| Postoperative cognitive dysfunction（POCD）is a common neurological complication after anesthesia surgery in elderly patients,mainly manifested by postoperative decreases in attention,memory,learning and executive abilities.The incidence of POCD is estimated to be 10%-30%and is higher in elderly patients.With the increase in the number of surgical procedures in the elderly population,POCD has attracted more attention.POCD can delay recovery,increase hospital stay and medical costs,and affect postoperative quality of survival and long-term survival.The pathophysiological mechanisms of POCD are unknown,and there are no effective preventive and curative measures.Vagus nerve stimulation（VNS）is a novel neuromodulation therapy that has been approved by the FDA for the treatment of epilepsy and depression.transcutaneous auricular vagus nerve stimulation（taVNS）is a non-invasive stimulation modality that achieves the same effects as invasive VNS by stimulating the ear branches of the vagus nerve.taVNS has been shown to affect a variety of physiological activities such as the function of the cholinergic system,inflammatory response,neuromodulation and cerebral blood oxygenation and blood flow.Some studies have also shown that taVNS can treat POCD in aged rats by alleviating central neuroinflammation.however,the effect of taVNS on POCD in clinically aged patients has not been reported,and the mechanism by which taVNS improves neuroinflammation in rats with POCD has not been clarified.In this study,we analyzed the incidence of POCD and changes in the function of the cholinergic system and the level of inflammatory factors in the blood of aged surgical patients and aged POCD model rats by performing taVNS;analyzed the behavioral changes,pathological changes in brain tissue,functional changes in the cholinergic system,and changes in neuronal death-related proteins in aged rats,and confirmed the taVNS at the human and animal levels The neuroprotective effects of taVNS were confirmed at human and animal levels.For the first time,we elucidated that taVNS can reduce neuroinflammation and improve POCD by enhancing the function of peripheral and central cholinergic system.Part one Effect of taVNS on POCD in elderly patients undergoing Total Knee ArthroplastyObjective:To perform taVNS in elderly patients undergoing Total Knee Arthroplasty,and to study the effect and mechanism of action of taVNS on POCD in elderly patients by observing the incidence of POCD and the levels of cholinesterase activity and inflammatory factors in peripheral blood.Methods:One hundred and twenty-four elderly patients who underwent knee arthroplasty at the Third Hospital of Hebei Medical University from April2021 to October 2021 were selected.The 124 consecutively recruited patients were divided into a sham stimulation group（SS group,n=62）and a taVNS group（taVNS group,n=62）according to the random number table method.The SS group received sham stimulation 1 h before induction of anesthesia;the taVNS group received taVNS 1 h before induction of anesthesia until the end of surgery.Patients in both groups underwent a series of neuropsychological tests 1 day before and 7 days after surgery,and venous blood was collected one day before and 1 day after surgery for serum acetylcholinesterase（ACh E）activity and serum butyrylcholinesterase activity（BCh E）,the inflammatory factors tumor necrosis factor alpha（TNF-α）,interleukin-1β（IL-1β）,IL-6,and high mobility group protein B1（HMGB1）,and brain injury factor S100calcium-binding protein B（S100B）.Basic information[age,sex,body mass index,years of education,ASA classification,preoperative coexisting diseases,and MMSE score],intraoperative indicators（anesthesia maintenance time,tourniquet time,blood loss,bradycardia,hypotension,tachycardia,hypertension）,and incidence of postoperative nausea and vomiting（PONV）were recorded for both groups.An additional 30 elderly healthy volunteers were recruited as non-operative controls（group C,n=30）and underwent two neuropsychological tests 8 days apart.The results of the neuropsychological tests at the above two time points in the SS group,taVNS group and Control group were recorded,and the incidence of POCD on postoperative day 7 was assessed using the composite Z score.Results:There were no statistically significant differences in age,gender,and MMSE scores among patients in the SS,taVNS,and Control groups.the differences in basic patient information,intraoperative indices,and PONV incidence between the SS and taVNS groups were not statistically significant（P>0.05）.119 of 124 patients completed neuropsychological testing 1 week after surgery.the incidence of POCD in the SS group was 27.1%（16/59）and the incidence of POCD in the taVNS group was 10%（6/60）,and the difference between the two groups was statistically significant（P<0.05）.The preoperative serum ACh E activity,BCh E activity,TNF-α,IL-1β,IL-6,HMGB1 and S100B levels were not significantly different between the taVNS and SS groups（P>0.05）.The postoperative ACh E and BCh E activities in the taVNS group decreased significantly compared with the preoperative levels,while the postoperative ACh E and BCh E activities in the SS group did not change significantly compared with the preoperative levels.The difference between the postoperative and preoperative values was significantly different between the two groups（P<0.05）.The postoperative levels of TNF-α,IL-6,HMGB1 and S100B increased significantly in both groups compared with the preoperative levels（P<0.05）.Compared with the SS group,the magnitude of the increase in IL-6,HMGB1 and S100B levels was significantly lower in the taVNS group（P<0.05）;the magnitude of the increase in TNF-αwas not significantly different（P<0.05）.The levels of IL-1βin the serum samples of most patients were below the low limit of detection and therefore could not be counted.Summary:taVNS reduced the incidence of POCD in elderly patients undergoing Total Knee Arthroplasty,as well as reduced serum ACh E and BCh E activities,inflammatory factor levels,and brain damage factor S100B levels in patients.Part two taVNS Alleviates Sevoflurane?Induced Cognitive Dysfunction in Aged Rats Via Activating Basal Forebrain Cholinergic NeuronsObjective:To establish a model of cognitive dysfunction in aged rats caused by sevoflurane and to investigate whether taVNS can improve cognitive impairment in model rats by activating the intracerebral cholinergic system and reducing hippocampal neuronal death and neuroinflammation.Methods:Older male SD rats（18 months old,500-600 g）were selected and divided into 5 groups:control group（Control group）,sevoflurane anesthesia group（Sev group）,sevoflurane anesthesia+taVNS group（Sev+taVNS group）,sevoflurane anesthesia+taVNS+immunotoxin 192-Ig G-saporin group（Sev+taVNS+192-sap group）and sevoflurane anesthesia+immunotoxin 192-Ig G-saporin group（Sev+192-sap group）.Rats were exposed to 2%sevoflurane for 5h to establish the POCD model.The taVNS was started24 h before sevoflurane inhalation for 30 min/day for 5 days.192-sap injection into the lateral ventricle one week before Morris water maze（MWM）training damaged the basal forebrain cholinergic neurons to explore the mechanism by which taVNS exerts neuroprotective effects.The cognitive function of the rats was evaluated by the Open field test（OFT）and MWM experiments;the number,death and loss of hippocampal neurons were observed by Nissl staining;the rate of hippocampal apoptosis and necroptosis was determined by flow cytometry;the activation of cholinergic neurons in the basal forebrain,the expression of apoptosis and necroptosis-related proteins in the hippocampus were evaluated by immunofluorescence staining;western blot analysis The expression of cholinergic-associated proteins in the basal forebrain and hippocampus,apoptosis and necroptosis-associated proteins in the hippocampus were detected by western blot analysis;hippocampal ACh E activity was determined by tissue activity kit;hippocampal ACh content was determined by enzyme-linked immunoassay kit.Results:1.Behavioral tests No statistically significant differences in OFT index results were observed among the five groups of rats（P>0.05）.Compared with the Control group,the Sev group,Sev+taVNS+192-sap group and Sev+192-sap group showed a significant decrease in the time spent in the target quadrant and the number of platform crossing（P<0.05）;there was no significant difference in the Sev+taVNS group.The time spent in the target quadrant and the number of platform crossings were significantly increased in the Sev+taVNS group compared with the Sev group（P<0.05）.The time spent in the target quadrant and the number of platform crossing were significantly decreased in the Sev+taVNS+192-sap group and Sev+192-sap compared with the Sev+taVNS group（P<0.05）.2.Nissl staining The neurons in the CA1 area of hippocampus in the Control group had regular morphology,neat arrangement,clear nucleus structure,uniform staining,and abundant niche vesicles;the neurons in the CA1area of hippocampus in the Sev group,Sev+taVNS+192-sap group,and Sev+192-sap group were disordered,and the nucleus was deeply stained and fixed,and the number of niche vesicles was reduced.The neurons in the CA1 region of the hippocampus of the taVNS group were slightly more neatly arranged,with a clearer nuclear structure and an increased number of niche vesicles in the cytoplasm.Compared with the Control group,the number of neurons in the hippocampal CA1 area was significantly reduced in the Sev group,Sev+taVNS+192-sap group and Sev+192-sap group（P<0.05）.The number of neurons in the hippocampal CA1 area was significantly increased in the Sev+taVNS group compared with the Sev group（P<0.05）.3.flow cytometry assay Compared with the Control group,the rates of apoptosis and necroptosis in hippocampal cells were significantly increased in the Sev group,Sev+taVNS+192-sap group and Sev+192-sap group（P<0.05）.Compared with the Sev group,the rates of apoptosis and necroptosis in hippocampal cells were significantly decreased in the Sev+taVNS group（P<0.05）.4.immunofluorescence assay（1）Cholinergic system indexes Compared with the Control group and Sev group,the number of Ch AT+cells and Ch AT+c-fos+cells in the forebrain basal nucleus area were significantly increased in the Sev+taVNS group（P<0.05）;the number of Ch AT+c-fos+cells was significantly increased in the Sev+taVNS+192-sap group（P<0.05）;the number of Ch AT+c-fos+cells was significantly increased in the Sev+192-sap group（P<0.05）.The number of Ch AT+cells in the basal nucleus region of the forebrain was significantly reduced（P<0.05）.The number of Ch AT+cells and the number of Ch AT+c-fos+cells in the forebrain basal nucleus area were significantly reduced in the Sev+taVNS+192-sap and Sev+192-sap groups compared with the taVNS group（P<0.05）.Compared with the Sev+taVNS+192-sap group,the number of Ch AT+cells and the number of Ch AT+c-fos+cells in the forebrain basal nucleus area were significantly reduced in the Sev+192-sap group（P<0.05）.（2）Neuronal death indicators Compared with the Control group,the proportion of cleaved Caspase-3+Neu N+cells and the proportion of p-MLKL+Neu N+cells in the hippocampal CA1 region in the Sev group,Sev+taVNS+192-sap group and Sev+192-sap group were increased to different degrees（P<0.05）;the proportion of cleaved Caspase-3+Neu N+cells in the Sev+taVNS group cleaved Caspase-3+Neu N+cell proportion increased,while p-MLKL+Neu N+cell proportion did not change significantly（P>0.05）.Compared with the Sev group,the proportion of cleaved Caspase-3+Neu N+cells and p-MLKL+Neu N+cells in the hippocampal CA1 region was significantly decreased in the Sev+taVNS group（P<0.05）.The proportion of cleaved Caspase-3+Neu N+cells and p-MLKL+Neu N+cells in Neu N+cells in the hippocampal CA1 region was significantly increased in both the Sev+taVNS+192-sap group and the Sev+192-sap group compared with the Sev+taVNS group（P<0.05）.（3）Neuroinflammatory indexes The number of Iba1+cells and the proportion of deramified g Iba1+cells in the hippocampal region were significantly increased in the Sev group,the Sev+taVNS+192-sap group and the Sev+192-sap group compared with the Control group（P<0.05）;the number of Iba1+cells was not significantly changed in the Sev+taVNS group,while the proportion of deramified Iba1+cells was significantly increased（P<0.05）.The number of Iba1+cells and the proportion of deramified Iba1+cells in the hippocampus were significantly reduced in the Sev+taVNS group compared with the Sev group（P<0.05）.Compared with the Sev+taVNS group,the number of Iba1+cells and the proportion of deramified Iba1+cells in the hippocampal region were significantly increased in the Sev+taVNS+192-sap group and the Sev+192-sap group（P<0.05）.5.western blot analysis（1）Cholinergic system indicators Compared with the Control group and Sev group,the forebrain basal nucleus and hippocampal Ch AT protein expression levels were significantly increased in the Sev+taVNS group,and significantly decreased in the forebrain basal nucleus and hippocampal Ch AT protein expression levels in the Sev+taVNS+192-sap group and Sev+192-sap group（P<0.05）.（2）Neuronal death indicators Compared with group Control,the expression levels of Bax,Caspase-3 and cleaved Caspase-3 proteins were significantly increased in the Sev group,Sev+taVNS+192-sap group and Sev+192-sap group（P<0.05）;the expression level of BCL-2 was significantly increased in the Sev+taVNS group（P<0.05）.Compared with the Sev group,the BCL-2 expression level was significantly increased in the Sev+taVNS group（P<0.05）,and the Bax,Caspase-3 and cleaved Caspase-3 protein expression levels were significantly decreased in the Sev+taVNS group（P<0.05）.Compared with the Sev+taVNS group,BCL-2expression levels were significantly decreased in the Sev+taVNS+192-sap group and Sev+192-sap group（P<0.05）,and Bax,Caspase-3 and cleaved Caspase-3 protein expression levels were significantly increased（P<0.05）.The expression levels of RIPK1,RIPK3,MLKL and p-MLKL proteins were significantly increased in the Sev group,Sev+taVNS+192-sap group and Sev+192-sap group compared with the Control group（P<0.05）.The expression levels of RIPK1,RIPK3,MLKL and p-MLKL proteins were significantly decreased in the Sev+taVNS group compared with the Sev group（P<0.05）.RIPK1,RIPK3,MLKL and p-MLKL protein expression levels were significantly increased in the Sev+taVNS+192-sap group and Sev+192-sap group compared with the Sev+taVNS group（P<0.05）.6.cholinergic kit assay Hippocampal ACh E activity was significantly lower in the Sev group compared with the Control group,Sev+taVNS group,Sev+taVNS+192-sap group and Sev+192-sap group（P<0.05）.Compared with the Control group and the Sev+taVNS group,the Hippocampal ACh content was significantly lower in the Sev group,Sev+taVNS+192-sap group,and Sev+192-sap group（P<0.05）.Summary:taVNS activated cholinergic pathways in the basal forebrain and hippocampus,reduced hippocampal neuronal apoptosis,necroptosis and microglia activation,and improved sevoflurane-mediated cognitive dysfunction in aged rats.Depletion of cholinergic neurons in the basal forebrain could block taVNS from exerting its effects of improving cognition,protecting hippocampal neurons and inhibiting microglia activation.Overall,taVNS may ameliorate sevoflurane-induced hippocampal neuronal damage and inflammatory responses by activating the basal forebrain cholinergic system. |
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