Localization of 70-kDa stress protein induction in gerbil brain after ischemia

Summary Induction of the 70-kDa heat shock protein, hsp70, has been demonstrated in brain following experimental stroke. In the present study, hsp70 was localized in gerbil brain at intervals after transient ischemia using a monoclonal antibody specific for stress-inducible forms of hsp70-related proteins. Induced immunoreactivity was found only in neurons, primarily in hippocampus, striatum, entorhinal cortex and some neocortical regions. Notably hsp70 accumulation was minimal in hippocampal CA1 neurons which die after brief ischemic episodes, but was most pronounced in dentate granule cells and CA3 neurons which are spared. The peak of CA3 immunoreactivity occurred at 48-h recirculation, at the onset of CA1 neuron loss at 2–4 days, demonstrating that hsp70 induction is also a component of this delayed hippocampal pathophysiology rather than a direct response to the metabolic disruption of the initial ischemic episode. These results suggest that hsp70 immunocytochemistry may serve as a marker for neuronal circuitry involved in proposed excitotoxic mechanisms after ischemia and other stresses. Control animals showed immunoreactivity in ependymal cells lining the ventricles, indicating a role for hsp70 in normal functioning of these specialized cells.     

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.     

实验性局灶性脑缺血再灌注后HSP70 mRNA的表达

目的：探讨脑缺血后HSP70mRNA表达变化,方法：采用沙土鼠短暂前脑缺血再灌注损伤模型。Northern blot定量检测HSP70m RNA表达,结果：沙土鼠前脑缺血6分钟再灌注后各时期HSP70 mRNA表达量增加（P＜0．05）,热休克预处理能增加沙土鼠短暂前脑缺血再灌注后各时期HSP70 mRNA表达（P＜0．05）,结论：沙土鼠前脑缺血6分钟再灌注后各时期HSP70 mRNA表达增加,热休克预处理增加HSP70mRNA表达。     

实验性局灶性脑缺血再灌注后HSP70mRNA的表达

目的探讨脑缺血后HSP     

Accumulation of exogenous polyamines in gerbil brain after ischemia

Regionally selective delayed neuronal degeneration is a characteristic sequel of cerebral ischemia. Recent evidence indicates that changes in brain polyamine metabolism may be critical for nerve cell survival after ischemia. Within hours after ischemia, intracellular putrescine levels are greatly increased and remain elevated for days, whereas only minor changes are noted in the levels of the polyamines spermine and spermidine. In contrast, the extracellular levels of all polyamines are low after ischemia. Injections of polyamines following ischemia, however, can protect neurons in the gerbil brain from delayed cell death, with spermine being the most potent of the polyamines. In the present study, therefore, we sought to determine if increased polyamine uptake occurs in the brain after ischemia. In the hippocampal slice preparation, temperature-dependent uptake was unique for spermine, but not for spermidine or putrescine. Uptake of [¹⁴C]spermine was transiently increased after ischemia, peaking at 150% of control by 12–13 h and subsiding by 24 h. Intravenous injections of [³H]spermidine resulted in a postischemic accumulation of this polyamine throughout the forebrain parenchyma. We conclude that:

1. Active cellular uptake of spermine is transiently increased early after ischemia;
2. A nonspecific accumulation of exogenous polyamines occurs early after ischemia probably owing to a compromised blood-brain barrier, and
3. The findings indicate that exogenous polyamines can exert their effect directly in the brain after ischemia.

     

Cerebral ischemia decreases endogenous calcium-dependent protein phosphorylation in gerbil brain

Acute forebrain ischemia in the gerbil produced a significant decrease in endogenous Ca2+-dependent hippocampal protein phosphorylation in comparison to sham and naive control animals. Inhibition of Ca2+-dependent phosphorylation was demonstrated as early as 2 h post-ischemia and shown to persist up to 7 days following the ischemic insult. The ischemia-induced loss of Ca2+-dependent phosphorylation was only seen in brain regions affected by the localized ischemia and not in non-ischemic brain regions from the same animal. These results suggest that an early event in the development of ischemia-induced neuronal death is a decrease in Ca2+-dependent protein phosphorylation.     

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)     

Brain-gut induction of heat shock protein (HSP) 70 mRNA by psychophysiological stress in rats

Restraint water-immersion stress-induced expression of heat shock protein (HSP)70 mRNA in the cerebral cortex and stomach of rats was evaluated by Northern blotting. Cerebral and gastric HSP70 mRNA significantly increased in the 6 h-stressed rats and the amount of mRNA measured as optical densities was highest in the 12 h-stressed rats. These data confirmed our previous observations and suggest that families of HSPs play a salient cytoprotective role in stress-vulnerable organs.     

Microvascular disturbances and edema formation after repetitive ischemia of gerbil brain

Summary Three transient episodes of 5 min ischemia spaced at 1-h intervals were produced in Mongolian gerbils by bilateral carotid artery occlusion with an implanted vascular occlusion device. The interval of 1 h was chosen to allow for the development of postischemic hypoperfusion between the ischemic episodes. Three minutes and 1 h after each ischemic episode, and 6 and 24 h after the third occlusion, Evan's blue (EB) was injected intravenously to trace circulating blood, and the number of perfused capillaries was determined in various brain regions by fluorescence microscopy. Brain edema was evaluated by measuring specific gravity in tissue samples taken from adjacent areas. Repetitive ischemia caused progressively increasing brain edema and a progressive reduction of the number of perfused capillaries. Immediately after each ischemic episode, transient recruitment of capillaries occurred, thus excluding noreflow as a main pathogenetic factor of microcirculatory disturbances. The pattern of microcirculation 6 and 24 h after the last occlusion revealed a redistribution of circulating blood, characterized by a reduction in the number of EB-filled capillaries associated with a noticeable dilatation of the larger vascular channels. Our studies suggest a close interrelationship between post-ischemic microcirculatory hypoperfusion and the development of brain edema, the degree and extent of which progresses with the repetition of ischemic episodes when they are carried out during the periods of hypoperfusion.     

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.     

Induction of heat shock hsp70 mRNA and HSP70 kDa protein in neurons in the ‘penumbra’ following focal cerebral ischemia in the rat

Induction of hsp70 heat shock protein (HSP70) and hsp70 mRNA was examined using adjacent sections in the same rat brain following permanent middle cerebral artery (MCA) occlusions. hsp70 mRNA was induced within 4 h of MCA occlusion and persisted for at least 24 h. Cellular resolution autoradiographs suggested that hsp70 mRNA was induced primarily in neurons in the periphery of ischemia both outside and inside of the infarction, with small amounts of hsp70 mRNA being induced in the core of the infarction. HSP70 protein was localized in neurons outside the infarction and in endothelial cells within the infarction at 24 h but not at 4 h following permanent MCA occlusions. It is proposed that the penumbra, one of the areas that can be rescued by pharmacological agents, can be defined anatomically as the volume of tissue outside the area of infarction in which HSP70 protein is expressed primarily in neurons.     

Hypothermia reduces 72-kDa heat-shock protein induction in rat brain after transient forebrain ischemia.

BACKGROUND AND PURPOSE: We examined the influence of concurrent moderate hypothermia (30 degrees C) and transient forebrain ischemia on the induction of 72-kDa heat-shock protein and neuronal damage in male Wistar rats. SUMMARY OF REPORT: Experimental groups included: normothermic with 8 minutes of transient forebrain ischemia (group 1, n = 7), hypothermic without ischemia (group 2, n = 9), and hypothermic (30 degrees C) with 8 minutes of transient forebrain ischemia (group 3, n = 5). Intense 72-kDa heat-shock protein immunoreactivity was demonstrated in rat forebrain 48 hours after induction of normothermic forebrain ischemia (group 1); it was not detected in the brain of animals subjected to hypothermia without ischemia (group 2), and hypothermia during ischemia (group 3) significantly inhibited its expression compared with that in normothermic ischemia animals (group 1). CONCLUSIONS: These observations suggest that 72-kDa heat-shock protein induction is not the mechanism by which moderate hypothermia protects against ischemic cell damage.     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain.

In situ hybridization was used to estimate regional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was increased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distribution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (1-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The wide-spread increase in HSP-70 mRNA suggests that reperfusion triggered expression in all previously ischemic regions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the ischemic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions undergoing injury, but is transient in surrounding regions that recover.     

Expression of Ca-dependent (classical) PKC mRNA isoforms after transient cerebral ischemia in gerbil hippocampus

Cerebral ischemia is known to modify the expression of genetic information in the brain. To complement this knowledge, in the present study we have estimated the expression of calcium- and phospholipid-dependent (classical) protein kinase C (c PKC) isoform mRNAs (α, βI and γ) at different time following ischemia. Forebrain cerebral ischemia was performed on Mongolian gerbils by 5 minutes bilateral occlusion of common carotid arteries. At the pointed time the cytoplasmic RNA was extracted from hippocampus and the expression of PKC mRNA quantified by RT PCR technique using GAPDH expression as an internal standard. Results indicate that only one γ isoform of cPKC mRNA expression becomes significantly modified in postischemic hippocampus. A transient increase up to 145% of control within the first 3 h was followed by its decline to 60–65% at a longer recirculation period. This lowered levels returned back to control at 72 h postischemic recovery. This result indicates that γ PKC could be particularly sensitive to ischemic insult and would react in accordance with the other early signals determining ischemic outcome.