腹腔注射氢生理盐水对急性马尾综合征大鼠神经功能的影响及相关机制初探

Preliminary study of mechanism and neuroprotective effects of hydrogen-rich saline intraperitoneal injection in rats with acute cauda equina symdrome

目的:初步探讨腹腔注射氢生理盐水对急性马尾综合征大鼠神经功能的影响及相关机制。方法:选取200～250g的SD大鼠168只,随机均分为3组:假手术组、对照组、实验组。假手术组仅行L5棘突和L5/6间隙黄韧带切除,对照组和实验组建立急性马尾综合征大鼠硅胶条压迫模型;实验组建模术后立刻行5ml/kg氢生理盐水腹腔注射,以后1次/8h腹腔注射,假手术组和对照组均行等量生理盐水腹腔注射。术后3h、6h、12h、24h、48h和72h分别于每组各取8只大鼠进行热板试验,评价行为学表现,而后麻醉下取T13~L1段脊髓、L4~L6段马尾神经及L5、L6双侧背根神经节(dorsal root ganglion,DRG)行丙二醛(MDA)含量检测,分析各时间点的变化。另于术后48h每组各取8只行多聚甲醛灌注后取T13~L1段脊髓、L4~L6段马尾神经及L5、L6双侧DRG,行HE染色观察组织形态,取马尾神经行神经纤维蛋白(NF)染色并计算阳性率,取脊髓及DRG行Caspases 3染色后计算阳性指数,行Tunel染色后计算凋亡指数。结果:由于麻醉苏醒时间不一致术后3h各组的缩足潜伏期数据不采用,6h^72h的缩足潜伏期实验组均低于对照组,对照组和实验组均高于假手术组(P<0.05)。术后对照组和实验组MDA水平在3h^48h均较同组前一时间点有明显增加(P<0.05),在48h和72h无明显增加(P>0.05),对照组在3h时增幅最大,实验组在24h增幅最大(P<0.05);术后3h^72h各时间点马尾、脊髓及背根神经节的MDA水平实验组均比对照组低,对照组和实验组均比假手术组低(P<0.01)。假手术组的NF染色阳性纤维率为100%,实验组为(59.08±6.25)%,对照组为(21.35±2.38)%,三组间两两比较有显著性差异(P<0.01)。对照组和实验组脊髓和DRG的Caspases 3阳性指数均明显大于假手术组(P<0.01);实验组的阳性指数均明显小于对照组(P<0.05)。对照组和实验组脊髓和DRG的Tunel凋亡指数均明显大于假手术组,实验组脊髓和DRG的凋亡指数均小于对照组(P<0.01)。结论:氢生理盐水可改善急性马尾综合征大鼠的行为学表现,减少神经纤维变性及神经元凋亡,其机制可能与氢对氧化应激反应具有抑制和延缓作用有关。

Objectives： To study the mechanism and neuroprotective effects of hydrogen-rich saline intraperi- toneal injection in rats with acute cauda equina syndrome. Methods： 168 SD rats weighing 200-250g were randomly allocated into three groups： sham-operation group, control group and experimental group. The L5 spinous process and ligamentum flavum between L5 and L6 vertebra was removed in sham-operation group, the rat models of acute cauda equina syndrome were made in control group and experimental group. Hydro- gen-rich saline was injected intraperiteonally in a 5ml/kg dose after operation immediately and then once ev- ery 8 hours in experimental group; normal saline was injected in sham-operation group and control group. 3h, 6h, 12h, 24h, 48h and 72h after operation, 8 rats from each group were respectively examined by the hot plate test, then their samples of IA-L6 cauda equine, T13-L1 spinal cord and dorsal root ganglia of L5 and L6 were harvested to analyze tissue concentrations of MDA. At 48h, the samples experienced HE, NF, Cas-pases 3 and Tune1 staining. By the above methods, animal ethology, oxidative stress, histomorphology and cell apoptosis were evaluated statistically. Results： The paw-withdrawal latency in 3h groups was unavailable due to different recovery time from anesthesia. From 6h to 72h, the paw-withdrawal latency in experimental group was lower than control group, and both control group and experimental group had higher latency than sham- operation group（P〈0.05）. The MDA level of cauda equine, spinal cord and dorsal root ganglia in control group and experimental group increased significantly at the time from 3h to 48h （P〈0.05） and showed no significant difference between the 48h and 72h （P〉0.05）. The MDA level increased at 3h in control group and at 24h in experimental group（P〈0.05）. The experimental group form 3h to 72h had lower MDA level, than control group, while control group or experimental group had higher MDA level than sham-operation groups（P〈0.01）. The pos- itive rate of NF staining was 100% in sham-operation group, （59.08±6.25）% in the experimental group, （21.35±2.38）% in the control group, pairwise comparisons among the three groups showed significant differ- ences （P〈0.01）. The positive index of Caspases 3 staining of spinal cord and dorsal root ganglia in control group or experimental group was higher than that in sham-operation group （P〈0.01）, while experimental group was lower than control group （P〈0.05）. Tunel apoptotie index of spinal cord and dorsal root ganglia in control group or experimental group was higher than that in sham-operation group, while experimental group was lower than control group（P〈0.01）. Conclusions： In acute cauda equina syndrome, Hydrogen-rich saline may improve ethology performance and reduce neurons apoptosis and degeneration of nerve fibers through restrained and deferred oxidative stress.