Metabolic effects of kynurenate during reversible forebrain ischemia studied by in vivoP-nuclear magnetic resonance spectroscopy

The metabolic effects of kynurenate, an endogenous excitatory amino acid antagonist, were studied by in vivo 31P-NMR spectroscopy before, during and after reversible forebrain ischemia in the rat. Kynurenate had no effect on cerebral metabolism before ischemia. During a 30-min ischemia, kynurenate protected against the decrease in phosphocreatine (up to -55 +/- 3% vs -73 +/- 3% in the reference group) and the increase in inorganic phosphate (up to +479 +/- 39% vs +805 +/- 66%), whereas there was no statistical difference in the decrease in intracellular pH (up to 6.37 +/- 0.05 vs 6.30 +/- 0.03) and ATP (up to -60 +/- 3% vs -60 +/- 7%). The recovery of PCr, Pi, and pHi to control levels during recirculation was faster in the treated group than in the reference group, whereas the time course of ATP recovery was similar in both groups. We conclude that kynurenate protects against neuronal loss, as previously reported, by mechanisms other than metabolic protection.     

Effects of kainate-induced seizures on cerebral metabolism: a combinedH and P NMR study in rat

The cerebral metabolic changes elicited by kainate-induced seizures in the rat were investigated by in vivo combined NMR spectroscopy of ³¹P and ¹H. Systemic injection of kainate induced no significant changes in cerebral ATP or PCr levels during up to 90 min of continuous, generalised seizures, and the cerebral³¹P spectra showed only a transient mild cerebral acidosis 30 min after kainate administration. In parallel with the changes in intracellular cerebral pH, the ¹H spectra showed a significant increase in lactate, which remained elevated throughout the seizures. These findings indicate that oxidative metabolism does not completely match the increased glycolysis during seizures though the energy homeostasis is maintained. This suggests that oxidative metabolism has a limited capacity to satisfy the brain's energy needs during the kainate-induced seizures, but that the different pathways of energy production in the brain cells can overcome this limitation. Thus the brain damage associated with this experimental model of epilepsy is not due to extended major failure of the energy supply.     

In vivo measurement of energy metabolism and the concomitant monitoring of electroencephalogram in experimental cerebral ischemia

The energy metabolites in rat brain in vivo were measured by using topical magnetic resonance (TMR) during the whole course of ischemia, in combination with the concomitant monitoring of electroencephalogram (EEG). Immediate loss of high energy phosphorus compounds, phosphocreatine (PCr) and ATP, resulted in the flattening of EEG after the induction of ischemia. PCr and ATP returned to almost normal level 30 min after recirculation of the ischemic brain, but EEG showed no recovery and the abnormality lasted for 12 h. The measurement of in vivo 31P-NMR is essential for the decision of the convalescence of cellular function in the brain.     

Selective in vivo fluorescence labelling of cholinergic neurons containing p75 in the rat basal forebrain

The cholinergic system of the rat basal forebrain is used as a model for the homologous region in humans which is highly susceptible to neuropathological alterations as in Alzheimer's disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75^NTR. This has been utilized for selective immunolesioning of cholinergic neurons after internalization of an immunotoxin composed of anti-p75^NTR and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in the basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75^NTR with the bright red fluorochrome carbocyanine 3 (Cy3). Three days after intraventricular injection of Cy3-192IgG the fluorescence microscopic analysis revealed a pattern of Cy3-labelled cells matching the distribution of cholinergic neurons. Apparently the marker was internalized within complexes of p75^NTR and Cy3-192IgG which were then retrogradely transported to the cholinergic perikarya of the basal forebrain. In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. The specificity of the in vivo staining was proven by subsequent immunolabelling of choline acetyltransferase (ChAT) with green fluorescent Cy2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-192IgG. In parallel experiments, digoxigenylated 192IgG was not detectable within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalized. On the other hand, digoxigenylated 192IgG was found to be an excellent immunocytochemical marker for p75^NTR as shown by double labelling including highly sensitive mouse antibodies directed against ChAT. Based on the present findings, future applications of the apparently non-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed.     

脑缺血选择性海马CA1区神经元损害的实验研究

采用Ｐｕｌｓｉｎｅｌｉ－Ｂｒｉｅｒｌｅｙ４血管阻塞脑缺血模型观察了大鼠全脑缺血２０ｍｉｎ再灌流８ｈ，ｃ－ｆｏｓ基因表达及再灌流７ｄ海马ＣＡ１区迟发性神经元损害。在缺血再灌流早期（８ｈ）海马ＣＡ１区极少ｃ－ｆｏｓ表达，而齿状回、海马ＣＡ３区、杏仁核大量ｃ－ｆｏｓ表达。缺血再灌流晚期（７ｄ）镀银染色显示海马ＣＡ１区神经元及其突触终末带呈黑色溃变相，而齿状回、海马ＣＡ３区、杏仁核呈金黄色正常相。相邻切片ＨＥ染色示缺血组海马ＣＡ１区核完整的锥体细胞数（５±２．６个／２００μｍ）与对照组（４０±２．９个／μｍ）比较差异有显著意义（Ｐ＜０．０１）。脑缺血诱导的ｃ－ｆｏｓ基因表达对于缺血易损海马ＣＡ１区迟发性神经元坏死可能起直接的调控作用。     

Moderate hypothermia mitigates neuronal damage in the rat brain when initiated several hours following transient cerebral ischemia

Intraischemic moderate hypothermia generally protects the brain against ischemic cell death, while hypothermia instigated several hours into the reperfusion phase is considered to be less effective. Here we report the effect of hypothermia (32.5°–33.5°C) of 5-h duration, initiated at 2, 6, 12, 24 and 36 h into the recirculation phase following 10 min of transient cerebral ischemia, on ischemic neuronal injury in the hippocampus and striatum of the rat. Hypothermia induced at 2 h, and 6 h postischemia reduces neuronal damage in the entire hippocampal CA1 region by approximately 50%. In the lateral CA1 region hypothermia induced at 12 h postischemia, significantly mitigates necrosis. When initiated at 2 h postischemia, but not later, protection was also observed in the striatum. Hypothermia induced 24 and 36 h postischemia was ineffective. A period of hypothermia of 5 h, initiated 2 h postischemia, was required for marked neuronal protection in the CA1 region, while 3.5-h hypothermia decreased neuronal damage by approximately 10% and 30 min hypothermia was ineffective. The clinical implications of the data are that extended period of hypothermia initiated long into the recovery phase following ischemia may prove beneficial. Hypothermia protects brain regions displaying rapid as well as delayed neuronal damage, and a minimal time of hypothermia is required for effective neuronal protection. Also, strict temperature control for up to 24 h postischemia may be required for proper assessment of the efficacy of cerebro-protective drugs.Supported by the Swedish Medical Research Council (grant no. 08644), The Medical Faculty at Lund University, The Segerfalk Foundation, The Crafoord Foundation, Åke Wibergs Foundation, and the CNPq (Brazilian Council for Development of Science and Technology)     

Decrease in N-acetylaspartate without commensurate accumulation of acetate infocal cerebral ischemia in rat

Abstract

N-acetylaspartate (NAA) is a plausible marker of neuronal viability which decreases in a variety of neurodestructive conditions. To elucidate the mechanism that leads to NAA decline in two different types of cerebral ischemia in rats, we simultaneously determined cortical concentrations of NAA and its hydrolytic metabolites, aspartate, and acetate by high-resolution 1H-NMR spectroscopy. NAA decreased almost linearly up to 24 h in both decapitation induced global cerebral ischemia, and in ischemic cortices of focal ischemia. Acetate was increased continuously for up to 24 h of global ischemia, while in focal cerebral ischemia it was increased transiently at 6 h. Aspartate did not show any change in global ischemia, while it was decreased in focal ischemia. Although NAA decreased similarly in the brain with global and focal ischemia, temporal changes of two NAA hydrolytic metabolites were different in each type of ischemia. The present results suggest hydrolytic degradation of NAA may be modified alternatively under each pathophysiologic condition. [Neurol Res 1999; 21: 771–774]     

Neuronal damage in the rat hippocampus in a new model of repeated reversible transient cerebral ischemia

The influence of the interval of the repeated reversible transient cerebral ischemia on the neuronal damage in the hippocampal CA1 sector was investigated in the rats using a 4-vessel occlusion (4-VO) model. A single 3-min 4-VO did not produce any significant neuronal damage in the hippocampal CA1 sector, whereas the rats subjected to three 3-min 4-VO at 1-h intervals revealed a very severe neuronal damage which was much more severe than that in the rats subjected to a single 9-min 4-VO. In contrast, the rats subjected to three 3-min 4-VO at 6-h intervals revealed only a mild neuronal damage. The degree of the neuronal damage in the rats subjected to three 3-min 4-VO at 5-min intervals was similar to that in the rats subjected to a single 9-min 4-VO. The present study indicates that even such a brief, non-lethal ischemia as 3-min 4-VO can produce a severe neuronal damage if it occurs repeatedly at 1-h intervals.     

Neurodegeneration in the rat hippocampus and striatum after middle cerebral artery occlusion

Animal models of ischemia are in wide use to elucidate the molecular mechanisms of brain injury that result from cardiovascular disease in humans. We have used the fluorescent, anionic dye, Fluoro-Jade, to examine cellular degeneration that occurs in association with the middle cerebral artery occlusion (MCAO) model. MCAO results in cortical infarction as well as damage to the hippocampus leading to a delayed form of death of hippocampal neurons. We examined brain sections at 6 h, 12 h, 1, 4, 7, 14 and 21 days after injury. Fluoro-Jade labeling of the striatum was seen over a protracted time-course, with degeneration beginning by 6 h after injury. Neuronal degeneration in the hippocampus, in contrast, occurs between 12 h and 7 days after injury with neuronal death reaching a peak at 4 days. GFAP/Fluoro-Jade double labeling revealed that the Fluoro-Jade positive staining at late time-points in the striatum included astrocytic cells. Together, the results show Fluoro-Jade to be a useful marker of cellular degeneration following ischemic injury. Further, the use of this dye has enabled us to demonstrate previously undescribed events of cellular injury resulting from ischemia.     

Glucose metabolism and neurogenesis in the gerbil hippocampus after transient forebrain ischemia

Recent evidence exists that glucose transporter 3 (GLUT3) plays an important role in the energy metabo-lism in the brain. Most previous studies have been conducted using focal or hypoxic ischemia models and have focused on changes in GLUT3 expression based on protein and mRNA levels rather than tissue levels. In the present study, we observed change in GLUT3 immunoreactivity in the adult gerbil hippocampus at various time points after 5 minutes of transient forebrain ischemia. In the sham-operated group, GLUT3 immunoreactivity in the hippocampal CA1 region was weak, in the pyramidal cells of the CA1 region in-creased in a time-dependent fashion 24 hours after ischemia, and in the hippocampal CA1 region decreased signiifcantly between 2 and 5 days after ischemia, with high level of GLUT3 immunoreactivity observed in the CA1 region 10 days after ischemia. In a double immunolfuorescence study using GLUT3 and gli-al-ifbrillary acidic protein (GFAP), we observed strong GLUT3 immunoreactivity in the astrocytes. GLUT3 immunoreactivity increased after ischemia and peaked 7 days in the dentate gyrus after ischemia/reperfu-sion. In a double immunolfuorescence study using GLUT3 and doublecortin (DCX), we observed low level of GLUT3 immunoreactivity in the differentiated neuroblasts of the subgranular zone of the dentate gyrus after ischemia. GLUT3 immunoreactivity in the sham-operated group was mainly detected in the subgran-ular zone of the dentate gyrus. These results suggest that the increase in GLUT3 immunoreactivity may be a compensatory mechanism to modulate glucose level in the hippocampal CA1 region and to promote adult neurogenesis in the dentate gyrus.     

A case of mitochondrial myopathy,lactic acidosis and complex I deficiency

Summary A 34-year-old man affected by exercise intolerance, mild proximal weakness and severe lactic acidosis is described. Muscle biopsy revealed mitochondrial abnormalities and an increase of cytochrome c oxidase histochemical reaction. Biochemical investigations on isolated muscle mitochondria as well as polarographic studies revealed a mitochondrial NADH-CoQ reductase (complex I) deficiency. Mitochondrial dysfunction was confirmed by ³¹P nuclear magnetic resonance spectroscopy. Immunological investigation showed a generalized reduction of all complex I polypeptides. Genetic analysis did not reveal mitochondrial DNA deletions. The biochemical defect was not present in the patient's muscle tissue culture. Metabolic measurements and functional evaluation showed a reduced mechanical efficiency during exercise.     

Early impairment and late recovery of synaptic transmission in the rat dentate gyrus following transient forebrain ischemia in vivo

Ischemic stroke causes various functional deficits in the brain such as memory impairment, and clinical reports have shown that the impaired brain functions may partially recover. However, there has been no experimental model suitable for studying cellular mechanisms of functional recovery following brain ischemia. Therefore, we investigated the long-term influence of transient forebrain ischemia on excitatory synaptic transmission in the rat dentate gyrus, a brain region relatively resistant to ischemia. Fifteen minutes of transient forebrain ischemia produced no apparent histological damage in dentate granule cells, but caused a significant reduction of basal synaptic potentials evoked by perforant path stimulation. Field excitatory postsynaptic potential remained reduced for at least 1 month after ischemia, while population spike recovered to control level in 1 month. The induction of long-term potentiation was also impaired after ischemia, but it showed faster recovery than basal synaptic potentials. In conclusion, we found that synaptic transmission in the dentate gyrus of the rat is impaired following transient forebrain ischemia, but has a potential to recover. These results may provide a good model for studying the mechanisms of impairment and recovery of brain function after transient ischemia.     

The density and distribution of ischemic brain injury in the rat following 2–10 min of forebrain ischemia

Summary The density and distribution of brain damage after 2–10 min of cerebral ischemia was studied in the rat. Ischemia was produced by a combination of carotid clamping and hypotension, followed by 1 week recovery. The brains were perfusion-fixed with formaldehyde, embedded in paraffin, subserially sectioned, and stained with acid fuchsin/cresyl violet. The number of necrotic neurons in the cerebral cortex, hippocampus, and caudate nucleus was assessed by direct visual counting.Somewhat unexpectedly, mild brain damage was observed in some animals already after 2 min, and more consistently after 4 min of ischemia. This damage affected CA4 and CA1 pyramids in the hippocampus, and neurons in the subiculum. Necrosis of neocortical cells began to appear after 4 min and CA3 hippocampal damage after 6 min of ischemia, while neurons in the caudoputamen were affected first after 8–10 min.Selective neuronal necrosis of the cerebral cortex worsened into infarction after higher doses of insult. Damage was worst over the superolateral convexity of the hemisphere, in the middle laminae of the cerebral cortex. The caudate nucleus showed geographically demarcated zones of selective neuronal necrosis, damage to neurons in the dorsolateral portion showing an all-or-none pattern. Other structures involved included the amygdaloid, the thalamic reticular nucleus, the septal nuclei, the pars reticularis of the substantia nigra, and the cerebellar vermis.Supported by the Swedish Medical Research Council (projects 12X-03020, 14X-263) and the National Institutes of Health of the United States Public Health Service (grant no. 5 R01 NS07838). Dr. Auer is the recipient of a Medical Research Council of Canada Fellowship.     

Apoptosis and protein expression after focal cerebral ischemia in rat

We used double staining histochemistry to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage (p53, Bax, MDM2, Gadd45), DNA repair (PCNA) and cell cycle proteins (cyclin A, cyclin D, cdk2, cdk4) in rats (n=6; control rats, n=5) subjected to transient (2 h) middle cerebral artery occlusion (MCAo) and 46 h of reperfusion. Few apoptotic cells were detected in the non-ischemic hemisphere of control rats. In ischemic animals, scattered apoptotic cells were present in the ischemic core and clustered apoptotic cells were present and localized to the inner boundary zone of the ischemic core. Proteins were preferentially localized to the cellular cytoplasm of control rats and in the non-ischemic hemisphere of rats subjected to MCAo. However, after MCAo these proteins were expressed and were preferentially localized to nuclei within the ischemic lesion. DNA damage induced proteins (wt-p53 and p53-response proteins) were preferentially expressed within apoptotic cells after ischemia. DNA repair proteins and cell cycle proteins were preferentially expressed within morphologically intact cells and in reversibly damaged cells in the ischemic areas. The selective expression of proteins associated with DNA damage, DNA repair and cell cycle observed in morphologically intact cells, ischemic injured cells and apoptotic cells suggests a differential role for these proteins in cell survival and apoptosis after stroke.     

Laser scanning and electron microscopic evidence for rapid and specific in vivo labelling of cholinergic neurons in the rat basal forebrain with fluorochromated antibodies

Recently developed methods for the selective labelling of cholinergic basal forebrain neurons containing the low-affinity neurotrophin receptor p75 (p75(NTR)) in vivo and in vitro are based on carbocyanine 3 (Cy3)-tagged antibodies directed against p75(NTR). The present study focuses on the maintenance of this neuronal label after injection of such fluorescent antibodies into the cerebral ventricle. One, 3, and 10 days after injection this marker exclusively stains neurons immunoreactive for the cholinergic markers choline acetyltransferase and vesicular acetylcholine transporter in the rat medial septum, diagonal band and nucleus basalis. Thirty days after injection the in vivo labelling was nearly abolished. Predominant labelling of lysosomes was shown by electron microscopic analysis following photoconversion of the Cy3-label to an electron-dense reaction product. The pre-labelling of cholinergic neurons might facilitate pharmacological and electrophysiological approaches in living slices and cell culture systems as well as detailed investigations focused on the transport of neurotrophins in vivo and in animals with experimentally altered p75(NTR) expression.     

Modulation of extracellular glutamate concentration by nitric oxide synthase inhibitor in rat transient forebrain ischemia

The purpose of the present study was to clarify the effect of topical administration of a nitric oxide synthase inhibitor on extracellular glutamate concentration in transient forebrain ischemia. Two microdialysis probes were inserted into the bilateral striata of Wistar rats. N^G-Nitro-l-arginine (l-NNA) with or withoutl-arginine was topically administered into the unilateral striatum through one of the microdialysis probes, while Ringer's solution was perfused into the contralateral striatum as the control, and 14 minutes of forebrain ischemia was applied. The extracellular glutamate concentration during ischemia and subsequent reperfusion was statistically significantly higher on the 100 μMl-NNA-perfused side than on the control side, but 1 MMl-NNA was ineffective. When 100 μMl-NNA was perfused together with 500 μMl-arginine, the glutamate concentration did not differ from that on the control side. Moreover, administration of 500 μMl-arginine significantly suppressed the glutamate elevation after reperfusion. The fact that the lower dose ofl-NNA increased the accumulation of glutamate during ischemia and reperfusion without altering the blood flow may indicate that nitric oxide affords protection against ischemic neuronal damage. However, since the higher dose ofl-NNA did not affect the glutamate concentration, it appears that the effect of nitric oxide on extracellular glutamate concentration in forebrain ischemia differs, depending on the degree of the inhibition of NOS activity.     

Neuronal survival is associated with 72-kDa heat shock protein expression after transient middle cerebral artery occlusion in the rat

Induction of the 72-kDa heat shock protein expression is thought to protect neurons against the subsequent effects of ischemia. However, it is not clear whether the induction of 72-kDa heat shock protein expression by an ischemic event improves neuronal survival. To address this question, we outlined the temporal profile of neuronal induction and expression of the 72-kDa heat shock protein in a model of transient focal ischemia in the rat. Fifty two adult Wistar rats were subjected to middle cerebral artery occlusion of 2 h duration. At 0.5, 3, 6, 9, 12, 24, 48, 96 and 168 h after reopening the artery, coronal brain sections were analyzed using both immunohistochemical methods and hematoxylin and eosin staining to determine the topographic and cellular distribution of the 72-kDa heat shock protein, as well as the extent of neuronal damage. Immunoreactivity to the 72-kDa heat shock protein was not detected in neurons that were destined to become necrotic, and were located in the ischemic core of the brain lesions. However, 72-kDa heat shock protein expression was evident in morphologically intact neurons located in the peripheral zone. The earliest neuronal expression of 72-kDa heat shock protein was detected in animals in which the 2 h occlusion of the middle cerebral artery was followed by 6 h recirculation; the intensity of the 72-kDa heat shock protein immunoreactivity peaked at 48 h, and progressively disappeared 7 days after the ischemic reperfusion event. These studies suggest that (1) 72-kDa heat shock protein is not expressed in morphologically intact neurons destined to become necrotic after 2 h of focal ischemia; (2) the 72-kDa heat shock protein is expressed only in morphologically intact neurons located at the periphery of the ischemic territory where they may be subjected to only sublethal stress; these neurons preserved their integrity 7 days after the ischemic episode. These data support the hypothesis that the expression of 72-kDa heat shock protein in ischemic brain may confer “protection” to the neurons.     

Evidence for a relationship between body mass and energy metabolism in the human brain

Cerebral energy metabolism has been suggested to have an important function in body weight regulation. We therefore examined whether there is a relationship between body mass and adenosine triphosphate (ATP) metabolism in the human brain. On the basis of our earlier findings indicating a neuroprotective preferential energy supply of the brain, as compared with peripheral muscle on experimentally induced hypoglycemia, we examined whether this physiological response is preserved also in low-weight and obese participants. We included 45 healthy male subjects with a body mass index (BMI) ranging from 17 to 44 kg/m². Each participant underwent a hypoglycemic glucose-clamp intervention, and the ATP metabolism, that is, the content of high-energy phosphates phosphocreatine (PCr) and ATP, was measured repeatedly by ³¹phosphor magnetic resonance spectroscopy (³¹P-MRS) in the cerebral cortex and skeletal muscle. Results show an inverse correlation between BMI and high-energy phosphate content in the brain (P<0.01), whereas there was no such relationship found between skeletal muscle and BMI. The hypoglycemic clamp intervention did not affect the ATP metabolism in both tissues. Our data show an inverse correlation between BMI and cerebral high-energy phosphate content in healthy humans, suggesting a close relationship between energetic supply of the brain and body weight regulation.     

Brain glycogen content and metabolism in subjects with type 1 diabetes and hypoglycemia unawareness

Supercompensated brain glycogen may contribute to the development of hypoglycemia unawareness in patients with type 1 diabetes by providing energy for the brain during periods of hypoglycemia. Our goal was to determine if brain glycogen content is elevated in patients with type 1 diabetes and hypoglycemia unawareness. We used in vivo ¹³C nuclear magnetic resonance spectroscopy in conjunction with [1-¹³C]glucose administration in five patients with type 1 diabetes and hypoglycemia unawareness and five age-, gender-, and body mass index-matched healthy volunteers to measure brain glycogen content and metabolism. Glucose and insulin were administered intravenously over ∼51 hours at a rate titrated to maintain a blood glucose concentration of 7 mmol/L. ¹³C-glycogen levels in the occipital lobe were measured at ∼5, 8, 13, 23, 32, 37, and 50 hours, during label wash-in and wash-out. Newly synthesized glycogen levels were higher in controls than in patients (P<0.0001) for matched average blood glucose and insulin levels, which may be due to higher brain glycogen content or faster turnover in controls. Metabolic modeling indicated lower brain glycogen content in patients than in controls (P=0.07), implying that glycogen supercompensation does not contribute to the development of hypoglycemia unawareness in humans with type 1 diabetes.     

Correlates of delayed neuronal damage and neuroprotection in a rat model of cardiac-arrest-induced cerebral ischemia

Numerous studies over the past three decades have used rodent models of cerebral ischemia. To measure the postischemic outcome, the majority of these studies used histopathology as the method of choice both quantitatively and qualitatively. No functional measure of postischemic outcome has been proved to correlate well with the histopathological one. The rat chest compression model of cardiac-arrest-induced global cerebral ischemia was used in the present study. Two separate measures of neuronal damage at 7 days postischemia were performed: (a) histologically, by counting normal pyramidal cell bodies in the mid-CA1 hippocampal region of the rat brain, in hematoxylin-eosin-stained, paraffin-embedded 6-microm sections, and (b) electrophysiologically, by counting the number of 400 microm hippocampal slices in which it was possible to evoke a normal (>/=10 mV) CA1 population spike by orthodromic stimulation of the Schaffer collaterals. The correlation between these two measures was tested in the following groups of rats: (a) control, untreated group, (b) MK-801-treated groups (0.03 to 1.0 mg/kg given i.p. shortly after ischemia), (c) diltiazem-treated (DILT) groups 1.0 to 30 mg/kg, given i.p. shortly after ischemia, and (d) a group treated with a combination of the two drugs together (0.1 mg/kg MK-801+3.0 mg/kg DILT given i.p. shortly after ischemia). The two measures of postischemic outcome were highly correlated in all groups studied. Both MK-801 and DILT exhibited a dose-dependent neuroprotective effect. When administered together, a synergy between the neuroprotective effect of MK-801 and DILT was observed. At the doses used, minimal or no side effects of either MK-801 or DILT were observed.