Long-term observations on calcium accumulation in postischemic gerbil brain

We studied delayed postischemic calcium accumulation and neuronal damage in the gerbil brain, using 45Ca autoradiography as a marker for detection of injured tissue and light microscopy. Transient cerebral ischemia was induced for 15 min. Sham-operated gerbils showed no abnormal calcium accumulation and neuronal damage throughout the brain. At 2 and 7 days following 15 min of ischemia, marked calcium accumulation and mild to severe neuronal damage were found in the selectively vulnerable areas such as neocortex, striatum, hippocampus and thalamus, and brainstem such as medial geniculate body, substantia nigra and inferior colliculus. After 1-2 months of recirculation, the calcium accumulation was not recognized in the brainstem. But, the accumulation was still detectable in the striatum, the hippocampus and the thalamus. Morphological study showed that marked proliferation of glia cells was rapid in the inferior colliculus and was relatively slow in the striatum and the hippocampus, although these structures were severely damaged after ischemia. The result suggests that the speed of restoration of injured tissue and the mechanisms for the damage after cerebral ischemia may be different between the selectively vulnerable areas and the brainstem. Furthermore, they suggest that 45Ca autoradiographic technique may provide a useful approach for diagnosis of the restoration of injured tissue at chronic stage following cerebral ischemia.     

Prevention of delayed neuronal death in gerbil hippocampus by a novel vinca alkaloid derivative (Vinconate)

We investigated the effect of vinconate, a novel vinca alkaloid derivative, on delayed neuronal death using Mongolian gerbils. The animals were allowed to survive for 7 d after 3 or 5 min of forebrain ischemia induced by bilateral occlusion of the common carotid arteries. Morphological changes and calcium (⁴⁵Ca) accumulation were evaluated in the CA1 sector of the hippocampus after ischemia. Vinconate (50, 100, and 300 mg/kg) showed protective effects against neuronal death in a dose-dependent manner when administered intraperitoneally (ip) 10 min before 5 min of ischemia. However, the administration of vinconate (100 and 300 mg/kg, ip) immediately after 5 min of ischemia showed no therapeutic effect, whereas a marked therapeutic effect of vinconate (50 and 100 mg/kg, ip) was observed when administered immediately after 3 min of ischemia. An anesthetic dose of pentobarbital (40 mg/kg, ip) also produced significant protection against neuronal death. Furthermore, a⁴⁵Ca autoradiographic study indicated that a marked calcium accumulation was found in the CA1 sector at 7 d after 5 min of ischemia, which was consistent with the extent of histological neuronal damage. When vinconate (100 and 300 mg/kg, ip) was administered 10 min before 5 min of ischemia, the abnormal calcium accumulation was not detected in the CA1 sector. These data indicate that suppression of abnormal neuronal activity may be owing to the antagonistic action of vinconate on calcium accumulation.     

Neuronal damage and calcium accumulation following repeated brief cerebral ischemia in the gerbil

We investigated the distribution of neuronal damage following brief cerebral transient ischemia and repeated ischemia at 1-h intervals in the gerbil, using light microscopy and 45Ca autoradiography as a marker for detection of ischemic damage. The animals were allowed to survive for 7 days after ischemia induced by bilateral carotid artery occlusion. Following 2-min ischemia, neuronal damage determined by abnormal calcium accumulation was not observed in the forebrain regions. Following 3-min ischemia, however, abnormal calcium accumulation was recognized only in the hippocampal CA1 sector and part of the striatum. Two 2-min ischemic insults caused extensive abnormal calcium accumulation in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the substantia nigra and the inferior colliculus. The ischemic insults were more severe than that of a single 3-min ischemia. However, three 1-min ischemic insults caused abnormal calcium accumulation only in the striatum. On the other hand, three 2-min ischemic insults caused severe abnormal calcium accumulation in the brain. The abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus. Gerbils subjected to three 3-min ischemic insults revealed most severe abnormal calcium accumulation. Marked calcium accumulation was seen not only in the above sites, but also spread in the neocortex, the septum and the hippocampal CA3 sector. Morphological study after transient or repeated ischemia indicated that the distribution and frequency of the neuronal damage was found in the sites corresponding to most of the regions of abnormal calcium accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: distribution and time course

An important feature of ischemic brain damage is the selective vulnerability of specific neuronal populations. We studied the distribution and time course of neuronal damage following transient cerebral ischemia in the gerbil, using light microscopy and 45Ca autoradiography. Following 5 min of ischemia, selective neuronal damage determined by abnormal 45Ca accumulation was recognized only in the hippocampal CA1 subfield and part of the inferior colliculus. Ischemia for 10 to 15 min caused extensive neuronal injury in the 3rd and 5th layers of neocortex, the striatum, the septum, the whole hippocampus, the thalamus, the medial geniculate body, the substantia nigra, and the inferior colliculus. Progression of the damage was rapid in the medial geniculate body and the inferior colliculus, moderate in the neocortex, striatum, septum, thalamus, and the substantia nigra, and was delayed in the hippocampal CA1 sector. However, the delayed damage of the hippocampus occurred earlier when the ischemia period was prolonged. Histological observation revealed neuronal loss in the identical sites of the 45Ca accumulation. This study revealed that the distribution and time course of selective neuronal damage by ischemia proceeded with different order of susceptibility and different speed of progression.     

Regional neuroprotective effects of pentobarbital on ischemia-induced brain damage

We investigated the neuroprotective effect of pentobarbital, a GABAA receptor-effector, on ischemic neuronal damage in the gerbils. The animals were allowed to survive for 7 days after 10-min ischemia induced by bilateral occlusion of the common carotid arteries. Morphological changes and abnormal calcium accumulation were evaluated in selectively vulnerable areas after ischemia. Pentobarbital (40 mg/kg, IP), administered 30 min prior to ischemia, significantly reduced neuronal cell loss in the neocortex, the striatum, and the hippocampal CA3 sector. However, pentobarbital failed to prevent the damage to the hippocampal CA1 sector and the thalamus. 45Ca autoradiographic study also revealed that a marked calcium accumulation was found in the selectively vulnerable regions after ischemia, which was consistent with the extent of histological neuronal damage. The abnormal calcium accumulation was reduced in the sites corresponding to most of the regions in which the protective effect of pentobarbital was found. The results suggest that ischemia-induced neuronal damage may be partly caused by an imbalance between excitatory and inhibitory input.     

Alterations of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate binding using autoradiography in the exo-focal postischemic brain areas of the rat.

We studied the alterations of calcium accumulation and intracellular signal transduction using autoradiography of the second messenger system in order to clarify the mechanisms of the delayed neuronal changes in the remote areas of rat brain after transient focal ischemia. Chronological changes of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate (IP3) binding sites were determined after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. One day after the ischemia, [3H]IP3 binding sites decreased significantly compared with the control values in these ischemic areas. Moreover, 3 days after the ischemia, 45Ca accumulation was first detected in the ipsilateral thalamus and the substantia nigra, which lay outside the ischemic areas. In the substantia nigra, a significant decrease of [3H]IP3 binding sites and concurrent 45Ca accumulation were observed. In the thalamus, however, there was not alteration until 1 week after the ischemia, and then [3H]IP3 binding sites increased significantly 2 weeks (P less than 0.05) and 4 weeks (P less than 0.01) after the ischemia. Based on the present study, we speculate that different mechanisms associated with signal transduction systems may be responsible for exo-focal postischemic delayed neuronal changes in the thalamus and the substantia nigra. The increase of [3H]IP3 binding sites of the thalamus in the chronic stage may be new evidence of plasticity related to neurotransmission.     

Repetitive transient forebrain ischemia in gerbils: delayed neuronal damage in the substantia nigra reticulata.

Repetitive cerebral ischemia results in severe neuronal damage in multiple regions of the brain including the hippocampus, striatum, thalamus, medial geniculate nucleus and the substantia nigra reticulata (SNr). We postulated that the damage in the SNr was delayed, resulting from a loss of striatal inhibitory input. We used the gerbil model of repetitive ischemia (3 min times 2 and 3 min times 3) to evaluate the extent of neuronal damage at 2, 3, 5 and 7 days after the ischemic insult. Silver degeneration stain was used for histological evaluation. Our results indicate that damage in the SNr begins after 48 h and is maximum at 7 days. This delay in onset of damage offers a window for pharmacological protection.     

Neuronal damage and calcium accumulation following transient cerebral ischemia in the rat

The purpose of this study was to examine the distribution of neuronal damage following transient cerebral ischemia in the rat model of four-vessel occlusion utilizing light microscopy as well as⁴⁵Ca-autoradiography. Transient ischemia was induced for 30 min. The animals were allowed to survive for 7 d after ischemia. In the animals subjected to ischemia, the most frequently and seriously damaged areas were the paramedian region of hippocampus, the hippocampal CA1 sector, and the dorsolateral part of striatum, followed by the inferior colliculus, the substantia nigra, the frontal cortex, and the thalamus, which were moderate damaged. Furthermore, the cerebellar Purkinje neurons, the hippocampal CA4 sector, the medial geniculate body, and the hippocampal CA3 sector were slightly affected.⁴⁵Ca-autoradiographyic study also revealed calcium accumulation in the identical sites of ischemic neuronal damage, except for the frontal cortex. Regional cerebral blood flow during 10 min of ischemia was severely decreased in selectively vulnerable areas. The blood flow in the medial geniculate body, the substantia nigra, the inferior colliculus, and the cerebellum was less pronounced than that in the selectively vulnerable areas. The present study demonstrates that transient cerebral ischemia can produce significant neuronal damage not only in the selectively vulnerable regions, but also in the brainstem.     

Differential effects of haloperidol on phencyclidine-induced reduction in substance P contents in rat brain regions

We investigated the effects of a schizophrenomimetic drug, phencyclidine (PCP), on substance P (SP) contents in the discrete rat brain areas using an enzyme-immunoassay for SP. The acute intraperitoneal (i.p.) administration of PCP (10 mg/kg), which is a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) type glutamate receptor and a dopamine uptake inhibitor, reduced the concentration of the peptide in the prefrontal cortex, limbic forebrain, striatum, and substantia nigra, but not in the ventral tegmental area, at 60 or 120 min postinjection. A selective noncompetitive NMDA antagonist, dizocilpine hydrogen maleate ((+)-MK-801) (1 mg/kg, i.p.), also caused a decrease in the SP content in the prefrontal cortex and limbic forebrain but failed to alter the content in the other areas studied 30 min thereafter. Dopamine agonists, methamphetamine (4.8 mg/kg, i.p.) and apomorphine (4.4 mg/kg, i.p.), diminished the SP contents in the striatum and substantia nigra 60 min after their injection without effects in the prefrontal cortex, limbic forebrain, and ventral tegmental area. Furthermore, pretreatment with haloperidol (1 mg/kg, i.p.), a D2 preferable dopamine receptor antagonist and a typical antipsychotic, blocked the ability of PCP to decrease the SP concentrations in the substantia nigra but not in the prefrontal cortex. PCP, therefore, might diminish the SP levels by NMDA receptor-mediated and dopamine-independent mechanisms in the prefrontal cortex and limbic forebrain, but by NMDA receptor-independent and dopamine-dependent mechanisms in the striatum and substantia nigra. The haloperidol-insensitive reduction of the frontal SP could be involved in certain neuroleptic-resistant symptoms of PCP-treated animals, PCP psychosis, or schizophrenia.     

Valproate enhances GABA turnover in the substantia nigra

The effects of the antiepileptic drug, valproate (VPA), on regional turnover of gamma-aminobutyric acid (GABA) in the rat brain were studied by determining the rate of GABA accumulation following complete inhibition of GABA degradation by aminooxyacetic acid (AOAA). VPA was administered at a dose of 200 mg/kg 5, 15 and 30 min prior to injection of AOAA, 100 mg/kg. In most of the 12 regions examined, VPA did not alter the AOAA-induced GABA accumulation. However, significant increases in GABA accumulation were found in corpus striatum and, more marked, in substantia nigra. Since the substantia nigra has been identified as a substrate for the anticonvulsant action of GABAergic drugs, the data may indicate that the effect of VPA on GABA synthesis rate in this region may be involved in its mechanism of anticonvulsant action.     

Vinconate prevents ischemic neuronal damage in the rat hippocampus

The neuroprotective effect of vinconate, a novel vinca alkaloid derivative, was examined in a rat model of forebrain ischemia induced by 4-vessel occlusion. Hippocampal cell loss was quantified histologically 3 days after 10 or 15 min of ischemia. Intraperitoneal application of vinconate (25 and 50 mg/kg) 10 min before and immediately after 10 min of ischemia significantly reduced the neuronal cell loss in the CA1 sector of the hippocampus. Protective effect of vinconate against 15 min of ischemia was reduced, but there was still significant protection at the higher dose. Autoradiography using 14C-vinconate showed that the drug easily penetrates the blood-brain barrier and distributes in the hippocampus. The result suggests that vinconate prevents ischemic neuronal damage by direct action on the hippocampal CA1 neurons.     

Emphasized selective vulnerability after repeated nonlethal cerebral ischemic insults in rats.

BACKGROUND AND PURPOSE: We examined the density and distribution of brain damage after repeated periods of nonlethal ischemic insult in rats in comparison with damage after single lethal periods of ischemic insult. METHODS: Transient cerebral ischemia was induced by four-vessel occlusion for 3, 10, 20, and 30 minutes, and 3-minute periods of ischemia were repeated two, three, or five times at 1-hour intervals, followed by 7 days of survival. RESULTS: Three minutes of ischemia produced no brain damage, but 10-30 minutes of ischemia produced neuronal damage, depending on the length of ischemia, to the selectively vulnerable forebrain regions such as hippocampal CA1 and CA4 subfields, neocortex, striatum, and ventral thalamus, as well as to the brain stem structures (medial geniculate body, substantia nigra, and inferior colliculus) and cerebellar Purkinje cells. Two 3-minute periods of ischemic insult produced neuronal damage to the hippocampal CA1 subfield. Three and five 3-minute insults produced neuronal damage extensively to the selectively vulnerable forebrain areas. An intense cumulative effect of damage was observed in the ventral thalamus, whereas the substantia nigra and the inferior colliculus were resistant to repeated ischemic insults. CONCLUSIONS: Our data indicate that the density and distribution of neuronal damage after repeated ischemic insults are altered as compared with after single ischemia.     

Calcium deposits in the thalamus following repeated cerebral ischemia and long-term survival in the gerbil

We investigated the long-term changes in the gerbil brain following three episodes of 2-min forebrain ischemia at 1-h intervals in comparison with a 6-min period of ischemia. The animals were sacrificed after 1 month and 6 months. Following either ischemic insult, the hippocampal CA1 region showed a loss of pyramidal neurons together with a diffuse calcium accumulation as shown by alizarin red S staining. Three 2-min ischemic insults additionally produced neuronal damage in the striatum and thalamus. The thalamic damage was accompanied by an accumulation of small calcium granules after 1 month and large calcium concretions after 6 months. Calcium staining in the striatum was weak. Thus, the thalamic neuronal damage was accompanied by an active process of calcification, which has not been described in experimental cerebral ischemia models. The observations show that repeated ischemic insults produce different long-term effects in different brain regions.     

Inhibitory influence of GABA on central serotonergic transmission. Raphé nuclei as the neuroanatomical site of the GABAergic inhibition of cerebral serotonergic neurons

Acute injection of the gamma-aminobutyric acid (GABA) mimetics progabide, aminooxyacetic acid, gamma-acetylenic GABA and dipropylacetamide reduced 5-hydroxytryptophan (5-HTP) accumulation in serotonergic nerve terminal regions (prefrontal cortex, olfactory tubercle, septum, striatum, hypothalamus, hippocampus, substantia nigra, cerebellum and spinal cord) as well as in corresponding cell body areas (raphé dorsalis, medianus, pontis and magnus). This effect was antagonized by bicuculline. The inhibition of serotonin (5-HT) synthesis induced by a single progabide administration was accentuated on repeated treatment in the striatum, prefrontal cortex and cerebellum but was similar to that seen after acute treatment in the other areas. Local infusion of high concentrations of GABA or GABA mimetics into the striatum, septum or substantia nigra failed to modify 5-HTP accumulation in these areas. Cerebral hemitransection antagonized the ability of progabide (1200 mg/kg i.p.) to diminish 5-HTP accumulation in the striatum, hippocampus and prefrontal cortex. Intra-raphé dorsalis infusion of muscimol (0.1-100 ng) or GABA (1-100 micrograms) decreased 5-HT synthesis in the corresponding projection areas (e.g. striatum, substantia nigra, cortex) but not in the hippocampus or cerebellum. Conversely, intra-raphé medianus infusion of these drugs diminished 5-HTP accumulation in the corresponding projection areas (e.g. hippocampus, septum, cortex) but not in the striatum or cerebellum. Intra-raphé dorsalis or medianus injection of GABA antagonists (bicuculline, picrotoxinin, RU-5135) was without effect on cerebral 5-HT synthesis but antagonized the diminution of the amine synthesis observed in corresponding projection areas after intra-raphé dorsalis or medianus infusion of muscimol or GABA. These results suggest that GABA exerts an inhibitory (non-tonic) control over central serotonergic neurons which is mediated via GABA receptors located in the raphé nuclei.     

Effects of felbamate on brain polyamine changes following transient cerebral ischemia in the Mongolian gerbil

We sought to determine whether treatment with felbamate was capable to reduce the accumulation of putrescine induced by transient forebrain ischemia in the Mongolian gerbil. Gerbils underwent 10 min ligation of common carotid arteries followed by recirculation. Immediately after the release of the arterial occlusion, felbamate (75 and 150 mg kg(-1) i.p.) was administered. Putrescine and polyamine levels were measured in hippocampus and striatum at 1, 8, 24 and 48 h after recirculation. Putrescine levels appeared enhanced already 8 h after the release of the arterial occlusion and kept increasing up to 48 h in the hippocampus and striatum. No significant changes in spermidine levels during recirculation were detected. Conversely, spermine appeared to decrease in the hippocampus while it did not show changes in the striatum. Felbamate significantly reduced the ischemia induced changes in putrescine brain content only at the dose of 150 mg kg(-1) i.p.     

Autoradiographic analysis of second messenger and neurotransmitter receptor bindings in the strionigral system of the postischemic rat brain.

We studied the postischemic alterations of second messenger and receptor systems focusing on the strionigral pathway in order to clarify the mechanism of the delayed neuronal changes in remote areas of the rat brain after transient focal ischemia. Chronological changes of [3H]forskolin and [3H]SCH 23390 binding sites and 45Ca accumulation were determined by using autoradiographic methods after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen (CPu-L) and to the cerebral cortex, both supplied by the occluded MCA. After the ischemia, [3H]forskolin binding sites were found to be markedly decreased in the early stage in the CPu-L, the ischemic focus in this model, but reduction of the dopamine D-1 receptor sites was first detected there 1 day after the ischemia. On the contrary, in the exo-focal remote areas, there was no alteration of either [3H]forskolin or D-1 receptor binding sites on day 1. However, 3 days after the ischemia, marked reduction of both these binding sites was first observed in the ipsilateral substantia nigra, which had not been directly affected by the original ischemic insult. These postischemic delayed phenomena observed in the substantia nigra developed concurrently with abnormal 45Ca accumulation. These results suggest that strionigral terminal degeneration in the substantia nigra is caused by precedent ischemic damage of the ipsilateral caudate putamen and that intracellular signal transduction including both second messenger and receptor systems may be involved prior to the neuronal damage in the exo-focal postischemic brain areas.     

Intranigral GR-113808, a selective 5-HT4 receptor antagonist, attenuates morphine-stimulated dopamine release in the rat striatum

GR-113808, a potent and selective 5-HT₄ receptor antagonist, was infused through a microdialysis probe into the striatum and nucleus accumbens of awake rats, and basal and morphine-stimulated extracellular concentrations of dopamine (DA) were measured in these regions. At 1 and 10 μM GR-113808 did not effect the extracellular concentrations of DA in either region and 100 μM significantly reduced dialysate DA only in the striatum. A subcutaneous dose of 5 mg/kg morphine significantly raised extracellular concentrations of DA in the striatum and nucleus accumbens from 60 to 120 min after injection and the effect was not modified by 10 μM GR-113808 infused through the probe 20 min before and for 60 min after morphine. Bilateral injections of GR-113808 (1, 2.5 and 10 μg/0.5 μl) in the substantia nigra pars compacta did not affect dialysate DA in the striatum, except for a significant increase 120 min after the injection of 10 μg but the highest dose of GR-113808 prevented the increase of striatal DA caused by 5 mg/kg morphine s.c. The results suggest that 5-HT₄ receptors in the substantia nigra modulate the activity of the dopaminergic nigrostriatal system only when the neurons are activated.     

Alteration of protein kinase C activity in the postischemic rat brain areas using in vitro [3H]phorbol 12,13-dibutyrate autoradiography

Summary Chronological changes of protein kinase C (PKC) activity were measured using in vitro [³H]phorbol 12,13-dibutyrate (PDBu) autoradiography to investigate the postischemic alteration of this second messenger system in the rat brain. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 hours of recirculation, [³H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [³H]PDBu binding sites was first detected in the ipsilateral thalamus and the substantia nigra, which both areas had not been directly affected by the original ischemic insult. This postischemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with⁴⁵Ca accumulation, which was detected there in our previous study. These results suggest that alteration of second messenger (PKC) pathways may be involved not only in the ischemic foci, but also in neuronal degeneration of the exo-focal remote areas in relation to the disruption of intracellular calcium homeostasis which plays a key role in the pathogenesis of postischemic neuronal damage and that marked alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.     

GABA Agonist "Muscimol" Is Neuroprotective in Repetitive Transient Forebrain Ischemia in Gerbils

The damaging effects from transient forebrain ischemia may be a result of excessive excitability or loss of inhibitory influences. In the brain, GABA acts as the major inhibitory neurotransmitter and its loss may be an important factor leading to delayed neuronal damage in the substantia nigra reticulata (SNr). In this study, we looked at the protective effects of muscimol, a GABA A agonist in a gerbil model of repetitive forebrain ischemia. For cerebral ischemia, we used three episodes of 2 min with a reperfusion period of 1 h between the insults. Histological evaluations were done 7 days after the insult using silver degeneration staining. Muscimol was infused into the third ventricle continuously for 7 days beginning just prior to the insult. There were a total of 20 animals, 12 treated with muscimol and the other 8 serving as controls. At 7 days, there was significant protection in the cortex (P = 0.007), hippocampus [CA1 (P = 0.01), CA4 (P = 0.015)], substantia nigra reticulata (P = 0.007), striatum (P = 0.049), and thalamus (P = 0.012). All statistical comparisons were done using nonparametric tests (Mann-Whitney U test). Our study shows that potentiation of inhibitory mechanisms may be important mechanisms of neuronal protection from the effects of repetitive ischemia and the effects are not limited to the SNr. Further studies are needed to better understand their mechanism of action.     

Exo-focal postischemic neuronal death in the rat brain

We describe delayed neuronal damage in ipsilateral areas remote from the ischemic area of rat brain after transient focal ischemia induced by embolization of the right middle cerebral artery (MCA). After 15, 30, 60 and 90 min of MCA occlusion, recirculation was achieved by removal of the embolus. Chronological changes in the distribution of the neuronal damage were determined by using the 45Ca autoradiographic technique and the histological method, and the mechanism involved was investigated by measuring local cerebral glucose metabolism. Depending on the duration of ischemia, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. Moreover, 3 days after ischemic insult, 45Ca had accumulated in the ipsilateral substantia nigra and ventral posterior nucleus of the thalamus. Histological examination revealed that the neurons in both areas suffered damage and were selectively reduced in number. Cerebral glucose utilization decreased in the thalamus, but increased approximately 30% (P less than 0.01) in the substantia nigra compared with the value in the corresponding contralateral area. Both areas lie outside the ischemic area, but have transsynaptic connections with the ischemic focus. Based on the present study, we suggest that the mechanisms of delayed neuronal death in these two remote areas may not be identical, but that this phenomenon may be caused by a transsynaptic process associated with the ischemic focus.