大鼠局灶性脑缺血β淀粉样前体蛋白mRNA在缺血半暗带的表达变化

目的 :研究大鼠局灶性脑缺血不同缺血时间皮质半暗带和中心区淀粉样前体蛋白 (APP)在转录水平表达规律。方法 :用线栓法建立大鼠局灶性脑缺血模型 ,剥取缺血半暗带及中心区皮质组织 ,采用半定量逆转录 聚合酶链式反应 (RT PCR) ,测定APPmRNA水平的变化。结果 :半暗带APPmRNA在缺血后 48h升高 ,缺血 72h达到高峰 ,缺血 1周后仍高于正常。缺血中心区APPmRNA在缺血后 72h和 96h高于正常水平。结论 :APPmRNA在缺血半暗带的表达上调 ,有可能加重缺血损伤。     

大鼠局灶性脑缺血损伤中IGF-I mRNA表达

目的　观察局灶性脑缺血损伤中 IGF- I m RNA的表达特点 ,探讨其调控机制。方法　采用自体血凝块注入颈内动脉的方法制作大鼠局灶性脑缺血 2 h、4 h、6 h、12 h、2 4 h、4 8h模型 ,应用原位杂交及 RT- PCR方法 ,检测缺血中心区及半暗带区 IGF- I m RNA的表达。结果　局灶性脑缺血损伤时 ,缺血中心区及半暗带区 IGF- I m R-NA表达增加 ,尤以缺血半暗带区增加明显。结论　 IGF- I对局灶性脑缺血损伤具有保护作用。     

17β-雌二醇对大鼠脑缺血再灌注损伤脑组织HSP70表达的影响

目的观察雌二醇对大鼠脑缺血再灌注损伤脑组织HSP70表达的影响。方法72只大鼠随机分为假手术组(8只)、实验对照组及雌二醇治疗组,后两组又进一步分为局灶性脑缺血2h再灌注及3h、6h、12h、24h再灌注组,每时间点8只。线栓法建立大鼠局灶性脑缺血/再灌注损伤模型,采用石蜡切片HE及免疫组化染色法检测脑组织损伤及HSP70的表达情况。结果假手术组大鼠未见HSP70的表达;雌二醇治疗组的脑组织缺血半暗带区损伤程度较实验对照组明显减轻;随着再灌注时间的延长,治疗组半暗带区皮质HSP70的表达较对照组上调,且呈先上升后下降趋势,在12h为表达高峰;而纹状体区其表达呈进行性下调趋势。结论17β-雌二醇具有减少脑缺血/再灌注损伤的作用,而半暗带区皮质脑保护蛋白HSP70的表达升高可能其是重要机制之一。     

17β-雌二醇对大鼠脑缺血再灌注损伤脑组织HSP70表达的影响

目的 观察雌二醇对大鼠脑缺血再灌注损伤脑组织Hsp-70表达的影响.方法 72只大鼠随机分为假手术组（8只）、实验对照组及雌二醇治疗组,后两组又进一步分为局灶性脑缺血2h/再灌注3h、6h、12h、24h 组,每时间点8只.线栓法建立大鼠局灶性脑缺血/再灌注损伤模型,采用石蜡切片HE及免疫组化染色法检测脑组织损伤及hsp-70的表达情况.结果 假手术组大鼠未见Hsp-70的表达; 雌二醇治疗组的脑组织缺血半暗带区损伤程度较实验对照组明显减轻;随着再灌注时间的延长,治疗组半暗带区皮质hsp-70的表达较对照组上调,且呈先上升后下降趋势,在12h 为表达高峰;而纹状体区其表达呈进行性下调趋势.结论 17-β雌二醇具有减少脑缺血/再灌注损伤的作用,而半暗带区皮质脑保护蛋白Hsp-70的表达升高可能其是重要机制之一.     

大鼠脑缺血再灌注半暗带β淀粉样前蛋白mRNA表达与梗死体积的关系

为了探讨大鼠局灶性脑缺血再灌注缺血半暗带β淀粉样前蛋白(APP)转录水平与缺血时间及梗死体积的相互关系,用插线法建立大鼠局灶性脑缺血再灌注模型,剥取缺血半暗带皮质组织,采用半定量逆转录-聚合酶链式反应(RT-PCR),测定永久性缺血48 h和不同缺血时间再灌注48 h后,APPmRNA水平的变化.结果显示,梗死体积随再灌注前缺血时间的延长而增大,皮质半暗带缺血30 min再灌注48 h APPmRNA表达升高;缺血60min和缺血120 min再灌注48 h APPmRNA升高明显;缺血180 min再灌注48 h和永久性缺血48 h APPmRNA达到高峰.提示缺血半暗带APPmRNA的表达随再灌注前缺血时间延长而增加并与梗死体积有一定的相关性,早期再灌注可减少其表达.((GFDAl))     

亚低温对大鼠局灶性脑缺血再灌注后不同脑区iNOS表达的影响

目的 探讨亚低温对大鼠局灶性脑缺血再灌注后不同脑区诱导型一氧化氮合酶(iNOS)表达的影响.方法 雄性SD大鼠,随机分为假手术组、常温缺血组和亚低温组.采用线栓法制作大脑中动脉闭塞再灌注模型,于缺血后48h,观察不同组间组织形态学变化,检测不同脑区iNOS蛋白表达、iNOS活性和产物NO含量.结果 常温缺血后48h,纹状体和皮质均检测到iNOS活性升高和免疫阳性反应,且皮质缺血半暗带区iNOS免疫反应明显强于纹状体和皮质缺血核心区.亚低温明显缩小梗死面积,抑制皮质和纹状体iNOS活性,明显下调半暗带区iNOS蛋白表达,减少NO产生.结论亚低温可能通过减少半暗带区iNOS蛋白表达,抑制iNOS活性,减少NO产生而起到脑保护作用.     

大鼠局灶性脑缺血半暗带葡萄糖转运子3 mRNA的表达

目的 研究大鼠局灶性脑缺血不同缺血时间皮质半暗带和中心区葡萄糖转运子3（GLUT3）转录水平的表达规律。方法 用插线法建立大鼠局灶性脑缺血模型，剥取缺血半暗带及中心区皮质组织，采用逆转录-聚合酶链反应（RT-PCR），测定GLUT3mRNA水平的变化。结果 缺血半暗带GLUT3mRNA在缺血3小时升高，24小时达到高峰，96小时后基本恢复正常。缺血中心区GLUT3mRNA在缺血后3小时有一短暂的升高，随后迅速下降呈低水平表达。结论 GLUT3在缺血半暗带的表达上调，有可能是机体对缺血损伤的保护性反应。     

大鼠脑缺血再灌注半暗带β淀粉样前蛋白mRNA表达与梗死体积的关系

为了探讨大鼠局灶性脑缺血再灌注缺血半暗带β淀粉样前蛋白(APP)转录水平与缺血时间及梗死体积的相互关系，用插线法建立大鼠局灶性脑缺血再灌注模型，剥取缺血半暗带皮质组织，采用半定量逆转录－聚合酶链式反应（RT－PCR），测定永久性缺血48h和不同缺血时间再灌注48h后，APPmRNA水平的变化。结果显示，梗死体积随再灌注前缺血时间的延长而增大，皮质半暗带缺血30min再灌注48h APPmRNA表达升高；缺血60min和缺血120min再灌注48h APPmRNA升高明显；缺血180min再灌注48h和永久性缺血48h APPmRNA达到高峰。提示缺血半暗带APPmRNA的表达随再灌注前缺血时间延长而增加并与梗死体积有一定的相关性，早期再灌注可减少其表达。（（GFDA1））。     

大鼠局灶性脑缺血再灌注后BDNF mRNA及其蛋白的表达变化

目的 观察大鼠脑缺血再灌注后缺血半暗带皮质脑源性神经营养因子(BDNF)mRNA和蛋白的表达变化.方法 采用线栓法制作局灶性脑缺血大鼠(MCAO)模型,用原位杂交法和免疫组化法观察脑缺血后3、6、12h及1、3、7d共6个时间点BDNF mRNA及其蛋白的表达变化.结果 缺血半暗带BDNF mRNA及其蛋白的表达均于缺血再灌注后3h开始明显上升,6h表达继续增强,12h达高峰,3d后表达开始减弱(P＜0.05或0.01),7d后降至基础水平.结论 脑缺血再灌注后缺血半暗带BDNFmRNA及蛋白表达均明显增加,提示其可能参与了脑缺血后神经保护.     

大鼠局灶脑缺血酪氨酸激酶B表达图像分析

目的 观察局灶脑缺血大鼠酪氨酸酶B(TrkB)阳性神经元的表达,探讨脑缺血损伤与TrkB的关系。方法 制作大鼠局灶脑缺血模型,应用免疫组化方法观察不同缺血时间、不同脑区TrkB阳性神经元数的动态改变,并进行图像分析。结果 正常脑组织内即存在着一定数量的TrkB阳性神经元,脑缺血损伤后,在额、顶叶皮质及尾壳核外侧部的变性坏死区中心,TrkB阳性神经元缺失,而变性坏死区周边的半暗带区TrkB阳性神经元自缺血6h开始明显增多,且持续于整个缺血期。其它部位TrkB表达也不同程度增强。方差分析显示,与正常对照组比较,除假手术组外(P>O.05).缺血组各组均差异显著(P<0.01)。结论 脑缺血损伤后TrkB的表达有显著改变,考虑与神经元的损伤修复机制有关。     

大鼠局灶性脑缺血再灌注后FADD mRNA及其蛋白的表达变化

目的观察大鼠脑缺血再灌注后缺血半暗带皮质内Fas死亡结构域相关蛋白（FADD）mRNA及蛋白的表达变化。方法用半定量的逆转录PCR（RT-PCR）法检测缺血2h再灌注不同时间点缺血半暗带皮质内FADD mRNA的表达,Western blot检测FADD蛋白表达的变化。结果缺血半暗带脑皮质内FADD mRNA及其蛋白的表达于缺血灌注后3h明显升高,再灌注后12h达高峰（P＜0．01）,至再灌注后24h明显下降。结论脑缺血再灌注后缺血半暗带皮质内FADD mRNA及蛋白表达均明显增加,提示FADD可能在脑缺血再灌注损伤中发挥重要作用。     

大鼠短暂性脑缺血后葡萄糖转运子3表达变化

目的 研究大鼠短暂性脑缺血葡萄糖转运子3（GLUT3）转录水平表达规律。方法 用插线法建立大鼠短暂性脑缺血模型。剥取缺血半暗带及中心区皮质组织,采用半定量逆转录-聚合酶链式反应（RT-PCR）,测定不同再灌注时间GLUT3mRNA水平的变化。结果 再灌注3h缺血半暗区GLUT3mRNA3h升高,48h达到高峰,再灌注1周后仍高于正常。结论 再灌注后,缺血半暗带GLUT3的表达明显上调,有可能是机体抗损伤性反应。     

Hydroxyl radical formation is greater in striatal core than in penumbra in a rat model of ischemic stroke

Although hydroxyl radical ((*)OH) formation has been implicated in the pathophysiological changes of ischemic stroke, (*)OH production in the core and penumbra regions is not clear. It is extremely important to distinguish penumbra from ischemic core in focal cerebral ischemia studies, because the penumbra contains viable tissue, which can be salvaged by appropriate treatment. This study evaluated (*)OH production in both core and penumbra regions of ischemic striatum during ischemia and reperfusion. Microdialysis probes were placed in striatal tissue of rats subjected to the middle cerebral artery occlusion model of ischemic stroke. The (*)OH-trapping agent 4-hydroxybenzoic acid (4-HBA) was administered by both i.v. and probe infusion. Dialysate levels of the 4-HBA oxidation products, 3,4-dihydroxybenzoic acid (3,4-DHBA), were determined by HPLC-ECD. After microdialysis probe delivery of 4-HBA, (*)OH production was significantly increased in the striatal core during both ischemia and reperfusion. Penumbra (*)OH production increased only during reperfusion. Alterations of 3,4-DHBA concentration in dialysate following i.v. 4-HBA administration were likely related to alterations in tissue blood flow. The findings were confirmed by a greater oxidation of dihydroethidium in the ischemic core than in the penumbra as determined by fluorescent microscopy. The findings of (*)OH production in ischemic striatum are the opposite of those reported for ischemic cortex and suggest critical regional variations in (*)OH production that may have significant clinical implications in the treatment of ischemic stroke.     

Caspase-dependent and caspase-independent signalling of apoptosis in the penumbra following middle cerebral artery occlusion in the adult rat

Transient focal ischaemia by middle cerebral artery occlusion (MCAO) may produce cell death, but the mechanisms leading to cell death differ in the infarct core and in the penumbra, the immediate zone surrounding the infarct core. In the present study, transient focal ischaemia to adult rats was produced by intraluminal occlusion of the middle cerebral artery for 1 h followed by 0 h (n=6), 1 h (n=10), 4 h (n=8), 6 h (n=2) and 12 h (n=3) of reperfusion. The present model of ischaemia causes a large cortico-striatal infarct extending through the mediolateral cortex and dorsolateral striatum at 12 h. The expression and subcellular distribution of several proteins involved in apoptosis have been examined in the penumbra and in the infarct core by using combined methods of immunohistochemistry, cell subfractionation and Western blotting. Transient focal ischaemia by MCAO results in activation of complex signal pathways for cell death in the penumbra. Increased expression of Bcl-2 and Bax, but not of Bcl-x, occurs in the penumbra at the time when Bax translocates from the cytosol to the mitochondria, cytochrome c is released to the cytoplasm and active caspase-3 is expressed. Bax translocation, cytochrome c release and active caspase-3 are observed at 4 h, but not at 1 h, following reperfusion, and together indicate activation of the caspase-dependent pathway of apoptosis in the penumbra. In contrast, reduced Bax expression but not Bax translocation and cytochrome c release occurs in the infarct core, thus suggesting apoptosis signals restricted to the penumbra. In addition, increased expression of an apoptosis-inducing factor in the cytoplasm and nuclei of selected cells shows, for the first time, activation of the caspase-independent mitochondrial pathway in the penumbra following transient focal ischaemia and reperfusion.     

NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke

Reactive oxygen species play a role in neuronal damage following cerebral ischemia-reperfusion. We tested whether activity of the superoxide-generating enzyme, NADPH-oxidase, is enhanced in cerebral arteries within, adjacent and distant from the ischemic core. The right middle cerebral artery (MCA) of conscious rats was temporarily occluded by perivascular injection of endothelin-1 to induce stroke (ET-1; n=19). Control rats were injected with saline (n=9). At 24 h or 72 h post-administration of ET-1, the MCA and its branches within the ipsilateral penumbra and infarcted core, corresponding arteries in the contralateral hemisphere, and basilar artery were excised. Anatomically similar arteries were excised from saline-injected rats. At 24 h after stroke, NADPH-stimulated superoxide production by arteries from the infarcted core did not differ from levels generated by arteries from control rats, whereas levels were significantly lower 72 h after stroke. However, at both time points after stroke, superoxide production by arteries from the ischemic penumbra was 8-fold greater than levels generated by arteries from control rats. Surprisingly, even in the non-ischemic arteries from the contralateral hemisphere and in the basilar artery, superoxide production was increased approximately 4- to 6-fold at 24 h, but had returned to normal 72 h after stroke. The NADPH-oxidase inhibitor, diphenyleneiodonium, virtually abolished superoxide production by all arteries. Thus, the activity of NADPH-oxidase is enhanced in cerebral arteries from the ischemic penumbra at 24 h and 72 h following cerebral ischemia. Additionally, NADPH-oxidase activity is temporarily enhanced after cerebral ischemia within arteries from non-ischemic parts of the brain.     

大鼠局灶性脑缺血/再灌注损伤后Caspase-1的表达

目的研究Caspase-1在大鼠脑缺血／再灌注损伤中的作用。方法用Longa法制备大鼠大脑中动脉缺血（2h）／再灌注模型，HE染色观察梗死灶的形成，分别用TUNEL染色及免疫组化技术检测鼠脑缺血中心区及半暗带凋亡细胞与Caspase-1的表达。结果在缺血中心区Caspase-1及凋亡细胞主要见于缺血再灌注损伤早期；在缺血半暗带凋亡细胞与Caspase-1于缺血再灌注损伤早期表达不明显，于缺血再灌注24-48h则明显表达。结论细胞凋亡机制参与了缺血后迟发性神经元死亡，Caspase-1参与了其损伤过程。     

Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum

Pathological processes, including cerebral ischemia, can enhance neurogenesis in the adult brain, but the fate of the newborn neurons that are produced and their role in brain repair are obscure. To determine if ischemia-induced neuronal proliferation is associated with migration of nascent neurons toward ischemic lesions, we mapped the migration of cells labeled by cell proliferation markers and antibodies against neuronal marker proteins, for up to 2 weeks after a 90-min episode of focal cerebral ischemia caused by occlusion of the middle cerebral artery. Doublecortin-immunoreactive cells in the rostral subventricular zone, but not the dentate gyrus, migrated into the ischemic penumbra of the adjacent striatum and, via the rostral migratory stream and lateral cortical stream, into the penumbra of ischemic cortex. These results indicate that after cerebral ischemia, new neurons are directed toward sites of brain injury, where they might be in a position to participate in brain repair and functional recovery.