Activation of autophagy improves neuron injury after the restoration of spontaneous circulation from ventricular fibrillation in wistar rats

Abstract

Objective To investigate the effects of activation of autophagy on cerebral injury after cardiopulmonmy resuscitation (CPR) in Wistar rats.Methods At first 36 healthy adult male Wistar rats were induced to suffer ventricular fibrillation (VF) by an external transthoracic alternating electricity current shock for 7 minutes and then received CPR.Before VF (0) and at 1 h,2 h,4 h,8 h and 12 hours after the restoration of spontaneous circulation (ROSC) from VF,cerebral cortex were harvested to determine the protein levels of beclin-1 and LC3 Ⅱ by Weston blot.Thereafter,another 60 healthy adult male Wistar rats were divided into autophagy activator rapamycine group (Rapamycine group,n =20)、autophagy inhibitor 3-Methyladenine group (3-MA group,n =20) and normal saline group (control group,n =20) to observe the effects of autophagy activator on the levels of beclin-1,LC3 Ⅱ and the formation of autophagic vacuole and LC3 dots in the cerebral cortex after ROSC.The neurologic deficit score (NDS) was used to evaluate the neurologic function at 24 h,48 h and 72 h respectively after ROSC.The numbers of viable neurons and apoptotic neurons in the parietal cortex were counted by HE staining and TUNEL staining after 72 h.Oneway ANOVA was used for the levels of beclin-1,LC3 Ⅱ and neurons counting.Rank sum test was used for NDS.Results The levels of beclin-1 and LC3-Ⅱ were significantly lower at 2h and 4 h after ROSC than those before VF (P ＜ 0.05).The levels of beclin-1 and the conversion of LC3-Ⅱ in cerebral cortex at 2 and 4 h after ROSC in the Rapamycine group were significantly higher than those in control group and 3-MA group (P ＜ 0.05).The numbers of autophagic vacuole in the Rapamycine group at 2 and 4 h after ROSC were significantly higher than those in control group and 3-MA group.The number of viable neurons at 72 h after ROSC in the Rapamycine group was (19 ± 5/400) pixs,(16-± 4/400) pixs and was higher than (16 ±4/400) pixs in control group and (15 ±3/400) pixs in 3-MA group (P ＜0.05).The number of the TUNEL positive cells in the Rapamycine group was (12-± 4/400) pixs and was lower than (15 ± 6/400)pixs in the control group and (17 ± 5/400) pixs in 3-MA group (P ＜ 0.05).The NDS scores of animals in control group and 3-MA group at 24,48 and 72 h after ROSC were lower than those in Rapamycine group.Conclusions The levels of beclin-1 and LC3-Ⅱ in the cerebral cortex neurons were attenuated in Wistar rats after ROSC.The promotion of beclinq and LC3-Ⅱ levels by rapamycine can decrease the number of apoptotic neurons,preserve viable neurons and improve neurologic function. Key words: Ventricular fibrillation ; Cardiopulmonary resuscitation ; cerebral injury ; Autophagy; Rapamycine; 3-Methyladenine; beclin-1; LC3-Ⅱ