Role Of NADPH-oxidase-mediated Down-regulation Of Hippocampal Parvalbumin In Postoperative Cognitive Dysfunction In Aged Mice

Objective:Postoperative cognitive dysfunction(POCD)is a common complication in central nervous system in patients after anesthesia and surgery,especially in the elderly.It has been suggested parvalbumin(PV)interneurons are crucial for cognitive processes,whereas the dysfunction of PV interneurons is involved in various major neuropsychiatric disorders.Our previous study has shown that the down-regulation of PV in the hippocampus plays a key role in the pathogenesis of isoflurane anesthesia-induced cognitive impairments in aged mice.However,the alteration and the role of PV in POCD remain largely unknown.PV interneurons are vulnerable to redox dysregulation and oxidative stress due to their high metabolic requirements.Recent studies have demonstrated that nicotinamide adenine dinucleotide phosphate(NADPH)oxidase-dependent production of superoxide results in the down-regulation of PV,which is involved in a variety of brain disorders.Herein,we hypothesized that surgical trauma can activate NADPH oxidase,then induce overproduction of reactive oxygen species(ROS),subsequently down-regulate expression of PV,and ultimately result in cognitive impairments.The present study aimed to investigate the role of the alteration of hippocampal PV expression in the pathogenesis of POCD,and to study whether NADPH oxidase is involved in this process.Methods;Sixteen-month-old C57BL/6 mice undergoing isoflurane anesthesia and exploratory laparotomy were used to establish a model of POCD.There were 3 experiments in the project.In experiment 1,the animals were randomly divided into 2 groups(n=13):control group and surgery group.The open field test and fear conditioning test were performed with 8 mice in each group at 6 d and 7 d post-surgery,respectively.The hippocampus and cortex of the rest 5 mice in each grousp without behavioral tests were harvested for the determination of PV,gp91phox and p22phox(two key subunits of NADPH oxidase)levels by Western blot at 7 d post-surgery.In experiment 2,the mice were randomly divided into 2 large groups to receive chronic treatment with apocynin(APO;a NADPH oxidase inhibitor)and a single dose of APO,respectively.Each large group was divided into 4 small groups(for chronic treatment with APO groups,n=18;for a single dose of APO groups,n=10):contro1+vehicle group,control+APO group,surgery+vehicle group and surgery+APO group.In the chronic treatment with APO groups,mice were given subcutaneous(sc)injection of 5 mg/kg APO(or the same volume of vehicle)1 h after the surgery(or at the same time point)and once daily for subsequent 7 consecutive days.In the single dose of APO groups,20 mg/kg APO(or the same volume of vehicle)was injected sc just before the anesthesia(or at the same time point).In the chronic treatment with APO groups,the open field test and fear conditioning test were performed with 8 mice in each small group at 6 d and 7 d post-surgery,respectively.The brains of the rest 10 mice in each group without behavioral tests were harvested at 7 d post-surgery,in which 5 mice were used for Western blot(hippocanpus)and the other 5 mice for immunofluorescence(brain).The hippocampal levels of PV,gp91phox,p22Phox,IL-1?,and the markers of oxidative stress were determined at 7 d post-surgery.The brains of mice in the single dose of APO groups were harvested for the same determination 24 h after the surgery.Experiment 3 is an in vitro study.The primary hippocampal neurons cultured for 14 d were treated with lipopolysaccharide(LPS)or/and APO.The neurons were randomly divided into 4 groups:control group,controll+APO group,LPS group and LPS+APO group.After drug treatment for 24 h,the neurons were collected to determine the levels of PV,gp91phox,p22phox and postsynaptic density 95(PSD95;an excitatory postsynaptic marker)by immunofluoresence.Results:In experiment 1,compared with the control group,the freezing time was decreased in the context fear conditioning test,the levels of PV were decreased in the hippocampus(P ? 0.05)but not in the cortex(P ? 0.05),and the levels of gp91phox andp22phox were increased in both hippocampus and cortex(P ? 0.05)in the surgery group.In experiment 2,in the chronic treatment with APO groups,the freezing time was decreased,the levels of PV were decreased,and the levels of gp9lphox,p22phox,IL-1? and the markers of oxidative stress were increased inthe surgery+vehicle group compared with the control+vehicle group(P ? 0.05).Compared with the surgery+vehicle group,the freezing time was increased,the levels of PV were increased,and the levels of gp91phox,p22phox,IL-1? and the markers of oxidative stress were decreased in the surgery+APO group(P ? 0.05).The biochemical results in the single dose of APO groups were in consistent with those in the chronic treatment with APO groups.In eeperiment 3,compared with the control group,the levels of P V were decreased,the levels of gp91phox and p22phox were increased,and the levels of PSD95 were reduced in the LPS group(P<0.05).Compared with the LPS group,the levels of PV were increased,the levels of gp91phox and p22phox were decreased,and the levels of PSD95 were increased in the LPS+APO group(P<0.05).Conclusion:The down-regulation of PV in the hippocampus is involved in the development of POCD in aged mice,which is probably mediated by NADPH oxidase-derived ROS.