Promoting effect of 810 nm low-level laser on axonal regeneration of neurons in mice with spinal cord injury and its related mechanism / 中华创伤杂志

ABSTRACT

Objective To investigate the effect of 810 nm low-level laser on neuronal axonal regeneration of mice with spinal cord injury and its related mechanism.Methods In vivo experiment:20 Balb/c mice were randomly divided into the spinal cord injury group(SCI group)and the 810 nm low-level laser irradiation group(low-level laser group)after spinal cord injury according to the random number table method,with each group containing ten mice.A mice SCI model was established through clamp injury and the low-level laser group continuously irradiated the damaged area with weak 810 nm low-level laser with selected parameters(continuous wave with wave length 810 nm,power density 2 mW/cm2,spot are 4.5 cm2,irradiation time 50 minutes,energy 6000J/cm2).Then immunofluorescence staining was used to observe the M1 macrophage marker-inducible nitric oxide synthase(iNOS),the M2 macrophage marker arginase 1(Arg-1)and the universal marker F4/80 of macrophages after 14 days.Furthermore,in the in vitro experiment,standardized low-level laser-macrophage irradiation model was established.Another 20 Balb/c mice were used to obtain primary bone marrow-derived macrophages which were induced into M1 macrophages using lipopolysaccharide(LPS)and interferon-gamma(INF-γ).The M1 macrophages were randomly divided into the M1 macrophage group(M1 group)and the low-level laser therapy group(M1 + low-level laser group)equally according to the random number table method.The M1 group was not treated,and the M1 + low-level laser group was treated with low-level laser of selected parameters.RT-qPCR and ELISA were used to detect the expression of interleukin-1 receptor antagonist(IL-1RA)and interleukin-10(IL-10)in M1 macrophages 24 hours after irradiation.Western blot was used to analyze the expression of iNOS,Arg-1,differentiation antigen cluster 206(CD206),protein kinase B(AKT),phosphorylated protein kinase B(p-AKT),cyclic adenosine response element binding protein(CREB)and phosphorylated cyclic adenosine response element binding protein(p-CREB)in M1 macrophages 48 hours after irradiation.Dorsal root gangtion neurons(DRG)were cultured in two groups of macrophage conditioned medium,and the length of DRG axon growth was measured 48 h later to evaluate the effect of low-level laser on neuronal axon growth.Results In the in vivo experiment,compared with mice with spinal cord injury alone,the fluorescence intensity of F4/80+ iNOS+ in the spinal cord injury area decreased(1.00±0.08vs. 0.06±0.04)(P＜ 0.05)and the fluorescence intensity of F4/80 + Arg-1 + increased after low-level laser(1.00±0.07vs.2.15±0.12)(P＜0.01).In the in vitro experiment,compared with the M1 group,the expression of the M1 macrophage marker iNOS in the M1 + low-level laser group decreased(1.00±0.11 vs.0.08±0.01)(P＜ 0.01);the M2 macrophage marker Arg-1(1.00±0.14vs.2.44±0.16)(P＜0.01),and the expression of CD206(1.00±0.12 vs.1.83±0.05)(P＜0.01)increased.In addition,IL-1RA expression was increased in the M1 + low-level laser group compared with the M1 group(RT-qPCR:1.00±0.00vs.2.27±0.22)(P＜0.01)(ELISA:1435.58±100.48vs.2006.12±123.91(P＜0.05);IL-10 expression was also increased in the M1 +low-level laser group compared with the M1 group(RT-qPCR:1.00±0.00 vs. 3.45±0,56)(P＜0.05)(ELISA:137.13±4.20 vs.188.29±8.49)(P＜ 0,01);compared with the M1 group,the macrophage polarization pathway protein in the M1 + low-level laser group increased,AKT(1.07±0.12vs.1.74±0.04)(P＜0.01),p-AKT(1.00±0.12 vs.1.64±0.15)(P＜0.05),p-CREB(1.00±0.10vs.2.12±0.18)(P＜0.01).Compared with the M1 group,the conditioned medium of the M1 + low-level laser group significantly promoted DRG axon growth(567.66±63.59 vs.1068.95±130.14)(P＜ 0,05).Conclusions The 810 nm low-level laser irradiation can promote neuronal axon regeneration of mice with spinal cord injury,which may be related to the regulation of macrophage polarization phenotype by low-level laser through AKT/CREB pathway.