经典的小鼠大脑中动脉闭塞再灌注模型的建立和评价

目的:建立经典的小鼠大脑中动脉闭塞再灌注模型。方法:以头端涂硅胶的尼龙线自左颈外动脉向颈内动脉插入至大脑中动脉起始部,阻断血流2h后拔出线栓再灌注。通过神经功能评分和氯化三苯基四氮唑(tetrazoliumchloride,TTC)染色对模型进行评价。结果:小鼠缺血2h后,出现自主活动时向右旋转等功能障碍表现,TTC染色显示出梗死范围。再灌后22hTTC改变加重,神经病学评分同前。结论:该模型可以控制缺血和再灌注时间,是研究脑栓塞病理生理等的经典动物模型。     

限制性再灌注对脑缺血再灌注损伤的保护作用

目的 探究限制性再灌注对小鼠大脑中动脉闭塞(MCAO)模型缺血再灌注损伤的保护作用。方法 采用雄性C57BL/6J小鼠进行实验观察,其中假手术组7只,左颈总动脉结扎组(LCO组) 10只,通常再灌注组(ecMCAO组) 17只,限制性再灌注组(ccMCAO组) 22只。LCO组小鼠进行左侧颈总动脉结扎手术,24 h后行神经损伤评分和组织学评估。ecMCAO组小鼠经颈外动脉线栓造模,ccMCAO小鼠经颈总动脉线栓造模,在MCAO造模后2 h再灌注。小鼠在麻醉苏醒即刻与再灌注24 h,分别进行神经损伤评分,然后取材评估组织损伤。干湿重法测量脑组织含水量,并判断是否存在颅内出血。结果 LCO组小鼠各项指标与假手术组无明显差异。ecMCAO组小鼠再灌注24 h后神经损伤评分显著高于ccMCAO组(P 0.05)。ecMCAO组小鼠脑组织损伤体积与脑组织含水量大于ccMCAO组(均P 0.05)。脑出血率趋势ecMCAO组小鼠高于ccMCAO组,但无统计学意义。结论 C57 BL6/J小鼠大脑对侧代偿功能较好。与ecMCAO相比,ccMCAO模型再灌注后引起的组织与功能损伤较轻,说明限制性再灌注对小鼠脑缺血再灌注损伤有保护作用。     

颈内动脉线栓法建立小鼠局灶性脑缺血再灌注模型

目的 建立重复性强，稳定性好的小鼠大脑中动脉缺血模型，为以后对转基因小鼠的研究做好技术准备。方法 选择BALB/c和昆明白两种小鼠进行栓塞试验，观察小鼠品系、体重、线栓规格及术后温度对梗死结果的影响。结果 小鼠品系、小鼠体重与线栓规格是否匹配、术后温度是否保护恒定均影响小鼠线栓模型的稳定性。结论 必须严格控制上述各种因素以建立稳定的小鼠局灶性脑缺血模型，为神经科学研究提供可靠的技术支持。     

小鼠大脑中动脉缺血再灌注后缺血区及周边区星形胶质细胞的变化

目的 探讨小鼠大脑中动脉缺血再灌注后脑梗死体积、星形胶质细胞(AS)病理形态及其蛋白表达的变化.方法 选取69只成年健康雄性KM小鼠,将小鼠随机分为脑缺血2h再灌注1、4、10、24、48、72 h模型组(n=9)以及假手术组(n=6,仅结扎右侧颈总动脉)和正常组(n=9).采用线栓法制备小鼠大脑中动脉局灶性脑缺血再灌注模型.应用2,3,5-氯化三苯基四氮唑(TTC)染色法观察脑梗死的部位与体积;免疫组化法观察脑缺血中心区与周边区不同再灌注时间点胶质纤维酸性蛋白(GFAP)的表达.结果 缺血再灌注1h开始出现脑梗死灶,且再灌注24 h时梗死体积最大,之后逐渐缩小(F=745.534,P=0.000).小鼠脑缺血再灌注后缺血中心区AS肿胀、肥大进而较快发生凋亡,其GFAP表达水平低于周边区(P＜0.05);而缺血周边区GFAP阳性表达的AS出现反应性活化,进而发生形态学改变;缺血周边区及对侧相应脑组织区域GFAP的表达量均随再灌注时间的延长呈现增加趋势(P＜0.05).结论 AS反应性活化可能是一种缺血后的全脑保护性防御机制,尤其是在缺血周边区,其活化程度影响脑组织的存活与修复,提示在脑缺血再灌注过程中AS反应性活化可能在脑损伤后可塑性形态及功能改变中发挥重要作用.     

大鼠局灶性脑缺血再灌注后凋亡诱导因子的表达与神经元凋亡的关系

目的 探讨大鼠局灶性脑缺血再灌注后凋亡诱导因子(AIF)的表达变化及与细胞凋亡的关系.方法 将Wistar大鼠分为假手术组(6只)和模型组(30只).模型组大鼠采用线栓法建立大脑中动脉闭塞模型,假手术组大鼠插线较模型组浅,不造成大脑中动脉闭塞.观察假手术组及模型组缺血再灌注后6 h、24 h、48 h、72 h和7 d缺血半暗带AIF的表达,同时利用TUNEL法观察对应区域细胞凋亡的动态变化规律.结果模型组脑缺血再灌注6 h在缺血半暗带区AIF阳性细胞显著增加,再灌注48 h达到高峰[(130.47±11.32)个];各时相点均可见细胞凋亡,凋亡细胞数以再灌注48 h最多f(118.53±11.67)个];各组问比较差异均有统计学意义(JP<0.05).结论 局灶性脑缺血再灌注可致AIF表达增加.并引起细胞凋亡.     

白藜芦醇对缺血再灌注小鼠神经干细胞增殖作用的研究

目的研究白藜芦醇对小鼠缺血再灌注后海马区神经干细胞增殖的影响,探讨其促进缺血再灌注后神经功能恢复的机制。方法 15只雄性BALB/c小鼠随机分为缺血再灌注组、白藜芦醇组及假手术组,采用大脑中动脉线栓法制作局灶性脑缺血模型,Brdu标记增殖的干细胞,免疫组化法测定增殖细胞数。结果白藜芦醇组与缺血再灌注组相比Brdu阳性细胞数存在差异。结论白藜芦醇可以促进小鼠缺血再灌注后海马区神经干细胞的增殖,可能在其促进缺血后神经功能恢复中发挥作用。     

大鼠脑局灶缺血／再灌注对外周血单个核CD34^＋细胞的影响

目的本研究探讨大鼠脑局灶缺血/再灌注是否影响外周血单个核CD34+细胞的数量.方法采用大鼠大脑中动脉(MCA)缺血/再灌注线栓法模型,将大鼠随机分为3组对照组,假手术组,动物模型组.取再灌注后1、3、6、12 h和1、2、3、4、7、14 d,10个观察点,测外周血单个核CD34+表达情况.结果大鼠脑缺血/再灌注模型中外周血单个核CD34+细胞在再灌注后1 h～2 d无明显变化,在脑缺血/再灌注后3～7 d明显减少,14 d后恢复.结论大鼠脑缺血/再灌注模型再灌注后1 h～2 d再灌注后外周血单个核CD34+细胞没有变化,在脑缺血/再灌注后3～7 d明显减少.     

改良线栓法制备大鼠局灶性脑缺血／再灌注模型的实验研究

目的建立一种简便可靠、创伤较小的大鼠局灶性脑缺血／再灌注模型。方法对ZeaLonga线栓法进行适当改进,制’作大鼠右侧大脑中动脉闭塞2h后再灌注模型（实验组,n=40）。于建模前及建模后不同时间,评估大鼠的神经功能,并取脑组织进行病理组织学检查。另取10只大鼠作为假手术对照组。结果实验组40只大鼠中,共死亡13只（32．5％）,余27只建模成功。再灌注后4、12、24、32和64h,实验组的神经功能缺乏评分明显高于假手术对照组（P〈0．01）;术中栓线插入长度与大鼠体质量呈正相关（P〈0．01）;实验组大鼠的右侧大脑中动脉支配区域脑组织有明显的缺血性损伤改变。结论本实验采用改良线栓法制备的大鼠大脑中动脉栓塞／再灌注模型简便易行、创伤小且稳定性好,是用于研究局灶性脑缺血／再灌注损伤较为理想的动物模型。     

两种局灶性脑缺血再灌注模型的比较

目的比较两种大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)局灶性脑缺血再灌注模型,寻找一种更加理想、稳定和可靠的模型。方法将22只SD大鼠随机分为颈外动脉插线组和颈总动脉插线组,采用线栓法建立大鼠脑缺血再灌注模型,MCAO2h再灌注24h,采用TTC染色和NF-κB免疫组化染色,并分别测定脑梗死体积和NF-κB表达量。结果两种插线方法都能造成大鼠局灶性脑梗死,颈外动脉插线组再灌注状态优于颈总动脉插线组,且脑梗死体积和NF-κB表达量均高于颈总动脉插线组。结论颈外动脉插线闭塞大脑中动脉是一种较为理想和可靠的大鼠MCAO局灶性脑缺血再灌注模型制作方法。     

可重复性小鼠局灶性脑缺血/再灌注模型的探讨

目的介绍一种标准的小鼠局灶性脑缺血/再灌注模型的制作方法,并观察不同脑缺血/再灌注时间脑梗死体积和脑水肿的变化。方法用腔内线栓法制作脑缺血/再灌注动物模型,用TTC染色法进行脑大体观察,用甲酚紫染色法观察脑切片梗死灶,用脑血流激光多普勒监测脑血流的变化,用ImageJ软件计算脑梗死体积和脑水肿。结果当线栓封闭大脑中动脉时,脑血流就会急剧下降至最低水平,拔出线栓后脑血流迅速上升至缺血前水平。脑缺血后,脑片上呈现明显的梗死灶,脑梗死体积和脑水肿的大小较恒定。脑缺血90min再灌注24h组梗死体积、脑水肿体积、脑水肿百分数及神经功能缺损程度均显著大于脑缺血30min再灌注24h组(P<0.001)。脑缺血30min/再灌注72h脑水肿非常明显(72.6±4.3)mm3,再灌注7d时脑水肿开始减退,仅为(50.9±4.1)mm3,再灌注30d时脑容积出现萎缩,脑水肿呈负值(-20.1±1.8)mm3。结论该小鼠局灶性脑缺血/再灌注模型具有重复性好、容易操作的优点。脑缺血30min就可造成不可逆性脑损害,脑水肿在再灌注72h即达到高峰。     

Effects of tissue type plasminogen activator in embolic versus mechanical models of focal cerebral ischemia in rats.

Tissue type plasminogen activator (tPA) can be effective therapy for embolic stroke by restoring cerebral perfusion. However, a recent experimental study showed that tPA increased infarct size in a mouse model of transient focal ischemia, suggesting a possible adverse effect of tPA on ischemic tissue per se. In this report, the effects of tPA in two rat models of cerebral ischemia were compared. In experiment 1, rats were subjected to focal ischemia via injection of autologous clots into the middle cerebral artery territory. Two hours after clot injection, rats were treated with 10 mg/kg tPA or normal saline. Perfusion-sensitive computed tomography scanning showed that tPA restored cerebral perfusion in this thromboembolic model. Treatment with tPA significantly reduced ischemic lesion volumes measured at 24 hours by >60%. In experiment 2, three groups of rats were subjected to focal ischemia via a mechanical approach in which a silicon-coated filament was used intraluminally to occlude the origin of the middle cerebral artery. In two groups, the filament was withdrawn after 2 hours to allow for reperfusion, and then rats were randomly treated with 10 mg/kg tPA or normal saline. In the third group, rats were not treated and the filament was not withdrawn so that permanent focal ischemia was present. In this experiment, tPA did not significantly alter lesion volumes after 2 hours of transient focal ischemia. In contrast, permanent ischemia significantly increased lesion volumes by 55% compared with transient ischemia. These results indicate that in these rat models of focal cerebral ischemia, tPA did not have detectable negative effects. Other potentially negative effects of tPA may be dependent on choice of animal species and model systems.     

使用"夹心TTC染色法"可显著提高脑缺血模型中梗死灶的检测效率

目的 优化红四氮唑(TTC)染色法,以提高对脑缺血模型中梗死灶的位置及面积的精确判定程度.方法 采用线栓法建立Balb/c小鼠的大脑中动脉阻塞(MCAO)模型,缺血5h后断头取脑,利用"夹心TTC染色法"对梗死灶进行判定.结果 传统TTC染色仅有单侧脑片染色效果较好而另侧染色效果很差,相比之下,"夹心TTC染色法"染出的脑片整体颜色鲜红、均一,两侧染色程度均十分良好,能够明确判定出梗死灶.结论 优化后的"TTC夹心染色法"适合于梗死灶位置以及面积的精确判定,可显著提高脑缺血模型中梗死灶的检测效率.     

Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice.

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.     

Neuroprotection in ischemic mouse brain induced by stem cell-derived brain implants.

Protective mechanisms of the brain may reduce the extent of injury after focal cerebral ischemia. Here, we explored in a mouse model of focal cerebral ischemia potential synergistic neuroprotective effects of two mediators of neuroprotection: (i) neuronal or glial precursor cells and (ii) the inhibitory neuromodulator adenosine. Embryonic stem (ES) cells, engineered to release adenosine by biallelic disruption of the adenosine kinase gene, and respective wild-type cells were induced to differentiate into either neural or glial precursor cells and were injected into the striatum of mice 1 week before middle cerebral artery occlusion. All stem cell-derived graft recipients were characterized by a significant reduction in infarct volume, an effect that was augmented by the release of adenosine. Neuroprotection was strongest in adenosine-releasing glial precursor cell recipients, which were characterized by an 85% reduction of the infarct area. Graft-mediated neuroprotection correlated with a significant improvement of general and focal neurologic scores. Histologic analysis before and after ischemia revealed clusters of implanted cells within the striatum of all treated mice. We conclude that ES cell derived adenosine-releasing brain implants provide neuroprotection by synergism of endogenous precursor cell-mediated effects and paracrine adenosine release.     

PKH26和DAPI联合示踪骨髓干细胞在脑缺血模型小鼠脑内迁移的实验研究

目的 探讨PKH26和DAPI联合标记骨髓干细胞在脑内迁移过程中的示踪作用.方法 用PKH26和DAPI联合标记从Balb/c小鼠骨髓中分离出的骨髓单个核细胞(BMMCs),用改良电凝法制备局灶性脑缺血模型后,将标记的BMMCs经尾静脉移植入脑缺血小鼠体内,对照组经尾静脉注入PBS.移植2w后取脑组织,通过激光共聚焦荧光显微镜观察BMMCs向脑内迁移的情况.结果 实验组小鼠脑组织内可发现PKH26和DAPI双标的神经样细胞.结论 PKH26和DAPI可以联合标记向脑缺血模型小鼠脑组织迁移的骨髓干细胞,为体内示踪骨髓干细胞脑内迁移提供更加准确的实验方法.     

大鼠脑局灶缺血后AC133抗原和AC133mRNA的表达

目的:探讨AC133抗原及AC133 mRNA在缺血再灌注脑组织中的表达。方法:将成年SD雄性大鼠随机分为3组:正常组,假手术组,脑缺血再灌注组。采用大鼠大脑中动脉缺血再灌注线栓法模型。取缺血再灌注2、3、47、d作为观察点。用免疫组化法检测脑组织AC133抗原表达,RT-PCR法检测AC133基因的表达。结果:大脑缺血再灌注后4 d,AC133抗原在缺血大脑半球梗死区周围的部分中、小血管内皮细胞胞质染色阳性。而再灌注后2、3和7 d无阳性染色。脑缺血再灌注37、d缺血脑组织AC133 mRNA上调。结论:大鼠脑局灶缺血后AC133在基因和蛋白水平上均有不同程度的表达,AC133可能参与大脑局灶缺血后血管内皮的功能。AC133 Expression in Brain of Ischemia-Reperfused Rat HU Xiang-Shu,ZHOU Dong,LUO Zu-Ming Department ofNeurology,Guangdong 999 Brain Hospital,Guangzhou 510510,China     

c—fos反义寡聚核苷酸对脑缺血性损伤和电针抗损伤作用的影响

用c-fos反义寡聚核苷酸脑内微量注射、TTC染色、c-Fos免疫组织化学和电针等技术和方法,探讨即早反应基因c-fos在大鼠局灶性脑缺血（MCAO）模型的脑损伤中和电针抗局灶性脑缺血脑损伤中所起的作用。实验结果表明,局灶性脑缺血可引起c-fos在缺血侧皮质的大量表达,电针能部分抑制这种表达,使脑缺血梗死灶体积减小。在缺血中心区注射c-fos反义寡聚核苷酸后,脑内c-fos的表达基本上被完全阻断,导致脑梗死灶的体积明显增大,电针抗脑缺血脑损伤的作用也被取消,提示脑缺血后,脑内的c-fos适度表达可能对脑损伤有一定的保护作用。电针可能部分抑制了脑内c-fos表达,调整了缺血后的c-fos表达的程度,对脑缺血损伤起一定的保护作用。     

局灶性脑缺血大鼠脑内mdr1/P-glycoprotein表达的变化

目的观察大鼠局灶性脑缺血损害后脑内mdr1／P—glycoprotein的表达变化。方法大鼠大脑中动脉栓塞法(MCAO法)制作局灶性脑缺血模型,脑切片免疫组织化学染色检测mdr-1／P—glycoprotein在脑内的表达部位及表达时程的变化。结果 mdr-1／P—glycoprootein在缺血侧皮层和纹状体的血管内皮细胞表达增多,并出现在同侧损伤部位的神经元,其在损伤后2h开始出现,6h达到高峰,之后开始下降,到24h不能被检测到。结论大脑中动脉阻塞后可以诱导缺血损伤侧的血管内皮细胞P—glycoprotein过量表达,而同侧的神经元短暂表达P-glyco- protein．     

Anti-inlfammatory properties of lipoxin A4 protect against diabetes mellitus complicated by focal cerebral ischemia/reperfusion injury

Lipoxin A4 can alleviate cerebral ischemia/reperfusion injury by reducing the inlfammatory reaction, but it is currently unclear whether it has a protective effect on diabetes mellitus complicated by focal cerebral ischemia/reperfusion injury. In this study, we established rat models of diabetes mellitus using an intraperitoneal injection of streptozotocin. We then induced focal cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery for 2 hours and reperfusion for 24 hours. After administration of lipoxin A4via the lateral ventricle, infarction volume was reduced, the expression levels of pro-inlfammatory factors tumor necrosis factor alpha and nuclear fac-tor-kappa B in the cerebral cortex were decreased, and neurological functioning was improved. These ifndings suggest that lipoxin A4 has strong neuroprotective effects in diabetes mellitus complicated by focal cerebral ischemia/reperfusion injury and that the underlying mech-anism is related to the anti-inlfammatory action of lipoxin A4.     

米诺环素对大鼠局灶性脑缺血再灌注后ICAM-1表达的影响

目的观察米诺环素对大鼠短暂性脑缺血再灌注后细胞间黏附分子1表达的影响。方法采用改良线栓法制备大鼠大脑中动脉缺血2h再灌注24h模型,随机分为假手术组、缺血再灌注模型组、米诺环素处理组和米诺环素预处理组,每组16只。模型成功后观测各组大鼠的神经行为变化、脑梗死体积以及HE染色计数缺血区中性粒细胞的浸润数目,应用免疫组化方法检测细胞间黏附分子-1的表达情况。结果米诺环素能明显改善大鼠局灶性脑缺血再灌注引起的神经行为障碍,减少脑梗死体积,减少脑缺血引起细胞间黏附分子1的表达,抑制中性粒细胞的浸润。结论米诺环素对大鼠局灶脑缺血再灌足损伤有保护作用,抑制黏附分子可能是一种机制。