Differential induction of mRNA species encoding several classes of stress proteins following focal cerebral ischemia in rats

We report here the time-dependent expression of several classes of HSP mRNAs following focal cerebral ischemia in rats. HSP70, GRP78, HSP27, HSP47 and HSP47 have been reported to possess distinct functions under normal and/or stress conditions. These different classes of HSP mRNAs were differentially induced by ischemia, as determined by Northern blot analysis. Messenger RNAs of the HSP70 family proteins were induced within 4h after ischemia d then rapidly decreased, whereas HSP27 and HSP47 mRNAs reached a maximum level of expression at 24 h and 48 h after ischemic treatment, respectively. In situ hybridization showed that the expression of inducible HSP70 mRNA was observed predominantly in regions adjacent to the ischemic core except during the early periods of ischemia. HSP27 mRNA was expressed over a broad area of the ipsilateral cerebral neocortex except for the ischemic center 24 h after ischemia. The unique induction kinetics for each HSP mRNA species may reflect their distinct roles in the brain during various physiological stresses. We will also discuss that stress proteins may be involved in the central nervous system after ischemia in two important aspects: early protection against stress and restoration of damaged lesions in the brain at later stages after ischemia.     

大鼠局灶性脑缺血再灌流后HSP70表达及其mRNA变化

目的 探讨热休克蛋白７０（ＨＳＰ７０）在局灶脑缺血再灌流后的变化和作用。方法 采用线栓法制备大脑中动脉缺国知再灌流模型。应用免疫组化方法观察ＨＳＰ７０的组织学分布,利用ＲＴ－ＰＣＲ方法检测缺血皮层与基底节区脑组织ＨＳＰ７０ｍＲＮＡ相对含量。结果 基底节区ＨＳＰ７０阳性细胞较少,持续时间短,眼层区ＨＳＰ７０阳性细胞较多,持续时间长。ＲＴ－ＰＣＲ结果表明ＨＳＰ７０ｍＲＮＡ相对含量与免疫组化结果相一致。     

Overexpression of heat shock protein 27 reduces cortical damage after cerebral ischemia

Heat shock protein 27 (HSP27) has a major role in mediating survival responses to a range of central nervous system insults, functioning as a protein chaperone, an antioxidant, and through inhibition of cell death pathways. We have used transgenic mice overexpressing HSP27 (HSP27tg) to examine the role of HSP27 in cerebral ischemia, using model of permanent middle cerebral artery occlusion (MCAO). Infarct size was evaluated using multislice T₂-weighted anatomical magnetic resonance imaging (MRI) after 24 h. A significant reduction of 30% in infarct size was detected in HSP27tg animals compared with wild-type (WT) littermates. To gain some insight into the mechanisms contributing to cell death and its attenuation by HSP27, we monitored the effect of induction of c-jun and ATF3 on tissue survival in MCAO and their effects on the expression of endogenous mouse HSP25 and HSP70. It is important that, the c-jun induction seen at 4 h tended to be localized to regions that were salvageable in HSP27tg mice but became infarcted in WT animals. Our results provide support for the powerful neuroprotective effects of HSP27 in cerebral ischemia.     

Amyloid precursor protein accumulates in regions of neurodegeneration following focal cerebral ischemia in the rat

The distribution of beta-amyloid precursor protein (APP) was examined immunocytochemically in rats subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery. At 4 and 7 days post-occlusion, APP immunoreactivity was preferentially localized within axonal swellings, dystrophic neurites and neuronal perikarya all along the periphery of the infarct. Immunolabeling was observed with antibodies generated against N-terminal, midregion, and C-terminal domains of APP. No immunoreactivity was observed with antisera directed against beta-amyloid protein (beta A4) itself. This pathological accumulation of APP is consistent with alterations of APP recently described in other models of neurodegeneration and implies a role for this protein in the response to CNS injury.     

Temporal and spatial distribution of heat shock mRNA and protein (hsp70) in the rabbit cerebellum in response to hyperthermia

We have previously investigated the expression of hsp70 genes in the hyperthermic rabbit brain at the mRNA level by Northern blot and in situ hybridization procedures. Our studies have now been extended to the protein level utilizing Western blot and immunocytochemistry. Using an antibody which is specific to inducible hsp70, a prominent induction of hsp70 protein in glial cells of hyperthermic animals was noted. In particular, Bergmann glial cells in the cerebellum are strongly immunoreactive while adjacent Purkinje neurons are immunonegative. Extension of our in situ hybridization studies to a time course analysis revealed that the initial glial induction events were followed by a delayed accumulation of inducible hsp70 mRNA in Purkinje neurons at 10 hr post-heat shock. In control animals, high levels of constitutively expressed hsc70 mRNA and protein were observed in Purkinje neurons. Similar hsc70 and hsp70 mRNA observations were also made in neurons of the deep cerebellar nuclei and in motor neurons of the spinal cord. Our results suggest that these neuronal cell types accmulate hsp70 mRNA in response to hyperthermic treatment; however, the response is delayed when compared to the rapid response seen in glial cells. The high constitutive levels of hsc70 in certain neuronal cell types may play a role in the initial dampening of the hsp70 induction response in these cells. © 1993 Wiley-Liss, Inc.     

Expression of c-fos and inducible hsp-70 mRNA following a transient episode of focal ischemia that had non-lethal effects on the rat brain

Expression of c-fos and inducible hsp-70 mRNA was studied with in situ hybridization techniques at different times following an episode of middle cerebral artery (MCA) occlusion not resulting in any apparent lethal effect on the rat brain. hsp-70 and c-fos mRNA were found in the ipsilateral striatum and adjacent cortex. In the striatum, levels of hsp-70 mRNA increased from 1 to 2 and 4 h of reperfusion, whereas levels of c-fos mRNA decreased from 1 to 4 h of reperfusion. These results demonstrate that following non-lethal focal ischemia the brain areas within the MCA territory show high c-fos and hsp-70 mRNA expression response, illustrating the concomitant induction of these mRNAs in cells that survive the ischemic insult.     

Induction of heat shock (stress) protein 70 and its mRNA in the normal and light-damaged rat retina after whole body hyperthermia

In situ hybridization and immunocytochemistry were used to investigate the distribution of the 70 kDa heat shock or stress protein (hsp70) and its mRNA in specific layers of the retina of adult rats at 0, 4, 18, and 48 or 50 hr after a brief whole body hyperthermic treatment. Induction of hsp70 mRNA was noted in the photoreceptor layer of the retina within 4 hr after hyperthermia. Pronounced accumulation of inducible hsp70 immunoreactivity was observed in cytoplasmic extensions of the photoreceptor cells, especially the inner segment zone which attained peak levels at the 18 hr time point. Selective destruction of photoreceptors by light damage prior to hyperthermia inhibited the post-hyperthermic rise in newly synthesized retinal hsp70. Our results suggest that the photoreceptor cell layer is the primary site of synthesis of hsp70 in the rat retina and that the greatest increase in hsp70 immunoreactivity following such a hyperthermic stress occurs in that layer. This stress response of the photoreceptors is discussed in relation to their location and function in the retina. © 1994 Wiley-Liss, Inc.     

Unchanged balance between levels of mRNA encoding AMPA glutamate receptor subtypes following global cerebral ischemia in the rat.

Transient global ischemia leads to glutamate mediated delayed neuronal death in the CAI but not in the CA3 region of the rat hippocampus, and changes in AMPA receptor subunit composition has been proposed to cause a difference in excitatory input to the CAI and CA3 regions. In situ hybridization with riboprobes for AMPA receptor subtype GluR1–4 mRNA was performed on sections from the brain of sham operated and ischemic rats in two models (neck cuff and 4-vessel occlusion combined with hypotension) with identical results: the content of the GluR1–3 mRNA species was down regulated in the hippocampal regions CAI and CA3 but only weak changes were observed in the dentate gyrus. The down regulation observed in CA1 was non-selective among GluR1–3, i.e. all GluR mRNA species showed approximately the same degree of down regulation. A change in calcium permeability of the AMPA channels mediated by a shift in channel sub-unit composition and corroborating an increased calcium influx is thus not supported by these findings.     

Temporal profile of nestin expression after focal cerebral ischemia in adult rat

Nestin is an intermediate filament protein, transiently and abundantly expressed early in embryogenesis, e.g., in neuroepithelial cells, radial glia, germinal matrix cells and vascular cells. In the adult rat brain, nestin is only present in endothelial and select subventricular cells. We tested the hypothesis that after an experimental stroke, nestin expression is induced in glial cells and neurons. We measured the temporal profile of nestin expression after induction of focal cerebral ischemia in adult rats. Brain from rats (n=24) subjected to 2 h of transient middle cerebral artery occlusion (MCAo) and 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 7 days and 28 days (n=3, per time point) of reperfusion, and control sham operated (n=3) rats were processed for Western blotting to quantify nestin. Another set of brains from rats (n=28), subjected to 2 h of MCAo and 6 h, 12 h, 2 days, 7 days, 14 days, 21 days, and 28 days (n=4, per time point, except n=8 at 2 days) of reperfusion, and control sham operated (n=3) and normal (n=2) rats were processed by single and double labeled immunohistochemistry for cellular identification of nestin expression. By Western blotting, nestin within ischemic tissue increased slightly as early as 6 h, peaked at 7 days, and expression persisted for at least 4 weeks after 2 h of MCAo. By immunohistochemistry, nestin was expressed in astrocytes in the ischemic core from 6 to 12 h after MCAo. Nestin immunoreactivity was present in large numbers of astrocytes, and in scattered oligodendroglia and monocytes/macrophages in both the inner and outer boundary zones to the ischemic core at 1–7 days after MCAo. Nestin expression in glial cells declined at longer durations of survival, although for least 4 weeks after MCAo the nestin immunoreactivity delineated the boundary zone adjacent to the ischemic core. Nestin expression was present in some neurons localized to the outer boundary zone of the ischemic lesion in the cortex and striatum, and in most ependymal cells in the ventricular and subventricular zone (VZ/SVZ) from day 2 after MCAo and onward. The expression of nestin increased throughout the microvasculature in both the ischemic core and the boundary zone in all ischemic rats after 12 h of reperfusion. After stroke, nestin immunoreactivity in glial, neuronal and ependymal cells is suggestive of a protein expression pattern found in developing brain.     

巴曲酶的神经保护作用—缺血再灌注后不同时程热休克蛋白70及病理组织学变化的相关性研究

本文研究目的为巴曲酶是否影响热休克蛋白起到神经保护作用。用中大脑动脉(MCA)线栓法缺血再灌注大鼠模型。Wistar大鼠共51只。发现:在再灌注1h、2h、3h对照组与巴曲酶组(8BU/kg ip)HSP70均呈轻度表达,从再灌注12h起表达显著,至再灌注后24h达高峰,至再灌注后6d仅见于坏死灶周围,至再灌注后14d恢复至假手术组水平。巴曲酶组的HSP70表达变化在时程上与对照组一致,但在再灌注12h起至6d较对照组显著,同时相应时间点的MCA血供区皮层神经细胞有缺血变性者巴曲酶组少而轻。本文结果提示巴曲酶的神经保护作用,可能与它能影响HSP70蛋白合成的调控机制有关。     

Monocyte chemoattractant protein-1 expressed in neurons and astrocytes during focal ischemia in mice

Focal cerebral ischemia elicits an inflammatory response characterized by the infiltration and accumulation of leukocytes, as well as the secretion of inflammatory mediators (Clark et al., Brain Res. Bull., 35 (1994) 387-392; Garcia et al., Am. J. Pathol., 144 (1994) 188-199; Wang et al., J. Neurochem. 71 (1998) 1194-1204). Leukocytes eliminate microbial invaders and necrotizing tissue debris, and can also turn against surrounding healthy tissue and exacerbate tissue injury (Furie and Randolph, Am. J. Pathol., 146 (1995) 1287-1301; Kochanek and Hallenbeck, Stroke 23 (1992) 1367-1379). Inflammatory mediators are considered to play an important role in attracting and stimulating leukocytes (Weiss, N. Engl. J. Med., 320 (1989) 365-376). Monocyte chemoattractant protein-1 (MCP-1) functions as an inflammatory mediator, whose source and role in focal cerebral ischemia is worth studying. MCP-1, a potent chemoattractant factor, may play an important role in ischemia-induced inflammatory response. The aim of the present study is to determine the time course and cell type of MCP-1 protein expression after permanent focal ischemia in mice. ELISA and immunohistochemical staining were used to detect the expression of MCP-1 protein after 0 h, 2 h, 4 h, 12 h, 1 day, 2 days, 3 days, 5 days and 7 days of middle cerebral artery occlusion (n=3-5 in each group). Double-labeled fluorescent staining was used to examine the cellular localization of MCP-1. The results demonstrated that MCP-1 expression was mainly observed in the ischemic core after 12 h of middle cerebral artery occlusion, then gradually increased and extended to the ischemic perifocal area. MCP-1 expression peaked at 2 days and 3 days, and gradually decreased after 5 days of MCAO. Double-labeled immunostaining for MCP-1 and neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP) showed that MCP-1 positive neurons were observed as early as 12 h of ischemia, while MCP-1 positive astrocytes were observed after 2 days of ischemia. These results support the functional role of MCP-1 in ischemic brain injury and reveal a distinct temporal and spatial expression of MCP-1 in cells believed to be neurons and astrocytes.     

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.     

Morphological consequences of early reperfusion following thrombotic or mechanical occlusion of the rat middle cerebral artery

Summary The early morphological consequences of recirculation following middle cerebral artery (MCA) occlusion were studied in two rat models. The proximal MCA was occluded for 1 h by either a surgical clip or platelet thrombus; subsequently, 1 h of recirculation was facilitated. Following clip occlusion and recirculation, mild astrocytic swelling, especially around blood vessels, was detected in reperfused cortical and striatal areas. Neuronal changes included slight chromatin clumping and dilation of the rough endoplasmic reticulum. In contrast, severe structural abnormalities were detected following recanalization of the thrombosed MCA segment. Marked astrocytic swelling of cell bodies and perivascular processes with neuropil vacuolation were commonly seen. A heterogeneous pattern of neuronal alterations, including a high frequency of dense shrunken neurons surrounded by swollen astrocytic processes was documented in cortical and striatal regions. Severe neuronal changes were documented in brain regions exhibiting a wellperfused microcirculation. Vascular endothelial cells contained large numbers of pinocytotic vesicles associated with luminal and abluminal surfaces. Pronounced and rapid morphological changes evolve with reperfusion when thrombotic and ischemic events occur simultaneously. The basis for these rapid parenchymal changes following vascular thrombosis may involve acute alterations in cerebral microvascular permeability which exacerbate ischemic consequences.Supported by USPHS grants NS-05820 and NS-23244, National Parkinson Foundation Grant YR 660068, and by the American Heart Association Grant-in-Aid 87-1012, with funds contributed by the Florida Affiliate. W. Dalton Dietrich is an Established Investigator of the American Heart Association.     

NMDA receptors mediate heat shock protein induction in the mouse brain following administration of the ibotenic acid analogue AMAA

Expression of inducible heat shock protein-70 (HSP-70) and hsp-70 mRNA were studied in the adult mouse brain following systemic administration of the ibotenic acid analogue (±)-2-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMAA), which is a potent N-methyl- d-aspartate (NMDA) agonist. At the dose of 20 mg/kg, AMAA produced excitatory behaviours in adult mice but overt convulsions were not seen. This treatment did not result in any detectable morphological brain damage at 4 days following administration. At 2.5 h and 5 h following treatment induction of hsp-70 mRNA expression was found in the pyramidal cell layers of CA1 and, to a lesser extent, CA3 fields of hippocampal Ammon's horn, amygdala, olfactory lobes, tenia tecta, hypothalamic nuclei and a superficial layer of cingulate, frontal and retrosplenial cortices. The presence of HSP-70 was detected by immunohistochemistry at 24 h following drug administration in those regions previously showing hsp-70 mRNA induction. AMAA-induced hsp-70 mRNA expression was prevented by pre-treatment with the non-competitive NMDA antagonist MK-801. These results suggest that NMDA receptors are involved in the stress response induced by AMAA.     

Protective effect of a new nonpeptidyl mimetic of SOD,M40401, against focal cerebral ischemia in the rat

We tested the neuroprotective effects of M40401, a new, low molecular weight (511.4 Da) maganese superoxide dismutase mimetic, against 90 min of middle cerebral artery occlusion (MCAO) in male Wistar rats. Animals received a single injection of vehicle (n=8), 1 mg/kg (n=6), or 3 mg/kg (n=7) 30 min before MCAO. Total lesion volume was reduced only in the group receiving 3 mg/kg M40401 (163.5+/-18.7 versus 43.4+/-7.0 mm(3), for vehicle and M40401, respectively; P<0.05), with almost complete reduction of lesion volume in the cortex but little protection in the basal ganglia. Neurological score was also improved in this group. The dose of 1 mg/kg M40401 had smaller and inconsistent effects on lesion parameters. Administration of a single dose of 3 mg/kg M40401 at 60 min of MCAO or at the end of MCAO (90 min) failed to significantly reduce lesion volume. A single dose of M40401 plus prolonged infusion into the post-MCAO period also failed to decrease lesion volume significantly. These data indicate that M40401 protects cerebral tissue from ischemic insult when administered before MCAO, probably by limiting damage mediated by detrimental actions of superoxide anion.     

大鼠大脑中动脉阻断可逆性局灶脑缺血模型的研究

采用普通外科手术，以一顶端贫成光滑圆球的4—0单丝尼龙线，可逆性阻断大鼠一侧大脑中动脉（MCA）的血流，制备局灶性脑缺血及再灌注模型。通过观察动物的神经行为学、脑电生理学及病理形态学变化情况，对该模型的可靠性进行客观评价。结果表明这种改良栓线法制备的模型操作简单，与临床缺血性脑卒中的发病情况相近似，适用于局灶性脑缺血及再灌注损伤机制的研究以及治疗手段的评价。     

Behavioral changes and expression of heat shock protein hsp-70 mRNA, brain-derived neurotrophic factor mRNA, and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid

Kainic acid-induced seizures in rats represent an established animal model for human temporal lobe epilepsy. However, it is well-known that behavioral responses to the systemic administration of kainic acid are inconsistent between animals. In this study, we examined the relationship between expression of genes, neuropathological damage, and behavioral changes (seizure intensity and body temperature) in rats after systemic administration of kainic acid. The considerable differences in the response to kainic acid-induced seizures were observed in rats after a single administration of kainic acid (12 mg/kg i.p.). There was no detection of the expression of heat shock protein hsp-70 mRNA and HSP-70 protein in brain of vehicle-treated controls and in animals exhibiting weak behavioral changes (stage 1–2). A moderate expression of hsp-70 mRNA was detected throughout all regions (the pyramidal cell layers of CA1–3 and dentate gyrus) of the hippocampus, the basolateral, lateral, central and medial amygdala, the piriform cortex, and the central medial thalamic nucleus of rats that developed moderate seizures (stage 3–4). Marked expression of hsp-70 mRNA was detected in the all regions (cingulate, parietal, somatosensory, insular, entorhinal, piriform cortices) of cerebral cortex and all regions of hippocampus, and the central medial thalamic nucleus of the rats that developed severe seizures (stage 4–5). In addition, marked HSP-70 immunoreactivity was detected in the pyramidal cell layers of CA1 and CA3 regions of hippocampus, all regions (cingulate, parietal, somatosensory, insular, piriform cortices) of cerebral cortex, and the striatum of rats that developed severe seizures (stage 4–5). Furthermore, a marked expression of cyclooxygenase-2 (COX-2) mRNA and brain-derived neurotrophic factor (BDNF) mRNA levels by kainic acid-induced behavioral seizures (stage 3–4 or stage 4–5) was detected in all hippocampal pyramidal cell layers, granule layers of dentate gyrus, piriform cortex, neocortex, and amygdala. The present study suggest that the behavioral changes (seizure intensity and body temperature) and neuropathological damage after systemic administration of kainic acid are inconsistent between animals, and that these behavioral changes (severity of kainic acid-induced limbic seizures) might be correlated with gene expression of hsp-70 mRNA, COX-2 mRNA, and BDNF mRNA in rat brain.