Protective Effects Of Midazolam On Ketamine Induced Autophagy And Apoptosis In Fetal Rat Neurons

Anesthesia is the key link in the clinical diagnosis and treatment of animals.However,anesthesia also has certain side effects,such as neurodegeneration,which has now aroused widespread concern.Ketamine is one of the commonly used intravenous anesthetics in clinical practice,and it is favored for its good analgesic effect,but at the same time its neurotoxicity can not be ignored.It has been reported that some anesthetics including midazolam can protect neurons,but the neuroprotective mechanism of midazolam is still unclear.We also do not know whether midazolam can reduce ketamine-induced damage.Therefore,in order to understand the effects of ketamine anesthesia on embryos,pregnant mice were anaesthetized by ketamine and fetal rats were used to study the effects of ketamine on fetal rat brain neurons and midazolam during ketamine anesthesia.Play a protective role.For the future to find relatively safe anesthesia provide a theoretical basis.It can also provide some theoretical support for the anaesthesia of pregnant animals,and try to avoid excessive damage caused by anesthesia to young children.The pregnant rats were randomly divided into 4 groups.On the 19 th day of pregnancy,the rats of the control group(C)were not subjected to any treatment.The rats of the ketamine group(K)were injected intramuscularly with ketamine;midazolam(Group M)were injected intramuscularly with midazolam;midazolam + ketamine(K + M group)rats were injected intramuscularly with ketamine and midazolam for caesarean section and fetal rats were removed.Western-blot was used to detect autophagic and apoptosis-related protein changes,such as autophagy marker light chain 3(LC3)and autophagy-related gene 4(ATG4),autophagy-related gene 5(ATG5),Bcl-2 homeodomain protein(Beclin-1),cleaved caspase-3(c-caspase-3),autophagy P62 protein(P62),B-cell lymphoma-2(Bcl-2)and B-cell lymphoma/leukocyte gene accompanying protein x(Bax).Oxidative stress-related kits were used to detect reactive oxygen species(ROS),total antioxidant capacity(T-AOC),and malondialdehyde(MDA).The CCK-8 kit was used to detect the optimal concentration of ketamine,midazolam,and hydrogen peroxide on PC12 neurons.Then the cell model of oxidative stress was established,and the autophagy enhancer(Rapa)and inhibitor(3-MA)were added for vitro validation.The mRFP-EGFP-LC3 dual-fluorescence autophagic indicator system was used for the study of LC3 tracking and changes in autophagic flow.Flow cytometry was used to detect the effect of ketamine on the apoptosis of PC12 cells and the role of midazolam in it.Then we measured the change of autophagy and apoptosis related protein by Western-blot,and re verified the in vivo results.Results are as follows:1.Ketamine induced apoptosis of fetal rat hippocampus and PC12 cells by increasing the expression of c-caspase-3 and Bax and decreasing the expression of Bcl-2.2.Ketamine also increased the expression of LC3 II,Beclin-1,and ATG5,decreased the expression of ATG4 and P62,and ultimately induced autophagy in rat hippocampus and PC12 cells.3.Ketamine promotes the production of ROS and MDA and reduces T-AOC.4.Compared with K group,the autophagy and apoptosis-related proteins were significantly different in K + M group(P<0.05).5.Compared with group C,there was no significant change in autophagy and apoptosis related proteins in group M.In conclusion,ketamine anesthesia leads to autophagy and apoptosis in the rat hippocampus.Ketamine also induces apoptotic pathways and activation of autophagy through ROS production,thereby inducing toxicity in neurons.Midazolam improves the toxic effects of ketamine on nerve cells by reducing ROS production.