电刺激嗅球对脑缺血再灌注大鼠内源性神经干细胞增殖、迁移及分化的影响

The effect of electrical stimulation of olfactory bulb on neurogenesis after ischemia/reperfusion injury in rats

目的探讨嗅球电刺激对脑缺血再灌注大鼠脑内神经发生的影响。方法将健康雌性SD大鼠随机分为假手术组、脑缺血再灌注组、电刺激组、假刺激组,其中脑缺血再灌注组、电刺激组,以线栓法建立大鼠右侧大脑中动脉缺血(middle cerebral artery occlusion,MCAO)/再灌注模型,假手术组仅分离出颈内动脉;电刺激组在右侧嗅球内埋置双极电极,于MCAO再灌注后对嗅球进行电刺激,假刺激组只埋置电极,不进行电刺激;以5-溴脱氧尿嘧啶(Brdu)标记内源性神经干细胞(NSC)。假手术组于术后48h、1周,脑缺血再灌注组于缺血再灌注后48h、1周,电刺激组及假刺激组于刺激后48h、1周分别行Brdu免疫组化染色,观察各组脑室下区(SVZ)区NSC增殖情况;假手术组、缺血再灌注组、电刺激组、假刺激组4组大鼠在电刺激组行电刺激后腹腔注射Brdu 50mg/kg,2次/d,连续2d,4周后处死,分别取脑切片,进行免疫组化和免疫荧光双标染色,观察各组NSC迁移和分化情况。结果缺血再灌注组(37.67±1.97)、假刺激组(36.5±2.35)和电刺激组(43.67±1.63)大鼠48h后SVZ区Brdu阳性NSC细胞数较假手术组(15.5±1.52)明显增多(F=57.21,P<0.05),缺血再灌注组(41.17±2.94)、假刺激组(41.83±2.14)和电刺激组(47.67±2.34)1周后Brdu阳性细胞数较假手术组(15.5±1.52)增多(F=73.62,P<0.01),电刺激组在各时间点均较其他组Brdu阳性NSC细胞数增多(P<0.01)。缺血并注射Brdu 4周后缺血侧皮质区电刺激组Brdu阳性NSC细胞数(57.17±2.4)较缺血再灌注组(46.83±2.48)和假刺激组(45.83±2.14)增多(F=161.50,P<0.01);免疫荧光双标染色示电刺激组Brdu阳性NSC细胞中Brdu/神经元核抗原(Neun)双阳性细胞比例约为(74.67±3.61)%,Brdu/胶质纤维酸性蛋白(GFAP)双阳性细胞比例约为(38.83±3.36)%,与缺血再灌注组和假刺激组比较差异均无统计学意义(均P>0.05)。结论电刺激嗅球可能促进脑缺血大鼠SVZ区NSC增殖,并可能促进新生NSC向缺血皮质区迁移,增加缺血皮质新生神经元数量,但SVZ区新生NSC的分化不受影响。

Objective To investigate the effect of olfactory bulb（OB）electrical stimulation on neurogenesis of rats suffered from ischemia/reperfusion injury.Methods Healthy adult female Sprague-Dawley（SD）rats were randomly divided into the sham-operation group,the ischemia/reperfusion（I/R）group,the stimulation group,and the sham-stimulation group.Cerebral ischemia/reperfusion in the I/R group and the stimulation/sham-stimulation group was induced by intraluminal right middle cerebral artery occlusion（MCAO）.Arteries in the sham-operation group were dissected but not occluded.Electrical stimulation was performed via a bipolar electrode implanted in the right OB of rats,and rats in the sham-stimulation group were implanted electrodes but not stimulated.Bromodeoxyuridine（Brdu）was injected intraperitoneally to label endogenous neural stem cells（NSC）.The animals in all groups were sacrificed at 48 h,1 week after corresponding intervention.Immunohistochemistry staining was used to detect the proliferation of NSC in subventricular zone（SVZ）.After injection with Brdu,the rats were sacrificed 4weeks after stimulation.Fluorescent double staining for Brdu/Neuronal nuclei antigen（Neun）and Brdu/Glial fibrillary acidic protein（GFAP）was performed in the brain slices finally.Results In SVZ of rats,Brdu-positive cells began to increase 48 hafter ischemia in the I/R group and the stimulation group.There were more Brdu positive cells in the stimulation group than the I/R group at 48h（43.67±1.63,37.67±1.97;P〈0.05）and 7d（47.67±2.34,41.17±2.94;P〈0.01）after ischemia/reperfusion in rats.Four weeks after stimulation,the Brdu-positive cells were significantly increased in cortex of the stimulation group（57.17±2.4）than I/R group（46.83±2.48）and sham-operation group（45.83±2.14）（P 0.01）.Fluorescence double staining showed that stimulation of OB had no effect on the differentiation of NSC into neurons or gliocytes（P〉0.05）.Conclusions These observations demonstrated that electrical stimulation of OB might enhance proliferation of NSC in SVZ and promote migration of NSC to ischemic cortex,but OB stimulation has no effect on differentiation of NSC.