Role Of Microglial P2X7R/NLRP3/caspase-1 Axis In Postoperative Cognitive Dysfunction

Part I: Effects of surgery with different inhaled anesthetics on postoperative cognitive function Objective: To compare the postoperative cognitive function and neuroinflammation in mice undergoing surgery with three commonly used inhaled anesthetics : isoflurane,sevoflurane,and desflurane.Methods : Eight-week-old C57BL/6J mice were randomized into four groups: control group(Con),isoflurane + surgery group(Iso),sevoflurane + surgery group(Sev),and desflurane + surgery group(Des),12 in each group.The experiment was started after the mice were acclimated for three days in the animal room.On the first day,each group of mice inhaled the above three different inhalation anesthetics,the inhalation concentration was 1.5 MAC,30 min later,the right carotid artery exploration was performed,the operation time was about 15 min,and then the inhalation anesthesia was continued,and the total inhalation time was 2 hours.After the end,wait for the mice to wake up and put them in the animal room to continue feeding.On days 15-18,Barnes maze training was started for 4 consecutive days;on the 19 th and 26 th days,the Barnes maze was used to test the short-term and long-term spatial memory ability.On day 27 and 28,a fear conditioning experiment was performed to observe the context and tone memory.The hippocampus tissue was treated with the same conditions at 6h and 7d postoperatively.Immunofluorescence staining was used to observe the activation of microglia,P2X7 with Iba-1,GFAP and Neu N co-locolized Immunofluorescence were also performed.Western blot and q PCR were used to detect hippocampal P2X7 R,NLRP3 and caspase-1 protein and m RNA expression,IL-1β content was detected by ELISA.Results: On Days 3 and 4 of the Barnes Maze training phase,the time to find the target hole was significantly shortened in all mice compared to Day 1(p<0.05),while there was a significantly decline in short-term and long-term memory test in the isoflurane andsevoflurane anesthesia group(p<0.05).However,in the context-related memory test,the freezing time of the isoflurane and sevoflurane groups were significantly shortened(p<0.05),while the desflurane group did not change significantly(p > 0.05).Immunofluorescence staining of Iba-1 labeled microglia showed that the activation of microglia in the isoflurane group was significantly higher than that in the control group(p<0.05).The ELISA results showed that the isoflurane group of IL-1β were higher than the control group and desflurane group(p<0.05).In the results of western blot and q PCR,P2X7 R,NLRP3 and caspase-1(p20)were significantly increased in the isoflurane group compared with the control group(p<0.05).Conclusion: After surgery with three different inhalation anesthesia,cognitive function had a significantly decline in isoflurane and sevoflurane group,and with microglial activation,P2X7 R and NLRP3 inflammasome activation,and increased IL-1β release.Part II: Role of microglia P2X7R/NLRP3/caspase-1 axis in postoperative cognitive dysfunctionObjective: To evaluate the role of P2X7R/NLRP3/caspase-1 axis in postoperative cognitive dysfunctionMethods: C57BL/6J mice(8 weeks old)were randomly divided into four groups: control group(Con),BBG group,surgery group(Surg),BBG+ surgery group(BBG+Surg),with12 rats in each group.The experiment was started after the mice were acclimated for three days in the animal room.5 days before the operation,the Con group and the Surg group were intraperitoneally injected with 1 ml of normal saline per day,and the BBG group and the BBG+Surg group were intraperitoneally injected with BBG 50 mg/kg.On the day of surgery,mice in each group were induced by inhalating 3% isoflurane,and then maintaining the inhalation concentration of 1.5 MAC,30 minutes later,the right carotid artery exploration was performed,and then the inhalation anesthesia was continued for a total inhalation time of 2 hours.After the end,wait for the mice to wake up and put them in the animal room to continue feeding.On days 15-18,Barns maze training was started for 4 consecutive days;on day 19 and 26,the Barns maze was used to test the short-term and long-term spatial memory ability.On day 27 and 28,fear conditioning experiment was performed to observe the context and tone memory of the mouse.At 6h and 7d after surgery,the hippocampus tissue was harvested and use Immunofluorescence staining to observe the microglial activation.Western blot was used to detect P2X7,NLRP3,and caspase-1 expression in hippocampus;q PCR was used to detect m RNA expression;ELISA was used to detect hippocampus IL-1β content.Then,it was further confirmed by the P2X7 knockout mouse.Results: The time to find the target hole on the 3rd and 4th day of the Barnes Maze training was significantly shorter than the first day(p<0.05).During the training period,BBG effectively shortened the time for the mice after anesthesia to correctly find the target hole(P<0.05).In the tests of short-term and long-term memory performed 1 and 7days after training,BBG also effectively shortened the time for mice to find the target hole correctly(p<0.05).In the fear conditioning experiments,BBG effectively prevented the decreased freezing time caused by anesthesia and surgery(p<0.05).Immunofluorescence staining showed that the expression of Iba-1 in hippocampus of mice increased significantly under the anesthesia and surgery,and the effect of increased expression of Iba-1 was significantly inhibited by BBG(p <0.05).Similarly,the increased concentration of IL-1β in hippocampus caused by anesthesia and surgery was also blocked by BBG(p < 0.05).At 6h and 7 days after anesthesia and surgery,the expression of P2X7 and inflammasome related proteins or m RNA in hippocampus was significantly increased,while the P2X7 inhibitor BBG significantly inhibited P2X7 and caspase-1related proteins or m RNA Expression(p <0.05).Compared with the control group,Barnes maze training,short-term and long-term memory tests,and fear conditioning test results in P2X7 knockout mice showed that even if anesthesia and surgery were performed,the difference was not statistically significant(p> 0.05).Similarly,the concentration of IL-1β in hippocampus of P2X7 knockout mice receiving anesthesia and surgery was also not significantly different from that of the control group(p>0.05).Conclusion: The P2X7R/NLRP3/caspase-1 axis mediates postoperative cognitive dysfunction caused by isoflurane anesthesia and surgery.