The Role Of Hippocampal GABA Ergic Neurons In Postoperative Cognitive Dysfunction In Aged Mice

Background Postoperative cognitive dysfunction(POCD)refers to central nervous system disorders after surgery,mainly manifested as cognitive deficits,social disorders,personality changes,etc.There are many risk factors for POCD,including age,anesthesia,operation types,postoperative pain,sleep disorders,low education,and so on.Among them,advanced age is an independent risk factor for POCD.Epidemiological studies report that the incidence of POCD in non-cardiac patients is about 25.8% 7 days after surgery.However,the incidence of POCD in elderly patients is nearly 25-40%,and it may still reach 9.9% 3 months after surgery.POCD significantly affects patients’ outcomes and increases the burden on the family and society.Therefore,how to effectively prevent and treat POCD has become an important medical issue that the international community pays attention to.Multiple neurotransmitter receptors have been demonstrated to be associated with memory deficits.γ-aminobutyric acid(GABA)the major inhibitory neurotransmitter in the central nervous system,regulates learning,memory,and synaptic plasticity.GABA receptors(GABARs)which comprise different subunits,and different combinations of GABARs have shown different localizations and distinct physiological and pharmacological characteristics.In particular,the α5-subunit-containing subtype of GABAARs(α5GABAARs),which makes up 20-25% of the hippocampal GABAARs,are specifically localized to extra-synaptic regions of hippocampal pyramidal neurons,and are mainly involved in mediating tonic inhibition,as well as being implicated in the processing of memory.Furthermore,the increase of α5GABAARs activity causes profound memory blockade.At the same time,a reduction in the expressions or functions of α5GABAARs improves certain memory performance.Orser et al.found that exogenous administration of IL-1β up-regulated the expressions of α5GABAARs in the hippocampus,increased the tonic current in the CA1 region,and then impaired cognitive functions in mice.Therefore,it is necessary to investigate the roles of the GABAergic neurons in POCD in aged mice.Recent studies showed that neuroinflammation is a common factor contributing to neurodegenerative diseases and associated with the hippocampal-dependent memory deficits after anesthesia and surgery.Our previous studies have confirmed that anesthesia and surgery can cause neuroinflammation,thereby reducing the expressions of synapse-related proteins and leading to deficits of hippocampal-dependent learning and memory.Neuroinflammation is a dynamic,multi-stage physiological response,mainly manifesting as the activation of natural immune cells in the central nervous system,accompanied by the release of a variety of pro-inflammatory factors(like IL-1β,IL-6 and TNF-α)that ultimately leads to changes of homeostasis in the central microenvironment.Studies have demonstrated that MAPK signaling pathway may be involved in regulating the transport of GABAA receptors,and changes in synaptic plasticity such as LTP,which could be responsible for learning and memory dysfunctions.However,the exact mechanisms underlying how neuroinflammation causes memory deficits is not well understood and no treatments that are available to effectively reverse or prevent memory deficits after anesthesia and surgery.Therefore,it is necessary to explore the down-stream mediators of neuroinflammation that induce memory deficits.We hypothesized that anesthesia and surgery may induce neuroinflammation in the hippocampus,disrupt the functions of the GABAergic neurons,and ultimately result in cognitive dysfunctions.In this study,laparotomy was used to simulate clinical abdominal surgery and to establish the model of POCD.Cognitive behavioral tests,molecular biological techniques,nuclear magnetic resonance and electrophysiological techniques were used to explore the roles of the GABAergic neurons in the hippocampus in POCD and its related mechanisms.Methods and Results 1.The expressions of GABA and α5GABAARs in the hippocampus were altered in POCD in aged mice.Methods: 16-month-old C57BL/6J female mice were randomly divided into the Control,Anesthesia and Anesthesia+Laparotomy groups.Novel object recognition test and fear condition test were performed 7 days after surgery to examine the hippocampal-dependent learning and memory.After the behavioral tests,hippocampal slices were prepared to record the long-term potentiation(LTP)of evoked field postsynaptic potentials(f PSPs)in the stratum radiatum in the CA1 region following electrical stimulation of the Schaffer collateral pathway.Nuclear magnetic resonance(NMR)was used to detect concentrations of Glutamate and GABA in the hippocampus.Quantitative real-time PCR technology was used to detect the transcription levels of α5,α1 and β3 subunits at 1,3,7 and 10 days after surgery.Western blot was used to detect the protein expressions of GAT-3,GAD65 and α5GABAARs.Results: The results showed that compared with the Control group,the hippocampal-dependent learning and memory was obviously disrupted in the Anesthesia+Laparotomy mice,while no difference was found in the Anesthesia group.And there was a remarkable increase in the amplitude of f PSPs(% of baseline)in the Control and Anesthesia slices after high-frequency-stimulation(HFS).In contrast,LTP was impaired and increased slightly in the Anesthesia+Laparotomy slices.Next,the NMR results showed that the levels of GABA were clearly decreased in the Anesthesia+Laparotomy group,while there was no difference in the levels of glutamate among the three groups.Then,the quantitative real-time PCR showed that there was no significant difference at any time point of α1 and β3 subunit levels,while the α5 subunit level was increased at 1 day and continued to increase at 3,7 and 10 days after surgery.Last,the expressions of GAT-3 and GAD65 were evidently decreased after surgery,and at the same time,the levels of surface α5GABAARs were up-regulated in the Anesthesia+Laparotomy mice.2.Pharmacological blockade of α5GABAARs could reverse POCD in aged mice.Methods: 16-month-old C57BL/6J female mice were randomly divided into the Control+Vehicle,Control+L655,708,Anesthesia+Vehicle,Anesthesia+L655,708,Anesthesia+Laparotomy+Vehicle and Anesthesia+Laparotomy+L655,708 groups.L655,708 was administrated to examine whether blockade of α5GABAARs could reverse POCD in aged mice.7 days after surgery,LTP was recorded.After recording the stable baseline,L655,708 was perfused for 10 min,then HFS was used to induce LTP and recorded for another 60 min.Results The results showed that L655,708 effectively alleviate hippocampal-dependent learning and memory deficits in the Anesthesia+Laparotomy+L655,708 mice.And the amplitude of f PSPs in the Anesthesia+Laparotomy+L655,708 mice was increased after application of L655,708.3.Activation of the P38-MAPK signaling pathway induces GABAergic neurons dysfunction in POCD in aged mice.Methods: 16-month-old C57BL/6J female mice were randomly divided into the Control,Anesthesia and Anesthesia+Laparotomy groups.Immunofluorescence was used to examine the morphology of microglia.Cytokine expressions of IL-1β,IL-6 and TNF-α were detected.The protein levels of P38,p-P38,JNK1/2,p-JNK1/2,ERK1/2 and p-ERK1/2 were evaluated using western blot.Then,behavioral tests were performed to examine whether SB203,580 could reverse POCD by blocking the P38-MAPK signaling pathway in aged mice.7 days after surgery,LTP was recorded.After recording the stable baseline,SB203,580 was perfused for 30 min,then HFS was used to induce LTP and recorded for another 60 min.Western blot was used to detect the protein levels of P38,p-P38,JNK1/2,p-JNK1/2,ERK1/2,p-ERK1/2 and surface α5GABAARs after using SB203,580.Results: The results showed that compared with the Control group,the morphology of microglia in the Anesthesia+Laparotomy mice was clearly activated,while no change was found in the Anesthesia mice.The MSD results showed that the levels of IL-1β and IL-6 were obvious up-regulated in the Anesthesia+Laparotomy group,but the expression of TNF-α was increased both in the Anesthesia and Anesthesia+Laparotomy groups.Compared with the Control group,the expression of p-P38 protein was obviously increased in the Anesthesia+Laparotomy mice.No statistical difference was observed in the protein levels of P38,JNK1/2,p-JNK1/2,ERK1/2 and p-ERK1/2.However,no difference was found in the Anesthesia group.SB203,580 effectively alleviate hippocampal-dependent learning and memory deficits in the Anesthesia+Laparotomy+SB203,580 mice.And the amplitude of f PSPs in the Anesthesia+Laparotomy+SB203,580 mice was increased after application of SB203,580.At the same time,a qualitative decrease in p-P38 protein and surface α5GABAARs protein was observed in the Anesthesia+Laparotomy+SB203,580 mice after using SB203,580,but there was no difference was found in the expressions of P38,ERK1/2,p-ERK1/2,JNK1/2 and p-JNK1/2.And at the same time,no difference was found in the Anesthesia mice.Statistical analysis All results are presented as mean ± SEM.An unpaired Student’s t-test was used to compare two groups.For multiple groups,One-way ANOVA or Two-way ANOVA was applied.Graph Pad Prism 7.0 was used for all analyses,and p < 0.05 was considered statistically significant in this study.Conclusion In our study,we found that hippocampal-dependent memory was disrupted by anesthesia and surgery rather than by anesthesia alone in aged mice.Anesthesia and surgery caused neuroinflammation in the hippocampus,which consequently induced dysfunctions of the GABAergic neurons and impaired LTP,eventually resulted in POCD in aged mice.Summary In this study,the results revealed that disruption of the GABAergic neurons in the hippocampus is a possible mechanism for POCD induced by anesthesia and surgery in aged mice.The levels of IL-1β,IL-6 and TNF-α were absolutely up-regulated in the hippocampus after anesthesia and surgery,which further increased the expressions of surface α5GABAARs especially through activating the P38-MAPK signaling pathway.Interventions targeting at the α5GABAARs or P38-MAPK signaling pathway can effectively alleviate POCD in aged mice.Therefore,our research may provide a new viewpoint for exploring the mechanisms of POCD,and α5GABAARs may serve as a potential target for preventing or treating POCD.