Photobiomodulation and different macrophages phenotypes during muscle tissue repair

Abstract

Macrophages play a very important role in the conduction of several regenerative processes mainly due to their plasticity and multiple functions. In the muscle repair process, while M1 macrophages regulate the inflammatory and proliferative phases, M2 (anti-inflammatory) macrophages direct the differentiation and remodelling phases, leading to tissue regeneration. The aim of this study was to evaluate the effect of red and near infrared (NIR) photobiomodulation (PBM) on macrophage phenotypes and correlate these findings with the repair process following acute muscle injury. Wistar rats were divided into 4 groups: control; muscle injury; muscle injury + red PBM; and muscle injury + NIR PBM. After 2, 4 and 7 days, the tibialis anterior muscle was processed for analysis. Macrophages phenotypic profile was evaluated by immunohistochemistry and correlated with the different stages of the skeletal muscle repair by the qualitative and quantitative morphological analysis as well as by the evaluation of IL-6, TNF-α and TGF-β mRNA expression. Photobiomodulation at both wavelengths was able to decrease the number of CD68⁺ (M1) macrophages 2 days after muscle injury and increase the number of CD163⁺ (M2) macrophages 7 days after injury. However, only NIR treatment was able to increase the number of CD206⁺ M2 macrophages (Day 2) and TGF-β mRNA expression (Day 2, 4 and 7), favouring the repair process more expressivelly. Treatment with PBM was able to modulate the inflammation phase, optimize the transition from the inflammatory to the regeneration phase (mainly with NIR light) and improve the final step of regeneration, enhancing tissue repair.

Keywords: macrophages; muscle; photobiomodulation; regeneration; regenerative medicine.

Figure 1

Morphological evaluation of injury, injury + PBM 660 nm and injury + PBM 780 nm groups after 2, 4 and 7 d. The injury group exhibited more myonecrosis (*) and inflammatory cells in comparison to the PBM groups. PBM 780 nm was associated with largest number of mature vessels. Both PBM treatments were able to promote the formation of immature muscle fibres (H&E staining, original magnification: 200×). PBM, photobiomodulation

Figure 2

Quantitative analysis of morphological parameters evaluated in injury, PBM 660 nm and PBM 780 nm groups after 2, 4 and 7 d. Myonecrosis (A), number of inflammatory cells (B), number of blood vessels (C) and number of immature muscle fibres (D). Data expressed as mean ± SEM. (ANOVA/Tukey's test; *P < .05; **P < .01; ***P < .001; ****P < .0001 compared to the injury group without PBM). PBM, photobiomodulation

Figure 3

Immunohistochemical evaluation of CD68⁺ macrophages infiltration. Representative images (A) and quantitative analysis (B). The number of CD68⁺ cells was evaluated in injured muscle with or without PBM treatment in 3 different periods (2, 4 and 7 d). A significant decrease was found in the PBM 660 and 780 nm groups in comparison to the injury group (P < .01 and P < .05, respectively) on Day 2 (Original magnification: 400×). Data expressed as mean ± SEM. ANOVA/Tukey's test; *P < .05; **P < .01. PBM, photobiomodulation

Figure 4

Immunohistochemical evaluation of CD206⁺ macrophages. Representative images (A) and quantitative analysis (B). The number of CD206⁺ cells was evaluated in injured muscle with or without PBM treatment in different periods (2, 4 and 7 d). A significant increase was found in the PBM 780 nm group in comparison to both the injury and PBM 660 nm groups (P < .001 and .001, respectively) (Original magnification: 400×). Data expressed as mean ± SEM. ANOVA/Tukey's test; ***P < .001. PBM, photobiomodulation

Figure 5

Immunohistochemical evaluation of CD163⁺ macrophages infiltration. Representative images (A) and quantitative analysis (B). The number of CD163⁺ cells was evaluated in injured muscle with or without PBM treatment in different periods (2, 4 and 7 d). A significant increase was found in the PBM 780 nm group on Day 2 in comparison to the PBM 660 nm group (P < .05). On Day 7, a significant increase was found in the PBM 660 and 780 nm groups in comparison to the injury group (P < .01 and P < .05, respectively. (Original magnification: 400×). Data expressed as mean ± SEM. ANOVA/Tukey's test; *P < .05; **P < .01. PBM, photobiomodulation

Figure 6

IL‐6,TNF‐α and TGF‐β mRNA expression in control, injury, PBM 660 nm and PBM 780 nm 2, 4 and 7 d after injury. Data expressed as mean ± SEM (ANOVA/Tukey's test; *P < .05; **P < .01; ***P < .001). PBM, photobiomodulation