Effects Of Autophagy On Isoflurane Induced Neuronal Apoptosis In The Developing Rat Hippocampus

Isoflurane,as a commonly used clinical inhaled anesthetic,have been found that it could induce abnormal neuronal apoptosis in the brain during “rapid growth period” and could also cause long-term cognitive dysfunction.Our group has been working on the neurotoxicity of isoflurane for many years.We have obtained much experimental data about isoflurane induced behavioral changes and neuronal apoptosis in young rats,and we also found that isoflurane could not only induce apoptosis but also cause autophagy.The relationship between autophagy and apoptosis is a hot issue in today’s research field.Therefore,it is meaningful to clarify the effect of autophagy on isoflurane induced neuronal apoptosis and the molecular mechanisms behind of this.It will provide a theoretical basis for anesthetic toxicity.In this study,postnatal 7 days(PD7)Sprague-Dawley(SD)rats were recruited,combining with autophagy tool-drugs,to determine the effect of autophagy on isoflurane induced hippocampus neuronal apoptosis,and to clarify the relationship between anesthetics induced neuronal apoptosis and autophagy.A total of 120 PD7 SD rats were randomly divided into three groups: isoflurane anesthesia group(ISO),rapamycin pretreatment group(ISO+RAP)and 3-methylpurine pretreatment group(ISO+3-MA).Group ISO+RAP and group ISO+3-MA were intraperitoneal injection with 20 mg/kg rapamycin and 20 mg/kg 3-MA respectively.Before(30 min prior)exposure to isoflurane,rats in each group were inhaled 1.5% isoflurane mixed 60% oxygen for 0 h(CON),2 h,3 h,4 h and 6 h respectively,and CON group only inhaled 60% oxygen for 6 h.The relationship between autophagy and apoptosis induced by isoflurane was investigated,using hematoxylin eosin(HE)staining,terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining,LC3 II immune-histochemical staining and Western blot methods,from morphological and biologic changes.The results were as follows:HE staining showed that: in control group,the hippocampal nerve cells arranged closely,had less apoptosis;ISO group showed obvious neuronal apoptosis after exposed to isoflurane for 3 h or longer by a time-dependent manner;There was a significant apoptosis in the ISO+RAP group after exposed to isoflurane for 4 h or longer;In the ISO+3-MA group,the different degrees of apoptosis were found at different time point,especially at 4 h and 6 h.TUNEL staining showed that:Comparing with the control group,the apoptosis in the ISO group was significantly increased after3 h;In the ISO+3-MA group apoptosis was found at all assessment time points,while in the ISO+RAP group the obvious apoptosis was only found at 4 h and 6 h.Comparing among the groups: The apoptosis rate in the ISO+RAP group was lower at 4 h(P < 0.05)and 6 h(P < 0.05);Compared with the ISO group,the apoptosis rate in the ISO+3-MA group was increasedsignificantly at 2 h(P < 0.05),3 h(P < 0.05)and 4 h(P < 0.05).The expression of cleaved-caspase-3 blotting were consistent with the results of TUNEL.LC-3 II immunohistochemistry showed that: Compared with the control group,the ratio of positive cells in ISO group at 3 h was higher,the ratio of LC-3 II positive cells in the ISO+3-MA group was lower but at 6 h;The ISO+RAP group showed more positive cells at 3 and 4 h.The expression of LC-3 II protein showed that: Comparing with the control group,the expression of LC-3 II in the ISO group was significantly increased at 3 h;it was always lower significantly in the ISO+3-MA group during the assessment time;the expression of LC-3 II in the ISO+RAP group was significantly increased at 2 h,3 h and 4 h.Comparing among the groups,the expression levels in the ISO+RAP group were significantly higher than that in the ISO group at 2 h(P < 0.05),and higher than that in ISO+3-MA group at 2 h(P < 0.05)and 3 h(P < 0.05)respectively.The Beclin-1 levels were consistent with the LC-3 II basically.Bcl-2 blotting results showed that: Compared with the control group,the Bcl-2 levels in the ISO group and in the ISO+3-MA group were significantly decreased after exposed to isoflurane for 3 h or longer;the ISO+RAP group was significantly decreased only at the time of 4 h and 6 h.Comparing among groups,ISO group and ISO+3-MA group were lower than the ISO+RAP group at 3 h(P < 0.05,P < 0.05),4 h(P < 0.05,P < 0.05)and6 h(P < 0.05,P < 0.05);at 2 h,ISO+3-MA group was significantly lower than that of ISO+RAP group(P < 0.05)and ISO group(P < 0.05).The results showed that PD7 SD pups inhaled 1.5% isoflurane could induce autophagy and apoptosis of hippocampal neurons,and autophagy appeared earlier than apoptosis.Autophagy caused by isoflurane has an inhibitory effect on neuronal apoptosis.That is to say,isoflurane induced hippocampal neuronal autophagy could reduce its neurotoxicity and have a neuroprotection.In addition,inhibiting autophagy could promote isoflurane induced hippocampal neuronal apoptosis,but activating autophagy could mitigate isoflurane induced hippocampal neuronal apoptosis.