Effects Of Sevoflurane On The Glymphatic Function In Mice Brain By Increasing The Expression Of Aquaporin 4

Background: The glymphatic system is a paravalvular space between the cerebral vascular system and astrocytes.This system plays a vital role in maintaining normal brain neurological function by transporting cerebrospinal fluid and eliminating waste products.It plays a role similar to that of the glymphatic system in the brain.Therefore,it is named the glial-lymphatic system.The glymphatic system delivers nutrients and drugs to the brain parenchyma and rapidly removes metabolic waste products such as lactic acid and beta-amyloid(A?)from deep within the brain.Dysfunction of the glymphatic system leads to the accumulation of toxic metabolites in the brain parenchyma,triggering neuroinflammation that contributes to cognitive dysfunction.Moreover,glymphatic system function also has an immunomodulatory role and may be involved in neuroinflammatory responses.Scholars believe that glymphatic dysfunction is associated with many neurodegenerative diseases,including Alzheimer's disease.The typical pathological feature of Alzheimer's disease is the aggregation of A? in the perivascular interstitial,vessel wall,and intercellular spaces.Numerous studies have also shown that there is an increase or accumulation of A? in the cerebrospinal fluid and brain in patients with postoperative cognitive impairment.Therefore,we hypothesize a causal relationship between dysfunction of the glymphatic system and pathological mechanisms of postoperative cognitive dysfunction.Perioperative anesthesia and surgical trauma may affect the glymphatic function,causing barriers to the excretion of metabolic substances,affecting brain function,and leading to postoperative cognitive dysfunction.Numerous clinical studies have demonstrated that sevoflurane as an inhalation anesthetic agent commonly used in clinical anesthesia has a significant relationship with postoperative cognitive dysfunction.Therefore,this experiment investigates the effect of sevoflurane on the function of the brain's glymphatic system.Methods: The first part was animal experiments.Firstly,we studied the effect of sevoflurane on mice's behavior and glymphatic function.The study aims to discover the relationship between mice behavioral changes and the glymphatic function.The position navigation test of the Morris water maze(reference memory test)was used to familiarize the mice with the experimental conditions four times a day before the operation.On the day of surgery,mice were divided into two groups: the control group and sevoflurane group,and Evans Blue was injected into the cisterna magna of mice in both groups.The control group mice were then kept awake and free to move,while the sevoflurane group was sevoflurane(4%)anesthetized for four hours.Following that,all groups of mice were kept fully awake and free to move for 4 hours.Subsequently,open-field tests and Morris water maze tests were performed on the mice in both groups.After the behavioral tests were completed,the mice were immediately euthanized.The brains were dissected to observe the amount of Evans blue on the surface of the brain and speculate on the effect of sevoflurane on the glymphatic function.Next,the effects of a novel aquaporin 4 inhibitor(TGN-020)on the glymphatic system and mice's behavior were assessed.The results of this experiment showed that TGN-020 exerted only specific inhibition of AQP4 in the short term,did not change the behavior of mice,and was non-toxic and harmless to the central nervous system.Therefore,the TGN group was not set up anymore in the subsequent experiment.Finally,we investigated how the effect of TGN-020 on the glymphatic function during the sevoflurane anesthesia state and whether it ameliorates the behavioral changes caused by sevoflurane anesthesia.Mice were divided into three groups: control group,sevoflurane group,and sevoflurane + TGN group.Evans blue was injected into cisterna magna to observe the functional changes of the glymphatic system.The Morris water maze test and the open field experiment were used to study sevoflurane's effects on cognitive function or other behavioral aspects.The total fluorescence content within the three groups' brains was observed by in vivo imaging with near-infrared fluorescence imaging;the Evans blue content in the serum of the three groups was measured by immunofluorescence spectrophotometry to detect the amount of Evans blue secreted to blood circulation via the glymphatic system.The expression of IL-6 in the three sera groups was detected by the enzyme-linked immunoassay technique.After that,the expression of water channelprotein 4 in the brain was detected by fluorescence immunology.The second part is the cellular experimental part.Primary astrocytes were used to explore the mechanism related to the increase of glymphatic function by sevoflurane.The expression of aquaporin 4 was detected by western blotting and cellular immunofluorescence.The expression of protein kinase C(PKC)in astrocytes was observed by western blotting.The enzyme-linked immunoassay technique detected the expression of IL-6 in astrocytes.The amount of superoxide dismutase(SOD)was detected by nitrogen blue tetrazolium chromogenic assay.The cell membrane clamp technique observed sevoflurane and TGN-020 on the conductivity of total potassium channels in astrocytes.The final statistical comparison between the experimental and control groups was performed statistically by one-way analysis of variance(ANOVA).Results: 1.The effects of 4 hours of 4 percent sevoflurane anesthesia showed a significant increase in glymphatic activity and significant behavioral changes during the early post-anesthetic recovery time(4 hours after awakening).2.Puncture of the cerebellar cisterna magna significantly increased IL-6 expression in mice,while sevoflurane caused minor inflammation.3.Sevoflurane increased aquaporin 4 and PKC expression in the brain,decreased SOD content,and weakened superoxide scavenging.In addition,sevoflurane reduced the conductivity of potassium channels in astrocytes.4.During 4% sevoflurane anesthesia,TGN-020 still exerted glymphatic function inhibition and improved behavioral abnormalities induced by sevoflurane anesthesia.It also ameliorated sevoflurane-induced decrease in the conductivity of potassium channels in astrocytes.5.TGN-020,a novel aquaporin 4 inhibitor,significantly inhibited glymphatic function.Within 8 hours after receiving a single dose of TGN-020,mice showed no significant behavioral changes.Conclusion: 1.Sevoflurane enhanced glymphatic function,the mechanism of which is involved with the upregulation of aquaporin 4 and PKC expression in astrocytes.2.Enhanced glymphatic function induced by sevoflurane may cause excessive surgical inflammation into the brain via cerebrospinal fluid,causing neuroinflammation and leading to postoperative behavioral changes in mice.3.TGN-020,a novel aquaporin 4-specific inhibitor,inhibited the sevoflurane-induced glymphatic hyperfunction and improved behavioral changes in the mice model.