羟丁酸钠对小鼠脑缺血再灌注损伤的影响

Effects of Sodium Gamma-hydroxybutyrate on Focal Cerebral Ischemia-Reperfusion Injury in Presenile Mice

目的观察羟丁酸钠（Sodium Oxybate,SO）对早老龄小鼠局灶性脑缺血再灌注损伤的影响,为羟丁酸钠的脑保护作用提供实验依据。方法采用早老龄小鼠左侧颈总动脉阻断法制作局灶性脑缺血再灌注损伤的模型。动物分为5组,每组16只：假手术（Sham）组：手术操作同其它组,但不夹闭左侧颈总动脉;②NS组：腹腔注射生理盐水;③羟丁酸钠50mg/kg（SO50）组;④羟丁酸钠100mg/kg（SO100）组;⑤羟丁酸钠200mg/kg（SO200）组。、②组于缺血前30min腹腔给生理盐水,③~⑤组于缺血前30min腹腔给SO一次,以后各组每日给药一次。手术后动物分为两部分进行实验,其中每组6只于术后24h断头,制备脑组织匀浆,用于MDA含量、SOD活性的测定。另10只分别于术后24h和48h分别进行神经学评分,术后第3、4d进行避暗实验,术后第5、6、7d进行水迷宫测试。水迷宫测试后将小鼠麻醉,进行海马CA1区神经元组织形态学检查。结果神经症状评分：术后24h和48h,NS组小鼠的神经症状评分高于假手术（Sham）组（P〈0.01）和各给药组,其中SO100组SO200组与NS组相比差异有显著意义（P〈0.05或0.01）;②避暗试验：NS组小鼠潜伏期（Latent period）明显较假手术组缩短、错误次数（Wrong Times）增加（P〈0.01）;SO各组避暗潜伏期比NS组长、比Sham组缩短,错误次数比NS组少,比Sham组多。其中,SO100和SO200组与NS组的差异有显著意义（P〈0.05或0.01）;③水迷宫试验：NS组小鼠比Sham组到达时间延长、错误次数增加,SO各组比NS组到达时间缩短、错误次数少,但比Sham组到达时间延长、错误次数增加,其中,SO100和SO200组与NS组的差异有显著意义（P〈0.05或0.01）;④MDA含量、SOD活性：术后24h NS组小鼠的MDA含量高于Sham组（P〈0.01）,SOD活性低于Sham组（P〈0.01）。SO三剂量均能明显减少MDA含量和增加SOD活性,其中SO100组、SO200组MDA含量低于NS组（P〈0.01）;⑤海马组织学：NS组小鼠海马CA1区细胞肿胀、变形,乃致核固缩、核溶解,细胞坏死或缺失。SO能不同程度地减轻海马CA1区神经元的损伤,减少坏死细胞数目（P〈0.05或0.01）。结论羟丁酸钠能剂量依赖性地改善早老龄小鼠局灶性脑缺血再灌注损伤后的神经症状和学习记忆能力下降,减轻海马CA1区锥体细胞的损害,表明羟丁酸钠对脑缺血再灌注损伤具有保护作用。这一保护作用可能与提高早老龄小鼠脑缺血再灌注损伤后的SOD活力、减少MDA的合成有关。

Objective To investigate the effects of sodium gamma-hydroxybutyrate on focal cerebral ischemia-reperfusion injury in presenile mice.The extent of ischemia injury was assessed by behavioural and histological and biochemistrical endpoints.Methods The occlusion of lateral carotid artery of presenile mouse was used to make the cerebral ischemia-reperfusion models.The presenile mice were randomly divided into 5 groups：① Group 1（Sham）,presenile mice underwent lateral carotid arteries exposure only;②Group 2（NS）,presenile mice were exposed to ischemia of 5 min duration and injected intraperitoneally（ip）NS 30 min prior to the induction of ischemia;③Group 3（SO50）,sodium gamma-hydroxybutyrate 50 mg/kg ip;④Group 4（SO100）,sodium gamma-hydroxybutyrate 100 mg/kg ip;⑤Group 5（SO200）,sodium gamma-hydroxybutyrate 200 mg/kg ip;Group 3~5 gerbils were exposed to ischemia of 20 min and treated with sodium gamma-hydroxybutyrate 30 min before brain ischemia,Later,Group 2 presenile mice were treated with NS once every day.Group 3~5 presenile mice were administered with sodium gamma-hydroxybutyrate once every day.Step-through test was carried out on days 3 and 4 following ischemia/sham occlusion surgery.Water-maze test was carried out on days 5~7 following ischemia/sham operation.At 7 days postischemia the animals were sacrificed,hippocampus CA1 area was used to carry out the histological evaluation.Results ①Step-through test：On days 3 after surgery,the NS mice showed significant reduce in analytics activity over sham mice（P〈0.01）,the exploring activity of the SO groups were higher than the NS group.There were significant differences between SO100、SO200 groups and NS group（P〈0.05）.At 7 days the locomotor activities of all groups of mice were lower than that on days 3.There still were significant differences between SO groups and NS group（P〈0.05 or 0.01）;② Step-through test：On days 4、5 and 6 after surgery,the NS mice displayed significant decline in learning and working memory over sham mice（P〈0.01）,SO can increase learning and working memory.There were significant differences between SO100、SO200 groups and NS group（P〈0.05 or 0.01）;③ the CA1 region of the hippocampus histological results：Extensive CA1 region of hippocampus damage occurred in the NS group.SO attenuated neuronal injury in the CA1 zone and reduced necrosis cell counts in different extent（P〈0.05 or 0.01）;④ Activities of SOD and contents of MDA in the cerebrum：The activities of SOD in cortex、hippocampus and striatum significantly declined and the contents of MDA significantly increased after cerebral ischemia reperfusion in mice.SO can protect the activities of SOD and decrease the contents of MDA in the mice（P〈0.01 or 0.05）.Conclusion Sodium gamma-hydroxybutyrate significantly ameliorated learning and working memory ability in the Water-maze and Step-through test,reduced the extent of CA1 hippocampal pyramidal cells injury after transient global cerebral ischemia in mice.Sodium gamma-hydroxybutyrate also markly protected SOD activities,reduced the contents of MDA.Moreover,the effects of sodium gamma-hydroxybutyrate gradually potentized with the increase of dose.These results suggest that sodium gamma-hydroxybutyrate provides significant protective effect against cerebral ischemia-reperfusion injury by reducing brain energy failure and lipid peroxidation.