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Stroke. 1996;27:1393-1398

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(Stroke. 1996;27:1393-1398.)
© 1996 American Heart Association, Inc.

Articles

Cerebroprotective Effects of Aminoguanidine in a Rodent Model of Stroke

Kevin M. Cockroft, MD; Malcolm Meistrell, III, BS; Gary A. Zimmerman, MD; Donald Risucci, PhD; Ona Bloom, BS; Anthony Cerami, PhD Kevin J. Tracey, MD

the Laboratory of Biomedical Science, The Picower Institute for Medical Research (K.M.C., M.M., G.A.Z., O.B., A.C., K.J.T.), and the Department of Surgery (Neurosurgery), North Shore University Hospital (D.R., K.J.T.), Manhasset, NY; and the Division of Neurosurgery, The New York Hospital—Cornell Medical Center, New York, NY (K.M.Z., G.A.Z., K.J.T.).

Correspondence to Kevin J. Tracey, Laboratory of Biomedical Science, Picower Institute for Medical Research, 350 Community Dr, Manhasset, NY 11030. E-mail ktracey@picower.edu.


*    Abstract
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 *Abstract
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Background and Purpose During a cerebral infarction, a complex cascade of cytotoxic events ultimately determines the volume of brain cell loss. The studies presented here demonstrate that aminoguanidine, an experimental therapeutic currently in clinical trials to prevent diabetic complications, is cerebroprotective in focal cerebral infarction.

Methods Adult Lewis rats (n=6 to 12 per group) were anesthetized with ketamine and subjected to focal cerebral infarction by tandem permanent occlusion of the right middle cerebral artery and ipsilateral common carotid artery (CCA), followed by temporary occlusion of the contralateral CCA. Infarct volume (cortical) was assessed 24 hours after the onset of ischemia by planimetric analysis of coronal brain slices stained with tetrazolium.

Results Aminoguanidine (320 mg/kg IP) administered 15 minutes after the onset of ischemia resulted in a significant reduction of infarct volume (7.6±2.6% of hemisphere in controls versus 1.3±0.2% of hemisphere in aminoguanidine-treated rats; P<.05). Administration of aminoguanidine conferred significant cerebroprotection even when administered 1 or 2 hours after the onset of ischemia (88% and 85% reduction from control, respectively; P<.05). Cerebroprotection by aminoguanidine was independent of systemic physiological variables known to influence stroke size (eg, temperature, mean arterial blood pressure, blood glucose, and arterial pH, PCO2, and PO2).

Conclusions These results indicate that the stroke-reducing properties of aminoguanidine are dose and time dependent, with substantial cerebroprotection persisting even with drug delivery up to 2 hours after the onset of ischemia. It is now plausible to pursue development of aminoguanidine as an experimental therapeutic in stroke, and possible mechanisms of these cerebroprotective effects are under consideration.


Key Words: cerebral ischemia • glycation end products, advanced • neuroprotection • polyamines • rats


*    Introduction
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During the evolution of a cerebral infarction (stroke), a core of densely ischemic tissue becomes rapidly and irreversibly damaged. Cellular damage in the surrounding area, termed the "ischemic penumbra," progresses more slowly. This progressive cell loss can be prevented with experimental therapeutics that inhibit the cytotoxic effects of glutamate, NO, cytokines, free radicals, and polyamines in the penumbra.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 While investigating the molecular basis for enhanced cell death in the penumbra of diabetic stroke, we discovered that AGEs, a class of modified proteins implicated in the pathogenesis of other diabetic complications, are cerebrotoxic in the ischemic penumbra.23 As an additional control in these experiments, we administered aminoguanidine, a small-molecule inhibitor of AGE cross-linking reactions, and observed that it effectively abrogated the cerebrotoxicity of AGEs during focal cerebral ischemia.23

Somewhat unexpectedly, however, aminoguanidinewas also found to be cerebroprotective during focal ischemia in normal nondiabetic animals, independent of exogenous AGEs.24 These observations stimulated the present analysis of aminoguanidine efficacy in a nondiabetic animal model of focal cerebral infarction. Aminoguanidine is a nontoxic small molecule currently in phase III clinical trials for the prevention of the chronic tissue complications of diabetes mellitus. It has high bioavailability after oral or intraperitoneal administration and is excreted in the urine largely unchanged; the estimated serum half-life is 4.4 hours.25 In animal models, aminoguanidine prevents complications of diabetic proteinuria,26 27 28 retinopathy,29 and neuropathy30 by suppressing the accumulation and cross-linking of AGEs. In addition to these "anti-AGE" effects, aminoguanidine possesses other pharmacological activities. For example, it is an effective inhibitor of NOS (with modest selectivity for the inducible isoform),31 32 and it is also an effective inhibitor of tissue PAO, an enzyme that produces toxic, reactive aldehyde metabolites by oxidation of biogenic polyamines.33

The present studies address whether aminoguanidine can provide neuroprotection in a clinically relevant model of focal stroke. The results show significant cerebroprotection from aminoguanidine administered within the critical period up to 2 hours after the onset of focal cerebral ischemia. The molecular basis for this cerebroprotective effect remains uncertain, but discussion reveals that inhibition of PAO can contribute to the neuroprotection of aminoguanidine.


*    Materials and Methods
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 *Materials and Methods
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All animal protocols were reviewed and approved by the animal care and use committees of the Picower Institute and North Shore University Hospital, in conformity with guidelines from the National Institutes of Health.

Animal Model of Focal Cerebral Infarction
The triple-vessel model of focal cerebral infarction used here has previously been described in detail.23 Briefly, adult male Lewis rats (250 to 400 g) were anesthetized with ketamine (120 mg/kg IM) and subjected to focal cerebral infarction by tandem occlusion of the right CCA and the ipsilateral MCA, followed by temporary occlusion of the left CCA. After 60 minutes the animals were returned to their cages for 24 hours with free access to food and water. Rectal temperature was monitored continuously with a digital thermometer and thermostatically maintained at 36°C to 37°C by use of a heat pad. In all experiments, temperature thermostasis was maintained throughout the period of experimental manipulation. Temperature thermostasis was discontinued only after animals had recovered completely from anesthesia and had no requirement for exogenous heat to maintain rectal temperature.

Measurement of Infarct Volume
The technique for calculating infarct volume has also been described previously.23 Briefly, 24 hours after MCAs were severed, the animals were killed; brains were removed, sectioned into 1-mm coronal slices, and stained with TTC for 30 minutes at 37°C. Infarct size was determined by planimetry, and data were expressed as a volume percentage of the hemisphere. In separate experiments, the zone of infarction determined by TTC staining was verified by histological analysis of serial brain sections stained with hematoxylin and eosin from controls and aminoguanidine-treated animals. Moreover, aminoguanidine added to cell cultures of both primary neurons and the non-neuronal murine cell line L929 did not interfere with metabolism of tetrazolium to formazan (data not shown).

Experimental Protocol
Aminoguanidine (Alteon Inc) was dissolved in sterile normal saline vehicle (NaCl 154 mmol/L). Aminoguanidine (160 mg/kg or 320 mg/kg) was administered intraperitoneally 15 minutes after the onset of ischemia. Control animals received an equivalent volume of vehicle only. In subsequent experiments, aminoguanidine (320 mg/kg IP) was administered at 60, 120, and 180 minutes after the onset of ischemia. Physiological variables known to influence stroke size were assessed in a parallel group of animals receiving either aminoguanidine (320 mg/kg) or normal saline (control group) 60 minutes after the onset of ischemia. Physiological parameters were measured at baseline (before MCA occlusion), at the time of MCA severing, and at hourly intervals thereafter for 2 hours.

Statistical Analysis
Among animals receiving aminoguanidine or vehicle 15 minutes after the onset of ischemia, mean infarct volume (% hemisphere) was compared across dosage groups (control versus 160 mg/kg aminoguanidine versus 320 mg/kg aminoguanidine) by one-way ANOVA, with simple a priori contrasts, separately comparing controls with each treatment group. A two (control versus 320 mg/kg aminoguanidine) by three (15- versus 60- versus 120-minute interval) factorial ANOVA compared mean infarct volume across groups defined by treatment and time interval. Simple a priori contrasts compared controls with treated subjects, whereas a priori polynomial contrasts were used to compare time intervals. Post hoc comparisons for simple main effects used separate Student's t tests with Dunn-Bonferroni adjustment to keep the overall {alpha} level at .05. Changes over time for each physiological variable were compared across treatment and control groups by a two-group repeated measures ANOVA.


*    Results
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Cerebroprotective Effects of Aminoguanidine Are Dose Dependent
In this model of cerebral infarction, cell loss is largely confined to the cortical zone, with sparing of the basal ganglia, as shown in Fig 1Down. Cortical infarct volumes were significantly attenuated by aminoguanidine (320 mg/kg) given 15 minutes after the onset of ischemia, resulting in an 83% reduction in infarct volume (P<.05 versus control) (Fig 2Down, top). A regression analysis of aminoguanidine dose and infarct volume revealed a statistically significant linear regression (P=.007), indicating that the cerebroprotective effects of aminoguanidine are dose dependent even when it is administered 15 minutes after the onset of focal cerebral ischemia. Because the volume of cortical loss is small, and the basal ganglia are spared in this model, within 12 hours after the onset of stroke, control animals were noted to exhibit normal grooming, ambulation, and feeding without somnolence, seizure, or motor paralysis. Assessment of animals treated with neuroprotective doses of aminoguanidine revealed no functional differences in spontaneous activity, grooming, and feeding.



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  		Figure 1. Typical pattern of cerebral infarction observed at five stereotactic levels in animals receiving either vehicle or aminoguanidine as noted. Note that the aminoguanidine-treated animals show reduced areas of infarction at each stereotactic level and that infarction is present on fewer stereotactic sections.




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  		Figure 2. Top, Dose-response of aminoguanidine cerebroprotection. Animals received either normal saline vehicle (154 mmol/L NaCl, n=7) or aminoguanidine (160 mg/kg, n=12, or 320 mg/kg, n=7) intraperitoneally, 15 minutes after the onset of ischemia. Data shown are infarct volumes as a percentage of ipsilateral hemisphere (mean±SE). *P<.05 by ANOVA. Bottom, Time course of aminoguanidine cerebroprotection. Control animals received vehicle alone (dark bar, n=28). Experimental animals received 320 mg/kg aminoguanidine at 15, 60, and 120 minutes after the onset of ischemia (light bars; n=12, 6, and 8, respectively). Animals treated with aminoguanidine (320 mg/kg) 180 minutes after the onset of ischemia did not show a significant reduction in infarct volume (data not shown). Note that aminoguanidine provided significant cerebroprotection even when administered up to 2 hours after the onset of ischemia. *P<.05 vs controls; Student's t test with Dunn-Bonferroni adjustment.

Cerebroprotective Effects of Aminoguanidine Persist up to 2 Hours After the Onset of Focal Cerebral Ischemia
We next sought to determine the time interval over which the administration of aminoguanidine would continue to provide cerebroprotection. Accordingly, aminoguanidine (320 mg/kg IP) was administered to animals at increasing intervals (60, 120, and 180 minutes) after stroke. The results (Fig 2Up, bottom) demonstrate a significant reduction in infarct volume with delivery of aminoguanidine both at 60 minutes (88%) and 120 minutes (85%) after the onset of ischemia (P<.05). No significant protective effect was noted with administration of aminoguanidine 180 minutes after the onset of ischemia (not shown). These results demonstrate that aminoguanidine retains its cerebroprotective activity even when administered up to 2 hours after the onset of focal cerebral ischemia.

Administration of Aminoguanidine Did Not Significantly Alter Physiological Parameters
To address whether the cerebroprotective mechanism was dependent on alterations of cardiovascular physiology, we measured the effect of aminoguanidine on a number of physiological parameters that are capable of influencing stroke volume, including temperature, blood pressure, blood glucose, and arterial pH, PCO2, and PO2.1 2 34 35 36 37 These parameters were measured in parallel groups of animals subjected to craniotomy and focal cerebral ischemia, followed by intraperitoneal administration of either aminoguanidine (320 mg/kg) or normal saline vehicle alone (control group) 60 minutes after the onset of ischemia. Physiological parameters were measured at baseline (before MCA occlusion), again when the MCA was severed, and at hourly intervals thereafter for 2 hours. In agreement with previous experience,23 24 animals in both the control and aminoguanidine-treated groups experienced a transient period of hypotension immediately after MCA occlusion; these temporary effects normalized within 1 hour. Significant differences were not observed in the hemodynamic or metabolic responses to stroke in animals receiving aminoguanidine compared with controls (data not shown). These results indicate that cerebroprotection provided by aminoguanidine is not mediated by changes in systemic cardiovascular parameters, suggesting that the mechanism of cerebroprotection occurs via another local effect of aminoguanidine.

Brain Histology
We performed histological analysis of hematoxylin and eosin–stained brain sections obtained from aminoguanidine-treated and control animals subjected to focal stroke. As expected from previous experience with this model,23 the zone of histological necrosis correlated with infarct volume measured with the TTC staining method. In sections obtained from vehicle-treated animals, we observed diffuse pallor, hypereosinophilic neurons, a modest acute inflammatory cell infiltrate, and pyknotic nuclei within the infarct zone. There was no evidence of significant hemorrhage. Examination of sections obtained from aminoguanidine-treated animals revealed similar findings. In agreement with the volume measurements obtained using TTC, we observed reduced infarct size in animals given aminoguanidine, but there were no significant differences from controls with respect to the other histological findings of pallor, hypereosinophilic neurons, and a modest inflammatory cell infiltrate.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The results presented here indicate that aminoguanidine provides dose-dependent cerebroprotection in a rodent model of stroke when delivered within a clinically relevant therapeutic window of at least 2 hours after the onset of brain ischemia. This model of focal cerebral infarction has been widely used to assess the potential role of experimental therapeutics for stroke because it is both reproducible and responsive to pharmacological agents.8 9 23 24 38 39 40 41 42 43 44 45 46 The observed cerebroprotective effects cannot be attributed to a preferential reduction of cerebral edema formation because edema contributes to only 15% to 20% of infarct volume in this model, but aminoguanidine conferred an 85% protective effect. Moreover, the small size of the cortical infarct in this model causes no significant functional impairment, and as expected aminoguanidine treatment is associated with normal neurological function. It will be of interest to assess the functional consequences of aminoguanidine neuroprotection in larger animals and humans.

Aminoguanidine is presently undergoing clinical evaluation in diabetic patients as an inhibitor of the nonenzymatic synthesis and cross-linking of AGEs. Elevated AGE levels are found in serum and tissues of diabetic patients, where they have been implicated in the pathogenesis of the chronic tissue complications of diabetes.47 48 During stroke, AGEs enhance cerebrotoxicity by activating macrophages and other cells, with the resultant production of secondary cytotoxins.49 50 51 52 53 54 55 56 For instance, binding of AGEs to specific cellular receptors for AGE-modified proteins increased macrophage expression of mRNA for tumor necrosis factor, interleukin-1, and interleukin-6, and it increased endothelial cell production of reactive oxygen intermediates.50 51 52 57 These cytokines and reactive oxygen species can contribute to enhanced tissue injury during focal brain ischemia.58 59 60 61 62 63 Whereas increased AGE levels can contribute to increased cerebrotoxicity seen in diabetic stroke,23 the role of AGEs during cerebral infarction in normal (nondiabetic) stroke is undefined. AGE levels are considerably reduced in nondiabetic compared with diabetic patients, and the biological activity of these low "physiological" AGE levels is limited.47 48 Since normal basal AGE levels do not contribute significantly to the pathogenesis of stroke in nondiabetic animals, we believe that the cerebroprotective effects of aminoguanidine are not attributable principally to inhibition of AGEs.

Aminoguanidine is also an inhibitor of NOS, with some selectivity for the inducible isoform.31 32 NO has been implicated as a mediator of neuronal cell death in cerebral infarction because enhanced NO production in acute stroke leads to increased infarct size, and this effect is prevented by NOS inhibitors.4 5 6 7 62 64 65 66 67 68 Although it is plausible that iNOS inhibition by aminoguanidine contributes to the cerebroprotection observed in the present study, two lines of evidence published recently suggest that this is not the mechanism of action operating in the present study. First, using a similar rat model of focal cerebral infarction, Iadecola and coworkers67 68 found that iNOS and cNOS expression did not increase during the first 24 hours after the onset of ischemia. When they administered aminoguanidine 24 hours after the onset of ischemia, it effectively inhibited NOS activity and reduced stroke volume measured 72 hours after the onset of ischemia.67 68 This represents an important difference from the present study, however, because we administered aminoguanidine within 2 hours and measured stroke volume 24 hours after the onset of ischemia, before iNOS expression increased. Since we observed cerebroprotection during the period before iNOS induction in an earlier stage of stroke maturation, it is unlikely that the cerebroprotection of aminoguanidine was due to iNOS inhibition. Second, we previously reported that the doses of aminoguanidine used here did not inhibit iNOS-dependent regulation of cerebral blood flow during focal stroke.23 When considered together, these results suggest that iNOS inhibition by aminoguanidine can account for the late protection observed in the model of Iadecola et al67 68 but not for the protection observed in the present study within the first 2 hours after stroke.

It is plausible that the mechanism of aminoguanidine neuroprotection in stroke is by inhibition of the activity of brain PAO involved in the oxidative metabolism of biogenic polyamines. Previous work has implicated polyamine metabolism in the pathogenesis of cerebral ischemia. For instance, stroke is associated with increased levels of brain-tissue spermine and spermidine, enhanced polyamine oxidation, and upregulation of ornithine decarboxylase activity.1 20 21 22 69 70 71 A polyamine binding site has been identified on the N-methyl-D-aspartate receptor, and it has been suggested that receptor-ligand interaction at this site can increase neuronal cytotoxicity during ischemia by increasing glutamate toxicity.21 70 73 It has also been suggested that reactive aldehyde intermediates produced by the oxidation of the polyamines can directly mediate cytotoxicity in brain74 and that the protective effects of aminoguanidine during stroke may be due to inhibition of toxic aldehyde intermediates. In support of this possibility, aminoguanidine blocked polyamine toxicity in BHK-21/C13 cells maintained in media containing PAO activity because it prevented the formation of cytotoxins from polyamine oxidation.33 We previously reported that systemic administration of aminoguanidine (in doses used here) prevents brain necrosis in animals receiving polyamines by stereotactic microinjection into brain cortex in vivo.74 Consideration of the facts that brain contains large amounts of PAO and that the products of this enzyme are cytotoxins suggests that the mechanism of aminoguanidine cerebroprotection is via inhibition of production of cytotoxins. It is likely that the cerebroprotective effects of aminoguanidine in the present focal stroke model are due, at least in part, to blocking the synthesis of toxic polyamine metabolites by inhibiting brain PAO activity.74 75

Despite uncertainty about whether the mechanism of aminoguanidine-mediated cerebroprotection is due to inhibition of PAO or combined interaction with another pathway(s), relatively few pharmacological agents have been found to possess significant cerebroprotection when administered up to 2 hours after the onset of cerebral ischemia. Moreover, aminoguanidine is well absorbed after oral or intravenous administration and is relatively nontoxic. The significant cerebroprotective effects of aminoguanidine now suggest that it is plausible to investigate further the use of aminoguanidine in early stroke. It is anticipated that future experimental and clinical studies will provide a better understanding of the underlying cerebroprotective mechanism of aminoguanidine and possibly add an additional therapeutic target in stroke pathogenesis.


*    Selected Abbreviations and Acronyms
 
AGE = advanced glycation end product
CCA = common carotid artery
cNOS = constitutive nitric oxide synthase
iNOS = inducible nitric oxide synthase
MCA = middle cerebral artery
NO(S) = nitric oxide (synthase)
PAO = polyamine oxidase
TTC = 2,3,5-triphenyltetrazolium chloride


*    Acknowledgments
 
This work was supported in part by National Institutes of Health grant RO1-DK49283 (Dr Tracey), a faculty fellowship award from the American College of Surgeons (Dr Tracey), and institutional funding from The Picower Institute.


*    Footnotes
 
The authors acknowledge the following financial conflicts of interest regarding this article: The study was supported in part by research funding supplied to The Picower Institute for Medical Research by a for-profit entity, Alteon Inc, which correspondingly enjoys certain rights to the results of that research. Dr Anthony Cerami is a full-time employee of The Picower Institute, a not-for-profit 501(c)(3) tax-exempt medical research organization, and is a member of Alteon's Board of Directors. Dr Cerami is also a member of the Board of Scientific Advisors, a stockholder, and a paid consultant of Alteon Inc. The value of licenses to patents on which Dr Cerami has coinventorship status and which have been assigned or otherwise licensed to Alteon Inc for commercial development may be affected by the information reported in this article, particularly as such information relates to the use of aminoguanidine.

Received January 15, 1996; revision received April 15, 1996; accepted April 23, 1996.


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 *References
down arrowIntroduction 
 down arrowReferences 
 

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Editorial Comment

Giora Feuerstein, MD, MSc, Guest Editor

Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
In this brief communication I found most remarkable data on the efficacy of the agent aminoguanidine in reducing infarct size at a "clinically relevant" (rat model) time frame—2 hours after permanent brain ischemia.

Aminoguanidine, a simple, "nonbrand" chemical, has demonstrated outstanding capacity to reduce infarct size 85% to 90% up to 2 hours after ischemia. At the doses administered, aminoguanidine had no apparent cardiovascular, metabolic, or respiratory effect, nor did it change temperature. Such data may make it compelling to "fast track" the agent for pharmaceutical development for treatment of stroke even though the mechanism of action is entirely unclear. (Inhibition of NO synthesis, polyamine metabolism? Other?) However, my experience in this complicated business of drug development for stroke raises the flag of caution, which in this particular case points to several good reasons for "cooling the jets."

First, the efficacy demonstrated by aminoguanidine was shown in a very mild model in which the ischemic volume was much less than that commonly produced in standard rat PMCAO models; this is also reflected in the lack of neurological/behavioral deficits. Therefore, it is difficult to associate this model with clinically relevant human stroke since by definition only functional deficits qualify for diagnosis of stroke. Therefore, aminoguanidine effects as reported in this paper seem to work in a situation that is unlikely to qualify as or represent the common ischemic stroke. Second, while aminoguanidine had dramatic neuroprotective action at 2 hours (85%), it was ineffective at 3 hours; no data are provided on the {alpha} or ß errors of this statement, but if the agent indeed had absolutely no effect at 3 hours, it is unlikely to be useful for the majority of stroke patients by the same argument that qualifies its early therapeutic potential. Third, a complete loss of efficacy within 120 to 180 minutes from almost complete protection is hard to reconcile with the traditional evolutionary nature of the ischemic process (mild injury usually produces late manifestations),1R unless in this "triple vessel" model a particular damaging mechanism "kicks in" at this special time frame. Is this the case in humans? Finally, while some "dose-response" relationships were attempted (although this attempt was on the "short side"), it is critical to extend the dosing regimen, because efficacy is linked to safety and such relationships are explored by the "therapeutic index." In my opinion, two doses that are twofold apart are more likely to qualify as a one-dose effect than as a dose-response.

My extended comment to this paper is written with the hope that this study will stimulate the much-needed rigorous, pharmacologically sound, and mechanistically based research to address all possible aspects and meet the gold standards of stroke research in view of developing aminoguanidine for treatment of human stroke. Such research plans should include relevant functional measures, additional qualifying neuroprotection data (direct neuron inspection), complete dose-response (at log intervals), and studies in more than one species. Better insights on the mechanism of aminoguanidine actions would be most useful for further rationale design and direction of preclinical and clinical investigations.


*    Selected Abbreviations and Acronyms
 
AGE = advanced glycation end product
CCA = common carotid artery
cNOS = constitutive nitric oxide synthase
iNOS = inducible nitric oxide synthase
MCA = middle cerebral artery
NO(S) = nitric oxide (synthase)
PAO = polyamine oxidase
TTC = 2,3,5-triphenyltetrazolium chloride


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. Du C, Hu R, Csemansky CA, Hsu CY, Choi DW. Very delayed infarction after mild focal cerebral ischemia: a role for apoptosis? J Cereb Blood Flow Metab. 1996;16:196-201.



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