甘露醇对脑缺血／再灌注损伤后细胞凋亡影响的实验研究

Evaluation of the anti-apoptotic effect of mannitol on cerebral ischemia/reperfusion injury in mice

目的进一步验证昆明小鼠脑缺血／再灌注损伤（isehemia／reperfusioninjury，I／RI）实验动物模型的实用性和脑I／RI后甘露醇干预的最佳时机。方法健康昆明小鼠36只，制备全脑I／RI模型后，按随机数字表法分为6组（每组6只）：假手术组（A组）、对照组即I／RI组（B组）和缺血，再灌注（ischemia／reperfusion，I／R）即刻、30min、1、1．5h甘露醇干预组（分别为C、D、E和F组）。再灌注24h后取脑组织，对各组海马CAl区细胞的病理形态学变化[苏木精咿红（HE）染色和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminaldeoxynucleotidyltransferasemediatednickendlabeling，TUNEL）技术]、存活细胞数和变性细胞率作出检测。结果脑I／R即刻甘露醇干预组锥体细胞大多保持正常形态，细胞结构完好，细胞间质无水肿表现；存活细胞数为（23．01±1．23）个／100μm，与假手术组（24．00±0．75）个／100μm差异无统计学意义（P〉0．05）；变性细胞率（8．26％）较对照组（30．59％）明显减少（P〈0．05），与假手术组（7．45％）差异无统计学意义（P〉0．05）；TUNEL检测细胞凋亡指数（apoptosisindex，AI）值（6．50％）明显低于对照组（28．00％）（P〈O．05），与假手术组（5．00％）比较，差异无统计学意义（P〉0．05）。再灌注30min以上甘露醇再干预，神经元损伤不能改善。结论脑I／R早期血压梯度建立后及时给予甘露醇建立反相的渗透压梯度，可以有效防止细胞水肿的形成，并能阻断由此引发的不可逆细胞损害。本实验观察结果再次提示临床心跳骤停抢救中，在自主心跳恢复的同时即应开始甘露醇脱水治疗。

Objective To further verify the practicality of the experimental animal model induced by mice cerebral ischemia-reperfusion injuryand to investigate the optimal time point of mannitol intervention in cerebral ischemia-reperfusion injury. Methods Thirty-six mice were randomly divided into six groups（n=6）： sham operation group （group A）, control group（ischemia- reperfusion injury group, group B）, and mannitol intervention group at 0, 30 min, 1 h and 1.5 h after reperfusion （group C, D, E, F）. Brains were removed twenty-four hours after reperfusion. Morphological changes, ceils survival and cellular degeneration in CA1 area of hippocampus were studied in each group. Results Ingroup C, most hippocampal pyramidal cells remained normal morphologically, intact structurally and no intercellular edema . The amount of viable cells was （23.01±1.23） per 100 μm in group A and no difference compared with group A （24.00±0.75）. The frequency of cellular degeneration was 8.26%, significantly less than that of group 13 （30.59%,P〈0.05）. And no difference compared with group A （7.45%）. The apoptosis index （AI） was 6.50%, significantly less than that of group B （28.00% ,P〈0.05）. No difference of eomlSared with that of group A （5.00%）. Mannitol intervention beginning over 30 minutes after reperfusion failed to alleviate neuron injury. Conclusions Inverted osmotic pressure gradient by aggressive mannitol intervention can effectively prevent intracellular edema and avoid the subsequent irreversible cellular injury after the establishment of blood pressure gradient at the early stage of cerebral ischemia-reperfusion. The results suggest that during cardiac resuscitation, mannitol dehydration treatment should be initiated simultaneously at the recovery of autonomous heart rate.