Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain.

A complementary DNA (cDNA) library was constructed with a plasmid vector from cerebral cortices of gerbils at 8 h of reperfusion after 10 min of bilateral common carotid ligation. After the 3rd screening of this cDNA library with a human genomic DNA probe for HSP70 (pH2.3), 4 cDNA clones were isolated (named pGA3' pGB1' pGD3 and pGE4' respectively). Southern and Northern blot analysis, and partial nucleotide sequence analysis indicated that pGA3 and pGE4 were the HSP70 cDNA clones, and that pGB1 and pGD3 were the HSC70 cDNA clones, which selectively recognized HSP70 or HSC70 mRNA, respectively. HSP70 mRNA is present in a very small amount in normal controls, and is greatly induced after the transient ischaemia. HSC70 mRNA is constitutively expressed in a normal gerbil brain, but is still inducible. In situ hybridization study demonstrated that the HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells in the sham control, and that the mRNA was greatly induced in the cells of hippocampus, dentate gyrus, medial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arcuate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of sham control, and was slightly induced after 8 h of reperfusion. Our results indicate that HSP70 and HSC70 cDNA clones were first isolated from post-ischaemic gerbil brain, and selectively recognize HSP70 or HSC70 mRNA, respectively. A regional difference in the induction of the HSP70 and HSC70 mRNA in post-ischaemic gerbil brain was observed by in situ hybridization.     

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.     

Synthesis of heat shock proteins in rat brain cortex after transient ischemia

Cell-free protein synthesis and two-dimensional gel autoradiography were used to characterize early postischemic protein synthesis in rat neocortex. Severe forebrain ischemia was induced for 30 min (four-vessel occlusion model) and followed by 3 h of recirculation. Polysomes were isolated from the cerebral cortex, translated in vitro in a reticulocyte system, and analyzed by two-dimensional gel electrophoresis. The translation products of postischemic polysomes included a major new protein family (70 kDa) with multiple isoelectric variants that was found to comigrate with the 68- to 70-kDa "heat shock" protein synthesized from polysomes of hyperthermic rats. Two other stress proteins (93 and 110 kDa) also appeared to be synthesized in increased amounts after ischemia. A complement of proteins that was indistinguishable from that of controls was also synthesized after ischemia, indicating that messenger ribonucleic acid coding for most brain proteins is preserved after ischemia and is bound to polysomes.     

Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain

Abstract

A complementary DNA (cDNA) library was constructed with a plasmid vector from cerebral cortices of gerbils at 8 h of reperfusion after 10 min of bilateral common carotid ligation. After the 3rd screening of this cDNA library with a human genomic DNA probe for HSP70 (pH2.3), 4 cDNA clones were isolated (named pGAy pGBy pGD3 and pGE^ respectively). Southern and Northern blot analysisand partial nucleotide sequence analysis indicated that pGA3 and pGE4 were the HSP70 cDNA clones, and that pGB1 and pGD3 were the HSC70 cDNA clones; which selectively recognized HSP70 or HSC70 mRNA, respectively. HSP70 mRNA is present in a very small amount in normal controls, and is greatly induced after the transient ischaemia. HSC70 mRNA is constitutively expressed in a normal gerbil brain, but is still inducible. In situ hybridization study demonstrated that the HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells in the sham controland that the mRNA was greatly induced in the cells of hippocampus, dentate gyrusmedial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arcuate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of sham control and was slightly induced after 8 h of reperfusion. Our results indicate that HSP70 and HSC70 cDNA clones were first isolated from post-ischaemic gerbil brain, and selectively recognize HSP70 or HSC70 mRNA, respectively. A regional difference in the induction of the HSP70 and HSC70 mRNA in post-ischaemic gerbil brain was observed by in situ hybridization. [Neurol Res 1992; 14: 000-000]     

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.     

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.     

N omega-nitro-L-arginine-methyl ester inhibits electrocortical recovery subsequent to transient global brain ischemia in Mongolian gerbil.

Electrocorticographic (ECoG) activity remains isoelectric for about 15 min after transient (10 min) bilateral carotid arteries occlusion in mongolian gerbils. In this model of global forebrain ischemia N omega-Nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, significantly delays the recovery of ECoG amplitude. Thus, the present experiments suggest that NO is involved in the cerebrovascular physiological response to brain ischemia.     

Changes in expression of mRNAs in the gerbil hippocampus following transient forebrain ischemia

The phenomenon of delayed neuronal death in CA1 neurons following brief duration of global ischemia has eluded definitive explanation. Using a differential display technique, we examined changes in expression of mRNAs in the hippocampus following 5-min cerebral ischemia in Mongolian gerbils. Under pentobarbital anesthesia, gerbils were sacrificed by decapitation at 6 h (n = 20) and 2 days (n = 20) after ischemia, and sham-operated gerbils (n = 20) were sacrificed at 6 h after surgery. Total RNA was isolated from the hippocampal samples in each group for the differential display analysis. The mRNAs were classified into three patterns; gradual disappearance, decrease and recovery, and new appearance. Representative mRNAs in three patterns were subcloned and sequenced partly. An mRNA in the gradual disappearance pattern showed homologous with neuronal pentraxin. In situ hybridization and Northern blot analyses of neuronal pentraxin revealed the gradual disappearance pattern. An mRNA in the decrease and recovery pattern showed homologous with 14-3-3 protein gamma-subtype, and an mRNA in the new appearance pattern showed no homology in the data base. The differential display analysis is a useful technique with which to investigate changes in expression of mRNAs following transient cerebral ischemia. The novel mRNA may be involved in the treatment of cerebral ischemia. Further studies are necessary for this point.     

Changes in expression of mRNAs in the gerbil hippocampus following transient forebrain ischemia

Abstract

The phenomenon of delayed neuronal death in CA 1 neurons following brief duration of global ischemia has eluded definitive explanation. Using a differential display technique, we examined changes in expression ofmRNAs in the hippocampus following 5-min cerebral ischemia in Mongolian gerbils.. Under pentobarbital anesthesia, gerbils were sacrificed by decapitation at 6 h (n = 20) and 2 days (n = 20) after ischemia, and sham-operated gerbils (n = 20) were sacrificed at 6 h after surgery. Total RNA was isolated from the hippocampal samples in each group for the differential display analysis. The mRNAs were classified into three patterns; gradual disappearance, decrease and recovery, and new appearance. Representative mRNAs in three patterns were subcloned and sequenced partly. An mRNA in the gradual disappearance pattern showed homologous with neuronal pentraxin. In situ hybridization and Northern blot analyses of neuronal pentraxin revealed the gradual disappearance pattern. An mRNA in the decrease and recovery pattern showed homologous with 14-3-3 protein y-subtype, and an mRNA in the new e appearance pattern showed no homology in the data base. The differential display analysis is a useful technique with which to investigate changes in expression ofmRNAs following transient cerebral ischemia. The novel mRNA may be involved in the treatment of cerebral ischemia. Further studies are necessary for this point. [Neural Res 2000; 22: 825-831]     

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

Cerebral ischemia induces the expression of a number of proteins that may have an important influence on cellular injury. The purpose of this study was to compare the regional effects of hypoxia-ischemia on the expression of the proto-oncogene, c-fos, and the heat shock protein-70 (HSP-70) gene in developing brain. Unilateral hypoxia-ischemia was produced in the brain of immature rats (7, 15, and 23 days after birth) using a combination of carotid artery ligation and systemic hypoxia (8% O2). After recovery for 2 and 24 h, the regional expression of c-fos and HSP-70 mRNA was determined using in situ hybridization. Littermates were permitted to recover for 1 week for assessment of histologic injury. Hypoxia-ischemia increased the expression of both c-fos and HSP-70 mRNA, but the topography of expression varied with the age of the animal as well as the mRNA species. In the 7-day-old group, expression of c-fos at 2 h increased in multiple regions of the ipsilateral hemisphere in nearly one-half of the animals, while HSP-70 mRNA was not expressed until 24 h and, then, predominantly in the hippocampus. In 15- and 23-day-old rats, expression of c-fos was increased at 2 h in the entorhinal cortex and in the dendritic field of the upper blade of the hippocampal dentate gyrus, while HSP-70 mRNA was prominently expressed in neocortex and the cell layers of the hippocampus. Interestingly, the strong expression of HSP-70 mRNA in dentate granule cells did not occur in the innermost layer of cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia.

Sulfated glycoprotein-2 (SGP-2) is emerging as a prominent marker of neurodegeneration in mammalian brain. Regulation of brain SGP-2 was studied in adult male Wistar rats subjected to 30 min of forebrain ischemia by four vessel occlusion. By 3 days after the ischemic insult, SGP-2 RNA levels were increased two fold in caudate nucleus and hippocampus. SGP-2 protein levels assessed by immunoblots were markedly increased in both brain regions following ischemia. GFAP RNA levels also increased over 5 fold in caudate nucleus and hippocampus following the ischemic insult. Despite significant elevations in GFAP RNA, protein levels of GFAP assessed by immunoblot were only marginally affected. The elevated expression of SGP-2 in rodent brain following this and other experimental lesion paradigms (e.g., excitotoxic lesions, deafferentation) suggest some general involvement of SGP-2 in neurodegeneration and remodelling following neuronal injury.     

沙土鼠脑缺血后HSP70表达变化研究

目的研究脑缺血后ＨＳＰ７０表达变化。方法采用沙土鼠短暂前脑缺血再灌损伤模型,光镜观察缺血再灌后神经细胞损伤情况,Ｎｏｒｔｈｅｒｎｂｌｏｔ和免疫组化方法分别检测脑缺血后不同时期额叶ＨＳＰ７０ｍＲＮＡ及蛋白表达。结果沙土鼠脑缺血后各期ＨＳＰ７０ｍＲＮＡ表达增加（Ｐ＜０．０５）,而ＨＳＰ７０蛋白仅在再灌后１ｄ有少量表达（Ｐ＜０．０１）。缺血神经细胞在再灌后７ｄ大多出现损伤改变。结论沙土鼠脑缺血后,虽有ＨＳＰ７０转录增加,却存在着ＨＳＰ７０的翻译障碍。ＨＳＰ７０翻译障碍可能是导致神经细胞损伤的重要原因之一。     

Opioid peptide levels in gerbil brain after transient ischemia: lasting depletion of hippocampal dynorphin

Peptides derived from each of the 3 endogenous opioid precursors were measured in gerbil brain regions at various times after transient bilateral carotid artery occlusion using radioimmunoassays specific for beta-endorphin-, met-enkephalin-, and dynorphin A-related peptides. Lasting changes were observed only in the hippocampus. The most striking effect was on dynorphin A immunoreactivity, which was reduced by 30-40% as early as 1 hour after recirculation and remained at 50% of the control level for at least 1 week. In some experiments dynorphin levels showed a transient recovery at 24 hours. These results demonstrate a unique sensitivity of the dynorphin-containing dentate granule cell-mossy fiber pathway to transient ischemia. Although these cells remain histologically intact, the decrease in dynorphin level precedes and continues during the delayed loss of hippocampal CA1 neurons characteristic of this model and further defines the selective vulnerability of hippocampal circuitry following ischemia. These observations clearly identify the hippocampus as a well-defined brain region in which further studies of the postischemic pathophysiology of endogenous opioid peptides may provide a rational basis for evaluating the place of opiate pharmacology in stroke treatment.     

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.     

Induction by synaptic zinc of heat shock protein-70 in hippocampus after kainate seizures

Following seizures, heat shock protein (HSP)-70 is induced in various brain regions. Since zinc that can induce HSP-70 in various cell systems is enriched in certain glutamatergic terminals and translocates to postsynaptic neurons with seizures, we examined the possibility that HSP-70 induction in the epileptic brain is mediated by synaptic zinc. Adult rats were injected intraperitoneally with kainate to induce seizures. Seizures were halted 3 h after the kainate administration by the injection of phenytoin. Staining of brain sections with zinc-specific fluorescent dye TFL at 24 h after the kainate injection revealed a one-to-one correlation between dense TFL fluorescence and acidophilic neuronal degeneration in the hippocampus. Subsequent staining with anti-HSP-70 antibody, however, revealed that more numerous neurons than degenerating neurons exhibited HSP-70 immunoreactivity. Most of the HSP-70(+) neurons were not stained with acid fuchsin but exhibited mild zinc fluorescence in the cytoplasm. Intraventricular injection of CaEDTA attenuated neuronal death as well as the HSP-70 induction in a dose-dependent manner. Supporting the specificity of zinc rather than calcium as the inducer of HSP-70 in neurons, exposure to zinc but not to a calcium ionophore or excitotoxins increased expression of HSP-70 mRNA and protein in cultured cortical neurons. The present results suggest that not only selective neuronal death, but also HSP-70 induction in neurons after seizures, is mediated by the translocation of endogenous synaptic zinc.     

Monitoring of functional changes after transient ischemia in gerbil cochlea

Ischemia and reperfusion are involved in numerous sensorineural pathologies. A model of reversible cochlear ischemia has been designed in Mongolian gerbil. Selective labyrinthine ischemia of variable duration (4–10 min) was achieved through a posterior transcranial approach. Ischemia and reperfusion were controlled with the help of laser Doppler velocimetry. Functional changes were monitored every 1–10 s throughout experiments, using cochlear potentials and otoacoustic emissions. After interruption of blood flow, all signals rapidly began to decay. In contrast to cochlear potentials, otoacoustic emissions always exhibited a plateau before reaching noise floor only after ≈4–5 min. Upon ischemia release, cochlear blood flow recovered instantly and completely and cochlear potentials rapidly improved in most cases, in contrast to otoacoustic emissions that underwent a delayed decay after immediate partial recovery. The phase and group latency of otoacoustic emissions exhibited only small changes throughout ischemia and reperfusion, suggesting adaptive rather than damaging mechanisms. Cochlear function returned to normal after 5 min 30 s ischemia but longer complete ischemia sometimes led to irreversible damage despite the systematic presence of some recovery just after ischemia release. This behavior suggests that reperfusion in itself can be deleterious to a sensorineural organ and this model can be useful for identifying the noxious mechanisms of ischemia and reperfusion. © 1997 Elsevier Science B.V. All rights reserved.     

LY178002 reduces rat brain damage after transient global forebrain ischemia

Several feasible mechanisms have been proposed as sources of neuronal damage from ischemia and subsequent reperfusion. Included among these are oxidative damage caused by free radical production and lipid peroxidation and products derived from phospholipid breakdown. A series of 4-thiazolidinone compounds represented by LY178002 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene-4-thiazolidinon e) have been described as inhibitors of multiple enzymes in the arachidonic acid cascade, including fatty acid cyclooxygenase, 5-lipoxygenase, and phospholipase A2. Accordingly, we evaluated LY178002 in a four-vessel occlusion model of global forebrain ischemia with reperfusion. A 2-hour pretreatment of 11 male Wistar rats with 150 mg/kg LY178002 significantly protected against striatal (p = 0.0007) and hippocampal CA1 (p = 0.006) damage after 30 minutes of global ischemia. Similar protection was observed for the striatum (p = 0.005) and hippocampal CA1 layer (p = 0.025) after pretreatment of 13 rats with 50 mg/kg LY178002. We further evaluated LY178002 as a possible inhibitor of lipid peroxidation because part of its chemical structure incorporates the aromatic backbone of the known antioxidant butylated hydroxytoluene. We found LY178002 to be a potent inhibitor of iron-dependent lipid peroxidation. Few substances possessing a single pharmacological activity have been found to be of significant therapeutic benefit in global ischemia of 30 minutes' duration because the mechanisms that lead to cell death in response to ischemia are likely to be multifactorial. Thus, the efficacy of LY178002 in this model may be due to its ability to inhibit multiple sources of damage.