短暂脑缺血后成年和老年大鼠大脑髓鞘相关蛋白基因表达变化

目的探讨短暂脑缺血后成年和老年大鼠大脑髓鞘相关蛋白基因表达变化及其意义。方法以线栓法制作成年和老年SD大鼠短暂脑缺血模型（阻塞60min再灌注1d、7d和14d）；采用5末端标记地高辛的寡核苷酸探针荧光原位核酸分子杂交检测短暂脑缺血大鼠大脑梗死中心区、梗死周边区和梗死对侧区PLP mRNA和MyT1 mRNA照射后表达变化，并计数阳性细胞数量。结果（1）短暂脑缺血后成年和老年大鼠大脑皮质梗死中心区PLP mRNA和MyT1 mRNA阳性细胞数明显减少，两组之间比较无显著差异。（2）短暂脑缺血后早期成年和老年大鼠梗死周边区PLP mRNA和MyT1 mRNA阳性细胞数无明显变化，之后（7d和14d）逐渐增加，成年大鼠PLP mRNA和MyT1 mRNA阳性细胞数增加更明显。结论成年大鼠短暂脑缺血后急性期梗死周边区髓鞘相关蛋白基因表达强于老年大鼠，提示年龄是影响少突胶质前体细胞参与脑缺血后损伤修复的因素之一。     

局灶性脑缺血再灌注大鼠大脑NG2和O4表达变化

目的:探讨脑缺血再灌注大鼠早期大脑少突胶质祖细胞及未成熟少突胶质细胞变化及意义。方法:以线栓法制作SD大鼠局灶性脑缺血再灌注模型(阻塞90min再灌注1d、3d和7d);用免疫荧光组织化学法检测脑缺血再灌注后早期大鼠大脑梗死中心区、梗塞周边区和缺血对侧NG2和O4阳性细胞数量。结果:脑缺血再灌注后各时间点梗死中心区NG2和O4阳性细胞数明显减少;梗塞周边区NG2和O4阳性细胞数随着再灌注时间延长而逐渐增加,再灌注3d和7d增加显著;脑梗死对侧区NG2和O4阳性细胞数无明显变化。结论:成年SD大鼠脑缺血再灌注后梗塞周边区少突胶质祖细胞及未成熟少突胶质细胞增多,可能参与缺血损伤的修复过程。     

NBQX对脑缺血再灌注后成年大鼠大脑少突胶质前体细胞的保护作用

目的 探讨脑缺血再灌注后成年大鼠大脑少突胶质前体细胞的变化及MK-801和NBQX对其保护作用.方法 以线栓法制作成年SD大鼠局灶性脑缺血再灌注模型(阻塞90min再灌注1d、7d和14d),分别向其大脑皮质缺血区立体定向注射5mmol/L 的MK-801和50 mmol/L的NBQX各1μl,用免疫荧光组织化学法检测脑缺血再灌注后成年大鼠早期大脑梗死中心区、梗死周边区和缺血对侧NG2和O4阳性细胞数量.结果 脑缺血再灌注后成年大鼠大脑皮质梗死中心区NG2 和O4阳性细胞逐渐减少, NBQX组大鼠NG2和O4阳性细胞数减少的幅度较少.脑缺血再灌注后梗死周边区NG2和O4阳性细胞数逐渐增加,NBQX组大鼠增加更明显,而MK-801组与生理盐水组无明显差别.结论 成年SD大鼠脑缺血再灌注后梗死周边区少突胶质前体细胞增多.NBQX对少突胶质前体细胞早期的缺血性损伤有保护作用.     

大鼠局灶性脑缺血后少突胶质前体细胞激活及髓鞘再生的实验研究

目的探讨脑缺血再灌注大鼠少突胶质前体细胞(oligodendrocyte progenitor cells,OPCs)及髓鞘的表达变化。方法线栓法建立大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型,免疫组化方法检测脑缺血再灌注后不同时间点1d、1w和2w不同脑区(梗死中心区、梗死周边区和梗死对侧区)OPCs特异性细胞标志物NG2的阳性细胞数及和髓鞘标志物碱性髓鞘蛋白(myelin basic protein,MBP)的表达变化。结果脑缺血后梗死中心区NG2阳性细胞数和MBP的表达随着再灌注时间延长而逐渐减少;梗死周边区NG2阳性细胞数在1w～2w增加,MBP的表达在24h～1w内降低,2w恢复到正常水平;梗死对侧区NG2阳性细胞数和MBP的表达无明显变化。梗死周边区OPCs细胞呈"单极"或"双极"分裂状,并从梗死灶的外带迁移到内带,提示OPCs细胞激活、增生并发生迁移。结论脑缺血再灌注后梗死周边区NG2细胞增多,使得一度缺失的成熟少突胶质细胞及髓鞘得到补充,提示NG2细胞可能参与缺血损伤的修复过程。     

雌性和雄性大鼠脑缺血再灌注神经干细胞增殖的比较研究

目的探讨雌性和雄性成年大鼠脑缺血再灌注海马齿状回颗粒下层(subgranular zone,SGZ)、侧脑室室下层(subventricular zone,SVZ)和梗死皮质周边神经干细胞(neural stem cell,NSC)增殖的差异性.方法雌性和雄性成年大鼠各16只,制作线栓法大脑中动脉闭塞2 h后再灌注模型,用免疫组织化学方法动态检测海马齿状回SGZ、SVZ和梗死皮质周边5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)标记的阳性细胞数的变化,并采用HPIAS图像分析系统进行对比分析.结果雄性大鼠SGZ、SVZ及梗死周边区域BrdU阳性细胞在缺血再灌注3 d开始增加,7 d达到高峰,11d后开始下降,18 d进一步减少.而雌性大鼠则是3 d开始增加,7 d明显增加,11d达到高峰,18 d才开始减少,且各时间点雌性大鼠的BrdU阳性细胞数均明显高于雄性大鼠.结论脑缺血再灌注雌雄成年大鼠均有NSC增殖,雌性大鼠NSC增殖较雄性大鼠更为明显.     

氯化锂-匹罗卡品致(疒间)大鼠脑髓鞘转录因子的表达及其意义

目的探讨氯化锂-匹罗卡品致疒间大鼠脑髓鞘转录因子1(MyT1)的表达及其意义.方法给SD大鼠先后腹腔注射氯化锂、匹罗卡品,制成癫疒间动物模型;用免疫荧光组化法检测癫疒间大鼠癫疒间发作后不同时间大脑皮质和海马CA1区MyT1阳性细胞数.结果与对照组相比,癫疒间后1 d组大鼠海马CA1区MyT1阳性细胞数显著减少(P＜0.05),癫疒间后其他各时间组大鼠脑皮质和海马CA1区MyT1阳性细胞数均有明显的增加,其中癫疒间后7 d组MyT1阳性细胞数最多(P＜0.01,P＜0.05).结论氯化锂-匹罗卡品致疒间大鼠早期大脑MyT1表达增加,并有时程性变化.     

N-Methyl-D-Aspartate受体拮抗剂对脑缺血后成年大鼠神经干细胞内源性激活的实验研究

目的　研究N - Methyl- D- Aspartate(NMDA)受体拮抗剂MK- 80 1对脑缺血后神经干细胞(NSC)激活的作用。方法　将4 0只SD大鼠分成对照组和实验组,两组大鼠均采用传统线栓法作成大脑中动脉缺血再灌注模型,实验组大鼠腹腔注射MK- 80 1,对照组腹腔注射生理盐水,通过免疫组织化学技术标记鼠脑海马齿状回颗粒细胞层(SGZ)、室管膜下层(SVZ)及梗死皮质周边区注射后第3、7、11、18天的Brdu、Nestin阳性细胞数。结果　对照组大鼠Brdu、Nestin阳性细胞7d在SGZ出现一小高峰,然后迅速下降,11d阳性细胞甚少,梗死皮质周边区更少;而实验组Brdu、Nestin阳性细胞3d在SVZ明显表达,7～11d在SGZ区达高峰,并可持续至18d,同样梗死皮质区Brdu、Nestin阳性细胞7～18d表达明显,两组比较,有统计学意义(P<0 .0 1)。结论　NMDA受体拮抗剂MK- 80 1在脑缺血后,能促进NSC的增殖、分化。     

锂-匹罗卡品致癫大鼠早期大脑皮质MyT1 mRNA表达变化

目的 探讨氟化锂-匹罗卡品致癫大鼠脑髓鞘转录因子1基因表达变化及其意义。方法 以氯化锂、匹罗卡品对雄性成年SD大鼠先后腹腔注射，制成癫痫持续状态动物模型；采用5′末端标记地高辛的寡核苷酸探针荧光原位核酸分子杂交检测癫性发作后早期大鼠大脑皮质MyT1 mRNA阳性细胞数量。结果 与对照组相比，癫痫后1d组大鼠脑皮质MyT1 mRNA阳性细胞数减少（P＜0.05）；其他各组大鼠脑皮质MyT1 mRNA阳性细胞数都有明显的增加，其中癫痫后7d和14d组MyT1mRNA阳性细胞数都有非常显著的增加（P＜0.05，P＜0.01）。结论 氯化锂-匹罗卡品致癫大鼠早期大脑MyT1 mRNA表达增加，并有时程性变化，提示与早期脑损伤修复有关。     

大鼠脑缺血再灌注损伤后脑组织STAT3变化的免疫组织化学研究

目的 探讨信号转导和转录激活子（ＳＴＡＴ）３在大鼠局灶性脑缺血再灌注损伤中的表达及其与缺血性神经细胞损伤的关系方法 用ＡＢＣ免疫组化方法观察大鼠局灶性脑缺血再灌注损伤后脑组织中的ＳＴＡＴ３蛋白免疫反应阳性细胞分布。结果 正常和假手术大鼠脑内以及脑缺血后的非缺血半球脑组织中未发现有ＳＴＡＴ３免疫反应阳性细胞,脑缺血再灌注损伤后１２小时在栓塞侧梗死区可见少量ＳＴＡＴ３免疫阳性细胞,２４小时后阳性细胞显著增多达高峰,在缺血侧纹状体和缺血皮质周边区表达最明显,１周后梗死周边区少数神经细胞仍有阳性表达。差异有显著意义（Ｐ〈０．０１）。结论 ＳＴＡＴ３活化及超量表达可能介导了缺血神经细胞信号转导过程,并参与了脑缺血神经细胞损伤与修复的病理生理过程。     

大鼠局灶脑缺血后AC133抗原表达的初探

目的探讨AC133抗原在脑缺血再灌注脑组织中是否表达.方法采用大鼠大脑中动脉缺血再灌注线栓法模型,将成年SD雄性大鼠随机分为(1)正常组,(2)假手术组,(3)动物模型组,分别取缺血再灌注2、3、4、7 d作为观察点,每个观察点6只大鼠,共36只大鼠,根据神经功能评分纳入实验,并采用免疫组化染色法检测脑组织AC133抗原表达.结果大脑缺血再灌注后4 d AC133蛋白在大脑半球梗死区周围的部分中、小血管内皮细胞胞浆染色阳性,而再灌注后2、3、7 d无阳性表达.结论大鼠脑缺血再灌注4 d脑梗死边缘区域部分血管内皮细胞AC133抗原表达阳性.AC133抗原可能参与大脑局灶缺血后的血管内皮功能.     

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。     

Insulin-like growth factor-1 is an endogenous mediator of focal ischemia-induced neural progenitor proliferation

The adult mammalian brain contains resident neural progenitors in the subgranular zone of the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricles. The proliferation of neural progenitors increases after focal cerebral ischemia in both of these regions, but the mechanisms that promote ischemia-induced neural progenitor proliferation are not yet understood. We hypothesize that diffusible factors from the ischemic area play a role in this process as the DG is remote from the area of infarction. In this study, we observed that the peak of neural progenitor proliferation in the ipsilateral DG was between day 2 and day 4 of reperfusion after transient middle cerebral artery occlusion in adult spontaneously hypertensive rats. GeneChip and real-time PCR analysis showed a three- to 102-fold increase in the expression of 15 diffusible, mitogenic factors in the ischemic cortex at 3 days of reperfusion. Of these, insulin-like growth factor-1 (IGF-1) showed increased protein expression in the activated astrocytes in the ischemic penumbra. In addition, the progenitors in both the SVZ and DG showed IGF-1 receptor expression. Inhibiting IGF-1 activity by introcerebroventricular infusion of IGF-1 antibody significantly prevented the ischemia-induced neural progenitor proliferation. These results indicate that IGF-1 formed in the ischemic penumbra might be one of the diffusible factors that mediate post-ischemic neural progenitor proliferation.     

Induction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in rat brain after focal ischemia/reperfusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, has been recently identified to be involved in the initiation of neuronal apoptosis. To investigate the serial changes and cellular localization of GAPDH expression, and its role in ischemia/reperfusion-induced neuronal apoptosis, the authors analyzed immunohistochemically brain areas of rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Nuclear overexpression of GAPDH was noted in the ischemic core area after 2 hours of MCAO without reperfusion. During the subsequent reperfusion, nuclear accumulation of GAPDH in this area decreased in a time-dependent manner. However, cytoplasmic and nuclear GAPDH immunoreactivity was detected in neurons of the penumbra area of the parietal cortex, in rats subjected to 2-hour MCAO followed by 3-hour reperfusion. The increase of nuclear GAPDH immunoreactivity was persistently noted up to 48 hours of reperfusion, whereas cytoplasmic immunoreactivity correlated inversely with the duration of reperfusion. Moreover, double staining revealed colocalization of nuclear GAPDH and TUNEL in the penumbra area. The authors' study demonstrated that overexpression of GAPDH and nuclear translocation occurred in both the ischemic core and penumbra area soon after focal ischemia. These processes could be viewed as an early marker of ischemia/reperfusion-induced apoptotic neuronal death. The results suggest that GAPDH may play a critical role in the progression and spread of ischemic neuronal damage.     

氯化锂-匹罗卡品致痫大鼠脑髓鞘转录因子的表达及其意义

目的探讨氯化锂-匹罗卡品致     

Expression of IL-6 in the ischemic penumbra

We examined the expression of IL-6 within the ischemic penumbra at various time points after transient (3 h) middle cerebral occlusion (MCA-O) in rats. The animals were killed at 1, 3, 7 or 14 days following operation. Coronal brain sections were processed for immunohistochemistry with antibodies against GFAP, OX 42, IL-6 and Nissl-staining. Glial activation within the penumbra started on day one after ischemia and persisted up to day 14. Expression of IL-6 was not present in sham-operated controls. One day after MCA-O there were several IL-6-positive cells in the penumbra. This expression of IL-6 increased on day 3 and remained elevated up to day 14. According to the shape of the IL-6-positive cells they seem to be microglia and neurons. The present results demonstrate a longlasting expression of IL-6 in the ischemic penumbra.     

Endoplasmic reticulum in the penumbra following middle cerebral artery occlusion in the rabbit

Caspase-12 has been localized to endoplasmic reticulum (ER) and showed to involve ER stress-induced apoptosis. In the present work we investigated the temporospatial alterations of caspase-12 immunoreactivity in the penumbra following cerebral ischemia/reperfusion in rabbit. Transient cerebral ischemia was produced by intraluminal occlusion of the middle cerebral artery for 2 h followed by 1 h, 6 h, 1 day, 3 days, 7 days and 14 days of reperfusion. Caspase-12 immunohistochemistry was first increased in the penumbra 1 h after reperfusion, with a peak at day 1 to day 3, and then gradually decreased to basal level at day 14. The number of TUNEL-positive cells and ultrastructural observation of brain sections in the penumbra showed a similar change at the same time points. ER mediated by caspase-12 participated in apoptosis induced by cerebral ischemia/reperfusion injury, which may provide a new area for therapeutic intervention to ameliorate outcomes following cerebral ischemia.     

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.