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.     

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.     

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)     

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.     

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.     

Postischemic glucose utilization in rat hippocampal layers

The influence on hippocampal glucose utilization was determined in male Wistar rats 7 days after a 10-min forebrain ischemia. Ischemia was induced by clamping of the carotid arteries and lowering blood pressure to 40 mm Hg. Despite severe neuronal damage as assessed by histological techniques, local cerebral glucose utilization (LCGU) was significantly increased in the pyramidal and radiatum layer of the CA1 sector, while in layers of the CA2, CA3 and CA4 sector and dentate gyrus. LCGU was reduced compared to non-ischemic controls. The increases in LCGU are suggested to reflect long-lasting hyperexcitation in the selectively vulnerable CA1 sector, implicating a correlation between cellular hypermetabolism and neuronal damage.     

Brain regional glucose use during Soman-induced seizures

The (14C)-2-deoxyglucose procedure was used to determine the effects of the potent acetylcholinesterase inhibitor Soman on regional metabolism in the brain. Groups of rats were given 112 micrograms/kg Soman, 84 micrograms/kg Soman, or saline i.m., and 15 min later the (14C)-2-deoxyglucose mapping procedure was initiated. All animals given 112 micrograms/kg Soman and 2 of 6 given 84 micrograms/kg Soman developed seizures that continued throughout the mapping procedure. Very high rates of glucose use occurred in most of the brain regions studied during seizures. The most striking increases occurred in substantia nigra, septum, outer layer of dentate gyrus of the hippocampus, hippocampal body, frontal cortex, caudate, ventral thalamus, parietal cortex, medial geniculate and interpeduncular nucleus. Only the inferior colliculus, superior olivary nucleus and lateral habenula were unaffected by the seizures. The mid layers of cerebral cortex rostral to superior colliculus showed marked reductions in glucose use which may represent inhibition of neuronal activity or functional failure from depleted energy reserves. The animals given 84 micrograms/kg i.m. that did not have seizures had regional glucose use patterns similar to the controls. The results indicate that the brain damage observed by others in Soman treated rats may be in part due to the excessive neuronal stimulation that occurs during the prolonged Soman-induced seizure.     

Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil

Summary Regional cerebral protein synthesis following brief ischemia was investigated in the Mongolian gerbil, utilizing l-[methyl-¹⁴C]methionine autoradiography. Transient ischemia was induced for 1,2 or 3 min. At various recirculation periods up to 48 h, animals received a single dose of l-[methyl-¹⁴C]-methionine and then were terminated 35 min later. Sham-operated animals showed a normal pattern of amino acid incorporation into the proteins of the brain. Following 1-min ischemia, the pattern of protein synthesis was similar to that in the sham-operated gerbils. Ischemia for 2 min, however, caused marked inhibition of protein synthesis in the neocortex, striatum, hippocampal CA1 sector and the thalamus at 1 h of recirculation. Extensive recovery of protein synthesis was found in the neocortex, the striatum, the hippocampal CA1 sector and the thalamus at 5–24 h of recirculation, but, a slight inhibition was detectable in the hippocampal CA1 sector in one of six animals. This inhibition had fully recovered at 48 h of recirculation. Following 3-min ischemia, severe impairment of protein synthesis was found in the neocortex, striatum, the whole hippocampus and the thalamus. After 5–24 h of recirculation, the protein synthesis in these regions had gradually recovered, except that complete lack of amino acid incorporation was seen in the hippocampal CA1 subfield. This impairment of protein synthesis in the hippocampal CA1 sector was not recovered at 48h of recirculation. Morphological study indicated that 2-min ischemia did not produce any significant neuronal damage in the brain, whereas gerbils subjected to 3-min ischemia revealed a mild neuronal damage in the hippocampal CA1 sector. The present study indicates that even non-lethal ischemia can produce a severe inhibition of protein synthesis in the selectively vulnerable regions during the early stage of recirculation.     

Sequential alteration of [H]rolipram binding in gerbil brain after transient cerebral ischemia

The time course of rolipram (Ca²⁺/calmodulin independent cyclic adenosine monophosphate inhibitor) binding sites changes following gerbil transient forebrain ischemia was determined using receptor autoradiography. Gerbils subjected to 10-min ischemia revealed a significant reduction in rolipram binding in most selectively vulnerable regions early in the recirculation (1–5 h). Marked reduction in the rolipram binding was seen in the selectively vulnerable areas 48 h or 7 days after ischemia. Thereafter, the rolipram binding in the hippocampal CA1 and CA3 sectors, which were most vulnerable to ischemia, was severely reduced up to 1 month after recirculation. In contrast, the reduction of the rolipram binding activity in other regions recovered to sham-operated level or showed a slight recovery. Interestingly, the dentate gyrus, which was resistant to ischemia, also exhibited a significant reduction of the rolipram binding activity up to I month after ischemia. Eight months after ischemia, the hippocampal CA 1 and CA3 sectors showed severe shrinkage and marked reduction in the rolipram binding. Other regions exhibited no significant reduction in the rolipram binding except for a slight reduction in the thalamus. These results demonstrate that transient cerebral ischemia causes severe reduction in rolipram binding sites in selectively vulnerable areas, and this reduction precedes the neuronal cell loss. These findings may reflect the alteration of an intracellular phosphodiesterase activity after ischemia.     

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [³H]nimodipine and [³H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [³H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [³H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [³H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [³H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [³H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [³H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [³H]nimodipine and [³H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.     

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.     

Chronic maintenance of presynaptic terminals in gliotic hippocampus following ischemia

Following brief cerebral ischemia, neurons are selectively damaged and die, whereas glial cells and blood vessels survive. This phenomenon of selective vulnerability is well illustrated in the hippocampal CA1 region. Five min of forebrain ischemia in the Mongolian gerbil produced selective neuronal necrosis in the hippocampal CA1 sector. After destruction and loss of CA1 neurons, a remarkable glial reaction (gliosis) was seen. The thickness of the CA1 subfield remained unchanged until 1 month after ischemia and then gradually shrank over several months. Ultrastructural observation of this region revealed persistent maintenance of presynaptic structures. Numerous presynaptic terminals containing synaptic vesicles were scattered throughout the gliotic scar tissue. These presynaptic terminals were apposed to degenerative structures which seemed most likely to be remnants of dendrites. In another group of animals, at one month following ischemic damage in the CA1 sector, the CA3 neurons were destroyed by kainic acid injection. In these animals, numerous degenerating presynaptic boutons were seen in the CA1 sector when fixed 4 days following kainate injection. These results indicate that even in gliotic tissue, presynaptic terminals can survive and maintain their structural characteristics although neuronal cell bodies are almost absent.     

Brain ischemia alters the density of binding sites for glibenclamide, a specific blocker of ATP-sensitive K+ channels

Transient ischemia in normoglycemic animals leads to delayed neuronal damage which is confined to selectively vulnerable regions. In at least one of these, the CA1 sector of the hippocampus, cell death is preceded by neuronal hyperactivity, presumed to be caused by loss of inhibitory control. Hyperglycemic subjects develop postischemic seizures, and show enhanced damage. The ATP-sensitive K+ channel, which may be important in inhibitory control, is the target of antidiabetic sulfonylureas. We determined densities of sulfonylurea binding sites in rat brain after forebrain ischemia. Normoglycemic animals showed a decrease of glibenclamide receptor binding in the CA3 field, hilus and dentate gyrus of the hippocampus after 1 day of recovery. After 4 days of recovery, levels of sulfonylurea binding sites decreased mainly in the CA1 field and in the hilus, as well as in the substantia nigra. After 1 day of recovery, hyperglycemic animals did not show any significant variations of densities of sites compared to control animals. It is proposed that reduction of inhibitory control by ATP-sensitive K+ channels may be associated with delayed neuronal death.     

Postischemic alteration of muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in selectively vulnerable areas: an autoradiographic study of gerbil brain.

We performed receptor autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of a voltage dependent L-type calcium channel blocker 1 h-1 month after transient cerebral ischemia in the gerbil brain. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Transient ischemia was induced for 10 min. [3H]QNB and [3H]CHA binding showed no significant alteration in selectively vulnerable areas at an early stage (1-24 h) of recirculation. However, the dentate molecular layer which was resistant to ischemia revealed a significant decrease in the [3H]CHA binding sites 24 h after ischemia. Thereafter, the [3H]QNB and [3H]CHA binding showed significant reduction in most of selectively vulnerable areas. Marked reduction was especially found in the dorsolateral part of striatum and the hippocampal CA1 sector which was the most vulnerable to ischemia. In contrast, [3H]PN200-110 binding showed a transient elevation in the hippocampal CA1 sector, the dentate molecular layer and the thalamus 1 h of recirculation. However, the striatum and neocortex revealed no alteration in the [3H]PN200-110 binding. Thereafter, the reduction in the [3H]PN200-110 binding was seen only in the dorsolateral part of the striatum and the hippocampal CA1 sector. The results suggest that transient cerebral ischemia can cause the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of L-type calcium channel blocker in most of selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic and functional mapping of the neural network subserving inferior collicular seizure generalization

The sensory-motor portion of the inferior collicular cortex is capable of seizure genesis that is characterized initially by coincident wild running behaviors and localized electrographic afterdischarge. With repeated stimulations, this seizure activity spreads into the forebrain, producing generalized tonic-clonic or myoclonic seizure activity. In order to characterize the neural network subserving this caudal-rostral seizure generalization, three mapping techniques were used: 2-deoxyglucose (2-DG) utilization, c-fos expression and local anesthetic microinjection. Kindled seizure generalization from the inferior collicular cortex produced a global increase in 2-DG accumulation, while relative 2-DG increases were found in the inferior collicular cortex, dorsal lateral lemniscus, dorsal central gray, peripeduncular nucleus, medial geniculate nucleus, substantia nigra, entopeduncular nucleus, ventroposterior and centromedian thalamus and tenia tectum, as well as the perirhinal, somatosensory and frontal cortices. Kindled seizure generalization also increased c-fos-like immunoreactivity (FLI) in the inferior collicular cortex, cuneiform nucleus, dorsal lateral nucleus of the lateral lemniscus, peripeduncular nucleus, caudal central gray, dentate gyrus of the hippocampus, rhinal fissure area of the perirhinal cortex and the frontal cortex. Microinjections of procaine into the amygdala, perirhinal cortex, entopeduncular nucleus, substantia nigra, peripeduncular nucleus, dorsal central gray, and pontine reticular nucleus all prevented generalized seizure behaviors, but had no effect on the wild running seizures. Conversely, procaine microinjection into the area of the cuneiform nucleus/pedunculopontine tegmental nucleus prevented the wild running seizure but did not block the generalized seizure activity. Neither wild running, nor generalized seizures were altered following procaine microinjections into the anterior thalamus, sub-thalamus, lateral hypothalamus, hippocampus or deep superior colliculus. Thus, specific forebrain sites form a widespread neural network that mediates the generalization of seizure activity from the inferior collicular cortex into the forebrain.     

Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat.

The connections of the laterodorsal tegmental nucleus (LDTg) have been investigated using anterograde and retrograde lectin tracers with immunocytochemical detection. Inputs to LDTg were found from frontal cortex, diagonal band, preoptic areas, lateral hypothalamus, lateral mamillary nucleus, lateral habenula; the interpeduncular nucleus, ventral tegmental area, substantia nigra and retrorubral fields; the medial terminal nucleus, interstitial nucleus, supraoculomotor central grey, medial pretectum, nucleus of the posterior commissure, paramedian pontine reticular formation, paraabducens and paratrochlear region; the parabrachial nuclei and nucleus of the tractus solitarius. Terminal labelling from PHA-L injections of LDTg was found in infralimbic, cingulate and hippocampal cortex, lateral septum, septofimbrial and triangular nuclei, horizontal limb of diagonal band and preoptic areas; in the anterior, mediodorsal, reuniens, centrolateral, parafascicular, paraventricular and laterodorsal thalamic nuclei, rostral reticular thalamic nucleus, and zona incerta; the lateral habenula and the lateral hypothalamus. A number of brainstem structures apparently associated with visual functions were also innervated, mainly the superior colliculus, medial pretectum, medial terminal nucleus, paramedian pontine reticular formation, inferior olive, supraoculomotor, paraabducens and supragenual regions, prepositus hypoglossi and nucleus of the posterior commissure. Also innervated were substantia nigra compacta, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, dorsal and medial raphe, pedunculopontine tegmental region, parabrachial nuclei, and nucleus of the tractus solitarius. These findings suggest the LDTg to be a highly differentiated part of the ascending "reticular activating" system, concerned not only with specific cortical and thalamic regions, especially those associated with the limbic system, but also with the basal ganglia, and visual (particularly oculomotor) mechanisms. Additional links with the habenula-interpeduncular system are discussed in this context.     

Protective effect of pitavastatin,a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor,on ischemia-induced neuronal damage

Abstract

We investigated the neuroprotective effects of a novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (pitavastatin) on ischemic neuronal damage in gerbils using immunohistochemistry. The animals were allowed to survive for 14 days after 5 min of ischemia induced by bilateral occlusion of the common carotid arteries. Five days after ischemia, severe neuronal cell loss was observed in the hippocampal CA1 sector. Prophylactic treatment with pitavastatin dose-dependently prevented the hippocampal CA1 neuronal cell loss 5 days after ischemia. Immunohistochemical study did not show the change of nNOS and iNOS expression in the hippocampus except for, in a few regions, up to 1 day after ischemia. Thereafter, the expression of iNOS was observed in the hippocampal CA1 sector 5 and 14 days after ischemia. In contrast, the expression of nNOS and eNOS gradually decreased in the hippocampal CA1 sector up to 14 days after ischemia. Prophylactic treatment with pitavastatin also prevented the expression of iNOS and the decrease of eNOS expression and the number of nNOS-positive cells in the hippocampal CA1 sector 5 days after ischemia. However, prophylactic treatment with pitavastatin at a dose of 10 mg kg^-1 did not change the immunoreactivity of iNOS and nNOS in the hippocampus at an early phase after ischemia. In contrast, this drug prevented the reduction of eNOS immunoreactivity in the hippocampal CA1 neurons at an early phase after ischemia. These findings demonstrate that the HMG-CoA reductase inhibitor pitavastatin can protect hippocampal CA1 neurons after transient forebrain ischemia through up-regulation of eNOS expression in this region. Thus pharmacological modulation of eNOS expression may offer a novel therapeutic strategy for cerebral ischemic stroke.     

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.     

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.