Preischemic blood glucose supply to the brain modulates HSP(72) synthesis and neuronal damage in gerbils.

Preischemic hyperglycemia is known to aggravate brain damage caused by transient forebrain ischemia. Because heat shock proteins (HSPs) 72 have been proposed to play a protective role against ischemic neuronal injury, we studied the HSP(72) mRNA expression and protein synthesis in gerbils subjected to 10 min bilateral carotid occlusion under normoglycemic, hyperglycemic and fasting conditions. HSP(72) mRNA expression and HSP(72) synthesis were studied using in situ hybridization and immunostaining, respectively. After 8 h of blood recirculation, HSP(72) mRNAs were expressed in all the hippocampal subfields of the three different groups, with higher expression in the hyperglycemic gerbils. After 48 h of reperfusion, HSP(72) mRNAs had almost completely disappeared in the hyper- and normoglycemic groups, and were more strongly expressed in the CA(1) neurons of the fasted group. At this time, fasted gerbils exhibited intense HSP(72) immunoreactivity in the CA(1), whereas an absence of immunoreactivity was observed in that area in the other groups. Finally, ischemia was also associated with marked astrocytic activation, as evidenced by GFAP immunostaining. Overall results indicate that preischemic differences in blood glucose supply to the brain are related to HSP(72) mRNA expression (in terms of duration) and to HSP(72) protein induction (in terms of intensity) in the vulnerable CA(1) neurons of the hippocampus. Ability of CA(1) neurons to synthesize HSP(72) proteins was associated with higher neuronal survival in the fasted group after 48 h of reflow, suggesting a protective role of HSP(72), even though evaluation of neuronal damage at 7 days indicated that neuronal death was mainly delayed in the time.     

Synergistic induction of HSP40 and HSC70 in the mouse hippocampal neurons after cerebral ischemia and ischemic tolerance in gerbil hippocampus.

An ischemia-induced gene was screened using a differential display technique in mouse transient forebrain ischemia. One of the ischemia-responsive clones was found to encode mouse hsp40. HSP40 has a critical regulatory function in the HSC70 ATPase activity. Expression of hsp40 mRNA was low in the nonischemic mouse hippocampus, but it was significantly upregulated 4 hr after ischemia by Northern blot analysis. In situ hybridization analysis revealed hsp40 mRNA induction in the neuron. HSP40 protein expression was also enhanced in the pyramidal and dentate granular neurons from 2 to 4 days after ischemia. The temporal expression and distribution profile of HSC70 protein was similar to that of HSP40, and both proteins were colocalized in ischemic hippocampal neurons. In the gerbil transient forebrain ischemia model, both HSP40 and HSC70 proteins were expressed strongly in ischemia-resistant CA3 neurons and dentate granule cells 1 day after 5 min ischemia, but were not expressed in vulnerable CA1 neurons. However, both proteins were in parallel expressed in the tolerance-acquired CA1 neurons. Based on the current observation that both HSP40 and HSC70 proteins were synergistically expressed in the ischemia-resistant and tolerance-acquired neurons, cochaperone HSP40 may play a significant role against postischemic neuronal response and lead to cell survival through interaction with simultaneously induced HSC70.     

Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain.

Thresholds of induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brain were investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brain, HSP70 mRNA was little present, while HSC70 mRNA was present in most cell populations. A 0.5-min occlusion of bilateral common carotid arteries did not affect the amount of HSP70 and HSC70 mRNAs. The selective induction of HSC70 mRNA was observed in dentate granule cells at 1 h, and in most cells of hippocampus especially dentate gyrus at 3 h after 1 min of ischemia when induction of HSP70 mRNA was not evident in the identical brain. The selective induction diminished by 2 days. However, after 2 min of ischemia, HSP70 and HSC70 mRNAs were induced together in hippocampal cells from 1 h of the reperfusion, and the co-induction prolonged in CA1 cells until 2 days. Body temperatures monitored at rectum increased after the reperfusion with a peak at 30 min. The degree of increase of the body temperature was significantly higher in the case after 2-min ischemia than in the cases after 0.5- and 1-min ischemia. Although HSP70 and HSC70 mRNAs are generally co-induced in stressful conditions, our results suggest the different thresholds of the induction between HSP70 and HSC70 mRNAs after transient brain ischemia. The selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA may relate to the differences of the duration of ischemia and the degree of the increase of body temperature after ischemia.     

Distributions of heat shock protein-70 mRNAs and heat shock cognate protein-70 mRNAs after transient global ischemia in gerbil brain.

Distributions of heat shock protein (HSP)-70 mRNAs and heat shock cognate protein (HSC)-70 mRNAs after 10 min of transient global ischemia were investigated in gerbil forebrain by in situ hybridization using cloned cDNA probes selective for the mRNAs. Expression of HSP70 immunoreactivity was also examined in the same brains. In hippocampal CA1 neuronal cells, in which only a minimal induction of immunoreactive HSP70 protein was found, the strong hybridization for HSP70 mRNA disappeared at around 2 days before the death of CA1 cells became evident. Furthermore, in hippocampal CA3 cells, a striking induction of HSP70 mRNA was sustained even at 2 days along with a prominent accumulation of HSP70 immunoreactivity. In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most neuronal cells, especially dense in CA3 cells, of the sham brain. A co-induction of HSP70 and HSC70 mRNAs was observed in several cell populations after the reperfusion with a peak at 8 h, although the magnitude of HSC70 mRNA induction was lower than that of HSP70 mRNA, particularly in CA1 cells. The expression of HSC70 mRNA in CA1 cells also disappeared at around 2 days. All the induced signals of HSP70 and HSC70 mRNAs in other cell populations were diminished and returned to the sham level, respectively, by 7 days. These results are the first to show the time courses of distribution of HSP70 and HSC70 mRNAs and the immunoreactive HSP70 protein in the same gerbil brain after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporospatial expression of HSP72 and c-JUN, and DNA fragmentation in goat hippocampus after global cerebral ischemia

The role of gene induction (expression of HSP72 and c-JUN proteins) and delayed ischemic cell death (in situ labeling of DNA fragmentation) have been investigated in the goat hippocampus after transient global cerebral ischemia. The animals were subjected to 20-min ischemia (bilateral occlusion of the external carotid arteries plus bilateral jugular vein compression) and allowed to reperfuse for 2 h, and then 1, 3, and 7 days. Histological signs of cell loss were not found in the hippocampus at 2 h, 1 day, or 3 days of reperfusion. However, such an ischemic insult produced extensive, selective, and delayed degeneration in the hippocampus, as 68% of the neurons in CA1 had died at 7 days, but cell loss was not detected in CA3 and dentate gyrus fields. Concomitantly, a high percentage of TUNEL-positive CA1 neurons (60+/-9%, mean +/- SEM) was seen at 7 days, but not at the earlier time points. Mild induction of HSP72 was detected in the goat hippocampus after ischemia. The maximum percentage of HSP72-positive neurons (10-15%) was shown at 3 days of reperfusion and was concentrated mainly in the CA3 field, subiculum, and hilus, rather than in the CA1 field, whereas HSP72 expression was hardly detected at 7 days. At this later time point, scattered induction of nuclear c-JUN was found in a few neurons. The results show that: 1) postischemic delayed neuronal death selectively affects the CA1 field in the goat hippocampus, a phenomenon which seems to take longer to develop than in previously reported rodent models; and 2) postischemic expression of c-JUN does not appear to be related to cell death or survival, while the inability of most CA1 neurons to express HSP72 could contribute to neuronal death.     

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.     

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.     

Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia

Several investigations have postulated evidence of the involvement of apoptosis in delayed neuronal death following brief periods of global cerebral ischemia. Apoptosis may be closely linked to mitochondrial dysfunction. Heat shock protein (HSP) 60 and HSP10 are mitochondrial matrix proteins induced by stress and form the chaperonin complex that is implicated in protein folding and assembly within the mitochondria. This study investigated the induction of these mitochondrial stress protein genes in the hippocampal CA1 region and less vulnerable regions following transient forebrain ischemia. In situ hybridization analysis revealed that the induction pattern of HSP60 mRNA was identical to that of HSP10 mRNA throughout the entire ischemic course. No changes occurred in the expression of both mRNAs after 2 min ischemia. Strong induction of both mRNAs occurred in the CA1 region after 10 min ischemia and persisted until 1 d after reperfusion. In contrast, induction of both mRNAs in the less vulnerable regions was terminated by 1 d after reperfusion. These results demonstrate that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in regions vulnerable to transient forebrain ischemia.     

沙鼠脑缺血耐受的组织学变化及HSP在其中的作用

目的 :观察脑缺血耐受时的组织学变化及 HSP在其中的作用。方法 :通过 HE染色观察脑缺血耐受时的组织学变化 ,并通过免疫组化染色 ,了解 HSP70及 HSP2 7在其中的作用。结果 :一次性 5分钟缺血后 7天海马 CA1区神经元大多坏死 ,若在缺血前给予 2分钟的缺血预处理 ,该区神经元大多保留 ,表现出明显的保护作用。只给一次性 5分钟缺血 ,海马 CA1区神经元无 HSP70染色。若在缺血前给予预处理 ,海马 CA1区神经元可见明显 HSP70染色。而HSP2 7主要在胶质细胞表达 ,海马区的神经元未见其表达。结论 :缺血前给予预处理对以后的缺血有保护作用 ;在缺血耐受过程中 ,HSP70表达出一定的保护作用。     

Comparative study on Cu,Zn-SOD immunoreactivity and protein levels in the adult and aged hippocampal CA1 region after ischemia-reperfusion

In the present study, we investigated chronological changes in Cu,Zn-superoxide dismutase (SOD1) immunoreactivity and its protein levels in the hippocampal CA1 region of adult and aged gerbils after transient forebrain ischemia to compare ischemia-related changes in SOD1 in adult and aged gerbils. Delayed neuronal death in the CA1 region at 4 days after ischemic insult was prominent in adult gerbils compared to that in aged gerbils. In sham-operated gerbils, SOD1 immunoreactivity and protein level in the aged group were significantly higher than that in the adult group. At 12 h after ischemia-reperfusion, SOD1 immunoreactivity and protein level were increased in both the groups. At 1 day after ischemia, SOD1 immunoreactivity and protein level in the adult group were significantly increased: the SOD1 immunoreactivity was increased in non-pyramidal cells as well as pyramidal cells. At this time after ischemia, SOD1 immunoreactivity and protein level in the aged group were decreased: the immunoreactivity was decreased significantly in pyramidal cells. At 4 days after ischemia, SOD1 immunoreactivity was detected only in non-pyramidal cells of the CA1 region in both the groups. These results suggest that SOD1 in the gerbil hippocampal CA1 region is higher in sham-aged group than that in sham adult one, and that different changes in SOD1 in CA1 pyramidal cells after ischemia in adult and aged gerbils may indicate different processes in delayed neuronal death with time after ischemic insult.     

'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.     

Induction of the HSP110/105 family in the rat hippocampus in cerebral ischemia and ischemic tolerance.

Recently, the authors isolated a novel gene of the HSP110 family, ischemia responsive protein 94 kDa (irp94), and demonstrated the expression of this gene after transient forebrain ischemia. In the current study, the authors investigated the expression profiles of all HSP110 family members including hsp110/105 and osp94/apg-1, after transient forebrain ischemia using rat four-vessel occlusion model. Among three members of the HSP110 family, induction of hsp110/105 was the most prominent after ischemia. hsp110/105 mRNA expression was clearly enhanced from 4 to 24 hours after a 6-minute or longer ischemic period. First, hsp110/105 mRNA expression was induced in the dentate gyrus, and later in the pyramidal layer. HSP110/105 protein expression also was enhanced by a 6-minute or longer period of ischemia. Profiles of HSP110/105 expression after ischemia were similar to those of inducible HSP70. After transient forebrain ischemia for 10 minutes, HSP110/105 protein was induced in the dentate gyrus and the CA3 pyramidal layer, but not in the CA1 pyramidal neurons. However, 6 minutes of ischemia induced the HSP110/105 protein, as well as the HSP70 protein, in the CA1 region. CA1 pyramidal neurons expressing HSP110/105 acquired tolerance against subsequent severe ischemia. In conclusion, HSP110/105 showed the most prominent induction after ischemia among the three HSP110 gene family members. Colocalization of HSP110/105 and HSP70 in the CA1 neurons that acquired tolerance suggested that induced HSP110/105 might contribute to ischemic tolerance together with HSP70.     

Differential expressions of glycine transporter 1 and three glutamate transporter mRNA in the hippocampus of gerbils with transient forebrain ischemia.

The extracellular concentrations of glutamate and its co-agonist for the N-methyl-d-aspartate (NMDA) receptor, glycine, may be under the control of amino acid transporters in the ischemic brain. However, there is little information on changes in glycine and glutamate transporters in the hippocampal CA1 field of gerbils with transient forebrain ischemia. This study investigated the spatial and temporal expressions of glycine transporter 1 (GLYT1) and three glutamate transporter (excitatory amino acid carrier 1, EAAC1; glutamate/aspartate transporter, GLAST; glutamate transporter 1, GLT1) mRNA in the gerbil hippocampus after 3 minutes of ischemia. The GLYT1 mRNA was transiently upregulated by the second day after ischemia in astrocytelike cells in close vicinity to hippocampal CA1 pyramidal neurons, possibly to reduce glycine concentration in the local extracellular spaces. The EAAC1 mRNA was abundantly expressed in almost all pyramidal neurons and dentate granule cells in the control gerbil hippocampus, whereas the expression level in CA1 pyramidal neurons started to decrease by the fourth day after ischemia in synchrony with degeneration of the CA1 neurons. The GLAST and GLT1 mRNA were rather intensely expressed in the dentate gyrus and CA3 field of the control hippocampus, respectively, but they were weakly expressed in the CA1 field before and after ischemia. As GLAST and GLT1 play a major role in the control of extracellular glutamate concentration, the paucity of these transporters in the CA1 field may account for the vulnerability of CA1 neurons to ischemia, provided that the functional GLAST and GLT1 proteins are also less in the CA1 field than in the CA3 field. This study suggests that the amino acid transporters play pivotal roles in the process of delayed neuronal death in the hippocampal CA1 field.     

Neuronal induction of 72-kDa heat shock protein following methamphetamine-induced hyperthermia in the mouse hippocampus

By means of an immunohistochemical technique, we examined the neuronal induction of 72-kDa heat shock protein (HSP72) in response to methamphetamine-induced hyperthermia in the mouse hippocampus. Strong HSP72 immunoreactivity (ir) was found in the neurons of hippocampus proper, particularly in the CA1/2 and medial CA3 subfields, at 10 h after drug injection. By 18 h, those neurons still revealed HSP72-ir, while neurons of the dentate gyrus also appeared positive for HSP72. At this stage, intense HSP72-ir was first detected in non-neuronal cells, i.e. glial and vascular endothelial cells. At 24 h, no apparent HSP72-ir was found in the hippocampal neurons, while only non-neuronal cells still revealed immunoreactivity for HSP72. In addition, no morphological evidence of cell degeneration or loss was noted in the CA1 sector or other hippocampal regions at 5 days after hyperthermic insult. In conclusion, (1) methamphetamine-induced hyperthermia per se is a stressful stimulant causing neuronal induction of HSP72 in the hippocampus neurons, particularly of CA1/2 and medial CA3 sectors, but does not prove fatal to the cells; (2) there is a cell type-specific difference in response to hyperthermic insult by inducing HSP72 and the timing of the induction response in the hippocampal formation; and (3) the animals that underwent drug-induced hyperthermia may be useful as an experimental model for the study of the protective mechanism of heat shock proteins against subsequent harmful stimuli.     

Clomethiazole (ZENDRA, CMZ) improves hind limb motor function and reduces neuronal damage after severe spinal cord injury in rat

The neuroprotective role of the expression of heat shock protein (HSP) and immediate early gene remains unclear. Using immunoelectron microscopy, we examined the ultrastructural integrity of the neurons with expression of c-Fos, c-Jun and HSP70 in gerbils after transient cerebral ischemia and repefusion. Induction of c-Fos and c-Jun was observed in the CA3 region resistant to ischemia, while HSP70 was expressed not only in the CA3 but also in the vulnerable CA1 region. With immunoelectron microscopy, the expression of c-Fos/c-Jun and HSP70 was observed in the neurons which retained neuronal integrity except for mitochondrial swelling and polyribosomal disaggregation. In contrast, the CA1 neurons without immunoreaction for HSP70 showed cytoplasmic vacuoles and parallel stacking of rough endoplasmic reticulum, the features associated with the process of delayed neuronal death. These findings suggested that c-Fos and c-Jun were induced selectively in reversibly damaged neurons, whereas HSP70 was up-regulated even in neurons with irreversible damage, but was more preferentially and intensely expressed in neurons with reversible damage. Received: 23 January 1998 / Revised, accepted: 12 June 1998     

‘Ischemic tolerance’ phenomenon detected in various brain regions

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the ‘ischemic tolerance’ phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of ‘ischemic tolerance’.     

Global ischemia induces NMDA receptor-mediated c-fos expression in neurons resistant to injury in gerbil hippocampus.

The induction of c-fos protein-like immunoreactivity (CFPLI) was examined in the hippocampus of gerbils at several time points after transient global ischemia. c-Fos protein induction was largely confined to the dentate gyrus, CA3 and CA4 regions from 2 to 8 h after transient bilateral carotid occlusion. Little CFPLI was seen in the CA1 subfield, which is disproportionately sensitive to injury after global ischemia. c-Fos induction was completely blocked by pretreatment with MK-801 (3 mg/kg). Our results show that c-fos expression after global ischemia is NMDA receptor mediated, and mainly found in hippocampal neurons resistant to ischemic injury.