Comparação do efeito do laser em baixa intensidade (660 e 780 nm) sobre a polarização de macrófagos e a modulação da expressão e síntese de citosinas envolvidas no reparo muscular

Abstract

The interactions between the muscle and the macrophages which invade afteran injury occurs are crucial to the evolution of this tissue repair. In this sense, low intensity laser therapy (LLLT) has been widely used in the treatment of these lesions at the clinical and experimental level, but little is known about its effects on macrophages and their products. The aim of this study was to compare the effects of red and infrared LLLT on the role of macrophages in muscle repair. Initially an evaluation of the dose-response effect of irradiation with the two wavelengths (in 6 combinations of dosimetric parameters) on the gene and protein expression of IL-6, TNF-α, IL-1β by macrophages with an M1 (pro-inflammatory) profile was done. After choosing the two parameter combinations that best modulated the expression of the three cytokines,an evaluation of the effect of macrophage supernatants with an M1 profile, irradiated with these two parameter combinations,on the differentiation of myogenic precursor cells was done. Cultures of the macrophage lineage J774 were therefore activated by the inflammatory phenotype M1 and irradiated by two wavelengths (660nm and 780nm) with 3 different parameter combinations (20mW; 5J/cm2; 0,2J; 40 mW; 10J/cm2; 0,4J; or 70 mW; 17,5J/cm2; 1J). An analysis of the expression of the cytokines IL-6, TNF-α, IL-1β by means of qRT-PCR was performed. The culture supernatant was used (24 hours after irradiation) to assess the protein production of the three cytokines (ELISA) and for treating myogenic precursor cells (C2C12) in order to analyze their differentiation via myogenin immunoblot. Non-irradiated macrophages served as controls. Three independent experiments were performed, each using the afore mentioned methodology. In vivo experiments evaluated the phenotypic profile of macrophages present in the acute muscle injuries treated with the two wavelengths,as well as the development of tissue repair in both situations. For the analysis in vivo, Wistar rats were divided into 04 groups: control (n = 5); muscle injury without treatment (n = 15); muscle injury treated with red LLLT (n = 15) and muscle injury treated with infrared LLLT(n = 15). After 2, 4, and 7 days, animals were sacrificed and the tibialis anterior muscle was removed and processed for analysis using immunohistochemistry. In the in vivo experiments, the phenotypic profile of macrophages present in the acute muscle injuries treated with the two wavelengths, as well as tissue repair development in both situations,was evaluated. Immunohistochemical expression of total macrophages (CD68+), M2 profile (CD206+ and CD163+), was correlated with the different phases of the skeletal muscle repair process. Qualitative and quantitative morphological analyses of total inflammatory cells, myonecrosis, immature blood vessels and muscle fibers were done. All results were statistically analyzed. The in vitro results showed that the infrared LLLT was more efficient than the red LLLT in decreasing gene expression and protein production of IL-6 and TNF-α by M1 phenotype macrophages irradiated with these lasers; irradiation with 660nm LLLT reduced the myogenin expression in myoblasts treated with these macrophage supernatants. The in vivo results also showed that, when compared to the untreated group, within 2 days, red and infrared LLLT irradiation were able to reduce myonecrosis, the number of inflammatory cells and the number of CD68+ macrophages and also increase angiogenesis.During this period only irradiation with 780nm laser generated an increase in the number of macrophages with a M2 profile (CD206+ and CD163+). In 4 days, only the irradiation with the 780nm LLLT was able to reduce myonecrosis, inflammatory cells, and further enhance angiogenesis, but both LLLTs reduced the number of CD68+ cells. In 7 days, both LLLTs decreased inflammatory cells and increased angiogenesis and the number of CD163+ cells. In this period, only the 780nm LLLT increased the number of immature fibers and CD206+ cells. The sum of the results of the evaluations in vitro and in vivo, shows that irradiation with infrared laser can generate superior effects to those of the red laser when treating acute muscle injuries; this modulation may be linked to its greater ability to modulate the activation and polarization of macrophages.