OF CYCLOOXYGENASE
AFTER FOCAL ISCHEMIA . Two enzymes that are intimately
related to free radical production are upregulated during the 6- to 24-h period after temporary focal ischemia,
as they are after global ischemia. Both iNOS (479) and
COX-2 (837) are upregulated in the penumbral region
during this period. The former is in endothelial cells
and invading neutrophils (479, 836), whereas the latter
is in neurons (836). Cyclooxygenase-2 (prostaglandin H
synthase) generates superoxide radicals (837). The upregulation of these enzymes might lead to NO and
superoxide synthesis during the later phases of reperfusion. Although they are in different cell types, they
might well react to form peroxynitrite because both NO
and superoxide can diffuse for several hundred microns (74). Further evidence for communication between the two processes is that about one-half of the
enhanced COX-2 activity, although not the enzyme upregulation, results from the action of the iNOS (836).
This was determined both by pharmacological and
knockout studies.
D) SUMMARY. The studies definitively show that free
radical production is elevated during both global and
focal ischemia, for at least 2 h after focal ischemia and
very probably for at least 6 –12 h. Levels are elevated 24 h
after global ischemia in vulnerable cells. Nitric oxide
production is elevated to the low micromolar range during global and focal ischemia; it is also elevated after focal
ischemia; no measurements have been made after global
ischemia. Peroxynitrite is produced during or after both
focal and global ischemia. Superoxide levels of 25 mM and
NO levels of 1–15 mM have been measured. They should
(74), and apparently do, yield a high level of peroxynitrite
formation.
Critical questions that remain include the time
course of free radical generation throughout the postischemic period and further localization and quantitation.
1475
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PETER LIPTON
after focal ischemia (488, 1046). Neutrophils oxidize
NADPH to generate superoxide and thus are important
potential free radical donors during and after focal ischemia. In addition, neutrophils synthesize iNOS 1–2 days
after focal ischemia (480). Evidence as to their role in free
radical production is conflicting as discussed in section
III. Neutrophils may play a priming role in activation of
free radical production. There is no evidence concerning
the roles of neutrophils in global ischemia free radical
production.
E) MECHANISMS OF NO AND PEROXYNITRITE GENERATION. Nitric oxide is generated by neuronal or endothelial NOS in
an oxygen-dependent reaction that is activated by Ca21/
calmodulin in most neurons and endothelial cells (477,
996). The Ca21/calmodulin directly activates the enzyme
and may also remove a phosphorylation-induced block by
activating calcineurin (235). Activation of NOS by the
Ca21/calmodulin system has not been explicitly demonstrated by measuring, directly or indirectly, effects of
appropriate agents on NO production during ischemia.
The conclusion that the Ca21/calmodulin system is important is based on the importance of Ca21 in NOS-mediated
glutamate toxicity, as well as the ability of calmodulin
inhibitors and calcineurin inhibitors to prevent such toxicity (235). The latter studies are not persuasive, however,
because calcineurin inhibitors prevent NMDA toxicity in
cerebellar cultures where NOS is not mediating the toxicity (26). Thus proving the involvement of calmodulin
remains an issue.
Both neuronal (1294) and inducible (478) NOS are
upregulated after focal ischemia, and iNOS is also upregulated in glia 1–2 days after global ischemia (303, 477).
There is no explicit demonstration that these cause NO
accumulation, but it must be considered very likely.
Peroxynitrite is generated by the reaction between
superoxide and NO
2
O2
2 z 1 NO z 3 ONOO
It can be protonated to produce the very reactive
peroxynitrous acid that dissociates into OHz and various
nitrogen/oxygen species (74). The unprotonated form,
which is about 75% of total at pH 7.4, is also very reactive,
with a half-life in biological systems of ;1–2 s and long
diffusion distances of ;100 mm. There is good evidence
that both the acid and the anion participate fairly equally
in most reactions (1013). One or other of the forms readily
crosses cell membranes so that peroxynitrite, like superoxide, can act in cells other than those in which it is
generated (74, 1162).
Direct evidence for peroxynitrite formation was described above in all forms of ischemia, as accumulation of
nitrotyrosine. As well-argued by Beckman (74), the peroxynitrite formation occurs because the rate constant for
this reaction is very high (6.7 3 109 M21 z s21) so that NO
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sanoid production occurs at 6 – 48 h and so constitutes a
major source of delayed free radical production.
C) ALTERED MITOCHONDRIAL FUNCTION. Generation of free
radicals by mitochondria has been well reviewed (740,
1142). The organelles normally generate free radicals at a
rapid rate (117, 1142), but one which is handled by normally functioning cells. When the electron carriers become highly reduced, as much as 2% of the electron flow
leads to direct single-electron reduction of oxygen and
formation of superoxide (117, 336, 561). This very probably occurs during focal ischemia, and low-flow global
ischemia when the members of the respiratory chain are
in a relatively reduced form. Accumulation of Ca21 (1143)
and opening of the MTP (617, 710, 1275) are among other
factors that may well occur at some point after ischemia,
and which very probably cause mitochondrial free radical
production by mechanisms that are not yet known (see
sect. IVB).
Despite the great potential for mitochondria as a
source of free radicals, there is only one study that actually demonstrates their involvement (908). Direct measurement of free radical concentration, using the salicylate trap, revealed a steady increase during 15-min 2-VO
and for the following 1-h reperfusion. This was completely prevented when mitochondrial respiration was
blocked by including rotenone in the dialysate. It was
restored if the rotenone block was by-passed by succinate, further implicating mitochondrial electron transport
as the source. The free radical production was also prevented by haloperidol, which blocks respiration at complex I (908). As the authors point out, these results do not
exclude other possible sources of free radicals, particularly prostaglandin metabolism, because salicylate is a
potent inhibitor of cyclooxygenase and so could have
been blocking that pathway while measuring free radical
production.
Free radical production from electron transport also
occurred in cultured neurons exposed to NMDA (282).
The authors suggested this was due to accumulation of
Ca21. Consistent with this idea, the combination of elevated Ca21 (2.5 mM) and ADP potently activated OHz
production in isolated mitochondria (287), with the OHz
acting as a positive feedback to enhance OHz production.
Although the mechanism was not determined, the conditions approximate conditions after ischemia and after
glutamate application.
Knockouts of mitochondrial SOD (Mn-dependent
SOD) enhance the extent of the lesion after focal ischemia
(786), whereas overexpression of the same enzyme decreases infarct size (562). If this enzyme selectively inactivates mitochondrially generated O2
2 z, then mitochondria
are definitely a source. However, this selectivity has not
been established (74).
D) NEUTROPHIL ACCUMULATION AND ACTIVATION. As discussed in section III, these cells adhere to the vessel walls
and invade the parenchyma to a limited extent during and
Volume 79
CEREBRAL ISCHEMIA ON NEURONS
October 1999
O2
2 1 2H 2 O 3 1 H 2 O 2
Fe 21 1 H 2 O 2 3 Fe 31 1 OH 2 1 OH z
Brain iron is ;63 mM/g dry tissue, a concentration of ;12
mM. Most of this is in heme enzymes or bound to ferritin.
Iron appears to be delocalized from the proteins in the
postischemic phase and possibly also during ischemia
(1205), probably as a result of lowered pH (488) or superoxide-induced reduction of Fe31 (692). One hour of anoxia in cortical homogenates tripled the amount of delocalized iron (118). The increase in free iron should greatly
enhance OHz production, and iron conjugation has been
used somewhat successfully to reduce ischemic damage
(46, 1205), probably by blocking this reaction. Decreased
pH is an important initiator of iron delocalization and
hence OHz production.
In contrast to this scenario, it has been argued that
the Fenton reaction is of little importance. The reactivity
of OHz may paradoxically render it less harmful than other
free radicals because it will oxidize many small organic
molecules and so be effectively “inactivated” with regard
to damaging important macromolecules (74). Furthermore, the superoxide-driven Fenton reaction is very slow,
even in the presence of iron, and it has been argued that
superoxide would be largely metabolized by SOD and also
by NO to peroxynitrite (74). Although sound, these considerations have not been verified experimentally and
may not be applicable; lowered pH does actually enhance
damage (see sect. VID) arguing for the importance of the
Fenton reaction. However, protonated ONOO2 may well
be more damaging than ONOO2 itself, providing an alternate way by which lowered pH could enhance damage
(1013). At this stage, the importance of the Fenton reaction needs to be established. The efficacy of iron chelation
suggests it is important.
H) CONCLUSION. Free radicals including NO, and also
peroxynitrite, certainly rise as a result of ischemia. Despite the multiplicity of possible pathways, the only explicit demonstration of a mechanism for free radical formation is via the mitochondrial redox chain during and
for 45 min after global ischemia. Monoamine oxidase
acting on released monoamines very probably releases
H2O2, which could then be metabolized to OHz in the
Fenton reaction. This may be an important source of early
free radical production, but it does not appear to contribute to damage. Specific demonstrations of XO-induced
free radical generation, arachidonic acid metabolite-induced free radical generation, calmodulin-induced NO
production, and neutrophil-induced superoxide formation
from NADPH are lacking at this time. The fact that inhibitors of iNOS and of COX-2, as well as iNOS knockouts,
attenuate damage suggests strongly that synthesis of
these two enzymes generates NO and superoxide 12 h to
several days after ischemia, but no measurements have
been made.
There is good evidence that ONOO2 is formed from
increased NO and superoxide. Indirect evidence suggests
that it can also be formed after activation of NOS alone,
using basal levels of superoxide, or from superoxide generation alone.
Peroxynitrite, superoxide, and OHz represent three
very active species. It is, in fact, quite difficult to determine which of these contributes to the measured elevations in free radicals described in section VA1 because
most of the standard measuring techniques for free radicals also respond to peroxynitrite. Lucigenin fluorescence
is quite specific to OHz, but PBN will also respond to
peroxynitrite or its acid (1013) as will the hydroxylation
of salicylate to DBA (809).
3. Evidence that free radicals are involved in ischemic
cell death
Free radicals are generated during and after both
global and focal ischemia. If they are damaging, then
conditions which reduce their accumulation should ameliorate damage.
Studies in which free radical or NO/ONOO2 accumulation is attenuated, or enhanced, strongly suggest these
species make critical contributions to ischemic cell death.
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can effectively compete with the very active SOD for
superoxide when it rises to 1–2 mM, as it does in ischemia.
Peroxynitrite formation is not expected in the absence of
NO generation unless superoxide generation is high
enough to effectively saturate the competing SOD reaction. Mass action would then allow significant formation
of peroxynitrite. This may, in principle, occur endogenously and does appear to occur when superoxide is
artifactually generated in cultured PC6 cells (562).
F) MONOAMINE ACCUMULATION. There is quite strong evidence that H2O2 is produced from accumulated catecholamines (51) via monoamine oxidase (MAO) in the 5
min after 15-min global ischemia in rat (1053). Measurements were indirect, but rapid oxidation of glutathione
was blocked by MAO inhibitors, suggesting MAO activation of H2O2 production. This certainly makes sense given
the large release of monoamines during ischemia. Blockade of MAO was not protective (1053), indicating that this
early H2O2 production was not damaging.
G) INTERACTIONS BETWEEN DIFFERENT SPECIES: FORMATION OF
OH FREE RADICAL. Superoxide and NO are usually the first
free radicals formed, but superoxide is not very reactive.
The formation of ONOO2 is likely to mediate much of its
toxicity. In addition, though, the reaction products of
superoxide, OHz or HO2z, are very reactive and more likely
to mediate toxic effects (413). Formation of OHz is
strongly favored by the decreased pH that prevails during
and after ischemia (75, 488) and also by free iron (413), as
shown below in the Fenton reaction
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PETER LIPTON
ischemia, probably because of the more extensive involvement of the vasculature in this insult. It almost completely blocked the twofold increase in TBAR that usually
occurred 24 h after 3-h MCA occlusion and reduced infarct area by ;25% (1154). Temperature was well-regulated. Superoxide dismutase (30), SOD conjugated with
polyethylene glycol (679), and SOD entrapped in liposomes (492) consistently reduced infarct volumes by 25–
40% in well temperature-controlled focal ischemia studies. These studies have been pursued and confirmed in a
very sophisticated series, principally by Chan and colleagues (573, 1246), using mouse mutants. Overexpression of Cu/Zn SOD by about threefold reduced the infarct
volume in two different models of temporary focal ischemia in mouse. The penumbra was significantly preserved
24 h after 1-h cortical ischemia (573) and 3 h after a 3-h
proximal occlusion (26%) (1246). In both cases, there was
also preservation of scavenging systems (reduced glutathione and/or ascorbate), strongly indicating that the mutants were more effectively dissipating free radicals.
Overexpression of the mitochondrial Mn SOD, at a level
twice its normal value, reduced infarct volume by ;20%
24 h after 1-h focal ischemia (562). The similar effect of
the two overexpression mutants suggests that mitochondrial and cytosolic SOD might both act on the same pool
of superoxide.
Uric acid, a very effective scavenger of peroxynitrite
and OHz, reduced the infarct measured 24 h after 2-h MCA
occlusion by 75% when injected either before or immediately after the ischemia (1266). This dramatic effect,
which extended to the striatal core of the infarct, suggests
the importance of peroxynitrite in damage to that region.
Inhibition of COX-2 to levels that almost completely block
excess generation of prostaglandins decreases infarct size
by ;30%, suggesting that delayed development of the
infarct is free radical dependent (837).
II) Permanent ischemia. There are apparently conflicting data on effects during permanent lesions; however, they can be resolved. The mice in which overexpression of Cu/Zn SOD prevented the temporary focal
ischemic damage described above were not at all protected when subjected to 24-h permanent focal ischemia
(172), suggesting free radicals play no role. On the other
hand, the spin trap PBN reduced infarct size by .50% at
the end of 48-h transient or permanent MCA occlusion,
even when administered 3–12 h after the onset of the
ischemia (160, 1298). This discrepancy may be readily
resolved. Permanent ischemia is protected by inhibiting
NOS (see sect. VA), implying damage is mediated by peroxynitrite. This might have been formed from excessive
NO production and residual superoxide even when superoxide production was attenuated in the mutants. On the
other hand, PBN should scavenge free radicals produced
as intermediates in reactions of peroxynitrite with targets
such as amino acid side chains or scavenge peroxynitrite
or peroxynitrous acid themselves (1013).
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Unfortunately, most studies are not accompanied by measurements of changes in free radical accumulation during
or after the ischemia. That makes it very difficult to derive
any quantitative relationship between the level of free
radical increase and damage.
A) GLOBAL ISCHEMIA. Several free radical scavenger systems protect against global ischemia in the gerbil. a-Tocopherol rescued ;75% of the cells that normally died 7
days after 5-min ischemia (424); the animals were on
heating pads, but their temperatures were not monitored.
A similar degree of protection was afforded by the spin
trap PBN when injected for 2 days starting right after the
5-min ischemia (1270). The PBN had no measurable effect
on body temperature, although at higher concentrations it
did strongly lower temperature, so there is a small suspicion that temperature may be a factor even at the lower
concentrations. When infused into ventricles just before
the ischemia, human recombinant Cu/Zn SOD attenuated
damage in CA1 (1157) as did the XO inhibitor and general
free radical scavenger oxypurinol (40 mg/kg), when injected intravenously. Temperature was not regulated in
this study (903), but subsequent studies in rat pups (8
days) showed that the very similar drug, allopurinol, at
higher doses did not lower body temperature (865). Intraventricular SOD (1135, 1136) protected against damage
from three successive 2-min ischemic episodes, and as
predicted, if free radicals are involved, the combined
administration of SOD and catalase was more effective
than either enzyme alone (1135, 1136).
Free radicals also play a major role in damage in the
rat. The membrane-permeant free radical scavenger
U-101033E attenuated damage in CA1 by 50% after 20-min
2-VO; temperature was unaffected for at least 70 min after
the ischemia (1059). This protection contrasts with earlier
results with another free radical scavenger that was found
not to penetrate the blood-brain barrier (72). The OHz
scavenger MCI-186 reduced CA1 pyramidal cell damage
by 70% 3 days after 10-min 4-VO in a well temperaturecontrolled study (1238). This reduction in damage was
associated with about a 50% reduction in OH radicals in
that region, as measured by the salicylate-DHBA method.
Oral administration of a flavinoid antioxidant, (2)-catechin, for 2 wk before and 1 wk after completely protected
CA1 in gerbil hippocampus against 5-min ischemia (496).
There was an associated 25% increase in the free radical
scavenging activity of cortical homogenates. Temperature
was not controlled in the study. Fivefold overexpression
of the cytosolic Cu/Zn SOD reduced CA1 damage in rat by
;50% 3 days after 10-min 2-VO (173), further implicating
free radicals in the damage. The implication is supported
by the protective effect of the COX-2 inhibitor SC-58125.
This reduces damage 14 days after ischemia by ;35%,
even when given 1 h after ischemia (836).
B) FOCAL ISCHEMIA. I) Temporary ischemia. Even
the blood-brain-barrier-impermeant scavenger U-74006F
strongly protected against damage in temporary focal
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CEREBRAL ISCHEMIA ON NEURONS
4. Role of NO and peroxynitrite in ischemic cell death
A) NO. Interpretive difficulties in early studies were
caused by conflicting effects of endothelial NOS activation and neuronal NOS (nNOS) activation (230). It is now
clear that nNOS activity is damaging during and after
ischemia in all major models and that endothelial cell
NOS activation is beneficial. Although more sophisticated
measurements may later sort out subtleties, the current
evidence for this conclusion is quite compelling (861).
I) Focal ischemia. In focal ischemia, relatively specific inhibitors of nNOS, when used at levels that do not
decrease blood flow or prevent the vessel response to
ACh, are strongly protective against permanent (1263)
and transient (308, 1296) ischemia in well-controlled studies. ARL-17477 reduced the infarct volume measured 7
days after 2-h MCA occlusion by 55%, at a dose that had
no effect on blood flow and 7-nitroindazole (7-NI), and
another more long-lasting inhibitor, TRIM, decreased infarct by ;45% 24 h after 2-h MCA occlusion. 7-Nitroindazole decreased infarct volume by 25% after 24-h permanent ischemia (1263). The reduction may have been
smaller than with ARL because the occlusion was permanent rather than transient or, more probably, because the
lifetime of the 7-NI is only ;2 h. In one study (1296), the
drug was fully effective when injected at the start of
reperfusion. In addition to effects of these pharmacological agents, nNOS knockout mice showed 40% reductions
in infarct size measured after 24- and 72-h permanent
MCA occlusion (474) and measured 24 h after temporary
ischemia (423).
Inducible NOS knockout mice showed about a 20%
decrease in infarct size measured after 96 h, but not 24 h,
of permanent focal ischemia (482). These studies are
consistent with the delayed (24 – 48 h) increase in iNOS, in
invading neutrophils. They indicate that the late progress
of damage may be due to production of NO from this
source. These studies are more persuasive than effects of
the iNOS blocker aminoguanidine (799) because the latter
also inhibits other potentially damaging enzymes.
Thus there is strong evidence that NOS is very important in focal ischemic damage.
II) Global ischemia. Neuronal NOS knockout mice
showed greatly reduced damage 72 h after 10-min global
ischemia (868). A normal ischemic effect of 85% cell loss
was reduced to a 32% cell loss in the CA1 pyramidal cell
layer. This is a substantial effect and argues for an important role of NO generation. Earlier pharmacological studies did not support a role for NO in global ischemic
damage because they showed no protection against CA1
damage in either rat or cat (138, 585). However, when the
specific inhibitor of nNOS 7-NI was used in well temperature-controlled studies, it reduced damage 7 days after
20-min 2-VO in the rat by ;35% (808). The level of inhibition of NO production is not known, but the result
indicates that NO generation during the first 2 h of reper-
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The time window for damaging free radical action of
between 3 and 12 h after the onset of ischemia has been
further refined in a nice study on a mixed permanent/
temporary model of thrombotic occlusion (1099). A shortlasting OHz scavenger, EPC-k-1, whose brain turnover half
time is ;1 h was very protective when administered at 3 h
after the onset of ischemia, but not at 0 or 6 h. This study
very nicely pinpoints the first damaging action of OHz to
between 3 and 6 h after the start of ischemia.
Further evidence that free radicals are important in
permanent focal ischemia comes from the effects of altered dietary vitamin E in a well temperature-controlled
study. Forty-eight hours after proximal MCA occlusion,
the lesion size was twice as large in rats that had been fed
a vitamin E-deprived diet as compared with those on a
vitamin E-supplemented diet. Lesion size correlated well
with blood levels of vitamin E in the two populations
(542). This is a dramatic effect, indicating the great importance of free radical scavenging in preventing permanent focal ischemic damage.
C) ENDOGENOUS SOD CHANGES IN VULNERABLE REGIONS. Given
the apparent importance of free radical changes in damage, it is extremely interesting that levels of both forms of
SOD decrease in the vulnerable CA1 and CA4 regions 1
day after 10-min global ischemia in gerbil (734). Levels are
unchanged or elevated in CA3 and dentate gyrus, two
regions which are not vulnerable. There is a similarly
selective downregulation of the predominantly mitochondrial Mn SOD after 5- to 10-min 4-VO in rat (680). It is clear
from other systems that downregulation of these enzymes
by using antisense to Cu/Zn SOD hastens apoptotic cell
death (393, 967) and also increases the size of infarcts
resulting from focal ischemia (601, 786). Thus the enzyme
decrease following ischemia might well contribute to the
vulnerability of CA1 pyramidal cells. This is rendered
more likely by the quite dramatic selectivity of the superoxide increase that occurs 24 h after a global insult in rat.
Although COX-2, a likely perpetrator of the delayed rise,
increases throughout the hippocampus (806), superoxide
only rises in the CA1 pyramidal layer (173).
D) SUMMARY. Various factors that should reduce free
radical buildup provide reasonable but not complete protection against global ischemia in rats and gerbils. Protection against temporary focal ischemic damage ranges
from reasonable (25% reduction in infarct size) to very
strong when uric acid or the spin trap PBN were used.
PBN was also very protective against permanent focal
ischemia. Damage from permanent ischemia was also
very sensitive to dietary vitamin E, again indicating a
dependence on free radicals. Unfortunately, none of these
studies included measurements of free radical levels, so
the degree of free radical suppression is not known. Studies in which actual free radical levels are measured and in
which the full time course of protection is measured are
needed to help determine the mechanism of free radical
damage.
1479
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PETER LIPTON
subjected to BDNF withdrawal, there was induction of
nNOS and an increase in protein nitrosotyrosines. Cell
death was prevented by blockers of NOS or by the cellpermeant free radical scavenger manganese TBAP (310).
This again suggests that peroxynitrite formation is important. In a similar vein, toxicity caused by the NO donor
Na1 nitroprusside, or by SIN-1, is very strongly attenuated
by SOD (237), providing strong evidence that NO is toxic
as a result of its conversion to ONOO2.
A study in newborn piglets is probably the most
direct demonstration that peroxynitrite formation is important in brain ischemia, although it does not directly
demonstrate a role in cell death. During hypoxia in the
piglets, there is an increase in PBN spin adducts, indicating increased free radicals or ONOO2 and also in lipid
peroxidation, as revealed by conjugated dienes. These are
both prevented by pretreatment with the NOS inhibitor
L-NNA (843). This strongly suggests that peroxynitrite,
formed from NO and existing superoxide, is the active
agent in this case.
The fact that either free radical scavengers or NOS
inhibition block global and focal damage is consistent
with peroxynitrite being the active agent. Another explanation for these results is that NO and a superoxidederived product are independently acting on a molecule
or function and that both actions are necessary for ischemic damage. This seems less likely than the involvement
of the one peroxynitrite species.
It is worth considering why peroxynitrite might be
the damaging agent in so many cases. Although NO alone
is not necessarily toxic, superoxide or its products, H2O2,
or the OHz radical, certainly can be (630). One possibility
is that ONOO2 attacks specific molecules that are critical.
There is no particular precedent for this. For example,
single-strand DNA breaks, which seem very important as
activators of PARP (see below), are induced very rapidly
by ONOO2 (1093), O2
2 or OHz (1112), or H2O2 (999). It is
more likely that the reason is quantitative rather than
qualitative. If superoxide (and probably therefore OHz or
H2O2) generation is fast enough, toxicity is likely to be
independent of NO; if it is not, then NO and peroxynitrite
formation will become important. This could be tested.
C) CONCLUSION. Overall, the studies are quite persuasive in ascribing a major role to free radical generation in
ischemic cell death. A large number of agents that prevent
free radical buildup, or NO buildup, strongly protect
against global ischemia and against penumbral damage in
permanent or temporary focal ischemia. In several of the
studies, temperature has been well regulated. There are,
though, critical details that need to be understood.
The degree of protection is variable, depending on
the agents used to prevent accumulation. The basis for
this is unknown. It could be that treatments have different
efficacies on free radical or NO buildup; measurements
are required to resolve this. It could be that different
species (e.g., OHz, ONOO2) have different efficacies in
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fusion contributes to damage. Greater protection may
have been achieved if the 7-NI was injected before the
ischemia as NO is generated during global ischemia.
III) Cell cultures. Very dramatic effects are manifested in cell cultures where delayed cell death after
60-min in vitro ischemia is almost completely blocked in
cultures from mutant mice lacking nNOS (237) and also
by NOS inhibition (704).
IV) Localization of nNOS. In the resting state, almost
all nNOS in the forebrain is localized to interneurons (261,
956, 1159, 1198). It is possible that a few pyramidal cell
neurons contain nNOS, but several studies, referenced
above, show very clearly that most nNOS neurons in
forebrain structures are interneurons. The interneuron
processes with nNOS are very ramified among the parenchyma, with good evidence that nNOS-containing processes are very close to non-nNOS-containing cells (956)
and so could readily affect them (236). Thus it seems
likely that interneurons, which are generally much less
vulnerable to ischemia, must receive the (presumed Ca21)
signal to enhance NO production, release it in the vicinity
of other neurons and, in this way, help cause toxicity.
Added to this, there is a major and very rapid increase in the number of nNOS-positive neurons during
focal ischemia that peaks within 4 h (1294). This no doubt
reflects de novo synthesis of the enzyme in these neurons
(as mRNA also goes up) and may well contribute to the
damage. The cells that nNOS is in are not clear, although
in one micrograph there was quite extensive involvement
of what appear to be principal neurons (1294). The role of
this process has not been pursued since the original publication.
B) PEROXYNITRITE. Peroxynitrite is currently considered
to be the major mediator of NO toxicity (107, 231, 678,
1093). Evidence for this is only indirect because there are
no reliably specific scavengers. Urate, which has been
used in some cases (1095), and does react with peroxynitrite, is not necessarily specific (74) and is not very reactive; it fails to prevent oxidation of protein thiol groups
(1162). On the other hand, its actual effects in smooth
muscle are most consistent with a selective blockade of
peroxynitrite action (1095), so the compound could become useful with more control studies.
The experimental evidence that peroxynitrite is important comprises a large number of studies showing that
both NO and superoxide are necessary for damage, even
in situations where the primary event is activation of NO
synthesis. Other studies show the much greater potency
of peroxynitrite than NO.
A very elegant study by Xia et al. (1228), in a nonneuronal culture, demonstrated that A-23187-induced cell
death did not occur when NO production was activated in
the absence of superoxide production, or vice versa, but
did occur when both NO and superoxide were generated
by the NOS. Cell death could be blocked either by inhibiting NOS or by scavenging superoxide. In motor neurons
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
different systems and that they are differentially affected
by inhibiting NO or superoxide accumulation. Details of
buildup and targets are really necessary to get at this next
level of understanding.
5. Molecular targets of free radical action
6. DNA cleavage and activation of PARP
Activation of the enzyme PARP (or PARS) appears to
play a critical role in damage following transient focal
ischemia. The basic findings, from at least four different
groups, are that blockade of PARP using either of two
different classes of drug (683, 1098), one of which is
highly specific (683, 1280), and also using knockout mice
(298, 305) greatly decreases infarct size measured by
different staining techniques 24 h after beginning 90-min
to 2-h transient ischemia. Decreases in infarct size range
from ;45 to 85%, the latter of which, in the mouse, and
the rat (1280) is the largest effect of any drug or knockout
treatment yet noted. In at least one case, the drug GPI6150 is effective when given 1 h after 2-h ischemia (1280).
Only one group has studied permanent ischemia, and
although data are not presented, there is said to be little
elevation of polyadenine-ribosylated protein and little
protection against infarct by inhibition of PARP (305).
This is a dramatic difference between the two forms of
ischemia. There are no reports of measurements in global
ischemia.
Cortical cell cultures from Parp2/2 mice were completely protected against cell death after 60-min in vitro
ischemia (298). Cell death following 5-min exposure of
cultured cortical neurons to 0.5 mM NMDA is essentially
completely blocked by the quite specific PARP inhibitor
DPQ (298).
Two major issues are how the PARP is activated and
what its activity may be doing to cause damage.
A) ACTIVATION OF PARP. Poly(ADP-ribose) polymerase is
a nuclear enzyme that is strongly activated by singlestranded DNA breaks; there are no other known activators (88, 1093). It is extremely likely that these breaks are
caused by peroxynitrite (1093), although free radicals
such as superoxide or hydroxyl are also capable of doing
this and almost surely do so following ischemia in heart
muscle (1112). Hydrogen peroxide has the same effect
(999).
Such single-strand breaks, detected by DNA polymerase I-mediated biotin-dATP nick translation (PANT) have
been observed in both core and penumbra neurons early
during reperfusion after 1-h focal ischemia (182). Production of the single-strand breaks seems to require reperfusion. There were no breaks at the end of 60-min focal
ischemia, yet there were a sizable number after just 1 min
of reperfusion (182). This requirement may derive from
the large spikelike increase in free radical formation that
occurs at the onset of reperfusion after 1-h MCA occlusion (893). The requirement for reperfusion is consistent
with the lack of involvement of PARP in permanent ischemia damage. The very rapid production of single-strand
DNA breaks that was observed is also consistent with the
early appearance of poly(ADP-ribose), within 5 min of
terminating 2-h focal ischemia (305). It would be very
important to know if blockade of free radical or NO
buildup diminished the number of single-strand breaks
and also the extent of PARP activation, which can be
measured (305).
The importance of peroxynitrite in the strand breaks
is suggested by work in several model systems including
thymocytes (983), smooth muscle cells, and macrophages
(1094), where 25–50 mM peroxynitrite, probably acting in
its protonated form, causes strand breaks and PARP activation, followed by cell death, with timely kinetics
(1304). Nitric oxide is very ineffective in this process
(1093).
B) MECHANISM OF PARP TOXICITY. This has not been well
defined. The PARP-catalyzed reaction involves the cleavage of NAD, and one commonly considered basis for
toxicity is depletion of NAD, with resultant inhibition of
mitochondrial function. This would cause depletion of
ATP and possibly the opening of the MTP. Single-stranded
DNA breaks lead to this sequence (absent any measurements of MPT) in several systems, and ATP and NAD are
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Free radicals affect the structures of both lipids and
proteins. They interact strongly with unsaturated bonds in
lipids, leading to a chain reaction formation of peroxides,
hydroperoxides, and aldehydes (488, 561). They activate
phospholipase (PL) A2, possibly because the peroxidation
makes the lipids better substrates (1163). Probably as a
result of the formation of aldehydes, they markedly reduce membrane fluidity (186). Free radicals oxidize protein side chains, forming carbonyl groups (336, 1067) or
disulfides, and can also act as reductants, causing SH
formation from S-S bonds (336).
Quite short exposures (10 –90 min) strongly inhibit
several very important enzymes, including plasmalemma
Na1 (300, 446, 684) and Ca21 (1017) pumps, creatine
kinase (1267, 1271), and several mitochondrial dehydrogenases (1290). In the case of the Na1-K1-ATPase, oxidation by free radicals makes the enzyme susceptible to
calpain-mediated proteolysis (1307). Peroxynitrite is
equally toxic, permanently inactivating the cytochrome b
and aa3 complexes of neuronal cultures 24 h after a short
exposure to 500 mM concentrations; smaller concentrations are also effective (107). Free radicals and peroxynitrite also produce single-strand breaks in DNA (999,
1093).
Any or many of these effects may account for damaging effects of free radicals, but in no case yet has a
specific molecular change been tied to free radicals, and
also been shown to be instrumental in damage. The most
clear-cut change is probably DNA cleavage, as discussed
in section VA6.
1481
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PETER LIPTON
7. Gene-mediated effects of free radicals: effects on
NFkB, cytokines, and CD-95 ligand
A) NFKB. There is accumulating evidence in many
tissues, including brain, that free radical damage following ischemia is at least partially mediated by the cytosolically located transcription factor NFkB. NFkB is activated by the inactivation of an inhibitory protein, IkB,
which occurs in two major steps. Phosphorylation is mediated by effects of free radicals (1000) and other pathways (56, 747, 1005) on IkB kinases (254). Subsequent to
this, the phosphorylated IkB is broken down by the 20S
proteasome and rendered inactive (56). Two active subunits of NFkB are then translocated to the nucleus. NFkB
activates synthesis of many proteins including iNOS and
COX-2 (1005); the latter has been particularly nicely demonstrated during hypoxia, where it was shown that the
p65 unit is responsible (995). It also upregulates the CD-95
ligand (1173), which is upregulated after global ischemia.
It may upregulate proapoptotic BCl-xshort after global
ischemia, but this is speculative (264). Although the NFkB
pathway can be antiapoptotic in some conditions (1005,
1186), there is evidence that the overall pathway is damaging after ischemia. The evidence for this, and its mediation by free radicals, is much stronger in noncerebral
tissues such as liver (1309), vascular endothelium (464),
and heart (176) than it is in brain, but there is emerging
evidence that it may be instrumental in the latter also.
NFkB is activated and translocated to the nuclei of
hippocampal neurons after 30-min 4-VO global ischemia in
rats (206); this occurs in all neuronal populations 24 h after
ischemia, but only in the soon-to-die CA1 population 72 h
after the ischemia. The delayed translocation is blocked by
the antioxidant LY-231617 (207), indicating that it is mediated by free radicals. However, somewhat surprisingly, the
early translocation is not blocked by the antioxidant, suggesting another pathway is responsible for that process,
probably a kinase cascade (640, 1005). This differs from
other tissues such as liver (1309), cerebral blood vessels
(464), and heart (176), where early postischemic activation
of NFkB is prevented by free radical scavengers. The antioxidant was very protective against global ischemic damage
(207), consistent with, but certainly not proving, the involvement of NFkB in damage.
NFkB is activated in the core and penumbra 1 day
after 90-min temporary focal ischemia of the cortex (984).
The specific proteasome inhibitor PS-519 strongly attenuated damage measured 24 h after 2-h ischemia (902). As
discussed previously, this might reflect damage via proteolysis. However, because the proteasome pathway is required for NFkB activation, the result may reflect the
importance of NFkB in focal damage. If so, it shows that
the ischemic core is most susceptible to damage via this
system.
Aspirin and salicylate, which directly block activation of NFkB, protect strongly against glutamate toxicity
in cultures and seem to do so by blocking NFkB. Effects
are not mediated by reducing Ca21 entry or by inhibiting
prostaglandin metabolism and are strongly correlated
with inhibition of the activation of NFkB by 50 mM glutamate (395).
NFkB is likely to be instrumental in upregulation of
iNOS and COX-2, which are damaging in both focal and
global ischemia, and also of cell adhesion molecules that
are damaging in focal ischemia. However, it is not at all
established that this is its mode of action. The nearcomplete blockade of glutamate toxicity, which is not
thought to be mediated by either of the enzymes or adhesion molecules, certainly suggests alternate modes of
action.
B) CD-95 LIGAND. The 45-kDa CD-95 ligand is upregulated after focal ischemia and is a reasonable candidate
for initiation of apoptosis, as discussed previously. This
ligand is upregulated in 12 h by H2O2 in cultured microglial cells, and the mRNA is upregulated after hypoxia/
reoxygenation (no measurements were made of protein)
(1173). In the former case, the upregulation coincides
with activation of NFkB; this and the fact that the CD-95
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almost completely depleted if there is enough strand
breakage (88, 1094). Hydrogen peroxide very likely
causes cell death by this mechanism in several cases (216,
999).
The evidence for such an energy metabolism-mediated cell death in temporary focal ischemia is not great. In
general, effects of PARP inhibitors on ATP changes have
not been measured. In one study, there was a 65% fall in
NAD in the ischemic territory, and both PARP knockouts
and a PARP inhibitor 3-aminobenzamide attenuated this
fall by ;40% (305). This result is somewhat difficult to
interpret because measurements were made at 24 h, by
which time there were a lot of dead cells. Thus the cell
saving by PARP inhibition could be responsible for the
higher NAD levels. Measurements before significant cell
death would be more useful. The other difficulty is that
there is no evidence for dramatic falls in ATP in temporary focal ischemia. It is possible that the small reductions
in ATP may be damaging over a long period, as discussed
in section IVB. Effects of PARP inhibition on these ATP
levels need to be measured. Alternatively, ATP may fall
dramatically for a short period during reperfusion, and
this may trigger damage.
Thus, at this stage, there is no well-established mechanism for the damaging effects of PARP after ischemia.
Possibilities other than inhibited energy metabolism include additional effects of NAD(H) depletion and resultant metabolite changes, or effects on histones and other
nuclear proteins as a result of the transient ADP ribosylation (it lasts ;3 h; Ref. 305). Alternatively, this ADPribosylation appears to be a critical step in apoptosis in
many model systems (1049) and could certainly play such
a role in ischemia.
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CEREBRAL ISCHEMIA ON NEURONS
8. Summary: possible mechanisms
of free radical-mediated cell death after ischemia
The studies described above make it quite clear that
free radical production makes a major contribution to
ischemic cell death. Six likely mechanisms of action are
described here.
1) As described above, activation of PARP which
probably, but not surely, results from free radical/peroxynitrite-mediated cleavage of DNA may well mediate a
lethal effect of free radicals after temporary focal ischemia. The very large saving produced by uric acid (1266),
which is a good peroxynitrite scavenger, is consistent
with this because PARP inhibitors also greatly reduce the
size of the lesion (1280).
2) Synthesis of CD-95 ligand via activation of NFkB
might be a mechanism by which free radicals cause apoptotic cell damage, as may be the increase in TNF-a.
However, the pathways by which the CD-95 ligand and
TNF-a are produced after focal ischemia have not yet
been determined, so this is very speculative. Free radicals
appear to initiate apoptosis in several systems, so there
may also be other pathways by which they do so in
ischemia.
3) Other known functional targets of free radicals
that could well lead to cell death include increased plasmalemma permeabilities (201, 849), prolonged inhibition
of protein synthesis in (vascular endothelial) cells via
effects on eIF-4-like proteins (513), and major changes in
mitochondrial function, including opening of the MTP, as
discussed extensively in section IVB. There is, though, no
evidence positively identifying the importance of these
phenomena in ischemic cell death.
4) Effects of free radicals on Ca21 homeostasis could
be important. After 60-min focal ischemia, cytoplasmic
Ca21 remains elevated for at least 30 min. This elevation
is prevented if ventricles are perfused with SOD at high
levels (30), suggesting that free radicals affect some aspect(s) of Ca21 homeostasis, possibly by elevating Ca21
fluxes through existing or new pathways, or possibly by
inhibiting Ca21-ATPase. This could reflect the same underlying process as the apparent dependency of postglobal ischemic mitochondrial Ca21 accumulation on NOS
activation (1208).
5) Inhibiting NOS during focal ischemia prevents the
normal fall in pH in the penumbra, suggesting that NO
synthesis in some way causes the pH drop (943). The
mechanism of this somewhat surprising effect is not
clear, but if it does do this, it could certainly be a way that
NO enhances damage (see sect. VID). One mechanism for
this may be the effect described immediately below,
where NO contributes to the release of glutamate in focal
ischemia. Glutamate generally acidifies neurons (159).
6) Nitric oxide (possibly via peroxynitrite production) appears to play an important role in the accumulation of glutamate in the core of a focal lesion and the
subsequent spread of glutamate to the penumbra and the
resulting intraischemic depolarizations. Release of glutamate in the core, and subsequent intraischemic depolarizations, were greatly attenuated in nNOS knockout mice
(1031). The mechanism may be the peroxynitrite-mediated inhibition of glutamate transporters (1132).
Of course, because of their wide-spread effects on
different proteins, free radicals might alter protein or
proteins whose importance has yet to be shown.
9. Summary: apoptosis or necrosis
Exogenous free radicals are able to cause necrosis
(111), or apoptosis (393, 502, 788, 789, 967, 1134), in
cultured neurons or neuronal-like cells; at a much more
extreme level, free radicals (largely OHz) generated by the
xanthine/XO/Fe21 system in the intact brain caused dramatic cellular degeneration within 2 h, with neurons appearing as in ischemic-cell change and the blood-brain
barrier becoming highly permeable to Evans blue. Infarct
developed within 24 h (174). Thus whether free radicals
selectively promote apoptosis or necrosis after ischemia
may well be a function of the level of free radical production, as seen in cultures (111).
When excess free radicals were produced as a result
of 2/2 SOD null mutants in mice, the region that became
more vulnerable showed a greater number of apoptotic
neurons than other regions (601), suggesting free radicals
caused apoptosis. However, this could have arisen from
the fact that the insult intensity in the newly vulnerable
region (the former peri-infarct zone) was low. It would be
very useful to explicitly count the effect of free radical, or
NO, on attenuation of apoptotic and necrotic neurons in
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ligand gene has a putative NFkB binding region suggests
that the transcription factor may mediate activation of the
CD-95 ligand (1173). If this mechanism also pertains after
cerebral ischemia, it would be an important way by which
free radicals mediate apoptotic cell death. Further work
along these lines is necessary.
C) ROLE OF CYTOKINES. Cytokines that are upregulated
early in ischemia and reperfusion, particularly TNF-a and
IL-1, are strong activators of NFkB (1039). Thus they,
along with free radicals generated from other sources, are
potential activators of the transcription factor. The mechanism by which cytokines activate NFkB is not fully
known, but it does appear likely that free radicals generated by the cytokines mediate the effect because several
free radical scavengers strongly attenuate the effect (381,
1000). However, other pathways activated by the cytokines, such as ceramide synthesis and MEKK kinase cascades (1005, 1039, 1186), may also be important. It is not
currently known what is responsible for the ischemic
upregulation of cytokines TNF-a or IL-1 (747).
At this stage, the actual activators of NFkB during
and after ischemia are not known.
1483
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PETER LIPTON
regions that were protected by free radical scavengers or
by SOD overexpression to determine whether free radicals preferentially induce one or the other forms of cell
death. They certainly have the potential to induce both
forms.
10. Summary: timing of damaging
free radical actions
shows that the delayed production of free radicals by this
enzyme contributes significantly to damage.
Overall, more data in which the time windows of
efficacies of different agents are better ascertained, and in
which the time they remain in the brain is measured,
would help sort out the times at which free radicals are
acting and thus help to determine what they are doing.
The most likely bases for prolonged maintenance are
COX-2 and iNOS inductions and possible ongoing production by mitochondria. These possibilities can be tested.
B. Ca21-Dependent Proteases
1. Inhibitor specificities
There is strong, albeit not very plentiful, evidence
that the Ca21-activated neutral cysteine protease calpain
plays an extremely important role in focal ischemic damage and very possibly in global damage (66, 379, 750, 971).
This evidence is based on cell-permeant inhibitors of
calpain that have been developed in the last few years (66,
715) and relies on their specificity. Knockout animals
have not yet been made. Inhibitor specificity is somewhat
problematic because there are at least three currently
known alternate proteolytic systems that might play a role
in ischemia. These are caspases and cathepsins, both of
which play or may well play roles in ischemic cell death,
and also the 20S proteasome.
The current generation of cell-permeant intracellular
protease inhibitors are aldehyde-linked di- or tripeptides
in which the peptides mimic the substrate specificity of
the protease (715). Amino acid sequences that most potently inhibit calpain contain two hydrophobic residues.
Val-Phe (MDL-28170) and Leu-aminobutyrate (AK-275).
These are the two inhibitors that have been shown to
possess the greatest potency in focal ischemia (67, 715). A
much less cell-permeant molecule, leupeptin, was very
protective in global ischemia (641).
Caspases have very different substrate specificities
from calpain, and caspase inhibitors have two or three
hydrophic amino acids but they must be accompanied by
an aspartate. It is thus considered extremely unlikely that
the calpain inhibitors will inhibit caspases. This being
said, there is only one reported study in which it was
shown explicitly that E64 and antipain, inhibitors that are
similar but not identical to MDL-28170 and AK-275, have
no effect on caspase-1 (ICE) (1214). One indirect but quite
strong piece of evidence that calpain inhibitors do not
affect caspases is that MDL-28170 does not prevent apoptosis in thymocytes when the apoptosis is triggered by
insults that do not require protein synthesis (1064). This is
very likely a caspase-mediated event.
Some three or four hydrophobic amino acid sequences that effectively block calpain (inhibitory constant ;1027) also block proteasomal activity, although at
100-fold lower potency (749); potency may be greater in
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The data suggest that free radicals persist for well
beyond 24 h after the start of the insult in temporary focal
ischemia; they have been measured explicitly for at least
6 h in the penumbra and the core, and iNOS and COX-2
are induced between 6 and 24 h of reperfusion, suggesting
there will be prolonged production of free radicals and
peroxynitrite. The latter accumulates for at least 3 h in the
penumbra; later measurements are not reported. Free
radicals have not been measured more than 3 h after the
start of permanent focal ischemia but may well persist.
Thus free radicals persist in the tissue for many hours and
possibly days. What is important is when these free radicals are actually damaging. One can envision their being
important early, perhaps as a trigger, as occurs in sympathetic ganglia following NGF withdrawal (239), perhaps to
set off synthesis of NFkB and resulting downstream effects or to alter Ca21 homeostasis. One can then envision
them acting later, effecting damage of important molecules involved in ICC. They seem to play such a role in
staurosporine-induced apoptosis in neurons so that delayed addition of scavengers is very effective (618). Unfortunately, there is little available information, since
most inhibitors are added early in the insult and are often
maintained. The duration of their efficacy is often not
known (e.g., Ref. 207). In permanent focal ischemia, there
is an important window during which inhibitors are effective, between 3 and 6 or 12 h after the ischemia. There
may be others, later. No equivalent information exists for
temporary ischemia.
Free radicals and NO are elevated early during and
after global ischemia and, again, most drugs are added
very early so the window of activity has not been determined. However, there is reasonably good evidence that a
delayed activation of free radicals, 24 – 48 h after ischemia, plays a major role in the delayed damage in CA1.
Inducible NOS is increased in CA1 glia between 24 and
48 h after the insult, and COX-2 is increased between 6
and 24 h. There is a large increase in superoxide in CA1,
along with a reduction in SOD 24 h after the ischemia.
Thus there is certainly a conspiracy to effectively produce
a delayed increase in free radicals in the CA1 pyramidal
region. One effect may be upregulation of NFkB, which is
greatly elevated in CA1 some time between 24 and 72 h
after ischemia. Pharmacological evidence that all these
changes are functionally important is not impressive, being confined to the efficacy of COX-2 inhibition which
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
2. Regulation of calpain
Although quite high concentrations of Ca21 are necessary to initially activate the isolated enzymes m- and
M-calpain (10 mM and 1 mM, respectively), these concentrations are lowered by binding to polyphosphoinositides,
particularly phosphatidylinositol 4,5-bisphosphate (PIP2)
at the cell membrane and by products of lecithin breakdown (37). The mean affinity constant for Ca21 activation
of m-calpain is reduced from ;10 mM to ;100 nM, and the
sensitivity of M-calpain is also increased (971, 972, 1087,
1240). The Ca21 sensitivity is also greatly increased by a
membrane protein that has been found in several tissues,
including the erythrocyte and brain (979). Once activated
by Ca21, the isolated enzyme often undergoes autoproteolysis, and the smaller protein is active at normal cell
levels of Ca21 (622). Another mode of activation may
include changes in calpastatin, but little is known about
this. Thus calpains may well be activated by quite small
increases in Ca21.
Even without mechanisms of increasing its sensitivity, at least m-calpain can be expected to be activated
during ischemia as both in situ (1048) and in slices (397)
cytosolic Ca21 appears to rise into the tens of micromolar
range.
An important unresolved issue is how the enzyme
might be inactivated after ischemia.
3. Localization of calpain and calpastatin
The localization of calpain may well be relevant to
selective vulnerability of neuronal populations. m-Calpain
is widely distributed throughout the neuron, including
spines, dendrites, cell somata, and axons (892). In rabbit
hippocampus, it is primarily localized to pyramidal neurons, whereas M-calpain, the less sensitive isozyme, is
primarily localized to inhibitory interneurons (344). This
is interesting because of the great resistance that interneurons show to ischemic damage. The localization of
calpastatin is interesting in this regard also. Although it is
present in most cells, it is at a far lower concentration in
(vulnerable) CA1 pyramidal cells than (resistant) CA3
pyramidal cells (344).
4. Increased calpain activity during and
after ischemia
A) GLOBAL ISCHEMIA. Many measurements of calpain
activity are actually measurements of spectrin breakdown
into a fragment that is characteristic of calpain action
(although all proteases have not been tested). Early studies showed spectrin proteolysis 30 min after 10-min global
ischemia in CA1 of gerbil that was inhibited by leupeptin
(641). However, for reasons that remain somewhat of a
mystery, more recent immunohistochemical studies have
revealed that the protease activation is biphasic. Five
minutes after ischemia there is spectrin breakdown in
CA3 and in cortex, but not in CA1 of hippocampus (952).
After 10-min ischemia, there is a small amount of proteolysis in CA1 (973), but not until 24 h after ischemia is
there a large spectrin breakdown in CA1 (952, 973, 1258).
The very delayed nature of calpain activation is difficult to
understand because Ca21 certainly seems to rise in CA1,
and previous studies had demonstrated an early calpain
increase. If this holds up, and it has so far been demonstrated by two different groups, it suggests that delayed
calpain activation may be more important to global ischemic damage than early activation (which occurs in nonvulnerable neurons). This could be tested with appropriately timed inhibitor studies.
Finally, there is a very large increase (to 40%) in
spectrin breakdown around the time of cell degeneration
(1258). If this in fact precedes cell death, it indicates a
very strong calpain activation at a critical time, but the
detailed timing is not yet known.
A direct measure of calpain autolysis indicated activation in whole rabbit brain during the first 10 min of
global ischemia (823).
B) FOCAL ISCHEMIA. There was a large increase in spec-
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situ (1033). Thus, at the doses used in vivo (;10 mg/kg or
30 mM to get strong protection), it is possible that the
calpain inhibitors are also blocking proteasomal activity.
Neither of the dipeptides used against ischemia has been
tested against proteasomal activity yet. Leupeptin may
also inhibit proteasomes; it blocks the trypsinlike activity
of proteasomes (but not the chymotrypsin-like activity)
(408). This is an important issue because a specific proteasomal inhibitor has recently been found to provide
strong protection in focal ischemia (902). This issue
would be best resolved by examining effects of calpain
inhibitors with widely different specificities for proteasomal action (749, 1033) or by using the peptide calpain
inhibitors at very low doses.
The protease that is most likely to be confounding
the results is cathepsin B which, although it is a serine
protease, has a very similar substrate specificity to calpain (715) and is thus inhibited by MDL-28170, and by
leupeptin with potencies that are similar to those for
calpain (66, 657, 744, 1191). There is at least one cellpermeant inhibitor that is much more effective against
cathepsin than against calpain, ZYA-CHN2 (224), but this
has not yet been used in ischemia. This would be a good
way to test whether the inhibitors are acting on cathepsins.
Another way to attain reliable calpain specificity has
been to develop a cell-permeant inhibitor that blocks at
the Ca21 binding site of calpain. This has been done
(1190), and although it is effective in cell culture (1064), it
is unfortunately not effective in brain slices because of
penetration problems (Lipton and Zhang, unpublished
data) and has not been studied in vivo. It is likely to be
ineffective there too.
1485
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PETER LIPTON
5. Mechanism of calpain activation during and
after ischemia
The mechanism of calpain activation is not clearly
established. Although increased intracellular Ca21 is of
course the most likely mechanism, it is notable that Ca21
levels needed to activate the m-form are very high unless
it is bound to phosphoinositides or to specific proteins.
In liver cells, calpain was activated twofold during
anoxia, without an increase in intracellular Ca21. Activation appeared to be dependent on PLA2 breakdown (37,
38). This is consistent with the ability of phosphatidylcholine breakdown products to activate calpain (37).
In global ischemia, most of the early and delayed
calpain upregulation is blocked by MK-801, suggesting
that Ca21 entering via NMDA receptors is important
(952). This is consistent with either direct activation or
activation via PLA2 since the latter is Ca21 dependent.
6. Involvement of calpain in ischemic damage
As far as tested, calpain inhibitors appear to reduce
cell death very effectively. In the global gerbil model,
leupeptin infusion into the ventricles prevented the early
breakdown in spectrin and almost completely prevented
cell death in CA1 7 days later (641). Temperature effects
were not measured. This is the only reported study in
global ischemia.
In focal ischemia, superfusing the cell-permeant calpain inhibitor AK-275 into cortical tissue at various times
before during and after a temporary distal MCA/CCA occlusion reduced the size of the cortical infarct by 50 –70%
(67). The drug was very effective when administered 3 h
after starting the ischemia but not 4 h after ischemia. This
is a massive reduction in infarct volume and suggests that
calpain plays a crucial role in the process, possibly even
in damage to the core of the infarct. A similar level of
protection was found against 3-h temporary focal ischemia where another dipeptide/aldehyde inhibitor, MDL-
28170, reduced infarct size by ;70% when administered
intravenously within 30 min of starting ischemia. The drug
was still very effective when administered up to 6 h after
starting the ischemia (3 h into the reperfusion) (715). In
both studies, damage was only measured 24 h after occlusion so that very long-term protective effects were not
tested. Temperature was quite well controlled in one
study (67), where no effect of inhibitor on temperature
was noted for at least 1 h after MCA occlusion, but was
not discussed in the other study (715). The fact that the
two inhibitors are very similar certainly suggests that
temperature did not vary in that study either.
Cell death following in vitro ischemia in cerebrocortical cultures was attenuated by the cell-permeant calpain
inhibitor that has almost no effect on cathepsins (1190),
strongly implicating calpain in cell death in cultures. In
conclusion, the effects of calpain inhibition on focal lesion size are more profound than all other ways of intervening. Unless these drugs are protecting by inhibiting
cathepsin B activity, or proteasome activity, the results
show a major contribution of calpain to cell damage in
transient focal ischemia. There are, though, only very few
studies, particularly in global ischemia.
7. Possible sites of calpain action
The apparent importance of calpain in focal ischemic
cell death, and probably global also, makes knowing its
targets very important.
A) SPECIFIC TARGETS. The enzyme attacks many cytoskeletal proteins, including MAP2 (900, 1291), spectrin,
tubulin (971), and very probably ankyrin. It leads to loosening of the postsynaptic density (269) and participates in
the breakdown of microtubules in an unknown way, as
evaluated from effects of MDL-28170 and calpain inhibitor
1 (1291). Hence, dissolution of critical cytoskeletal elements may well be how it participates in cell death.
Opening of the MTP is another possible site of action.
In hepatocytes, mitochondrial calpain was activated by
increased intramitochondrial Ca21 and by oxidative
stress generated by tert-butyl hydroperoxide, leading to
opening of the MTP (7).
Another possible site of action is eIF-4, which is
broken down by calpain to one-third its normal level
during 20-min global ischemia in the rabbit (820, 822).
However, a role for global protein synthesis inhibition in
focal ischemic death is quite unlikely, so this is probably
not its mode of action. It may well be important in global
ischemia.
Calpain may play a role in activating autophagocytosis. The activated form of m-calpain was identified immunocytochemically on lysosomal membranes after global
ischemia in gerbils (1240), before the formation of the
autophagic vacuoles (834).
B) APOPTOSIS. Calpain appears to play a critical role in
apoptosis in many cases, as judged both by its upregula-
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trin proteolysis during 3 h of focal ischemia in regions that
later developed infarct. This had begun by the time of the
first measurement, at 1 h. There was a delayed increase in
the peri-infarct regions, where individual cell death generally occurs. That increase was noted after 21-h reperfusion and was as large as the increase in the infarcted
regions (455). Because cell damage was developing in the
lesion throughout the study, it is not clear whether the
calpain activation preceded the damage, although the
significant proteolysis by 1–3 h in core and penumbra
certainly preceded major cell death.
C) BRAIN SLICES. In rat brain slices, where calpain inhibitor-sensitive MAP2 proteolysis and microtubule dissociations were used to monitor calpain activity, there was
a clear calpain activation during the first 5 min of in vitro
ischemia, with continued activity in the reperfusion period (1291).
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CEREBRAL ISCHEMIA ON NEURONS
8. Conclusions
The existing evidence strongly suggests involvement
of calpain in ischemic cell death; protections by AK-275
and MDL-28170 against temporary focal ischemia are the
most profound reported with any protectant except for
one set of studies on the PARP knockout mice, and the
protection by leupeptin against global ischemia in gerbil
was also very profound. There are some caveats, however. Although temperature control was good during the
occlusion in the case of AK-275, there are no reports of
temperature control during the study of global ischemia.
This is of concern. A second concern is the specificity of
the agents for calpain. Although it is very unlikely that
they act on caspases, given the marked differences in
targets and hence inhibitor construction, the inhibitors
may well act on cathepsins, or on proteasomes. It will be
necessary to judiciously use newly developed selective
inhibitors against these different proteolytic systems to
firmly establish the importance of calpains in ischemia.
The inhibitor studies with AK-275 and MDL-28170
indicate that the lethal calpain action in temporary focal
ischemia is somewhere between 3 and 6 h after the start
of ischemia, a time consistent with its involvement in
early events of apoptosis and in development of necrotic
death. This is quite similar to the time at which free
radicals are critical in permanent focal damage. If the two
events are comparable, then a critical time for damage
development is the 3- to 6-h period after the onset of focal
ischemia. Although there are no timed inhibitor studies in
global ischemia, there is certainly an apparent dramatic
upregulation of calpain activity in vulnerable CA1 at ;24
h. This is the same time as the appearance of significant
free radical changes in this region and suggests that this
period may well be a critical one for development of
global ischemic damage. As with free radicals, it would be
very useful to have studies in which the periods during
which drugs effectively prevent damage became known.
This necessitates knowing the period for which an injected drug is efficacious.
At this stage of our understanding the most likely
damaging action of calpain is on apoptotic biochemistry.
Effects on the cytoskeleton may mediate necrosis but the
role of the cytoskeleton in necrotic cell death is not yet
well established; the same is true for effects on protein
synthesis. The other known functional effects of calpain
do not seem likely to be lethal. It would be interesting to
see whether calpain inhibition selectively prevented apoptotic cell death.
C. Phospholipid Metabolism
Breakdown of phospholipids could, in principle,
cause major changes in membrane function and also
changes in signaling capabilities resulting from loss of
phosphoinositides. Furthermore, the breakdown leads to
major increases in FFA production and hence, also, free
radical formation. All these are potentially damaging. Cytidine diphosphate choline, which can act as a phospholipid precursor, appears to attenuate ischemic damage
(1014), highlighting the potential importance of phospho-
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tion and the effects of calpain inhibitors. For example,
T-cell apoptosis induced by anti-CD3 monoclonal antibody was blocked by calpain inhibitors, and there was a
calpain blocker-sensitive breakdown of spectrin to the
150-kDa fragment during the process (720). This calpainmediated cleavage of spectrin occurs after several different apoptotic stimuli in different types of cultured cells
including hippocampal neurons (511, 720). There are
many instances in which cell-permeant calpain inhibitors
including calpeptin in neuroblastoma (78), MDL-28170 in
cultured hippocampal cells (511), calpeptin and MDL28170 in thymocytes and metamyelocytes (1065), and leupeptin in T cells (989), prevent apoptosis. One of the
studies (1065) very nicely demonstrated the autoproteolysis of calpain I, showing calpain activity is turned on,
using an antibody that was specific to the native form.
In thymocytes, calpain inhibition by the Ca21 binding
site-specific inhibitor PD-150656 produced a clear inhibition of all aspects of apoptosis when apoptosis required
protein synthesis (for example, when induced by dexamethasone), but not when protein synthesis was unnecessary for apoptosis (1064), suggesting calpain is involved
in a relatively early event in apoptosis.
Taken together, these results provide a picture of a
strong involvement of calpain in apoptosis.
The role of calpain is not known and may be quite
complex. It required protein synthesis to be activated in
staurosporine-treated neuronal glial culture (909) where
its inhibition not only prevented cell death but also DNA
breakdown. A possible site of action is actin breakdown,
a feature of apoptosis in most cell types. In chick neurons
deprived of growth factor (1169), calpain inhibitors I and
II blocked both apoptosis and actin proteolysis. The effect
on actin is almost undoubtedly indirect. Prevalent forms
of actin are not substrates for calpain (269, 971), whereas
several actin-binding proteins are substrates (971).
C) A CAVEAT. Although this section has focused on
damaging effects of calpain, the enzyme also appears to
play an important role in remodeling dendritic structure
after injury. In cultured neurons, NMDA induces reversible dendritic varicosities that result, at least in part, from
disassembled microtubules. The reestablishment of normal dendritic structure was blocked by calpain inhibitors,
implying that calpain helps in recovery from morphological damage (313). It is not clear if these effects relate to
ischemia, but the results point out that calpain may be
beneficial in reestablishing neuronal function during late
phases of recovery. This is important when considering
therapeutic aspects of the drug.
1487
1488
PETER LIPTON
lipid breakdown in damage. However, the dearth of good
inhibitors makes study of this topic very difficult.
1. Changes in FFA and phosholipids in ischemia
percentage decreases in the more plentiful phospholipids
are too small to reliably measure.
E) INOSITOL TRISPHOSPHATE CHANGES. These are very small
and last no more than 30 s in vivo or in vitro despite the
large falls in phosphoinositides (662, 663, 674, 1082).
There are large and prolonged increases of inositol monophosphate and inositol bisphosphate in vivo (663, 1082)
and of IP2 in slices (674). The reason that the increases in
inositol trisphoshate are small and unsustained is very
probably because the low ATP levels make it impossible
to regenerate the substrate, PIP2 (487, 663).
F) SUMMARY. Changes in arachidonic acid both during
and after global and focal insults are substantial and
should contribute to superoxide production. They may be
prolonged in vulnerable tissue. Decreases in phosphoinositides could lead to deficits in signaling pathways.
2. Mechanisms of net phospholipid breakdown
A) PHOSPHOLIPASES THAT ARE INVOLVED. Measurements of
reactants and products showed that the FFA increases
during the first 2 min largely result from phospholipase
C-mediated breakdown of the phosphoinositides, PIP2
and PIP, and resultant lipolysis of the diacylglycerols (3,
487, 554, 1082, 1083). This was confirmed by the action of
the (quite nonspecific) phospholipase C inhibitor, phenylmethylsulfonyl fluoride (PMSF), which largely blocked
the FFA increase during that time (1153). Phospholipase
A2 activation might contribute somewhat to production of
22:4 and palmitic acid (16:0) during this early time (3,
554). Release of FFA later than 2 min is basically unaffected by PMSF and so must largely be due to ongoing
activity of PLA2 or other lipases (3, 487, 1083).
B) PHOSPHOLIPASE ACTIVATION. I) Activation of PLC.
Brain PAF may be responsible for increased PLC activity.
The PAF activity increases 10-fold at the end of 10-min
global ischemia in gerbils, and regional effects on exogenous PAF binding suggest that the largest increase is in
the hippocampus (266). The mechanism of the increase in
PAF is not known. It may be synthesized by “remodeling,”
which requires activation of PLA2 followed by the action
of 1-alkyl GPC acyltransferase (54). Both these enzymes
can be activated by Ca21, kinase-mediated phosphorylations, and G protein activation (54, 55, 250), events which
are increased by ischemia. De novo synthesis of PAF is
also quite possible, since this should be strongly activated
by the decrease in ATP levels; the key enzyme is inhibited
by 0.5–1 mM MgATP (55).
In normoxic neuronal cultures (1268), PAF activates
PLC via PAF receptors and G protein activation (166,
661). This pathway remains to be established in ischemia.
Certainly activation of PLC seems far too early to result
from neurotransmitter release and binding to a metabotropic receptor.
II) Activation of PLA2. Possible mechanisms of PLA2
activation have been nicely reviewed (250, 738). Pres-
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A) FFA IN GLOBAL ISCHEMIA. There is a very rapid increase
in FFA during global ischemia, first noted about 25 years
ago by Bazan (69) and since reported by a large number of
workers in all models and species tested. Levels of total
FFA rise approximately fourfold during the first 5 min of
ischemia (69, 945, 1083) and reach a plateau after ;15 min
at 8 –10 times basal values (69, 945, 1262). There are
marked differences in the percentage increases of different FFA. With an average of five different studies between
the 1970s and 1990s, arachidonic acid (20:4) increased
from ;30 to 300 mM, 22:6 rose from ;10 to 30 mM, and
oleic acid (18:1) went from ;40 to 100 mM. Of the two
principle saturated fatty acids, palimitic (16:0) rose from
;60 to 180 mM and stearic (18:0) went from ;50 to 350
mM (3, 945, 1034, 1261, 1262). Thus there are very sizable
concentrations of FFA at the end of ischemia.
B) MAINTENANCE OF FFA AFTER GLOBAL ISCHEMIC INSULTS. The
oxidative metabolism of arachidonic acid, which is
where free radicals are generated, will be greatest during reoxygenation. In the rat, fatty acid levels in whole
brain or cortex largely resolve by 30-min postischemia,
and in most studies, they completely recover by 60 –90
min (574, 945, 1260, 1262). However, in gerbils, levels
are much elevated as long as 90 min after 10-min ischemia (869); FFA and arachidonic acid in the CA1 region
remained elevated for at least 1 day after 5 min of
ischemia (4). Unfortunately, this delayed elevation has
only been studied once. It suggests a prolonged generation of FFA in a vulnerable region, and this could be
important in damage. Consistent with this, PLA2 activity was elevated 10 min after 10-min ischemia in gerbils
(960), and it might stay elevated; this has not been
measured.
C) FFA IN FOCAL ISCHEMIA. Total FFA were increased 4- to
10-fold between the first 15 min and the end of 60 min of
MCA occlusion (1292). As with global ischemia, the largest percentage increases were in 20:4, 22:6, and 18:0
(1292). If ischemia was reversed after 1 h, FFA returned
toward baseline, and there was then a large secondary
increase at 16 and 24 h of reperfusion. In this case, 18:0
rose to a very high level, but arachidonic acid (20:4) was
not elevated (1292). Thus the contribution of this increase
to free radical generation would probably not be great.
D) CHANGES IN PHOSPHOLIPIDS. Both PIP2 and phosphatidylinositol 4-monophosphate (PIP) fell by ;60% during
2–3 min of ischemia (487). However, despite the large
increases in FFA, there were generally no measurable
decreases in other phospholipids (3, 945). An exception
was a report showing a 20% decrease in phosphatidylcholine after 10-min global ischemia in the gerbil (1133). The
absence of other measured falls is probably because the
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CEREBRAL ISCHEMIA ON NEURONS
3. Mitochondrial phospholipid changes
Mitochondrial lipid changes are profound and are
different from those in the cell as a whole. During 30 min
of 4-VO, mitochondrial contents of all FFA, saturated and
unsaturated, increased severalfold with no apparent preference for specific FFA (1079, 1080). The increased level
of FFA is maintained for at least 60 min of reperfusion.
Unlike cells as a whole, there is a measurable decrease in
phospholipid contents of the mitochondria (1079).
The basis for the phospholipid breakdown is not
known, but the high mitochondrial contents of phosphatidylcholine and phosphatidylethanolamine, and their measured breakdown (1079), suggests that PLA2 is activated,
very probably by the early accumulation of Ca21. Phospholipase A2 activity in the mitochondrial fraction of gerbil forebrain 10 min after 10-min ischemia is two times
normal (960).
4. Role of phospholipid changes in ischemic
cell damage
Whether these dramatic changes in cellular phospholipid metabolism actually play a role in cell death has been
very difficult to resolve.
A) EFFECTS OF ARACHIDONIC ACID. This would be expected
to be a major source of free radicals. However, effects of
blocking arachidonic acid metabolism, with its subsequent free radical production, have not been reported,
except for retina where they are protective against anoxic
damage. Inhibition of COX-2, which is upregulated in
neurons several hours after ischemia, is also protective in
temporary focal and global ischemia, indicating that delayed metabolism of arachidonic acid is damaging. In the
absence of more pharmacological studies, one can only
speculate as to possible effects of early arachidonic acid
increase.
On the basis of in vitro studies, the early rise in
arachidonic acid (200 mM) is more than adequate to account for the early inhibition of mitochondrial respiration.
Furthermore, at 500 mM, which is about twice the level
during and shortly after ischemia, arachidonic acid exposure causes profound swelling of cortical slices within 40
min, very probably as a result of free radical formation
(171, 175). It is possible that the rapid ECC seen in some
models is a result of such a process. However, it is more
surprising that such swelling is generally not seen in
ischemia despite the large increase in arachidonic acid.
Between 1 and 50 mM, arachidonic acid inhibits glutamate uptake on the Na1-coupled transporters. This
should increase ambient glutamate and may thus be damaging.
B) EFFECTS OF PAF. Platelet-activating factor may well
play an important role in ischemic damage (666). In the
gerbil, there is a ninefold increase in PAF during and right
after ischemia, which may persist (this has not been
measured). In the rat, there is a small increase in PAF in
a microdialysate during ischemia, but an extremely large
increase ;2 h after the end of 20-min 4-VO (901).
Platelet-activating factor antagonists strongly reduce
the increase in FFA that occurs after global ischemia in
whole tissue (869) and in mitochondria (1080), implying
that PAF causes these. Platelet-activating factor does rapidly increase cytosolic Ca21 about twofold in cultured
neurons and other cell types, by a currently unknown
mechanism (98, 661, 1269), but it is not clear that this
accounts for its very large FFA mobilization. In principle,
though, it could certainly enhance damage.
The PAF antagonist BN-52021 (25 mg/kg) modestly
reduced neuronal damage in rat hippocampus by ;20% 7
days after 10-min 2-VO (921) in a study in which temperature was well controlled. The antagonist strongly reduced infarct volume during permanent MCA occlusion,
with no effect on blood flow (97). It also strongly protected neuronal cultures against NMDA receptor-mediated glutamate toxicity (921).
These experiments are all consistent with the conclusion that PAF production may act to enhance fatty acid
production during ischemia, and possibly Ca21 accumulation, and in these two ways enhance the probability of
cell death. Platelet-activating factor may also activate
transcription factors. It activates expression of COX-2 in
cultured rabbit cornea where the effect is mediated by an
increase in cytosolic Ca21 (68), whereas in alveolar macrophages, the effect also requires lipopolysaccharides
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ently, it is not known which form of the enzyme is activated by ischemia. The Ca21-dependent form should be
elevated by the rapid rise in cytosolic Ca21 levels. The
Ca21-independent form of the enzyme is strongly activated in cardiac ischemia (436), and its inhibition is protective (988). The same could be occurring in brain, although the Ca21-independent form in cardiac tissue is
unique; it primarily targets plasmologens that are the
principal lipids of the cardiac sarcolemma.
C) DECREASED RESYNTHESIS OF PHOSPHOLIPID. During global
ischemia, the large rise in FFA is coincident with the large
fall in ATP (553), and it is possible that the FFA increase
occurs because resynthesis of phospholipid is blocked by
the lowered ATP (1084). This receives support from effects of intracerebrally injected CDP-choline, a precursor
for phosphatidylcholine synthesis. This very strongly attenuated the early appearance of FFA in the gerbil and rat
(268, 1133). Thus, at the least, phospholipid resynthesis
plays a major role in the net breakdown during and
shortly after ischemia.
The very delayed increase in FFA discussed above,
;1 day after temporary focal ischemia, is correlated with
a reduction in a major phospholipid-synthesizing enzyme,
lysophospholipid:acyl-CoA acyltransferase (1281). The
mechanism of the activity loss is not known, but it could
lead to a change in membrane properties.
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PETER LIPTON
5. Conclusions
Ischemia causes profound changes in phospholipid
metabolism with large increases in FFA in mitochondria
and in the rest of the cell. The bases for the increased
mobilization have not been completely determined but
very probably involve activation of PLC and PLA2 and also
decreased resynthesis of phospholipid due to low ATP.
Whether or not these changes in FFA and phospholipids help cause functional damage and cell death has not
been established to date. This is partly because there are
no satisfactory inhibitors, although informative studies
could still be carried out with the current crop, such as
PLA2 inhibitors and cyclooxygenase inhibitors. The protective effects of compounds that enhance phospholipid
synthesis suggest the importance of the phospholipid
breakdown, but the studies are not complete. The protective effects of PAF inhibitors, combined with their strong
inhibition of total and mitochondrial FFA production, also
suggest the importance of phospholipid changes, perhaps
in the mitochondria. However, PAF may be damaging at
the level of the genome rather than by mobilizing FFA.
Reasonable mechanisms of damage by net phospholipid breakdown include altered membrane function, free
radical generation, activation of calpain, or in the case of
PAF, genetic mechanisms.
Overall, there is good reason to think that the very
dramatic changes in phospholipid metabolism are important in ischemic cell death. However, studies have not yet
established this.
D. Long-Term and Short-Term Changes in Protein
Kinase and Phosphatases
Permanent, or long-term, inactivation of protein kinases or phosphatases could lead to initiation of apoptosis or could lead to the permanent alteration of proteins
involved in cell membrane or mitochondrial function, the
cytoskeleton or protein synthesis. Such effects could thus
make a major contribution to ischemic cell damage.
1. PKC
Five minutes of global ischemia in rat does not affect
overall PKC activity, nor does it cause substantial damage. However, 10-min ischemia causes damage and
causes major reductions in soluble and particulate enzyme activity in hippocampus (35), striatum (161, 1211),
and cortex (227). Decreases of ;30 –50% occur within
minutes or a few hours and persist for several days. At
present, the basis for this change in PKC activity is not
known. There is no measurable decrease in total PKC, and
there is a major shift of immunoreactivity from the cytosol to the membrane (161, 163).
Enzyme activities in focal ischemia have not been
reported; there is a small (10%) reduction in total PKC in
the ischemic core, measured as binding sites for phorbol
esters, 3 h after 90-min focal ischemia (797) but no reports
of activity changes.
There are no direct studies linking the very profound
PKC changes in global ischemia to ischemic cell death.
However, there is an important study linking such PKC
changes to glutamate-induced cell death in cultured neurons (283). Glutamate is not toxic in young cultures (,8
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(1114). It is not clear that this action is important in
ischemia.
C) EFFECTS OF CHANGES IN PHOSPHOLIPID CONTENT. I) Phosphoinositides. Although levels of triphosphoinositides
and diphosphoinositides in the cortex recover completely
after 5-min ischemia in gerbils, those in CA1 region of
hippocampus stay very low. They remain at 30 and 20% of
normal throughout the period during which there is delayed neuronal degeneration (4). The reason that levels
remain low is not clear, possibly a prolonged activation of
PLC, but the effect of the large decrease may be to prevent signaling mechanisms that rely on polyphosphoinositides (238). Some of these, for example, activation of
phosphatidylinositol 39-hydroxykinases, are critical in
preventing apoptosis in the face of neurotrophin withdrawal (232). Thus this phenomenon is potentially very
important in enhancing apoptotic death. More work is
needed here.
II) Phospholipids and membrane structure. There is
quite good evidence that depletion of membrane phospholipids, and presumed loss of membrane integrity, is a
major factor in ischemic damage to liver (314, 1069), but
this is not the case for brain where the loss in phospholipid content in nonmitochondrial membrane is very
small. However, when CDP-choline was given systemically for several days after 20- or 30-min 4-VO, it greatly
ameliorated neurological deficits measured 10 days later.
Neurological status was assessed on a 0 –5 scale (5
worst), and the CDP-choline improved the score from 2.5
to ,1 (516). Also, CDP-choline administration, when combined with a low dose of MK-801 which was not normally
protective, reduced infarct volume 7 days after 90-min
focal ischemia by ;50% (855). These protective effects
may be mediated by enhancing synthesis of phospholipid
in the brain, since the combination of choline and cytidine
does do this in striatal slices (992). If so, it would point to
the importance of phospholipid depletion in cell death
and, hence, the likely importance of membrane damage,
although the effect may be in an organelle other than the
plasmalemma (e.g., mitochondria). Again, more studies
are required to follow up these important possibilities.
III) Phospholipids and calpain. In liver cells, phospholipid breakdown very probably leads to activation of
calpain (37). This would be a very potent mechanism by
which the change in phospholipid metabolism could lead
to damage. There is no evidence yet as to whether this is
important in brain tissue.
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
1491
structure is the basis for the ischemic change in CaMKII
activity and immunoreactivity, it is notable that it is extremely long lasting.
As with PKC, CaMKII suffers long-term inhibition
during glutamate toxicity in cultured cells (199, 779), and
also following the application of A-23187 (779), suggesting
the inhibition is Ca21 mediated. Unlike PKC, there are, as
of yet, no studies that establish the importance of the
decrease in CaMKII in a cell death paradigm.
In this vein, it would be most interesting to know the
effects on cell viability of combining PKC and CaMKII
inhibition, as actually occurs after ischemia. The resultant
(presumed) reduced level of phosphorylation, possibly
localized to specific cell regions, could surely have profound effects.
2. CaMKII
3. Other protein kinases
There is also a profound, rapid, and prolonged decrease in CaMKII following ischemia. Activity actually
increases about fourfold after 2-min ischemia (1273) but
is reduced by ;50% in both particulate and cytosolic
fractions, at the end of 5-min global ischemia in rat, and
stays reduced for at least 24 h. Ten minutes of ischemia
reduces activity by ;70% (35, 447, 1204). Twenty minutes
of 4-VO led to equal falls in activity in CA1, CA3, and
dentate gyrus 12 h after ischemia. There was partial recovery in the two nonvulnerable regions but no recovery
over the next days in CA1 (780).
Ca21/calmodulin-dependent protein kinase II was
also strongly inactivated in both the core and the penumbra during focal ischemia. This occurred within 5 min and
was maintained throughout ischemia. After reperfusion,
activity was restored when ischemia was short (5 min)
but not if ischemia was longer (30 and 60 min) (418).
Thus, in both global and focal ischemia, there is a very
good correlation between permanent inhibition of
CaMKII and cell death.
As for PKC, the mechanism of the activity decrease is
not yet known. After global ischemia, there is a major
decrease in immunoreactivity (200, 469, 780) and a
marked shift of remaining immunoreactivity from the cytosol to the particulate fraction of the tissue (1019), as
with PKC. The shift is largely to the postsynaptic density
(469, 600). This persists in CA1 (469). Although there may
be a small decrease in the total amount of CaMKII, that is
not the major reason for the loss of activity, or immunoreactivity (200, 469). Thus the loss of activity and movements must reflect structural changes in the kinase (200,
1019). The changes are accompanied by a large decrease
in ATP affinity and in the ability of an ATP analog to bind
to the enzyme, indicating a structural change in the ATP
binding region of the protein (200). This conclusion is
supported by phosphopeptide mapping, which reveals regions in CaMKII from ischemic tissue that become inaccessible to autophosphorylation (1019). If this change in
In experiments where PKC and CaMKII were both
strongly inactivated by ischemia there was no change in
PKA (35).
However, there does appear to be an important
downregulation of tyrosine kinase activity. After a 10-min
global insult in rat, basal tyrosine phosphorylation was
upregulated in nonvulnerable regions but was the same or
was downregulated in vulnerable regions (468), a pattern
that persisted for at least 24 h. The same microanatomy
pertained for casein kinase II. The activity decreased by
25–50% in CA1 of hippocampus and striatum within 1 h of
global ischemia while activity increased in nonvulnerable
regions, CA3 and neocortex, within 15 min and remained
elevated (467).
Thus two kinase systems whose level of phosphorylation reflect trophic effects on cells are significantly
lower in vulnerable regions of the brain after global ischemia. This is certainly consistent with a role in damage.
4. Calcineurin
As measured immunohistochemically, the calmodulin-dependent phosphatase begins to decay in hippocampal CA1 somata and dendrites ;1.5 days after 20 min of
4-VO. This is before the major phase of cell death, which
occurs ;12 h later (780). The ramifications of this are not
obvious at present.
5. Short-term activation of protein kinases and
cell death
The long-term effects of protein kinase decreases,
which may well be damaging, need to be contrasted with
acute effects, determined by introducing inhibitors of the
kinases just before an insult. Kinase activation is damaging in this early phase.
Inhibition of tyrosine kinases (571) strongly protected against 5-min global ischemia in gerbil; inhibition
of PKC with staurosporine strongly protected against
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cell divisions) but is toxic in older cultures. The differential toxicity was not related to differences in glutamate
effects on cytosolic Ca21. However, it was strongly correlated with an absence of PKC inhibition in the younger
cultures. Protein kinase C was inhibited by 50% in older
cultures in a Ca21-dependent manner. However, it was
not blocked in young ones. Furthermore, when excitatory
amino acids were combined with artifactual inhibition of
PKC in these younger cultures, there was major cell
death. Thus it was concluded that the combination of a
Ca21 increase and PKC inactivation was necessary, and
sufficient, for cell death (283). This ascribes a key role in
damage to the PKC inhibition, and it is anticipated that
similar studies will be carried out using ischemia as the
insult.
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PETER LIPTON
6. Conclusions
Almost all of the protein kinases show activity decreases after global ischemia, whose timing and localization are consistent with their importance in cell death.
The mechanisms of the kinase activity decreases are
not currently known. Although direct evidence for involvement in cell death is lacking, there is good evidence
that a similar decrease in PKC is damaging in excitotoxicity. Certainly, the ability of staurosporine to induce
apoptosis is consistent with the possibility that inhibition
of multiple kinases may lead to apoptosis, but it is not
firmly established that this staurosporine effect is mediated by its inhibition of kinases. Knowing the effects of
prolonged multiple kinase inhibitions or multiple kinase
knockouts on survival of cells in normoxic conditions
would help determine whether the ischemic kinase
changes mediate cell death.
If the kinase downregulation is important, then it
becomes very important to understand why this occurs,
including what causes it, and what the changes in the
kinases are. Regarding the former, it would be very interesting to know the involvement of free radicals, calpain,
or phospholipid breakdown in the loss of activity.
E. Summary
Several potential perpetrators of the molecular
changes causing cell death have been considered in this
section. On the basis of measured changes and effects of
pharmacological agents, free radicals and calpain must be
considered very likely mediators of cell death in temporary focal and global ischemia models, although there are
important caveats, which were pointed out in the summaries at the end of each section. The most important one
for free radicals is that most “scavenger studies” do not
measure or correlate protective effects with changes in
free radical production. For calpain, the most important
caveat is the paucity of studies and the important question
of inhibitor specificities. In both cases, whether temperature is controlled well in all the studies and for long
enough is also of some concern. Free radicals also appear
to be important in permanent focal ischemia, but there
are no studies of effects of calpain inhibition on that
insult. Phospholipid and protein kinase changes are large
and a priori seem likely to contribute to cell death. However, there are no really solid pharmacological or knockout studies implicating them as of yet.
The fact that inhibition of either calpain, or free
radical accumulation, are (very probably) strongly protective in all models implies that both calpain activation and
free radical generation are necessary for damage. This
receives (very indirect but intriguing) support from the
apparent coincident activation of both these variables ;1
day after global ischemia. The basis for this could be
either that two or more independent macromolecular
changes are required to produce the critical functional
changes (say in mitochondria) or that free radicals and
calpain are synergistic at the molecular level. The fact
that free radical action tends to inhibit calpain (402)
argues against the latter, but free radical sensitization of
the Na1-K1-ATPase to calpain sets a precedent for such
synergism (1307). The combined effects of calpain inhibition and free radical scavengers might be very protective.
VI. ACTIVATORS OF PROCESSES CAUSING
FUNCTIONAL CHANGES
This section considers ionic and molecular changes
that may be activating the damaging processes discussed
in section V.
A. Ca21
Increases in Ca21 concentration in appropriate compartments can effect all the changes that were discussed
in section V. The ion has generally been considered to be
a major effector of necrotic cell death during ischemia
(614), and this has received a major boost from the fact
that Ca21 very clearly plays a critical role in the normal
cell death caused by ischemia in cell culture systems
(376). The goal of this section is to critically evaluate the
evidence that Ca21 also plays a crucial role in vivo. Although the evidence is consistent with such a role, there
is little solid demonstration of it.
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5-min ischemia in the gerbil and 20-min 4-VO in the rat
(425), and inhibition of PKC and CaMKII during the insult
protected against ischemia in cultured neurons (409, 705).
These studies imply that there is early activation of
the protein kinases, as has been explicitly shown for
tyrosine kinase phosphorylation of MAP2 (157) via MAP
kinase kinase (MEK), CaMKII as discussed above (1273),
as well as others (1257), and that these are important in
initiating damage. The targets of these kinases are not yet
established. Some of these include activation of PLA2,
which will activate PAF synthesis (907), enhancement of
Ca21 entry through NMDA receptors (691), and, in the
case of tyrosine kinase, ischemic release of glutamate; the
latter is strongly inhibited by genistein during global ischemia (907). The mechanism of this effect may well be
MAP kinase phosphorylation of synapsin. Inhibition of
MEK with PD-98059 blocks ischemia-induced neural injury in culture and coincidentally blocks glutamate release as well as phosphorylation of synapsin (924).
Gene activation may also be an important mediator
of the early kinase activations; this has not yet been
determined.
Volume 79
CEREBRAL ISCHEMIA ON NEURONS
October 1999
1. Changes in cell Ca21 during ischemia
21
A) INCREASES IN TOTAL CELL CA
fura 2 (803). The increase was not quantified. The large
increase noted by Silver and Erecinska (1048) also occurs
in slices where, using a low-affinity Ca21 dye in a dual
wavelength mode, a rapid increase from 60 nM to 24 mM
was shown (397).
Cytosolic Ca21 was also measured in cortex during 60
min of MCA occlusion in the rat, using surface fluorimetry of
indo 1 (1151). Unfortunately, although the authors described
how they corrected for NADH and reflectance changes, no
actual data were given, and others who tried to reproduce
these results after making appropriate corrections were unable to show any 400/506-nm fluorescence changes when
Ca21 moved into the cell (660). It was noted that accurate
measurements were very difficult because indo fluorescence
was smaller than the basal fluorescence of the tissue and
also because the method for correcting the NADH fluorescence changes was not accurate enough. In the absence of
data showing the NADH and indo fluorescences, it must be
considered that the earlier studies may have been contaminated by artifact and thus, unfortunately, they must be regarded as questionable at this stage. This is unfortunate
since they are the only studies of cytosolic Ca21 during focal
ischemia.
The only other measurement of cytosolic Ca21 is
indirect but useful; the loss of tissue staining by a calmodulin antibody that binds only to calmodulin that is not
complexed with Ca21 was measured (242). Loss of staining corresponds to increased cytosolic Ca21, and there
was a near-maximal decrease of staining within 2 h of
starting focal ischemia in the core of the lesion and within
4 h in the penumbra, qualitatively indicating increases in
cytosolic Ca21 that occurred more rapidly in focus than
penumbra.
There is an increase in cytosolic Ca21 during in vitro
ischemia in gerbil (765, 769) and rat (95, 1288) hippocampal slices, measured by fluorimetric dyes. In the one case,
where actual concentrations were measured, using fura 2
and correcting for NADH fluorescence, cytosolic Ca21
rose from 180 to 600 nM after 5-min anoxia and to 1.5,
then 1.8 mM after 10 and 15 min (95). As pointed out,
when cytosolic Ca21 was measured with a low-affinity
dye, it rose to 24 mM. The higher values are more accurate
(476).
It has been difficult to measure effects in culture, but
there is now a perfusion setup that allows deoxygenation
of the culture medium at the same time as direct optical
studies (1108). Early studies show an increase to 1 mM
within 10 min in neuronal cultures.
C) SUMMARY. The only reliable measure of cytosolic
Ca21 in vivo during ischemia shows a rapid increase to 30
mM during global ischemia. Such increases are large and
would clearly activate all Ca21-dependent processes discussed, including M-calpain. However, there is a paucity
of studies, and more are quite urgently needed, particularly in focal ischemia where change in calmodulin binding is the only semidirect measure. Indirect evidence,
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. There are large increases in total intracellular Ca during ischemia in all
systems.
During global ischemia, extracellular Ca21 abruptly
falls to ;0.1 mM at the time of the anoxic depolarization,
60 –90 s after the start of ischemia (85, 1040, 1231). The
Ca21 is moving into the intracellular space and, with the
assumption the extracellular space is ;20% of total tissue
volume, the calculated rise in cell Ca21 is ;250 mM, about
a 25% increase in total cell Ca21 (532).
During focal ischemia, extracellular Ca21 decreases
to ;0.1 mM for at least 2 h in the core after ;10 min (430,
612). This occurs at a flow rates of ;15 ml z 100 g21 z
min21. In the penumbra, where flow is much higher,
extracellular Ca21 decreases are limited to the times of
the intraischemic depolarizations (612) and are thus very
much less than in the core. When studied over the course
of permanent 24-h focal ischemia, total Ca21 in the core of
the lesion gradually increased and was far greater than in
the penumbra (570, 937). In one case, total Ca21 in the
core rose at ;1 mM/h from the beginning of the insult
until ;24 h (937). It is not clear whether accumulation
preceded or followed cell death because the latter was
also increasing over this time. However, the earliest increases, at 2 h, are before any significant level of cell
death. This slow increase reached very profound levels
(;15 times control levels at 24 h).
There are decreases in extracellular Ca21 and increases in intracellular Ca21 during in vitro ischemia in
cortical and hippocampal slices that occur within the first
few minutes (6, 611, 686). There are quite delayed increases of Ca21 in neuronal cell cultures (376). The rise in
cultures may be delayed because the ATP fall is small,
only 30% after 60 min (see Table 1), or because the
cultures are from embryonic rat tissue (533). The changes
in slices follow similar kinetics and have similar properties to those in global insults (see below) and so are better
models for this phenomenon.
21
B) INCREASES IN CYTOSOLIC CA . A critical question in
terms of damage is the level to which cytosolic Ca21 rises
during and after ischemia. Unfortunately, there are only
two reported studies of cytosolic Ca21 in vivo, and in one
of these the technology is not reliable.
In an important study, Silver and Erecinska (1048)
used Ca21 microelectrodes and recorded very large increases in cytosolic Ca21 in CA1 and CA3 hippocampal
neurons during 4-VO or compression ischemia in the rat
(1048). There was a small, 10 –30 nM, increase within the
first minute of ischemia (1047), and within the next 2–3
min, cytosolic Ca21 rose from ;90 nM to 30 mM. This
increase seems very large, but the fact that it was delayed
by MK-801 tends to exclude artifact (1047, 1048). A delayed increase in cytosolic Ca21 of CA1 pyramidal cells
was also measured with dual-wavelength excitiation of
21
1493
1494
PETER LIPTON
such as spectrin breakdown, MAP2 breakdown, and NOS
activation, certainly strongly suggests an increase in cytosolic Ca21 during focal ischemia. However, there are
other possible explanations for each of these events, so
the conclusion is not firm.
Thus, although total cell Ca21 surely increases during
ischemia, more data are required to confirm the increase
in cytosolic Ca21, particularly in focal ischemia, and to
estimate its magnitude.
2. Mechanisms of Ca21 increases during ischemia
Na1 channel blocker lidocaine (10 mM) and the nonNMDA glutamate blocker CNQX (10 mM). Thus some of
the Ca21 that enters the cell during ischemia is taken up
by mitochondria, and this uptake is normally attenuated
by the efflux of Ca21 on 2Na1/Ca21 exchanger, which is
activated by the entering Na1 (1289). Thus the entering
Na1 effectively acts to increase cytosolic Ca21 during
ischemia. Whether or not this occurs in vivo is not known.
It ascribes an important role to entering Na1, and such a
role is evidenced in vivo by the very strong protection
afforded by Na1 channel blockers (see sect. VIIC).
Two other features emerge from the measurements
of cytosolic Ca21. There is fairly strong evidence that
Ca21-induced Ca21 release (CICR) from ER contributes
to the rise in cytosolic Ca21. Dantrolene, the specific
blocker of CICR, has the same effect as NMDA blockers in
ischemic hippocampal slices (1288) and in cultures
treated with cyanide (279). In vivo the evidence for CICR
is indirect in that dantrolene significantly protects gerbil
hippocampus against damage (1196). The in vitro studies
suggest that effects of NMDA-mediated Ca21 entry on
cytosolic Ca21 are greatly amplified by CICR. Such amplification occurs after Ca21 entry caused by high-frequency stimulation (14). The same amplification probably
occurs in vivo and accounts for the protective effect of
dantrolene.
The other feature of the results is the rapid inactivation of the NMDA receptors during global ischemia and
during in vitro ischemia in the slice. This is probably due
to dephosphorylation, since it was eliminated by okadaic
acid in the slice (686). The dephosphorylation is probably
a consequence of the precipitous ATP fall (696) and may
well explain the relative unimportance of NMDA-mediated Ca21 entry in global ischemic damage (see below).
Damage would presumably be greater, and more NMDA
dependent, if the receptors did not inactivate. Adenosine
59-triphosphate does not fall nearly as much in the penumbra of focal lesions, and this probably explains the far
greater importance of NMDA receptors in that insult,
although inactivation has not been tested in any way.
21
A) INTRACELLULAR LOCALIZATION OF ENTERING CA . Even if
21
cytosolic Ca
rises to 30 mM, most of the Ca21 that
enters the cells during ischemia will not be free but will be
on binding proteins or in organelles. The total initial
increase during global ischemia and in the ischemic core
is ;250 mM, and levels appear to rise continually in the
core. The amount on cytoplasmic binding proteins can
only be very roughly estimated because their dissociation
constants and concentrations are not at all precisely
known. The approximate concentration of Ca21 binding
sites in the cytoplasm is ;200 mM and the average dissociation constant of these proteins is 1–10 mM (50, 1217,
1224). Thus, if cytosolic Ca21 rises to 20 –30 mM, then the
proteins will be largely saturated so they will contain
;200 mM Ca21. A significant portion of the entering Ca21
will locate on nuclear binding proteins (917) as the free
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Cytosolic Ca21 could rise as a result of a net entry of
Ca across the plasmalemma or, transiently, due to liberation of Ca21 from intracellular stores.
In vivo the early increase in total cell Ca21 during
global ischemia is via NMDA receptors. D-2-Amino-5-phosphonovalerate (D-APV) reduced the fall in Ca21 in the
extracellular space during global ischemia by ;60 –70%
and slowed the fall so it took ;8 min (85). In accord with
this, the early increase in cytosolic Ca21 during the first 3
min of global ischemia was largely blocked by either
MK-801 or ketamine. However, by the end of 8-min ischemia, Ca21 levels were the same whether or not the
NMDA receptors were blocked (1047, 1048). Thus there is
an early influx of Ca21 via NMDA receptors that ceases
and that is replaced by influx through other pathways, or
release from intracellular stores. L-channel blockers did
not affect the increase in cytosolic Ca21, showing that the
latter does not result from Ca21 entry via those channels.
In vitro systems are far more accessible and provide
more information. In cortical cell cultures at least 80% of
the bulk Ca21 entry during in vitro ischemia is prevented
by NMDA blockers (376) (as is the increased cytosolic
Ca21, Refs. 884, 1199), and the Ca21 influx occurs at the
same time as the increase in extracellular glutamate
(376). Thus Ca21 entry and increased cytosolic Ca21 are
very largely mediated by NMDA receptors.
In the hippocampal slice, influx of Ca21 during the
first 2.5 min of in vitro ischemia is via NMDA receptors,
but these appear to inactivate after this time (686). Influx
during the next 2.5 min is via L channels (25%) and
Na1/Ca21 exchange (35%) and via an unidentified pathway (686). The very early rise in cytosolic Ca21, measured
using the high-affinity dye calcium-green I, was blocked
by MK-801, as expected from the total Ca21 measurements. Also, as in vivo, the rise in cytosolic Ca21 after the
first few minutes was unaffected by MK-801, and the final
level of cytosolic Ca21 after 10-min ischemia was independent of NMDA receptors (1289).
The later sustained cytosolic Ca21 increase in slices
was greatly attenuated when mitochondrial Ca21 efflux
was blocked by a novel inhibitor of the mitochondrial
Na1/Ca21 exchanger, CGP-37157 (10 mM) (1289). This
Ca21 efflux depends on Na1 influx during ischemia because the cytosolic Ca21 rise was similarly blocked by the
21
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
3. Postischemic changes in Ca21
There is an eclectic body of evidence that cytosolic
Ca21 levels are elevated in the postischemic period. This
could be very important in development of damage.
A) GLOBAL ISCHEMIA. There is a large increase in total
Ca21 in the vulnerable medial-CA1 region of the gerbil
hippocampus immediately after 5-min global ischemia.
This returns to normal by 30 min, although this area is
subsequently badly damaged (802). A small, 40%, elevation in free Ca21 has been noted 2– 8 h after 8-min global
ischemia in the rat, but these are very difficult measurements (1048).
Six to 24 h after 2-VO in rats there is a threefold
enhancement of the Ca21 entry from the extracellular
space during burst (15 Hz) firing (22), which is mediated
by NMDA receptors. Such firing is fairly normal for hippocampal cells, so a significant increase in cytosolic Ca21
may well occur. The result suggests prolonged hyperacti-
vation of NMDA receptors. This is supported by our studies of MAP2 proteolysis (Zhang and Lipton, unpublished
data) that show continual Ca21-dependent breakdown in
the postischemic period that is mediated by NMDA receptors. It would be of interest to see if very delayed application of MK-801 would attenuate cell death. The drug
generally does not do so if administered in the immediate
postischemic period (327, 819, 1023).
There is excess mitochondrial Ca21 accumulation
6 –24 h after global ischemia in gerbil (285) and rat (1272)
hippocampus and also during the first 6 h in gerbil (846).
The most straightforward explanation is that this reflects
increased cytosolic Ca21 leading to greater uptake via the
Ca21 uniporter, whose mean affinity constant is in the
micromolar range (400). This is suggested by the qualitative observation of elevated numbers of Ca21 deposits in
the cytoplasm between 2 and 6 h after ischemia (846). No
measurements were made after 6 h in that study. The
NMDA, AMPA, or voltage-dependent Ca21 channel dependencies of the elevated postischemic Ca21 have not been
checked.
B) FOCAL ISCHEMIA. There is a persistent elevation of
cytosolic Ca21, measured by antibody binding to calmodulin, out to 24 h after 1-to 2-h focal ischemia in the rat
(242), implying a prolonged elevation of cytosolic Ca21.
This is supported by measurements of total Ca21 levels
using 45Ca21. Calcium levels remain two- to threefold
elevated in the core throughout a 24-h reperfusion period,
despite some extrusion upon reoxygenation, and they are
elevated, but not as much, in the penumbra. In the latter
case, the increase does not occur until 6 h of reperfusion
(612).
The studies described in this section are almost anecdotal because they have not been followed up in a
satisfactory way. Nonetheless, they highlight the real possibility that elevated cytosolic Ca21 in the postischemic
period may contribute to damage. Coupled with the observation of delayed calpain activation in CA1 of gerbil,
described in section VB, they certainly become important.
4. Role of increased cytosolic Ca21 in ischemic
cell death
A) CELL CULTURES. N-methyl-D-aspartate antagonists almost completely block Ca21 entry and almost fully protect against anoxic/ischemic and CN-induced damage in
neuronal cultures, arguing that Ca21 entry, and thus probably cytosolic Ca21, is critical for damage (237, 376, 405,
603, 884). Although this may well be, it is notable that in
vitro ischemia induces cell death in zero Ca21 buffer
(377), and this is also NMDA dependent (376). It is dependent on extracellular Na1, showing that NMDA receptors
can mediate cell death via Na1 entry. The speed of that
process, and the fact that it is accompanied by more cell
swelling than normal, suggests that it proceeds by a different mechanism from the cell death in normal buffer
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nuclear Ca21 is likely to equilibrate (854), or nearly equilibrate (16), with the free Ca21 in the cytosol.
There may also be Ca21 accumulation in the ER and
mitochondria; recent studies show uptake into mitochondria even during ischemia in the hippocampal slice (1110).
Although the importance of NOS activation, and very
probable effects on other cytosolic proteins such as calpain and phospholipases, suggests that the rise in cytosolic Ca21 is important, changes in other structures may
also be important, for example, in activating genes and in
affecting mitochondrial integrity.
B) SUMMARY. There is very little known about mechanisms of Ca21 entry or accumulation in focal ischemia,
although there is almost undoubtedly significant entry in
the penumbra during the intraischemic depolarizations.
This has not been explicitly shown.
The early entry of Ca21 and buildup of cytosolic Ca21
in global ischemia is probably due to Ca21 entry via
NMDA receptors and its effect in eliciting CICR (14).
N-methyl-D-aspartate receptors appear to inactivate
within a few minutes, probably due to the profound ATP
decrease and resulting receptor dephosphorylation (686).
The steady-state increase in cytosolic Ca21 during ischemia, in slices, is largely due to Na1 entry and resultant
activation of 2Na1/Ca21 transporter-mediated efflux from
the mitochondria. This may pertain in vivo also, although
it has not been studied. An important unknown is whether
an augmented influx pathway contributes to the maintained cytosolic Ca21 after the NMDA receptors are inactivated. There is entry via L channels and plasmalemma
3Na1/Ca21 exchange in the slice, but these do not appear
to contribute to the elevated cytosolic Ca21 (1047, 1048,
1289). The maintained elevation could result simply from
the Na1-induced Ca21 release from the mitochondria coupled with somewhat impaired removal of Ca21 across the
plasmalemma due to lowered Ca21-ATPase.
1495
1496
PETER LIPTON
Ca21 entry does not contribute very much to cell Ca21
increases, especially after 2–3 min. Other sources of Ca21
may be leading to damage when the NMDA receptors are
blocked (see sect. VIB). Alternatively, because MK-801 and
other blockers of NMDA actually cause neuronal damage
in normoxia (299) and ischemia (1037), there could be a
damaging effect that is countering a protective effect.
However, the lack of protection does prevent using these
data as evidence that Ca21 is important.
II) Voltage-dependent Ca21 channels. Voltage-dependent Ca21 channel blockers offer significant protection
against global damage, without affecting postischemic
blood flow. Nimodipine (781, 842) and flunarizine (252)
protected against damage in the gerbil, with optimal protection occurring when the drug was present before or
right after the ischemia and for a long period after the
ischemia. Fifty to one hundred percent protection was
achieved, the latter when treatment was continued for
24 h. Although these results are impressive, they are
somewhat flawed because in those early studies temperature was not controlled, nor were effects of the blockers
on temperature measured. The N-channel blocker SNX111 (v-conotoxin MVIIA) (139, 1158) was protective when
added up to 24 h after 10- to 15-min 4-VO in the rat,
preventing ;50% of the damage. This was very probably
not due to prevention of glutamate release, since a conotoxin that was much more potent against glutamate release did not protect against damage. Core temperature
was controlled for 6 h after ischemia or drug treatment,
which was far better than in most studies, and was very
probably adequate.
If temperature depression was not a basis for the protection with the channel blockers, then the data suggest that
either there is a postischemic opening or upregulation of L
and (postsynaptic) N channels or that sensitivities of intracellular processes are altered so that normal Ca21 entry via
the L or N channels is damaging. The apparent involvement
of both channels suggests the latter may be the case. Alternatively, prolonged opening of both channels could help
account for the increased cell Ca21 in the postischemic
period. The issue is not yet resolved, but in either case, using
L and N channel blockers together should enhance protection; this should be tried.
III) Ca21 from intracellular sources. As described
previously, dantrolene provided significant protection
against cell death in gerbil global ischemia, in studies
where temperature was very well controlled (1196). There
are no known sites of dantrolene action aside from blocking Ca21 release from the ER (1196), so this result is
reasonable evidence that such Ca21 is damaging during
global ischemia.
As discussed in section VIIC, blockade of Na1 fluxes
is very protective in global ischemia. This might reflect
damage caused by Na1-induced release of Ca21 from the
mitochondria as seen in the hippocampal slice. There is
currently no evidence for this, but it could be tested by
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(376), and zero-Ca21 incubation has been shown to sensitize other tissues to free radical damage (315, 881). Thus
it is generally concluded that the normal ischemic damage
is Ca21 mediated and that the processes in zero Ca21 do
not occur when extracellular Ca21 is normal. This is
probably correct.
Neuronal cell death in organotypic cultures is partly
dependent on NMDA receptor activation (5, 923, 1073),
implying a Ca21 dependence as above. Successfully loading cells with the Ca21 chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-ethanesulfonic acid attenuated
damage. However, this was very probably due to blocking
glutamate release presynaptically rather than preventing
the cytosolic Ca21 increase in the pyramidal cells themselves (5). Thus it does not particularly support a direct
role for Ca21 in cell damage.
Another way to determine whether Ca21 is important in
damage is to determine whether Ca21-dependent processes
are involved. Consistent with the importance of Ca21, calpain and calmodulin antagonists both substantially, but not
completely, protect against ischemic damage 24 h after 30min ischemia in organotypic cultures (1085, 1190), and as
described previously, so do inhibitors of NOS, which is very
probably activated by Ca21/calmodulin.
Taken together, these studies present a good case for
the involvement of cytosolic Ca21 in cell death in cultures, although rigorous studies in which intracellular
buffers were used to show that cytosolic Ca21 and damage were causally related would be most persuasive.
21
B) IN VIVO: GLOBAL ISCHEMIA. I) NMDA. The Ca
mea21
surements show that NMDA-mediated Ca
entry accounts for the early increase in cytosolic Ca21 in rat;
measurements have not been made in the gerbil. The
actions of NMDA antagonists on cell death are considered
in some detail in section VIC because there are major
artifact problems, in particular a very large and prolonged
(12– 48 h) hypothermic effect. When these are taken into
account, as they have been in very careful studies on
gerbil (454, 768) and rat (99, 135, 819, 1089), it appears
that MK-801 partially protects (;50%) against 5-min ischemia in the gerbil, providing the temperature is 37°C or
below. It does not protect if temperature is 38.5°C or
above (136, 454). However, even here, as discussed (454),
it is quite possible that protection is not due to block of
Ca21 entry because very high doses of MK-801 (10 mg/kg)
had to be used to get reliable protection. A dose of 3
mg/kg generally gives maximal MK-801 effects on Ca21.
Thus the protection may be mediated by blockade of Na1
channels (414), leaving no positive evidence that Ca21
entry is important in damage.
MK-801 does not appear to protect against 5- or 15min ischemia in rat (99, 819, 1158), except in a 2-VO model
in which hippocampal blood flow is only reduced to 20%
(rather than the usual 1–3%) (1089).
The lack of protection in no way eliminates the possibility that Ca21 is important in damage; NMDA-mediated
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CEREBRAL ISCHEMIA ON NEURONS
whether protection results from increased blood flow.
The results are not clear cut. There were no effects of
nimodipine on blood flow during temporary ischemia
(1150), but the drug caused a 50% hyperperfusion during
the postischemic period. The latter is probably not important because blood flow does not appear to be limiting in
the postischemic phase, but the possibility cannot be
ruled out. Nimodipine does not affect blood flow for at
least several hours if it is injected right after the onset of
ischemia (87, 262). However, it did strongly increase
blood flow to the core when it was preperfused for 2 h
(501), and this may account for the protection seen in the
three reported studies of permanent focal ischemia (96,
500, 1166).
The N-channel Ca21 blocker SNX-111 reduced infarct
volume, after 4-h permanent focal ischemia, by ;65%
(1103) and also reduced infarct by 45% after temporary
ischemia. This is a strong effect that is not mediated by
effects on blood flow, and it may well reflect blockade of
damaging Ca21 entry into the neurons as receptors are
present at significant concentrations on dendrites (761).
However, protection may also have resulted from reducing glutamate release (1103).
III) Summary. The protective effects of NMDA antagonists and Ca21 channel blockers are the only direct
tests of the role of Ca21 in damage. Thus they are very
important. The results are consistent with damaging Ca21
entry via NMDA receptors as well as L and N channels,
and hence, the conclusion that Ca21 is damaging. However, there are alternate explanations for the results that
cannot be discounted as well as instances where NMDA
antagonists are not protective. As discussed in section
VIC, the weight of the data indicates that the protective
effect of NMDA antagonists is not due to increased blood
flow, suggesting that Ca21 entering cells via NMDA receptors is indeed damaging. However, this is not completely
certain.
The involvement of NOS activation and calpain in
focal ischemic damage evidenced by mutational and pharmacological studies are both good evidence for the involvement of Ca21, although here too there are alternate
explanations. Calpain can be activated at normal cell
Ca21 levels by phospholipid breakdown and by activator
proteins. Although no Ca21-independent mechanisms for
nNOS activation have been demonstrated to date (505,
690), the enzyme is induced within 4 h of the onset of
focal ischemia (1294), and this could account for its toxicity. There are other possible mechanisms such as regulation by palmitoylation (505) and by removal of an endogenous inhibitor (506), but there is no serious evidence
for these.
The calmodulin antagonist trifluoperazine reduced
the lesion size in Wistar rats by 87% 24 h after 2-h temporary focal ischemia. This is the largest protective effect of
any drug that has been reported. The drug had no effects
on blood flow in the postischemic period (626). Unfortu-
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examining effects of the mitochondrial 2Na1/Ca21 exchange blocker CGP37157 on global ischemic damage.
IV) Summary. There is equivocal evidence, because
of the high MK-801 dose needed, that NMDA-mediated
Ca21 entry contributes to damage in gerbil (but no evidence in rat) and reasonable evidence that this may be
mediated by resulting CICR from ER. It is speculated that
Na1-induced Ca21 release from mitochondria might be
damaging and would explain the very potent effects of
Na1 channel blockers. The most persuasive evidence in
favor of Ca21 is that postischemic Ca21 entry via voltagedependent channels contributes to damage in both rat and
gerbil, providing effects of blockers were not contaminated by temperature artifact.
Reasonably good evidence for the importance of Ca21
comes from the role of Ca21-dependent processes in cell
death. The only calpain inhibitor studied has been leupeptin,
which is not at all specific for calpain over cathepsins or
proteasomes. Damage was strongly attenuated in the mouse
NOS knockout, and when a specific nNOS inhibitor was
used in rat, implicating NOS and hence Ca21. As discussed
in sections VB and VA, respectively, both calpain and NOS
could be activated by events other than increased Ca21.
21
C) IN VIVO: FOCAL ISCHEMIA. The evidence that Ca
is
important in damage is stronger for focal insults than it is
for global insults.
I) NMDA. Protective effects of NMDA blockade are
clear in both temporary and permanent ischemia (334,
782, 1102), providing temperature is maintained at 37°C or
below (753). This effect, which is limited to reducing the
size of the penumbra, is very robust, and both competitive
and noncompetitive antagonists produce a 30 –50% reduction in total infarct size. Unlike in global ischemia, NMDA
antagonists do not cause a postischemic temperature fall.
If antagonists are protecting by blocking Ca21 entry, the
results strongly implicate increased cell Ca21 in damage.
There are, though, other possible modes of action,
which are considered further in section VIC. The most
serious artifact is that NMDA antagonists may be protective by increasing blood flow rather than by reducing Ca21
entry (141, 1102), although the weight of evidence argues
against this.
In addition, there are focal models (thrombotic lesions or spontaneously hypertensive rats) in which
NMDA blockade is not effective at all (141, 970, 1102,
1234). Perhaps ATP levels fall further in these cases,
making the situation akin to global ischemia. At the least,
these data fail to support a role for Ca21 in damage in this
model. The absence of protection by MK-801 is rather
important clinically because so many human strokes are
thrombotic. It may help account for the poor performance
of NMDA antagonists in clinical trials, to date.
II) Effects of Ca21 channel blockers. L-channel blockers are protective against transient (156, 1150) and permanent (96, 500, 1166) focal lesions, reducing measured
infarct sizes by ;50% in all cases. A critical issue here is
1497
1498
PETER LIPTON
nately, the drug is not specific to calmodulin; it affects
Ca21 channels for example. However, calmodulin or another Ca21-dependent process is a very likely site of
action, so the result does provide strong evidence for the
importance of Ca21 in damage.
5. Possible mechanisms by which increased cytosolic
Ca21 causes cell death
6. Conclusions
A) EVALUATION OF EVIDENCE. Although the conclusion
that Ca21 is critical to ischemic cell damage is widely
accepted (615), one of the aims of this section was to
highlight the quite large uncertainties that remain as to
whether this is indeed so for in vivo ischemia.
There is undoubtedly Ca21 accumulation in brain
cells during global and focal ischemia. Certainly, it is
likely that this will lead to increased cytosolic Ca21 as it
does in slices. However, there are only two studies showing an increase in cytosolic Ca21 during global ischemia
and only one, indirectly, indicating an increase in cytosolic Ca21 during focal ischemia. Furthermore, only one of
the three studies is quantitative. There are several studies
showing increased total cell Ca21 during the 24 h after
global and focal ischemia, and it is reasonable to infer that
there is also increased cytosolic Ca21, despite the lack of
measurements.
A major way to demonstrate involvement of increased cytosolic Ca21 or total Ca21 in damage is to test
effects of blocking the increase. Unfortunately, most
drugs that should block Ca21 buildup either have very
small effects (NMDA blockers in global ischemia) or have
other potentially interfering effects. At this stage, the
most reliable pharmacological evidence is the attenuation
of damage by L- and N-type Ca21 channel blockers in the
period after global ischemia, indicating the importance of
postischemic Ca21, and also the prevention of damage by
NMDA blockers added during or after focal ischemia,
indicating the importance of Ca21 during permanent focal
ischemia and after temporary focal ischemia. There is,
though, a small but real possibility that these blockers act
by increasing blood flow (see sect. VIC). Another apparently discrepant result is that calbindin knockout mice
show much reduced cell damage after 12-min global ischemia. Thus eliminating a principal cytoplasmic Ca21 binding protein improves outcome. No other common Ca21binding proteins were upregulated in the knockouts so
that it seems that reducing immediate Ca21 buffering
reduces damage (589). This could be explained by more
rapid clearance of cytosolic Ca21 following the ischemia,
but it also may indicate that increased free Ca21 during
ischemia is not harmful. More detailed studies need to be
done.
The other way to demonstrate the importance of
Ca21 is to show that processes activated by increased
cytosolic Ca21 are involved in cell death. In this regard,
the very clear dependence of damage on NOS and the
almost certain dependency on calpain in focal ischemia
and global ischemia are very strong indicators of the
importance of Ca21. However, there are alternate explanations for the activation of these enzymes that do not
rely on activation by cytosolic Ca21. Inhibition of damage
by the calmodulin antagonist trifluoperazine is strong evidence for Ca21-mediated damage, although the processes
affected are not known. The concern here is the specificity of the drug and the fact that protection was so strong
that it would be well to see the study repeated. Evidence
was discussed in section IVB that mitochondrial Ca21
accumulation was damaging, but although this is quite
likely for glutamate toxicity, it is still only speculation for
ischemia. There is a suggestion that Ca21 may not be
involved in the apoptotic-like processes, from work on
ischemia in cell culture.
Putting the skepticism of the preceding paragraphs in
perspective, all the data are consistent with the conclusion that increased cytosolic Ca21 is critical in causing
damage. Although this variable has only been measured
well in one case, that is really because of the difficulty of
making such measurements, not because cytosolic Ca21
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These have been discussed in the earlier sections of
the review and so are just be noted here. They include
NOS activation, calpain activation, generation of free radicals via phospholipid metabolism or by transforming XD
to XO (in global ischemia), damaging mitochondria (282)
or of course in ways that have not yet been studied,
particularly those mediated by calmodulin that could well
involve effects on cytoskeletal integrity (517). Calcium
may also well be involved in upregulation of damaging
genes during and after ischemia, although this has not
been shown yet.
None of the inhibitor studies has addressed the question of whether Ca21-mediated damage (NMDA or L-channel dependent, for example) preferentially causes apoptosis or necrosis. However, an important study suggests that
apoptotic death may be Ca21 independent. When in vitro
ischemia in cell culture was prolonged to 90 min, neuronal death developed even in the presence of NMDA blockade (405). Unlike the normal cell death in the cultures,
this cell death was associated with increased TUNEL
labeling and was prevented by protein synthesis inhibitors (405), giving it apoptotic-like qualities. Death may
have been triggered by intracellular Ca21 release, or by
non-NMDA-mediated entry processes that are not manifested during 45-min ischemia (Ca21 entry during 45 min
is almost completely blocked by NMDA receptors, Ref.
376), but a serious alternative is that it may be Ca21
independent. Unfortunately, Ca21 levels were not measured in the studies. The result raises the possibility that
apoptotic-like death in focal ischemia and hypoxia/ischemia may occur in cells where Ca21 levels do not rise
enough to activate other processes.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
B. Zinc
It has been known for several years that elevated
levels of extracellular zinc, leading to elevated intracellular levels, are toxic to cultured neurons and other cell
types (summarized in Refs. 195, 1138). Furthermore, during kainate-induced seizures, there is an accumulation of
zinc in CA3 cells that are destined for death (335). An
important recent study took a significant step in linking
these earlier results to ischemic cell death by implying
that accumulation of zinc in vulnerable cells is an impor-
tant component of damage (595). There was a measurable
movement of zinc into vulnerable neurons within 30 min
after 10-min global ischemia in rat and a clear accumulation at 24 h. Zinc was present in all degenerating neurons
after 2 or more days. When looked at in detail, there was
a very strong overlap between zinc-containing neurons
(measured by fluorescence of TSQ) and eosinophilia, denoting ICC. Most importantly, it was shown that CaEDTA,
which is able to chelate zinc (but not Ca21), reduced zinc
accumulation by the neurons and protected against ischemic damage. Protection was significant after 3 days of
ischemia, but less substantial after 14 days, indicating that
the protection may not be permanent; zinc may be involved in increasing the rate of cell death by protease
activation, while not affecting the final outcome. Another
study demonstrated the same selective accumulation of
zinc, following 5-min ischemia in the gerbil, using another
fluorescent indicator, zinquin (1138). In this case, there
was an upregulation of the mRNA for the plasma membrane zinc transporter protein, involved in zinc extrusion
(867), which is a general response to zinc overload (1138),
but no apparent synthesis of the protein. Thus, similar to
the expression of several other putatively protective proteins discussed throughout the text, and particularly in
section VIII, CA1 shows mRNA expression but no protein
expression. This inability to express protective proteins
seems to be a lethal pattern that besets the CA1 pyramidal
cells. Overexpression of this protein protects cultured
cells from zinc neurotoxicity (1138).
The way zinc might cause, or help cause, neurotoxicity is not yet clear (195). It may activate proteases, many
of which are zinc dependent (1194).
It is somewhat surprising that CA1 cells, and not CA3
cells, accumulate zinc after ischemia. The zinc is thought
to be released presynaptically, and indeed, this has been
demonstrated after epilepsy (335). Mossy fiber terminals
have far more zinc in them than CA3 pyramidal cell
terminals, and indeed, it is this zinc that is notably released in epilepsy, with accumulation in CA3 cells and
(possibly) resulting damage (195, 335). Presumably, release of zinc during ischemia is different from its release
during neural firing, and so is restricted to CA3 cell terminals in hippocampus. This provides a very interesting
and unexpected insight into selective vulnerability. It is
possible that, rather than this property residing solely in
the postsynaptic cell (which is actually damaged), it resides to some extent in the presynaptic terminal’s ability
to release zinc.
C. Glutamate
Because of the likely importance of both Ca21 and
Na and protein kinases in cell death, and its own large
release during ischemia, the role of glutamate in ischemic
cell death has been intensively studied. The work began
1
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does not rise. The one direct measurement showed an
impressive increase in cytosolic Ca21. The pharmacological studies are not definitive because of potential artifacts and because cytosolic Ca21 could not be measured.
Results that bring the damaging role of Ca21 into
question are the quite minimal effects of NMDA blockers
in global ischemia and in some models of focal ischemia.
These may be explained by the very transient nature of
NMDA-mediated Ca21 accumulation during global ischemia. In the focal ischemia cases, other sources of Ca21
may overwhelm that from NMDA receptors, although
there is no evidence.
Calcium-dependent processes seem to play major
roles in cell damage, providing strong evidence that Ca21
is damaging. However, the results are not completely
conclusive because there are other methods of activating
these processes. These are less likely than Ca21 activation
but cannot be entirely dismissed.
Feasible studies that would help confirm or negate
the role of Ca21 include correlating measured cell Ca21
levels with cell death in the face of different manipulations, demonstrating that NOS activation and calpain activation really result from increased cytosolic Ca21 and, if
possible, showing that preventing the rise in Ca21 using
intracellular buffers prevents cell death. All of these are
very difficult. At present, the role of Ca21 in in vivo cell
death must be considered likely, but it remains to be
demonstrated more convincingly.
21
B) IMPORTANCE OF CA
IN THE POSTISCHEMIC PERIOD. The
21
timing of Ca -dependent damage is somewhat unexpected and important. Drugs that inhibit Ca21 entry are
protective in the postischemic period in both global and
focal ischemia indicating that elevated Ca21 in this period
is key to production of damage. This is consistent with the
efficacies of delayed application of calpain and NOS
blockers. These findings do not mean that Ca21 changes
during ischemia are unimportant. They may well set the
stage for the later alterations in Ca21 metabolism. Also,
they are certainly important during permanent focal ischemia because there is no reperfusion. If postischemic
Ca21 metabolism is indeed a key to damage, then determining why it is elevated becomes a very important goal.
Little is known of this at present.
1499
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PETER LIPTON
with the seminal finding by Meldrum and co-workers in
1984 (1052) that NMDA blockade attenuated the damage
following vessel occlusion in the rat. The question addressed in this section is the role that glutamate binding
to its receptors plays in ischemic cell death.
1. Release of glutamate
2. Mechanism of glutamate release during ischemia
There is some conflict over whether glutamate release is via standard exocytosis, cytosolic Ca21-dependent spontaneous vesicular release, or via reversed operation of the high-affinity Na1-dependent glutamate uptake
system. There is strong evidence for both spontaneous
and transporter-mediated release in brain slices, and
some evidence for classical exocytosis in vivo, although
predominantly during focal ischemia. The other two
mechanisms have not been studied in vivo.
When the mechanism is considered, it is relevant that
a wide variety of neurotransmitters show the same magnitude and kinetics of extracellular increase during in
vivo ischemia, including ACh, which has no transmembrane transport system (40, 51, 621).
A) IN VIVO. The microdialysate in which glutamate was
collected during global ischemia in gerbils was altered to
contain either zero Ca21 or zero Ca21-Co21. Removing the
Ca21 alone did not inhibit glutamate release (486), suggesting release was not via exocytosis. However, removing Ca21 and adding 10 mM Co21 did delay the glutamate
release by ;5 min, and attenuated the later release (274,
536). One explanation for these results is that there is
exocytotic release in global ischemia and the zero Ca21
dialysate did not effectively remove extracellular Ca21.
The other explanation is that the absence of inhibition by
zero Ca21 shows that there is no exocytotic release; Co21
could have blocked release by its ability to delay anoxic
depolarization for several minutes (188). There is quite
strong indirect evidence for exocytosis or spontaneous
vesicle release in that the accumulation of zinc in pyramidal cells shortly after 10-min global ischemia probably
arises from vesicles in the presynaptic terminals synapsing near the pyramidal cells (595).
Direct evidence for an early exocytotic release is
much stronger for focal ischemia, where removal of Ca21
from the dialysate did slow down the earliest (3– 6 min)
release of glutamate by 50%, while having no effect on
final release levels (1183). Also, the N-type Ca21 channel
blocker SNX-111 (1103) reduced maximal glutamate release during focal ischemia by ;50%. The occurrence of
exocytosis during focal ischemia, but not global, would be
consistent with the much smaller reduction in ATP during
focal ischemia (Table 1), since exocytosis requires ATP
(828). These studies did not differentiate between release
in core and penumbra, and there is likely to be a major
difference because there is no anoxic depolarization in
the penumbra. There may be exocytotic release in the
penumbra during the intraischemic depolarizations. As
discussed below, core release, but not penumbral, is
greatly inhibited in nNOS knockout mice (1031), again
indicating a major difference between the two regions.
B) IN VITRO. Accessibility allows more detailed studies
of release in vitro. There is absolutely no inhibition of
glutamate release from hippocampal slices during ischemia when extracellular Ca21 is removed (685, 888, 1109,
1306), even when 1 mM EGTA is included in the medium
(888). This eliminates the possibility that there is classical
exocytosis. There is, though, good evidence for both
spontaneous vesicular release and release via reversal of
the Na1-glutamate transporter.
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Normal levels of extracellular glutamate measured
by microdialysis are 1–5 mM (1183) and could themselves
activate NMDA receptors during the massive anoxic depolarization that accompanies ischemia (1074). However,
there is a substantial increase of extracellular glutamate
during ischemia in all systems, and this is likely to contribute strongly to any glutamate-mediated damage.
A) RELEASE OF GLUTAMATE IN VIVO. As first shown by
Benveniste and co-workers using microdialysis (84), there
is a severalfold increase in extracellular glutamate during
global ischemia, beginning within 1–2 min (84, 766). Glutamate rises to between 16 and 30 mM by 10 –15 min (51,
84, 766).
There is a similar rise during focal ischemia, beginning within 2 min of MCA occlusion (1183). Because of
the microdialysis technique itself and different probe
placements, there is a lot of variation between studies.
However, except for one case where levels were extraordinarily high, levels in the striatal core rise to between 50
and 90 mM and in the cortical core and/or penumbra rise
to between 30 and 50 mM; levels in the peri-infarct region
rise to only ;6 mM (52, 388, 778, 1028, 1183). Distinguishing between core and penumbral regions is very difficult
in these microprobe studies and leads to some variability.
During prolonged focal ischemia, the high extracellular
glutamate levels are maintained in the focus for many
hours; however, penumbral and peri-infarct glutamate fall
back to baseline after ;1 h (389, 778).
B) POSTISCHEMIC GLUTAMATE. After transient global ischemia, glutamate recovers to baseline in 5–20 min; thus
there is excess glutamate during the early phase of oxygen reperfusion (51, 84, 772). This is important because
glutamate itself is far more toxic in the presence of oxygen than in anoxia (277). There are scattered reports of
delayed increases in glutamate, which might be important. There was a twofold elevation of glutamate some 3 h
after a global insult (23), and there was a threefold increase in extracellular glutamate in the penumbra after
2-h focal ischemia, which remained for several hours
(727). The latter is consistent with the ability of NMDA
blockers to protect against focal ischemia when added
after the insult.
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CEREBRAL ISCHEMIA ON NEURONS
October 1999
3. Modulators of ischemic glutamate release
1
A) INWARD NA
FLUX. There is a large and early accumulation of Na in brain cells during anoxia in slices
(338) and during ischemia in vivo (417). Blockade of this
1
influx by Na1 channel blockers in slices (148, 1109) and in
vivo (245, 343, 388) decreases glutamate release by 50 –
75%. This is consistent with release by exocytosis or by
reversal of the Na1-glutamate transporter. Exocytosis
would be blocked because depolarization is attenuated
while reversal of the transporter would be blocked because intracellular Na1 rises less.
B) FREE RADICALS. Free radical scavengers, mannitol
and SOD, reduced the ischemic release of glutamate from
hippocampal slices by ;60% (888) and, in another study,
SOD almost completely blocked the extracellular glutamate accumulation measured after 5-h hypoxia in cultures (168). In vivo, combined SOD and catalase inhibited
glutamate (and GABA) release during 4-VO by 80% (857).
Although limited in number, these results suggest that
free radicals play a major role in activating the net glutamate release during ischemia.
The basis for this is unknown. Most attention has
focused on the strong inhibition by free radicals of glutamate uptake on the Na1-glutamate transporter in glia
(1177) and also on peroxynitrite inhibition of the three
major cloned transporter species GLT1, GLAST1 and
EAAC1 (1131). This has led to the suggestion that inhibition of uptake by free radicals accounts for the bulk of the
net ischemic glutamate accumulation (1177). There is no
direct evidence for this, but if it does, then the ischemic
glutamate release in vivo cannot be via the reversed transporter because this too would be inhibited by the free
radicals. Thus enhanced exocytosis or spontaneous release coupled with decreased uptake would be the mechanism of ischemic glutamate accumulation.
The other possible site of free radical action is enhanced glutamate release. Activation of spontaneous release by NO/peroxynitrite is not occurring in global ischemia because inhibition of NOS did not affect ischemic
release of glutamate in that model (1279). However, in
focal ischemia, NOS knockout mice release far less glutamate from the ischemic core than do normal mice
(1031), implying that NO (and very possibly peroxynitrite)
plays a major role in the ischemic release of glutamate.
Although the effect may be via enhanced exocytosis, it
may be by decreasing reuptake as discussed above.
C) TEMPERATURE. Ischemic glutamate release is remarkably sensitive to the temperature in focal and global ischemia and in cultures (52, 153, 373, 766, 885, 1308). It is
much more sensitive than, for example, dopamine release
(373). In both striatum and hippocampus, reductions of
2– 4°C, between 37 and 33°C, reduced glutamate release
from 4- to 10-fold. Even the smaller reduction bespeaks a
huge Q10, of ;20. The basis for this remarkable temperature dependency is not known, but it must be incorporated into any model of ischemic glutamate release. Oxygen-derived free radicals are much reduced during
hypothermic ischemia, lending some support to the idea
that free radical action is important (1300).
D) KINASE SYSTEMS. As discussed previously, inhibition
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There is an increase in miniature excitatory postsynaptic potential frequency during the first 5 min of anoxia.
It is blocked by dantrolene, suggesting it is activated by
Ca21 exiting from the ER via CICR (538). It is also
blocked by PLA2 and cyclooxygenase inhibitors (539).
Glutamate release during 2-VO in vivo is also blocked by
PLA2 inhibition (856, 905), indicating there may be spontaneous vesicular release in vivo. However, this is not
certain because PLA2 also blocks glutamate uptake in
hippocampal tissue (267). Thus inhibiting the enzyme
could reduce glutamate release by enhancing its uptake.
Although outward movement on the Na1-glutamate
transporter is widely discussed as a major mechanism for
ischemic glutamate release (827, 1096), there is only one
reported experiment that addresses the issue. The release
of glutamate from hippocampal slices between 5 and 15
min after the onset of ischemia was inhibited 75% by
preloading the slices (688) with two competitive inhibitors of the high-affinity glutamate transporter, t-PDC and
threo-hydroxy aspartate (957). Because inhibition is competitive, and because the competitive inhibitors only accumulate in the cell to about the same concentration as
the glutamate, this is undoubtedly an underestimate of the
total transporter-mediated release. Thus, at least in slices,
reversal of the transporter does represent a major efflux
pathway for glutamate.
C) SOURCES OF GLUTAMATE RELEASE. Cutting afferent axons in vivo strongly attenuates ischemic glutamate release
in CA1 of hippocampus, indicating that the release is very
probably from presynaptic rather than postsynaptic neuronal elements or glia (86, 764). In slices, the carriermediated ischemic glutamate release was not affected by
dihydrokainate (957), the transport inhibitor that is specific for the GLT-1 isoform of the transporter (39). Because this isoform is almost exclusively on glia, the result
strongly supports the conclusion that release is from neurons and not glia (957). Cultured glial cells do release
glutamate during in vitro ischemia (688), but changes in
transporters may occur in culture.
D) SUMMARY. There appears to be a strong exocytotic
component to release during focal ischemia, possibly because ATP levels do not fall terribly low, although this
may well be limited to the core of the lesion. The situation
is less well resolved for global ischemia where evidence
for exocytosis is not as strong. Neither reversed transport
on the Na1-glutamate carrier nor spontaneous vesicular
release has been tested in vivo, although there is strong
evidence that both occur in hippocampal slices. Current
evidence ascribes the major source of glutamate to presynaptic terminals.
1501
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PETER LIPTON
of tyrosine kinase with genistein almost completely
blocked glutamate release during global ischemia (907).
The mechanism may well be phosphorylation of synapsin
I via the MEK-MAP kinase system, as the ischemic activation of release in cultures is prevented by a MEK inhibitor (924). Another possibility is that MAP kinase activates
PLA2 and hence glutamate release (907).
4. Correlative evidence that extracellular glutamate is
damaging during and after ischemia
1
CHANNEL BLOCKADE.
5. Lesion studies showing that glutamate is damaging
during ischemia
Lesions of afferent glutamatergic pathways protect
against ischemic damage. In CA1, damage during global
ischemia is reduced by lesioning the CA3-CA1 pathway
(84), and also by obliterating the dentate granule cells,
which attenuates activation of the CA3 cells (509).
Even more persuasive, and very unexpectedly, blocking activity in the deep prepyriform cortex (area tempesta) with a local injection of 6-nitro-7-sulfamobenzoquinoxaline-2,3-dione (NBQX) protects CA1 of the rat against
4-VO global ischemic damage by ;70% (558). The area
tempesta is very important in seizure generation; during
locally generated seizures, it strongly activates pathways
to the dentate gyrus and CA3 cells of the hippocampus
(557). This rather startling result received further support
when the area tempesta was lesioned. This strongly pro-
tected against CA1 damage while also nearly eliminating
the ischemic increase in extracellular glutamate in CA1
(557). The implication of these results is that glutamate
release in CA1 contributes strongly to damage (although
MK-801 does not protect against damage in this model).
The way in which area tempesta activity during 4-VO
would lead to the release of glutamate is not yet apparent.
The simplest explanation is that seizurelike activity is
generated in that region and spreads to CA3 neurons that
release glutamate in CA1. Whether this can occur during
10 min of 4-VO is a puzzle; the EEG generally becomes
silent within ;40 s of starting the ischemia (935). Thus
how steady glutamate release would be affected is unknown. An initial burst of release might affect enzyme
activities in CA1. Despite the difficulty in explaining them,
the data are imposing and strongly suggest an involvement of glutamate in damage.
6. Involvement of NMDA receptors in damage
A) NMDA ANTAGONISTS IN GLOBAL ISCHEMIA. The excitement
following early studies that showed protective effects of
NMDA blockers (838, 1052) was soon dissipated by conflicting results in both gerbil, where profound and prolonged temperature reduction by NMDA antagonists (136)
was seen to be a major artifact, and in rat, where the early
results of Meldrum and co-workers (1052) showing protection could not be repeated by others (135). These
issues have now been largely resolved.
Variable results in gerbil, even with good temperature maintenance (135, 136, 363), have been explained by
a careful study (454) in which it was shown that the ability
of MK-801 to protect depends on the temperature that is
being maintained. MK-801 was unable to protect when
temperature during ischemia was maintained at 38.5°C,
but it did protect when temperature was maintained at
36.5°C, although very high doses of MK-801 were necessary (10 mg/kg) and damage was still substantial. This
result was supported by a very thorough study in which
telemetry was used to monitor and regulate brain temperature for days after the insult. MK-801 (10 mg/kg) was
clearly protective at 37°C (1282). The requirement for
such high doses of MK-801 suggests that the effect may be
on Na1 channels, so evidence for action of glutamate
from these studies is weak (454).
Even without temperature control, MK-801 does not
usually protect against 4-VO (5 or 15 min) or 2-VO in rats
(99, 135, 819), although doses as high as 10 mg/kg, which
were needed to protect in gerbil, have not been used. The
same lack of protection after 10-min 2-VO was seen with
a novel NMDA/glycine receptor antagonist, which significantly protected against focal ischemic lesions (1192).
The group led by Meldrum (1089), which has consistently reported moderate protective effects of competitive antagonists D-APV or D-APH, use a 2-VO model in
which blood flow in hippocampus is only depressed to
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Na1 channel blockers lidocaine, tetrodotoxin, BW-1003C87, BW-619C89, and lamotrigene all inhibit glutamate release and provide very
strong protection against focal and global ischemic damage (245, 343, 388, 693, 939). In at least one study, temperature was rigorously controlled (693). The blockers
also strongly attenuate damage to brain slices (338, 1195).
These results do not necessarily implicate glutamate release in damage because there are many other effects of
decreased Na1 entry that might be protective.
21
B) CA
CHANNEL BLOCKADE. The specific N-channel
21
Ca blocker SNX-111 (conotoxin MVIIA) reduced glutamate release during focal ischemia by 50% and infarct
volume after 4 h by ;65% (1103). Again no conclusion can
be drawn; blockade of N channels may protect by preventing postsynaptic Ca21 entry.
C) REDUCED TEMPERATURE. Both glutamate release and
neuronal damage are dramatically reduced by lowering
temperature 3– 4° during ischemia (52, 77, 92, 766). In a
very nice study it was shown that there was a strong
correlation between the reductions in glutamate release
and damage to CA1 cells in gerbil hippocampus, when
temperature was varied between 39 and 31°C (766). However, as with Na1, there are many other effects of temperature that could reduce damage, so this cannot be
taken to show that glutamate release is important in
damage.
A) NA
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CEREBRAL ISCHEMIA ON NEURONS
afforded in mice in which the NR2A was knocked out;
there was no effect of the knockout on blood flow during
ischemia (776). In this study, there was no protection
against permanent focal ischemia, only against transient,
2-h ischemia. In all these cases, only the penumbra was
protected. The core of the lesion was not protected even
if the drugs were injected before the insult or, like the
antisense or knockout, had exerted their effect before the
insult. Although very careful temporal studies have not
been done, NMDA antagonists are very effective when
added as much as 2 h after the start of permanent occlusion (873), or 15 min before the end of 1-h or 90-min
temporary occlusion (771, 833).
Focal ischemia does not produce nearly as large a
temperature fall as global ischemia, and protection by
NMDA antagonists is not due to temperature effects.
When skull muscle temperature was maintained constant
throughout 24 h, MK-801 reduced infarct size by almost
50% (334), and CGS-19755, the competitive blocker, reduced infarct size without affecting core temperature
(1102).
In contrast to these protective effects, MK-801 was
not able to reduce infarct size in spontaneously hypertensive rats (141, 970, 1234) and in a thrombotic stroke model
(1253). The drug was able to protect against selective
neuronal loss (peri-infarct region) in the latter case. The
contradictory results are considered below.
D) EFFECTS OF NMDA ANTAGONISTS ON BLOOD FLOW DURING
FOCAL ISCHEMIA. The simplest interpretation of the protection by NMDA antagonists in focal ischemia is that they
block NMDA receptor-mediated Ca21 entry into the neurons and/or intraischemic depolarizations (see sect. IIH).
However, MK-801 and other noncompetitive NMDA
blockers (891), as well as competitive NMDA blockers
(1102), enhance blood flow in control conditions, and two
studies demonstrated major increases in blood flow during ischemia, to the core (3-fold) (141) and to the penumbra/core (1102).
Although the above results suggest protection may
be due to effects on blood flow, they certainly do not
show it. In contrast, at least two studies show significant
(30 –50%) protection by MK-801 with no measurable
changes in blood flow (253, 875, 1149), and one study
shows similar protection when the NR2A receptor is
knocked out, again with no change in blood flow (776).
Furthermore, one careful study showed that MK-801 actually decreased blood flow to collaterals during vessel
occlusion (954).
E) POSTISCHEMIC INVOLVEMENT OF NMDA RECEPTORS. N-methyl-D-aspartate receptor antagonists are fully protective
when added 15 min before the end of a transient focal
ischemic insult, and they are also fully protective against
permanent ischemia when the antagonist is added 2 h
after the onset of ischemia. The protection during reperfusion is consistent with the secondary increase in extracellular glutamate at that time (727) and suggests that
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20%. This is a much milder insult than usual global ischemic events, which probably explains the discrepancy.
Protection has been shown in other studies also. Three
episodes of 5-min ischemia in rat were quite well protected by MK-801, with 50% recovery of damaged neurons
(629). This is consistent with the role of NMDA receptors
in the early increase in cytosolic Ca21 (1048), but unfortunately, temperature was only controlled for 30 min after
the last insult. The glycine-site blocker 7-chlorokynurenate, injected intraventricularly, provided reasonable
(50%) protection against 20-min 2-VO, but core temperature was only regulated until 30 min after ischemia (1222).
Overall, the evidence that NMDA blockade is protective during global ischemia is not at all compelling. The
protective effects that are seen involve very high levels of
MK-801, or the use of halothane, or do not adequately
control temperature. There is, though, an important caveat, that NMDA antagonists can be damaging.
B) DAMAGING EFFECTS OF NMDA ANTAGONISTS. MK-801 actually causes neuronal damage when administered during
normoxia, at the doses that are used for protection (15,
299, 853). Although frank damage has not been seen in
hippocampal CA1 (135), there may be minor damage that
sensitizes tissues to ischemia. More compellingly, Buchan
and co-workers have described actual damage enhancement by MK-801 during global ischemia in CA1, rather
than protection (135, 647), indicating that damaging actions may indeed compromise the interpretation of results
with this inhibitor.
This possibility was strongly reinforced in studies
where NMDA blockade was combined with hypothermia
(334, 1037). Hypothermia blocks glutamate release so that
only minimal protective effects of NMDA blockade would
be expected, because there is little glutamate to cause
damage. Thus an NMDA blocker during hypothermic ischemia would better reveal any damaging effect of the
blocker itself. Indeed, adding the competitive blocker
CGS-19775 did actually reverse the protection offered by
hypothermia during global ischemia (1037), showing quite
clearly that the blockade does enhance damage by some
mechanism.
These considerations mean that the absence of protection by NMDA blockers during ischemia in rats, and in
gerbils, must be interpreted with caution. In particular,
NMDA receptors may be involved in damage; the possibility cannot be excluded.
C) NMDA ANTAGONISTS IN FOCAL ISCHEMIA. MK-801 (141,
874, 1149), NMDA glycine-site antagonists (361), and competitive NMDA antagonists (144, 833, 991, 1102) all reduce
infarct size, or the area of significant cell necrosis, caused
by permanent (334) and transient (141, 833) focal ischemia. Antisense oligonucleotide to NMDA-R1, when injected at a level that reduced NMDA antagonist binding
sites by ;40%, strongly protected against 24-h MCA occlusion in the rat. It reduced infarct size somewhat more
than MK-801, by ;40% (1184). A similar protection was
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PETER LIPTON
7. Effects of AMPA/kainate and metabotropic
glutamate receptors on ischemic damage
A) AMPA RECEPTOR ANTAGONISTS DURING ISCHEMIA. At least
five studies very clearly show strong protective effects of
AMPA antagonists during permanent focal ischemia, in
cat (145) and rat or mouse, using NBQX and also newer
water-soluble AMPA antagonists YM90K, YM872, and
ZK200775 (759, 776, 1032, 1255). Temperature was well
controlled throughout the early ischemia in most of the
studies in rat. No studies have been described in which
AMPA receptor blockers have been applied before global
ischemia, nor have effects on temporary focal ischemia
been described.
The protection in focal ischemia might result from
attenuation of the intraischemic depolarizations (47, 759),
or from blockade of Na1 entry itself. The latter is consistent with effects in hippocampal slices where AMPA
blockade reduced the increase in cytosolic Ca21 by preventing Na1/Ca21 exchange at the mitochondrial membrane (1289). The protection by AMPA blockade may also
result from temperature reduction; temperature was not
explicitly maintained throughout the ischemia and, as
discussed below, at least some AMPA blockers do lower
temperature in the postischemic period.
B) POSTISCHEMIC INVOLVEMENT OF AMPA /KAINATE RECEP TORS IN GLOBAL AND FOCAL ISCHEMIC DAMAGE . NBQX attenuated damage when added at different times up to 12 h
after either 2-VO or 4-VO ischemia (140, 647, 819, 1176),
suggesting that delayed activation of AMPA receptors
was damaging. However, it has now been shown that
NBQX lowers core temperature and that this hypothermia quantitatively accounts for almost all the protection in gerbils, and very probably in rats (844). When
gerbils were treated three times with 30 mg/kg NBQX
between 50 and 90 min after 3-min ischemia, almost all
CA1 neurons were protected from the normally severe
neuronal loss (75%), measured 4 days later. However,
the drug caused a 1.5°C drop in temperature, relative to
control, that persisted at least 2 days. When the temperature drop was prevented for 24 h, the drug provided no protection. Furthermore, maintaining the temperature 1–1.5°C below normal for 24 h gave about the
same protection as afforded by NBQX (the latter maintained the lower temperature for 1–2 days longer). This
study is very persuasive, and it is impossible to conclude, at this time, that postischemic activation
of AMPA receptors is involved in promulgating the
damage.
The water-soluble quinoxalinedione ZK-200775 attenuated the infarct size by 45% when added 2 h after 90-min
temporary focal ischemia (1144) so that focal ischemic
damage is also able to be rescued by postischemic blockade of AMPA receptors. In this case, temperature was not
well monitored, so it is impossible to know whether the
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significant damage occurs during this time. It tends to
dissociate protection from prevention of intraischemic
depolarizations because the latter do not occur after the
transient insult is over. The prolonged glutamate toxicity
during permanent ischemia is somewhat surprising as
glutamate levels in penumbra fall back to baseline after
;1 h (389, 778); however, residual glutamate plus compromised energy metabolism (356, 839) probably make
the system very sensitive. The delayed protection by
NMDA antagonists in both models of focal ischemia suggests that preventing the action of cytosolic Ca21 at this
late time is effective. That is consistent with the efficacies
of delayed inhibition of calpain and NOS.
Most groups have found no protection by early postischemic application of NMDA antagonists in global ischemia (327, 819, 1023) despite implications that the receptor system is abnormally activated in that period (22, 457,
890). One study did show good protection when NMDA
antagonists were added 15 min and 5 h after 2-VO in rat
(167), but this was done by the group that has consistently
observed a protective effect of NMDA antagonists during
the ischemia and probably reflects the much milder, focal
ischemic-like insult. As discussed previously, it would be
interesting to test very delayed NMDA blockade (12–24 h)
in global ischemia as cell Ca21 is elevated in this period.
F) SUMMARY. The effects of NMDA antagonists are
clearly difficult to interpret unambiguously. In global ischemia, the antagonists may actually be protective; the observed lack of protection may result from antagonistinduced damage masking a protective effect that would
otherwise result from blockade of Ca21 entry. Although
this seems somewhat unlikely, it cannot be dismissed. At
the other pole, the protection by high levels of MK-801 in
gerbil may well reflect Na1 channel opening rather than
NMDA channel opening.
In focal ischemia, the lack of protection in the SHR
and thrombotic models has to be explained, as do the
conflicting data on the effects of antagonists on blood
flow.
A possible reconciliation of the first issue is that
NMDA blockade does indeed protect because it attenuates Ca21 entry and that it did not protect in SHR, and the
thrombotic model because the insult intensities were too
great (or, possibly that damaging effects of the antagonists were more significant in these models, Ref. 689).
N-methyl-D-aspartate blockade can fail to protect in vitro
systems when the insult intensity is very high (685).
It would be very useful to resolve the quite conflicting
data on blood flow changes. Some very good studies do
demonstrate protection by NMDA antagonists in the absence of blood flow changes, strongly suggesting that
NMDA damage in focal ischemia is via Ca21 entry into
neurons. However, the discrepancies between blood flow
changes measured in different studies need to be satisfactorily explained.
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
8. Other transmitters
Several other neurotransmitters including monoamines acetylcholine and GABA show quite massive
extracellular increases during ischemia, with similar
time courses to that of glutamate (51, 497, 621). Inhibition of GABA reuptake greatly improves recovery from
5-min global ischemia in gerbil, indicating a protective
effect of this transmitter. However, enhanced GABA
activity is strongly hypothermic (1010), and the protective effect seen during ischemia can be completely
explained by hypothermia induced by the uptake block
(497). There are reports of damaging effects of some
transmitters, particularly serotonin acting at 5-HT2 receptors (374) and dopamine acting at D2 receptors
(433). Because little at present is known about the
possible mechanisms of these effects, they will not be
considered further in this review. The multiple receptor
types for these transmitters means that much work is
required to sort out their true effects during ischemia.
They may be quite important.
9. Relationship of glutamate involvement in ischemia
to excitotoxicity: mechanisms of glutamate damage
in ischemia
A) GLUTAMATE LEVELS. The previous discussion establishes that glutamate is likely to play roles in focal damage
and may well play a role in global ischemic damage. The
question is the extent to which the action of glutamate in
ischemia mimics its action as an excitotoxic agent. That
is, is the role of ischemia simply to cause release of
glutamate, which then causes excitotoxic cell damage?
Concentrations of glutamate reached during global
ischemia and in the penumbra of a focal lesion are ;30
mM. These are of the order that will cause excitotoxic
damage in cultures during short exposures, equivalent to
global ischemia, when energy metabolism is compromised (356, 839). They also cause damage in cultures
during longer exposures when energy metabolism is normal (395), as in the penumbra of focal lesions.
B) GLOBAL ISCHEMIA. While conditions in the focal penumbra are compatible with the occurrence of excitoxicity, conditions during global ischemia are not. In cultures,
short exposures to glutamate are only toxic in the presence of oxygen (277); this is probably for at least two
reasons. Glutamate toxicity depends on free radical (282,
631, 883) and NO (236, 237, 1074) formation, and it also
appears to depend on mitochondrial uptake of entering
Ca21 and resulting mitochondrial damage (26, 142, 994,
1206). The former two require oxygen, and the latter
requires very active oxidative metabolism. Thus, although
glutamate may well contribute to global ischemia damage,
as evidenced by the studies in which area tempesta in the
endopyriform cortex was lesioned, it probably does not
do so by a purely excitotoxic mechanism. Either it contributes to damage because it enhances Na1 or Ca21 entry
or it acts via metabotropic receptors in some way.
C) FOCAL ISCHEMIA. In the focal ischemic penumbra,
oxygen levels are quite high, both during and certainly
after the insult, as are ATP levels (Table 1). Glutamate is
at levels that cause excitotoxic damage in cultures; unfortunately, there are no measurements of excitotoxic
levels in vivo. Thus conditions are appropriate for glutamate excitotoxicity. Furthermore, ischemic damage and
excitotoxic damage share many characteristics. Glutamate will cause both apoptosis and necrosis (919), as is
seen after focal ischemia. Also, excitotoxic damage in
cultures has very similar dependencies to those of focal
ischemic damage, including the time required for damage
development (12–24 h). It is dependent on NO formation
(236, 237, 1074), on free radical formation (282, 631, 883),
on calpain activation in most (125, 126, 158) but not all
(709) cell types, on calcineurin as defined by dependence
on both cyclosporin and FK-506 (26), and on PARP activation (298). Thus it seems quite likely that very similar
mechanisms are followed in the focal ischemic penumbra
and in excitotoxicity. If so, then mitochondrial damage
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protection was due to blockade of AMPA receptors on the
neurons.
Another mechanism, for very delayed damaging effects of AMPA/kainate receptors, soon before delayed
death, is suggested by recent studies from Zukin and
colleagues (384). They showed that the ratio of GluR2 to
GluR1 receptor mRNA was selectively decreased in the
CA1 pyramidal cell layer beginning 24 h after 5-min global
ischemia in the gerbil and that this became exaggerated in
the next 2 days. There were no ratio changes in CA3 and
DG. If followed by the same change in receptor protein
(which appears to be the case; R. S. Zukin, Princeton
Conference, 1998), this receptor change would increase
Ca21 permeability of the AMPA receptors. There is explicit evidence for the latter as the cytosolic Ca21 increase
elicited by AMPA (in the presence of cyclothiazide, which
prevents inactivation) is far greater in CA1 neurons 3 days
after ischemia than it is in control CA1 neurons or even
those taken 48 h after ischemia (384). It remains to be
shown that these currents are actually damaging. If they
are, this might help explain the delayed death; it might
also account for the increased cell Ca21 measured 24 h
after global ischemia (285, 1272).
C) METABOTROPIC RECEPTORS. There are no reported studies of the actions of metabotropic glutamate antagonists
in vivo. The combined group I/II agonist t-ACPD is protective in focal ischemia (193) as is the II/III agonist
(S)-4C3HPG in global ischemia (442), suggesting that the
net effect of metabotropic activation will be protective via
group II type receptors. However, this is difficult to assess
as prior addition of metabotropic agonists causes longterm changes that are probably not produced immediately
upon glutamate release. Antagonists are better suited to
this issue.
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PETER LIPTON
10. Conclusions
A) GLUTAMATE INVOLVEMENT IN GLOBAL ISCHEMIC DAMAGE.
Blocking glutamate release in CA1 by inactivating the area
tempesta of the endopyriform cortex is strongly protective against 4-VO. If those results stand up, and no artifactual explanations are found, they are extremely strong
evidence that glutamate release is a major effector of cell
damage in global ischemia. This creates a quite paradoxical situation, because pharmacological evidence for the
involvement of glutamate in global ischemic damage is
very weak. As discussed, there are no studies on metabotropic or AMPA/kainate receptors, and NMDA antagonism
gives no protection in the rat and very little or none in the
gerbil.
There seem to be four reasonable resolutions of this
paradox. The first is that good pharmacological studies
will reveal that AMPA/kainate or metabotropic receptors
do contribute to cell death. At least the former certainly
do contribute during intense insults in vitro, and they do
increase cytosolic Ca21 by releasing the ion from mito-
chondria during ischemia in slices. The second possibility
is that the absence of protection by NMDA antagonists,
principally MK-801, is misleading because the antagonists
also cause damage, which offsets their protective effects.
This was discussed at length, and there is reasonable
evidence that it is so. Indeed, the fact that NMDA antagonists are completely useless above 37°C may be because
they cause greater damage at higher temperatures. The
third possible explanation is that, because the intensity of
the insult is great, combined NMDA, AMPA (and perhaps
metabotropic) blockade is necessary to see any marked
reduction in damage. A final, and important, possibility is
that the effects of AT lesions on glutamate release are not
the basis for their protective effects so that glutamate is,
in fact, not important in damage. Resolution of this important issue awaits further studies.
A delayed role for glutamate is suggested by the
findings that AMPA receptors become far more Ca21 permeant in vulnerable regions between 1 and 3 days after
global ischemia (384). It remains to be shown that this
contributes to damage. In the limit, it is possible that this
is actually the key toxic event in global ischemia and that
preceding events are damaging because they eventually
cause this change in AMPA receptors, which then leads to
classical excitotoxicity. There are no pertinent studies in
focal ischemia.
B) GLUTAMATE INVOLVEMENT IN FOCAL ISCHEMIC DAMAGE.
Both NMDA receptor antagonists and AMPA receptor
antagonists strongly attenuate damage in the penumbra in
permanent and temporary ischemia in most, but not all,
cases. The weight of evidence indicates that NMDA protection is independent of actions on blood flow, and it is
certainly independent of effects on temperature. These
possible artifactual effects of AMPA antagonists have not
been as well controlled or tested, so there is more ambiguity in the conclusion that AMPA receptors contribute to
damage.
Glutamate-mediated damage may result from the intraischemic depolarizations, which activate damaging ion
fluxes through voltage-dependent and receptor-mediated
channels, as has been argued by Hossmann (463). Alternatively, damage may be independent of the depolarizations and due to receptor effects on Ca21 and Na1 fluxes.
It is likely that both mechanisms are operative and that
the depolarizations enhance receptor-mediated influx.
The fact that NMDA antagonists are effective when added
near the end of temporary focal ischemia, after which
there are no intraischemic depolarizations, shows that
major glutamate damage continues without them. Ionmediated mechanisms of glutamate action very probably
resemble those in classical excitotoxicity because conditions are similar, but the importance of NMDA-mediated
TNF-a activation shows that there are additional effects
in ischemia.
The core of the infarct is immune to protection by
glutamate antagonists (although a combined treatment
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may be important in focal ischemic damage, as discussed
in section IVB.
This being said, there are still major differences between glutamate toxicity and ischemic damage that necessitate great caution in moving from one to the other. In
particular, PARP activation does not seem to be involved
in damage following permanent focal ischemia; this differs from glutamate toxicity. Furthermore, focal ischemia
is a more complex insult than excitotoxicity in cultures.
Not only is there a primary inhibition of oxidative phosphorylation, but there is induction of important enzymes
and cytokines in focal ischemia (see sect. VIII), and there
is also the involvement of the vasculature and, in particular, leukocyte accumulation. Blocking cytokine production and blocking leukocyte production both greatly alleviate focal damage, the latter only in temporary ischemia.
In permanent focal ischemia, MK-801 reduces the
production of the cytokine TNF-a by 60%. Both MK-801
and treatments that independently neutralize TNF-a action reduce infarct size by ;50% (90, 636). This activation
of cytokine production may be a major basis for the
NMDA-mediated damage in permanent focal ischemia.
This mechanism has not yet been tested in temporary
ischemia; it is almost undoubtedly not operative in excitotoxicity.
A very important question that emerges from these
considerations is why focal damage is so dependent on
factors such as white blood cells and cytokines when
glutamate toxicity occurs in cultures without these. Possibly glutamate levels themselves are not elevated
enough, or for long enough in the penumbra, to cause
toxicity in vivo. As mentioned above, there are no existing
measures of the concentrations of glutamate that are
lethally excitotoxic in vivo.
Volume 79
October 1999
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CEREBRAL ISCHEMIA ON NEURONS
has not been tested), which highlights the role that glutamate plays in cerebral ischemic damage. All tissues suffer
ischemic damage, so ischemic brain cell death would
certainly occur in the absence of glutamate. Glutamate
appears to sensitize brain tissue to ischemia, making
short exposures (global ischemia), or prolonged but only
mildly hypoxic exposures (penumbra of focal ischemia)
lethal. The core of the focal lesion is presumably so
metabolically compromised for so long in all the paradigms that are used that damage occurs without glutamate.
D. pH
1. pH fall during ischemia
A) GLOBAL ISCHEMIA. The extracellular space begins to
go acid within 20 s of beginning anoxia or global ischemia
(845, 1057), well before the anoxic depolarization (717,
845). Intracellular pH follows a similar time course, beginning to fall within ;1 min and reaching a minimal
value after ;2 min (297, 419, 1086).
The magnitude of the fall in pHo during global ischemia is ;1 unit. As seen in Table 3, the average pHi in rat
falls from ;7.0 to 6.4, although in the one study of gerbils
it fell further. Tissue lactate rises from ;4 mM (793) to
between 8 and 15 mM (550, 551, 793).
B) FOCAL ISCHEMIA. At the core, where blood flow was
reduced to 25% at 10 min and to 10% by 3 and 4 h, pHi fell
to 6.6, 6.2, and 6.1 at those times. In the penumbra, where
flow was reduced to 40%, there was no significant pHi
drop at 3 h and a slight drop (to pH 6.75) at 4 h (755, 943).
C) CELLULAR LOCUS OF PHi CHANGES. This is an important
issue that has not been well resolved since few of the
studies measure changes in different compartments. In
the limit, all pH changes could be in glia, which would
greatly affect interpretations. Several groups have noted
multiple pH profiles during ischemia using 31P-NMR (108,
System
2-VO; 10 mina (402)
CA; 10 minb,f (633)
4-VO; 5–30 mina,f (1210)
Complete; 5–30 minc (609)
Complete; 60 sd (296)
2-VO; 5, 10, 15 mina (465)
2-VO; 1, 2, 3, 5, 15 mind
(419)
2-VO; 15 mind,f,g (419)
4-VO; 8 minc (1048)
Complete in dog; 2, 12
mina (297)
Gerbil; 5 mine (784)
Total Brain
pHi
Neuronal
pHi
Glial
pHi
6.4h
7.3h
EC pH
6.5
6.4
6.8
6.8
6.8,
6.5,6.5
6.8,6.6,
6.5,6.5
6.2
6.3
6.7,
6.2
6.0
Measurement methods are as follows: a 31P-NMR; bneutral red;
intracellular pH (pHi) electrode; dextracellular pH electrode, measurement of total CO2, HCO3 and extracellular space, and calculation of pHi
(550); eumbelliferone fluorescence. fParadigm causes irreversible damage in region where pH was actually measured. gHead temperature
control. hThese compartmentalized values are inferred, not measured
directly. Reference numbers are given in parentheses.
c
326, 1086), but relevant compartments have not been able
to be determined. In one study, where the pHi fall was
measured in neurons with microelectrodes (1048), pHi fell
to 6.3 in both CA1 (vulnerable) and CA3 (ischemia-resistant) cells. This is similar to pHi falls measured in whole
tissue (Table 3), suggesting strongly that the latter do
reflect neuronal changes.
2. Postischemic recovery of pH
Because acidity exacerbates free radical damage, the
pH profile during reperfusion is a very important parameter. Unfortunately, there is a lot of variability in reported
recovery times, possibly because little attention has been
paid to this quantity. In most cases, 50 and 95% recovery
times for pH range between 2 and 8 min and 5 and 30 min,
respectively (419, 633, 1057, 1086, 1128, 1210), but there
are several cases where measured recovery is more prolonged (297, 1048). In one case, pHi remained 0.3 units
below normal for several hours (1128), and in another,
when localized pHi was measured with umbelliferone
after 5-min ischemia in gerbil, there was a rapid recovery
to pH 6.8, but a rather slow (2 h) recovery from 6.8 to 7.1
that was peculiar to CA1, the most vulnerable region
(784). Whether these are functionally important is not
known.
3. pH changes in brain slices and cell cultures
Resting pHi is high, ;7.4, in hippocampal and cortical
slices when they are in bicarbonate buffer (124, 531, 634).
When extracellular glucose is 10 mM, pHi falls to between
6.7 and 6.5 at the end of 10-min anoxia (531, 913). No
studies have been reported for other glucose concentra-
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As illustrated in Table 3, intracellular pH (pHi) falls
by 0.5–1 unit during cerebral ischemia. This fall is very
rapid, and the question is the extent to which this contributes to ischemic damage (1042, 1045, 1120). Unlike
glutamate, these pH changes alone do not damage neurons, as discussed below, so that the question becomes
whether they exacerbate the damage.
Although the weight of evidence suggests that the pHi
fall is damaging, the issue is not completely resolved. One
difficulty is the absence of drugs that alter the pH change
during ischemia. Thus its role can only be tested by
metabolic manipulations that also alter other variables.
Another difficulty is that pHi and extracellular pH (pHo)
are very closely linked (748, 815, 1057) so that manipulations generally affect both these variables very similarly.
Because the two pH changes may have opposite effects
(see below), interpretations have to be made cautiously.
3. Minimum pH during different global
ischemia paradigms
TABLE
1508
PETER LIPTON
tions. There are no reported measurements for neuronal
cultures.
4. Mechanism of fall in pH
5. Role of pH fall during ischemia
The major strategy to get at this critical question has
been to vary the pH change during ischemia by manipulating the blood glucose levels. The simple idea is that
hypoglycemia will attenuate the decrease in pH, whereas
hyperglycemia will enhance it so that if the normal pHi
decrease is contributing to damage, then hypoglycemia
should be protective. Hyperglycemia might enhance damage if the normal fall in pH does not saturate the system.
Showing the protective effects of hypoglycemia is the
most critical to the hypothesis that the normal fall in pH
is damaging; unfortunately, these results are the least
conclusive.
Another strategy, which has only been used to date in
focal ischemia, is to test the effects of buffering the
normal decrease in pH. These approaches will be described in the following sections.
A) BUFFERING PH CHANGE DURING FOCAL ISCHEMIA. An alkaline trisaminomethane buffer was infused into the ventricles during focal ischemia in cats. The concentration was
adjusted to almost completely prevent the extracellular
acidification during the insult. The treatment reduced the
infarct volume by 40% (796). Although only pHo was explicitly maintained, and pHi was not measured, the buffer
readily crosses the cell membrane (796) so very probably
buffered pHi also. The simple conclusion from this is that
the reduction in pHi during focal ischemia makes a major
contribution to development of damage. This needs to be
repeated by other groups and examined further. It represents, in principle, a very useful approach. A caveat is that
the buffer may have been acting elsewhere (for example
on NMDA receptors or free radicals), although there is no
evidence that this is so.
B) EFFECTS OF HYPOGLYCEMIA. Hypoglycemia has been
induced either by fasting or by insulin. The results with
insulin, which attenuated the pH fall from 0.7 to 0.3 units
(550), are too complex to interpret because of effects of
insulin itself (346, 643, 1076, 1175).
Forty-eight hours of fasting decreased blood glucose
by 40% and decreased lactate production during 4-VO by
50%, suggesting a smaller fall in pHi (711). Twenty-four
hours of fasting reduced blood glucose by only 20% but
sharply reduced the increase normally seen during and at
the end of ischemia (260). The pH nadir during 16-min
ischemia in cats was 6.3 rather than 6.0 (198).
These fasting regimes did reduce the neuronal damage following global and hypoxic/ischemic insults (260,
375, 711). Food-deprived animals were very strongly protected against 30-min 4-VO, a fairly profound insult (711),
and were also almost completely protected against early
infarct after 25-min hypoxia/ischemia, when compared
with controls which suffered major damage (375). These
protections correlated with the attenuation of the pH fall,
but protection may have been due to other effects of
starvation. [The low insulin caused by starvation would, if
anything, exacerbate damage, so it is not a complication
(1174).] Attempts were made to distinguish possible protective effects of the long-term starvation from the effects
of the hypoglycemia at the time of the insult by injecting
glucose just before the insult (375). Unfortunately, the
results were ambiguous. However, it was shown that the
protective effects of hypoglycemia were not due to ketosis (375), nor to reduced release of glutamate. Fasted rats
showed much less severe striatal infarction and penumbral damage 24 h after 15-min hypoxia/ischemia, despite
actually releasing more glutamate during ischemia (260).
As expected, ATP levels fell more rapidly in hypoglycemic
conditions (550), and protection occurred in spite of this.
These results are consistent with the fall in pH being
damaging, but other possible effects of the starvation
have not yet been satisfactorily excluded.
C) EFFECTS OF HYPERGLYCEMIA. Since the early serendipitous findings of Myers and Yamaguchi (795), and followup work by that group (792–794), a large number of
studies have very clearly established that moderate hyperglycemia, in which glucose levels are elevated to
16 –20 mM, greatly enhances damage after global ischemia
(255, 522, 552, 651, 932) and both permanent and temporary focal ischemia (221, 240, 1178, 1256). Individual cellular necrosis (255, 649, 650, 932) as well as infarct devel-
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The early decrease in pHi (553) probably results from
the immediate blockade of oxidative phosphorylation and
continued flux through the glycolytic pathway (358). The
mechanism for the maintained decrease in pHi has not
been studied fully but has generally been assumed to be
the activation of glycolysis stemming from the lowered
ATP. Certainly this is an important determinant; the pHi
fall usually correlates with increased lactate concentration as blood glucose is increased (550). However, there
are indications that the regulation is more complex
(1045). Although there is controversy (550), several
groups have reported that moderate hyperglycemia has
little or no effect on the pHi change during ischemia, even
though lactate production is increased about twofold
(214, 465, 610). This suggests a homeostatic mechanism is
in play. A second consideration is that the inhibitor of
NOS, NG-nitro-L-arginine methyl ester, prevented the fall
in pH in the penumbra of a focal ischemia lesion at a
concentration that did not affect cortical blood flow, or
mitochondrial redox state (943). There is no good explanation for this quite surprising but strong effect, although
it may well be relevant that free radicals lead to decreased
pH in cardiac tissue (1225). These anomalies in pH regulation suggest that transporter activities, or unknown parameters, are altered by the ischemic insults and modulate the pH change.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
old pHi for damage and thus that damage is a continuous
function of the pHi fall during ischemia.
E) OTHER EXPLANATIONS FOR ENHANCED HYPERGLYCEMIC DAMAGE. In principle, there are several ways by which hyperglycemia might be damaging other than exaggerating the
fall in neuronal pH. These need to be considered.
I) Seizures. There are almost always seizures following hyperglycemic insults and they could, in principle, be
the reason that damage is exacerbated. Although cell
death has been anecdotally dissociated from the occurrence of seizures (651), the glucose threshold for inducing
seizure activity after global ischemia (12–16 mM) is about
the same as the glucose threshold for significant aggravation of necrotic cell damage (650). The strongest experimental argument against this explanation for the enhanced damage is that the damage enhancement caused
by elevated CO2 was not associated with an increase in
seizure activity (552). To the extent that effect of hypercapnia represents the effect of lowered pH, as it very
probably does, these studies show convincingly that seizures do not account for the enhancement of damage by
reduced pH.
II) Effects on vasculature and infarct formation.
Damaging effects of hyperglycemia, and lowered pH, may,
in principle, be mediated by effects on the vasculature or
on glia. This remains a possibility particularly because no
in vitro models have been found in which hyperglycemia
enhances damage. In brain slices, increased glucose in the
bathing medium always improves recovery of synaptic
transmission (60, 532, 686, 1119), probably because it
maintains ATP levels and retards anoxic depolarization.
Although there are no data directly demonstrating a vascular or glial site of damage, severe hyperglycemia does
lead to infarct formation even after global insults (522).
This suggests that processes may be occurring that are
qualitatively different from those occurring during normoglycemic ischemia. On the other hand, in a careful study
of effects of hyperglycemic ischemia on small vessel patency, Siesjo and co-workers (648) found no effect of the
hyperglycemia, eliminating the possibility that decreased
pH caused postischemic obstruction. The broader vascular issue needs to be well resolved before it can be concluded that hyperglycemia is damaging by decreasing pH
in neurons.
III) Other metabolic changes. Other metabolic
changes caused by hyperglycemia and hypercapnia would
seemingly protect against, rather than enhance, ischemic
damage. Hypercapnia in vivo reduces net Ca21 entry into
cells during ischemia and delays anoxic depolarization
(613). The ATP fall and anoxic depolarization are retarded
by hyperglycemia; these slowings should be protective
(1178). However, it has been argued by one group that it
is precisely the attenuation of the ATP fall that makes
hyperglycemia damaging (465), because it decreases formation of adenosine, which it very probably does. Adenosine is protective in several ischemic models (21, 904),
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opment (522, 794, 932) are both enhanced. The most
straightforward and consistent interpretation is that the
increased damage results from a larger than normal decrease in pH.
Supporting this conclusion, profound hypercapnia
during normoglycemic global ischemia, with CO2 levels
chosen so that pHi fell to about the same level as during
the hyperglycemic ischemia, increased cell damage to
about the same degree as hyperglycemic ischemia (552).
D) RELATIONSHIP BETWEEN DEGREE OF PH REDUCTION AND
EXTENT OF DAMAGE DURING HYPERGLYCEMIC ISCHEMIA: IS THERE A
THRESHOLD FOR DAMAGE? This is a tricky but very important
question because if there is a threshold, it argues that the
pH fall in normoglycemic conditions is not enhancing
damage, contradicting the inference from the hypoglycemic studies. Results from several groups agree that elevating blood glucose to 20 mM or above enhances damage
at the same time as it accentuates the rise in lactate and
the fall in pH (609, 793, 1057). In general, the rapid falls in
pHi during ischemia in normoglycemic conditions are
enhanced by 0.3– 0.6 units at 20 mM glucose. Intracellular
pH falls to between 5.9 and 6.2 (633, 1057, 1210). Larger
increases in glucose produce larger average decreases in
pHi during ischemia (1210); microelectrodes identified a
profound decrease, to 5.3, in astrocytes, when glucose
was high enough (50 mM) to lower pHo to below 6.2.
Neuronal pH fell to ;6.0 (607).
The increased damage due to 20 mM glucose is thus
associated with pHi decreases that are 0.3–0.6 units greater
than normal. If the normal drop in pHi is causing damage, as
suggested by the fasting studies, then smaller increments in
this drop, produced by smaller increments in blood glucose,
should also increase damage. However, Siesjo and co-workers (651) found that damage was only measurably enhanced
when blood glucose was elevated to ;15 mM (651); pHi at
this point fell ;0.3 units more than normal (550). If pHi
during ischemia is a continuous function of blood glucose,
these results indicate that there is a pHi threshold for damage and thus that the normal decrease in pHi is not enough
to be enhancing damage, opposing the conclusions from the
studies of hypoglycemia.
Indirect measurements of pHi by Siesjo and co-workers (550, 1057) suggested pHi was indeed a continuous
function of glucose and thus supported the threshold
concept. However, other studies suggested that pHi is a
discontinuous function of glucose (and lactate) levels.
Kraig et al. (610) noted a nonlinear relationship in which
pHo fell to 6.8 when blood glucose was anywhere below
;15 mM, and fell to 6.1 when blood glucose rose to 18 mM
or greater (610), about the level at which damage begins
to be manifested. Furthermore, NMR studies of pHi in rat
(465) and gerbil (214) showed that glucose levels had to
be elevated to 15–20 mM before pHi fell more than normal. These quite direct studies are persuasive and are
consistent with the conclusion that there is not a thresh-
1509
1510
PETER LIPTON
6. Protective effects of decreased pHo in vitro
Although evidence that the pH fall is damaging in
vivo is quite strong, artifactually reducing pHo is protective against both glutamate and ischemia (518, 1118, 1119)
in vitro. The basis for the protection is not completely
known.
Reduced pHo attenuates NMDA-mediated Ca21 entry
(290, 1097, 1107, 1120). This is almost undoubtedly an
extracellular action as it occurs in excised patches (1107).
It would be very protective in cell culture where NMDAmediated Ca21 entry is the major source of damage. However, this cannot be the whole story as MK-801 combined
with acidity is far more protective than MK-801 alone
(518). Furthermore, low pH buffers are also protective
against ischemia in nonneural tissues (109, 229, 1021),
where there are no NMDA receptors. In liver cells, protective effects of extracellular acidosis are due to the
resulting intracellular acidity (123) which, at pH 6.4, very
strongly inhibits the protease activity induced by anoxia
in isolated hepatocytes (123).
If these protective effects are operative in vivo, then
normal damage is occurring despite them, and they must
be outweighed by other, damaging, effects of decreased
pH. This issue has been discussed from several points of
view, in some cases maintaining that the fall in pH must
be protective (1045, 1120). This is very difficult to accept
based on the evidence discussed in the previous sections,
and at this stage, it seems most likely that the net roles of
pH in vitro and in vivo are quite different. This is readily
explicable if in vitro effects are primarily on NMDA-mediated entry because the latter is more important in culture than in vivo. Indeed, it is particularly unimportant in
global ischemia, where effects of pH are most profound.
Another obvious possibility is that damaging effects in
vivo result from effects on the vasculature, which would
not be significant in vitro. This does not seem likely, as
discussed above. Certainly, the protective effects of lowered pH in vitro indicate multiple effects of pH during
ischemia. However, at this stage, the weight of the evidence is that lowered pH is damaging in vivo.
Unlike NMDA, AMPA/kainate receptor-mediated cell
death is actually greatly accentuated by reducing the pH
in cortical cultures from 7.4 to 6.6 (741). Furthermore, the
AMPA/kainate component of death resulting from in vitro
ischemia is also enhanced by the lowered pH (741). To the
extent that AMPA/kainate receptors mediate cell death in
vivo, these studies provide further evidence that the decreased pH is likely to be damaging.
7. Possible mechanisms of pH-mediated damage
A) EFFECTS OF LOW PHI ALONE. The pH values reached
during global and focal ischemia, even in the presence of
20 mM glucose, are not adequate to kill neurons so that
reduced pH certainly cannot be considered to be the
lethal activator of damage. Lowering pHi to 6.5 for 75 min
in vivo, using hypercapnia, produced no histological damage measured 1– 4 wk after the incident (209); it required
several hours of exposure to pH 6.2 to produce delayed
damage (817). The pH had to be lowered to ;4.5 to get a
delayed damage from short (10 min) exposures (608, 815,
817).
In vitro systems are more sensitive to lowered pH
than the in vivo systems described above; however, this is
probably not relevant to in vivo ischemia. Lowering pHo
to 6.7 for only 30 min caused delayed degeneration in
hippocampal slice cultures, probably as a result of free
radical production (1024). Just this condition could arise
during recovery from global ischemia, and in the penumbra of focal ischemic damage, but at present, because of
the measurements showing the far smaller sensitivity in
vivo, it has to be assumed that this phenomenon is restricted to culture systems.
B) LOW PH COMBINED WITH ISCHEMIA. There are at least
four reasonable explanations for the damaging effect of
the lowered pHi during ischemia, but there is little evidence at present for any of these.
Lowered pHi could increase intracellular Na1 by ac-
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probably via action on the A1 receptor (904). Although
unexpected, this idea cannot really be dismissed at this
stage, as is discussed in section VIIIA.
IV) Immediate effects of glucose infusion. Recent
studies by Schurr and colleagues (A. Schurr, R. S.
Payne, M. Tseng, and J. Miller, personal communication) cast a novel perspective on the effects of hyperglycemia. In a cardiac arrest global model, they find
that although hyperglycemia greatly exacerbates cell
damage when ischemia is induced within 15 min of the
glucose injection, it actually reduces damage when the
ischemia occurs 2 h after the glucose injection. Blood
glucose levels are actually higher at the later time point
(22–35 mM). If this holds up it is very important. It
would indicate that damaging effects of hyperglycemia
may result from a rapid (hormonal?) response to the
injection rather than from the elevated glucose levels.
F) CONCLUSION. The effects of hypoglycemia and hyperglycemia, as well as of hypercapnia and of pHo buffers,
are consistent with the conclusion that the fall in pHi (or
pHo) during ischemia is a significant determinant of the
degree of cell death that follows global and focal ischemia. However, there are alternate explanations of the
effects of all these agents, except perhaps elevated CO2.
None of these alternate explanations negates the conclusion that the pH drop is important at this stage, but they
show that the question is not yet resolved. All the manipulations used to show the importance of pHi very probably affect pHo as well as pHi, so that the former could be
the damaging change. However, as seen below, extracellular acidity is likely to be protective so that the damaging
change is very likely to be pHi.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
times, by transient denaturation or other means. For example, endonucleases are activated by a pH of ;6.6 (288).
Of these possible mechanisms, the most likely to
cause damage is effects on free radical production. This
could well lead to a continuous relationship between pH
and damage, as appears to be the case. Maintaining cytosolic Ca21 at elevated levels after ischemia also constitutes a viable mechanism for damage enhancement and
needs to be considered.
8. Conclusions
Most of the existing data are consistent with the
hypothesis that the fall in pHi during ischemia is damaging. There are, though, points of contention in all three of
the major experimental approaches: use of hypoglycemia
and hyperglycemia in global ischemia and use of hyperglycemia in focal ischemia. These have to be resolved, but
until that time, it is prudent to conclude that the fall in pHi
probably (strongly) exacerbates damage during ischemia.
This is despite specific protective effects of small decreases in pHo (and possibly pHi).
The cellular site of action of low pH is not completely
resolved, and the apparently opposite effects in culture
and in vivo suggest that the vasculature may be important,
either because of breakdown of the blood-brain barrier or
activation of production of free radical or other toxic
substance. This being said, at this stage a site of action on
neurons, or glia, is in no way ruled out.
The mechanism by which decreased pH exacerbates
damage is not known. At this stage, effects on free radical
production, which appear to exist but have not yet been
extensively investigated, and effects on BDNF production, appear the most likely and are quite reasonable.
Certainly, the latter might explain the difference between
in vitro and in vivo results, since there is no particular
evidence that endogenous neurotrophin production exerts a protective effect in cultures, whereas there is good
correlative evidence for effects in vivo (see sect. VIIIC).
At this stage, it should be assumed that if the pH
change during ischemia in vivo was prevented, damage
might well be very severely attenuated. This very strong
effect of pH is suggested by the fact that hyperglycemia
and elevated CO2 enhance damage, despite delaying anoxic depolarization and attenuating the normal fall in
ATP, two events that are generally considered to exacerbate damage. A caveat here is the possibility that damage
after glucose injection is due to another, immediate effect
of the hyperglycemia, rather than the decreased pH; this
was discussed above. Hypoglycemia attenuates damage
despite enhancing the fall in ATP.
VII. INITIATORS OF ISCHEMIC DAMAGE
The primary event during ischemia is inhibition of the
electron transport chain and hence oxidative phosphory-
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tivation of Na1/H1 exchange, as apparently occurs in the
heart after ischemia when extracellular H1 is washed out
during reperfusion. Blockers of Na1/H1 exchange markedly reduce Na1 accumulation and infarct size (225, 911).
This has not been well tested in brain.
Another possible mechanism is enhancement of free
radical formation, which is likely to result from lowered
pH via iron delocalization and the Fenton reaction (725,
930). There is some evidence for such a mechanism. Focal
ischemic damage was exacerbated by preexisting hyperglycemia and, concomitantly, there was more than a twofold enhancement of brain free radical production in the
hyperglycemic animals. Free radical production was measured by salicylate microdialysis (1197). This is a profound effect. It is also quite possible that protonated
ONOO2 is more damaging than ONOO2 itself if so lowered pH would enhance damage in this way (1013). There
is one study testing whether free radical scavengers abolish the enhanced damage seen in hyperglycemia. The free
radical scavenger from Upjohn, Tirilizad, markedly improved the acidity-induced decrement in metabolic function measured 3 h after 30-min ischemia, without affecting
the large fall in pH, to 5.6, that occurred during this insult
(569). Although far too few in number, these studies
indicate that pH action may be mediated by enhancement
of free radical production.
Another possible mechanism of damage is suppression of neurotrophin synthesis. Glucose (20 mM) completely suppresses the activation of the immediate early
gene, c-fos (213), and both hyperglycemia and hypercapnia almost completely block the increased transcription
of BDNF mRNA that normally occurs in CA3 and the
dentate gyrus after 20-min global ischemia (1146). Those
regions are normally not vulnerable and synthesize
BDNF. It is possible that pH-induced vulnerability results
from failure to synthesize BDNF or other neurotrophins.
It would be of interest to see whether hypoglycemia allowed BDNF to be synthesized in CA1 of hippocampus
after ischemia; it usually is not.
Another mechanism, suggested by studies on cortical
cultures, is that the lowered pH may decrease the ability
of cells to lower cytosolic Ca21 after ischemia (741). This
effect is extremely strong. When pHo was adjusted to 6.6,
cytosolic Ca21 was maintained at ;50% over baseline
values for at least 100 min after a short exposure to
kainate or (AMPA-cyclothiazide), with no sign of decay at
that time (741). At pH 7.4, it returned within a few minutes. The basis for this is unknown at present, and it
promises to be difficult to work out as, for example, the
same phenomenon does not occur when cytosolic Ca21 is
elevated by NMDA. However, the effect may well occur in
vivo, and it would clearly maintain a potentially toxic
situation for a very prolonged period.
Finally, less specifically at this stage, pH may act by
altering key enzymes or structural proteins at critical
1511
1512
PETER LIPTON
A. Decreased ATP
1. Decrease in ATP and PCr
Decreases in ATP during anoxic/ischemic insults are
in Table 1. Not illustrated in Table 1 is the fact that the
ATP fall in global ischemia and, to an extent, in slices is
quite precipitous. Levels fall from ;90% to close to their
lowest values within about a 1- to 2-min time interval (553,
676, 717). This fall follows a significant depletion of the
PCr (PCr) stores; for example, the fall in ATP during
global ischemia only begins after PCr has declined to
between 10 and 20% of control values (553). This precipitous decline in ATP is predicted by analysis of the PCr
kinase-catalyzed equilibrium (1170)
ADP 1 PCr 1 H 1 7ATP 1 Cr
where Cr is creatine, and the fairly long delay is due to the
quite robust nature of the PCr kinase system in brain
tissue. Enzyme levels are high, particularly in synaptic
regions (339), and resting PCr levels are about twice as
high as ATP levels (294, 676).
2. Evidence that decreased ATP is important
in cell death
The decrease in ATP has many consequences that
might activate cell death, primary of which is inhibition of
the Na1-K1-ATPase with subsequent effects on intracellular Na1/K1 and membrane potential. Other important
consequences of the ATP fall may include activation of
glycolysis and the resulting fall in pH, activation of free
radical formation by increasing xanthine levels, and also
possible changes in phosphorylation states of different
enzymes and structural proteins. Transient inhibition of
the Ca21-ATPase in ER and in the plasma membrane may
also be important, although there is no direct evidence
that this is so in vivo. In addition, several key transporters, including Na1-H1 and the Na1-K1-Cl2 cotransporter,
require low levels of ATP for their activity (17, 385),
probably due to the importance of phosphorylation (17,
406) or to other events in the case of the Na1-H1 transporter (249). These might be adversely affected.
There are also protective effects. There is a 30-fold
increase in cAMP levels during global ischemia (783)
which is likely to come, at least in part, from adenosine
produced by the ATP breakdown (1215). There is little
work relating to how this increase affects damage and
that which exists is ambiguous. In the one direct study,
the phosphodiesterase inhibitor rolipram alleviated damage after 3-min global ischemia in the gerbil (540), indicating that cAMP may be beneficial, at least in this mild
insult. The only other relevant data concern type II
metabotropic glutamate agonists, which affect cAMP metabolism. These are protective when added before a
global ischemic insult (193, 910). Unfortunately, these
results are difficult to interpret. Type II receptors generally depress activation of adenylyl cyclase (436, 988), and
if this is the case, the results suggest cAMP is damaging,
contradicting the studies with rolipram. However, type
II-like metabotropic receptors also accentuate the production of cAMP that normally accompanies b-adrenergic
or adenosine receptor activation (1215). The very large
release of norepinephrine during ischemia (401), as well
as the released adenosine, may thus lead to a situation
wherein the type II receptors enhance cAMP accumulation. If so, then the above studies taken together would
indicate that cAMP production is protective in ischemia
(133). The situation remains unresolved at present, but
the weight of the evidence indicates a protective effect. If
so, this marks a protective effect of the ATP reduction.
Other more certainly protective effects of the ATP
decrease include dephosphorylation and inactivation of
NMDA (686), and probably L-type Ca21 (529, 616) channels, opening of ATP-sensitive K1 channels (444), and
production of adenosine (21).
Despite the strong likelihood that the fall in ATP is,
overall, damaging, there are only two reported direct
tests. In both of these, the PCr buffer system for ATP was
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lation. This is followed very rapidly by decreased levels of
high-energy phosphates, and a major consequence of the
fall in ATP [and possibly phosphocreatine (PCr) (1187)] is
inhibition of the Na1 pump with a subsequent increase in
intracellular Na1, decrease in intracellular K1, and a profound membrane depolarization that causes the macroscopic “anoxic depolarization” (717). The latter represents the sum of profound somatic depolarizations (1081,
1233). These changes are very dramatic during global
ischemia and also in the core of focal lesions. However,
changes are much less severe in the penumbra of focal
lesions. There are only minor changes in Na1 and K1, and
there is no anoxic depolarization. There are intraischemic
depolarizations in the penumbra during ischemia, in
which profound membrane depolarization and Na1/K1
changes occur for periods of several minutes. Levels of
ATP do not fall nearly as much as in global ischemia or
the core.
These events are very probably the basis of all ensuing damage, yet they do not, themselves, cause molecular
damage. In that sense, they are considered initiators of
ischemic damage.
The goal of this section is to evaluate the evidence
that the profound fall in ATP, the anoxic depolarization,
and the increased intracellular Na1 are responsible for
ischemic cell death in global ischemia. Other immediate
effects, such as free radical generation from the inhibited
electron transport chain and the decrease in pHi, have
been considered elsewhere.
The situation in focal ischemia is considered in section VIID.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
3. Regeneration of ATP after ischemia
It is possible that the recovery rate of ATP after short
lethal durations of global ischemia may be important in
development of damage, since a slow recovery rate will
significantly extend the short period of energy depriva-
tion. The rate in vivo has not been measured carefully, but
in slices, levels are back to their maximal levels after 10
min (533). The rate of recovery may be accelerated by the
lactic acid buildup during ischemia. Although this has not
been tested directly, brain slices recover far better from in
vitro ischemia when lactate is the postischemic substrate
than when glucose is the substrate (1009). The basis for
this may well be that glucose metabolism requires phosphorylation by hexokinase and so may be slowed during
conditions of low ATP. Measurements on ATP recoveries
have not yet been made, but it seems probable that the
resistance to damage is due to more rapid recovery of
ATP in the lactate-exposed slices. It is not known whether
this phenomenon occurs in vivo.
4. Conclusion
Although experimentally verifying the importance of
the ATP fall in damage may seem “academic,” it is not. It
is possible to envision a damage scenario in which the fall
in ATP is irrelevant (or protective) and where damage
stems from the blockade of the mitochondrial respiratory
chain. The latter could produce increases in free radicals
and in cytosolic Ca21 and, via opening of the MTP, might
also trigger apoptosis. The increased cytosolic Ca21 could
increase the spontaneous vesicular release of glutamate,
leading to further toxicity. The decreased oxidative phosphorylation would lead to a lowering of pH. The single set
of data opposing this possibility in vivo is that where
creatine preincubation was protective against metabolic
inhibitors (736). Although limited, these data are quite
persuasive in confirming that the fall in ATP is damaging.
However, they were carried out on chemical insults, not
ischemia. Further studies are very much necessary to
verify the very fundamental hypothesis that the fall in ATP
is a key initiator of ischemic damage.
B. Anoxic Depolarization
A very important review of early ion changes and
anoxic depolarization was published some 12 years ago
by Hansen (417), and there are more recent excellent
reviews (554, 717).
1. Characteristics of anoxic depolarization
Anoxic depolarization is a cardinal feature of anoxia/
ischemia in vivo (537, 604, 635, 1231) and in slices (58, 60,
611, 1038). It has not been observed in cell cultures. It occurs
between 60 and 180 s after onset of global ischemia in situ,
and between 1 and 12 min after the onset in slices. In the
latter it is very dependent on glucose levels in the incubation
medium (950) and on the level of anoxia. Anoxic depolarization is observed in the core of focal ischemic lesions (188)
but does not occur in the penumbra.
The phenomenon is characterized by a large (10 –30
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fortified by preexposure to creatine. Early studies were
done in the hippocampal slice preparation, where preincubation with 25 mM creatine built up a store of creatine
phosphate (676) that retarded the fall in ATP during anoxia (531, 532, 676). This in turn retarded the fall in
transmission (676), the anoxic depolarization (59; D. Lobner and P. Lipton, unpublished data) and strongly attenuated the Ca21 entry into the cells during 10-min anoxia
(532). In terms of long-term damage, the creatine preincubation quite dramatically reduced the damage to synaptic transmission (531) and protected against anoxic
damage to protein synthesis (165), measured 1–3 h after
the anoxic insult. It did not attenuate the anoxic decrease
in pHi, measured using the creatine kinase equilibrium
(531). Thus attenuating the fall in ATP significantly delayed the appearance of acute forms of damage. More
recent studies indicate the protective effects are achieved
at much lower creatine concentrations, of 1 mM (59).
In vivo, animals were kept on a regime of dietary
creatine for 2 wk and then exposed, not to ischemia, but
to levels of the mitochondrial inhibitors malonate or 3-nitropropionic acid, which lowered ATP by ;50% for several hours. The creatine diet elevated PCr by ;60%, far
less than in the slice studies, but it dramatically reduced
the size of the infarct produced by each of the mitochondrial inhibitors (736). This strongly suggests that maintaining ATP levels during the insult is protective. Measured effects of creatine showed it maintained ATP levels
relative to control and also decreased free radical and
peroxynitrite production. Unfortunately, those measurements were made at the time the infarct had developed in
the untreated tissue so that ATP and other metabolite
levels in healthy tissue were being compared with levels
in damaged tissue. Thus the differences may not reflect
effects of creatine during the time when damage was
developing. Measurements before infarct formation
would show whether the creatine pretreatment did, in
fact, attenuate the ATP fall and free radical production.
In contrast to these results, hyperglycemia increases
damage, despite maintaining ATP for a longer period
during the ischemia. This is thought to be because the
damaging effects of decreased pH overwhelm the marginal protection from the delayed fall in ATP, but this has
not been proven. Another explanation, based on the fact
that in some instances mild hyperglycemia was damaging
although it did not increase the fall in pH, is that ATP
maintenance is actually damaging (465). Although this
seems unlikely, there are several ways this might occur,
as discussed above.
1513
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PETER LIPTON
Volume 79
rats, the anoxic depolarization always occurred within 5 s
of the time at which the ATP suffered a precipitous fall
(between 60 and 90 s) (553). Average ATP values were
40% at the time of anoxic depolarization. In slices, there is
a good correlation between the buildup of PCr in creatinefortified buffers and the duration of anoxia required for
anoxic depolarization (59).
The other reason for ascribing a major role to pump
inhibition is that ouabain, the specific inhibitor of the
pump, leads to a massive depolarization that has the
appearance of anoxic depolarization. In fact, low concentrations of ouabain, which mimicked the levels of Na1K1-ATPase inhibition seen during anoxia, led to a large
depolarization at almost the same time as the anoxic
depolarization (R. Rader, T. Lanthorn, and P. Lipton, unpublished data). A similar but more rapid effect of a
higher (100 mM) dose of ouabain was observed by
Balestrino (57). A third reason to conclude that the anoxic
depolarization is due to pump inhibition is that it is essentially independent of extracellular Ca21 (1264), and
NMDA receptor activation (394, 611, 635, 934, 1231) and
only very slightly (1231) or not at all (635) dependent on
non-NMDA glutamate receptors. The ability of pump inhibition to cause the rapid depolarization is thought to be
due to the very high surface-to-volume ratios of neuronal
elements, which means that pump inhibition produces
rapid changes in ion concentrations (675, 677).
2. Mechanism of anoxic depolarization
If pump inhibition is indeed the basis for the anoxic
depolarization, the shift in K1 as well as any specific
permeability changes are likely to be responsible for the
majority of the depolarization (378). The electrogenic
contribution to membrane potential in vivo is not known
but is not generally very large (1115).
Glibenclamide, which blocks the ATP-sensitive K1
channels, moderately slows the increase in extracellular
K1, suggesting some of the K1 efflux is via this channel.
Excitable Na1 channel blockers such as tetrodotoxin,
lidocaine, and phenytoin both in situ (1229, 1231) and in
slices (1109, 1195) delay the anoxic depolarization from 2
to 6 min in situ and from 6 to 9 min during a relatively mild
insult in slices. The delay is probably a direct result of
blocking Na1 influx, and hence Na1/K1 exchange after
inhibition of the pump. By virtue of blocking the Na1
entry, the drugs also reduce the rate of fall in ATP (105,
338), and this will add to the delay in anoxic depolarization.
Exaggerating the ischemic reduction of pH, either by
hypercapnia in situ (552) or by extracellular H1 in slices
(1119), delays the anoxic depolarization dramatically. The
mechanism of this effect is not known. Presumably, a
critical ion channel is inhibited so that Na1/K1 exchange
is delayed.
The basis for the anoxic depolarization has not been
determined. There are three reasons for thinking that it is
largely due to inhibition of the Na1-K1 pump.
First, there is a consistent, and strong, correlation
between cell levels of ATP and the occurrence of the
anoxic depolarization. Conditions that increase the rate of
ATP depletion, such as hypoglycemia (613) and 0 mM
Ca21 buffer, which increases the rate of depletion of ATP
in hippocampal slices (686), shorten the time to anoxic
depolarization, whereas conditions that slow the rate of
ATP depletion, such as hyperglycemia (613, 950, 1040),
creatine preincubation (59), hypothermia and barbiturate
anesthetics (1168, 1233), all increase the time to anoxic
depolarization.
Several studies have established the correlation quite
quantitatively. In rats maintained at different core temperatures (38 and 28°C), or given different doses of anesthesia, ATP levels were between 13 and 18% of control at the
time of anoxic depolarization whether this was 73 s (normothermia), 125 s (high levels of anesthesia), or 242 s
(hypothermia) (1168). In another study, the very rapid
kinetics of ATP depletion and anoxic depolarization in
cortex were measured during cardiac arrest. In different
3. Ion fluxes and transmitters during
anoxic depolarization
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mV) extracellular negative direct-current shift that occurs
in both neuropil and cell body regions of the ischemic
tissue. This is invariably accompanied by precipitous decreases in extracellular Na1 (to ;50 mM), Cl2, and Ca21
(to ;0.1 mM), and by a rise in K1 (to ;60 mM) (611, 635,
717, 1231). There is a rise in extracellular impedance, or
extracellular marker concentration (537, 604), indicating
significant cellular swelling that is almost undoubtedly
due to isotonic entry of Na1 and Cl2 (417).
Although intracellular ion changes cannot be measured with the same temporal precision as extracellular
ion changes using electrodes, there are large increases in
intracellular Cl2 in hypoglossal neurons at about the time
of depolarization (508), and there is an approximate doubling in the level of intracellular Na1 after 5 min of anoxia
in the hippocampal slice (338). These almost certainly
reflect the acute changes seen extracellularly.
There is a large depolarization of pyramidal cells (to
near 0 mV) at the time of the anoxic depolarization in
slices (934) and in situ (1233). This is associated with a
slight increase in membrane resistance in situ and a 50%
or more decrease in resistance in the slice (858, 934). The
basis for this difference is not known (1233), but it is clear
that no huge membrane resistance change can account
for the massive depolarization. Thus it probably results
from the ion changes rather than membrane permeability
changes. The concomitant failure of transmission in the
presynaptic terminals of the Schaffer collaterals in the
hippocampal slice (see below) shows that the depolarization affects neuropil elements also.
October 1999
CEREBRAL ISCHEMIA ON NEURONS
4. Role of anoxic depolarization in cell death
C. Increased Cell Na1
1. Evidence for a role in cell damage
There is a large increase in cell Na1 during the first
few minutes of anoxia or ischemia that coincides with the
anoxic depolarization.
Drugs that block Na1 fluxes and prevent these ion
changes are always strongly protective in both permanent
focal (388, 939, 1058) and global (226, 245, 343, 693, 1036)
ischemia. Most of the drugs used are quite specific for
Na1 channels, although they may, of course, have other
undocumented effects. They include lamotrigene and related compounds (245, 388, 1058), tetrodotoxin (693), and
lidocaine (343). Temperature appeared to be very well
controlled throughout the permanent focal ischemia studies (388, 1058) and was well controlled, or shown to be
unaffected by the drug during, and for ;15 min after the
global insults (226, 343, 693). It was not maintained or
measured after this so that it is possible that drugs, which
are potent inhibitors of Na1 channels and hence neural
activity, may have affected temperature during the hours
after ischemia. There is no strong reason to think this is
the case. Thus Na1 entry is very likely to be a major cause
of damage.
Because of the large Na1 entry during ischemia, and
the glutamate release at that time, one would expect the
effects of Na1 entry to be exerted early during the insult.
This may be true. However, the Na1 channel blockers
discussed above are very effective in preventing damage
from global ischemia when applied 15 min (226) after the
end of global ischemia and even 2 h after the insult (245).
This quite surprising result suggests that Na1 entry is
(also) causing damage in the postischemic period. Drug
effects cannot be due to seizure blockade since postischemic seizures are not present in rats. It has not been
possible to isolate the action of the drugs to the beginning
of the insults, which would allow determination of
whether the early entry of Na1 is also damaging. It may
well be. However, it is clear that delayed Na1 entry is
damaging.
2. Possible mechanisms of Na1-induced damage
There are several ways that Na1 might be damaging
during the acute phase of the insult. These have been
discussed previously and include causing glutamate release, increasing cytosolic Ca21, and helping to deplete
ATP levels via activation of the Na1-K1 pump. The latter
is well-illustrated by the fact that tetrodotoxin and lidocaine slow the rate of the ATP fall during anoxia (105,
338). Entry of Na1 (and Cl2) will cause intracellular
edema, but there is no particular reason to think this is
damaging.
The postischemic involvement of Na1 channels in
global insults means that a very important effect of ischemia
is to enhance the damaging efficacy of Na1 channels for a
prolonged period. One possibility is that their open probability increases either due to a change in channel properties
or to a small maintained depolarization. A similar effect is
indicated in studies of glutamate toxicity. In cultured striatal
neurons, a continual cycle of Na1 entry, glutamate release,
and NMDA-mediated activation of NOS persists for many
hours after an initial 5-min glutamate exposure and accounts
for the development of damage. Free radical damage from
NO elevation is the actual damaging step (1074). This “vicious cycle” (1074) may result from damage to any one of
the components, but a reasonable target is the Na1 channel,
and indeed, damage is prevented with tetrodotoxin. Such a
mechanism might well be operative in the penumbra of focal
ischemia because damage there is sensitive to delayed
NMDA blockade (771, 833) (see sect. VIID). However, global
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This is very difficult to determine. Damage is strongly
correlated with the anoxic depolarization, in that artifactually delaying the anoxic depolarization in one of several
ways almost always increases the duration of anoxia or
ischemia that is necessary for damage. This is so for
pharmacological agents (60, 1109), temperature variations (805), age (950), osmolarity of anoxic solution (472),
and comparisons between different brain regions (58).
Furthermore, there are massive biochemical changes and
ionic changes at the time of the anoxic depolarization,
including the very large increase in FFA during global
ischemia (553). The reason it is hard to conclude that the
anoxic depolarization itself contributes to the damage is
because the anoxic depolarization is coincident with, and
very probably dependent on, the decrease in ATP levels,
and increase in Na1 entry, two factors which almost
undoubtedly strongly activate damaging events. There is
currently no way to prevent the anoxic depolarization
without affecting the fall in ATP and rise in Na1 also. The
main argument that the anoxic depolarization is important is that blockers of voltage-dependent Na1 channels
are protective, suggesting that depolarization-induced
opening of these channels is important. However, as seen
immediately below, this protection is manifested when
the Na1 channel blockers are added several hours after
the anoxic depolarization is terminated. To the extent that
the anoxic depolarization is critical for glutamate release,
it is very probably important in damage. However, the
involvement of the anoxic depolarization in this release is
not clear.
The fact that CO2 exacerbates damage while greatly
slowing the anoxic depolarization shows that the anoxic
depolarization is not essential for damage. It is clearly a
marker for profound metabolic change but may not itself
contribute to the damage; that is, damage might be just as
great if there were no anoxic depolarization.
1515
1516
PETER LIPTON
D. Events in Focal Ischemia
The core of the focal insult, which experiences the
large changes in ATP, Na1, and membrane potential, is
not protected by any manipulations affecting Na1 entry
so that it is not possible to determine whether these
events are damaging, although it seems very likely that
the fall in ATP and membrane depolarization are important. It is not clear why Na1 entry blockers do not ameliorate damage.
The penumbra does not suffer the massive ATP loss,
anoxic depolarization, and Na1 entry, so the role of these
changes in damage is moot. The minor loss in ATP may
well be critical, but to show this explicitly, it would need
to be prevented, and this is extremely difficult over such
a long duration. The Na1 channel blockers prevent damage in the penumbra of permanent focal ischemia when
added 1 h after the start of ischemia; no studies are
reported for temporary ischemia (388, 939, 1058). Damage
in the penumbra is very sensitive to glutamate, and the
channel blockers do prevent glutamate release, so this
could account for their effects. Alternatively, or in addition, it is quite possible that the vicious cycle described in
section VIIC might well be responsible for Na1-dependent
damage; damage in the penumbra is prevented by NMDA
blockade, both early during permanent ischemia and soon
after temporary ischemia (771, 833). Neither the importance of Na1 entry nor the timing of any effect has been
studied for temporary focal ischemia.
E. Summary
The fall in ATP, the anoxic depolarization, and the
initial rise in cytosolic Na1 are three very early ischemic
changes in global ischemia and in the core of focal ischemia that are closely interrelated. They are all quite
strongly correlated with damage in that they occur early,
and when they are blocked, damage is almost invariably
attenuated. (The only time this is not true is during hyperglycemia or with elevated CO2 where damage is en-
hanced despite the fact that all these processes are
slowed. These results are very probably an indication of
the importance of the pH change in damage.)
It is difficult to know with any certainty whether the
ATP fall and the anoxic depolarization are indeed contributing to the damage in global ischemia. Blockade of the
fall in ATP with creatine incubation is very protective
against early functional damage in slices and in a chemical in vivo model of energy deprivation, but this has not
yet been shown in an in vivo global ischemic model. If it
were it would show the importance of the fall in ATP.
There is no independent evidence that the anoxic depolarization is critical either; the correlations between anoxic depolarization onset and damage may well be explained by the effects of the different conditions on the
fall in ATP or on entry of Na1. There is no explicit
demonstration of delaying anoxic depolarization without
delaying the fall in ATP or the rise in Na1.
There is strong evidence that Na1 entry is important.
However, here too, the results are not simple. If Na1 entry
during the insult was damaging, this would strongly implicate the pathway comprising ATP decrease, membrane
depolarization, and massive Na1 entry. However, the
identified damaging Na1 entry appears to be after the
initial dramatic changes so that its direct cause is neither
the large decrease in ATP nor the anoxic depolarization.
The early Na1 entry may also be damaging, but this has
not been directly demonstrated.
Thus experimental evidence for the sequence of ATP
fall, membrane depolarization, and massive Na1 entry as
a principle way by which ischemic damage is initiated in
global ischemia is woefully lacking. Certainly many conditions that slow the fall in ATP and the anoxic depolarization are protective. However, they also doubtless slow
other sequelae of energy metabolism inhibition, such as
the pH fall and free radical generation.
At this stage, it remains possible that mitochondrial
changes are sufficient, without mediation by ATP, as discussed above. Certainly, it is difficult to accept that the
fall in ATP and the anoxic depolarization may simply be
epiphenomena; however, experiments need to be done to
show otherwise.
Sodium channel blocker studies do show an extremely important role for Na1 entry in damage, both in
global ischemia and in the penumbra of focal ischemia.
Damage is strongly attenuated by several channel blockers, and effects of temperature have been reasonably well
controlled. However, this damaging Na1 entry occurs at a
relatively late stage, well after the massive Na1 influx in
global ischemia has subsided. It most likely results from a
long-term membrane change, possibly in the Na1 channels themselves. The origin of this change is not known,
but its apparent importance in damage makes it extremely
important to study.
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ischemic damage is generally not attenuated by postischemic NMDA blockade (except when the ischemia is mild,
Ref. 167) so that the same mechanism cannot be operating
there. However, if Na1 channels are altered, then continual
Na1 elevation with resultant cytosolic Ca21 elevation might
well lead to damage in the same way. This seems the most
likely mechanism for the Na1-dependent damage.
This does not obviate the possibility that the early
massive entry of Na1 is also damaging. There are clearly
many mechanisms by which it might help set in motion
events that lead to the delayed Na1 toxicity. However
absent the delayed entry, it would not cause cell death,
because blocking the delayed entry later strongly attenuates cell death.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
VIII. GENE ACTIVATION AND TOLERANCE
A. Immediate Early Genes
1. Gene activation
There is a rapid transcription of a large number of
immediate early genes including c-fos, c-jun, junB, krox20, and zif-268 within the first 30 min after global ischemia, during or after focal ischemic insults (19, 212, 456,
485, 556, 567, 575, 1061, 1223), and after hypoxia/ischemia
(270). There are good recent reviews that detail these
increases and their localizations (8, 323, 593), and there is
much discussion as to whether they play protective, destructive, or no roles (8, 566, 1259). Overall, this issue has
not been completely resolved, but certainly the nature of
the proteins that are eventually changed suggests some of
the early genes are important. The mechanisms of these
early gene activations are not known. Synthesis is significantly blocked by MK-801 in gerbil global (1223) and rat
focal (212) ischemia, suggesting that Ca21 entry is important. Other factors have not been identified, although free
radicals are certainly able to activate immediate early
genes (864), as is PAF (70).
2. Localization of protein increases
Far more important than the mRNA synthesis is
whether proteins are synthesized. In gerbils, transcription
of several early genes increases equally in all hippocampal
regions (567, 1148, 1223); however, although the proteins
are upregulated in several regions, there is almost no
expression of the proteins in the vulnerable CA1 cells.
Levels sometimes fall below baseline (567). There are no
studies that directly address whether there is a causal
relationship between the lack of synthesis of the proteins
and the ensuing cell death. Certainly, there are situations
in which cells go on to die despite immediate early gene
expression. For example, in the young rat exposed to
15-min carotid occlusion and 8% O2, there is an expression of several immediate early gene proteins within an
hour, throughout a region of the brain in which there is
massive delayed cell death (270). Still, this does not obviate the possibility that synthesis of one or more immediate early gene products is critical for recovery, at least
in global ischemia. Studies with controlled gene knockouts are necessary to determine the roles of these early
genes.
B. Heat Shock Proteins
1. Pattern of heat shock protein expression
after ischemia
The pattern of heat shock protein expression after
global ischemia in the gerbil is very similar to that of the
immediate early genes. Messenger RNA for heat shock
protein (HSP) 70, HSC70 and HSP90 begin to rise within a
few minutes of the insult in all regions, and persist (485,
556, 1223). The protein is upregulated in nonvulnerable
neurons within a few hours but, as first noted by Nowack
(840), there is no increase in HSP in vulnerable CA1 (556,
567, 1148, 1167).
The rat is very different from the gerbil. After ischemia, HSP70 and HSP72 are expressed as strongly, or in
one case much more strongly, in CA1 than in CA3 and
dentate granule cells (681, 1050). Expression is absent in
all regions at 2 h but is very strong at 1 and 3 days after the
10-min insult. To the extent that damage is similar in the
gerbil and rat, this argues against a role for HSP as a
major determinant of cell death/cell protection.
Neurons in the ischemic core of a temporary MCA
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Ischemia activates synthesis of potentially protective
and potentially damaging proteins. The functional significance of some of these proteins is considered in this
section.
An underlying pattern, to which there are important
exceptions, is that the protective proteins are synthesized
in relatively nonvulnerable cells and are not synthesized
in vulnerable cells, or cells that will die (see Table 5). This
raises the possibility that the absence of these proteins
may be important in development of cell death. This is
supported by the phenomenon of tolerance, in which
normally vulnerable cell populations become resistant to
damage. Evidence suggests that tolerance results from
synthesis of protein that is normally not synthesized in the
vulnerable cells but that is synthesized in cells that do not
go on to die.
In contrast to the protective responses, at least four
putatively damaging proteins, COX-2, nNOS, iNOS, and
Bax, are synthesized, as are at least two cytokines, IL-1b
and TNF-a, which are largely damaging, and there is good
evidence that they greatly enhance cell death (185, 420,
477, 837). These damaging proteins are synthesized in
both global and focal insults.
Mechanisms of gene activation are extremely complex
in that they involve many signals and transcription factors.
In general, the mechanisms by which genes are activated by
ischemic conditions, and in postischemic periods, have not
been worked out, so I do not consider them in any detail.
Nevertheless, it is important to point out that Ca21, neurotrophins, PAF, as well as neurotransmitters acting at
metabotropic receptors are all capable of activating genes
via the MAP kinase system or other pathways. Much of this
is very nicely summarized elsewhere (345). Mitogen-activated protein kinase is hyperphosphorylated soon after
global ischemia in most regions (468) and so may well
mediate early effects; increased Ca21 may also do so by the
MAP kinase or by alternate pathways (510), as may free
radicals (864), for example as a result of NFkB (206, 597) or
AP1 activation (1018).
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PETER LIPTON
2. Mechanism of HSP increase
As for immediate early genes, induction of HSP by
ischemia is dependent on activation of NMDA receptors.
MK-801 blocked its production in carefully temperaturecontrolled studies in gerbil (546, 1148). Free radicals may
well play a part in the induction of the HSP; they do after
ischemia in liver (998). In that sense, free radicals may be
protective. Similarly, the inhibition of HSP (and immediate early gene) formation by MK-801 will tend to make
NMDA blockade damaging rather than beneficial.
3. Protection by HSP
Overexpression of HSP70 protects cultured neurons
against ischemic damage (2-h in vitro ischemia, 4-h recovery) (18) and also protects astroglia (871). The amount of
LDH release was reduced from 80 to ;18% of available
LDH. Thus HSP certainly has the potential to protect
against ischemic damage.
There is no well-accepted mechanism for how protection occurs. Expression of the protein inhibits the
activation of the transcription factor NFkB (317), which is
activated in ischemia and appears to be associated with
toxicity. This might well be the protective mechanism; it
has not been studied explicitly. The 70-kDa HSP also
protects against apoptosis, by blocking the downstream
actions of caspases on activation of PLA2, the cell nucleus, and other processes (499). This would certainly be
a protective mechanism in ischemia. The major biophysical action of the HSP70 and HSP90 classes of proteins
with their coactivator proteins is to bind to unfolded or
partially unfolded peptide chains, largely by binding to
hydrophobic regions of the protein, and to ensure that
proper folding occurs (440). To the extent that they are
protective against ischemia in this way, it suggests that
loss of protein structure is an important component of
ischemic cell damage. If so, this is a very important conclusion. Nothing is known about possible molecular targets of these protective actions.
C. Neurotrophins and Neurotrophic Factors
1. Pattern of expression after ischemia
Basal levels of neurotrophins are lower in CA1 than
in other hippocampal regions. This is consistent with their
importance in damage (599). There are several somewhat
conflicting reports of increases in mRNA for BDNF, trkB
receptors, and NT-3 in hippocampus that differ in their
descriptions of onset times and durations. However, message levels are certainly increased by global ischemia in
all regions (667, 754, 1100, 1139). However, as with other
putatively protective proteins that have been discussed,
protein levels do not rise in vulnerable regions. After
global ischemia in the rat, there is a small increase of
BDNF in dentate gyrus at 24 h, little change in CA3, and a
large decrease of BDNF in vulnerable CA1 and neocortex
(599). This appears to occur largely in neuropil, perhaps
in the dendrites (1239). This same large decrease is seen
in the gerbil at 24 h (321). Thus, considering the combination of low basal levels, and changes during ischemia,
the vulnerable CA1 pyramidal cells have a much lower
level of this neurotrophin than other regions at critical
times. Probably of equal importance, there is only a very
low density of trkB receptors on CA1 pyramidal cell somata compared with other regions of the hippocampus
(321). This contrasts with the population of parvalbuminstaining GABAergic neurons in the CA1 pyramidal cell
layer (321) which strongly express trkB. This may be the
basis for the survivability of these neurons. Receptors for
another protective factor, epidermal growth factor (889),
are reduced in CA1 pyramidal cells 24 h after ischemia
(321). Thus, to the extent that growth factors offer protection, CA1 pyramidal neurons are certainly vulnerable.
2. Protective effects of exogenous growth factors
Neurotrophic factors mitigate ischemic cell damage
in several different preparations. They are usually added
24 – 48 h before the insult to allow full expression of their
induced proteins and are maintained throughout the insult and recovery phase. However, such a preincubation is
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occlusion never show an HSP70 immunocytochemical
response and show only a very small mRNA response
even though they are morphologically intact for the first
6 h after the 2-h exposure. Neurons in the penumbra,
many of which recover, show a marked HSP70 production
(576, 655). Thus the correlation between HSP and resistance to damage is present in focal ischemia.
Ubiquitin, a low-molecular-weight HSP involved in
conjugation and breakdown of damaged proteins is dramatically decreased in both gerbil and rat hippocampus
within hours after ischemia and then recovers over the
next 1–3 days in all cell types except CA1 cells that are
destined for death (251, 703, 1241). This phenomenon may
be involved in damage, possibly by allowing buildup of
partially denatured proteins (703), but unfortunately,
there is no further evidence to this effect.
In summary, in the gerbil, the anatomical association
between HSP expression and recovery is consistent with
the conclusion that HSP is necessary for survival and is
involved in survival. Although HSP may be necessary, it is
certainly not sufficient in the rat, at least when expressed
1 day after the insult. CA1 cells die despite expressing
HSP. Perhaps the absence of expression in CA1 earlier
than 1 day after ischemia is critical, but there is no particular reason to think so. At the present time, experiments in which HSP are knocked out are necessary to test
their role in protection of nonvulnerable neurons. As is
shown in section VIIIF, their upregulation in vulnerable
cells partially overlaps the rescue of these cells.
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3. Damaging effects of exogenous growth factors
Unlike the in vivo cases described above, preexposure to exogenous BDNF actually exacerbates damage
from ischemia and from high levels of NMDA in mouse
cortical cultures (594). This is associated with increased
Ca21 influx (594). Free radical-induced necrosis is also
enhanced by BDNF in these cells (403), and the growth
factor may exacerbate mitochondrial free radical production (D. Lobner, personal communication). At least a
partial explanation for this toxicity is that the BDNF
preincubation leads to an increased number of nNOSpositive neurons in the culture (986). Other possible
mechanisms of damage include activation of MAP kinase
and subsequent activation of cytosolic PLA2 (345). At the
moment, this effect does not appear to play a role in in
vivo ischemia.
4. Mechanism of protection by growth factors
There is little known about how endogenous, or exogenous, growth factors might protect against ischemic
damage. Some growth factors may act by inhibiting Ca21
increases. Acidic fibroblast growth factor administered
soon before the insult delayed the early rise in cytosolic
Ca21 during in vitro ischemia in gerbil hippocampal slices
by ;8 min (767), certainly enough to afford protection
against the 5-min (or 10-min) insult in gerbils. It is not
known if this effect pertains in vivo. The quite rapid
effectiveness in this case, and when BDNF was administered just before focal ischemia, suggest that (some of
the) protection by growth factors in ischemia results directly from rapid effects of the factors such as phosphorylation (281), rather than from resultant gene activation.
Protection against NMDA toxicity by BDNF occurs rapidly, within 4 – 8 h. The protection against submaximal
doses is very profound and is associated with preventing
the decrease in PKC that normally occurs, and that has
been shown to be instrumental in damage (1130). The
mechanism is not known, because the mechanism for the
downregulation of PKC is not known. However, the fact
that PKC is also downregulated in ischemia suggests that
BDNF may protect in this way against ischemic damage.
Neurotrophins also exert a concerted set of changes
that reduce the increase in free radicals (737). This appears to be dependent on protein synthesis. Twenty-fourhour preexposure to BDNF increased SOD activity and
glutathione reductase activity in rat hippocampal cultures. This led to a marked decrease (;50%) in free
radical generation 30 min after exposure to glutamate, as
well as protection against glutamate toxicity (737). Such a
mechanism might help rescue postischemic neurons, although this effect has not been shown in vivo. There is a
maintenance and upregulation of SOD in nonvulnerable
neurons and a loss of SOD in CA1 pyramidal cells 1 day
after global ischemia (680, 734). This is likely to be important in differential sensitivity of the neuronal populations and may well result from the differences in BDNF
metabolism.
Maintenance of protein synthesis is mediated in part
by tyrosine kinase systems that are activated by neurotrophins or growth factors. As discussed previously, the
absence of protein synthesis in vulnerable neurons has
been suggested to result from the absence of neurotrophin actions (468).
5. Conclusions
Basal neurotrophin levels and changes of levels in
different cell populations, as well as known protective
effects of neurotrophins in ischemia and other challenges,
suggest that they may act as endogenous neuroprotectants. (There is no evidence for a relevant damaging
role in vivo.) If so, there are many possible sites, most
dominant of which are prevention of free radical accumulation, prevention of PKC degradation, and, possibly, reestablishment of protein synthesis. They may also attenuate delayed Ca21 increases, but this is very speculative.
A major gap at this stage is a demonstration that knocking
out BDNF (or other neurotrophin) or pharmacological
neutralization of BDNF (525) leads to increased sensitivity to ischemia in vivo.
D. Bcl-2/Bax System
1. Protein expression after ischemia
As with HSP, The Bcl-2 group of proteins is selectively upregulated in nonvulnerable regions of hippocampus after global ischemia. This occurs in both rat and
gerbil and in this respect is a more viable candidate than
HSP70 or HSP90 as a major protectant molecule. In-
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not necessary. BDNF protected against ischemic cell
death in CA1 pyramidal cells of the rat (73, 1139) and in
the GABAergic reticular nuclear cells of the thalamus
(559) after global ischemia. In one of the first two cases,
the BDNF was added only 90 s before the insult and was
maintained throughout recovery (73). The neurotrophin
also provided very strong protection against infarct in
young rats 7 days after 2.5-h hypoxia-ischemia, and in this
case it was administered right before the insult. It was
quite protective even when added immediately after the
insult (192). The degree of protection depends very
strongly on the presence of trkB receptors, and almost all
neurons containing these at high densities are protected
by the neurotrophin (192, 320). A causal relationship has
not been established however.
Acidic fibroblast growth factor protects CA1 cells of
the gerbil against global ischemia (702, 990), as does a
mixture of basic fibroblast growth factor and NGF (1026).
Trophic factors block apoptotic death (664), excitotoxic cell death (737), and ischemic cell death (112) in vitro.
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PETER LIPTON
2. Mechanism of protection against damage
Bcl-2 strongly protects against free radical-mediated
cell death that is probably necrotic in a way that is not yet
known (524). It also has profound protective effects on
mitochondrial function, greatly enhancing maximal Ca21
uptake and reducing the ability of increased mitochondrial Ca21 to impair respiration, presumably by preventing opening of the MTP (787). These effects are likely to
protect against necrotic cell death. Bcl-2 also strongly
protects against apoptosis. One way it does this is to
prevent cytochrome c release and thus prevent activation
of the ICE-like proteases (591, 774, 1066, 1250). It also
protects downstream from the mitochondrial release of
cytochrome c (940).
E. Gene Products That Enhance Damage
As discussed in previous sections, gene products that
should enhance damage are also expressed in the postischemic period, after transcription of their mRNA. These
are COX-2, iNOS, nNOS, and cytokines, particularly IL-1b
and TNF-a. c-Jun expression may also enhance damage.
Other proteins, discussed more fully elsewhere, include
Bax, Bcl-x short, and cell adhesion molecules.
1. COX-2 and iNOS
Cyclooxygenase-2, which activates prostaglandin metabolism and hence leads to generation of free radicals,
was upregulated at 6, 12, and 24 h after 2-h focal ischemia
and was found in neurons at the advancing edge of the
penumbra (837), consistent with its involvement in cell
death. When viewed immunohistochemically, the COX-2
was usually associated with shrunken neurons showing
the basic ICC morphology, suggesting its involvement in
necrosis. Enzyme activity itself was very probably upregulated, because prostaglandin levels (PGE2) were elevated
about threefold at the time that COX-2 levels were maximally elevated. Furthermore, the COX-2 activity quite
probably contributed to damage because doses of the
COX-2 inhibitor, NS-398, which blocked ischemic PGE2
production by 50% reduced infarct size by ;30% (837).
Cyclooxygenase-2 is also upregulated after global ischemia and, there too, its inhibition reduces damage (806).
There is also synthesis of iNOS (478). In focal ischemia this is largely in endothelial cells or invading neutrophils (477). The time course closely follows that of
COX-2 (837). As was the case for COX-2, an inhibitor of
this enzyme, aminoguanidine, which does not block
nNOS, protected against both transient and permanent
focal ischemia, reducing infarct size some 35% (479, 481).
Aminoguanidine also inhibits polyamine oxidase (208), so
it is not completely specific. However, iNOS knockout
mice have markedly reduced delayed development of the
infarct, showing that the enzyme is involved in the continuing progression of damage after 48 h.
Inducible NOS was also elevated within 1–3 days
after 10-min global cerebral ischemia in rats, shortly before pyramidal cells died. The elevation was in the active
glia (303, 477). No pharmacological studies have been
done to see if the enzyme induction is important in damage, but it certainly may be.
There is a fundamental interaction between these
enzymes in that about one-half of the enhanced COX-2
activity after focal ischemia results from the action of the
iNOS (836), presumably via NO. This was determined by
both pharmacological inhibition and knockout of iNOS. It
is also likely that the superoxide produced by prostaglandin metabolism interacts with the NO from iNOS to form
peroxynitrite, creating another interaction between these
two enzymes. Syntheses of both these enzymes, and particularly COX-2, seem to be very important in full development of both focal and global ischemic damage.
The most likely basis for the increased activities of
these enzymes is NFkB activation, discussed fully in section VA, although studies have not addressed this issue
explicitly, either in terms of whether NFkB activation is
enough or is appropriately timed. Cytokines, probably via
NFkB and increased Ca21 (68), are able to activate COX-2
synthesis, as is PAF, via increased intracellular Ca21 (68).
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creased Bcl-2 and Bcl-x long were expressed in CA3 and
dentate granule cells of rat hippocampus within 2– 4 h of
15-min global ischemia but were not expressed in CA1.
This was despite the induction of mRNA in all regions
(181). A similar pattern was observed in gerbil after 5-min
ischemia (420). Six hours of focal ischemia led to a decrease in expression of Bcl-2 and BCl-x in neurons of the
penumbra, the great majority of which go on to die (364).
In contrast to the protective members of the Bclfamily, the “killer” protein Bax was upregulated in CA1 of
both rat and gerbil after the global ischemic paradigms
(185, 420) as was the Bax-like protein Bcl-short (264), and
both were expressed during development of ischemic
damage. Bax was not increased in CA3 but was increased,
along with the Bcl-2, in nonvulnerable dentate granule
cells. Bax was also upregulated in the penumbra of the
focal lesion (364).
Thus there is a dramatic downregulation of Bcl-2-like
proteins and upregulation of Bax-like proteins in vulnerable tissues. In dentate gyrus, which is nonvulnerable, the
increase in Bax is accompanied by an increase in BC1–2,
which probably neutralizes it. There is every reason to
believe that elevated Bax/Bcl-2 will be damaging and that
the elevation of Bcl-2 in nonvulnerable regions may well
be protective. Overexpression of Bcl-2 strongly protects
against anoxic/ischemic damage in focal ischemia (721),
global ischemia (27), and cultures (791). Overexpression
of Bax leads to apoptotic cell death in many systems.
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CEREBRAL ISCHEMIA ON NEURONS
2. Cytokines
that of IL-1b, although the cell types in which the two
cytokines are expressed are somewhat different.
Tumor necrosis factor-a has actually been shown to
be protective in vitro, and when added many hours before
ischemia in vivo (190), suggesting it may engender longterm protective effects, as discussed in the section on
ischemic tolerance. Indeed, TNF-a receptor null mice
have increased susceptibility to ischemic injury (131).
This may well reflect the protein synthesis-dependent
anti-apoptotic protective effect that is induced by TNF-a
in many of its target cells (43, 71, 1160, 1186). This effect
may be sustained in basal conditions by circulating
TNF-a. In contrast to these artifactual effects, there is a
large body of evidence indicating that cytokine that is
endogenously released during ischemia is toxic in both
transient and permanent focal ischemia. Damage is
strongly attenuated by antibodies in the circulation (636),
or in the cortex/ventricles (746, 1248), or by binding proteins in cortex (812) and also by a drug that prevents
TNF-a synthesis in brain by inhibiting its translation quite
specifically (746). The level of protection reached a 75%
reduction in infarct volume, which is extremely large.
Thus, as for IL-1b, TNF-a is upregulated quite soon
after the onset of focal ischemia and within hours of
starting global ischemia. It is shown to be damaging in
both temporary and permanent focal ischemia. Unfortunately, there are no reports on whether or not cytokines
are damaging in global ischemia.
C) MECHANISMS OF UPREGULATION OF CYTOKINES. Preexisting
cytokines are either released from cells, such as macrophages, or are synthesized in response to the ischemic
insult. Their synthesis is strongly coupled so that each
activates synthesis of the other, and this could account
for the appearance of both cytokines. Alternatively, their
synthesis could be activated independently. Sequelae of
ischemia that are known to activate cytokine synthesis
(and release) include free radicals, possibly via activating
NFkB and activation of MAP kinase (747). At this stage,
however, the trigger for synthesis or release is not known
(747). More distally MK-801, at levels which decreased
infarct size by ;50% (3 mg/kg), reduced the TNF-a production by 60% measured after 12 h of permanent MCA
occlusion (90). This suggests that NMDA receptor activation, presumably via increased cell Ca21, is a major trigger
for TNF-a production. Thus much of the damaging action
of NMDA receptor activation may be mediated by activation of TNF-a synthesis. This may well be very important,
and it would be interesting to know whether IL-1b synthesis is also modulated in this way.
D) MECHANISMS OF DAMAGE BY THE CYTOKINES. These molecules strongly interact with each other and influence a
very large number of signal transduction mechanisms
including those affecting gene activation (83). Thus determining how the cytokines enhance damage is a formidable task that has only just begun to be tackled. It is, of
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Two major proinflammatory cytokines, IL-1b and
TNF-a, appear to play very important roles in focal ischemic damage and may play a role in global damage (241).
A) INTERLEUKIN. Immunoreactive IL-1b increases very
rapidly, within 15 min of the onset of focal ischemia,
maximizing by 2 h. It is seen mainly in endothelial cells
and microglia (1295). Messenger RNA for IL-1b is elevated
in all cell types within 2– 8 h of global ischemia in rats, but
the protein is mainly expressed in glial cells. All cells
showed increased mRNA for the receptor for IL, but the
protein was largely expressed in neurons, with some in
endothelial cells (974). Thus IL and its receptors are
upregulated in a timely fashion.
There is good evidence implicating IL-1b in permanent focal ischemic damage. The natural peptide antagonist to the Il-1b receptor, IL-1ra, decreased neuronal death
in the peri-infarct zone of a permanent focal lesion (352)
and reduced infarct size after 24-h permanent ischemia by
50% (946). Furthermore, an antibody to the IL-1b receptor
antagonist, which is upregulated by ischemia, exacerbated damage (687). This argues that this antagonist is
generally helping to reduce damage, presumably by attenuating the IL-1b damaging action. In transgenic mice overexpressing the receptor antagonist, the lesion size after
24-h permanent ischemia was reduced by ;50% (1249).
These quite dramatic savings were not mediated by reduced temperatures. This effect is not mediated by blocking white cell accumulation, since the latter is only protective in temporary, and not permanent, focal ischemia
(203).
Surprisingly, when injected directly into brain, the
antagonist had to be put into the striatal core, even to
protect the cortex (1075). This suggests that IL-1b effects
are being exerted in or near the core of the lesion and that
resulting damage moves out into cortex. One such mechanism would be via spread of glutamate or K1 (and possibly resulting intraischemic depolarization). Interleukin-1 does lead to glutamate release in cerebral tissue
(968). Thus the antagonist may block core release of
glutamate; this should be measured. Core release is
blocked in nNOS knockouts (1031), and these two events
may be related.
B) TNF-a. Tumor necrosis factor-a appears in the circulation within 20 min of initiating focal ischemia and
persists for at least 2 h of reperfusion (636). This may be
independent of its synthesis. It is upregulated in microglia
and macrophages within 30 min of initiating focal ischemia, and its levels peak by 8 h (154). The receptor,
TNFR1, which mediates toxic effects and possesses the
“death domain,” is upregulated by ;6 h (116). Tumor
necrosis factor-a is also upregulated ;6 h after 5- to
10-min global ischemia in the gerbil (975) and within 1.5 h
after global ischemia in the mouse, largely in microglia
(1155). Thus the time course of expression is similar to
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PETER LIPTON
activated and, in determining the mechanism of their
action. There is a great need for studies in which TNF-a
and IL-1b actions are blocked and where measurements
of free radical production, NO production, and glutamate
release are then made.
3. c-Jun
There is reasonable evidence that c-Jun protein expression well after the ischemia might initiate damage,
perhaps by apoptosis. There is a massive expression of
c-Jun 24 h after 15-min hypoxia/ischemia in adult rats that
is restricted to cell populations that are destined for death
(270, 271). This synthesis also occurs after status epilepticus (271). A less detailed study reports a delayed induction of mRNA for c-Jun (and c-fos) in CA1 pyramidal cells
of the rat 24 – 48 h after 20 min of 4-VO (1203). The basis
for the late increase in c-Jun is not known but certainly
could be delayed free radical production, possibly via
COX-2 and/or NOS upregulation.
Evidence that this could be toxic comes from sympathetic neuron cultures. c-Jun was activated after NGF
withdrawal, and when a c-Jun dominant negative mutant
was introduced into these cells it prevented the cell death
that normally occurred after the NGF withdrawal. Constitutive expression of c-Jun caused apoptosis in these cells
even in the presence of NGF (415). Although clearly not
definitive, these results indicate the potential importance
of delayed expression of a gene that regulates transcription factors. The c-Jun target(s) is not known.
F. Ischemic Tolerance
1. Definition of phenomenon
Nonlethal short exposures to ischemia in both rats
and gerbils are extremely neuroprotective. When the animals are challenged between 2 and 4 days later with
ischemic insults that are usually lethal, there is almost no
damage in global ischemia and much-reduced damage in
focal ischemia (64, 445, 584, 586, 681, 846, 847). Similar
protection is provided by short periods of thermal stress
(196, 587), cortical spreading depression (733), and mechanical manipulation (372). Tolerance induction cuts
across ischemic models in that 5 min of 4-VO global
ischemia in rat provided very substantial protection
against subsequent focal ischemia, 4 days later. Infarct
volume after 3 h of temporary MCA occlusion was reduced, from 69 to 23 mm3 (732).
In an important study it was shown that induced
tolerance in CA1 did not alter the initial profound response to ischemia, which included dissolution of microtubules, blockade of protein synthesis, and disaggregation
of polysomes (348); rather, it allowed recovery from these
changes in the CA1 pyramidal cells. Understanding what
factor(s) mediates tolerance will provide major insight
into what is normally causing damage.
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course, a very important issue because it should give
insight into basic mechanisms of ischemic cell damage.
I) Vascular effects. One well-worked out effect of the
cytokines is upregulation of endothelial cell adhesion
molecules, in response to ischemia (1035), and the (resulting) accumulation of neutrophils in ischemia (352).
This may mediate the action of TNF-a in temporary ischemia, where there is good evidence that neutrophil accumulation is damaging, but it cannot mediate the effect in
permanent ischemia where the evidence strongly indicates that neutrophil accumulation does not exacerbate
damage. The same is true for IL-1b, which is very effective
against permanent ischemia. Other effects on the vasculature are certainly possible, particularly because circulating antibodies to TNF-a very effectively prevented
damage in temporary focal ischemia. However, intracerebrally injected antibodies or inhibitors are protective in
many studies, and it seems likely that circulating TNF-a is
damaging after uptake into the brain; there is significant
pinocytosis after focal ischemia (895, 898).
II) Generation of free radicals. Cyclooxygenase-2
synthesis and iNOS synthesis (477) are induced by both
cytokines, and might certainly mediate their effects, by
enhancing free radical production. Interleukin-1b is particularly effective in activating iNOS in cultured vascular
endothelial cells (113).
Tumor necrosis factor-a toxicity is strongly attenuated by overexpression of Mn-SOD in some tumor cells
(1221), as is ischemic damage. This indicates that mitochondrial generation of free radicals may be a mechanism
of damage. This idea is supported by a good study, again
in tumor cells (1003). Tumor necrosis factor-a affected
complex I of the mitochondrial electron transport chain
and enhanced free radical production via ubiquinone.
Toxicity could be strongly alleviated by blocking electron
transport through that site, and also by free radical scavengers. Toxicity was enhanced by blocking electron transport at a more distal site which should enhance free
radical production. These results are compatible with
what is known about mitochondria in ischemic damage. It
is not known why the TNF-a affects the mitochondria.
III) Apoptosis. Tumor necrosis factor-a is a potent
activator of apoptosis via the TNF1 receptor, which is
upregulated after ischemia. Although TNF-a activation of
apoptosis requires inhibition of protein synthesis in several systems (1160), this is not the case for neurons, at
least in culture (311), so that this is a viable mechanism
for TNF-a action after ischemia. The putative apoptotic
death after ischemia is blocked by protein synthesis inhibitors.
E) SUMMARY. The profound protection by the antagonists, particularly those to TNF-a, suggests that the cytokines are responsible for a great deal of the damage in
focal ischemia. Studies of effects on global ischemic damage are still needed. Further work is necessary to determine how early cytokine release and later synthesis are
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CEREBRAL ISCHEMIA ON NEURONS
2. Proteins that may be responsible for tolerance
of (measurable) HSP mRNA synthesis. In a third study,
anisomycin added at the time of the initial short ischemia
(and present for ;2 h later) largely inhibited the increased HSP synthesis. However, it did not affect tolerance (546). All these results could be explained by a very
nonlinear relationship between HSP and tolerance where
small changes in HSP produce the large protective effects,
but there is no particular reason to think that such a
relationship exists.
Although the above results militate against a role for
prior induction of HSP in tolerance, another study shows
tolerance may be associated with an increased ability of
CA1 pyramidal cells to synthesize HSP in response to the
second insult. Heat shock protein was greatly enhanced in
CA1 3 h after 3.5-min ischemia in gerbil, when tolerance
had been induced by 2-min prior ischemia (28). If this HSP
synthesis is, indeed, conferring the tolerance, then the
issue becomes what is allowing its synthesis to be activated after ischemia in the tolerant brain.
B) BCL-2. At present, Bcl-2 is the least-flawed candidate for mediating tolerance. There is a good correlation
between the time courses of appearance of Bcl-2 and of
tolerance. Bcl-2 appeared in CA1 30 h after 2-min ischemia in gerbil. It was not present at 12 h and was maximal
at 4 days (1029). Tolerance to ischemic cell death due to
5-min ischemia followed a very similar time course (545).
However, more detailed studies might reveal discrepancies, as they did for HSP.
C) NEUROTROPHINS. Although neurotrophins seem reasonable candidates a priori, there is a quite persuasive
study that argues strongly that they do not have a role
(445). Expression of NGF and BDNF mRNA are very
slightly upregulated by (generally lethal) 6-min ischemia.
However, if the 6-min ischemia is preceded by a 3-min
conditioning pulse that induces tolerance, then the neurotrophin mRNA are drastically downregulated 1 day after the 6-min ischemia. Without measuring the protein, no
firm inferences can be drawn, but this result suggests that
the neurotrophins are rendered less potent by the tolerance-inducing stimulus.
D) SUMMARY. A very notable feature of these studies is
that while ;2 min of ischemia leads to induction of
protective proteins in CA1, 5 min does not, although it
does in other cell types. This key event may well be a
critical feature of damage development in vulnerable
cells. Presumably events occur in CA1 after a short duration of ischemia that make it impossible for those cells to
synthesize protective proteins. The most likely locale for
this is at the level of global inhibition of protein synthesis.
Synthesis inhibition in CA1 is certainly more long-lasting
than in other regions and may well be more profound at
early times; this has not been rigorously tested. If this is
the basis for the inability to synthesize protective proteins, then inhibition of global protein synthesis assumes
a dominant role in development of cell death after global
ischemia.
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There are four generically different explanations for
tolerance: 1) tolerance may provide vulnerable cells with
a recovery mechanism that other cells already have, 2)
tolerance may provide vulnerable and nonvulnerable cells
with a new recovery mechanism, 3) tolerance may reduce
the initial insult in vulnerable cells so they can recover
using normal mechanisms, and 4) tolerance may remove
a normally lethal mechanism such as upregulation of
COX.
Because tolerance takes several days to become
maximal, and lasts for 7 days (543, 545, 584, 998), it seems
very likely that it involves the synthesis of new protein.
The importance of synthesis directly after the short preconditioning bout has been nicely shown for tolerance in
focal ischemia where administration of cycloheximide
before the 10-min focal ischemia completely prevented
the induction of tolerance to prolonged MCA occlusion
(64). Later additions of cycloheximide had no effect. The
mechanistic focus has thus been on protective proteins
that might be induced by the nonlethal stresses and that
might then protect vulnerable tissues during a major insult. The “protective” stress-related proteins discussed
earlier in this section thus all become attractive candidates; they are induced after brief insults, show a time
course of expression that quite closely parallels the time
course of tolerance induction, and are protective when
administered artifactually. Furthermore, all these proteins are expressed in nonvulnerable regions following
ischemia so may well be acting protectively when expressed endogenously.
A) HSP. The most widespread hypothesis had been
that induction of HSP by the preexposure protected during the later ischemia, since there appeared to be strong
correlations between HSP expression before the second
ischemia and tolerance in gerbils (547, 584) and rats (681).
However, more detailed examination has weakened the
correlation.
First, as discussed above, HSP does rise in CA1 of rat
hippocampus 1 day after lethal ischemic exposures (681)
so that its induction by prior insult seems unnecessary.
However, it is possible that HSP is not present early
enough to protect CA1, unless it is induced by tolerance.
There are more empirical arguments against HSP involvement. The correlation between HSP elevation and the
degree of tolerance is very nonlinear (584). One day after
the short (2 min) ischemia in gerbils there is substantial
tolerance with very little HSP induction. At 4 days, there
is better tolerance than at 2 days, despite the fact that
HSP levels are about the same as those two times. Along
the same lines is a careful study by Abe and Nowack (1).
Two-minute preischemia protected 86% of the neurons
against 5-min ischemia 4 days later, but HSP mRNA had
only increased in 46% of the neurons at the time of the
second ischemia. Thus tolerance occurred in the absence
1523
1524
PETER LIPTON
3. Mechanism of induction of tolerance
4. Cellular mechanisms of tolerance
The few existing studies show that tolerance appears
to be associated with an increased ability to buffer cytosolic Ca21 loads. Ohta et al. (846) showed that mitochondria in tolerant neurons (in vivo) accumulate more Ca21
than normal CA1 pyramidal layer neurons within the first
15 min after global ischemia, whereas cytoplasm has
much less total Ca21. Over the next several hours, the
cells in tolerant animals lose their mitochondrial and
cytoplasmic Ca21, whereas those in normally vulnerable
neurons maintain elevated mitochondrial Ca21 and also
elevated cytosolic Ca21 (846). Thus mitochondria in tolerant neurons show much better buffering of entering
Ca21 and, overall, lower levels of total Ca21 in the cytoplasm during the postischemic period. The basis for the
latter may be the early buffering by the tolerant mitochondria, but it may also be because the short, toleranceinducing, ischemic exposures induce a twofold increase
in plasmalemma Ca21-ATPase (846). Interestingly, all the
properties associated with the tolerant CA1 neurons are
shown by CA3 neurons, whether or not they have been
exposed to the short ischemic episodes, confirming that
tolerance represents the acquisition of properties shown
by less vulnerable cells. In a study by a different group,
2-min ischemia 2 days before the insult reduced the fluo-
rescence increase of the Ca21 dye rhod 2-AM in CA1 cells
by 50% when excised slices were exposed to in vitro
ischemia (1030). Although the relationship between the
two studies was not discussed, it is very probable that the
increased buffering by the mitochondria plus the enhanced Ca21-ATPase activity, noted in vivo in the first
study, is the cause of the reduced cytosolic Ca21 accumulation noted in both studies.
Thus the ischemia-tolerant neurons appear to be able
to buffer entering Ca21 better than normal using the
mitochondria, and also to eliminate Ca21 from the mitochondria far more quickly after the ischemia. As mentioned above, at least the first of these effects is similar to
those caused by overexpression of Bcl-2 (787). This is
consistent with the observed presence of Bcl-2 expression in tolerant cells. It does not prove that the Ca21
effect, or enhanced Bcl–2, is the basis for tolerance.
To the extent that it explains tolerance, this result is
consistent with the third generic mechanism, described at
the beginning of the section. Such an effect might well
reduce the damage due to increased cytosolic Ca21 in
vulnerable cells as distinct from targeting the recovery
process. The result also highlights the importance of Ca21
in damage. Bcl-2 is not normally upregulated until 2– 4 h
after a global ischemic insult. Thus the result suggests
that effective buffering of cytosolic Ca21, or uptake into
mitochondria without causing damage, as late as 4 h after
ischemia will protect neurons. The corollary is that elevated cytosolic Ca21, or the inability of mitochondria to
appropriately handle Ca21, is damaging well after the
ischemia, implying that cytosolic Ca21 levels remain
somewhat elevated and damaging when insults are toxic.
G. Summary
A large number of putatively protective genes are
expressed after global ischemia, mainly in cells that are
not destined to die. Although appealing as a correlation,
and teleologically, there is no evidence, yet, that this
endogenous production of growth factors, Bcl-2, or HSP
(or immediate early genes) actually protects cells from
the insults, nor is this known for focal ischemia. Creative
studies with knockouts will be necessary to establish this.
The possibility will be supported by the tolerance phenomenon if it can be clearly shown that tolerance results
from synthesis of one of these classes of molecules.
Vulnerable cells, in global or focal ischemia, suffer from
the absence of synthesis of these protective proteins, or in
some cases from their downregulation. This might well be
contributing to development of cell death, and this would be
well demonstrated if tolerance occurs as a result of their
synthesis. Why these proteins, as well as immediate early
genes, are not synthesized in vulnerable cells is unknown. It
could result from the general downregulation of synthesis,
although this is not a compelling argument. Certainly in
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There is now evidence that IL-1a or IL-1b (847), or
TNF-a (813), elevation may induce, but not mediate, the
tolerance. Evidence is more complete for the interleukins.
Interleukin-1a and IL-1b are moderately elevated in
blood after 2- to 3-min global ischemia. Maintenance of
IL1-ra, the IL receptor antagonist, during the interval between the short ischemia and the usually lethal ischemia
completely prevented the induction of tolerance. Furthermore, injection of IL-1b or IL-1a for 3 days, in the absence
of an inducing ischemia, led to very strong tolerance
against the 5-min ischemia. If this is substantiated, then
IL-1a may be acting to induce synthesis of stress-related
protein such as Bcl-2, or HSP, or may be acting to allow
the synthesis of HSP during ischemia, as discussed above.
This should be able to be determined and to provide
insight into the species responsible for tolerance.
Tumor necrosis factor-a may also mediate tolerance,
in focal ischemia, although the data are not nearly as
strong as for the action of IL-1b in global ischemia described above. Injection of TNF-a into the cortex 2 days
before 24-h MCA occlusion reduced the size of the infarct
(813).
These results highlight the complex role that the
cytokines play in ischemic damage. Not only do they
enhance damage during a normally damaging insult but,
presumably by activating genes, they induce tolerance by
increasing synthesis of protective proteins several days
after nonlethal insults.
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
IX. FINAL CONCLUSIONS
A. Mechanisms of Ischemic Damage
The major focus of the review has been to evaluate
the likelihood that different ion, metabolite, macromolecular, and functional changes participate in causing cell
death in the major models of ischemia. This section is
devoted to synthesizing the information into reasonable
hypotheses for cell death in global and focal ischemia, the
two models that have been considered most intensively.
There is no attempt to summarize the material in each
section because each change was covered very thoroughly and summarized. Here the “bottom lines” are used
to try and describe reasonable mechanisms for cell death.
Tables 4 and 5 reflect much of what has been discussed
in the review and are very useful in constructing hypotheses
for cell damage. Table 4 lists the results of most of the
pharmacological, knockout, and transgenic studies that
have been used to determine which variables are involved in
ischemic cell death. Table 5 lists changes in, and basal levels
of, variables that are different in vulnerable (CA1 hippocampal pyramidal cells) and nonvulnerable cells, and thus that
might be responsible for the great vulnerability of the CA1
pyramidal cells in global ischemia.
1. Mechanism of cell death after global ischemia
A) PARTICIPATION OF DIFFERENT PROCESSES IN CELL DEATH.
There is reasonably strong causal evidence, derived from
pharmacological or other types of intervention, that certain changes are critical for development of damage after
global ischemia. Much of this is apparent from Figure 1
and Table 4. These include 1) decreased pH, 2) entry of
Na1 in postischemic period, 3) postischemic Ca21 entering via voltage-dependent Ca21 channels, 4) calpain, 5)
free radical action, 6) NO generation, 7) caspase activation, and 8) accumulation of zinc.
The level of confidence in these conclusions was
discussed in the main text, but it is great enough to
incorporate them into a model for damage with reasonable certainty.
There are other changes in ions and metabolites that
are very probably damaging, but for which explicit evidence in ischemia is very weak or lacking. These include
1) decreased ATP during ischemia, 2) increased cytosolic
Ca21 during ischemia, 3) increased cytoplasmic Na1 during ischemia, 4) increased FFA during ischemia, and 5)
increased extracellular glutamate.
There is evidence that certain macromolecular
changes are also important. In this case the studies do not
establish causality, but they do show appropriate changes
in the amounts or activities of the molecules which, combined with what is known of their effects, suggests
strongly that they will be important in damage. Many of
these are indicated in Table 5. They include 1) increase in
Bax/Bcl-2; 2) decrease of SOD 1 day after the insult; 3)
increased levels of TNF-a; 4) increased iNOS in glia starting 1 day after the insult; 5) failure to synthesize protective proteins such as growth factors, HSP, and Bcl-2; 6)
loss of BDNF; and 7) prolonged diminution in CaMKII and
PKC activities.
Another change that is certainly a possible basis for
damage, but for which evidence is too restricted at
present, is the loss of mitochondrial synthesis of cytochrome aa3.
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global ischemia synthesis in all regions is very low for the
first 6–12 h, the times at which these proteins are being
synthesized. Thus determining the basis for the differential
synthesis of protective proteins is an outstanding task. In
particular, what is the reason that CA1 cells are unable to
synthesize so many of these proteins that are upregulated by
other cells (Table 5)? Lack of synthesis of these proteins is
not as clearly correlated with cell damage in focal ischemia.
This may be simply due to experimental conditions; appearance of the proteins needs to be correlated with cell viability
in a very mixed population of dead and living cells.
At the opposite pole, and of equal if not more importance, it now appears very likely that induction of a set of
putatively damaging genes is a critical factor in the sensitivity of the penumbra to focal ischemic challenges. It may also
be very important in global ischemia, but this has not been
shown. Cyclooxygenase-2 and iNOS, which are clearly capable of causing damage, are upregulated, and their inhibition ameliorates damage. Interleukin-1b and TNF-a are upregulated, as well as being released, and although these
cytokines can be protective when added before ischemia,
they appear to be very damaging when released during or
after ischemia. At present, the pharmacological interventions have been limited to focal ischemia and, for interleukin, to only permanent ischemia. However, many of the
proteins are activated in global ischemia and, when it is
done, pharmacology in that model may well be revealing.
The basis for activation of these genes needs to be determined, as does the mechanism by which the cytokines are
exerting their damage. It is likely that a vicious cycle may
develop in which free radicals activate synthesis of proteins
that beget free radicals.
Early observations on the cellular changes involved
in tolerance have shed light on a role that the protective
proteins might play; they might act to enhance the Ca21
buffering capabilities of the mitochondria and the cell
membrane. If so, then the latter are important sites of
vulnerability during ischemia.
An interesting result from this series of studies is that
NMDA receptor activation is required for synthesis of immediate early genes and HSP in global ischemia. To the
extent that these are beneficial, and there is reason to think
they are, this would render NMDA antagonists harmful. This
is notable with respect to clinical trials.
1525
1526
TABLE
PETER LIPTON
Volume 79
4. Agents that mitigate ischemic cell death
Agent
Antileukocyte adhesion agents
Matrix metalloproteinase inhibitor
Caspase-3 inhibitors
Antibody to CD95 ligand
CDP-choline
Reducing free radical accumulation in various ways
NADPH oxidase knockout
nNOS inhibitors or knockouts
iNOS inhibitors and knockouts
Calpain inhibitors
PAF antagonist
L- and N-type Ca21 channel antagonists
Calmodulin antagonist (trifluoperazine)
NMDA antagonists
AMPA/kainate antagonist
Hypoglycemia
Interleukin-1 antagonism
TNF-a antagonism
Block of polyamine oxidase; reduces 3-aminopropanal (498)†
Protection and Notes
Temporary focal*
Permanent focal
Global
Temporary focal
Mild temporary focal
Temporary focal
Temporary focal
Global
Global (rat and gerbil)
Temporary focal
Permanent focal
Temporary focal
Temporary focal
Permanent focal
Global (rat and gerbil)
Temporary focal*
Temporary focal
Global (rat)
Temporary and permanent focal
Temporary focal
Global
Permanent focal
Global
Global
Temporary focal
Temporary focal
Permanent focal
Global (gerbil only)
Global
Permanent focal
Global
Hypoxia/ischemia
Permanent focal
Permanent focal
Temporary focal
Temporary focal
Permanent focal
111
11
11
11
111
11
111 (Synergy with MK-801)
Functional recovery
1 to 11
11 to 111
111
111
111
111
11
111
11
11
11
111
111(Temperature not monitored)
111
1
11 (Postischemic)
111(1) (Drug specificity poor)
111
111
11 (Very high doses needed)
111 (In area tempesta)
111
11
11
111
111(1)
111(1)
1111
111 (Core protected)
S20 proteasome inhibition
Agents selected are those that affect known processes as distinct from those that provide protection in an unknown fashion (e.g., BDNF, adenosine).
All the studies, with the one noted exception, included reasonable temperature control and so are very probably free of artifact. References are not given.
All the studies were described in text, and antagonists are presented in order they were discussed in text. For focal ischemia: 1 is 10–15% reduction in
infarct; 11 is ,45% reduction; 111 is .45% reduction; 111(1) is 75% reduction or greater. For global ischemia: 1 is ,20% reduction in CA1 cell death;
11 is ,50% reduction, and 111 is .50% reduction. *Indicates agent was tested in other form of focal ischemia and was ineffective. †Reference given
because not discussed in text. nNOS, neuronal nitric oxide synthase; PARP, poly(ADP-ribose) polymerase; COX-2, cyclooxygenase-2; iNOS, inducible nitric
oxide synthase; PAF, platelet-activating factor; NMDA, N-methyl-D-aspartate; CDP-choline, cytidine diphosphate choline; TNF-a, tumor necrosis factor-a.
Finally, there is recent evidence, which suggests but
far from proves, that microglial activation plays a role in
global ischemic damage.
All of the above are within the class of damage
inducers; they are factors that will ultimately affect macromolecular structure, and hence cell function. Although
there is some uncertainty about the time at which these
different changes become important, because measurements are often not directed at this, it is reasonably
certain that they all play an important role. It is notable
how many processes are involved in cell death, and it is
quite certain that more will be discovered. It is obviously
difficult to construct a unique model for damage when so
many variables are involved. However, it is important to
try and do this, both for initiation of apoptotic change and
for necrotic cell death. In the latter case, the question is
how the inducers, and particularly the perpetrators, cause
the prolonged macromolecular and functional changes
associated with cell death.
I) Early events. Global ischemia is characterized by a
short and what surely must be characterized as a tumultuous period during electron transport blockade. The loss
of oxygen and glucose initiate a great many intracellular
ion and metabolite changes that are both very rapid and
very large. These include decreased ATP, increased
cAMP, decreased pH, increased cytosolic Ca21, increased
cytosolic Na1, membrane depolarization, free radical production, release of glutamate (and other transmitters),
increases in FFA, and decreases in phospholipids, in particular phosphatidylinositides. Although these clearly set
in motion the prolonged development of apoptotic and
necrotic damage and cell death, it has been almost im-
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PARP inhibitors
COX-2 inhibitor
Insult
CEREBRAL ISCHEMIA ON NEURONS
October 1999
TABLE
1527
5. Potential bases for selective vulnerability of CA1 pyramidal cells
Beneficial Changes in Other Cells Which Do Not Occur in CA1
Increased Caspase mRNA levels (826)
Protein synthesis permanently depressed (790, 1113)
Decreased SOD after 24 h (680, 734, 887)
Maintained elevation of free fatty acids (4)
Basal calpastatin levels low (344)
Large sustained decrease in PIP and PIP2
Persistent decreased activity and translocation of CaMKII (780)
Sustained decrease in tyrosine kinase and casein kinase (467, 468)
Elevation of calcium/lowering of ATP at 24 h (432)
Zinc accumulation (595, 1138)
Delayed decrease in GluR2/GluR1 receptor ratio (384)
Slow recovery of pH (784)
Prolonged elevation of NFkB (206)
Permanent loss of ubiquitin (251, 703, 1241)
Loss of BDNF (599)
cJun mRNA elevation 24–48 h after ischemia (1203)
Selective vulnerability of CA1 cells to superoxide free radical,
in organotypic culture (1212)
Synthesis of immediate early gene protein products (567)
Synthesis of BDNF (599)
Preferential synthesis of heat shock protein (gerbil only) (840, 1121)
Synthesis of Bcl-2 (181)
Differences between responses of vulnerable (CA1 pyramidal) and nonvulnerable (CA3 or dentate granule) cells in hippocampus are shown
that might reasonably account for vulnerability of CA1 cells to global ischemia. SOD, superoxide dismutase; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; CaMKII, Ca21/calmodulin-dependent protein kinase II; GluR, glutamate receptor. Reference
numbers are given in parentheses.
possible to explicitly demonstrate in a satisfactory way
that any one of them is involved. Thus one has to work
backwards from what is known about the later processes
that are important.
Inhibitor studies strongly suggest that important sequelae of the early maelstrom is maintenance of damaging
conditions that include the continued generation of free
radicals and the maintained increase in cytosolic Ca21
and Na1.
Continued free radical generation is postulated to
result from mitochondrial damage, which generates free
radicals and which is amplified by a cycle involving free
radical activation of NFkB, TNF-a, and IL-1 synthesis, and
further activation of mitochondrial free radical generation. This system, and activation of NFkB in particular,
leads to delayed elevation of COX-2 and iNOS, which
enhance free radical and peroxynitrite production.
Increased Ca21 and Na1 are likely to be maintained
by long-term changes in voltage-dependent channels or
NMDA receptors, based on studies of the efficacies of
inhibitors. These could result from calpain-mediated
cleavage (439), from free radical attacks, or from other
persistent modifications. The increased Na1 might well
enhance the increased Ca21 via the 3Na1/Ca21 exchanger. The elevated Ca21 will contribute to ongoing
mitochondrial damage, partly by activating the MPT.
Other key molecular damages are also maintained, including inactivation of protein kinases and decreases in phosphoinositides. These are likely to be very important in
preventing critical cell signaling. The bases for the initial
inactivations are not known but could also be proteases
or free radicals. The profound inhibition of protein synthesis in vulnerable cells ensures that channels, kinases,
and possibly key enzymes of PIP metabolism are not
resynthesized after the initial inactivations.
The overall postulate is that these prolonged ionic
and macromolecular changes result from the early events,
particularly the increased Ca21 and free radical production (but possibly from other causes also), and lead to the
subsequent major functional changes. As summarized below in section IXA3, explicit evidence for much of this is
lacking.
II) Apoptosis. Despite the absence of an easily identifiable apoptotic morphology, the biochemical and pharmacological evidence strongly suggests that the apoptotic
program plays an important role in cell death. The most
likely initiators of the process are one or more of the
following: CD-95 ligand, TNF-a, the cyclin D1, the prolonged decrease in several protein kinases, and decreased
amounts of neurotrophin or ability of neurotrophin to
signal. The question then is what causes these changes. A
reasonable basis for CD-95 ligand and TNF-a is the continual generation of free radicals discussed above with
NFkB activating the appropriate genes. The upregulation
of cyclins may result from the increased MEK/MAP kinase
activity (1129), which in turn results from increased cytosolic Ca21, or from the upregulation of c-Jun (1218),
which may in turn result from free radical action or the
MEK/MAP kinase pathway. The basis for the prolonged
protein kinase inhibitions is not known but may well
reflect the inhibition of global protein synthesis as discussed above, and the lowered phosphoinositides would
contribute to the “growth factor withdrawal” syndrome.
The way in which the ensuing “post global-ischemic”
apoptosis differs from more standard sequences is not
known. The protection by caspase inhibitors, although
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Detrimental Changes in CA1
1528
PETER LIPTON
ysis due to cathepsin upregulation or proteasome activity
(which has not been tested), the cell undergoes the phase
change that characterizes ICC.
There is reasonably good evidence for autophagocytosis in the gerbil, and this may well represent a parallel
process that is continuing along with those described
above. It may well be associated with the proliferation of
ER, since the autophagic vacuoles appear at the time the
ER proliferation falls away. Some suggestions have been
made for how it is induced, but much more work is
needed to define that process.
B) SUMMARY. The key events in necrotic cell death are
postulated to be the persistent generation of free radicals,
cytosolic Ca21, and Na1 (acting largely to maintain Ca21
but possibly, also, to maintain extracellular glutamate
levels) in the vulnerable cells, brought about by several
factors. Over time, these lead to the somewhat explosive
increase in free radicals and calpain which, together, act
on several cell functions to cause excessive damage and,
ultimately, a massive change in cell morphology via a
“phase change.” The process is enabled by the persistent
inhibition of protein synthesis and (probably as a consequence) the persistent downregulation of signaling systems.
Activation of an apoptotic cascade occurs in parallel
with the necrotic processes. The activation results from
one of several defined stimuli and leads to caspase activation. The resultant proteolysis adds to the necrotic
processes described above to exacerbate damage; normal
apoptotic morphology is precluded by the dominance of
necrotic processes, evidenced by the relatively small saving effects of caspase inhibitors. Autophagocytotic breakdown may well contribute to the cell death process in the
gerbil.
A key feature of ischemic death is that many different
inhibitors alleviate much of the cell death, suggesting that
death requires the combined effects of several activators
and perpetrators. On reflection, this is perhaps not terribly surprising for relatively mild insults such as are being
considered here. As discussed in section IXB, there is a
clear homeostatic force that tends to maintain a cell in a
viable state. In order for a mild insult to overcome this, it
is reasonable that many consequences of that insult will
need to work together.
2. Mechanism of cell death after focal ischemia
Unless otherwise stated, all of the discussion pertains to cell death in the penumbra of the focal lesion.
Little is known about the core because attempts to prevent damage have been almost universally unsuccessful.
A) PARTICIPATION OF DIFFERENT PROCESSES IN CELL DEATH. As
in global ischemia, there is a set of variables whose involvement in focal ischemic damage has been established
by experiments in which causality has been tested. Many
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not great, indicates that proteolytic actions of caspases
contribute to the death of the neurons. Assessment of
changes in specific protein targets, both intranuclear and
extranuclear, is required to get a full idea of the apoptotic
contribution. The paucity of apoptotic nuclear morphology may result from activition of other endonucleases or
enzymes by ischemia, which prevent the normal chromatin condensation, as indicated by the abnormal cleavage
pattern of the DNA.
III) Necrosis. The progression of damage is characterized by a fairly dramatic, delayed, coincidence of
changes about a day after the ischemia. These include
decreased levels of SOD in the CA1 pyramidal cells, increased superoxide in the same cells, appearance of induced COX-2 in these cells and iNOS in adjoining astroglia, a change in the subunit composition of AMPA
receptors which renders them far more permeable to
Ca21, and also a major activation of calpain. The reduced
SOD may result simply from reduced global protein synthesis; it is likely to be instrumental in the increase of free
radicals. The change in AMPA receptor composition results from action at the genome, but the mechanism is
unknown. Calpain activation may result from a delayed
rise in Ca21 but could also result from long-term changes
in calpastatin or in activators of the enzyme. All these
events appear to be damaging (although there is no explicit evidence for iNOS) so that it is reasonable to assume that this is a key event in the development of
necrotic cell death (it may also be important in the initiation of apoptotic processes as both calpain and free
radicals are important). Although not explicitly measured
at this late time, the prolonged elevation of Ca21 should
also maintain activity of nNOS and, hence, peroxynitrite
production. The altered AMPA receptor composition is
likely to exacerbate the increase in cytosolic Ca21. Thus
the stage is set for quite massive attacks on different cell
functions.
The calpain increase could be responsible for the
delayed disorganization of microtubules (in rats), which
adds to the damage done by the blockade of transport
initiated by damage to motor proteins.
Mitochondrial dysfunction is likely to be exacerbated
by the increased production of free radicals/peroxynitrite
(particularly because it is the mitochondrial SOD that is
downregulated). The free radicals will damage electron
transport and also sensitize the MTP to Ca21. If cytosolic
Ca21 levels increase, via the altered AMPA receptors, it
will greatly exaggerate these changes. The delayed reduction in cytochrome aa3 should also inhibit mitochondrial
function.
Excess free radical/peroxynitrite production, along
with diminished protein synthesis and intracellular transport, possible activation of PLA2 by Ca21, might well
cause membrane dysfunction and possible leakiness, although this has not been tested.
At some stage, possibly aided by a massive proteol-
Volume 79
October 1999
CEREBRAL ISCHEMIA ON NEURONS
strongly as neurons are in global ischemia. One noted
manifestation of this is the absence of the anoxic depolarization in the penumbra, but there may well be other
differences that arise from the smaller fall. For example,
protein kinases may not be as strongly inactivated (this
has not yet been measured). On the other hand, the
reasonably high flow leads to a large production of free
radicals in the region. It is very likely that penumbral
damage relies on glutamate released from the core of the
lesion; this substitutes for the profound ATP fall by leading to the intraischemic depolarizations and Ca21 entry.
C) PERMANENT ISCHEMIA: EARLY EVENTS. Unlike global ischemia, where there are drastic short-lived early events,
permanent focal ischemia is characterized by changes
that last many hours and which, judging from times at
which inhibitors are active, do not complete their damaging effects in the penumbra for several hours. These
critical events include production of free radicals, release
of glutamate largely from the core of the lesion that then
diffuses to the penumbra, the resulting increase in cytosolic Ca21 (which has not been measured but which is a
very likely result of glutamate action and the intraischemic depolarizations), and the resulting activation of NO
synthesis. The strong dependence of damage on NMDA
and AMPA receptors suggests that the elevation of Ca21
due to glutamate is a major factor in development of
penumbral damage, with the glutamate largely emanating
from the lesion core. This makes sense because the insult
is so mild in the penumbra (25% reduction in ATP) that
events there would not, in all probability, be able to
initiate major damage. On the other hand, as noted from
the anoxic depolarization in the core, major changes in
ions including Ca21 and Na1, and hence glutamate release, will occur there. Free radicals and NO (probably via
peroxynitrite) also play a major role in the damage as
seen from the very strong protective effects of free radical
scavengers in focal ischemia. The major importance of
decreased pH and of PAF in focal ischemic damage also
speaks to the importance of free radicals.
D) PERMANENT ISCHEMIA: APOPTOSIS. Although caspase inhibitors have not been used, morphology and caspase
cleavage show that apoptosis is prevalent in permanent
focal ischemia. The potential activators of apoptosis are
the same as described for global ischemia, although decreases in protein kinases have not been studied, so they
may not be a possibility. Unlike global ischemia, protective effects of cytokine blockade have actually been demonstrated, so there is a larger probability that they are
important. The reason that apoptotic nuclei develop in
focal ischemia while they are essentially nonexistent in
global ischemia is obviously not known. One explanation
is that cells take more divergent paths in focal ischemia,
because of local flow heterogeneity. Thus the apoptotic
program may not be as contaminated by events associated with necrosis.
E) PERMANENT ISCHEMIA: NECROSIS. The ongoing produc-
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of these variables overlap with those that are important in
global ischemia, but there are additional ones.
I) Permanent focal ischemia. Variables involved in
permanent focal ischemia include 1) decreased pH, 2)
Ca21 entry via NMDA receptors and/or membrane depolarization, 3) Na1 entry and/or membrane depolarization
via AMPA/kainate receptors, 4) Na1 entry, 5) free radical
generation, 6) NO generation, 7) PAF, 8) IL-1b, 9) TNF-a,
10) delayed iNOS induction, 11) COX-2 induction, and
12) matrix metalloproteinase.
II) Temporary focal ischemia. Several important
variables that have been tested in permanent ischemia
have not been studied in temporary ischemia, including
decreased pH, interleukins, PAF, matrix metalloproteinases, and AMPA/kainate antagonists. At this stage, there is
no reason to think that they are not important in temporary ischemia, since both insults include the same early
ischemic period so that all the processes in permanent
ischemia are probably active in temporary ischemia. On
the other hand, there are two factors that are important in
temporary ischemia that do not play a role in permanent
ischemia: 1) PARP activation and 2) leukocyte adhesion.
Two other enzymes have not yet been tested in permanent ischemia. They are calpain activation and caspase
activation. There is an increased Bax/Bcl-2 in permanent
ischemia that is probably related to cell death, but this has
not been directly tested.
Several changes that are quite dramatic following
global ischemia have not been investigated in focal ischemia and so cannot yet be included with confidence in any
model for damage. These include the decreased activity of
SOD, the decreased synthesis of cytochromes aa3, and
the decreased ratio of GluR2/GluR1 AMPA/kainate receptors.
B) GENERAL FEATURES OF THE CELL DEATH PROCESS: COMPARISON WITH GLOBAL ISCHEMIA. The damage process has several
features that distinguish it from global ischemia.
Damage generally develops quickly, with significant
cell death occurring within 12–24 h. The long lag period
does not exist. There is a small amount of more slowly
developing cell death, between 2 and 3 days after the
onset of ischemia. It may reflect a lag period for cells in
the mildly energy-deprived periphery of the penumbra, or
it may be relatively rapid cell death occurring in response
to the further reduction in blood flow with time in the
penumbra. Short periods of focal ischemia (30 min) do
lead to very slowly developing cell death. Another major
difference is the appearance of a more classical apoptotic
morphology in many neurons. Although these differences
may reflect the different cell types that are being analyzed
(the hippocampal pyramidal neurons are not included in
analyses of focal ischemic damage), they are more likely
to result from the fact that the insult is more prolonged,
but milder (ATP falls only by 25% in the penumbra).
The small drop in ATP in the penumbra means that
the penumbral neurons will not be directly affected as
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PETER LIPTON
are not protecting via their actions on proteasomes.) Mitochondria are damaged throughout the reperfusion period and accumulate Ca21. The calmodulin blocker trifluoperazine is also very protective, and although this is not
specific, other actions (on Ca21 channels) are also largely
on Ca21-mediated events. Thus it is likely that cytosolic
Ca21 is maintained elevated despite the absence of the
intraischemic depolarizations in the postischemic period.
As with global ischemia, this is postulated to arise from
alterations in Ca21 channels or, perhaps, from NMDA
channel changes; NMDA antagonists are very protective
when applied postischemically.
G) TEMPORARY FOCAL ISCHEMIA: APOPTOSIS AND NECROSIS. The
candidates for activation of apoptosis are the same as in
global and permanent focal ischemia at this stage. As for
permanent ischemia, there is currently no evidence that
protein kinases are inhibited. They need to be measured.
There is direct evidence for the importance of the FAS
ligand (CD-95L), but it has not been shown that it explicitly affects apoptosis.
Necrosis is thought to result from the combined,
concerted, and self-perpetuating effects of free radicals
on mitochondria, cytoskeleton, possibly plasma membrane, and possibly on cytoplasmic protein structure, all
leading to the phase change associated with ischemic cell
death and homogenizing cell change.
H) CELL DEATH IN THE CORE OF THE FOCAL LESION. Little is
known about mechanisms of death in this region, because
it is almost impossible to prevent with pharmacological
agents. There are large increases in cell Ca21 and Na1, a
maintained depolarization, and a maintained decrease in
ATP for several hours in temporary ischemia, as well as a
prolonged large elevation of glutamate. Existing evidence
suggests that free radical levels are low, at least early in
ischemia. There are features of the process that provide
clues as to the mechanism.
1) The degree of apoptotic cell death is much smaller
than in the penumbra; this is probably a function of the
prolonged maintenance of low ATP levels (25% normal).
2) Core damage is not prevented by the agents that
prevent penumbral damage. This could be because damage results from loss of ATP and rundown of ion gradients
with subsequent arrest of important processes, organelle
and cell dissolution; that is, it is almost exclusively a
dissipative process. Alternatively, the reason it cannot be
protected may be that the insult is so intense that synergism between different processes may not be necessary
for damage as it is in the penumbra and in global ischemia. Thus more than one pharmacological agent may
have to be used at once to obtain protection; for example,
both calpain and other protease inhibitors or NOS inhibitors may need to be coapplied. Of all the agents used, uric
acid, which is a very effective free radical and peroxynitrite scavenger eliminated almost all core damage,
whereas PARP and calpain inhibitors certainly prevented
some damage there. This suggests that damage is medi-
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tion of free radicals and prolonged elevation of Ca21 are
very probably the critical factors in damage. Inducible
NOS is induced in endothelial cells and leukocytes, and
COX-2 is induced in neurons; thus, much as in global
ischemia, there is self-generating mechanism for maintaining free radicals. It seems very likely that this involves
the cytokine/NFkB system, although this has not been
shown explicitly. There is good evidence for mitochondrial damage as well, which should contribute to the free
radical generation. On the basis of inhibitor studies, these
actions become lethal after ;6 h. Although no studies
have been done with calpain inhibitors, there does seem
to be a maintained elevation of Ca21 and a continuing
breakdown of MAP2 that is most likely to result from
calpain action. Thus, as in global ischemia but perhaps
accelerated by the continual hypoperfusion and high levels of free radicals, there is likely to be continued attack
on proteins and lipids of the mitochondria, cell membrane, and cytoskeleton, and it is these, along with possible direct breakdown or denaturation of cytosolic proteins, that lead to the ICC and homogenizing change that
characterize necrosis in this model.
F) TEMPORARY FOCAL ISCHEMIA. Unlike other forms of
ischemia, damage in temporary focal ischemia is strongly
dependent on the activation of PARP and, also, on accumulation of neutrophils in the ischemic tissue. Early
events are clearly the same as during permanent ischemia, and development of damage is dependent on
NMDA/AMPA as well as free radical/NO generation;
COX-2 and iNOS are increased at later times. However,
full development of damage appears to depend on the
accumulation of neutrophils in the postischemic period
and also on activation of the PARP. Blocking either of
these is profoundly protective.
Poly(ADP-ribose) polymerase is activated immediately upon reperfusion by a burst of free radicals that may
depend, to an extent, on the presence of leukocytes as
discussed previously. The most likely explanation for
their requirement is that they are necessary to boost the
production of free radicals in the reoxygenation period,
when electron transport is no longer blocked by the anoxia. It would be very interesting to assess the effect of
the agents that block neutrophil adhesion on the activation of PARP. Poly(ADP-ribose) polymerase may be damaging by reducing mitochondrial oxidative phosphorylation, but this seems unlikely as ATP levels in the
postischemic period do not fall greatly. It seems more
likely that the large-scale polyadenylation of the nuclear
proteins in some way exacerbates either apoptosis or free
radical production.
Development of damage depends strongly on calpain
in the postischemic period since inhibitors are extremely
protective at that time. (There is some concern here about
inhibitor specificity as proteasome inhibition is also very
protective, although the fact that this protection is restricted to the lesion core indicates that calpain blockers
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CEREBRAL ISCHEMIA ON NEURONS
ated by agents that act in the penumbra but that very
effective inhibition is necessary in the core.
3) Core damage does seem to be prevented by a
proteasome inhibitor. This is a somewhat surprising result; the inhibitor actually extended penumbral damage
somewhat (possibly due to the normally anti-apoptotic
effect of NFkB). It seems quite unlikely that the effect in
the core is due to blockade of NFkB because damage
there seems unlikely to require synthesis of new protein;
the insult is so intense. Thus the result suggests that
activation of proteasome S20 normally occurs and contributes to cell death by causing breakdown of protein.
Overall, the evidence is very strong that free radical
and peroxynitrite production are major determinants of
ischemic damage and that Ca21, via activation of calpain
and possibly other events such as mitochondrial accumulation and phospholipase activation, is very likely to be
important both during and, particularly, after ischemia. A
great number of effects of knockouts and pharmacological agents are consistent with the conclusion that these
two agents represent the major perpetrators of cell death.
However, beyond this there is a very large number of
extremely important unresolved issues, some of which
are apparent from reading the previous two sections.
Some of the major issues are discussed below.
A) BASES FOR MORPHOLOGICAL CHANGES: TARGETS OF PERPETRATORS. As was almost brutally apparent in the previous
two sections, there is essentially no knowledge of what
the protein changes are that follow the action of the
perpetrators and produce the morphological changes associated with the end stages. These will presumably be
worked out for apoptotic change by the workers in that
field, but it is necessary to determine what they are in
ischemia. They will presumably be a mixture of caspasemediated changes and those mediated by necrotic events.
Not knowing these changes makes it extremely difficult to determine what the critical functional changes
are. Thus, although changes in those functions have been
made clear through the text, the way they might cause
final damage is largely unknown because the nature of
that damage is unknown.
Thus it is critical to understand the protein composition and structure of the end stages; this is a very
difficult task.
B) ROLE OF PLASMALEMMAL DAMAGE. A priori the plasmalemma seems a very likely target of the perpetrators, and
one where damage could readily lead to necrotic cell
death. However, there is really no work that has been
aimed at assessing plasmalemmal function at critical periods of damage development.
C) THE APOPTOTIC SIGNAL. Despite the morphological
quagmire it is clear that caspase activation is likely to play
a major role in most types of ischemic cell death. Deter-
mining the signal, and its origin, which triggers the apoptotic sequence causing caspase activation is an important goal. The recent findings of cyclin D1 and FAS ligand
upregulations are exciting developments along this line.
The wide-spectrum kinase inhibitions are also possible
initiators of apoptosis. Several apoptotic signals, particularly those controlled by Ca21 and protein kinase B, are
mediated downstream by dephosphorylation of the proapoptotic protein BAD (1251). It would be very informative to know whether ischemic apoptosis is associated
with changes in its phosphorylation state, which can be
measured.
D) TIMING OF ACTIONS. In trying to describe the mechanism of cell death, it becomes apparent that a much firmer
knowledge of when different events are critical for damage is required. This applies to free radical changes, calpain activity, Ca21 changes, and pH changes. Required
studies are very difficult, and they have been started to
some extent, but discreet time windows of pharmacological actions need to be established. Simply knowing that
calpain is important is not enough to reconstruct the
mechanism of damage.
E) DAMAGE IN THE CORE. It is very important to initiate
studies aimed at determining the basis for damage in the
core of the focal lesion, where energy metabolism is very
strongly inhibited for a prolonged period. This does not, in
general, respond to treatments that are quite successful in
the penumbra, so it constitutes a particular challenge.
However, it is a critical clinical issue. The brain slice
might present a good model here, if used judiciously, as
described by Newman et al. (824). Conditions resembling
core can be created quite readily, and damage can be
studied in detail.
F) GLIA AND ENDOTHELIUM. The damaging roles of glia
and of the endothelium/blood-brain barrier need to be
explored more fully. There are hints of importance (synthesis of iNOS by glia in global ischemia, the protective
action of metalloproteinase-9 inhibition), but these have
not been rigorously explored.
G) OUTSTANDING MECHANISTIC ISSUES. The full explanation
of damage is not known for any of the variables. However,
certain of these mechanistic questions are both important
and intriguing.
These include the basis for the prolonged inhibition
of protein kinases in global ischemia (it has not been
tested in other models); the basis for maintained inhibition of protein synthesis; the basis for cytokine increases
in all forms of ischemia.
H) LACK OF CERTAINTY THAT SPECIFIC EVENTS ARE INVOLVED IN
DAMAGE. It is clear throughout the text that there are major
uncertainties about whether almost all of the proposed
inducers are actually involved in damage. These uncertainties stem from ambiguities in interpretation of pharmacological or metabolic studies or from the difficulties
of making appropriate measurements. Examples of the
former include pH, glutamate, cytosolic Ca21, and early
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3. Unresolved issues: future studies
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PETER LIPTON
also provide qualitatively new insights into the damage
process.
J) EXPERIMENTAL CAUTIONS AND APPROACHES. In trying to
solidify the involvement of different factors, it is essential
that studies be done with appropriate temperature controls, and also with very good measurements of local
blood flows during ischemia. Uncertainties in the contributions of these quantities to measured effects of drugs or
knockouts have contributed to the uncertainty in several
of the interpretations.
Appropriate use of in vitro systems can be very valuable in mechanistic studies, but end points have to be very
well defined. For example, it is probably not useful to
measure effects of agents on loss of synaptic transmission
in hippocampal slices, since there is really no understanding of the mechanisms underlying the loss of transmission. Similarly, in cell culture, studying necrotic cell death
is not very fruitful as, here again, there is little understanding of how it relates to the process in vivo. It would
be much more fruitful in both the in vitro models to
concentrate studies on specific processes, for example,
cytoskeletal changes, apoptosis etc. In all cases, care
should be taken to relate results to in vivo conditions.
B. A Final Viewpoint: Attractors, Cellular
Instability, and Cell Death
There are three aspects of ischemic cell death which
suggest that a formalism that is applied to dynamical
systems, in which attractors constitute stable states in a
multidimensional space, will provide a useful framework.
The first is that there is a very clear insult threshold.
Global ischemia and focal ischemia will only cause cell
death if they last a certain length of time (;4 and 30 min,
respectively). The second aspect of ischemic damage is
that there is an early profound damage, lasting ;12 h,
whether the insult is subthreshold or superthreshold. This
includes profound inhibition of protein synthesis, cytoskeletal disruption (in gerbil), and vacuolization of normal ER and Golgi structures. If the insult is subthreshold,
cells recover, whereas if it is superthreshold, they do not.
These observations suggest that ischemic cell death occurs when restorative processes that are in place following an insult are unable to effectively compete with the
degenerative processes induced by the insult. The third
aspect of ischemic cell death which suggests the formalism is appropriate is that the end stages of ischemic
damage are metastable states that are very different from
the normal state of the cell.
These properties suggest that ischemic cell death can
be treated semiformally, based on the formalism used for
describing stable and unstable states of dynamical systems in terms of attractors, as developed by Kauffman
(555) and others in which the emergent properties of
networks are manifested. This kind of analysis has re-
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entry of Na1. Measurements can be difficult either because of the technology or because appropriate pharmacological agents are not available. Examples of both these
include cytosolic Ca21, ATP, changes in kinases, and
membrane depolarization. Thus a major task is to develop
definitive ways of establishing the involvements of these
changes.
Evidence for most of the perpetrators of damage,
calpain, free radical action, PARP, is much stronger, although there are minor problems with each. Very few
studies have been done with calpain inhibitors, and none
in permanent ischemia. Further work, with more specific
inhibitors, and with more detailed consideration of timing, will be very valuable. The scavengers and blockers of
free radicals have, in general, not been used along with
measurements of free radical changes; this and the quite
large variability in results is somewhat unsettling but,
overall, it must be considered that the involvements of
free radicals, NO and PARP (for temporary ischemia), are
about the best established of all the perpetrators. Despite
the high likelihood that the changes are important, evidence for the involvement of phospholipid breakdown is
quite weak, due largely to the absence of appropriate
inhibitors.
I) IS THERE MORE TO KNOW? As discussed above, there are
clearly many major remaining issues that urgently need to
be resolved. However, this particular question is directed
at the validity of the basic model described here, in which
free radicals and Ca21 along (possibly) with permanent
kinase inhibition and apoptosis, are the major effectors of
cell death. There is no question but that more molecular
changes associated with ischemia will be found and that
more protective agents will be found. The issue is
whether or not all of these will be able to be fit into this
paradigm. It is important to differentiate findings that
initiate new insight from those that are consistent with
the current paradigm. With regard to the former, it seems
possible that intensive study of the basis and importance
of functional changes: mitochondria, plasmalemma, cytoskeleton, protein synthesis, and apoptosis initiation,
may provide results that allow new paradigms to be created. This will require learning what macromolecular
changes are responsible for the key functional changes.
Once this is done, then reasonable, detailed, mechanisms
for damage can be created, from which new insights will
certainly emerge.
New insights might emerge in a different way. There
are two protective agents that have profound effects in
focal ischemia but for which the actual basis for protection is not easily discerned. These are inhibitors of PARP
and polyamine oxidase inhibition, which seems to act by
lowering levels of 3-aminopropanol (498). These are different from all other protective effects that have been
discussed, because it is not clear at all why the decrease
in polyadenylation or in 3-aminopropanal would be protective. Perhaps understanding one or both of these will
Volume 79
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CEREBRAL ISCHEMIA ON NEURONS
variables similarly. This is clearly unacceptable. For example, while an altered level of phosphorylation, or the
loss, of a component of the protein synthesis system may
be extremely important, similar effects on glycogen phosphorylase are likely to be inconsequential (93). This is
reflected in the fact that there is a great asymmetry in cell
component space. Large changes in inconsequential parameters will not move the cell out of an attractor,
whereas small changes in key parameters will.
It is anticipated that more directed and quantitative
application of this formalism to cell biochemistry and
physiology (93) will lead to new insights about the relationships between cell stability and ischemic damage.
ADDENDUM
This review was completed in February 1999. Since that
time there have been many important publications. In this addendum I include those that appear to add important new information to the ideas that were discussed in the body of the
review. The new papers are discussed with regard to the sections of the review to which they are most appropriate.
SECTION II
Section IIL: Effects of ischemia on the vasculature. Del
Zoppo and co-workers (A9) have continued their work on the
roles of the matrix zinc-metalloproteinases (MMP) that degrade
laminin and collagen and have now shown that the other principal MMP, MMP2, is hugely activated in the lesion core. There
is a fourfold increase in activity within the first hour of MCAO in
monkeys, and this persists during 7 days of reperfusion, during
which time damage is developing. The mechanisms of activation
are not known, although there are complex controls on these
enzymes. It is also not established that the activation is causing
damage. However, it seems likely that local vascular disturbances, and changes in glial-endothelial relationships, may be
very important. In the context of the latter, another recent
publication demonstrated that astroglia appear to be severely
damaged several hours before neurons in permanent focal ischemia, as judged by loss of immunoreactivity of glial markers
within the first few hours of ischemia (A14). It is possible that
disruption of endothelial-glial relationships due to MMP action
could be significant here, rather than the more obvious suspects
such as low pH. The glial changes may influence neuronal
damage, but this is not established.
Section IIM: Effects of temperature on damage. Among the
many possible bases for the protective effects of hypothermia,
decreased glutamate release has been prominently considered.
A careful study by Yamamoto et al. (A23) strongly negates this
possibility. They artifactually increased extracellular glutamate
levels in CA1 hippocampus during global ischemia in gerbils at
31°C to levels that were actually higher than those that occurred
during ischemia at 37°C. Thus they overrode the inhibition of
release. Despite this, damage was still just as strongly attenuated by the hypothermia.
SECTION III
Section IIIB: Apoptotic cell change. Biochemical changes
associated with apoptosis. MacManus et al. (A16) have now
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cently been applied to movements between stable states
in very complex networks of protein kinase-driven biochemical reactions (93), rendering it particularly applicable to ischemic perturbations.
The cell state is defined by its position in a multidimensional “cell component space” whose axes are the
concentrations of all species within the cell, including
small molecules, ions, macromolecules, phosphorylated
macromolecules, and macromolecular complexes or polymers. The state of the cell is thus uniquely represented by
a point in cell component space. The particular concentrations of the components will lead to a constellation of
rates of reaction between, and catalyzed by, the components and hence to movement through the component
space as concentrations change. According to this, the
cell’s steady state is a stable point in cell component
space, where concentrations do not change as a result of
the ongoing reactions, or there is a short cycling between
different points in the space. The dynamical theory shows
that such steady states lie at the nadirs of “attractors”
(555). The latter are regions in the space (basins, troughs)
within which the cell will always move to that steady
state.
Physiologically, the attractors represent the homeostatic mechanisms of the cell. Ischemia moves the cell to
a new point in cell component space. Within this formalism, subthreshold ischemic insults are not large enough to
move the cell out of its normal attractor. Put more physiologically, homeostatic mechanisms are able to overcome the processes set in motion by the insult. (These
homeostatic mechanisms may be complex, involving synthesis of new protein for example). In contrast, a superthreshold insult constitutes a major impact that puts the
cell outside the attractor for a normal cell. In this case, the
cell will take a new trajectory through component space,
involving new sets of reactions, toward other attractors.
For a lethal insult, the end stages of cell damage will lie at
the nadir of these other attractors. These stages are considered to be within attractors because they are stable
enough to be viewed in large numbers of micrographs, so
they must persist for a significant time.
In this formalism, the several end stages, including
apoptotic cell change, are represented by different attractors, and the particular constellation of component concentrations at the end of a superthreshold insult will
determine which pathway through cell component space
is followed and hence which attractor is found (which end
stage is formed). The fact that different end stages are
associated with the same insult can be explained if different cells exit the normal attractor with different concentrations of their components. They would be at a
different spot in the space and thus take different trajectories through it. This seems a very reasonable possibility
given different initial states of cells and different proximities to glutamate release.
As discussed so far, the formalism tends to treat all
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PETER LIPTON
SECTION IV
Section IVA: Altered membrane transport properties. Membrane damage. A new study in which the phospholipid precursor, citicoline (cytidine 59-diphosphocholine), was used in an
embolus model of permanent focal ischemia revealed very protective effects, lending support to the idea that membrane damage may be a key component of cell death (A1). Citicoline, at 500
mg/kg, injected intraperitoneally 45 min after embolization reduced the infarct size 5 days later by 80%; in both penumbra and
core of the lesion, 250 mg/kg gave only marginal protection.
Protection in the core is unusual and encouraging.
Section IVB: Mitochondrial damage. Wieloch and co-workers (A18) have used a cyclosporin derivative that does not
inhibit calcineurin to examine the role of the MPT in focal
ischemic damage. They showed that it attenuates damage 48 h
after 2-h MCAO in rat (A18). The derivative is N-methyl-Val-4cyclosporin, and it was delivered at the start of reperfusion and
24 h later at 10 mg/kg. It reduced infarct size in cortex by 55%
and also in the striatum, by 75%. This is a very dramatic decrease, particularly in the core of the lesion, and suggests that
the MPT is a very important component of cell death in both
core and penumbra. It supplements earlier studies showing
protective effects of cyclosporin A that were equivocal because
the latter also blocks calcineurin. Still, caution is necessary here
until it is clear that the MPT is the effective target of the
cyclosporin derivative.
Mitochondria in apoptosis. A key step in apoptosis is
release of cytochrome c from mitochondria to cytosol. This
appears to occur in ischemia (A8), but its basis is not known.
Chan and co-workers (A7) demonstrated that the cytochrome c
release is increased in MnSOD 2/1 mice, which have ;25% the
normal complement of the mitochondrial SOD. They suggest
that free radical generation is a normal (possibly partial) trigger
for cytochrome c release. This is certainly a possibility. If so,
then cytochrome c release should be attenuated in brain overexpressing MnSOD.
SECTION V
Section VA: Free radical and peroxynitrite actions. There
is increasing evidence for superoxide production in vessel lumens, certainly during reperfusion after temporary MCAO, and
there is evidence that scavenging these radicals is highly protective. This adds some impetus to the importance of vascular
changes in causing the pathology of temporary focal ischemia,
either by white cell accumulation or vessel pathology. During 60
min of reperfusion, there was an increasing amount of superoxide radical in the lumens of the small vessels of the ischemic
zone (A19), as measured using SOD-inhibitable DAB deposition.
That this is damaging is suggested by the large number of
studies showing that exogenous SOD, in the vasculature, is
protective against ischemia (e.g., Refs. 30, 492, 679, 1154 in main
text) as well as recent data showing that extracellular SOD
knock-outs show greatly enhanced damage after transient focal
ischemia (A20).
Section VA: Free radicals and peroxynitrite actions. The
very important role of nitric oxide in the cell death following
temporary focal ischemia has been reaffirmed and expanded
substantiated by using both the neuronal NOS inhibitor 7-nitroindazole (7-NI) and the more general inhibitor L-NAME. Both
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published their study showing that the nucleosomal DNA fragments that are produced after either global or focal ischemia are
different from those in classical apoptosis or in cardiac ischemia. Rather than being blunt ended, they have a staggered end
with recessed 39-strand. Thus endonuclease activity in ischemia
is clearly different from that during classical apoptosis. This
suggests alternate methods of activation from the classical
caspase 3 activation of the endonuclease CAD (Ref. 301 in main
text). This may be one manifestation of “nonclassical” apoptosis
occurring during ischemia.
Triggers for apoptosis. The work demonstrating that
CD-95L mRNA and protein is upregulated ;12 h after 90-min
transient focal ischemia in rats and showing that mutant lpr
mice, lacking normal CD-95, have a 60% reduced infarct volume has now been published (A17). In this paper there is
quite nice evidence that the CD-95 system is involved in
apoptotic death in that the mutant mice showed no cell death
in the penumbral region. This death normally has a strongly
apoptotic morphology. These authors hypothesized that CD95L was synthesized after activation of the MAPK-JNK pathway, with c-Jun expression and phosphorylation being a key
step. Certainly, c-Jun and Jun phosphorylation were upregulated at the appropriate time, the latter in penumbra. Furthermore, FK-506, the calcineurin inhibitor, blocked c-Jun phosphorylation and upregulation of CD-95L and reduced the
infarct volume. Although more work is clearly required in this
area to establish causality, these results are really quite exciting in strongly implicating one possible apoptotic pathway
following temporary focal ischemia.
Indeed, there are several new reports demonstrating that
calcineurin inhibition is protective in transient focal ischemia,
reducing infarct sizes by 40 –70%. These studies explicitly
showed that protection did not arise from inhibition of nitric
oxide synthase (A21) and that the FK-506 (in one case) was
acting on calcineurin rather than another FKBP (A2). The site(s)
of calcineurin action has not been identified but clearly may
include blocking the CD-95 system.
Involvement of caspases in apoptosis. Caspase-3 inhibitors
are only mildly effective at protecting against many forms of
focal ischemia. One reason is suggested by studies of permanent
focal ischemia where a large upregulation of caspase-8 by cleavage of the procaspase form hours in the pyramidal cells of layer
V was seen after 6 h. Caspase-3 was elevated only in the layer
II/III cells (A22). This is interesting from several points of view.
One of these is that different populations respond differently;
another is that caspase-8 activation is generally associated with
the CD95/ligand system (as well as other death domain ligands)
and lends further fuel to the idea that this ligand system is
involved in apoptosis.
Morphological changes in global ischemia. Two studies
from Colbourne and co-workers (A4, A5) reemphasize the lack
of apoptotic morphology following very mild to somewhat intense global insults in the gerbil. There were no nuclear condensations as expected in apoptosis, and organelles were systematically dilated until ischemic cell change occurred. This fuels
the conflict over the extent of apoptosis in global ischemia,
where biochemical studies implicate the process despite lack of
good morphological evidence.
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CEREBRAL ISCHEMIA ON NEURONS
activity after 3-h ischemia and reperfusion for 24 h in Wistar rats
(A13). The basis for the increase is not known. There is almost
no immune product in the control state, although there is known
to be calpain. Presumably, the particular antibody is only able to
recognize calpain in a certain state (perhaps membrane bound),
and the amount of calpain in that state increases with time.
Although somewhat unsatisfactory because the form of the
antigen is not completely known, the result indicates that calpain activity is changing during development of the infarct, and
this may have to do with the delayed proteolysis that occurs and
was discussed in the main text (sect. VB).
SECTION VIII
Section VIIIE: Gene products that enhance damage. Inflammatory response. To the many components of the inflammatory
response that appear important for cell death is added another.
Interferon regulatory factor 1 (1RF1), a transcription factor, is
upregulated 12 h after the start of permanent focal ischemia in
the mouse (A11). Most excitingly, the IRF 2/2 mouse shows a
great reduction in infarct volume, which also occurs within the
striatal core. The heterozygote shows a 15% infarct volume
reduction, while the homozygous knockout shows a 40% decrease in infarct volume, and almost complete protection in the
striatum. Thus, with the use knock-out technology, a transcription factor has been shown to be crucial for ischemic cell death.
This is an exciting development.
Human studies. Certainly one of the major goals is to
apply research on ischemia to human disease. Although therapy has been difficult to achieve, recent studies indicate the
relevance of the animal models. In particular, elevated COX-2
has been observed in infarct regions of brains of people who
died 1–2 days after stroke. It was observed in invading neutrophils, vascular cells, and neurons in the damaged tissue
(A10). In addition to this, another enzyme that is very important in temporary focal ischemic damage in rodents, PARP,
appears to be upregulated in brain tissue removed within
1–1.5 days of cardiac arrest. There are greatly elevated levels
of poly(ADP-ribose), after these essentially global ischemic
insults, in neurons (A15).
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A4. COLBOURNE, F., H. LI, AND A. M. BUCHAN. Continuing postischemic neuronal death in CA3. Influence of ischemia duration and
cytoprotective doses of NBQX and SNX-111 in rats. Stroke 30:
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A6. ELIASSON, M. J. L., Z. HUANG, R. J. FERRANTE, M. SASAMATA,
M. E. MOLLIVER, S. H. SNYDER, AND M. A. MOSKOWITZ. Neuronal nitric oxide synthase activation and peroxynitrite formation in
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these, injected before the 3-h insult, reduced infarct in Wistar
rats by ;70% (A3); temperature effects were excluded.
An elegant study by Moskowitz and co-workers (A6) was
aimed at localizing the NO production and the peroxynitrite
production within the focal ischemic lesion. They found that
NO, measured as citrulline accumulation, was largely produced
in the peri-infarct region while peroxynitrite, measured as 3-nitrotyrosine, was increased in the infarct (A6). They thus concluded that the peroxynitrite, rather than the NO, was the major
toxic species. Presumably, NO diffuses from where it is formed,
as indicated by citrulline deposit, and encounters high levels of
superoxide in the infarct region. It reacts to form the peroxynitrite there, and this may well lead to much of the damage in the
infarct. This adds to several papers, discussed in the body of the
text, that have identified sites of generation of different components of the free radical/peroxynitrite system. The issue is not
yet resolved.
Unlike in focal ischemia, there is a paucity of data on the
role of NO in global ischemia. A report from Lei et al. (A12)
shows that nitric oxide is not elevated after 5-min ischemia in
the gerbil but is elevated for at least 3 h after 10- and 15-min
ischemia. There are actual decreases in NO during the ischemias. The elevations are completely blocked by either 7-NI
or by L-NAME, indicating they do arise from activation of
NOS. The results of NOS inhibition by 7-NI are rather paradoxical. There is some protection against 5-min ischemia,
where there was no measurable increase in NO, and there was
no protection against 10- and 15-min ischemia, where NO
rose! These results differ somewhat from those described in
the main text (Refs. 1126 and 850) where NO increases were
observed during the ischemia and after 5 min. In those cases
NO was measured directly, whereas in the current studies it
was measured as total nitrites/nitrates and so is somewhat
less direct, although is possibly more sensitive. Despite the
time course differences, the rather small protection afforded
by NOS inhibitors was evident in all the studies; in this case
there is almost none. This contrasts with focal ischemia, where
NOS inhibition is very protective and also, to some degree, with
studies of global ischemia in knock-out mice where protection is
more substantive (see sect. VA4 in text). The poor pharmacological
protection in global ischemia occurs despite the fact that the 7-NI
knocked out all NO production, measured as total nitrites/nitrates
(A12). Thus the role of NO production in global ischemia remains
quite unresolved.
Section VB: Ca21-dependent proteases. Until now there had
been only one study of effects of calpain inhibition in global
ischemia. Using a tripeptide calpain inhibitor trapped within
liposomes, Yokota et al. (A24) demonstrated ;40% protection
against ischemic damage following 5-min global ischemia in the
gerbil, although unfortunately temperature was not regulated
after the ischemia. Interestingly, they used increasing doses of
calpain, and the dose that gave 40% protection completely prevented spectrin proteolysis during the ischemia, indicating
nearly complete block of calpain activity. Thus what appears to
be fairly complete calpain blockade does not completely block
damage suggesting that the enzyme is less important than in
focal ischemia. There is no reason to think that the inhibitor in
this study was more specific than the others that have been used
so that as with all the studies to date, calpain is not unequivocally indicated as the damaging protease.
There is a steady increase in calpain immunohistochemical
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PETER LIPTON
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