Neuroprotective Effect Of Dexmedetomidine On Rat With Minimal Hepatic Encephalopathy And Its Mechanisms

Objective: The aim of this study was to investigate the effects of dexmedetomidine on sleep structure,neurobehavior and brain morphology in rats with minimal hepatic encephalopathy,to explore the relevant mechanisms,and to provide theoretical basis for the neuroprotective effect of dexmedetomidine on minimal hepatic encephalopathy.Methods:1.Experimental model: Healthy male SD rats,weighing 220-250 g,were tested with Morris water maze for 5d before modeling to determine the rats' escape latency.Then MHE was induced by intraperitoneal injection of TAA(200 mg/kg)every 24 h for two consecutive days.TAA-treated rats without HE symptoms were then subjected to behavioral test again to confirm whether theyhad MHE or not.If TAA-treated rats met the following criteria,they were assigned to the MHE group: the escape latency of Morris water maze was higher than that of the Con group.2.Part one: The 32 rats were randomly divided into 4 groups : Control group(group Con),TAA group(group TAA),dexmedetomidine group 1(group D1)and dexmedetomidine group 2(group D2).The Con group and TAA group were injected with 0.9% normal saline of the same volume.The samples were collected for detection at 24 hours after dexmedetomidine injection.We recorded and analyzed 24 h sleep EEG of rats by Multi-channel physiological signal acquisition and processing system.The movement and mental state were then tested with the open field tests.Moreover,the liver and brain histopathological changes,the levels of serum ammonia(Amm),alanine transaminase(ALT)and aspartate aminotransferase(AST)were measured after the above experiments.3.Part two: A total of 80 male Sprague–Dawley rats were randomly divided into 5 groups:group Con,group TAA,group D1,group D2 and Dexmedetomidine+Yohimbine treatment group(group DY).The expressions of NLRP3 inflammasome(including NLRP3 and Caspase-1 p20)in the prefrontal cortex of rats were measured by Western blotting and Immunofluorescence.Microglial activity was observed by immunofluorescence staining.In addition,enzyme-linked immunosorbent assay(ELISA)was performed to detect the levels of TNF-?,IL-1?,and IL-18.Results:Part one:1.After intraperitoneal injection of thioacetamide for two consecutive days,one rat died,and the remaining survivors were tested by Morris water maze method to detect the escape latency.The results showed that 79.17% rats had significantly increased on the escape latency compared with the normal group.After excluding the symptoms of overt hepatic encephalopathy,the model of MHE was established successfully.2.Hepatocytes appeared edema and necrosis with a large infiltration of inflammatory cells after administration of TAA.The inflammation and hepatocytes damage were significantly ameliorated after the rats treated with dexmedetomidine.Compared with Con group,the number of normal neurons in MHE group,D1 group and D2 group was significantly reduced(P<0.01 or<0.05).However,compared with MHE group,the neuronal damage of D1 group and D2 group were alleviated(P<0.01).Compared with Con group,the levels of Amm,ALT,and AST markedly increased in MHE group(P<0.01).Compared with MHE group,they significantly decreased in dexmedetomidine groups(P<0.01).3.Electroencephalography(EEG)results showed that,compared with Con group,the light slow wave sleep(SWS1)in MHE group increased significantly,while the deep slow wave sleep(SWS2)decreased,the difference wasstatistically significant(P<0.05 or P<0.05).During the sleep period of MHE rats,the awakening time increased,while the active period showed an increase in the light sleep time(SWS1).The sleep wake pattern was close to the inversion in MHE group.Compared with TAA group,the dexmedetomidine groups had shorter SWS1 time and longer SWS2 time,and the difference was statistically significant(P<0.01).The sleep structure was improved to a certain extent,which was manifested as significantly prolonged daytime sleep time and increased nighttime wakefulness time.This is similar to the daytime rhythm of normal rats.There was no significant difference in REM sleep among all four groups(P>0.05).4.The total distances in the open field test showed no significant difference in each group(P>0.05).The central area distances,central area time and rearing frequencies in MHE group were less than those in Con group(P<0.01).Compared with the TAA group,these indicators were increased by dexmedetomidine treatment(P<0.01).All these results suggested that MHE rats induced by TAA might have anxiety-like behavior.Part two:1.Western blot analysis was performed to measure the protein levels of NLRP3 and Caspase-1 in prefrontal cortex.The expression of NLRP3 and Caspase-1p20 in the MHE group was significantly increased than that in Con group.Howerer,the expression of those two proteins in dexmedetomidine groups were significantly decreased compared with MHE and DY groups(P<0.01).2.Immunofluorescence results showed that TAA induced an up-regulation of the microglial activation marker Iba-1.The number of microglial cells was also increased,and their filopodia retracted in MHE group.Compared to MHE and DY groups,microglial activation was inhibited in dexmedetomidine groups.3.Compared with Con group,the levels of IL-1?,TNF-?,and IL-18 in the prefrontal cortex in MHE group increased significantly(P<0.01).Compared with MHE group and DY group,the levels of inflammatory factors in D1 group and D2 group decreased significantly(P<0.01).4.Immunofluorescence results further showed that compared with Con group,NLRP3 expression was significantly up-regulated in the prefrontal cortex of TAA group,while NLRP3 expression was down-regulated after treatment with dex.The effect of dexmedetomidine was significantly inhibited by yohimbine treatment.Conclusion:1.The rats with minimal hepatic encephalopathy induced by TAA(intraperitoneal injection,ip)showed the EEG structural changes of sleep disorders,neurobehavioral abnormalities and brain injuries.Dexmedetomidine could regulate rats' abnormal sleep structure and neurobehavioral and improve their liver function and brain injury;2.The neuroprotective effect of dexmedetomidine on MHE rats might bepartly related to decreasing microglial activation,reducing neuroinflammation and inhibiting activated NLRP3/Caspase1 signaling pathway after the activation of alpha 2 adrenergic receptor.