异氟烷对发育期大脑的神经毒性及细胞周期的影响

Effects of Isoflurane on neurotoxicity and cell cycle in developing brain

目的探讨异氟烷对发育期大脑的神经元毒性和细胞周期的影响及其相关作用机制。方法将32只7日龄C57BL/6J小鼠随机分为4组:对照组自主吸入空气,异氟烷2h组吸入1.3%异氟烷2 h,异氟烷4 h组吸入1.3%异氟烷4 h,异氟烷6 h组吸入1.3%异氟烷6 h。实验结束后6 h,取对照组和异氟烷6 h组小鼠脑组织,免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(Caspase-3)表达情况,FJB方法检测神经元变性情况。另取4组小鼠新鲜海马组织提取蛋白,利用Western blot免疫印迹方法检测其细胞周期蛋白CyclinB1的表达情况。结果异氟烷6 h组海马区Caspase-3阳性细胞数及FJB阳性细胞数均明显高于对照组(P均<0.05)。异氟烷4 h组、6 h组CyclinB1表达量均明显高于对照组(P均<0.05)。结论异氟烷可通过影响细胞周期蛋白CyclinB1表达进而引起发育期大脑海马区神经元的凋亡,并且具有一定的时间依赖性。

Objective It is to investigate the effects and mechanisms of isoflurane on neurotoxicity and cell cycle in developing brain. Methods 34 seven-day-old C57 BL /6J mice were randomly divided into C,A1,A2,A3 four groups： The control group（ group C） inhaled air independent,Exposure to 1. 3%（ volume fraction） of isoflurane 2 h（ group A1）; Exposure to1. 3%（ volume fraction） of isoflurane 4 h（ group A2）; Exposure to 1. 3%（ volume fraction） of isoflurane 6 h（ group A3）.After the end of anesthesia for 6 hours,five mice taken respectively from the control group and experimental group. The brain tissue after cardiac perfusion with 4% paraformaldehyde were gotten to detect the hippocampus of mouse cysteine aspartic proteinase 3（ Caspase-3） expression by immunohistochemistry. Detecting the degeneration of neurons in hippocampus by using the method of Fluoro-Jade B staining. 6 mice from each groups respectively were selected and killed to get rapid extraction of hippocampal protein. Using Western blot immunoblotting method to detect the cell cycle protein B1（ CyclinB1） expression in the hippocampus of mice. Results Compared with the control group,the number of Caspase-3 positive cells and the number of degenerated neurons of the hippocampus in hippocampus of group A3 mice increased obviously（ P〈0. 05）; Compared with the control group,the expression of CyclinB1 in group A2 and group A3 increased obviously（ P〈0. 05）,while in group A1 there was no significant difference（ P〈0. 05）. Conclusion The study found that exposure to 1. 3% isoflurane for a period can increase the expression of CyclinB1 in hippocampal neurons,then caused the changes of neuronal cell cycle and lead to apoptosis.