实验性脑缺血再灌流后不同时程热休克蛋白基因表达的变化

为探讨脑缺血再灌流后热休克蛋白(HSP70)基因表达的变化,采用原位杂交和免疫组化方法检测了脑缺血2h再灌流后不同时程应激蛋白—热休克蛋白(hsp70)mRNA和蛋白表达的变化。结果显示再灌流后早期即可见hsp70mRNA的蛋白表达增加,以18～24h阳性染色最强,HSP70阳性细胞主要分布于缺血周围半暗带区,提示HSP70蛋白表达增加可抵御缺血性脑损伤,对神经元具有保护作用。     

脑缺血再灌注后热休克蛋白70、c-fos的表达及柘树制剂的神经保护作用

目的 观察局灶脑缺血再灌注后热休克蛋白（HSP）70、c-fos的表达及其与细胞调亡的关系，探讨柘树制剂对脑缺血后神经细胞损伤的保护作用。方法 采用改良Longa法制作大鼠局灶脑缺血冉灌注模型。柘树制剂预处理组（柘树组）大鼠在实验前灌服柘树制剂2ml每日3次，连用5d。在缺血再灌注不同时点（1h、6h、12h、24h、3d、7d）将大鼠处死取腑，进行HSP70及c-fos免疫组化染色、c-fos mRNA原位杂交、原位末端标记（TUNEL）及HE染色，许对其阳性结果进行半定量分析。结果脑缺血再灌注能诱导HSP70及c-fos的表达。缺血冉灌注6h绀HSP70存缺血侧皮质及基底节开始表达，24h达高峰。缺血再灌注1h组c-fos即有表达，6h达高峰，后逐渐下降。细胞凋亡于缺血再灌注6h最显著。柘树组HSP70及c-fos表达的阳性细胞数均较缺血再灌注组明监增加，两组比较差异均有显著性（均P〈0．01），而TUNEL阳性细胞数明显减少。结论 HSP70及c=-ros均参与了脑缺血的病理生理过程，柘树制剂对脑缺吡冉灌注损伤有保护作用。     

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.     

戊四氮致痫大鼠脑内热休克蛋白70的表达

目的：探讨热休克蛋白70（HSP70）在癫痫大鼠脑内的表达情况及其意义。方法：采用戊四氮致痫模型。应用免疫组织化学及常规清理检查的方法进行研究。结果：在正常大鼠脑内未见HSP70免疫反应（IR）阳性细胞,成四氮致痫12小时后脑内开始出现HSP70IR阳性细胞,24小对IR达高峰．3天后开始下降．7天后消失。HSP70主要在边缘系统（尤其是海马的CA1、CA3、CA4区）、大脑皮质（尤其是颞叶皮质,梨状皮质）等区域表达。同时常规病理检查发现,上述区域散在出现受损的异常神经元。结论：癫痫发作可诱导HSP70在大鼠脑内广泛表达。HSP70表达可作为神经元受损的一个早期指标。     

伽玛刀照射正常大鼠海马组织的放射生物学研究

目的研究伽玛刀照射对正常大鼠海马组织的放射生物学作用,探讨伽玛刀作用于正常脑组织的机制,为立体定向放射外科的开展提供理论依据。方法利用自行设计的伽玛刀动物定向头架,应用不同剂量的伽玛射线对大鼠海马组织进行照射,原位杂交观察c-fosmRNA和HSP70mRNA表达,免疫组化观察FOS、HSP70、GFAP和bcl-2蛋白表达。结果c-fosmRNA在照射后3h、7d出现2个高峰,HSP70mRNA在照射后1~3h即有表达。FOS、HSP70、GFAP和bcl-2蛋白表达随照射时间和部位的不同而各有自己的特点。结论自行设计的伽玛刀动物定向头架定位准确、可靠。c-fos和HSP70表达说明伽玛刀照射虽局限于海马,但引起的反应却是强烈的。bcl-2具有抑制电离辐射所致细胞凋亡作用,GFAP阳性细胞反应可作为脑组织损伤的一种标志。     

Expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis

Respiring neonatal rat cerebrocortical slices were exposed for 30 min to toxic concentrations of N-methyl-d-aspartate (NMDA; 100 μM, 500 μM and 1000 μM). In situ hybridization was used to study c-fos and hsp70 mRNA before, during, and for 8 h after NMDA exposure. Cell swelling and nuclear morphology were assessed using Cresyl violet (Nissl) staining. Possible evidence for apoptosis was examined using in situ terminal transferase d-UTP nick-end labeling (TUNEL) staining and agarose–gel electrophoresis of extracted slice DNA. After NMDA administration c-fos and hsp70 mRNA expression increased, with maxima occurring, respectively, at 1 h and 4 h after NMDA exposure. When treatment with dizocilpine (MK-801; 10 μM), a non-competitive NMDA antagonist, was started before NMDA exposures, expression of both c-fos and hsp70 mRNA was decreased to values near control, indicating that activation of NMDA receptors induces both genes. Only a minority of induced cells expressed FOS protein and no HSP70 protein expression was seen. These apparent failures of translation might be related to the stress response. Histologically, 1000 μM NMDA produced substantial necrosis, with no evidence of apoptosis. Evidence for apoptosis was found at the two lower NMDA concentrations, which produced TUNEL-positive fragmented nuclei and faint ladder patterns in DNA electrophoresis. Dizocilpine pre-treatment blocked NMDA-induced necrosis and attenuated TUNEL-positive staining in slice parenchyma. TUNEL-positive staining with a different morphology was found in the injury layer, a region 50-μm thick where mechanical trauma was inflicted when slices were cut from brain. When slices received dizocilpine immediately after decapitation, TUNEL-positive staining no longer occurred in the injury layer, in agreement with previous cell culture studies that implicated NMDA receptor activation after mechanical trauma to neurons. We conclude that at the toxic doses studied, NMDA receptor activation results primarily in necrosis. However, data at low NMDA concentrations are consistent with a small amount of apoptosis.     

Genes associated with pro-apoptotic and protective mechanisms are affected differently on exposure of neuronal cell cultures to arsenite. No indication for endoplasmic reticulum stress despite activation of grp78 and gadd153 expression

The effect of arsenite exposure on cell viability, protein synthesis, energy metabolism and the expression of genes coding for cytoplasmic (hsp70) and endoplasmic reticulum (ER; gadd153, grp78, grp94) stress proteins was investigated in primary neuronal cell cultures. Furthermore, signs of ER stress were evaluated by investigating xbp1 mRNA processing. Arsenite levels of 30 and 100 microM induced severe cell injury. Protein synthesis was reduced to below 20% of control in cultures exposed to 30 and 100 microM arsenite for 1 h, and it remained markedly suppressed until 24 h of exposure. Arsenite induced a transient inhibition of energy metabolism after 1 h of exposure, but energy state recovered completely after 3 h. Arsenite exposure affected the expression and translation of genes coding for HSP70 and GRP78, GRP94, GADD153 to different extents. While hsp70 mRNA levels rose drastically, approximally 550-fold after 6 h exposure, HSP70 protein levels did not change over the first 6 h. On the other hand, gadd153 mRNA levels rose only approximately 14-fold after 6 h exposure, while GADD153 protein levels were markedly increased after 3 and 6 h exposure. HSP70 protein levels were markedly increased and GADD153 protein levels decreased to almost control levels in cultures left in arsenite solution for 24 h, i.e. when only a small fraction of cells had escaped arsenite toxicity. Arsenite exposure of neurons thus induced an imbalance between pro-apoptotic and survival-activating pathways. Despite the marked increase in gadd153 mRNA levels, we did not observe signs of xbp1 processing in arsenite exposed cultures, indicating that arsenite did not produce ER stress.     

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.     

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.     

Heat-shock pretreatment prevents suppression of long-term potentiation induced by scopolamine in rat hippocampal CA1 synapses

We examined the effect of heat-shock pretreatment on long-term potentiation (LTP) in the CA1 hippocampal slices of the rat using the muscarinic blocker scopolamine as the LTP (memory) suppressor. Time course study using immunohistochemical techniques indicated peak expression of HSP70 16 h after heat-shock treatment. Focusing on that time point we found tetanic stimulation (at 100 Hz) induced LTP of 191.1+/-12.2% in control slices (n=7), which was suppressed by scopolamine to 114.5+/-2.8 %. Heat-shock pretreatment successfully prevented such suppression (216.6+/-38.2% and 190.2+/-10.6% with and without scopolamine, respectively, n=7). Both HSP expression and LTP responses were relatively small taken either 2 or 48 h after heat-shock or sham pretreatment. These results suggest that the induction of HSPs is time-dependent and can prevent scopolamine-mediated LTP suppression.