Sustained induction of heme oxygenase-1 in the traumatized spinal cord

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.     

Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage

The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.     

Relationship of lead-induced proteins to stress response proteins in astroglial cells

Astroglial cells are resistant to cell death and morphologic damage following lead (Pb) exposure at concentrations which elicit detrimental effects in neurons. A possible explanation may be that astroglial cells respond to Pb by increasing the expression of specific proteins, such as heat-shock proteins (HSPs), which confer resistance to low levels of Pb. However, there has been relatively limited information regarding the ability of Pb to evoke the synthesis of HSPs. In the current study, pulse-labeling of cultured astroglial proteins with [³H]-leucine was used to evaluate the nature of Pb-induced changes in protein expression. The effect of Pb on newly synthesized proteins was compared to the response elicited by heat-shock and oxidative injury. Immunoblot analysis was utilized to examine alterations in levels of various stress proteins including HSP27, HSP70, HSP90, and heme oxygenase-1 (HO-1). Even though Pb induced the synthesis of proteins with estimated molecular weights of 23 kDa, 32 kDa, 70 kDa, and 90 kDa, the accumulation of HSPs other than HO-1 was not observed. Hyperthermia and treatment with Na arsenite both resulted in enhanced expression of HSP70 and HO-1. In addition, exposure to hydrogen peroxide (H₂O₂), cadmium (Cd), and lipopolysaccharide (LPS) stimulated a rise in HO-1 levels. Although cellular insult failed to elicit an increase in either HSP27 or HSP90, cultured astroglia expressed readily detectable levels of both these proteins. Furthermore, Pb exposure resulted in the development of crosstolerance to subsequent injury by treatment with either Cd or H₂O₂. The results of this study indicate that Pb triggers a less conventional stress response in astroglial cells, which may provide enhanced resistance to the toxic effects of Pb. © 1995 Wiley-Liss, Inc.     

Induction of cell death by L-alpha-aminoadipic acid exposure in cultured rat astrocytes: relationship to protein synthesis

The excitotoxin, L-alpha-aminoadipic acid (L-AAA), kills primary astrocytes in the brain. The mechanism underlying the induction of cell death is not well understood although many possible mechanisms are theorized. Previous studies have reported that astrocytes die after prolonged exposure to L-AAA suggesting a delayed programmed cell death and apoptosis. In this study rat cortical astrocytes exposed to continuous 1 mM L-AAA exposure for 24-, 48-, or 72 hours demonstrated increased DNA laddering, a characteristic of apoptosis. Unexpectedly, this was not ameliorated by the presence of cycloheximide at 0.1 microg/ml medium. Because of our interest in cytoprotective heat shock proteins induced by excitoxic stress, we studied the effect of prolonged exposure of L-AAA on the synthesis of stress proteins and protein synthesis in rat cortical astrocytes. Protein synthesis as measured by [35S]-methionine labeling showed a marked and significant decrease in incorporation of radiolabel after 24 hours of exposure to L-AAA and prior to induction of significant cell death noted at 48- and 72 hours of L-AAA exposure. The inhibition of protein synthesis was partially reversible at 24 hours if cells were labeled in medium without L-AAA during the radiolabeling period. Heat shock or stress proteins, HSP70 and heme oxygenase-1 (HO-1), were analyzed after a 24 hour exposure to L-AAA and showed no significant induction of HSP70 or HO-1. The findings suggest that the prolonged inhibition of protein synthesis and associated lack of induction of HSP70 and HO-1 synthesis contributed to apoptotic cell death induced by the excitoxin L-AAA.     

Stress protein expression in the Alzheimer-diseased choroid plexus

Abnormal patterns of stress protein expression are found in the cerebral cortex and hippocampus of Alzheimer (AD) subjects. In this study, expression of various stress proteins in the Alzheimer-diseased choroid plexus (CP) was assessed immunohistochemically. We observed decreased HO-1 immunoreactivity in the AD CP, commensurate with our earlier report of suppressed HO-1 protein levels in AD cerebrospinal fluid (Schipper et al., Neurology 54:1297-1304, 2000). Heat shock protein (HSP) 90 was up-regulated in the AD CP relative to controls. There was a trend towards increased expression of HSP60, a mitochondrial stress protein; this is compatible with mitochondrial pathology recently documented in AD CP. Up-regulation of HSP90, a steroid receptor chaperone, in the AD CP may indicate abnormal hormone receptor expression in this secretory tissue. Glucose-regulated protein (GRP) 78 and 94 immunostaining was diminished in AD CP, implicating possible derangements in glucose or calcium homeostasis. Oxidative stress, per se, is probably not responsible for our observations because: i) there were no noticeable differences in the expression of HSP 70, ubiquitin, and alpha-B crystallin in the AD CP; and ii) augmentation, rather than the noted suppression, of HO-1 immunoreactivity would have been expected.     

Differential upregulation of heme oxygenase-1 (HSP32) in glial cells after oxidative stress and in demyelinating disorders

Oxidative stress is implicated in the pathogenesis of demyelinating disorders and inflammatory responses. Heme oxygenase-1 (HO-1; HSP32) is a small heat shock protein (HSP) with enzymatic activity, which is inducible by oxidative stress. In this study we analyzed autopsy and biopsy brain samples of patients with multiple sclerosis (MS) and ADEM (acute disseminated leucoencephalomyelits) and spinal cord lesions of mouse EAE (experimental autoimmune encephalomyelitis), which was actively induced by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55) peptide, for the presence of HO-1. HO-1 was observed in glial cells during different stages: (1) during acute phases of mainly inflammatory diseases (EAE and ADEM) expression of HO-1 was prominent in microglia/macrophages and astrocytes, and upregulation correlated with inflammation, and (2) in early MS lesions HO-1 was expressed in oligodendrocytes. Furthermore, in glial cell cultures, we can show that upregulation of HO-1 in oligodendrocytes was paralleled by severe morphological damage. Oligodendrocytes underwent apoptotic cell death at a concentration of hydrogen peroxide (50–200 μM) which did not affect astrocytes or microglia. Using oligodendroglial OLN-93 cells, we demonstrate that oxidative stress led to mitochondrial impairment and the disorganization of the microtubule network. Zinc protoporphyrin, an inhibitor of HO-1, augmented the cytotoxic consequences of hydrogen peroxide in OLN-93 cells. Hence, the presence of HO-1 in EAE, ADEM, and MS points to the involvement of oxidative stress and a role of HO-1 in the pathogenesis of the diseases. The data suggest that stress-induced HO-1 initially plays a protective role, while its chronic upregulation, might contribute to oligodendroglial cell death rather than providing protection.     

Involvement of the endothelin receptor subtype A in neuronal pathogenesis after traumatic brain injury

Endothelin-1 (ET-1) is a 21 amino acid peptide that has been closely linked to cerebral vasospasm and more recently to oxidative stress after traumatic brain injury. In this study, we have examined the effects of the endothelin receptor subtype A antagonist, Ro 61-1790, on acute cortical neuronal injury and delayed neuronal death in the cerebellum after mild traumatic brain injury. Rats were administered Ro 61-1790 or vehicle for 24 h after injury and euthanized at 1 day, 3 days, or 7 days. Heat shock protein70 (HSP70), a marker of neuronal stress/injury, was immunolocalized in the cortex. Induction of heme oxygenase-1 (HO-1) and enhanced immunoexpression of the complement C3bi receptor, both of which are indicators of cerebellar glial reactivity, and Purkinje cell loss were evaluated in the cerebellum. There was maximal induction of HSP70 in cortical neurons at 24 h postinjury in all animals. Drug treated animals showed significantly fewer HSP70 labeled cortical neurons at this time point. There were fewer reactive glia in the cerebellum of drug treated animals as compared to vehicle controls at 3 days postinjury. However, at 7 days postinjury glial reactivity and Purkinje cell loss were similar in both groups. These findings demonstrate that Ro 61-1790, when administered for the first 24 h postinjury, limits acute neuronal injury in the cortex, transiently influences glial reactivity in the cerebellum, and does not attenuate delayed Purkinje cell death. The latter finding may reflect the duration of infusion of the drug.     

Expression of Heat Shock Protein-70 and Limbic Seizure-induced Neuronal Death in the Rat Brain

The effect of MK-801, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, on the kainic acid-induced expression of the inducible heat shock protein 70 kDa (HSP70) and on neuronal death in the rat hippocampus was investigated. HSP70 is expressed in ?80% of the pyramidal neurons in the CA1 field 1 day after kainic acid injection. The majority of these HSP70-immunopositive neurons exhibited swelling and a hollow appearance in the perikaryon, indicating that they had been injured following kainic acid-elicited limbic seizures. Four days after administration of kainic acid, 87% of the pyramidal neurons in the CA1 field were dead. When a single dose of MK-801 was administered 1 h before kainic acid injection, the number of rats suffering with seizures was reduced, the severity of limbic seizures was attenuated and seizure onset was delayed. Neither HSP70 expression on day 1 nor neuronal loss on day 4 in the CA1 pyramidal cell layer was observed in these animals. A considerable number of HSP70-immunopositive neurons was detected in the dentate hilus, however, and somewhat fewer in the CA3a and CA3c subfields on day 1. Severe neuronal damage in these regions followed on day 4. Interestingly, little HSP70 expression or neuronal loss was observed in the CA3b subfield in these same animals. When a single dose of MK-801 was given 4 h after kainic acid treatment, HSP70 expression was partially blocked; 18% of neurons expressed HSP70 on day 1 and 37% on day 4 in CA1 pyramidal neurons in comparison to the kainic acid controls. About 50% neuronal death was detected in the CA1 pyramidal cell layer 4 days after kainic acid treatment followed by MK-801. When the animals were treated with MK-801 4 h after kainic acid treatment followed by additional daily administration for 3 days, a negligible number of pyramidal neurons expressed HSP70, and the survival of pyramidal cells was significantly increased in the CA1 field. Limbic seizure-induced HSP70 expression not only indicates neuronal injury in the pyramidal cell layer of the hippocampus but also predicts delayed neuronal death, at least in the case of the CA1 field of animals that suffered stage IV—V seizures.     

Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood

The initial aim of this study was to determine if the HSP70 (the main inducible heat shock protein), HO-1 (heme oxygenase-1, HSP32) and HSP47 (a collagen chaperone) stress proteins were induced in the same focal regions of rat brain following experimental subarachnoid hemorrhage (SAH). The next objective was to determine whether anti-oxidants prevented the stress gene expression in the focal regions. Lysed blood (150 microliter) was injected into the subarachnoid space of adult, female Sprague-Dawley rats via the cisterna magna. Animals were sacrificed 24 h later. Immunocytochemistry showed focal regions of stress gene induction in most animals (13/21), HSP70 and HO-1 proteins being expressed in neurons, microglia and astrocytes and HSP47 being expressed in microglia. Co-induction of the same three stress proteins was observed in focal areas in the striatum and cerebellum as well. In the 13 animals with focal regions of stress gene induction there were 8.1+/-1.8 foci in cortex, 5.5+/-0.9 foci in striatum, and 11.7+/-7.3 foci in cerebellum in the brain of each animal. The focal regions of stress gene induction varied in size from 200 micrometer to 7 mm in diameter. Systemic administration of the tirilazad-like anti-oxidants U101033E (n=8) and U74389G (n=7) completely blocked stress protein induction in focal brain regions normally produced by cisternal injections of lysed blood. There were fewer drug treated animals (0/15) with focal areas of stress gene induction compared to non-drug (13/21) treated animals following the cisternal lysed blood injections (p<0.01 using Fisher's probability test). This study shows that anti-oxidants prevent focal regions of injury as assessed by heat shock protein expression in a rat model of SAH.     

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)     

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.     

Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and αB crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in αB crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not αB crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.     

Neuroprotective preconditioning of rat brain cultures with ethanol: potential transduction by PKC isoforms and focal adhesion kinase upstream of increases in effector heat shock proteins

Preconditioning rat hippocampal-entorhinocortical (HEC) slice or cerebellar cell cultures with moderate concentrations of ethanol (20-30 mm) neuroprotects against pro-inflammatory proteins such as HIV-1 glycoprotein 120 (gp120) or amyloid-β. The neuroprotective mechanism of ethanol is unclear, but it conceivably involves sensors→transducers→effectors, analogous to other preconditioning modalities. We initially found that the preconditioning augmented two likely heat shock protein (HSP) 'effectors', HSP70 and HSP27, and that precluding HSP upregulation abolished neuroprotection. Here we examined whether pro-survival kinases are transducers potentially leading to HSP effectors. In cerebellar cultures, protein kinase C (PKC) activity increased modestly after 2 days of 30 mm ethanol and was significantly induced after 6 days, when neuroprotection against gp120 becomes manifest. After 4 and particularly after 6 days of preconditioning, immunoblots showed highly elevated PKCε levels and moderately increased PKCα and PKCδ, accompanied by increased membrane translocation (activation) of these isoforms. Also, at the latter preconditioning duration, focal adhesion kinase (FAK), an important actin-associated kinase, and its Y397-phosphorylated form (p-FAK) were elevated, along with parallel increases in HSP27, S85p-HSP27 and HSP70. Furthermore, while confirming increased HSP27 and HSP70 in HEC slices ethanol-preconditioned for 6 days, we detected elevations in PKC isoforms, FAK, p-FAK and p-HSP27 in these organotypic cultures. Importantly, PKC inhibition with GF109203X suppressed FAK, HSP70 and HSP27 amplification/activation in ethanol-preconditioned cerebellar cultures, indicating that PKC is an upstream transducer of FAK and the HSP effectors. Neuroprotection associated with increases in HSP27/HSP70 from ethanol preconditioning entails upregulation/activation of PKC isoforms and FAK, the latter kinase implicating actin cytoskeletal prosurvival pathways in brain preconditioning.     

Correlation between HSP70 and c-F0S expression and apoptosis in rats undergolng transient focal cerebral ischemla and reperfusion

OBJECTIVE To compare the induction of c-Fos and HSP70 and the presence of apoptosis and the influence of Ginkgo biloba extract (EGb761) in transient focal cerebral ischemic reperfusion rats.BACKGROUND Proto ancogene activation and induction of heat shock protein (HSP) occur in response to cerebral ischemia, but the correlation between these proteins and apoptosis remains uncertain. METHODS Healthy wistar rats were randomized to the normal control group(Group A, n=4), the Sham-operated control group(Group B, n=4), the ischemia and reperfusion group(Group C, n=24), the EGb761 pre-treated ischemia and reperfusion group(Group D, n=24). The rats of Group C and Group D were subjected to transient left middle cerebral artery occlusion (MCAO) as described by Zea longa for 1 hour. RESULTS Immunohistochemical analysis revealed no c-Fos or HSP70-immunoreactivity in Group A and Group B rats. However, in Group C rats, c-Fos was expressed in ipsilateral superficial cortical layers at 1 hour after reperfusion. At 6 hours, c-Fos immunoreactivities were increased in the ipsilateral cortex and were present in the contralateral cortex, while HSP70 were induced beginning in the ipsilateral neurons of MCA distribution. At 12 hours, the expression of c-Fos reached top in superficial cortical layers. At 24 hours HSP70 immunoreactivities reached top both in ipsilateral cortex and in ipsilateral striatum. At 3 days after recirculation, HSP70 expression decreased. c-Fos expression disappeared at day 7 and HSP70 expression only occured in endothelial cells. TUNEL staining showed that there was no cell apoptosis in Gronp A or in Group B. However, in Group C, TUNEL-positive neurons were observed in the border of the penumbra-like area that surrounds the ischemic core at 6 hours following reperfusion and then the number of TUNEL-positive cells reduced gradually. The changes in expression of HSP70 and c-Fos at different time points in Group D was in accord with in Group C, but the number of positive cells and the immunoreactivities in Group D were more intense than in Group C. We found only several TUNEL-positive cells at 6 hours following reperfusion in Group D and no TUNE;L-positive cells were present at other time points.CONCLUSION The present results indicate that c-Fos was expressed from an earlier stage of reperfusion and the expression occured in bilateral cerebral cortex. In contrast, HSP70 induction began later and only occured in ipsilateral neurons of MCA distribution. Our results indicated that EGb761 could increase the expression of c Fos and HSP70 and reduce the apoptosis of neurons.     

The effect of ECr on HSP70 expression and morphologic changes following transient cerebral lschemia in rats

OBJECTIVE To investigate the effect of Cudrania tricuspidata root extract (ECr) on ischemic cerebral damage and the expression of the 70KDa heat shock protein (HSP70) following transient focal ischemia. BACKGROUND The role that ECr plays in cerebral ischemia has not been studied. METHODS Healthy wistar rats were randomized to the normal control group(Group A,n=4),the sham-operated control group(Group B,n=4),the ischemia and reperfusion group (Group C,n=24),the ischemia and reperfiusion after ECr pre- administration group(Group D,n=24).The rats of Group C and Group D were subjected to transient left middle cerebral artery occlusion (MCAO)as described by Zea Longa for 1 hour. Brain sections at the level of striatum were performed for HSP70 immunohistochemistry and morphology.HSP70 positive reactions and morphologic changes were semiquantiratively analyzedl. RESLULTS In the rats of Group A and Group B, there were scarcely HSP70 immunoreactivities either in cortex or in striatal neurons. In the ischemic brain regions for Group C rats,the HSP70 was induced in morphologically intact neurons and endothelial cells at hour 6 after recirculation, increased at hour12,peaked at day I, decreased at day 3,and HSP70 expression only occured in endothelial cells at day7. In Group D rats, the HSP70 was induced in the neurons of left MCA distribution at hour 1 after reperfusion, The changes in expression of HSP70 at different time points in Group D rats was in accord whth in Group C, but the number of HSP70 positive cells in Group D increased more, and HSP70 irnmunoreactivity in the HSP70-postive cells were more intense than in Group C. Histopathological study with HE staining showed no neuron pyknosis in Group A or in Group B. While pykrotic cells were present in the ipsilateral cortex and striatal neurons of MCA territory of Group C rats beginning at 6 hours after roper fusion. The change of histopathology in Group D was lighter at every time point. The number of pyknotic neurons in left MCA distribution was less than in Group C and there was no evident cell damage at 3 days and 7 days of reperfusion in Group D rats. CONCLUSION Our study demonstrated that transient focal cerebral ischenia could induce the HSP70 expression and induce neurons pyknosis.While ECr pre-treatment before transient focal ischemia in rats could increase the expression of HSP70 and reduce neuronal injury. These data suggests that might be able to enhance neuronal ischemic tolerance of the rats and might have prophylactic neuroprotective effect on ischemic cerebral damage in rats.     

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。     

热休克因子1及多种热休克蛋白在人良性脑膜瘤中的表达

目的探讨人良性脑膜瘤细胞中热休克因子1（HSF1）及热休克蛋白（HSP）HSP27、HSP70和HSP90的表达。方法应用免疫组化法和western blot法检测15例人脑膜瘤原代培养细胞中HSF1、HSP27、HSP70和HSP90蛋白的表达。结果15例人脑膜瘤中，HSF1蛋白以单体形式存在（分子量为80kD左右），阳性率为100％（15／15），HSP27、HSP70和HSP90阳性率分别为40％（6／15）、46．7％（7／15）和53．3％（8／15）。结论人良性脑膜瘤存在HSF1蛋白表达，而HSP27、HSP70和HSP90组成型表达水平低。     

Heat shock protein 27 shows a distinctive widespread spatial and temporal pattern of induction in CNS glial and neuronal cells compared to heat shock protein 70 and caspase 3 following kainate administration

Kainate-induced status epilepticus is associated with both apoptotic and necrotic cell death and induction of heat shock proteins (HSPs) in hippocampal and cortical regions of the rodent brain. In the present study we have examined the temporal, spatial and cellular expression patterns of mRNAs for the highly inducible HSPs, HSP70 and HSP27, together with the apoptotic marker, caspase 3 (CPP32) in rat brain after systemic administration of kainate. HSP70 mRNA was transiently induced in the forebrain by kainate, principally in the CA1, CA3 and hilar cells of the hippocampal formation, in piriform cortex and discrete thalamic nuclei. Maximal expression was seen at 8 h after kainate which then declined to background levels by 7 days. Labelling was predominantly neuronal. In contrast, HSP27 mRNA expression was more widespread. Intense labelling was observed in CA1, CA3 and the hilar region at 8 h after kainate but the expression profile for HSP27 mRNA expanded considerably with intense signals seen in corpus callosum, cortex and thalamus at 24 h post kainate. Emulsion autoradiographs indicated a predominantly glial localisation for HSP27 mRNA. In the hilus, a distinct subpopulation of interneurones were found to express HSP27 mRNA. CPP32 mRNA was upregulated in CA1, CA3 and hilus of the hippocampal formation and in piriform cortex. CPP32 mRNA expression was more restricted and similar in distribution to HSP70 mRNA being localised to neurones. The present study demonstrates the unique early expression of HSP27 mRNA by glial cells and distinct populations of neurones which extends beyond those in which HSP70 and CPP32 induction occurs with subsequent cell loss.