Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.     

Hyperthermia enhances spectrin breakdown in transient focal cerebral ischemia

Calpain-mediated spectrin degradation is triggered by cerebral ischemia and, when persistent, is thought to signal irreversible neuronal injury. Hyperthermia superimposed upon cerebral ischemia may exacerbate the injury process. In this study, we compared the extent of spectrin degradation in the brains of rats subjected to 1 h of transient proximal middle cerebral artery (MCA) clip-occlusion performed under conditions of cranial normothermia (37°C) or mild cranial hyperthermia (39°C). Immunocytochemical localization of spectrin breakdown products was achieved by the use of a rabbit polyclonal antibody which reacted selectively with calpain-generated fragments of brain spectrin. The perfusion times studied were 1, 4 or 24 h. Following normothermic MCA occlusion, spectrin immunoreactivity was present only occasionally and only in scattered cortical neurons immediately upon reperfusion and 1 h later; all normothermic brains showed sparse immunoreactivity at 4 h of reperfusion; and no immunoreactivity was detected at 24 h. By contrast, following hyperthermic MCA occlusion, moderate-to-intense immunostaining was present in cortical pyramidal neurons even immediately upon reperfusion and persisted at 1 h of reperfusion. At 4 and 24 h, most brains exhibited dense immunoreactivity associated with morphologically shrunken neurons. Following 24 h survival, semi-thick plastic sections revealed intact neuropil and only selective neuronal necrosis in normothermic rats. By contrast, pan-necrosis was evident 24 h after the hyperthermic ischemic insult. These results indicate that mild cranial hyperthermia superimposed upon transient focal ischemia markedly enhances calpain activation and spectrin degradation; this process appears to be an important mechanism by which hyperthermia exacerbates ischemic injury.     

Postischemic changes in the immunophilin FKBP12 in the rat brain

An immunosuppressant tacrolimus (FK506) protects against neuronal damage following cerebral ischemia. On the other hand, the major physiological role of the immunophilin FK506-binding protein-12 (FKBP12) is a modulation of intracellular calcium flux. Since an increase in intracellular calcium concentration is a major mediator of ischemic neuronal death, we investigated the changes in FKBP12 following cerebral ischemia in the rat. We induced focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery for 1 h, and global cerebral ischemia for 10 min by bilateral carotid artery occlusion combined with hypotension. The animals were killed at 4 h to 7 days after reperfusion. Immunohistochemistry was performed on paraffin sections using a monoclonal antibody raised against recombinant FKBP12. Immunoreactivity to FKBP12 in control brains was most pronounced in the CA1 subfield of the hippocampus and the striatum, the localization being primarily neuronal. Following focal ischemia, FKBP12 immunoreactivity decreased rapidly in the ischemic core by 4 h, but increased in surviving neurons in penumbra areas (4 h-7 days). Within an area of infarction, invading leukocytes and macrophages exhibited immunoreactivity to FKBP12 (3-7 days). Following global ischemia, FKBP12 immunoreactivity in CA1 neurons decreased after 1 day, and then it was lost between 2 and 7 days, although many CA1 neurons showed a transient increase in FKBP12 at 2 days. No FKBP12 immunoreactivity was observed in reactive glial cells. Thus, FKBP12 declined in dying neurons, whereas FKBP12 was upregulated in less severely injured neurons. The findings suggest that (1) FKBP12 plays an important role in the process of neuronal survival and death following cerebral ischemia, and (2) FKBP12 is involved in inflammatory reactions that occur within an area of infarction.     

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia

Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damages in the brain. In the present study, ischemia-induced changes in GLP-1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP-1R agonist exendin-4 (EX-4) against ischemic damage was studied. GLP-1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP-1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP-1R protein level was increased again at 10 days after I/R, and GLP-1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX-4 treatment attenuated ischemia-induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose-dependent manner. EX-4 treatment also induced the elevation of GLP-1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP-1R is altered in the ischemic region after an ischemic insult and that EX-4 protects against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglial activation against transient cerebral ischemic damage.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.     

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)     

Neuronal subclass-selective loss of pyruvate dehydrogenase immunoreactivity following canine cardiac arrest and resuscitation

Chronic impairment of aerobic energy metabolism accompanies global cerebral ischemia and reperfusion and likely contributes to delayed neuronal cell death. Reperfusion-dependent inhibition of pyruvate dehydrogenase complex (PDHC) enzyme activity has been described and proposed to be at least partially responsible for this metabolic abnormality. This study tested the hypothesis that global cerebral ischemia and reperfusion results in the loss of pyruvate dehydrogenase immunoreactivity and that such loss is associated with selective neuronal vulnerability to transient ischemia. Following 10 min canine cardiac arrest, resuscitation, and 2 or 24 h of restoration of spontaneous circulation, brains were either perfusion fixed for immunohistochemical analyses or biopsy samples were removed for Western immunoblot analyses of PDHC immunoreactivity. A significant decrease in immunoreactivity was observed in frontal cortex homogenates from both 2 and 24 h reperfused animals compared to samples from nonischemic control animals. These results were supported by confocal microscopic immunohistochemical determinations of pyruvate dehydrogenase immunoreactivity in the neuronal cell bodies located within different layers of the frontal cortex. Loss of immunoreactivity was greatest for pyramidal neurons located in layer V compared to neurons in layers IIIc/IV, which correlates with a greater vulnerability of layer V neurons to delayed death caused by transient global cerebral ischemia.     

Multi-focal delayed neuronal damage after transient regional ischemia--mechanism of vascular dementia

We describe multi-focal delayed neuronal death of rat brain after transient regional ischemia induced by embolization of the right middle cerebral artery (MCA). After sixty minutes 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 examination. Sixty minutes after MCA occlusion, 45Ca accumulation extended to the lateral segment of the caudate putamen and the cerebral cortex supplied by the occluded MCA. Moreover, three days after ischemic insult, 45Ca had accumulated in the ipsilateral thalamus and substantia nigra. Histological examination revealed that the neurons in both area suffered damage and were selectively reduced in number. Both areas lie outside the ischemic area, but have transsynaptic connections with the ischemic focus. We suggest that the postischemic delayed neuronal death in exo-focal remote areas may be caused by a transsynaptic process associated with the infarcted areas and that these delayed multi-focal brain damage may exacerbate clinical symptoms in the chronic stage of stroke.     

Apoptosis in the adult striatum after transient forebrain ischemia and the effects of ischemic severity

The mechanisms of neuronal injury after cerebral ischemia have been under active investigation. The medium-size neurons in the dorsal striatum die within 24 h after transient cerebral ischemia. Using electron microscopy, the present study examined the nature of neuronal death in the striatum of adult rats following transient forebrain ischemia and tested the hypothesis that the ischemic severity might influence the nature of cell death. After severe ischemia (approximately 21 min ischemic depolarization), most neurons in the dorsal striatum died with swollen organelles and small irregular chromatin clumps resembling necrosis. The tissue damage in the dorsomedial striatum was less severe than that in the dorsolateral striatum and approximately 5% of the neurons in this region died with large chromatin clumps and relatively intact organelles resembling apoptosis. Some neurons displayed a mixture of necrotic- and apoptotic-like appearance. In contrast, the neurons with large somata only exhibited mild ultrastructural changes. After moderate ischemia (approximately 15 min ischemic depolarization), the tissue damage was less severe and the process of necrosis was temporally prolonged compared with that after severe ischemia. The apoptotic-like neuronal death was observed not only in the dorsomedial (approximately 6%) but also in the dorsolateral striatum (approximately 7%). The neurons in the striatum showed transient reversible changes after mild ischemia (approximately 10 min ischemic depolarization). The present study demonstrates that both apoptosis and necrosis occur in the adult striatum following transient forebrain ischemia and apoptosis occurs in the regions with less severe ischemia. These results suggest that ischemic severity might be one of the contributing factors to necrosis or apoptosis following transient global ischemia.     

Distribution of Ca(2+)/calmodulin-dependent protein kinase I beta 2 in the central nervous system of the rat

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.     

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.     

Neuroprotection of Chrysanthemum indicum Linne against cerebral ischemia/reperfusion injury by anti-inflammatory effect in gerbils

In this study, we tried to verify the neuroprotective effect of Chrysanthemum indicum Linne(CIL) extract, which has been used as a botanical drug in East Asia, against ischemic damage and to explore the underlying mechanism involving the anti-inflammatory approach. A gerbil was given CIL extract for 7 consecutive days followed by bilateral carotid artery occlusion to make a cerebral ischemia/reperfusion model. Then, we found that CIL extracts protected pyramidal neurons in the hippocampal CA1 region(CA1) from ischemic damage using neuronal nucleus immunohistochemistry and Fluoro-Jade B histofluorescence. Accordingly, interleukin-13 immunoreactivities in the CA1 pyramidal neurons of CIL-pretreated animals were maintained or increased after cerebral ischemia/reperfusion. These findings indicate that the pre-treatment of CIL can attenuate neuronal damage/death in the brain after cerebral ischemia/reperfusion via an anti-inflammatory approach.     

Green tea polyphenol (-)-epigallocatechin gallate reduces neuronal cell damage and up-regulation of MMP-9 activity in hippocampal CA1 and CA2 areas following transient global cerebral ischemia

Previous studies have demonstrated that (-)-epigallocatechin gallate (EGCG), a green tea polyphenol, protects against ischemia and reperfusion-induced injury in many organ systems. Here, we test the hypothesis that part of EGCG's neuroprotective effects may involve a modulation of matrix metalloproteinases (MMPs) after cerebral ischemia. C57BL/6 mice were subjected to 20 min of transient global cerebral ischemia. EGCG (50 mg/kg) or vehicle (saline) was administered i.p. immediately after ischemia. Brains were examined 3 days after ischemia. The effects of EGCG on MMP (gelatinase) activity and neuronal damage in the hippocampus were assessed. Gelatin gel zymography showed induction of active forms of MMP-9 protein after transient global cerebral ischemia. In situ zymography showed that ischemic gelatinase activity occurred primarily in pyramidal neuronal areas after brain ischemia. Mice treated with EGCG showed significantly reduced gelatinase levels. Neuronal damage was evident in CA1 and CA2 pyramidal sectors, corresponding to TUNEL-positive signals. In EGCG-treated mice, delayed neuronal damage was significantly reduced compared with vehicle-treated mice. These results demonstrate that the green tea polyphenol EGCG suppresses MMP-9 activation and reduces the development of delayed neuronal death after transient global cerebral ischemia in mouse brain.     

Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in gerbils

We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.     

Ischemia-related changes in naive and mutant forms of ubiquitin and neuroprotective effects of ubiquitin in the hippocampus following experimental transient ischemic damage

Ubiquitin binds to short-lived proteins and denatured proteins produced by various forms of injury. The loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structure and function. The aim of the present study is to observe the chronological changes in ubiquitin naive form and its mutant form (ubiquitin⁺¹) in the hippocampal CA1 region (CA1) after transient cerebral ischemia in gerbils. Delayed neuronal death in the CA1 was confirmed 4 days after ischemic insult with NeuN immunohistochemistry. Ubiquitin immunoreactivity and protein level in the CA1 were lowest at 12 h after ischemia/reperfusion; thereafter, they were increased with time. Ubiquitin⁺¹ immunoreactivity and protein levels in the CA1 were slightly decreased at 3 h after ischemia/reperfusion, and they were significantly increased 1 day after ischemia/reperfusion. In addition, ubiquitin and ubiquitin⁺¹ immunoreaction was expressed in astrocytes after delayed neuronal death in the ischemic CA1. To elucidate the protective effect of ubiquitin on ischemic damage, the animals were treated with ubiquitin (1.5 mg/kg body weight) intravenously via the femoral vein. Ubiquitin treatment significantly reduced ischemia-induced locomotor hyperactivity, neuronal death and reactive gliosis such as astrocytes and microglia. In addition, 5 days after ubiquitin treatment in the ischemic group, ubiquitin immunoreactivity was similar to that in the ubiquitin-treated sham group, however, ubiquitin⁺¹ immunoreactivity was higher than that in the ubiquitin-treated sham group. These findings indicate that the depletion of ubiquitin and the accumulation of ubiquitin⁺¹ in CA1 pyramidal neurons after transient cerebral ischemia may inhibit ubiquitin proteolytic pathway and this leads to delayed neuronal death of CA1 pyramidal neurons directly or indirectly after transient cerebral ischemia.     

A new model of transient focal cerebral ischemia for inducing selective neuronal necrosis

Brief cerebral ischemia leads to selective neuronal necrosis (SNN), which is characterized by neuronal death with sparing of glial and vascular elements of the central nervous system. Understanding the pathophysiology of SNN may help elucidating the mechanisms and consequences of neuronal injury in humans following brief ischemia. Contrary to the presence of reproducible models of transient global ischemia, animal models of transient focal ischemia producing SNN are scarce and have important limitations such as causing ischemia in a vast area and inducing additional insults. In this study, we developed a practical mouse model of SNN without these limitations, by compressing the distal middle cerebral artery (MCA) with a blunted micropipette for 15 min. The success of compression was evaluated by monitoring the regional cerebral blood flow, and conventional histopathology and immunolabeling of the brain sections. Seven/fourteen days after ischemia, intracisternally administered propidium iodide labeled numerous necrotic cells in the frontoparietal cortex, which were mostly NeuN-positive, but were not immunolabeled with astrocytic markers (GFAP and S100), and showed neuronal morphology with hematoxylin–eosin staining, indicating that the model successfully induced ischemic injury limited to neurons. The model could become an important tool for investigating the long-term effects of brief ischemic events like transient ischemic attacks and could offer convenient reversible distal MCA occlusion for studies using intravital microscopy.     

Evidence for neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rats.

The levels of brain-derived neurotrophic factor (BDNF) vary between different forebrain areas and show region-specific changes after cerebral ischemia. The present study explores the possibility that the levels of endogenous BDNF determine the susceptibility to ischemic neuronal death. To block BDNF activity the authors used the TrkB-Fc fusion protein, which was infused intraventricularly in rats during 1 week before and 1 week after 5 or 30 minutes of global forebrain ischemia. Ischemic damage was quantified in the striatum and hippocampal formation after 1 week of reperfusion using immunocytochemistry and stereological procedures. After the 30-minute insult, there was a significantly lower number of surviving CA4 pyramidal neurons, neuropeptide Y-immunoreactive dentate hilar neurons, and choline acetyltransferase- and TrkA-positive, cholinergic striatal interneurons in the TrkB-Fc-infused rats as compared to controls. In contrast, the TrkB-Fc treatment did not influence survival of CA1 or CA3 pyramidal neurons or striatal projection neurons. Also, after the mild ischemic insult (5 minutes), neuronal death in the CA1 region was similar in the TrkB-Fc-treated and control groups. These results indicate that endogenous BDNF can protect certain neuronal populations against ischemic damage. It is conceivable, though, that efficient neuroprotection after brain insults is dependent not only on this factor but on the concerted action of a large number of neurotrophic molecules.     

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.     

Neuronal damage following non-lethal but repeated cerebral ischemia in the gerbil

Summary Brief, non-lethal transient forebrain ischemia in the gerbil can injure selectively vulnerable neurons when such ischemia is induced repeatedly. The influence of the number and interval of the ischemic insults on neuronal damage, as well as the time course of damage, following repeated 2-min forebrain ischemia were examined. A single 2-min forebrain ischemia were examined. A single 2-min ischemic insult caused no morphological neuronal damage. A moderate number of hippocampal CA1 neurons were destroyed following two ischemic insults with a 1-h interval, and destruction of almost all CA1 neurons resulted from three or five insults at 1-h intervals. Three and five insults also resulted in moderate to severe damage to the striatum and thalamus, depending on the number of episodes. Although three ischemic insults at 1-h intervals caused severe neuronal damage, this number of insults at 5-min and 4-h intervals caused destruction of relatively few neurons, and non neurons were destroyed at 12-h intervals. Following three ischemic insults at 1-h intervals, damage to the striatum, neocortex, hippocampal CA4 subfield and thalamus was observed at 6–24 h of survival, whereas damage to the hippocampal CA1 subfield appeared at 2–4 days. The results indicate that even a brief non-lethal ischemic insult can produce severe neuronal damage in selectively vulnerable regions when it is induced repeatedly at a certain interval. The severity of neuronal damage was dependent on the number and interval of ischemic episodes.