局部亚低温对大鼠脑缺血-再灌注后的神经保护作用

The protective effect of local mild hypothermia on oxidative DNA damage following focal cerebral ischemia/reperfusion in rats

目的探讨脑局部亚低温对脑缺血-再灌注大鼠模型的神经保护作用及其机制。方法健康雄性SD大鼠96只,随机将大鼠分为亚低温组和正常温度组,每组48只。每组中12只用于脑梗死体积的测定;36只用于DNA单链损伤的测定。每组再分为假手术组,缺血3h再灌注0.5、2、8、24、72h亚组,每个亚组6只大鼠。采用线栓法制作大鼠脑缺血-再灌注模型。使用冰帽控制基底核区温度在32～34℃,同时使用电热毯维持肛温在36.5～37.5℃。应用2,3,5-氯化三苯基四氮唑(TTC)染色测定脑梗死体积;观察再灌注72h两组大鼠存活率;行DNA多聚酶Ⅰ介导的生物素偶联腺嘌呤脱氧核苷酸的缺口平移标记(PANT)染色,检测DNA单链损伤。结果①生理学指标:亚低温组及正常温度组血糖、血压及血气差异无统计学意义。②72h生存率:亚低温组再灌注72h大鼠的生存率为92%,正常温度组为58%(χ2=6.75,P=0.027)。③梗死体积:亚低温组总的脑梗死体积,脑皮质、基底核区梗死体积分别为(61±28)、(20±17)、(42±14)mm3;正常温度组为(240±55)、(163±41)、(77±17)mm3,两组比较,总的脑梗死体积(P<0.05),皮质、基底核区脑梗死体积,差异均有统计学意义(P<0.01)。④DNA氧化损伤:PANT染色阳性细胞数于再灌注0.5h开始出现,亚低温组和正常体温组分别为(20±7)、(44±4)个/高倍视野(P<0.05);24h达高峰,亚低温组和正常体温组分别为(44±9)、(133±12)个/高倍视野(P<0.01)。亚低温可以减轻所有时间点的PANT染色阳性细胞数。结论局部亚低温可降低大鼠缺血-再灌注脑损伤,其机制可能与减轻缺血后DNA氧化损伤有关。

Objective To investigate the neuroprotective effect of local mild hypothermia in a rat model of focal cerebral ischemia/reperfusion and its mechanism. Methods Ninety-six SD rats were randomly allocated into hypothermia group and normal temperature group （n =48 in each group）. Twelve of them were only used to measure cerebral infarction volume, and 36 were only used to measure single-strand DNA damage. The rats in each group were redivided into subgroups of sham-operation, and 3-hour ischemia and reperfusion for 0. 5, 2, 8, 24 and 72 hours（ n =6 in each subgroup）. A focal cerebral ischemia/ reperfusion model was induced by an intraluminal suture method. Ice cap was used to control the temperature of basal nuclei area to 32 - 34℃, and at the same time rectal temperatures were maintained at 36.5 - 37.5 ℃ by using an electric blanket. The cerebral infarction volume was measured by TTC staining; the suvival rate of rats at 72 hours in both groups were observed; DNA single-strand breaks were evaluated by using positive DNA polymerase Ⅰ-mediated biotin-dATP nick-translation （PANT） staining. Results （1）physiological data：blood glucose, blood pressure, pH, PaO2 and PaCO2 showed no significant different. （2）The 72-hour survival rate： The survival rate after reperfusion for 72 hours in the mild hypotention group was 92%, and it was 58% in the normal temperature group （χ^2 = 6. 75,P = 0. 027）. （3）Infarction volume： The total cerebral infarction volume, the brain cortex and infarction volume in the basal nuclei areas were （61 ±28）, （20 ± 17） and （42 ± 14）mm^3 respectively in the hypothermia group; they were （ 240 ± 55 ）, （ 163 ± 41 ） and （77 ± 17 ） mm^3 in the normal temperature group. There were significant differences between the total cerebral infartion volume （ P 〈 0. 05 ）, cortex and infarction volume in the basal nuclei areas in both groups （P 〈 0. 01 ）. （4）Oxidative DNA damage： The numbers of positive cells of PANT staining began to appear at 0. 5 hour after reperfusion, and reached the peak at 24 hours, while hypothermia could reduce the numbers of positive cells of PANT staining at all time points. Conclusion The local mild hypothermia may reduce the focal cerebral ischemia/reperfusion injury, and its mechanism may be associated with the oxidative DNA damage after ischemia having been alleviated.