Proteomic Analysis Of The Model Of Cervical Spinal Cord Hemicontusion In Mice

BackgroundFor patients with spinal cord injury(SCI),there is currently no effective clinical method to fully achieve functional recovery.Exploring the mechanisms and methods of nerve regeneration and repair after spinal cord injury has important theoretical significance and clinical value.After spinal cord injury,the primary and secondary injuries will lead to the inflammation of local nerve tissue,the formation of scar or cavity,etc.Recent research shows that lipid deposition and related lipid metabolism activities play an important role in it,which may improve the local microenvironment of nerve regeneration by improving lipid metabolism.ObjectiveThe purpose of this study is to study the changes of lipid deposition,key pathways and molecular biological characteristics in the evolution of spinal cord injury disease through behavioral,histological and proteomics,and provide new ideas and means for nerve regeneration and repair after spinal cord injury.Materials and methodsThe model of C5 hemilateral spinal cord injury was established by controlling the speed of 300 mm/s contusion and the depth of 1.2 mm contusion with a 1.0 mm percussion head.The degree of injury and the change of course of disease were evaluated by observing the weight change of mice,rearing test and magnetic resonance examination.Oil red O staining and transmission electron microscopy were used to observe the deposition of lipids and lipid droplets in the spinal cord at different microscopic scales.Then select the spinal cord injury segments of mice in the 6 weeks after operation and sham-operation group for proteomic analysis,analyze the differential expression protein and use GO analysis and KEGG analysis to make functional annotation.Finally,targeted proteomics,protein immunoblotting and immunofluorescence staining were used to verify the expression of related proteins.ResultThe mechanical parameters of the established model of hemilateral spinal cord contusion in mice were stable,and the body weight and behavioral score tended to be stable at 6 weeks after operation.The lesions showed low signal under T2WI sequence,and fibrous scar formation,and the course of disease entered the chronic stage.At 6 weeks after operation,the injured side of the tissue section was slightly atrophic,stained with oil red O,with deep color and wide range,and serious lipid deposition.Under transmission electron microscope,the accumulation of lipid droplets was obvious,mainly small lipid droplets.At 6 weeks after spinal cord injury,it was the representative period of lipid deposition in the chronic phase of spinal cord injury.Proteomic analysis showed that there were differences in the expression of Apod,Apoe,Dhrs1,Hexb and other proteins,which were closely related to lipid metabolism.At 6 weeks after spinal cord injury,in the Western blot test,the high expression of TNF-α,CTSB,CTSZ and CD11b suggests the aggravation of neuroinflammation;The high expression of ADFP,APOE and NPC2 suggests that lipid metabolism is strengthened;The high expression of LC3B and Beclin-1 suggests enhanced autophagy.At the same time,the immunofluorescence was accompanied by the accumulation of lipid droplets and the high expression of APOE,ADFP and LC3B.ConclusionSix weeks after injury can serve as a typical time point for studying the pathological and physiological changes in the chronic phase of SCI.There is significant neuroinflammation in the chronic phase of SCI,accompanied by lipid deposition and enhanced autophagy.Autophagy may be involved in regulating lipid metabolism and neuroinflammation in the chronic phase of SCI.