Promoting effect of 810 nm low-level laser on axonal regeneration of neurons in mice with spinal cord injury and its related mechanism

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 6 000 J/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 ganglion 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±0.08 vs. 0.06±0.04)(P<0.05) and the fluorescence intensity of F4/80+ Arg-1+ increased after low-level laser (1.00±0.07 vs. 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±0.14 vs. 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±0.00 vs. 2.27±0.22) (P<0.01) (ELISA: 1 435.58±100.48 vs. 2 006.12±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±0.12 vs. 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±0.10 vs. 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. 1 068.95±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. Key words: Spinal cord injuries; Laser therapy; Macrophages; Nerve regeneration