Effect And Mechanism Of Aggregated Macrophages On Blood-spinal Cord Barrier Restoration After Spinal Cord Injury

Part Ⅰ M1 Macrophages Impair Tight Junctions between Endothelial Cells after Spinal Cord InjuryBackground: In the acute phase of spinal cord injury(SCI),endogenous angiogenesis appeared in the core of the injury,but the leakage of the blood-spinal cord barrier(BSCB)continued to the chronic phase,which indicated that the BSCB could not be effectively restored in the secondary injury phase of SCI.Tight junctions(TJs)between endothelial cells are an essential part of maintaining the normal barrier function of the BSCB,and their destruction can lead to leakage of the blood vessels.The continuous leakage of the BSCB suggests the presence of incomplete TJs between endothelial cells in the injury core after SCI,but the expression of tight junction proteins remains to be further explored.Blood-derived macrophages infiltrate into the parenchyma and gradually aggregate in the injury core after SCI.It has been demonstrated that aggregated macrophages have different functions due to M1 and M2 polarization and can phagocytose myelin debris to form myelin-laden macrophages(Mye),which will change the phenotype of macrophages.Nevertheless,the specific effects of macrophages on TJs between vascular endothelial cells have not been reported.Therefore,we aimed to determine the spatiotemporal correlation between macrophages and the expression of tight junction proteins in injured spinal cord tissue and further confirm the effects of macrophages with different polarizations on TJs between endothelial cells.Methods: The mouse spinal cord(T10)clamp model was used in this study.Tissue immunofluorescence was used to double-label vascular endothelial cell tight junction proteins ZO-1 and CLN-5 with the vascular endothelial cell marker CD31 to explore the spatiotemporal expression pattern of vascular endothelial tight junction proteins after SCI.Double-labeling ZO-1 or CLN-5 with the macrophage marker CD68 to clarify the spatiotemporal correlation between the expression of tight junction protein in vascular endothelial cells and macrophage aggregation after SCI.The leakage of BSCB at different time points after injury was assessed by intravenous injection of fluorescein isothiocyanate(FITC)-dextran combined with tissue immunofluorescence to detect the fluorescence intensity of FITC in spinal cord tissue.Clodronate liposomes were used to specifically deplete macrophages in vivo combined with tissue immunofluorescence to detect ZO-1,CLN-5,NF,and 5-HT to demonstrate the effect of macrophages on TJs of vascular endothelial cells and nerve regeneration after SCI.Basso Mouse scale(BMS)score and footprint analysis were performed to demonstrate the effect of macrophages depletion on motor function recovery after SCI.RAW264.7 macrophages polarization were induced in vitro.b End.3 endothelial cells were exposed to the conditioned medium of macrophages with different polarization states,using western blot and immunocytochemistry to detect ZO-1,CLN-5 and occludin(OCLN),combined with endothelial cell permeability assay to verify the different effects of polarized macrophages on endothelial TJs.Results: 1.Vascular restoration and BSCB leakage could occur simultaneously in the injury core during the chronic phase of SCI 2.From 7 days postinjury(dpi)to 28 dpi,accompanied by the aggregation of macrophages,the expression of claudin-5(CLN-5)and zonula occludens-1(ZO-1)in vascular endothelial cells in the injury core was significantly decreased.3.Depletion of macrophages reduces the destruction of TJs between vascular endothelial cells and the leakage of BSCB after SCI 4.Depletion of macrophages after SCI had no significant effect on nerve regeneration and motor function recovery in mice.5.M1 macrophages and myelin-laden macrophages can impair TJs between vascular endothelial cells in vitro.6.M1 macrophages can impair TJs between vascular endothelial cells by secreting IL-6 in vitro.Conclusions: Our study demonstrated that macrophages that aggregated in the injured core after SCI could impair TJs between vascular endothelial cells,causing BSCB leakage.M1 macrophages could impair TJs between endothelial cells by secreting IL-6.Part Ⅱ Tocilizumab Promotes Repair of Spinal Cord Injury by Facilitating the Restoration of Tight Junctions between Endothelial Cells and Nerve RegenerationBackground:Macrophages aggregate to the injury core and polarized after SCI.The first part of the present study confirmed that M1 macrophages can impair tight junctions(TJs)between endothelial cells by secreting IL-6,suggesting that blocking IL-6 may facilitate the restoration of TJs between vascular endothelial cells.Previous studies have demonstrated that the application of MR16-1 to block IL-6 receptor is beneficial to reduce the excessive inflammatory response after SCI,but the effect of blocking IL-6 receptor on the restoration of TJs between vascular endothelial cells remains to be explored.Tocilizumab is a humanized IL-6 receptor monoclonal antibody,and its application in neurological diseases has been reported in recent years,but the effect of tocilizumab on blood-spinal cord barrier(BSCB)repair and nerve regeneration after traumatic spinal cord injury is unclear.This study aimed to explore the effect of tocilizumab on the restoration of TJs between vascular endothelial cells and on nerve regeneration and functional recovery after SCI.Methods: In this study,the mouse spinal cord(T10)clamp model was used,and tocilizumab was continuously injected intrathecally after the model was established until the day of the collection.Tissue immunofluorescence was used to double-label zonula occludens-1(ZO-1)or claudin-5(CLN-5)and endothelial cell marker CD31 in spinal cord tissue at 14 and 28 days postinjurydp(dpi)to verify the effect of tocilizumab on the restoration of TJs between endothelial cells after SCI,and to evaluate the leakage of BSCB by detecting fibrinogen in spinal cord tissue at 14 dpi and 28 dpi by tissue immunofluorescence.Tissue immunofluorescence was used to double-label CD68 and PDGFR-β in spinal cord tissue at 14 dpi to demonstrate the effect of tocilizumab on macrophage aggregation and fibrotic scar formation after SCI.Tissue immunofluorescence was used to detect NF and 5-HT in spinal cord tissue at 14 dpi and 28 dpi to certify the effect of tocilizumab on nerve regeneration after SCI.Finally,Basso Mouse scale(BMS)score and footprint analysis were used to verify the effect of tocilizumab on the recovery of motor function in mice after SCI.Results: 1.Intrathecal injection of tocilizumab facilitates the restoration of TJs between vascular endothelial cells at 14 dpi and 28 dpi after SCI.2.Intrathecal injection of tocilizumab reduces the leakage of BSCB at 14 dpi and 28 dpi after SCI.3.Intrathecal injection of tocilizumab reduces the aggregation of macrophages and the formation of fibrotic scar in the injured core at 14 dpi after SCI.4.Intrathecal injection of tocilizumab facilitates nerve regeneration and motor function recovery in the chronic phase of SCI.Conclusions: Our study demonstrated that application of tocilizumab to antagonize IL-6 receptor can effectively facilitates the restoration of TJs between vascular endothelial cells and reduced the leakage of BSCB,which is beneficial to nerve regeneration and functional recovery after SCI.