1725
Improved Posthypoxic Recovery of Synaptic
Transmission in Gerbil Neocortical Slices
Treated With a Calpain Inhibitor
Ken-ichiro Hiramatsu, MD; Neal F. Kassell, MD; Kevin S. Lee, PhD
Background and Purpose: Among the various calcium-induced biologic events occurring in hypoxic
activation of the calcium-activated neutral proteinase (calpain) is a likely mediator of neuronal
degeneration. In this study, we assessed the protective effects of a calpain inhibitor (Cbz-Val-Phe-H)
against hypoxic damage to the neocortex.
Methods: An in vitro neocortical slice model from gerbils was used to study the delay to hypoxic
depolarization during hypoxia and the recovery of synaptic responses after hypoxia. These responses were
examined in control slices and slices treated with Cbz-Val-Phe-H.
Results: The delay to hypoxic depolarization did not differ between treated and control groups. In
contrast, synaptic recovery after a fixed period of hypoxia (15 minutes) was significantly improved in the
Cbz-Val-Phe-H-treated slices (P<.01). Concentrations of Cbz-Val-Phe-H of 50 ,umol/L or greater were
significantly more protective than a concentration of 20 gmol/L (P<.01).
Conclusions: The data indicate that calcium-activated proteolysis plays a critical role in hypoxic damage
to the neocortex and that calpain inhibitors may be useful therapeutic agents. (Stroke. 1993;24:1725 -1728.)
KEY WoRDs * calcium * calpain * neuronal damage * gerbils
neurons,
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It is widely held that increased intracellular calcium
concentrations play a pivotal role in neuronal
injury associated with hypoxia and ischemia.'2
However, the calcium-sensitive mechanisms responsible
for this form of neuronal damage are not well understood and need to be elucidated. Understanding the
roles of calcium-activated mechanisms will not only
provide insights into the pathophysiology of hypoxic/
ischemic damage but will also facilitate improvement of
therapeutic strategies for stroke.
Among the various calcium-sensitive biologic events in
neurons, calcium-activated proteolysis via the neutral
protease calpain is a likely candidate to participate in
hypoxic/ischemic neuronal degeneration. Calpain is activated by an appropriate signal (elevated calcium), and
sustained activation can exert deleterious effects, such as
the proteolysis of key cytoskeletal proteins. Interest in
this concept has recently been intensified by two sets of
observations. First, the levels of several calpain substrates, including neurofilament protein, microtubuleassociated protein 2, spectrin, and calcium/calmodulindependent kinase II, are reduced substantially after
hypoxia or ischemia.3-7 Second, calpain inhibitors attenuate both ischemic and hypoxic neuronal injury in the
CA1 area of the hippocampus,8-10 a selectively vulnerable
region of the central nervous system. Targeting calciumactivated proteolysis may therefore be a useful therapeutic approach for ischemic neuronal death.
One of the critical remaining issues concerning the
therapeutic utility of calpain inhibitors is whether their
Received December 18, 1992; final revision received June 4,
1993; accepted June 7, 1993.
From the Department of Neurological Surgery, University of
Virginia, Health Sciences Center, Charlottesville, Va.
Correspondence to Dr Kevin S. Lee, Box 420, Health Sciences
Center, University of Virginia, Charlottesville, VA 22908.
See Editorial Comment, page 1728
neuroprotective effects are specific to hippocampal neurons or whether their effects generalize to other brain
regions. To examine this issue, we employed an in vitro
model of hypoxic cell death that uses neocortical brain
slices.'1 The findings presented here indicate that a
membrane-permeable inhibitor of calpain, Cbz-ValPhe-H (MDL-28170),12 significantly enhances the posthypoxic recovery of synaptic transmission in the
neocortex.
Materials and Methods
Adult Mongolian gerbils (Meriones unguiculatus)
weighing 60 to 80 g were anesthetized with ether and
killed by decapitation. The basic procedures for preparing cortical brain slices were similar to those described
previously.13 Briefly, the brains were rapidly removed
and placed in cold, artificial cerebrospinal fluid (ACSF)
consisting of the following (in mmol/L): 124 NaCl, 3.3
KCl, 1.25 KH2PO4, 2.4 MgSO4, 2.0 CaCl2, 25.7
NaHCO3, 10 glucose. Using a razor blade, the brain
hemisphere was dissected into the superior-lateral
quadrant and then cut in a coronal plane at a thickness
of 400 um. Although damage to the cut surfaces occurs
in slice preparations, this damage is usually limited to
approximately 50 gm from the surface; recording electrodes are not positioned in this compromised portion
of the slices. Brain slices containing parietal cortex at
the level of the striatum were transferred to an interface-type holding chamber maintained at 35.5°C with a
humidified atmosphere of 95% 02/5% CO2. After a
posteuthanasia period of at least 1 hour, slices were
transferred individually to an identical recording chamber as required. One to four slices were studied from a
given preparation.
1726
A
Stroke Vol 24, No 11 November 1993
j
wB
C
D_>_
o12 mV
2 mec
FIG 1. Tracings show effects of transient hypoxia on evoked potentials. A, Prehypoxic waveform. The waveform of the
evoked potential consisted of early (e) and late (o) negativities. B, Same response elicited 1 minute after starting hypoxia.
The late component is drastically reduced. C, Response taken 1 minute and 45 seconds after beginning hypoxia. Both
components of the evoked response were completely eliminated; hypoxic depolarization was observed at this time. D,
Response recorded 15 minutes after reoxygenation. In this case, the response amplitude recovered to approximately
80% of baseline level.
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A bipolar stimulating electrode was positioned in
layer V of the slice, and stimuli were delivered once
every 15 seconds. A glass microelectrode filled with 3
mol/L NaCl (1 to 5 MfQ) was placed in layer III near the
position of the stimulating electrode to record the
evoked responses. The amplitude of evoked responses
was measured on-line using a computer with an analogto-digital acquisition board. Stimulation intensity was
adjusted to elicit responses of approximately 60% to
80% maximum, and responses were measured for at
least 15 minutes to verify the stability of the slice. A
second recording electrode of the same construction
was placed in layer III to record the DC potential. After
the stability of the preparation was established under
normoxic conditions, hypoxic conditions were achieved
by substituting 95% N2/5% CO2 for the normal 95%
02/5% CO2 in the atmosphere of the recording chamber. In the present studies, and in almost all reported
studies on brain slices, normoxic conditions (ie, 95%
02/5% C02) are actually hyperoxic. This level of oxygen
in the chamber is used because normal atmospheric air
is incapable of sustaining synaptic responses in most
brain slice preparations.
In the first set of experiments, hypoxic conditions
were initiated and maintained until the occurrence of
hypoxic depolarization (HD); at this time, oxygen flow
was immediately reestablished. Control slices were perfused with ACSF for the entire experiment (n=10).
Slices treated with the calpain inhibitor (n=10) were
perfused with ACSF containing 100 ,umol/L Cbz-ValPhe-H for 90 minutes before the hypoxic insult. CbzVal-Phe-H (MDL-28170) was kindly provided by Marion Merrell Dow Research Institute (Cincinnati, Ohio).
This particular calpain inhibitor was selected because of
its membrane permeability,12 inhibitory characteristics,12 and proven ability to limit hypoxic damage in the
hippocampus.9 The timing of the treatment was chosen
based on previous studies by Arai et al14 in which
calpain inhibitors were shown to block hypoxia-induced
proteolysis. The delay to HD was measured in all slices
(n=20 slices) for quantitative assessment. Statistical
analyses of the two groups were performed using the
unpaired Student's t test. A two-tailed value of P<.05
was considered to be statistically significant.
In a second set of experiments (n=56 slices), hypoxia
was sustained for a fixed period of 15 minutes. The
maximum amplitude of evoked potentials was obtained
before and 1 hour after hypoxia. In these experiments,
electrodes were repositioned after hypoxia to ensure the
recording of maximal responses. A recovery ratio for
synaptic responses was calculated by dividing the maximal posthypoxic response by the maximal prehypoxic
response and multiplying this value by 100. The magnitudes of the recovery ratios were then compared among
the different groups, which included slices treated with:
(1) ACSF only (control slices; n=26); (2) 20 gmol/L
Cbz-Val-Phe-H (n=9); (3) 50 ,umol/L Cbz-Val-Phe-H
(n=9); and (4) 100 ,umol/L Cbz-Val-Phe-H (n=12).
Recovery values were assessed by analysis of variance,
and statistical differences between experimental groups
were determined by Tukey's multiple comparison test
(P<.05 was considered significant). All data in the text
and figures are presented as mean+SEM.
Results
Evoked responses recorded in layer III of the parietal
cortex exhibited a waveform similar to those described
previously.11"3 Cortical evoked responses were characterized by a small, short-latency, nonsynaptic response
followed by a larger and longer-latency synaptic potential (Fig 1). There was no apparent change in the shape
or amplitude of the evoked waveform when ACSF
containing 100 ,mol/L Cbz-Val-Phe-H was perfused,
an observation that is consistent with the studies of
Arlinghaus et al.9
Transient hypoxia produced a characteristic sequence
of changes in electrophysiological responses (Fig 1). In
most slices the synaptic responses declined first, followed later by an elimination of the nonsynaptic component. The complete loss of the nonsynaptic component coincided temporally with the occurrence of
hypoxic depolarization. HD is a sudden, 10- to 17-mV
shift of the DC potential. As shown in Fig 2, the average
delays to HD in the control and the treated groups were
virtually identical (1.71±0.09 and 1.88±0.09 minutes,
respectively). In addition, the sequence of changes in
the waveform during hypoxia was not affected by the
drug treatment. After reoxygenation, the DC shift, the
early nonsynaptic component, and the late synaptic
component recovered in series. The DC potential recovered completely within 1 to 2 minutes in both groups.
The time course, sequence, and magnitude of recovery
of evoked potentials were similar in the Cbz-Val-PheH-treated and control groups.
In the second series of experiments, sustained hypoxia elicited the same set of intrahypoxic events as was
observed in the first series of experiments. HD occurred
within 1 to 3 minutes, and these depolarizations were
sustained until the end of hypoxia in slices from each
group. However, since hypoxic conditions were main-
Hiramatsu et al A Calpain Inhibitor in Hypoxic Slices
50- and 100-,mol/L groups were significantly greater than
that observed in the 20-,umol/L group (Fig 3).
2.25
E
2.00
c:
1.75
1727
c
0
N
0-
1.50
1.25
0
1.00
.X
0~
0
0.75
0.50
C)
E
0.25
0
Control
Cbz
(1 00 gM)
FIG 2. Bar graph depicts delay to hypoxic depolarization
in control slices and slices treated with Cbz-Val-Phe-H
(100 ,umol/L). There was no significant difference between these groups with respect to the delay to hypoxic
depolarization. Cbz indicates calpain inhibitor-treated
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group.
tained for a total of 15 minutes in this paradigm, the
depolarized state persisted for a total of 12 to 14
minutes. The recovery of DC potentials after reoxygenation was complete in all groups. In contrast, the
recovery of the evoked potentials was substantially
reduced after sustained hypoxia. In the control group, 5
of 26 slices exhibited no detectable recovery of the
synaptic potential 60 minutes after reoxygenation, and
the average amount of recovery was 20.0+2.6% of the
prehypoxic level.
Slices treated with Cbz-Val-Phe-H exhibited enhanced
recovery of synaptic potentials after prolonged hypoxia.
Treatment with 20, 50, or 100 ,umol/L of Cbz-Val-Phe-H
resulted in recovery levels of 41.4±5.9%, 67.4±4.2%, and
68.2±2.7%, respectively (Fig 3). All treated groups exhibited significantly enhanced recovery when compared with
the control group; recovery of synaptic responses in the
80-
1
cn
CO)
C
60-
0
o40-__
_
0
20-
Control
Cbz
Cbz
Cbz
(20 FM) (50 RM) (100 [xM)
FIG 3. Bar graph depicts effect of Cbz-Val-Phe-H on the
posthypoxic recovery of synaptic responses. The percent
recovery of the late negativity (synaptic component) of
the evoked waveform is shown for slices subjected to a
fixed period of hypoxia (1 5 minutes). Posthypoxic measurements were taken 60 minutes after reintroducing
oxygen into the chamber. Treatment with the calpain
inhibitor enhanced the posthypoxic recovery of responses. Values are mean+SEM. *P<.01 vs control;
#P<.01 vs 20 ,umol/L Cbz-Val-Phe-H (one-way analysis
of variance followed by Tukey's procedure). Cbz indicates calpain inhibitor-treated groups.
Discussion
Numerous studies have attempted to ameliorate ischemic and hypoxic neuronal damage by directly or indirectly limiting the elevation of intracellular calcium. The
results of these studies have been mixed.15 The limitations
of this approach probably stem from the multiple sources
that contribute to the elevation of intracellular calcium
after sustained neuronal depolarization. Calcium can enter neurons through a variety of different channels; in
addition, substantial levels of calcium can be released
from intracellular stores. It is therefore quite difficult, if
not impossible, to antagonize all of these different sources
of calcium. The present experiments indicate that interventions that target mechanisms activated subsequent to
intracellular calcium elevation represent a viable therapeutic strategy, and that calcium-activated proteolysis is a
critical component of the pathophysiological cascade leading to neuronal death.
The rationale for investigating calpain inhibitors as
potential therapeutic agents for neocortical damage is
based on several recent observations. First, key cytoskeletal proteins (ie, spectrin, microtubule-associated protein
2, and neurofilament proteins) are preferred substrates for
calpain. Second, substantial changes in the levels of these
cytoskeletal proteins have been observed after hypoxia.310,14 Presumably, the uncontrolled proteolysis of any or
all of these structural proteins would jeopardize continued
cellular viability. Finally, calpain inhibitors have been
shown to exert a neuroprotective effect against hypoxic
and ischemic damage in the hippocampal neurons.8-10 The
concept that treatment with a calpain inhibitor would be
neuroprotective therefore has a good theoretical and
mechanistic basis.
The in vitro neocortical slice model used here simulates
in vivo hypoxic neuronal insults, which can incur considerable morbidity in the cerebral cortex. The slice preparation is particularly useful because it facilitates both the
identification of mechanisms contributing to cellular damage and the evaluation of treatments designed to protect
against such damage. A common feature of hypoxic responses in both in vitro and in vivo systems is the precipitous and profound depolarization observed during the
intrahypoxic period, ie, HD. The onset of HD is closely
associated with a massive entry of calcium into neurons.1617 This is a critical step in the hypoxic response
because sustained calcium elevation triggers a cascade of
events that can ultimately lead to cell death. Treatments
prolonging the delay to HD have been shown to be useful
for limiting hypoxic neuropathologyl8; these treatments
are presumed to act by delaying the elevation of intracellular calcium. A complementary approach for achieving
neuroprotection would be to reduce the deleterious impact of high concentrations of intracellular calcium. By
attenuating harmful calcium-induced mechanisms, it
might be possible to limit the extent of cellular damage at
a later stage of the hypoxic cascade. A useful feature of the
in vitro slice model is that it permits one to discriminate
between therapeutic effects operating before or after
calcium entry into the cell. The present studies took
advantage of this feature to characterize the neuroprotective effects of a calpain inhibitor. The data presented here
indicate that doses ranging from 20 to 100 ,.tmol/L of
Cbz-Val-Phe-H are protective. However, no greater neuroprotective effect was obtained in the 100-,umol/L group
1728
Stroke Vol 24, No 11 November 1993
than was seen with the 50-,umol/L group. The reason for
this lack of a progressive effect is unclear. It is possible that
the protection achieved at 50 ,umol/L and 100 ,umol/L
Cbz-Val-Phe-H represents a maximal effect. Further studies will be required to clarify this issue.
In conclusion, our results demonstrate that treatment
with Cbz-Val-Phe-H effectively improved posthypoxic
synaptic recovery without altering the delay to HD.
These observations indicate that the neuroprotective
effect of the calpain inhibitor is not the result of delayed
calcium entry. Rather, it appears to operate by mitigating against the impact of elevated intracellular calcium.
These findings underscore the importance of calciumactivated proteolysis in the process of hypoxic/ischemic
cell death and suggest that calpain inhibitors may be a
useful therapeutic treatment for clinical situations involving ischemia of the central nervous system.
6.
7.
8.
9.
10.
11.
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Acknowledgments
12.
This study was supported by National Institutes of Health
grant 30671 to Dr Lee. We thank Dr Frank Schottler for
critical reading of this work.
13.
14.
References
1. Choi DW. Calcium-mediated neurotoxicity: relationship to specific
channel types and role in ischemic damage. Trends Neurosci. 1988;
11:465-469.
2. Siesjo BK, Bengtsson F. Calcium influx, calcium antagonists, and
calcium-related pathology in brain ischemia, hypoglycemia and
spreading depression. J Cereb Blood Flow Metab. 1989;9:127-140.
3. Seubert P, Lee K, Lynch G. Ischemia triggers NMDA receptorlinked cyotskeletal proteolysis in hippocampus. Brain Res. 1989;
492:366-370.
4. Yamamoto H, Fukunaga K, Lee K, Soderling TR. Ischemiainduced loss of brain calcium/calmodulin-dependent protein kinase
II. J Neurochem. 1992;58:1110-1117.
5. Taft WC, Tennes-Ress KA, Blair RE, Clifton GL, DeLorenzo RJ.
Cerebral ischemia decreases endogeneous calcium-dependent
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Kitagawa K, Matsumoto M, Niinobe M, Mikoshiba K, Hata R,
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Microtubule-associated protein 2 as a sensitive marker for cerebral
ischemic damage: immunohistological investigation of dendritic
damage. Neuroscience. 1989;31:401-411.
Arai A, Kessler M, Lee K, Lynch G. Calpain inhibitors improve the
recovery of synaptic transmission from hypoxia in hippocampal
slices. Brain Res. 1990;532:63-68.
Arlinghaus L, Mehdi S, Lee KS. Improved posthypoxic recovery
with a membrane-permeable calpain inhibitor. Eur J Pharmacol.
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Lee KS, Frank S, Vanderklish P, Arai A, Lynch G. Inhibition of
proteolysis protects hippocampal neurons from ischemia. Proc Natl
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Hiramatsu K, Kassell NF, Lee KS. Thermal sensitivity of hypoxic
responses in neocortical brain slices. Soc Neurosci Abstr. 1992;
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Mehdi S. Cell-penetrating inhibitors of calpain. Trends Biochem
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Lee KS. Sustained enhancement of evoked potentials following
brief, high-frequency stimulation of the cerebral cortex in vitro.
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Arai A, Vanderklish P, Kessler M, Lee K, Lynch G. A brief period
of hypoxia causes proteolysis of cytoskeletal proteins in hippocampal slices. Brain Res. 1991;555:276-280.
Buchan A. Advances in cerebral ischemia: experimental approaches.
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Somjen GG, Aitken PG, Balestrino M, Herreras 0, Kawasaki
K. Spreading depression-like depolarization and selective vulnerability of neurons: a brief review. Stroke. 1990;21(suppl 3):
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Editorial Comment
Calcium influx following the activation of postsynaptic glutamate receptors has been identified as a major
factor contributing to neuronal death after an ischemia
insult.12 Various experimental strategies have aimed at
the blockade of various glutamate receptors and the
prevention of calcium influx.3 However, this approach
may cause undesirable side effects, since it affects the
normal neuronal function that is also associated with
glutamate receptor activation.4 Other experimental approaches have aimed at modifying calcium cascades,
including the alteration of the activities of phospholipases, proteases, protein kinase C, and Ca 2+ /calmodulindependent protein kinase 11.5 Using well-defined, neocortical slices of adult Mongolian gerbils as a model
system, Hiramatsu et al now report that posthypoxically
induced synaptic transmission recovery was significantly
improved by Cbz-Val-Phe-H, a specific inhibitor of the
calcium-activated, neutral proteinase calpain.6 These
data support the notion that Ca 2+-associated protein
degradation is an important event among the calcium
cascades that would ultimately lead to synaptic dysfunction and neuronal death.7 Thus, further studies are
warranted to extend this in vitro provocative observation of this compound to its possible use as neuronal
protective agent in vivo so that therapeutic intervention
can be developed to ameliorate ischemic brain injury.
Pak H. Chan, PhD, Guest Editor
Departments of Neurosurgery and Neurology
CNS Injury and Edema Research Center
University of California
School of Medicine
San Francisco, Calif
References
1. Choi DW. Calcium-mediated neurotoxicity: relationship to specific
channel types and role in ischemic damage. Trends Neurosci. 1988;
11:465-469.
2. Siesjo B, Bengtsson F. Calcium fluxes, calcium antagonists, and
calcium-related pathology in brain ischemia, hypoglycemia, and
spreading depression: a unifying hypothesis. J Cereb Blood Flow
Metab. 1989;9:127-140.
3. Choi DW. The role of glutamate neurotoxicity in hypoxic ischemic
neuronal death. Ann Rev Neurosci. 1990;13:171-182.
4. Hirose K, Chan PH. Blockade of glutamate excitotoxicity and its
clinical applications. Neurochem Res. 1993;18:479-483.
5. Choi DW. Methods for antagonizing glutamate neurotoxicity. Cerebrovasc Brain Metab Rev. 1990;2:105-147.
6. Mehdi S. Cell-penetrating inhibitors of calpain. Trends Biochem Sci.
1991;16:150-153.
7. Lee KS, Frank S, Vanderklish P, Arai A, Lynch G. Inhibition of
proteolysis protects hippocampus neurons from ischemia. Proc Natl
Acad Sci USA. 1991;88:7233-7237.
Improved posthypoxic recovery of synaptic transmission in gerbil neocortical slices treated
with a calpain inhibitor.
K Hiramatsu, N F Kassell and K S Lee
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Stroke. 1993;24:1725-1728
doi: 10.1161/01.STR.24.11.1725
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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