Electrochemical Failure of the Brain Cortex Is More
Deleterious When it Is Accompanied by Low Perfusion
Jens P. Dreier, MD; Ilya V. Victorov, MD; Gabor C. Petzold, MD; Sebastian Major, MD;
Olaf Windmüller, MD; Francisco Fernández-Klett, MD; Mahesh Kandasamy, PhD;
Ulrich Dirnagl, MD; Josef Priller, MD
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Background and Purpose—Clinical and experimental evidence suggests that spreading depolarization facilitates neuronal
injury when its duration exceeds a certain time point, termed commitment point. We here investigated whether this
commitment point is shifted to an earlier period, when spreading depolarization is accompanied by a perfusion deficit.
Methods—Electrophysiological and cerebral blood flow changes were studied in a rat cranial window model followed by
histological and immunohistochemical analyses of cortical damage.
Results—In group 1, brain topical application of artificial cerebrospinal fluid (ACSF) with high K+ concentration ([K+]ACSF) for
1 hour allowed us to induce a depolarizing event of fixed duration with cerebral blood flow fluctuations around the baseline
(short-lasting initial hypoperfusions followed by hyperemia). In group 2, coapplication of the NO-scavenger hemoglobin
([Hb]ACSF) with high [K+]ACSF caused a depolarizing event of similar duration, to which a severe perfusion deficit was coupled
(=spreading ischemia). In group 3, intravenous coadministration of the L-type calcium channel antagonist nimodipine with
brain topical application of high [K+]ACSF/[Hb]ACSF caused spreading ischemia to revert to spreading hyperemia. Whereas
scattered neuronal injury occurred in the superficial cortical layers in the window areas of groups 1 and 3, necrosis of all
layers with partial loss of the tissue texture and microglial activation were observed in group 2.
Conclusions—The results suggest that electrochemical failure of the cortex is more deleterious when it is accompanied by
low perfusion. Thus, the commitment point of the cortex is not a universal value but depends on additional factors, such
as the level of perfusion. (Stroke. 2013;44:490-496.)
Key Words: neuroprotection
■
spreading depression
■
spreading ischemia
B
ack in 1947, Aristides Leão had coined the idea that electrically induced short-lasting spreading depolarization
(SD) in healthy, naïve tissue and the terminal SD succeeding
arrest of the circulation are of the same nature.1 Later, experimental work confirmed that essential biophysical features
of short-lasting SD correspond with those observed during
the initial stage of terminal SD2. Thus, the unifying theme
of all SDs seems to be the near-complete breakdown of the
physiological electrochemical gradients across the neuronal
membranes associated with loss of electric activity, neuronal
swelling, and distortion of dendritic spines.2–5 Nevertheless,
marked differences exist within this spectrum of near-complete sustained depolarizations.6,7 This applies, for example, to
the sensitivity of the depolarizations to N–methyl-D–aspartate
receptor antagonists,8–13 or the observation that terminal depolarization is associated with neuronal death in contrast to
short-lasting depolarizations in healthy, naïve tissue.14,15
■
subarachnoid hemorrhage
■
vasospasm
Since the end of the 1970s, experimental evidence has
been accumulating that events with features between SD in
healthy naïve tissue and terminal depolarization occur in the
ischemic penumbra after middle cerebral artery occlusion,
under hypoxia, hypoglycemia or in the presence of chemical
factors, such as potassium.16–20 It has been increasingly recognized that those depolarizations of intermediate duration are
associated with lesion progression, although the neurons seem
to recover transiently from the ionic imbalance.21–25 Hence,
it has been hypothesized that they entail triggers activating
intracellular signals and proteases, leading to cell death if the
depolarization outlasts a certain time point, termed commitment point.2,4 Possibly, this commitment point is not universal
but depends on neuroanatomical structure, neuron population,
developmental stage, and noxious condition.4
SD is observed as a large negative direct current (DC)
change. The duration of the DC negativity is an extracellular
Received April 11, 2012; final revision received November 6, 2012; accepted November 26, 2012.
From the Center for Stroke Research Berlin (J.P.D., S.M., U.D.), Department of Experimental Neurology (J.P.D., S.M., G.C.P., O.W., U.D.), Department
of Neurology (J.P.D., S.M., G.C.P.), Department of Neuropsychiatry (F.F.-K., M.K., J.P.), and Excellence Cluster NeuroCure (J.P.D., U.D., J.P.), Charité
University Medicine Berlin, Berlin, Germany; Department of Neurology, University of Bonn, Bonn, Germany (G.C.P.); and Laboratory of Experimental
Neurocytology, Brain Research Institute, Moscow, Russia (I.V.).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
112.660589/-/DC1.
Correspondence to Jens P. Dreier, MD, Department of Experimental Neurology, Charité University Medicine Berlin, Charitéplatz 1, 10117 Berlin,
Germany. E-mail [email protected]
© 2013 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.112.660589
490
Dreier et al Electrochemical Failure and Perfusion 491
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index for the duration of the cellular depolarization. Novel
technology for invasive full-band DC signals has recently
revealed that a spatio-temporally diverse spectrum from shortlasting to very prolonged large negative DC changes also
occurs in the injured human brain.26–28 It is, therefore, interesting to study the commitment point under different conditions
in animals. This might answer the questions whether the commitment point is a universal value or not, and whether it is
necessary to combine DC recordings with recordings of other
measures, such as cerebral blood flow (CBF), tissue partial
pressure of oxygen, or parameters of cerebral metabolism to
estimate the commitment point in patients.
Here, we used an animal model in which artificial cerebrospinal fluid (ACSF) containing high K+ concentration
([K+]ACSF) was applied topically to the brain. We specifically
tested whether; (1) high [K+]ACSF can induce a prolonged depolarizing event in vivo that is reversible on wash-out; and (2) the
depolarizing event is not associated with a marked persistent
CBF change. The high [K+]ACSF model is interesting because
it shares the drug resistance to N–methyl-D–aspartate receptor antagonists with terminal depolarization under anoxia,29
and it allows us to modify the hemodynamic response to the
depolarizing event. Thus, coapplication of high [K+]ACSF with
an NO-lowering agent, such as hemoglobin (Hb), converts the
hemodynamic response from spreading hyperemia to ischemia (=inverse neurovascular coupling), whereas intravenous
coapplication of the L-type calcium antagonist nimodipine
causes the spreading ischemia to revert to the normal hyperemic response.30,31 The same inverse neurovascular response
can also be produced by the coapplication of high [K+]ACSF
with an NO-synthase inhibitor, such as N–nitro-L–arginine,31
but not by the coapplication of high [K+]ACSF with other vasoconstrictors, such as endothelin-1 (ET-1).32 The antagonistic
effect of nimodipine on the inverse neurovascular response
is attributable to its antagonistic action on the vasoconstrictors, which are released during the SD process, as previously
shown in an in vitro model for spreading ischemia in the
isolated middle cerebral artery.30 This antagonistic effect of
nimodipine has been of particular clinical interest because,
using subdural opto-electrodes for DC electrocorticography
and laser-Doppler flowmetry, it was observed in patients
that the inverse neurovascular response to SD is a mechanism involved in delayed ischemic stroke after subarachnoid
hemorrhage.27 In previous clinical trials, prophylactic treatment with nimodipine was found to reduce the frequency and
severity of delayed ischemic stroke.33 This beneficial effect of
nimodipine was possibly because of its antagonistic effect on
the inverse neurovascular response,31 a hypothesis further supported by the observation that nimodipine had no antagonistic
effect on the proximal vasospasm after subarachnoid hemorrhage,34 another candidate pathogenesis underlying delayed
ischemic stroke. For a more comprehensive account of the
mechanisms involved in the inverse neurovascular response,
including the possible role of astrocytes, we would like to
refer the reader to a previous review.4 Notably, because of the
markedly enhanced energy demand, SD can induce pockets
of tissue hypoxia in most distant supply territories of cortical capillaries, even when the CBF response is hyperemic.5
Importantly, the tissue hypoxia is dramatically augmented
when, in addition to the increased energy demand, the CBF
response is inverted.27
The features of the high [K+]ACSF model as described above
enabled us to investigate in vivo whether electrochemical neuronal failure of similar duration, induced by identical conditions in the subarachnoid space, has a more deleterious effect
at a reduced level of perfusion or not. In an analogous fashion,
we could have reduced the partial pressure of oxygen under
high [K+]ACSF to tackle our question, but this would have led
to global side effects, and it would not have been possible to
induce an energy compromise of similar severity because the
animals would have died from cardiac arrest.
The results of the present study may have relevance for
aneurismal subarachnoid hemorrhage or the ischemic penumbra after cerebral vessel occlusion because SDs can induce
perfusion deficits under these conditions both in animals and
humans.27,31,35,36
Methods
Animals
All animal experiments were performed in compliance with the
Governmental Animal Care and Use Committee. For a detailed description of Methods, see the online-only Data Supplement. An open
cranial window was implanted as reported previously in 22 animals
under halothane anesthesia.21 The dura mater was removed, and
ACSF continuously applied brain topically.
Data Collection
CBF was monitored by laser-Doppler flowmetry, and the surface DCelectrocorticogram with a subdural Ag–AgCl electrode. In 4 animals
of group 1, changes of the extracellular K+ concentration ([K+]o) and
intracortical DC-electrocorticogram were recorded with K+-selective
microelectrodes.30
Experimental Paradigm
In all groups, a long-lasting depolarizing event was induced by increase of the K+ concentration in the ACSF ([K+]ACSF) from 3 to 250
mmol/L for 1 hour. In group 1, high [K+]ACSF was applied alone,
whereas it was coapplied with Hb ([Hb]ACSF, 2 mmol/L) in groups
2 and 3. In group 3, nimodipine was coadministered intravenously
at a concentration of 2 µg/kg per minute. Hb was freshly prepared
from heparinized arterial rat blood. The rise of [K+]ACSF determined
[Na+]ACSF. [Na+]ACSF was reduced simultaneously to 24.5 mmol/L,
whereas [Cl−]ACSF rose from 136 to 255.5 mmol/L. Hence, ACSF osmolarity increased from ≈330 mosmol/L to ≈570 mosmol/L.
Five rats for each of the 3 experimental groups and 3 controls
([K+]ACSF at 80 mmol/L) were perfused transcardially with formalin at
48 hours after the experiment. Paraffin-embedded brain sections were
stained with hematoxylin and eosin, assayed for terminal deoxyuridine triphosphate nick-end labeling (TUNEL), and double-immunostained using antibodies against the glial fibrillary acidic protein and
Iba1. Immunohistochemical staining was analyzed by laser scanning
confocal microcopy. Histological analysis of 2 nonoperated naïve rat
brains served as controls. Quantification of TUNEL-, glial fibrillary
acidic protein–, and Iba1-positive cells in the window areas was performed by a blinded investigator (M.K.) on 1 to 2 sections per animal
of groups 1 to 3 as well as controls using a stereological approach
(see Methods in the online-only Data Supplement).
Results
Changes of DC-Electrocorticogram and CBF
Mean arterial pressure and arterial Po2 remained within the
normal range in the 3 groups, whereas animals of groups 2
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and 3 mildly hyperventilated (Table I in the online-only Data
Supplement). In group 1 (n=9), [K+]ACSF (250 mmol/L) was
applied alone. Before the cluster of recurrent SDs, [K+]o rose
from 3.0 to 4.5±0.8 mmol/L in a cortical depth of 400 µm,
and CBF increased mildly from 100 to 123±23%. The first
SD in the cluster showed a negative intracortical DC shift of
−19.1±4.0 mV, which lasted for 100±34 seconds. The delay
between the 2 microelectrodes was 48±29 seconds, indicating propagation of SD. In a depth of 400 µm, the isolated
depolarizations occurred superimposed on an ultraslow positive potential of 10.4±1.6 mV, and then melted progressively
with each other, so that the negative intracortical DC amplitude of individual depolarizations decreased to −5.5±5.2 mV
as shown in the representative traces of 1 animal in Figure 1.
Subdurally, the ultraslow potential was negative during the
cluster (−25.2±6.1 mV). [K+]o rose transiently to 40.8±4.4
mmol/L during the first SD. It did not return fully to baseline before the next depolarization started. As a consequence,
baseline [K+]o rose slowly and steadily between the recurrent depolarizations, until it reached a plateau of 52.9±31.7
mmol/L. From this plateau, [K+]o rose transiently to 70.2±21.6
mmol/L with each depolarization (Figure 1). Coupled to each
SD, short-lasting initial hypoperfusion to 73±20% for 49±16
seconds was followed by short-lasting spreading hyperperfusion to 142±39%. In Figure 1, the CBF fluctuations are not
much larger than the low-frequency vascular fluctuations that
preceded the SD cluster. The cluster ended within 19.3±14.7
minutes after wash-out of high [K+]ACSF.
In group 2 (n=5), brain topical application of [K+]ACSF
(250 mmol/L)/HbACSF (2 mmol/L) without nimodipine did
not change CBF before the first SD started (102±20%). The
SDs were characterized by a negative subdural DC shift of
−26.2±4.0 mV, to which spreading ischemia was coupled
Figure 1. Representative traces of a cluster of spreading depolarizations induced by high [K+]ACSF in an animal of group 1. Note
melting of depolarizations. ACSF indicates artificial cerebrospinal
fluid; CBF, cerebral blood flow; and DC, direct current.
lasting for 72±21 minutes (Figure 2A). During spreading ischemia, CBF dropped to 21±7% (Figure 2A).
In group 3 (n=5), combined administration of nimodipine intravenously (2 µg/kg per minute) with topical [K+]ACSF
(250 mmol/L)/HbACSF (2 mmol/L) increased CBF to 164±11%
before the first SD started. This CBF level before the first SD
was significantly higher than in the other 2 groups (1-way
ANOVA with Bonferroni post hoc test; P<0.05). The cluster
of SDs was characterized by a negative subdural DC shift of
−19.9±4.0 mV. Coupled to single SDs, short-lasting initial
hypoperfusion with a drop from 164 to 119±16% for 61±30
seconds was followed by short-lasting spreading hyperperfusion rising to 198±26% (Figure 2B). During the depolarizing
event, the mean CBF level was 97±39% in group 1, 42±7% in
group 2, and 161±12% in group 3. All groups differed significantly from each other (1-way ANOVA with Bonferroni post
hoc test; P<0.05; Figure 2C).
Animals of group 2 showed hemiparesis at 48 hours after
spreading ischemia (average grade: 2.6±1.1 on a scale from
0 [no deficit] to 4 [severe paresis]),37 whereas no significant
neurological deficit was observed in animals of groups 1 and
3, and the 3 control animals, in which single SDs had occurred
in response to 80 mmol/L [K+]ACSF (0.2±0.4 in each group).
Histological Changes
Histological analysis revealed brain herniation in the window
area (Figure 2Aa and 2Ba) and a meningeal reaction with
local accumulation of neutrophils in all experimental animals.
Single neurons in the first cortical layer were surrounded
by edema in the control group. Brains derived from groups
1 (high [K+]ACSF) and 3 (high [K+]ACSF/[Hb]ACSF/nimodipine)
showed tissue necrosis in the first cortical layer with single
shrunken acidophilic cells. In the second cortical layer, the
pathological changes mainly consisted of neuronal shrinkage as well as perineuronal and perivascular swelling (edema;
Figure 3Ab). In these layers, 2 main types of damaged neurons were detected: shrunken nonscalloped and shrunken scalloped cells. The shrunken neurons contained nuclei of normal
size and form without signs of chromatin condensation. The
cytoplasm of these neurons was somewhat denser than normal and contained dispersed Nissl substance. Around the
cell body, there was a rim of pericellular swelling. Shrunken
scalloped neurons had highly compressed triangular bodies,
sometimes with marked proximal dendrite stems. These cells
were surrounded by a wide pericellular space. Deeper layers
were mostly free of cellular injury (Figures 2Ba and 3Ab).
Brains derived from group 2 showed a prominent inflammatory response consisting of Iba1-immunoreactive microglia
and macrophages in the window area (Figure 3Ba). In comparison, the inflammatory reaction was significantly reduced
in brains derived from group 3 (Figure 3Bb). Quantification of
Iba1-immunoreactive cells in the cortical area below the cranial window revealed significant differences between group 2
and all other groups (group 1: 1.1±0.4×104 cells/mm3; group 2:
2.8±0.2×104 cells/mm3; group 3: 0.8±0.3×104 cells/mm3; controls: 1.7±0.1×104 cells/mm3; 1-way ANOVA with Bonferroni
post hoc tests; P<0.001; Figure 3Ba, 3Bb, and 3C).
The density of glial fibrillary acidic protein-immunoreactive
cells was slightly higher in brains derived from group 2, but this
Dreier et al Electrochemical Failure and Perfusion 493
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Figure 2. Comparison of cerebral blood flow (CBF) and direct current (DC) potential recordings as well as histological outcome between
(A) an animal not treated with nimodipine and (B) another animal treated with nimodipine intravenously after topical administration of high
[K+]ACSF and [Hb]ACSF to the brain. Aa and Ba, Hematoxylin- and eosin-stained sections of the window areas. Note the necrotic region with
partial loss of the tissue texture in Aa (marked with an x). Ab and Bb, Top trace: CBF in the window area; bottom trace: DC potential.
Note the spreading depolarizations (SD) with spreading ischemia in Ab and the SDs with spreading hyperemia in Bb. Ac, Propagation
of the spreading ischemia is illustrated in the top 2 traces. Note that the DC negativity (lowest trace) precedes the CBF decrease. C,
The amplitude of the subdural negative DC shift was not statistically different between animals treated with high [K+]ACSF alone (high K+),
[Hb]ACSF plus high [K+]ACSF (Hb/high K+) and nimodipine plus [Hb]ACSF plus high [K+]ACSF (nimo/Hb/high K+) in contrast to the mean CBF level
during the depolarizing event, which differed significantly among the 3 groups (*1-way ANOVA with Bonferroni post hoc test; P<0.05).
Hb indicates hemoglobin.
difference did not reach statistical significance (1-way ANOVA
with Bonferroni post hoc test; Figure 3Ba, 3Bb, and 3D). Few
TUNEL-positive cells were found in the cortex below the
cranial window in brains derived from group 3 (Figure 4Ab),
whereas a high number of TUNEL-positive cells was detected
across all cortical layers in brains derived from group 2 (Figure
4Aa). Quantification of TUNEL-positive cells in the cortical
area below the cranial window demonstrated a significant difference between groups 2 and 3 (group 1: 0.8±0.2×104 cells/
mm3; group 2: 1.8±0.9×104 cells/mm3; group 3: 0.4±0.2×104
cells/mm3; controls: 1.0±0.3×104 cells/mm3; Kruskal–Wallis
1-way ANOVA on Ranks with Dunn post hoc test; P<0.05;
Figure 4B). In all experimental groups, astrocytes and microglia were mildly activated all over the brain (including the contralateral hemisphere) when compared with naïve animals.
Discussion
In the present study, we induced a depolarizing event by brain
topical application of high [K+]ACSF for 1 hour. The depolarizing event consisted of recurrent SDs melting with each other
as reported previously for SDs induced by high [K+]ACSF in
the rat hippocampus.20 Subarachnoid K+ concentrations of 250
mmol/L do not occur in natural disease conditions. However,
the model provides interesting information for the fundamental understanding of SD and cortical damage because it allows
us to determine a commitment point for a prolonged depolarizing event at relatively normal perfusion level. In the present
study, the duration of about 60 minutes was not sufficient to
cause damage in deeper cortical layers, although the intracortical K+ concentration reached a level comparable with that in
natural disease conditions. [K+]o rose to about 70 mmol/L in a
cortical depth of 400 µm similar to the level of [K+]o reported
previously for terminal SD induced by anoxia in vivo.38
However, it remains possible that the harmful effect of SD is
underestimated because it could be deleterious through loss of
intracellular potassium, and high [K+]ACSF may mitigate the K+
outward flux.39
The classic experimental approach to demonstrate the
harmful effect of SD has been to ignite it chemically outside
of the ischemic penumbra after middle cerebral artery occlusion. Those SDs then propagated into the penumbra, and it was
measured that they enlarged the ischemic core as evaluated
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Figure 3. Impact of nimodipine treatment on neuronal cell loss and gliosis induced by high [K+]ACSF/[Hb]ACSF. Aa, Hematoxylin and eosin
staining of the cortex of an animal from group 2 (high [K+]ACSF/[Hb]ACSF). Note the extensive necrosis in superficial and deeper cortical
layers. Neurons with condensed nuclei are indicated by triangles. Ab, Hematoxylin and eosin staining of the cortex of an animal from
group 3 (high [K+]ACSF/[Hb]ACSF/nimodipine). Pathological changes are mostly restricted to the superficial cortical layers with perivascular
swelling and edema (arrows), shrunken nonscalloped neurons (open triangle), and shrunken scalloped neurons (closed triangle).
B, Double immunohistochemical staining for glial fibrillary acidic protein (GFAP) and Iba1 reveals numerous Iba1-immunoreactive
microglia and macrophages in brains derived from group 2 (Ba) and less Iba1-positive cells in brains obtained from group 3 (Bb). Reactive
GFAP-positive astrocytes are detected in both groups (Ba and Bb). C, Quantification of Iba1-immunoreactive cells in the window area
shows a significant increase in the density of Iba1-positive cells in brains derived from group 2 compared with groups 1 and 3 (*1-way
ANOVA with Bonferroni post hoc tests; P<0.001). D, No significant differences in the densities of GFAP-positive astrocytes were detected
among the 3 groups. Scale bars=100 µm (A) and 20 µm (B). Hb indicates hemoglobin.
by continuous apparent diffusion coefficient of water mapping.22,23 A similar conclusion was reached using brain topical
application of ET-1 in rats.21 In this model, the vasoconstrictive
effect of different ET-1 concentrations can be titrated. An ET-1
concentration was chosen, at which 50% of animals developed a local low-flow area giving rise to spontaneous SDs.
Figure 4. Triphosphate nick-end labeling (TUNEL)-positive cells after spreading ischemia. A, Comparison of TUNEL-positive cells in the
window area between an animal with spreading depolarizations after topical administration of high [K+]ACSF and [Hb]ACSF (group 2; Aa) and
another animal treated with nimodipine intravenously (group 3; Ab). TUNEL-positive cell nuclei are visualized by the brown 3,3′-diaminobenzidine (DAB) stain. B, Quantification of TUNEL-positive cells in the window area reveals a significantly increased density of TUNELpositive cells in brains derived from group 2 compared with group 3 (*Kruskal–Wallis 1-way ANOVA on Ranks with Dunn post hoc test;
P<0.05). Scale bar=20 µm. Hb indicates hemoglobin.
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Subsequent histological analysis revealed selective neuronal
necrosis restricted to this area. In the remaining 50% of animals, the same ET-1 concentration induced a low-flow area,
but neither SD nor focal necrosis (=nonresponders). However,
when an SD was triggered chemically in nonresponders and
propagated into ET-1–exposed cortex, those animals also
developed focal necrosis. Surprisingly, local duration of this
SD, as evaluated by the large negative DC shift, was only little
longer in the ET-1–exposed cortex compared with the nonischemic cortex.21 However, the large negative DC shift was superimposed on a shallow ultraslow potential component with
opposing polarity between the 2 regions, being positive in the
nonischemic and negative in the ET-1–exposed cortex. It was
suggested that the negative ultraslow component was related
to cellular injury.20,26,40 In the present study, high [K+]ACSF alone
produced recurrent SDs superimposed on a similar ultraslow
potential component. Interestingly, the ultraslow potential was
negative at the surface but positive deep in the cortex. Cortical
damage was limited to the superficial cortical layers. This supports the idea that the polarity of the ultraslow potential may
be related to the damage. The generators of the ultraslow negativity are unknown but probably of non-neuronal origin, which
might include both astrocytic and noncellular mechanisms as
reviewed recently.41 If the negative ultraslow potential is of
noncellular origin, it should be a diffusion potential, that is,
attributable to ion gradients in the extracellular space caused
by spillover of the ion content of cells on membrane lysis.
Necrosis of all cortical layers only developed in group 2
(high [K+]ACSF/[Hb]ACSF), in which a severe perfusion deficit
was coupled to the depolarizing event. In contrast, the cortical
damage was limited to the surface when intravenous nimodipine caused the severe perfusion deficit to revert to hyperemic
flow responses in group 3. Topical application of [K+]ACSF (3
mmol/L)/HbACSF (2 mmol/L) to the brain was previously studied, and neither inverted the neurovascular response to SD31
nor resulted in significant neuronal injury.42 These observations support the concept that decreased oxidative substrate
supply shifts the commitment point to an earlier period. What
could explain this?
1.During SD, electrochemical failure is only near-complete. Preserved function of the Na,K-ATPase seems
to prevent the complete electrochemical failure. When
energy deprivation causes failure of the Na,K-ATPase,
complete electrochemical failure might ensue, resulting
in neuronal death.
2.Absence of perfusion could halt subcellular energydependent protective processes downstream of the electrochemical failure.
3.Although astrocytes themselves are more resistant to
energy deprivation than neurons because of their higher
anaerobic capabilities, energy deprivation may cause
astrocytes to lose important protective effects on neurons, and this may render neurons more vulnerable.40
This is further discussed in the Discussion in the onlineonly Data Supplement.
Clinical Implications
In patients with subarachnoid hemorrhage, prolonged SDs
may be attributable to the rise of potassium to up to 50 mmol/L
in the subarachnoid clot,43 endogenous ouabain-like factors,
glutamate release, hypoglycemia, hypoxia, hypoperfusion, or
other factors yet to be determined that interfere with excitability, extracellular clearance of metabolites, or Na,K-ATPase
function.42 Moreover, SDs could be modified by drugs, such
as sedatives, anticonvulsants, or the L-type calcium antagonist
nimodipine, which inhibits calcium influx into neurons.44 Such
factors may not only influence the duration of SD,15,31,45 but
also modify subcellular mechanisms, and may thus shift the
commitment point. The effect of attendant circumstances on
the commitment point implies that a multimodal recording in
patients, including measurements of DC electrocorticography,
CBF, tissue oxygen, and glucose, is probably superior to DC
electrocorticography alone to determine the tissue outcome.27,46
Sources of Funding
This study was supported by DFG DR 323/3-1, BMBF (Center for
Stroke Research Berlin 01 EO 0801, BCCN 01GQ1001C-B2, and
Kompetenznetz Schlaganfall to J.P. Dreier.) and DFG FOR1336/B3
(to J. Priller.).
Acknowledgments
The authors would like to thank Peggy Mex for excellent technical
assistance.
Disclosures
None.
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Electrochemical Failure of the Brain Cortex Is More Deleterious When it Is Accompanied
by Low Perfusion
Jens P. Dreier, Ilya V. Victorov, Gabor C. Petzold, Sebastian Major, Olaf Windmüller,
Francisco Fernández-Klett, Mahesh Kandasamy, Ulrich Dirnagl and Josef Priller
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Stroke. 2013;44:490-496; originally published online January 3, 2013;
doi: 10.1161/STROKEAHA.112.660589
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SUPPLEMENTAL MATERIAL.
Supplemental Methods:
Animals
All animal experiments were performed in compliance with the Governmental Animal Care and
Use Committee (Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit
Berlin (LAGetSi)). The animals were housed in groups (2-4 animals per cage) under a 12h
light/dark cycle with food and tap water available ad libitum. Twenty-two male Wistar rats (250
to 400g; Charles River Laboratories, Wilmington, MA, USA) were anesthetized with halothane
(1.5% in 30% O2 and 70% N2O) and breathed spontaneously. Body temperature was maintained
at 38.0±0.5°C. The tail artery was cannulated and continuously infused with saline solution.
Systemic arterial pressure (RFT Biomonitor, Zwönitz, Germany), endexpiratory pCO2 (Heyer
CO2 Monitor EGM I; Bad Ems, Germany) and arterial blood gases were monitored with a
Compact 1 Blood Gas Analyzer (AVL Medizintechnik GmbH, Bad Homburg, Germany).
An open cranial window of 4x7mm was implanted over the frontoparietal cortex using a
saline-cooled drill as reported previously14,28. The dura mater was removed, and ACSF
continuously applied brain topically. The physiological composition of the ACSF in mM was:
Na+ 152, K+ 3, Ca2+ 1.5, Mg2+ 1.25, HCO3- 24.5, Cl- 136, glucose 3.7, and urea 6.7. The ACSF
was equilibrated with a gas mixture containing 6.6% O2, 5.9% CO2, and 87.5% N2.
Data Collection and Statistical Analysis
CBF was monitored by laser-Doppler flowmetry (Perimed AB, Järfälla, Sweden). The surface
DC-electrocorticogram (ECoG) (bandpass: 0–45Hz) was measured with a subdural Ag-AgCl
electrode. In 4 animals of group 1, changes of the extracellular K+ concentration ([K+]o) and
intracortical DC-ECoG were recorded with two K+-selective microelectrodes in a cortical depth
of 400µm. K+-selective microelectrodes were prepared and tested as described previously from
double-barrelled thetaglass capillaries (Kugelstätter, Garching, Germany)22. Potassium
1
ionophore I-cocktail A (Fluka/Sigma-Aldrich, Steinheim, Germany) was used as ion exchanger.
Electrodes were connected to a custom-made differential amplifier. Analog-to-digital
conversion was performed using a Power 1401 (Cambridge Electronic Design Limited,
Cambridge, UK). Data were recorded continuously by using a personal computer and a chart
recorder (DASH IV, Astro-Med, Inc., West Warwick, RI).
After the experiments, the wounds were treated with lidocaine HCl gel (2%) and sutured.
Five rats per experimental group (groups 1-3) and three controls were allowed to awaken after
disconnection of the cranial window from the syringe pump. The opioid agent, buprenorphine
(0.5mg/kg body weight), was administered subcutaneously as postoperative analgesic. Animals
recovered quickly. They drank and groomed on the first postoperative day. Rat brains were
fixed by transcardial perfusion of the animals with modified Lillie fixative (absolute alcohol
(70%), 37% formalin (20%), glacial acetic acid (10%)) at 48 hours after the experiment. Before
perfusion fixation, rats were examined neurologically in a blinded fashion and graded according
to the scale of Menzies and colleagues1.
Data were analyzed by comparing absolute changes of [K+]o and intracortical and
subdural DC potentials as well as relative changes of CBF calculated in relation to baseline
(100%) at measurement onset. Data in text and figures are given as mean value ± standard
deviation. Statistical tests are mentioned in the results section. A P-value of <0.05 was
considered statistically significant.
Hemoglobin preparation
Hemoglobin was freshly prepared from heparinized arterial rat blood. Blood was centrifuged
(2500G, 5min, 4°C) and the plasma discarded. Cells were washed five times with three to four
volumes of cold 0.9%NaCl, and the buffy coat was removed. The cells were lysed by
sonication. The suspension of lysed cells was subjected to centrifugation (15,000G, 10min, 4°C)
and the pellet removed. The hemoglobin-containing supernatant was transferred by gel
2
chromatography to the ACSF (Bio-Gel P-6, BioRad, Hercules, CA). Both Hb concentration and
composition were measured using a radiometer (total [Hb]ACSF: 2.0 ± 0.4mM; oxy-HbACSF:
95.2%) (radiometer, ABL System 625; Radiometer A/S, Copenhagen, Denmark). The
hemoglobin concentration in our experiments was five times higher than that measured in
human cerebral hematomas2. The necessity for relatively high hemoglobin concentrations was
possibly related to the following factors: (i) small mammals exhibit higher ischemic thresholds
and better collateralization, compared with human subjects - species influences are supported by
the observation that the typical syndrome of delayed cerebral ischemia is not observed in
subarachnoid hemorrhage models in small animals3; (ii) the time of incubation with hemoglobin
was shorter in our experiments, compared with that after subarachnoid hemorrhage; (iii) the site
of hemoglobin application spared the base of the brain, so that spasm of basal arteries would not
contribute to the energy compromise. Moreover, the decreased NO availability after
subarachnoid hemorrhage may not only be caused by the NO scavenger hemoglobin but also by
the release of endogenous NO-synthase inhibitors, uncoupling of endothelial NO-synthase and
the destruction of nitrergic perivascular nerves4, 5.
Histology, Immunohistochemistry and TUNEL Assay
After perfusion each brain was refrigerated in situ in the same fixative overnight. Thereafter, the
brain was removed from the skull. Dissected brains were treated with 96% alcohol overnight
before being paraffin-embedded. Twelve-micrometer sagittal sections were mounted onto slides
coated with albumin and stained using hematoxylin and eosin. Five to 10 sections were obtained
every
200µm.
Neighboring
sections
were
stained
using
histochemical
and
immunohistochemical methods so that direct comparisons were possible. As reported
previously14,28, sections for immunohistochemistry were deparaffinized and rehydrated. Antigen
retrieval was performed by boiling sections in Tris-EDTA buffer (pH 9.0) for 30min. After
blocking in Tris-buffered saline containing 20% normal donkey serum and 0.3% Triton X-100,
3
primary antibodies against GFAP (1:500, mouse-anti-rat; Sigma, Seelze, Germany) and Iba1
(1:200, rabbit-anti-rat; Abcam, Cambridge, UK) were added overnight at 4oC. Alexa594conjugated donkey-anti-mouse and Alexa488-conjugated donkey-anti-rabbit antibodies
(Invitrogen, Eugene, Oregon, USA) were added at room temperature for 4h to visualize the
stainings, or an appropriate 3,3’-diaminobenzidine (DAB) staining kit was used (Vector
Laboratories, Burlingame, California, USA). Omission of primary antibodies served as negative
controls. A laser confocal scanning microscope (Leica, Solms, Germany) was used for image
acquisition. The TUNEL assay was performed using the Apoptag Kit (Intergen, Oxford, UK)
according to the manufacturer’s protocol. Omission of the terminal deoxynucleotidyl transferase
reaction served as negative control. All brain sections were carefully evaluated by a
neuropathologist (I.V.).
Morphometrical analysis
The densities of Iba1-, GFAP- or TUNEL-positive cells in the window areas were determined
for animals of groups 1-3 as well as controls by a blinded investigator (M.K.) using a
stereological approach with the optical fractionator probe. Typically, more than 50 frames of
100 × 100µm in the cortical area under the cranial window were analyzed in 1-2 brain sections
per animal.
Supplemental Discussion:
The role of astrocytes for neuronal survival during spreading depolarization
Notably, during spreading depolarization under normoxic and normoglycemic conditions,
astrocytes in contrast to neurons are functional. Their depolarization is produced passively by
the decline in the potassium transmembrane gradient following the release of potassium from
neurons. The astrocytic depolarization leads to a flux of negatively charged chloride ions into
the astrocytes. In turn, the chloride influx attracts potassium which follows chloride. In addition,
4
potassium enters astrocytes in exchange with sodium by activation of the Na,K-ATPase causing
a decline in intra-astrocytic sodium. Hence, during spreading depolarization under normoxic
and normoglycemic conditions, astrocytes buffer the changes produced by the neurons. But, this
compensatory action of astrocytes is hindered under ischemic conditions when the astrocytic
Na,K-ATPases lack ATP. Under such conditions, the intra-astrocytic sodium concentration
appears to increase markedly, as found in primary astrocyte cultures under simulated ischemia6,
and potassium appears to be spilled out instead of taken up7. Consistent with these notions,
astrocytes do not show significant volume changes in normoxic and normoglycemic tissue
during spreading depolarization8 but they swell substantially under ischemia9, 10. The loss of
astrocytic function under ischemia may be detrimental for neurons. Even the failure of
astrocytic function alone is in fact sufficient to cause neuronal death despite normoxic and
normoglycemic conditions. This was previously found when the aerobic metabolism in
astrocytes was selectively blocked by aconitase inhibitors11. These compounds induced a cluster
of spreading depolarizations with deleterious outcome after a few hours.
5
Supplemental Table S1 Physiological variables
Group 1 [K+]ACSF (250mM)
topically
Group 2 [K+]ACSF (250mM)
/ HbACSF (2mM)
topically
Group 3 [K+]ACSF (250mM)
/HbACSF (2mM)/
topically
/nimodipine
(2µg/kg/min) i.v.
Mean arterial pressure
(mmHg)
Arterial pO2
(mmHg)
Arterial pCO2
(mmHg)
Arterial pH
80.0 ± 7.3
119.9 ± 12.1
42.6 ± 2.2
7.40 ± 0.02
77.7 ± 7.2
117.5 ± 25.8
47.5 ± 3.5
7.36 ± 0.07
74.1 ± 6.9
139.4 ± 19.6
51.0 ± 3.4*
(P<0.05 versus group 1)
7.31 ± 0.04*
(P<0.05 versus group 1)
6
Supplemental References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
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pathological evaluation of a reproducible model. Neurosurgery. 1992;31:100-106; discussion
106-107
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vasospasm and hemoglobin: Clinical and experimental studies. In: Wilkins RH, ed. Cerebral
arterial spasm. Baltimore: Williams & Wilkins; 1980:166-172.
Megyesi JF, Vollrath B, Cook DA, Findlay JM. In vivo animal models of cerebral vasospasm: A
review. Neurosurgery. 2000;46:448-460; discussion 460-441
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synthase after experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab.
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cerebral vasospasm in a primate model of subarachnoid hemorrhage. JAMA : the journal of the
American Medical Association. 2005;293:1477-1484
Rose CR, Waxman SG, Ransom BR. Effects of glucose deprivation, chemical hypoxia, and
simulated ischemia on na+ homeostasis in rat spinal cord astrocytes. J Neurosci. 1998;18:35543562
Largo C, Cuevas P, Herreras O. Is glia disfunction the initial cause of neuronal death in ischemic
penumbra? Neurol Res. 1996;18:445-448
Zhou N, Gordon GR, Feighan D, MacVicar BA. Transient swelling, acidification, and
mitochondrial depolarization occurs in neurons but not astrocytes during spreading depression.
Cereb Cortex. 2010;20:2614-2624
Risher WC, Andrew RD, Kirov SA. Real-time passive volume responses of astrocytes to acute
osmotic and ischemic stress in cortical slices and in vivo revealed by two-photon microscopy.
Glia. 2009;57:207-221
Kimelberg HK. Astrocytic swelling in cerebral ischemia as a possible cause of injury and target
for therapy. Glia. 2005;50:389-397
Largo C, Cuevas P, Somjen GG, Martin del Rio R, Herreras O. The effect of depressing glial
function in rat brain in situ on ion homeostasis, synaptic transmission, and neuron survival. J
Neurosci. 1996;16:1219-1229
7