缝隙连接在失血性休克大鼠缺血性脑损伤延生复苏中的作用

The role of gap-junction in suspended animation for hemorrhagic shock with brain injury： experiment with rats

目的探讨缝隙连接在失血性休克大鼠缺血性脑损伤延生复苏中的作用。方法采用出血未控制失血性休克＋双侧颈动脉夹闭的方式制备模型。将制成模型的24只sD大鼠随机分为3组（n=8）：传统复苏组（Ⅰ组），低温延生复苏组（Ⅱ组）和甘珀酸延生复苏组（Ⅲ组）。模拟救援过程，将各动物模型分为创伤失血期（1期）、院前救援期（2期）、院内复苏期（3期）3个时期。分别记录各时点的INSP、±dp／dtmax、MAP、HR、RR，记录大鼠输血复苏后的存活时间。取大鼠左侧脑组织观察大体标本，并取0．2g测定ATP（化学发光法）、肿瘤坏死因子-α、白介素16（ELISA法）；取海马区脑组织0．2g测定凋亡细胞百分数。结果低温延生复苏可以显著增加复苏后的存活时间（P〈0．01），减少ATP的损耗（P〈0．05），减少炎症因子的释放（P〈0．05），显著减少海马区细胞的凋亡（P〈0．01）；而甘珀酸延生复苏虽然能够显著增加复苏后的存活时间（P〈0．01）、维持较高的ATP水平和减少海马区细胞的凋亡（P〈0．05），但不能减少所有炎症因子的释放。结论低温及甘珀酸延生复苏的效果显著优于传统复苏，缝隙连接阻断剂可产生与低温相似的脑保护效果，缝隙连接在失血性休克大鼠缺血性脑损伤延生复苏中具有重要作用。

Objective To investigate the role of gap-junction in suspended animation for hemorrhagic shock with brain injury. Methods Twenty-four SD rats were made into models of uncontrolled hemorrhagic shock and occlusion of bilateral common carotid arteries and randomly divided into 3 equal groups： traditional resuscitation group （Group Ⅰ ） undergoing traditional resuscitation, hypothermy resuscitation group （ Group Ⅱ ） undergoing temperature lowering by ice-cap, and carbenoxolone resuscitation group （ Group Ⅲ） undergoing injection of carbenoxolone 50 μg/200 g. The process was divided into 3 periods： traumatic hemorrhagic period （30 min）, pre-hospital treatment period （60 min）, and in-hospital cardiopuhnonary resuscitation period （60 min）. The levels of left ventricular systolic pressure （LVSP）, maximum change rate of left ventricular pressure rise and fall （ ± dp/dtmax ）, mean arterial pressure （MAP）, heart rate （HR）, and respiratory rate （RR） were recorded at the beginning and the end of traumatic hemorrhagic period （T1 and T2 ）, the end of pre-hospital treatment period （T3 ） , and the end of in-hospital cardiopuhnonary resuscitation period （T4）. The survival time was recorded after in-hospital cardiopuhnonary resuscitation period. Then the left brain was taken out and the hippocampal neurons apoptosis was observed by flow cytometry, chemiluminescence was used to detect the ATP, and IL-6 and tumor necrosis factor （TNF）-α were detected by enzyme linked immunosorbent assay （ELISA）. Results The survival time of Groups Ⅱ and Ⅲ were （2. 9 ±0. 6） h and （2.6 ± 1.0） h respectively, both significantly longer than that of Group Ⅱ （ 1.4 ±0. 3） h, both P 〈0. 01 ]. The apoptotic rates of hippocampal neurons of Groups Ⅱ and Ⅲ were （72 ± 6） % and （75±9） % respectively, both significantly lower than that of Group [ （ 83± 5 ） % ,P 〈 0.05 ]. The ATP levels in hippocampus of Groups lI and ]R were （2, 0 ± 0, 3 ） and （ 1.9 ± 0.4） pg/g respectively, both significantly higher than that of Group Ⅰ [ （ 1.4 ± 0. 5 pg/g, both P 〈0. 05 ）. The TNF-αand IL-6 levels of Groups Ⅱ were （ 1.7 ± 0. 3 ） pg,/g and （ 19 ± 3 ） pg,/g respectively, both significantly lower than those of Group Ⅰ [ （2.2 ± 0.6 ） and （24 ±3 ） pg,/g respectively, both P 〈 0. 05 ]. The IL-6 level of Group Ⅲ was （ 26± 4 ） pg/g, significantly higher than that of Group Ⅱ （ P 〈 0. 01 ）. There were no significant difference between Groups Ⅱ and Ⅲ in the values of survival time, hippoeampal neuron apoptosis, dissipation of ATP, and liberation of TNF-α（ all P 〉 0. 05 ）, but there was significant difference in IL-6 （P 〈 0. 01 ）. Conclusion Both hypothermy resuscitation and carbenoxolone resuscitation protect the brain of cerebral isehemia on hemorrhagic shock with brain injury, and suggest that Gap junctions play an important role in suspended animation to treat hemorrhagic shock with brain injury.