罗哌卡因预处理对乳鼠离体脑片过氧化氢损伤保护作用的研究

Protective effects of ropivacaine pretreatment on hydrogen peroxide damage in neonatal rat brain slices in vitro

目的分析罗哌卡因预处理对乳鼠离体脑片过氧化氢损伤的保护机制。方法取出生7 d的SD乳鼠脑皮质制备离体脑片,用2,3,5-氯化三苯基四氮唑(TTC)染色判断脑片活性后按随机法分为正常对照组(A组)、过氧化氢(H2O2)损伤组(B组)、罗哌卡因预处理损伤组(C组),C组于培养3 d时进行药物预处理24 h(罗哌卡因注射液浓度为8. 94 mg/ml);脑片培养至4 d时用700μmol/L过氧化氢复制损伤模型,损伤30 min后将3组转入正常培养液中孵育至7 d,A组不予任何处理。观察脑片细胞形态学变化、细胞超微结构变化,碘化丙啶(PI)染色比例,检测乳酸脱氢酶(LDH)漏出率,神经细胞标记物热休克蛋白70(HSP70)阳性表达的变化。结果 (1)脑片形态学观察:3 d肉眼可见脑片轻度水肿,3~5 d水肿逐渐消失,6 d镜下见典型的神经细胞及少量胶质细胞;7 d细胞生长最旺盛,之后凋亡逐渐占据主导。(2)苏木素—伊红(HE)染色观察细胞形态:A组神经元呈多边或梭形;B组较A组明显减少;C组神经元数量介于A组与B组之间,且组间存在差异。(3) PI染色:A组细胞几乎全部呈现胞核红色荧光;B组荧光明显减少,C组大量神经元出现荧光,轮廊不清。(4) LDH漏出率:LDH的活性:B组> C组> A组,且组间存在差异。(5) HSP70阳性表达的变化:B组可见大量HSP70表达,A组表达很少,C组介于A、B组之间。结论罗哌卡因预处理对乳鼠离体脑片的过氧化氢损伤有保护作用。

Objective To analyze the protective mechanism of ropivacaine pretreatment on hydrogen peroxide damage in isolated brain slices of neonatal rats.Methods Seven-day-old SD neonatal rat brain cortex was taken to prepare brain slices in vitro.The brain slices were determined by 2,3,5-triphenyltetrazolium chloride(TTC)staining and randomly divided into normal control group(A group),hydrogen peroxide(H2 O2)injury group(B group),ropivacaine pretreatment injury group(C group).Group C was pretreated for 24 hours at 3 days of culture(ropivacaine injection concentration was 8.94 mg/ml).Brain slices were cultured for 4 days with 700 micromol/L hydrogen peroxide to replicate the injury model.After 30 minutes of injury,three groups were incubated in normal culture medium for 7 days.Group A was not treated.Morphological changes,ultrastructural changes,propidium iodide(PI)staining ratio,leakage rate of lactate dehydrogenase(LDH)and positive expression of heat shock protein 70(HSP70)were observed.Results(1)Morphological observation of brain slices:mild edema was observed in the brain slices on the 3 rd day with naked eyes,and gradually disappeared on the 3 rd to 5 th day.Typical nerve cells and a few glial cells were observed under the 6 th day microscope.Cell growth was the most vigorous on the7 th day,and apoptosis gradually dominated.(2)Cell morphology was observed by hematoxylin-eosin(HE)staining:neurons in group A were polygonal or spindle-shaped;neurons in group B were significantly less than those in group A;neurons in group C were between group A and group B,and there were differences between groups.(3)PI staining:In group A,almost all cells showed red fluorescence of nucleus;in group B,fluorescence was significantly reduced,and in group C,a large number of neurons showed fluorescence,and the corridor was unclear.(4)Leakage rate of LDH:LDH activity:group B>group C>group A,and there were differences between groups.(5)Changes of HSP70 positive expression:A large number of HSP70 expression was observed in group B,but few in group A.Group C was between group A and group B.Conclusion Ropivacaine pretreatment has protective effect on hydrogen peroxide damage in isolated brain slices of neonatal rats.