The Effect Of Sepsis In Mice On Glymphatic Clearance Pathways And The Improvement Of Jiawei Buyang Huanwu Decoction

Objective: Sepsis-associated encephalopathy(SAE)is the common complication in septic patients,which is related to a higher mortality,long term cognitive function and quality of life.However,the precise mechanisms underlying sepsis-induced cognitive impairment remain largely to be elucidated and there are no major breakthroughs in clinical diagnosis and treatment.Therefore,the cecal ligation and puncture(CLP)method was used to construct a sepsis mouse model in this study,through the evaluation of behavioral tests in survival mice,the function of glymphatic clearance pathways were measured by using in vivo two-photon imaging and the related of brain metabolites were measured by targeted metabolomics.We systematically evaluated the mechanism of brain injury in SAE,and to provide fundamental basis for the treatment of SAE.At the same time,the therapeutic effect of Jiawei buyang huanwu Decoction(JBH-5)on SAE and the effect on the clearance of brain metabolites were initially evaluated.Method: 1.The effect of sepsis in mice on glymphatic clearance pathways.In this study,CLP operation was used to induce SAE in mice.The behavioral tests at day 11 and 30 after operation,the pathological changes of the brain tissue and the damage of neurons at day 30 after CLP were performed in mice with acute sepsis.Living body imaging of the cerebral blood vessel clearance pathway in day 30 of mice after CLP with two-photon fluorescence imaging technique.Immunofluorescence was used to detect the expression of Neun,microglia and astrocytes,polarization levels of aquaporin-4(AQP4)at 48 h and day 30 brain tissues of mice after CLP.Metabolomics was performed in day 30 brain tissues of mice with cognitive impairment after CLP by LC-MS / MS.2.The improvement of JBH-5 on sepsis encephalopathy in mice.The C57 / B6 male mice(9-10 W)were randomly divided into sham group,CLP group,positive control group(cefoperazone 0.2275 g/kg),JBH-5 high dose group(50 g/kg),JBH-5 medium dose group(25 g/kg),JBH-5 low dose group(12.5 g/kg).The SAE model of mice was established by CLP,the behavioral tests were performed at the different groups and the histopathologic changes were observed by HE and Nissl staining after day 30 of administration.Two-photon in vivo imaging technique was used to determine the function of perivascular clearance in mice and the changes of microglia and astrocytes and AQP4 in the brain of each group were detected by immunofluorescence.Result: 1.The effect of sepsis in mice on glymphatic clearance pathways.The survival rate of day 12 after CLP in mice was 35%,and the neurobehavioral score of CLP mice was significantly decreased at 24 hours after CLP(P < 0.05);the results of behavioral analysis in 11 and 30 days after CLP showed that the spatial memory cognitive ability of CLP mice was damaged.The HE and Nissl's staining in day 30 brain tissues of mice after CLP showed that the pathological changes of brain tissue and the number of hippocampal neurons was decreased in CLP mice.The results of two photon living imaging show that cerebrospinal fluid fluorescent tracers accumulated in the parenchyma and cerebrovascular interspaces of CLP mice and the clearance rate in brain parenchyma was significantly slowed down.The astrocytes in the brain of CLP mice showed increased activation in 48 h and day 30 after CLP,but the microglial cells were significantly activated in 48 h after CLP,and then changed into resting state of day 30 after CLP.The AQP4 mainly expressed in the astrocyte cytoplasm region,the polarization level was significantly lower in CLP group(P < 0.05).The targeted metabonomics analysis showed that there were 15 different metabolites in day 30 brain tissues of mice with cognitive impairment,among them,adenosine monophosphate(AMP),inosine 5'-monophosphate(IMP),xanthosine,guanosine and uric acid are involved in the purine metabolic pathway.The high level of uric acid may be a potential biomarker for the diagnosis and treatment of SAE.2.The improvement of JBH-5 on sepsis encephalopathy in mice.Compared with the CLP group,the survival rate of day 12 after CLP and the score of neurological behavior at 24 h after CLP were significantly higher in the positive control group and JBH-5 low dose group(P < 0.05).The behavioral analysis of day 30 after administration showed that the cognitive ability of spatial memory in JBH-5 low dose group was significantly improved.The pathological observation of brain tissue showed that the conditions of hippocampal neuron contraction,deep staining,and neuron cell reduction caused by CLP were improved after 30 days of administration.The clearance rate of fluorescent tracers in the brain parenchyma of JBH-5 group of mice was significantly increased(P < 0.05),which effectively reduce the accumulation of fluorescent tracers in the brain parenchyma and the perivascular space,meanwhile,the activation of glial fibrillary acidic portein(GFAP)in the brain of mice with JBH-5 was decreased(P < 0.05),and there was no significant change of ionized calcium-binding adapter molecule 1(Iba1)in each group.The expression of AQP4 in the JBH-5 group was mainly expressed in the end foot area of astrocytes,and compared with the CLP group,its polarization level was significantly improved(P < 0.05).Conclusion: 1.The spatial learning and memory was decline in survived mice with sepsis.2.The glymphatic clearance pathways of sepsis mice was significantly impaired,the astrocytes were continuously over-activated and AQP4 was depolarized.3.The astrocytes in mice with sepsis were transiently activated,but the astrocytes were continuously over-activated,meanwhile,the number of neurons were markedly decrease.4.The targeted metabonomics analysis showed that the disorder of purine metabolism in the CLP mice with cognitive impairment and the level of uric acid in the brain was increased.5.JBH-5 can significantly improve the cognitive function of spatial memory in mice after CLP.6.JBH-5 can improve the function of the glymphatic clearance pathways in sepsis mice and it can inhibit the excessive activation of astrocytes and reduce the level of AQP4 depolarization.