| Objective:To investigate the role and mechanism of NOD-like receptor protein 3(NLRP3)inflammasome in the central neuropathological alterations and cognitive dysfunction caused by repetitive mild traumatic brain injury(rmTBI)in rats,and to provide a reference for the prevention and treatment of cognitive dysfunction caused by rmTBI.Methods:Part Ⅰ:Forty-five 8-week-old Wister rats were divided into Control group,rmTBI+3 d group,rmTBI+14 d group,rmTBI+42 d group,and rmTBI+MCC950 group.The rmTBI rat model was first established using a single pendulum impact device.The rats in the Control group were anesthetized and placed on the impact platform without impact.The rats in the rmTBI+3 d,rmTBI+14 d,rmTBI+42 d and rmTBI+MCC950 groups were anesthetized and rapidly impacted five times at 24 h intervals for five consecutive days.At 6 h,12 h and 24 h from the last impact,MCC950(10 mg/kg)was injected intraperitoneally into rats in the rmTBI+MCC950 group,and equal amounts of saline were injected intraperitoneally into rats in the other groups.Then,the cognitive function of rats in each group was assessed by the open-field test and the three-chamber sociability test.HE staining and Nissl staining were used to observe the general histopathological changes in the hippocampal region of rmTBI rats,and the expression levels of NeuN,GFAP and Iba1 in the hippocampal region were detected by immunohistochemical staining.Finally,the expression levels of NLRP3 inflammasome and it mediated inflammatory pathway-related proteins(ASC,Caspase1 p20/Caspase-1,GSDMD-N/GSDMD,IL-1β and IL-18)in the hippocampus of rats were detected by immunoblotting.Part Ⅱ:Thirty-six 8-week-old Wister rats were divided into Control+Saline group,Control+MCC950 group,rmTBI+Saline group,and rmTBI+MCC950 group.Firstly,the rmTBI rat model was established according to the method in Part I.Secondly,the pathological changes of myelin sheath were observed by HE staining and solid green myelin staining,the expression levels of myelin and oligodendrocyte-related proteins in the corpus callosum(CC)and caudate putamen(CPu)regions were detected by immunohistochemical staining,and the ultrastructural changes of myelin were observed by transmission electron microscopy.Then,immunohistochemical staining was used to detect the expression levels of astrocytes marker proteins,microglia marker proteins,and inflammatory factors to infer the underlying mechanisms of myelinrelated pathological changes caused by rmTBI.Finally,the number of neurons and axonal damage were detected by NeuN immunohistochemistry and glycine silver staining.Results:Part Ⅰ:The results of the open field test showed that the rats spent less time in the central area of the open field,and the total distance of activity at day 3,14 and 42 after rmTBI.The results of the three-chamber sociability test showed that the rats in the rmTBI+3d group,rmTBI+14d group and rmTBI+42d group spent less time with new animals compared to the rats in the Control group.The HE staining results showed that the neuronal perimeters in the cortical area of rats in the rmTBI+3d group were widened compared with the Control group rats.Nissl staining and NeuN immunohistochemical staining showed that the number of neurons in the hippocampal area of rats was reduced at day 3,14 and 42 after rmTBI,with the most significant decrease in the hippocampal region of rats in the rmTBI+3 d group.Compared with rats in the Control group,the number of GFAP+astrocytes and Iba1+microglia in the hippocampal region of rats in the rmTBI+3d group and rmTBI+14d group increased,and the cytosolic volume of microglia increased and the number of protrusions decreased,especially in the rmTBI+3 d group rats.The results of immunoblotting showed that compared with rats in the Control group,NLRP3 inflammatory vesicles and their mediated inflammatory pathway proteins(ASC,Caspase-1 p20/Caspase-1,GSDMD-N/GSDMD,IL-1 β and IL-18)expression levels were increased and their expression levels peaked in the rmTBI+3 d group.Behavioral tests after MCC950 treatment revealed that rats in the rmTBI+MCC950 group spent increased time in the central area of the open field,increased total distance of activity,and increased time spent with new rats in the threechamber sociability experiment compared to the rmTBI+3d group.It was observed by HE staining results that the neuronal perimembranes were narrower in the cortical area of rats in the rmTBI+MCC950 group compared with rats in the rmTBI group;Nissl and NeuN immunohistochemical staining results showed that the number of neurons in the hippocampal area of rats in the rmTBI+MCC950 group was higher than that of rats in the rmTBI group;GFAP and Iba1 immunohistochemical results showed that,compared with GFAP and Iba1 immunohistochemistry results showed that the number of astrocytes and microglia increased,and the volume of microglia cytosol decreased and the length of protrusion became shorter compared with rmTBI group rats.Immunoblotting results showed that the expression levels of NLRP3 inflammatory vesicles and their mediated inflammatory pathway proteins(ASC,Caspase-1 p20/Caspase-1,GSDMD-N/GSDMD,IL-1 β and IL-18)in the hippocampus of rmTBI+MCC950 group rats were significantly lower than those of rmTBI group rats.Part II:In the rmTBI rat model,HE staining and myelin staining by solid green method showed that the number of myelin vacuolation in the CC region,the diameter of myelin vacuoles increased and the number of nerve bundles in the CPu region decreased in the rmTBI+Saline group of rats compared with the Control+Saline group;however,treatment with MCC950 decreased the number of myelin vacuolation in the CC area and the diameter of nerve bundles in the CPu area in the rmTBI+number and diameter of myelin vacuolation in the CC region and increased the number of nerve tracts in the CPu region in the MCC950 group of rats.Immunohistochemical staining for myelin and oligodendrocyte-associated proteins showed that the expression levels of MBP,CNP and MOG proteins and the number of Olig2+oligodendrocytes in the CC region of rats in the rmTBI+Saline group was decreased,and the number of MBP,CNP and MOG protein-positive nerve tracts in the CPu region was decreased compared to rats in the Control+Saline group.The relative densities of MBP,CNP and MOG proteins and the number of Olig2+oligodendrocytes in the CC region and the number of MBP,CNP and MOG protein-positive nerve tracts in the CPu region were significantly increased in the rmTBI+MCC950 group compared with the rmTBI+Saline group rats.The effect of rmTBI on glial cell activation and inflammatory factor expression levels was assessed by immunohistochemistry,and the results showed that the number of GFAP+astrocytes in the CC region,the number of Iba1+microglia and the number of IL-18+oligodendrocytes were increased in the rmTBI+Saline group compared with the Control+Saline group rats,and the micro After intraperitoneal injection of MCC950,the number of GFAP+astrocytes,Iba1+microglia,microglia cytosolic volume and IL-18+oligodendrocytes in the CC region of rats in the rmTBI+MCC950 group were significantly lower than those in the rmTBI+Saline group.In addition,the nerve fiber density was reduced and the number of NeuN+neurons was significantly lower in the rmTBI+Saline group rats compared with the Control+Saline group rats;however,the nerve fiber density and the number of neurons were significantly higher in the rmTBI+MCC950 group rats than in the rmTBI+Saline group after MCC950 treatment.Conclusions:1.rmTBI caused cognitive dysfunction in rats,and the histopathological examination showed that there were neuropathological changes in rats,such as neuron loss,activation of astrocytes and microglia,decrease of oligodendrocytes and myelin sheath injury.2.The ASC/Caspase-1/GSDMD/IL-1β pathway mediated by NLRP3 inflammasome is closely related to cognitive dysfunction in rats.3.MCC950 reverses the up-regulation of protein expression in the NLRP3/ASC/Caspase-1/GSDMD/IL-1β pathway by inhibiting NLRP3 inflammasome,and prevents rmTBI-induced glial cell activation,oligodendrocyte and myelin damage,and neuronal loss,finally alleviating cognitive dysfunction. |
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