Study On The Repair And Mechanism Of Rat Spinal Cord Injury By Transplanting Rat Cardiomyocyte Mitochondria Into Schwann Cell

Spinal Cord Injury(SCI)is generally an injury to the central nervous system caused by a traffic accident,fall,violence or sports-related injury.In recent years,the treatment of SCI has focused on symptom-specific medication and surgery.However,none of these treatments have been very effective.Schwann Cells(SCs)are the main glial cells of the peripheral nervous system,capable of supporting axonal growth and myelin formation through the secretion of various signalling molecules and neurotrophic factors.Due to the properties of SCs,there is an increasing amount of research into the transplantation of SCs into the peripheral nervous system for the repair of SCI,but there are still limitations and challenges in the clinical application of this treatment method.As research progresses,mitochondrial transplantation(MT)is being widely studied in a variety of diseases such as neurodegenerative diseases,toxic injuries and cardiovascular diseases,and is beginning to be considered as a new potential therapeutic approach.Therefore,in order to explore a more effective treatment for SCI,this study combined SCs transplantation with MT,which is to introduce mitochondria from rat cardiomyocytes into rat SCs,and to use Schwann cells transplanted with exogenous mitochondria for the treatment of SCI,so as to judge the therapeutic effect.The main studies were as follows.(1)Extraction of mitochondria from Wistar rat cardiomyocytes,which firstly required characterisation of the extracted mitochondria.The morphology was mainly observed by transmission electron microscopy.The results showed that the extracted cardiomyocyte mitochondria were of high purity and structural integrity,with intact internal ridge structures and clear boundaries between the inner and outer membranes.Further protein immunoblotting(Western Blot)experiments were used to verify the presence of mitochondria-specific proteins.Cardiomyocyte mitochondria were then introduced into rat SCs using a co-incubation method.The viability of the SCs transplanted into exogenous mitochondria was examined by CCK-8 assay,Enzyme-linked immunosorbent assay(ELISA),apoptosis assay,mitochondrial membrane potential assay and cell scratch assay.The results showed that the exogenous mitochondria into SCs could significantly promote the proliferation of SCs,and also promote the secretion of more neurotrophic factors produced by SCs.Flow cytometry assays revealed a significant decrease in apoptosis and a significant increase in mitochondrial membrane potential in SCs transplanted with exogenous mitochondria,both under normal culture conditions and under hypoglycaemic/reoxygenated conditions.The results of the cell scratching assay further demonstrated that transplantation of exogenous mitochondria significantly enhanced the migratory capacity of SCs.All these results suggest that transplantation of exogenous mitochondria can enhance the ability of SCs very well.Secondly,in this study,a mitochondrial stress test was performed using the Seahorse Cell Energy Metabolism Analyzer.The results of the test showed that the intracellular mitochondrial reserve respiratory capacity of the transplanted cardiomyocyte mitochondria was stronger and better able to cope with the damage environment in vivo.(2)A rat model of spinal cord injury at the thoracic 10(T10)segment was constructed and treated with cell transplantation one week after modelling.The repair effect was evaluated by BBB score,ELISA test for neurotrophic factor secretion,Western Blot test for inflammatory factor expression,pathological observation of spinal cord HE sections at the injury site,and tissue immunofluorescence test.The experimental results showed that transplantation of exogenous mitochondrial SCs inhibited the expression of pro-inflammatory factors,promoted the expression of anti-inflammatory factors and improved the microenvironment of the injured area,and the HE results showed that the spinal cord was more compact,the area of spinal cord cavity was reduced,and the infiltration of inflammatory cells was significantly reduced compared with other groups;the secretion of neurotrophic factors was higher,and at the same time,astrocyte activation and glial activation were inhibited.The secretion of neurotrophic factors was higher,while the activation of glial cells and the formation of glial scar were inhibited,which promoted the regeneration of neurons and could better repair the injured spinal cord.Further,the mechanism of action of NRG1/Erb B,a regulatory signalling pathway of the nervous system,was investigated.The experimental results showed that transplantation of SCs with exogenous mitochondria could better promote the expression of neuregulin 1(NRG1)in vivo,while the expression of its receptor protein tyrosine inase eceptor 2(Erb B2)was upregulated,thus promoting axonal growth and regeneration of injured neurons,resulting in higher myelin basic protein(MBP)expression and promoting remyelination of the injured spinal cord.This promoted axonal growth and regeneration of injured neurons,resulted in higher MBP expression,promoted remyelination of the injured spinal cord,and reduced inflammation and improved the microenvironment at the injury site by inhibiting nuclear factor kappa-B(NF-κB)expression.