The Effect Of 810-nm Low Level Laser Therapy On The Interaction Between Macrophages And Neurons After Spinal Cord Injury

Spinal cord injury(SCI)is a worldwide unsolved problem.Reducing secondary spinal cord injury has been a hot topic of research.Macrophages are the main cells involved in secondary spinal cord injury.Macrophages can be polarized into neurotoxic M1 macrophages and M2 macrophages secrete neurotrophic factors and promote axonal regeneration.Previous studies have confirmed that by regulating the polarization state of macrophages in the injured area,reducing the number of M1 macrophages and promoting the expression of M2 macrophages,can effectively promote the repair of spinal cord injury.However,the current methods for regulating macrophage polarization have various problems to implement effectively.Therefore,there is an urgent need to find a safer and more effective treatment.Low Level Laser Therapy(LLLT)refers to the use of low-power lasers for localized irradiation to achieve therapeutic effects.Due to its non-invasive,anti-inflammatory and pro-repair characteristics,it has been applied in many clinical fields.Our previous studies have confirmed that 810-nm LLLT can effectively promote the recovery of rat function after spinal cord injury and change the polarization state of macrophages as well.However,it is unclear whether LLLT can directly affect the macrophages,regulate its polarization state and increase the secretion of neurotrophic factors to promote the regeneration of axons,and involved mechanism is also unknown.In addition,recent studies have confirmed that there are interations between macrophages and neurons,and the polarization state of macrophages is also affected by the secretory state of neurons.The CCL2 secreted by neurons is the key factor which can change the polarization state of macrophages and promote the polarization of macrophages to M2.However,it is still unclear whether 810-nm LLLT can affect the interaction between macrophages and neurons.To explore the above problems,the following two parts of the experiment were carried out.Experiment 1:Effects of 810-nm LLLT on the secretion of neurotrophic factors by macrophages to promote axon regeneration and related mechanismsObjective:To study the effect of LLLT on the polarization state of M1 macrophages and the secretion of neurotrophic factors and its mechanism.Methods:An in vitro irradiation model of LLLT-M1 macrophages was established.Irradiation was carried out using an infrared light source(wavelength,810-nm;power density,2 mW/cm~2;irradiation area,4.5 cm~2;irradiation time,440 s)to obtain energy of4 J(2 mW/cm~2×4.5 cm~2×440 s).Using RT-qPCR and ELISA to evaluate the secretion level of brain-derived neurotrophic factor(BDNF),nerve growth factor(NGF),ciliary neurotrophic factor(CNTF)and leukemia inhibitory factor(LIF)in M1 macrophages.Using Western Blot to analyse the expression level of Inducible Nitric Oxide Synthase(iNOS),Arginase 1(Arg-1),Protein Kinase A(PKA),cAMP Response Element Binding(CREB),Phosphorylation-cAMP Response Element Binding(p-CREB),extracellular signal-regulated kinase(ERK)and phosphorylation-extracellular signal-regulated kinase(p-ERK)in M1 Macrophages.Dorsal root ganglion(DRG)neurons were cultured in macrophage conditioned medium and evaluated for length of DRG axon.After using the PKA inhibitor H-89,the expression level of p-CREB and the secretion of BDNF,NGF,CNTF and LIF in M1 macrophages were evaluated,and the length of axons of DRG were also analysed.Results:810-nm LLLT increased the secretion of BDNF,NGF,CNTF and LIF in M1macrophages,decreased the expression of iNOS in M1 macrophages,up-regulated the expression of Arg-1,and increased the expression of PKA and p-CREB and promote DRG axon growth.The PKA inhibitor H-89 significantly reduced LLLT-induced elevation of neurotrophic factors and expression of p-CREB in M1 macrophages,and decreased DRG axon length after pipetting.Conclusion:810-nm LLLT can down-regulate the expression of M1 macrophages and up-regulate the expression of M2 macrophages.LLLT can promote the secretion of neurotrophic factors of M1 macrophages through PKA-CREB pathway and increase the growth of axon.Experiment 2:The role of 810-nm LLLT in DRG-secreting CCL2 affecting macrophage polarizationObjective:To study the effect of LLLT on the DRG under simulated oxidative stress and the effect of its secretion changes on the polarization state of macrophages.Methods:The in vitro LLLT-DRG model was constructed to perform LLLT on neurons under simulated oxidative stress in spinal cord injury.The irradiation parameters are as in Experiment 1.The ROS probe was used to measure the content of ROS in the DRG,the survival of the neurons was measured using the CCK-8 method,and the axon regeneration of the neurons was observed using immunofluorescence.The secretion of CCL2 from DRG was determined by RT-qPCR and ELISA.After using conditioned medium culture macrophage,the expression of iNOS and Arg-1 in macrophages was determined by Western blot analysis.The expression of TNF-αand IL-1βwas determined by ELISA.After adding the neutralizing antibody of CCL2 to the obtained DRG conditioned medium after LLLT irradiation,the macrophages were cultured by pipetting to observe the effects on macrophage polarization and secretion.Results:Studies confirmed that 810-nm LLLT reduced ROS levels in neurons,increased neuronal survival under oxidative stress,and promoted neuronal axon regeneration.In addition,810-nm LLLT can promote the increase of CCL2 secretion expression under oxidative stress.By constructing a DRG supernatant-M1 macrophage adoptive culture model,it was found that the supernatant of DRG after 810-nm LLLT intervention can reduce the expression of iNOS the marker of M1 macrophage and the secretion of TNF-αand IL-1βin M1 macrophages;at the same time,it can up-regulate Arg-1,markers of M2 macrophages,in genetic level.However,this effect can be reduced by the addition of neutralizing antibodies to CCL2.Conclusion:810-nm LLLT can promote DRG survival and axonal regeneration under SCI oxidative stress,increase the secretion level of DRG CCL2,and this change can reduce the polarization of macrophages to M1,further indicating that 810-nm LLLT has a promoting effect on the crosstalk between macrophages and neurons.