亚低温对局灶性脑缺血-再灌注大鼠脑皮质脑源性神经营养因子表达及神经元凋亡的影响

Effect of mild hypothermia on brain-derived neurotrophic factor expression and neuron apoptosis in rat focal cerebral ischemia-reperfusion

目的观察局灶性脑缺血-再灌注后亚低温干预对大鼠脑源性神经营养因子表达及神经元凋亡的影响,并探讨脑源性神经营养因子在亚低温脑保护机制中的作用。方法采用线栓法制备成年雄性SD大鼠左侧大脑中动脉闭塞局灶性脑缺血-再灌注改良模型,缺血时间2h。随机分为常温缺血组和亚低温缺血组。常温时大鼠脑温控制于36.5℃～37.5℃,肛温为35.9℃～36.9℃;亚低温时脑温维持于32.5℃～33.5℃,肛温为32.2℃～33.1℃。两组大鼠分别于脑缺血-再灌注及亚低温干预后2、6、24和72h进行神经功能缺损评分,并同时行三苯基氯化四唑(TTC)染色、HE染色、TUNEL染色、免疫组化染色及免疫组化与TUNEL双重染色,从而评估大鼠神经功能缺损状况;检测脑梗死体积及脑源性神经营养因子表达水平;观察组织病理学变化和神经元凋亡数量。结果与常温缺血组相比,亚低温缺血组大鼠神经功能缺损评分低(P<0.01),脑梗死体积小(P<0.01),缺血灶周围脑皮质中的脑源性神经营养因子表达水平增高(P<0.01),而且神经元凋亡数量少(P<0.01)。在脑源性神经营养因子免疫组化染色呈阳性反应的神经元细胞核中,未发现TUNEL染色阳性者。结论亚低温干预治疗可促进缺血灶周围的脑皮质对脑源性神经营养因子的表达,从而抑制神经元凋亡,减少大鼠脑梗死体积,改善神经功能缺损体征。

Objective To observe the effects of mild hypothermia on the expression of brain-derived neurotrophic factor （BDNF） and neuron apoptosis after focal cerebral ischemia-reperfusion, and study the effect of BDNF on the neuroprotective mechanisms in mild hypothermia. Methods The modified focal cerebral ischemia-reperfusion models of adult male SD rats were established by occlusion of left middle cerebral artery for 2 hours. Then the model rats were randomizely divided into two groups ie. normothermic ischemic group and hypothermic ischemic group. In normothermic group, cerebral temperature and rectal temperature were controlled at 36.5℃～37.5℃ and 35.9℃～36.9℃;, respectively, while the cerebral and rectal temperature were controlled at 32.5℃～33.5℃ and 32.2℃～33.1℃ respectively in hypothermic group. The effect of focal cere bral ischemia-reperfusion and mild hypothermia in rats were observed at 2 h, 6 h, 24 h and 72 h after treatment. The neurological impairment was evaluated with Neurological Grading Scale. The infarction volume was measured with triphenyltetrazolium chloride （TTC） staining. The histopathological changes in brain were studied with HE staining. The amount of neuron apoptosis after focal ischemia was observed by TUNEL staining. The expressions of BDNF were measured with immunohistochemical staining or combined immunohistochemical and TUNEL staining. Results Compared to normothermic group, the neurological defect score, the infarction volume and the quantity of apoptotic neurons were significantly decreased （P〈0.01） and the quantity of BDNF positive neurons was significantly increased in mild hypothermic group （P〈 0.01）. None of positive TUNEL staining was found in the BDNF immunohistochemical staining positive neuron nucleus. Conclusion Intervention of mild hypothermia may upregulate the expression of BDNF on the cortex surrounding the ischemic focus and results in restraining the neuronal apoptosis, reducing the infarction volume and decreasing the neurological defect signs.