Kyuya Kogure

Protection of rat hippocampus against ischemic neuronal damage by pretreatment with sublethal ischemia

We examined whether preconditioning with sublethal ischemia protects against neuronal damage following subsequent lethal ischemic insults. Forebrain ischemia for 3 min in Wistar rats increased heat shock protein-70 immunoreactivity in the hippocampal CA1 subfield but produced no neuronal damage. Preconditioning with 3 min of ischemia followed by 3 days of reperfusion protected against hippocampal CA1 neuronal damage following 6 and 8 min of ischemia but not damage after 10 min of ischemia. The result strongly suggests that stress response induced by sublethal ischemia protects against ischemic brain damage.

Temporal profile of heat shock protein 70 synthesis in ischemic tolerance induced by preconditioning ischemia in rat hippocampus

We investigated the temporal profile of heat shock protein 70 induction in the rat hippocampus using immunohistochemistry to clarify the mechanism of ischemic tolerance following preconditioning with sublethal ischemia. Although a 6-min period of forebrain ischemia produced severe neuronal damage to the hippocampal CA1 subfield, preconditioning with 3 min of ischemia followed by three days of reperfusion protected against the CA1 neuronal damage after 6 min of ischemia. Immunohistochemical staining against heat shock protein 70 showed that the protein is induced in CA1 pyramidal cells one, three and seven days after 3 min of ischemia, the immunostaining being most intense after three days. Heat shock protein synthesis was observed in CA1, CA3 and dentate hilar neurons one and three days after 6 min of ischemia, both with and without preconditioning. In addition, the heat shock protein was stained in the CA1 2 h and seven days after 6 min of ischemia with preconditioning, but the intensity of staining was relatively weak at these time points. The results suggest that stress response induced by sublethal ischemia protects against ischemic neuronal damage, and that the induced stress response, including heat shock protein 70 synthesis during and immediately after the second ischemic episode, is correlated with the protection because late induction of the heat shock protein did not prevent neuronal death.

Altered gene expression in cerebral ischemia.

Background Using the techniques of molecular biology, recent experimental studies have shown that cerebral ischemia induces a variety of changes in gene expression in the brain. Summary of Review During the early postischemic stages, protein synthesis in the brain is generally suppressed, but specific genes are expressed and their corresponding proteins may be synthesized, such as immediate-early gene products (c-fos, c-jun, and zinc finger gene), heat-shock proteins, and amyloid precursor protein. The ability of neurons to induce such stress responses, which depends on both the severity of ischemia and the intrinsic nature of the neuronal populations, may be directly associated with neuronal death and survival after ischemia. Nerve growth factor and fibroblast growth factor are also induced after ischemia and may be related to repair processes, in which a role of glial cells is suggested. Postischemic events that may be associated with the altered gene expression include (1) induction of tolerance to ischemia after pretreatment with sublethal ischemia, (2) slow, progressive neuronal changes and the development of neuronal plasticity after ischemia, and (3) delayed neuronal changes in remote areas outside the cerebral ischemic focus. Conclusions Because a variety of harmful stresses, including ischemia, elicit the same stress response and because this response is induced when total protein synthesis in the brain is nearly completely suppressed, this response may be vital to cell survival and repair. A successful induction of this response may induce resistance and survival of neurons after ischemia. However, failure or abortion of the response and persistent stresses may lead to neuronal death and possibly long-term changes and degeneration.

Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil

We examined the effects of treatment with basic fibroblast growth factor (b-FGF) on hippocampal CA1 neuronal damage following 3 min of forebrain ischemia in the gerbil. Continuous infusion of b-FGF (24 or 240 ng/day over 4 days) using an implanted osmotic minipump into the lateral ventricle prevented CA1 neuronal damage in a dose-dependent manner.

An immunohistochemical study of copper/zinc superoxide dismutase and manganese superoxide dismutase in rat hippocampus after transient cerebral ischemia

We investigated the changes of copper/zinc superoxide dismutase (CuZn-SOD) and manganese superoxide dismutase (Mn-SOD) in the rat hippocampus after 10 min of cerebral ischemia induced by 4-vessel occlusion. The rats were allowed to survive for 4 h, 1 day, 3 days, and 7 days after ischemia. The distribution of SODs were determined by immunohistochemical staining with antibodies against rat CuZn-SOD and Mn-SOD. CA1 pyramidal neurons and granule cells of the dentate gyrus showed intense CuZn-SOD immunoreactivity, whereas CA3 and CA4 neurons showed weaker immunostaining than CA1 neurons in normal animals. The immunoreactivity was reduced by 4 h after ischemia in CA1, CA3, and CA4 neurons when no histological damage was observed. Mn-SOD immunostaining revealed more intense immunoreactivity in CA3 pyramidal neurons than in CA1 neurons in normal animals. Interneurons in the CA1 and CA3 regions and the dentate hilus also showed high Mn-SOD immunostaining. Although CA1 neurons lost Mn-SOD immunoreactivity by 1 day after ischemia, CA3 neurons and interneurons retained the immunoreactivity and preserved intact cell contour after ischemia. In addition, reactive glial cells, which were differentiated by immunocytochemical staining against glial fibrillary acidic protein for reactive astrocytes and histochemical staining for reactive microglial cells, were intensely stained for CuZn-SOD and Mn-SOD after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

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. The results suggest that the weak induction of HSP70 protein in CA1 cells, which may relate to the vulnerability of this cell population, is due to both translational and transcriptional deficits. The different roles under normal condition and the cooperative role in the recovery process from ischemic injury between HSP70 and HSC70, and the involvement of HSC70 in CA1 cell death, are suggested.

Acceleration of HSP70 and HSC70 heat shock gene expression following transient ischemia in the preconditioned gerbil hippocampus

To evaluate the mechanism of tolerance to ischemia, inductions of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs in gerbil hippocampus were compared with in situ hybridization between cases of a single 3.5-min period of forebrain ischemia and a 3.5-min period of ischemia 2 days after 2-min pretreatment with ischemia. Immunohistochemistry for HSP70 protein and morphological studies were also performed in the same brains up to 7 days after the reperfusion. Following a single 3.5-min period of ischemia, HSP70 and HSC70 mRNAs were induced in all hippocampal cells. However, the hippocampal CA1 cells produced only a minimum of HSP70 protein, and the cells were almost lost by 7 days. Following 3.5 min of ischemia after 2-min pretreatment, large populations of the CA1 cells survived at 7 days. The peak time of the HSP70 and HSC70 mRNA induction shifted to an earlier period of reperfusion in all hippocampal cells as compared with the case of a single episode of ischemia. The peak of HSP70 and HSC70 mRNA induction shifted from 1 day to 3 h in the CA1 cells. The CA1 cells produced strongly immunoreactive HSP70 from 3 hr to 2 days. These results suggest that pretreatment with an initial period of ischemia (for 2 min) accelerated HSP70 and HSC70 gene expression at the transcriptional level, ameliorated the translational disturbance of HSP70 mRNA to protein, and saved the CA1 cells from subsequent lethal ischemia (for 3.5 min). These changes of heat shock gene expression might play important roles in the acquisition of ischemic tolerance of hippocampal CA1 neurons.

Induction of tolerance to ischemia : alterations in second-messenger systems in the gerbil hippocampus

Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu), [3H]cyclic adenosine monophosphate (cAMP) and [3H]rolipram, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [3H]IP3 binding (p < 0.01 vs. control) and 15-25% reductions in [3H]forskolin (p < 0.01 vs. control), [3H]cAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CA1 pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [3H]IP3 and [3H]rolipram binding in CA1 showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively, in the early reperfusion period. This downregulation of the IP3 system may play a role in the protection against cell death.

Inhibition of ischaemic tolerance in the gerbil hippocampus by quercetin and anti-heat shock protein-70 antibody.

To clarify the role of heat shock protein-70 (HSP70) in ischaemic tolerance following pretreatment with sublethal cerebral ischaemia, we examined whether the induction of tolerance in the gerbil hippocampus is inhibited by quercetin, an inhibitor of HSP70 expression, or anti-HSP70 antibody. A 3 min period of forebrain ischaemia was induced following pretreatment with 2 min of ischaemia and 3 days of reperfusion. Quercetin or anti-HSP70 antibody was continuously infused into the left lateral ventricle using an implanted osmotic minipump started 3 h after or 2 h before the first ischaemia. The animals were killed 4 days after the second ischaemia for histological observations. Both agents produced no neuronal damage in the brain following a single 2 min period of ischaemia. The neuronal density of the CA1 hippocampus in animals subjected to treatment with quercetin and anti-HSP70 antibody was significantly lower than vehicle-treated animals but were significantly higher than animals with a single 3 min period of ischaemia. Thus, the present study showed that quercetin and anti-HSP70 antibody prevent the induction of ischaemic tolerance. The result suggests that HSP70 expression, at least in part, plays a role in the induction of ischaemic tolerance.

Distributions of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient focal ischemia in rat brain

The distribution of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNA after 30 min of middle cerebral artery (MCA) occlusion was investigated in rat brain by in situ hybridization using cloned cDNA probes selective for the mRNAs. While HSP70 mRNA was hardly present at caudate and dorsal hippocampal levels of the sham brain this mRNA was greatly induced in cells of the MCA territory 1 h after reperfusion. Although the maximum amount of induced HSP70 mRNA in the caudate was much smaller than that in the cortex the maximum induction in the caudate (3 h) preceded that in the cortex (8 h). In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most cells of the sham brain, and was especially dense in hippocampal CA3 cells. Further induction of HSC70 mRNA was observed after reperfusion in the same cell populations, as in the case of HSP70 mRNA. HSC70 mRNA levels were significantly reduced in the caudate at 8 h when small amounts of HSP70 mRNA were still elevated. In the ipsilateral granule cells of the dentate gyrus and hippocampal CA3 cells a slight but significant induction of HSC70 mRNA was observed from 1 h to 1 day, while obvious induction of HSP70 mRNA never occurred. All the induced signals of HSP70 and HSC70 mRNA were diminished or returned to the sham level by 7 days, except for HSC70 mRNA in the caudate. These results are the first observations of the distribution of HSP70 and HSC70 mRNA after transient focal ischemia of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)