| Spinal cord injury(SCI)is a serious traumatic disease with poor prognosis and high disability rate in the central nervous system.Once occurs,it will bring an enormous psychological and economic burden to patients and their families.The glial scar,which is the outcome of secondary spinal cord injury,is the main obstacle to the recovery of nerve function and axonal regeneration.Macrophages,the most important inflammatory cells in secondary injury,infiltrate the damaged area and polarize into a cytotoxic M1-type after SCI,inducing a serious inflammatory response.At the same time,the inflammatory reaction further promotes the proliferation of astrocytes and the secretion of extracellular matrix(ECM)mainly composed of chondroitin sulfate(CSPG),which is the key for a physical and chemical barrier hindering axonal regeneration.A large number of studies have used various treatments to reduce the formation of glial scars after SCI,such as drug treatment,cell transplantation,material transplantation.However,since there exists some deficiencies in these methods(narrow drug safety window,immune rejection and secondary damage to the human body,etc.),scientists still need to explore safer and more effective methods for SCI treatment.Low-level laser treatment(LLLT),which mainly refers to the interaction between low-level laser and biological tissue,has the advantages of non-invasive,anti-inflammatory,analgesic,and pro-repair.In recent years,LLLT has been widely used in the therapy of joint diseases,peripheral nerve injury,medical beauty,etc.Furthermore,a lot of studies have shown that 810 nm LLLT can significantly improve the motor function of spinal cord injured animals,promote neuronal survival,decrease the area of damage,reduce the number of M1 macrophages and inhibit the expression of inflammatory factors in the injured area.However,whether LLLT can affect the activation and secretion of astrocytes after SCI remains unknown.Besides,it is also not clear if macrophages assist the process that LLLT regulate astrocytes.In order to address above questions,we conducted the following two parts of research.Experiment 1:Effect of low-level laser therapy on glial scar after spinal cord injuryObjective:To study the effect of low-level laser therapy on gliar scar after spinal cord injuryMethods:40 mice were randomly divided into the spinal cord injury group and the spinal cord injury with low-level laser therapy group according to random number method.Low-level laser therapy was performed with 810nm low-level laser on the spinal cord injury area of mice through percutaneous irradiation.The laser wavelength was 810nm,the irradiation power was 150mW,the spot area was 0.3cm~2,and the time of single irradiation was 50 min,once a day for 7 days.Immunofluorescence staining was used to detect the existence of macrophages?astrocytes and CSPG at 7 days after spinal cord injury.Results:After low-level laser therapy,compared with the spinal cord injury group,the expression of iNOS in M1 macrophages was significantly decreased,and the expression of astrocyte markers GFAP and CSPG was significantly down-regulated,accompanied with the morphology and distribution changes of astrocytes.Conclusion:Low-level laser treatment can significantly regulate the activation of astrocytes and the expression of CSPG in the injured area after spinal cord injury,which accompanied with the change of macrophage polarization.Experiment 2:Effect of M1 macrophages on astrocytes under low-level laserObjective:To investigate the effect of M1 macrophages on astrocytes under low-level laser.Methods:An in vitro macrophage-astrocyte co-culture model was used.The astrocytes were divided into five groups:Ctrl group(blank control);M1 group(cultured in M1 macrophage-conditioned medium),M1+L group(cultured in M1 macrophage-conditioned medium and then irradiated with low-level laser),L-M1 group(cultured in irradiated M1 macrophage-conditioned medium),and L-M1+L group(cultured in irradiated M1 macrophage-conditioned medium and then irradiated with a low-level laser).The low-level laser parameters are:wavelength 810 nm,power density 2 mW/cm~2,spot area 4.5 cm2,irradiation time 440 s.Immunofluorescence and Western Blot were used to detect the polarization changes of macrophages in vitro.RT-qPCR,Western Blot and CCK8 were used to detect the activation status of astrocytes.RT-qPCR and ELISA were used to detect the inflammatory properties of astrocytes.Expression of factors(IL-1?,TNF-?)and CSPG core proteins(neurocan,NG2,aggrecan).Results:CCK8 assay showed that M1 conditioned medium increased the activity of astrocytes,while after weak laser intervention,the activities of three groups(M1+L,L-M1,L-M1+L)significantly reduced.Western Blot assay showed that M1 significantly promoted the expression of astrocyte GFAP and the classical activation pathway pSTAT3compared with the Ctrl group.Compared with the M1 group,the expression of GFAP and pSTAT3 in the L-M1 and L-M1+L groups.Significant inhibition.RT-qPCR and ELISA showed that compared with the Ctrl group,the expression of CSPG(neurocan,NG2,aggrecan)and inflammatory factors(IL-1?and TNF-?)in the M1 group significantly increased;compared with the M1 group,the expression of CSPG and inflammatory factors in the L-M1 group and the L-M1+L group significantly decreased at the gene and protein levels.At the same time,the multiple expression changes of astrocytes in M1+L group were not as obvious as those in L-M1 group and L-M1+L group.The primary macrophages were cultured with different conditioned mediums from astrocytes.Results showed that the astrocytes in the M1-astrocyte group promoted the expression of iNOS of macrophages,while decreased in L-M1+L-astrocyte group.Conclusion:Low-level laser therapy can significantly inhibit the activation of astrocytes by M1 macrophages and reduce the expression and secretion of CSPG and inflammatory cytokines in astrocytes.Futhermore,there was a positive feedback interaction between astrocytes and macrophages,and under LLLT,both changed in the direction of inhibiting inflammatory response. |
PDF Full Text Request