Effect And Mechanism Of Perlecan On Blood-Spinal Cord Barrier Repair After Spinal Cord Injury

Objective and Significance:Spinal cord injury(SCI)is a common disease of central nervous system(CNS)injury.Primary injury will lead to the destruction of the blood-spinal cord barrier(BSCB),and the infiltration of various inflammatory factors and inflammatory cells from blood through the damaged BSCB will induce and aggravate secondary injury,so the destruction of the BSCB is considered to be closely related to the secondary injury of the spinal cords.Current studies on the BSCB after SCI mainly focus on cellular components,such as endothelial tight junctions(TJs)repair,pericellular coverage and astrocytic AQP4 edema,but the changes and significance of the basement membranes(BMs)in the BSCB after SCI remain unclear.Previous studies have shown that Perlecan in the BMs of the blood-brain barrier(BBB)is more sensitive to stroke than other proteins such as Laminin and Collagen Ⅳ,and plays an important role in nerve injury and functional recovery.This suggests that Perlecan is sensitive to ischemic brain injury and has important implications for the function of the BBB.However,how is Perlecan expressed after SCI?What is its significance in maintaining BSCB function and in secondary injury and repair?None of these have been reported in the literature.In this study,mouse SCI model,CRISPR/Cas9-based synergistic activation mediator system(CRISPR/Cas9-SAM)lentivirus in situ and GRGDSP(integrin receptor antagonist)in vivo injection model were used to investigate the effect of Perlecan on the BSCB and nerve regeneration and repair after SCI and its related molecular mechanism.Methods:Western blotting,immunofluorescence and qPCR were used to detect the expression and localization of Perlecan at different time points after SCI.Perlecan overexpression was detected by western blotting,immunofluorescence and qPCR after in situ injection of CRISPR/Cas9-SAM lentivirus.The movement and bladder function were detected by BMS,footprint test,swimming test and bladder function score.Immunofluorescence staining and qPCR were used to detect the neuronal survival,axonal regeneration and neuroinflammatory reaction.The BSCB permeability of mice was detected by immunofluorescence and Evans blue.Western blotting,immunofluorescence and electron microscopy were used to detect the thickness of BMs and the expression of junctions proteins.The expression of integrinβ1/ROCK/MLC signaling pathway after Perlecan overexpression was detected by western blotting,qPCR,immunofluorescence and integrin receptor antagonist GRGDSP.Results:1.Perlecan was specifically localized in BMs,and its expression decreased and then increased after SCI.2.Perlecan overexpression promoted motor function recovery and nerve regeneration after SCI.3.Perlecan overexpression improved the recovery of BSCB function and reduced neuroinflammatory response after SCI.4.Perlecan reduced the breakdown of TJs through interacting with integrin β1 to inhibit ROCK/MLC pathway after SCI.Conclusion:We found that Perlecan was specifically localized in BMs in the intact spinal cord and underwent degradation/remodeling after SCI.After the CRISPR/Cas9-based SAM system was used to overexpress Perlecan,we found Perlecan improved locomotor recovery and neural regeneration and reduced BSCB permeability along with the neuroinflammatory response after SCI.Mechanismly,Perlecan inhibited stress fiber formation through interacting with integrin β1 and inhibiting downstream ROCK/MLC pathway,resulting in reduced TJs disassembly and improved BSCB integrity after SCI.