大鼠纹状体边缘区内神经降压肽和生长抑素免疫阳性反应的分布

用免疫组织化学方法研究了大鼠纹状体边缘区中神经降压肽和生长抑素免疫阳性反应的分布。神经降压肽和生长抑素免疫阳性纤维和胞体散在分布于纹状体中,但在边缘区中分布密集,形成一条明显的带,带的宽度和位置和边缘区一致。边缘区中可见神经降压肽和生长抑素免疫阳性胞体。本研究证明纹状体边缘区中存在密度较高的神经降压肽和生长抑素免疫阳性纤维和胞体,并推测和边缘区的学习记忆功能有关     

P物质受体在大鼠纹状体边缘区内的表达

我们以前的工作观察到纹状体边缘区内有密集的P物质纤维及终末分布,本用原位杂交和免疫组织化学方法研究了大鼠纹状体边缘区内P物质受体（SPR）的表达及分布,原位杂交结果发现P物质mRNA阳性杂交信号在纹状体内的分布不均匀,尾壳核内只有少量中等大小的阳性胞体,苍白球内只有少量较大的阳性胸体,而在尾壳核和苍白球之间的边缘区部位则可见许多中等大小的梭形阳性神经元胞体,并呈现密集的带关分布。免疫组织化学结果观察到P物质阳性神经元胞体在纹状体内的分布与原位杂交结果一致。推测大鼠纹体边缘区内可以合成P物质受体,具有接受和整合P物质神经递质的功能,推测边缘区内SPR神经元可能对SP递质的接受、调节有重要作用。     

细胞周期调控对大鼠全脑缺血后海马迟发性神经元死亡的影响

目的观察细胞周期调控对大鼠全脑缺血再灌流后海马区迟发性神经元死亡(delayed neuronal death,DND)以及星形胶质细胞的活化、增殖的影响.方法建立大鼠短暂性全脑缺血再灌流模型,利用尼氏染色、TUNEL、免疫组织化学方法观察再灌流后细胞周期素依赖的蛋白激酶(cyclin depedent kinase, CDK)抑制剂Olomoucine对海马DND以及星形胶质细胞活化增殖的影响.结果全脑缺血再灌流后3d、7d、30d海马神经元明显脱失,部分CA1、CA2区神经元凋亡;星形胶质细胞数目增多,GFAP表达上调,应用Olomoucine后TUNEL阳性神经元数目明显减少,幸存神经元数目增加;星形胶质细胞数目无明显增多,GFAP表达明显下调.结论 CDK抑制剂Olomoucine可有效抑制大鼠全脑缺血后海马神经元DND以及星形胶质细胞活化增殖.     

大鼠局灶性脑缺血后缺血边缘区NG_2细胞的表达

目的观察局灶性脑缺血后缺血边缘区海马和皮层NG2细胞的动态表达,探讨其在脑缺血神经损伤与修复过程中所起的作用。方法将大鼠随机分为假手术组(sham)和脑缺血再灌注组,脑缺血再灌注组采用线栓法制备大鼠大脑中动脉阻塞再灌流模型(MCAO),假手术组不插入线栓,采用免疫荧光组织化学法结合共聚焦显微镜成像观察sham组及脑缺血后3d,7d,30d不同时间点缺血边缘区的海马CA1区和皮层区NG2的动态表达情况。结果脑缺血再灌注后缺血边缘区海马和皮层NG2胶质细胞表达增加,缺血后7d最明显。结论脑缺血后缺血边缘区存在NG2细胞的增生和形态变化可能与脑缺血后损伤修复密切相关。     

甘珀酸干预对大鼠脑缺血再灌注损伤的影响

目的观察缝隙连接阻断剂甘珀酸对局灶性脑缺血/再灌注损伤的影响。方法采用大鼠大脑中动脉阻塞再灌流模型（MCAO）,将动物随机分为脑缺血60min再灌注（MCAO）组,脑缺血再灌注加甘珀酸干预（MCAO＋CBX）组和假手术组（sham）。采用尼氏染色显示脑梗死灶并计算梗死灶体积;应用免疫荧光与TUNEL染色法分别观察脑缺血后3d与7d不同时间点缺血边缘区胶质纤维酸性蛋白（GFAP）的表达和细胞凋亡情况。结果（1）缺血后3d、7d MCAO＋CBX组大鼠梗死体积小于MCAO组,3d、7d MCAO＋CBX组大鼠梗死体积较MCAO组分别缩小5%和4.6%;（2）缺血后3d、7d于缺血边缘区可见大量TUNEL阳性染色细胞,且MCAO组大鼠缺血边缘区细胞凋亡数目明显多于MCAO＋CBX大鼠（P〈0.001）;（3）缺血后3d和7d组缺血边缘区GFAP表达明显增强,3d的MCAO组与MCAO＋CBX组大鼠缺血边缘区GFAP的表达均较假手术组强（P〈0.05）,7d的MCAO＋CBX组大鼠缺血边缘区GFAP的表达较假手术组强（P〈0.001）,但明显弱于MCAO组大鼠（P〈0.01）;结论缝隙连接阻断剂甘珀酸可减少大鼠大脑中动脉阻塞后脑梗死体积,其机制可能与阻断缝隙连接后缺血边缘区神经元凋亡降低有关,星型胶质细胞的反应性变化参与了该过程。     

大鼠面部伤害性刺激后纹状体边缘区内原癌基因c-fos和NOS的表达

为了研究纹状体边缘区和痛觉的关系,用c-fos和NADPH－d双标记方法研究了大鼠面部伤害性刺激后c-fos蛋白（Fos）和NOS在纹状体边缘区的表达。面部伤害性刺激后30分钟,边缘区中即出现Fos表达,刺激后3小时,Fos表达达最高峰,而且主要在边缘区部位表达。正常大鼠纹状体边缘区中有密集的NOS阳性神经元及纤维,面部伤害性刺激3小时后,纹状体其余部位的NOS阳性胞体及纤维减少或消失,但边缘区中仍保留,并可见少数Fos和NOS双标记细胞,提示纹状体边缘区可能和面部痛觉的调制有关。     

大鼠脑缺血区细胞间粘附分子ICAM-1表达和LFA-1阳性细胞浸润的研究

研究粘附分子和白细胞与脑缺血／再灌流损伤的病理联系,运用原位杂交和免疫组化技术对36只SD大鼠脑缺血区细胞间粘附分子（ICAM－1）表达和淋巴细胞机能相关抗原（LFA－1）阳性细胞浸润进行了观察。结果显示,脑缺血区的毛细胞血管内皮细胞表达ICAM－1 mRNA发生于脑缺血1h,在脑缺血1h／再灌流8h达到高峰。而脑缺血区毛细血管ICAM－1蛋白质的表达则发生于脑缺血1h／再灌流2h,高峰出现于脑缺血1h／再灌流16h,LFA－1阳性细胞在脑缺血区的聚集发生在脑缺血1h,并随再灌流时间延长,其聚集数量逐渐增加。结果提示,脑缺血／再灌流能诱导缺血区的血管内皮细胞表达ICAM－1 mRNA和蛋白质,进而导致白细胞在脑缺血区的浸润,此可能是脑缺血／再灌流损伤的病理机制之一。     

大鼠脑缺血再灌注血管壁NOS和ICAM-1的表达

一氧化氮（NO）和一氧化氮合酶（NOS）与脑血管功能有重要关系,细胞间粘附分子1（ICAM－1）可由脑缺血／再灌注诱导产生并与脑组织损伤密切相关,本实验用免疫组织化学和NADPH－d酶组织化学方法,观察了SD大鼠实验性脑缺血再灌注内皮细胞ICAM－1和NOS的表达,结果显示正常对照组大鼠脑血管ICAM－1免疫组织化学显色为阴性或弱阳性反应,再灌注2h,ICAM－1阳性反应明显增强,与对照组相比,P＜0.01。随再灌注至16h,ICAM－1表达增加近一倍。脑缺血1h缺血侧侧脑血管壁开始出现NOS的阳性表达,与对照组相比,P＜0.01,再灌注2h,NOS表达最多,随后逐渐下降,结果提示脑缺血再灌注与ICAM－1和NOS表达升高有关。     

Smad4蛋白在不同龄大鼠睾丸的定位研究

探讨转化生长因子-β超家族肽类的细胞内信号转导分子Smad4蛋白在发育不同阶段大鼠睾丸的表达与分布。分别选用出生后3、7、14、28天以及成年大鼠,应用免疫组织化学ABC法结合葡萄糖氧化酶-DAB-硫酸镍铵增强技术及蛋白质免疫印迹技术,检测Smad4蛋白在大鼠睾丸的表达、定位和发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。结果显示,发育各个阶段的间质细胞中都有较强的表达,免疫阳性产物位于细胞质内,而各级生精细胞则无阳性反应,且随着大鼠睾丸发育阶段的变化而蛋白表达量逐渐增多,为TGF-β超家族成员在精子发生和发育过程中的分子机理提供了直接证据。     

大鼠局灶性脑缺血再灌注后免疫蛋白酶体的变化

目的观察大鼠局灶性脑缺血再灌注后脑组织免疫蛋白酶体LMP2和LMP7表达及其意义。方法线栓法制作SD大鼠大脑中动脉阻塞再灌注(middle cerebral artery occlusion,MCAO)模型,脑缺血1h再灌注72h。免疫荧光染色观察脑组织LMP2、LMP7、NF-κB、IL-1β、TNF-α表达和细胞分布。Western blot分析LMP2、LMP7、磷酸化NF-κB p65、IL-1β、TNF-α蛋白水平变化。结果 1局灶性脑缺血再灌注后上调LMP2和LPM7表达,尤其在梗死灶周边的皮层和纹状体区,与假手术组比较有显著性差异(P0.001)。2免疫荧光双标显示星形胶质细胞是LMP2主要来源细胞,而OX42阳性的小胶质细胞/巨噬细胞是LMP7主要来源细胞;而且,NF-κB、IL-1β、TNF-α与LMP2、LMP7具有一定程度的细胞共定位。3Western blot结果表明,脑缺血再灌注后NF-κB p65、IL-1β、TNF-α蛋白水平表达趋势与LMP2、LMP7变化相类似。结论局灶性脑缺血再灌注后免疫蛋白酶体LMP2和LMP7主要来源于免疫炎症相关的细胞,推测LMP2和LMP7可能参与调节缺血性脑卒中后脑神经炎症反应。     

Differential Changes in Pyridoxine 5′-Phosphate Oxidase Immunoreactivity and Protein Levels in the Somatosensory Cortex and Striatum of the Ischemic Gerbil Brain

Pyridoxal 5'-phosphate (PLP) is an important cofactor in a wide range of biochemical reactions, such as the metabolism of various amino acids, including GABA. PLP is synthesized by the oxidation of pyridoxine 5'-phosphate (PNP), which is catalyzed by PNP oxidase (PNPO). We observed the changes in cresyl violet-positive neurons, PNPO immunoreactivity and PNPO protein levels in the somatosensory cortex and striatum in gerbils after 5 min of transient forebrain ischemia. Cresyl violet-positive neurons showed condensed cytoplasm in the somatosensory cortex and lateral part of the striatum at 2 days after ischemia/reperfusion. PNPO immunoreactivity began to increase in neurons in layers III and V at 3 h after ischemia/reperfusion and this immunoreactivity was significantly increased at 12 h after ischemia/reperfusion. Thereafter, PNPO immunoreactivity decreased with time after ischemia/reperfusion. PNPO-immunoreactive neurons were only slightly detected in the lateral part of the striatum at 12 h after ischemia/reperfusion. In addition, the PNPO protein levels in both the somatosensory cortex and striatum homogenates peaked at 12 h after ischemia/reperfusion. These results indicate that PNPO is significantly increased in the ischemic somatosensory cortex and lateral part of the striatum, and this change in the level of PNPO may be associated with the neuronal damage induced by ischemia.     

Expression of NMDA neuroreceptors in experimental ischemia

The role of NMDA receptors in molecular mechanisms of neurotoxicity was investigated using rat models of global and focal cerebral ischemia. Expression of NR2A and NR2B receptor mRNAs up-regulated in cortex after 3 h of reperfusion following middle cerebral artery occlusion (MCAo). This effect was accompanied by an increase in NR2A and NR2B immunoreactivity. At six hours of reperfusion, drastic activation of NR2A mRNA expression was observed in the penumbra that returned to the control level at 24 h of reperfusion. The monitoring of NR2A autoantibodies in the blood of the experimental rats showed its reliable increase to the 5-6th day of reperfusion that maintained elevated to the 20th day of the experiment. The data indicate that NR2A and 2B receptor subunits and NR2A autoantibodies are biochemical markers of the neurotoxicity underlying cerebral ischemia.     

Calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart: influence of age

In the present study, we examined whether age-related reduction of ischemic preconditioning is related to calcitonin gene-related peptide (CGRP) release in the rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant decrease of cardiac function and a marked increase of creatine kinase (CK) release at 2, 6 and 20 months of age. Ischemic preconditioning and pretreatment with CGRP for 5 min significantly improved cardiac function and reduced CK release during reperfusion at 2 and 6 months of age but not at 20 months of age. The content of CGRP in the coronary effluent during ischemic preconditioning was significantly increased in the first cycle at 2, 6 months of age but not at 20 months of age. These results suggest that the protection afforded by ischemic preconditioning is decreased in aging hearts, and the age-related change may be related to reduction of the release and effect of CGRP in the rat heart.     

Comparison of Changes in GAD65 and GAD67 Immunoreactivity and Levels in the Gerbil Main Olfactory Bulb Induced by Transient Ischemia

In the present study, we investigated changes in glutamate decarboxylase 65 (GAD65) and GAD67 immunoreactivity and protein levels in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. GAD65 immunoreactivity in the sham-operated group was shown in neurons and neuropil except for the somata of granule cells. GAD65 immunoreactivity was increased in neurons in the external plexiform layer 60 days after ischemia, and in mitral cells 30 and 60 days after ischemia. GAD67 immunoreactivity in the sham-operated group was shown in periglomerular cells, neuron in the external plexiform layer and granule cells with neuropil. GAD67 immunoreactivity in periglomerular cells was increased 10, 45 and 60 days after ischemia. GAD67 immunoreactivity in neurons in the external plexiform layer was increased 10 and 15 days after ischemia. Mitral cells showed strong GAD67 immunoreactivity 10 days after ischemia. However, GAD67 immunoreactivity in the granule cells was not changed with time after ischemia. In Western blot analysis for GAD65 and GAD67 protein levels in the ischemic gerbil MOB, GAD65 level was not changed after ischemia; GAD67 level was increased 10 days after ischemia. These results suggest that transient ischemia causes changes in GAD65 and GAD67 immunoreactivity in the gerbil MOB, and this change may induce a malfunction in olfaction after an ischemic insult. Ki-Yeon Yoo and In Koo Hwang equally contributed to this article.     

Hyperoxidized Peroxiredoxins and Glyceraldehyde-3-Phosphate Dehydrogenase Immunoreactivity and Protein Levels are Changed in the Gerbil Hippocampal CA1 Region After Transient Forebrain Ischemia

Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO₃) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO₃) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO₃ immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO₃ immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO₃ immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO₃ immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO₃ immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. Prx-SO₃ and GAPDH-SO₃ protein levels in the ischemic CA1 region were also very high 12 h and 1 day after ischemia/reperfusion and returned to the level of the sham-operated group 3 days after ischemia/reperfusion. Their protein levels were increased again 5 days after ischemia/reperfusion. In conclusion, Prx-SO₃ and GAPDH-SO₃ immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.     

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.     

Evidence for calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart.

Previous studies have suggested that calcitonin gene-related peptide (CGRP) may play an important role in the mediation of ischemic preconditioning. In the present study, we examined the release of CGRP during ischemic preconditioning and the effect of preconditioning frequency on this effect in the isolated rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant cardiac dysfunction and an increase in the release of creatine kinase (CK) during reperfusion. Preconditioning with one, two or three cycles of 5-min ischemia and 5-min reperfusion caused a marked improvement of cardiac function and a decrease in the release of CK, and there was no difference in the degree of improvement among groups. The protective effects of ischemic preconditioning were abolished by the CGRP receptor antagonist CGRP(8-37). A single preconditioning cycle induced a significant increase in the release of CGRP in the coronary effluent. In the hearts treated with two or three preconditioning cycles, the level of CGRP was highest in the first cycle, and was gradually decreased with increasing number of cycles of preconditioning. These results suggest that the protective effects of ischemic preconditioning are mediated by endogenous CGRP in the isolated rat heart.     

Protection of calcitonin gene-related peptide-mediated preconditioning against coronary endothelial dysfunction induced by reperfusion in the isolated rat heart

This study was designed to explore the protective effect of ischemic preconditioning on reperfusion-induced coronary endothelial dysfunction, with a focus on the role of calcitonin gene-related peptide (CGRP) in this effect, in the isolated perfused rat heart. Thirty minutes of global ischemia and 30 min of reperfusion significantly decreased heart rate, left ventricular pressure, and its first derivative and impaired vasodilator responses to acetylcholine. Ischemia-reperfusion did not affect vasodilator responses to sodium nitroprusside. Preconditioning induced by three cycles of 5 min of ischemia and 5 min of reperfusion produced a significant improvement in cardiac function concomitantly with an amelioration of vasodilator responses to acetylcholine. The protective effects of ischemic preconditioning were abolished by CGRP(8-37) (10(-7) M) , the selective CGRP receptor antagonist. After pretreatment with capsaicin (50 mg x kg(-1), s.c.) to deplete endogenous CGRP, the preconditioning effect was absent. Pretreatment with exogenous CGRP (5 x 10(-9) M) for 5 min induced a preconditioning-like protection. The present study suggests that the cardioprotection of ischemic preconditioning is related to the preservation of the coronary endothelial cell, and that the protective effect of preconditioning is mediated by endogenous CGRP in the isolated perfused rat heart.     

Neuroprotective efficiency of NMDA receptor blockade in the striatum and CA3 hippocampus after various durations of cerebral ischemia in gerbils

The purpose of this study was to investigate neuroprotective efficiency of N-methyl D-aspartate (NMDA) receptor (NMDAR) blockade on the neuronal damage in the less studied and allegedly less affected CA3 hippocampus and striatum in the Mongolian gerbil model of global cerebral ischemia. The common carotid arteries of gerbils were occluded for 5, 10 or 15 minutes. Gerbils were given a low dose of non-competitive NMDA antagonist (MK-801, 3 mg/kg i.p.) or saline immediately after the occlusion in normothermic conditions. Neuronal damage was examined on 4th, 14th and 28th day after reperfusion. The effect of NMDAR blockade was followed in vivo by monitoring the neurological status of whole animals or at the cellular level by standard light- and confocal microscopy on brain slices. Increased duration of cerebral ischemia resulted in a progressive loss of striatal and CA3 hippocampal neurons. The most beneficial NMDAR blockade effect was observed when the neuronal damage was most severe - on the 28th day after 15-min ischemia. As judged by morphological and neurological data, the effect of ischemia is also apparent in the presumed less vulnerable regions (CA3 and striatum) which are functionally important in stroke plasticity. So, NMDAR blockade in normothermic conditions showed neuroprotective efficiency.