Background: Postoperative delirium(POD)is an acute neuropsychiatric syndrome characterized by fluctuating disturbances in attention,level of consciousness,and cognition defects following surgery,which is common in the elderly and associated with long-term cognitive impairment.However,the pathogenesis of POD is not fully identified,there is no standard knowledge and methods to reduce the occurrence of POD.Clinical and pre-clinical studies have shown that the abnormally activated reactive oxygen species(ROS)and the breakdown of blood-brain barrier(BBB)are positively correlated with the development of POD,but the association between these two factors and the potential mechanism is not clear.Cyclophilin A(CypA)is a specifically chemotactic leukocyte factor that can be secreted in response to ROS,which activates matrix metalloproteinase9(MMP9)and mediates BBB breakdown.This study was to investigate the correlation between ROS and BBB destruction in POD,and further explore the molecular mechanism of ROS regulating BBB permeability in response to anesthesia/surgery.We speculated that ROS may contribute to anesthesia/surgery-induced BBB damage and delirium-like behavior via the CypA/MMP9 pathway.Methods: In this experiment,16-month-old female C57/BL6 mice were subjected to laparotomy under 3% sevoflurane anesthesia to construct the POD model.The research was mainly divided into two parts:The first part was to study the role of ROS in regulating BBB permeability and inducing delirium-like behavior in aged mice after anesthesia and surgery.Mice were randomly divided into the following groups: control group,anesthesia/surgery group,control +NAC group,and anesthesia/surgery + NAC group,where NAC was used as a ROS scavenger.Mice in the anesthesia/surgery group and anesthesia/surgery + NAC group received laparotomy under 3% sevoflurane anesthesia,in the control + NAC group and anesthesia/surgery + NAC group were intraperitoneally injected with 90 mg/kg NAC at0.5 h before anesthesia,and in the control group and anesthesia/surgery group were intraperitoneally injected with the same volume of sterile normal saline.A battery of behavior tests(buried food test,open field test,and Y maze test)was used to evaluate behavioral changes at 24 h before and after anesthesia/surgery in mice: the buried food test to assess the natural tendency of mice to use olfactory cues for foraging,the open field test to measure the exploratory and general activity in mice,and the Y maze test to further assess spatial learning and memory ability in mice.Tissues were taken immediately at the end of behavior test,blood was collected from the eye socket and the concentration of S100β in the serum was detected by ELISA.Levels of tight junction proteins(ZO-1、occludin、claudin5),CypA,MMP9,postsynaptic density protein(PSD)-95,and synaptophysin in the prefrontal cortex were assessed by western blotting,the expression of ROS was measured by DHE staining,as well as IgG fluorescent staining to observe BBB leakage.The second part was to investigate whether ROS mediates BBB disruption through the CypA/MMP9 signaling pathway,thereby inducing delirium-like behavior in aged mice.Mice were randomly divided into the following groups: control group,anesthesia/surgery group,control + CsA group,and anesthesia/surgery + CsA group,where CsA was used as a CypA inhibitor.Mice in the anesthesia/surgery group and anesthesia/surgery + CsA group received laparotomy under 3% sevoflurane anesthesia,in the control + CsA group and anesthesia/surgery + CsA group received intraperitoneal injection of 10 mg/kg CsA at 0.5 h before anesthesia,and in the control group and anesthesia/surgery group received1% DMSO with the same volume.A battery of behavior tests was used to evaluate behavioral changes at 24 h before and after anesthesia/surgery in mice: the buried food test to assess the natural tendency of mice to use olfactory cues for foraging,the open field test to measure the exploratory and general activity,and the Y maze test to further assess spatial learning and memory ability in mice.Tissues were taken immediately at the end of behavior test,blood was collected from the eye socket and the concentration of S100β in the serum was detected by ELISA.Levels of tight junction proteins(ZO-1、occludin、claudin5),CypA,MMP9,PSD-95,and synaptophysin in the prefrontal cortex were assessed by western blotting,the expression of ROS was measured by DHE staining,as well as IgG fluorescent staining to observe BBB leakage.Results:Result 1: Compared with the control group,the A/S group showed increased levels of ROS,decreased expression of Tj proteins(claudin-5,occludin,ZO-1),increased release of CypA/MMP9,decreased levels of PSD95 and SYN in the prefrontal cortex,and the leakage of S100β in serum and IgG in the cerebral cortex were increased.Compared with the A/S group,the A/S + NAC group showed decreased ROS levels,increased Tj proteins expression,decreased CypA/MMP9 release,increased PSD95 and SYN expression,and decreased leakage of both S100β and IgG.However,the expression of above molecules in the A/S + NAC group was not significantly different compared to the control group.Compared with the control group,in the A/S group mice,the latency to find the hidden food and the latency to the center area were increased,and the duration in the center area,the time spent in the novel arm,and the percentage of novel arm entries were significantly decreased.Compared with the A/S group,in the A/S + NAC group mice,the latency to find the hidden food and the latency to the center area were decreased,and the duration in the center area,the time spent in the novel arm,and the percentage of novel arm entries were significantly increased.There was no significant difference in the behavior tests between A/S + NAC group and control group.Result 2: Compared with the control group,the A/S group showed increased levels of ROS,decreased expression of TJ proteins,increased release of CypA/MMP9,decreased levels of PSD95 and SYN in the prefrontal cortex,and the leakage of S100β in serum and IgG in the cerebral cortex were increased.Compared with the A/S group,the A/S + CsA group showed no significant ROS levels changes,increased TJ proteins expression,decreased CypA/MMP9 release,increased PSD95 and SYN expression,and decreased leakage of both S100β and IgG.However,the expression of above molecules in the A/S+ CsA group showed no significantly difference compared to the control group.Compared with the control group,in the A/S group mice,the latency to find the hidden food and the latency to the center area were increased,and the duration in the center area,the time spent in the novel arm,and the percentage of novel arm entries were significantly decreased.Compared with the A/S group,in the A/S + CsA group mice,the latency to find the hidden food and the latency to the center area were decreased,and the duration in the center area,the time spent in the novel arm,and the percentage of novel arm entries were significantly increased.There was no significant difference in the behavior tests between A/S + CsA group and control group.Conclusion: Anesthesia/surgery induced the release of ROS,and then activated the expression of CypA/MMP9 cascade,resulting in BBB dysfunction and delirium-like behavior in aged mice. |