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.


| INTRODUCTION
Acute muscle injuries provide a good model for the study of the modulating effect of immune cells on the tissue repair process. 1 Immediately after an acute injury, muscle tissue undergoes the rapid invasion of inflammatory cells, mainly neutrophils and macrophages. 1 Macrophages constitute the majority of intramuscular leucocytes and, besides removing tissue debris through phagocytosis, these cells synthesize growth factors, chemokines and cytokines that modulate all phases of muscle repair. 1,2 Under the microenvironment stimuli provided mainly by infiltrated neutrophils (ie, presence of Th1 mediators, such as IFN-γ and TNF-α) in the initial steps after an acute muscle injury, macrophages are activated and acquire a proinflammatory phenotype, classically known as M1 (CD68 high , CD206 − and CD163 − ) and characterized by enhanced phagocytic activity and production of proinflammatory mediators as IL-1β, IL-6, TNF-α, IL-12 and IL-23. 1,[3][4][5] Other mediators released by M1 macrophages, such as IL-6, IL-1, VEGF and IL-13, also stimulate angiogenesis and the proliferation of myogenic precursor cells. 1,4,5 M1 cell surface marker CD68 is a receptor for oxidized low-density lipoproteins that activate phagocytosis and increase the production of proinflammatory cytokines when specifically connected. Normal muscle tissue does not express CD68 + macrophages. 3,4 After approximately 3 days, other macrophage phenotypes, identified as M2 (CD68 low , CD206 + and CD163 + ) or alternatively activated macrophages, become more numerous in the damaged tissue and persist until 7 days after injury. 1 M2 macrophages produce antiinflammatory cytokines and growth factors as TGF-β and IL-10 as well as enzymes that are important to angiogenesis, fibroblast proliferation and the differentiation of myogenic precursor cells. 1,4,5 M2 macrophage surface marker CD206 is a mannose receptor that internalizes sugar moieties on molecules in inflamed tissue, such as myeloperoxidase. 3 CD163 is a specific receptor for hemoglobin and haptaglobin complexes. 3 Specific binding to the both receptors triggers the expression of anti-inflammatory cytokines, such as IL-10 and TGF-β, leading to the deactivation of M1 macrophages 1,3,4 and enabling the predominance of M2 macrophages at the injury site during the transition from the proliferative stage to the differentiation and growth stage of myogenesis. 1,3,4 It is well accepted that although there are different degrees of differentiation between the populations of macrophages that inhabit the muscle tissue after an injury, the coordinated activation of proinflammatory or anti-inflammatory macrophage predominance in each step of the muscle repair process is essential to the resolution of the inflammatory process and regeneration of the muscle tissue. 1,4,5 The modulation of macrophage plasticity is considered so important that macrophage-based therapeutic interventions are currently emphasized in regenerative medicine to improve the healing process and avoid undesirable effects associated with altered macrophage function. 6,7 Among the therapeutic interventions for the treatment of muscle injuries, photobiomodulation (PBM) has been extensively investigated (for review, see 8 ). Photobiomodulation consists of the use of low-power non-thermal light using a source (such as laser or LED) to modulate inflammation and healing (see [9][10][11] and references therein). The most common spectral regions used in PBM are the red (600-700 nm) and near infrared (NIR, 780-110 nm) wavelengths, [9][10][11] both of which achieve greater tissue penetration compared to other wavelengths due to the lower absorption and scattering by tissue chromophores. [9][10][11] Regarding the muscle tissue repair process, the use of NIR PBM is more common, but both red and NIR therapies are reported to decrease myonecrosis and the infiltration of inflammatory cells 12,13 as well as increase the number of immature muscle fibres, leading to better organized muscle tissue. 8,[12][13][14][15] In a time-dependent manner, red and NIR therapies are also able to modulate the gene expression of mediators, such as TNF-α, 15,16 IL1-β, 17 IL-6 18 and TGF-β 16,19 as well as genes involved in the differentiation of myogenic stem cells, such as MyoD 18,20 and myogenin 18,20 during the muscle repair process.
As macrophages are the main source of cytokines, chemokines and growth factors that guide muscle repair, it is important to investigate whether PBM modulates the different macrophage phenotypes during the progression of the repair process. Thus, the aim of the present study was to compare the effect of red and NIR PBM on the muscle repair process following an acute injury and correlate the findings with the presence of macrophage phenotypes, mRNA expression of IL-6, TNF-α and TGF-β, and the evolution of tissue repair after different experimental periods.

| MATERIALS AND METHODS
All animal procedures were performed in accordance with the guidelines of the National Council for the Control of Animal Experimentation and received approval from the Animal Research Ethics Committee (certificate number: 0017/2014). Fifty male Wistar rats (Rattus norvegicus: var. albinus, Rodentia, Mammalia) were kept under controlled temperature (22°C) and relative humidity (40%), with a 12-hour light/dark cycle. The animals were offered solid ration and water ad libitum throughout the experimental period. The 2-month old (200 ± 15 g) animals were randomly divided into 4 experimental groups: (i) control group (n = 5, not subjected to injury or PBM); (ii) injury group (n = 15, subjected to cryoinjury and not treated with PBM); (iii) injury + PBM 660 nm group (n = 15, cryoinjury and treatment with red PBM λ = 660 nm); and (iv) injury + PBM 780 nm (n = 15, cryoinjury and treatment with NIR PBM λ = 780 nm). Animals in Groups 2, 3 and 4 (n = 5) and Group 1 (n = 1) were euthanized on Days 2, 4 and 7 following the induction of injury for analysis.

| Injury procedure
The cryoinjury procedure was performed using a previously described method. 16,18 The surgical procedures were performed SOUZA ET AL.
| 4923 under anesthesia with 10% ketamine HCl (Dopalen; Vetbrands, São Paulo, Brazil) and 2% xylazine (Anasedan; Vetbrands) (100 and 10 mg/kg, respectively). The tibialis anterior (TA) muscle was surgically exposed by a 15-mm-long longitudinal skin incision over the central portion of the muscle. The cryoinjury procedure consisted of applying the flat end of a metal rod (3 mm in diameter), which had previously been cooled in liquid nitrogen, directly to the ventral surface of the exposed muscle for 10 seconds. After the area had thawed, the procedure was repeated for an additional 10 seconds, followed by the suturing of the incision ( Figure S2A).

| PBM treatment protocol
Photobiomodulation treatment was initiated 2 hours after the cryoinjury procedure and was performed daily with a 24-hour interval between sessions. [16][17][18] Photobiomodulation was performed with aluminium-gallium-indium-phosphide (AlGaInP) and aluminium-galliumarsenide (AlGaAs) diode lasers (Twin Laser; MM Optics, São Carlos, SP, Brazil) operating at a wavelength of 660 and 780 nm, respectively. The dosimetric parameters are described in Table 1. The laser beam was applied in contact with the skin surface over the cryoinjury area at an angle of 90°between the emitter and skin to prevent reflection. Irradiation was applied to 8 points ( Figure S2B

| Morphological analysis
Muscle samples were fixed in 10% buffered formalin (pH 7.4), embedded in paraffin and sectioned with a microtome (Leica RM2125, Nussloch, Germany). The sections were stained with hematoxylin-eosin (H&E) and examined under a light microscope (Zeiss, Axioplan 2, Germany). Five areas representing at least 70% of the injury were photographed with a 20× objective (magnification: 200×) in each section. Morphological aspects relevant to muscle repair, such as myonecrosis, inflammatory infiltrate, blood vessels and immature muscle fibres, were quantitatively and qualitatively evaluated using the Image J cell count software plug-in (National Institutes of Health, USA) by an experienced pathologist with no knowledge of the experimental groups. 13,21,22 The results of the 5 areas of each section were summed. Three samples from each group were examined and the data were subjected to statistical analysis. and the data were subjected to statistical analysis.

| cDNA synthesis and real-time PCR analysis
Total RNA was isolated from TA muscles using cold Trizol reagent (Invitrogen, CA, USA), following the manufacturer's instructions. RNA quantity and integrity were assessed using spectrophotometry

| Statistical analysis
Statistical analysis was performed with GraphPad Prism 7 software (San Diego, CA, USA). Data were expressed as mean values ± standard error of the mean (SEM). Statistical differences were evaluated using 1-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Results were considered significant when P < .05. Figure 1 shows the morphological aspects of each group in each period evaluated. The control group had skeletal muscle with normal morphology (polygonal fibres with multiple peripheral nuclei) and no signs of injury ( Figure S1). Figure 2 displays the results of the quantitative analysis of the parameters described above.

| Myonecrosis
All groups subjected to injury exhibited a higher level of myonecrosis  Figure 2A). Seven days after treatment, a low degree of myonecrosis was found in all groups, with no significant differences among the groups (Figure 2A).

| Inflammatory cells
No inflammatory cells were found throughout the experimental period in the control group ( Figure S1). On Day 2 (Figure 1), an acute inflammatory process was found in all groups subjected to cryoin-

| Blood vessels
Well-vascularized muscle tissue was found in the control group (Figure S1). An increased number of blood vessels was found during the phases of muscle repair and was evident on Day 7 in all groups subjected to cryoinjury, indicating the repair process ( Figure 2C). On Days 2 and 7, only the PBM 780 nm group exhibited increased number of blood vessels in relation to the injury group, indicating the occurrence of a more preserved tissue (P = .0077 and P = .0277, respectively; Figure 2C). No differences were found between the injury group and PBM 660 nm group or between the 2 PBM groups.

| Immature muscle fibres
As expected, no immature fibres were found on Day 2 in any of the groups subjected to cryoinjury, as the initial phase of tissue repair was characterized by an acute inflammatory process ( Figure 2D). On

| CD206 + macrophages
No CD206 + macrophages were observed in the control group (Figure S1B). In the injury group, the number of CD206 + macrophages reached a peak on Day 4 ( Figure 4). The PBM 780 nm group showed an increase in the infiltration of CD206 + macrophages on Day 2 compared to the injury group and PBM 660 nm group (P = .0001 and P = .0009, respectively; Figure 4). The number of CD206 + macrophages remained high until Day 4 in the PBM 780 nm. Four and 7 days after injury, no significant differences were found in the number of CD206 + macrophages among the groups subjected to cryoinjury (Figure 4).

| CD163 + macrophages
No CD163 + macrophages were found in the control group (Figure S1). Two days after injury, all groups subjected to cryoinjury exhibited a small number of CD163 + macrophages ( Figure 5), but

IL-6
The control group was used as reference to evaluate the relative mRNA expression level of each gene. Differences in TNF-α, TGF-β and IL-6 mRNA expression were found in all periods.

| IL-6
IL-6 mRNA expression in the injured group reached its peak on Day 2 ( Figure 6). Both PBM treatments were able to decrease the mRNA expression of IL-6. However, the reduction was more evident in the

| TNF-α
The highest TNF-α mRNA expression level was found in the injury group on Day 2. TNF-α mRNA expression was significantly lower in the PBM 660 nm group compared to both the injury and PBM 780 nm groups (P = .0073 and P = .0262, respectively), with no significant difference between the latter 2 groups ( Figure 6). No differences were found among the groups on Days 4 and 7.

| TGF-β
The highest TGF-β mRNA expression level in the injury group was  The experimental model used in the present study was able to induce a muscle repair profile similar to that previously described. 1,2 Cryoinjury, which can be considered an acute injury 1,12,13,16,17,21  and small number of CD206 + and CD163 + (M2) macrophages were also found 2 days after injury, which is in accordance with previous descriptions (for review, see 1 ). Here, the proinflammatory cytokines IL-6 and TNF-α mRNA were also highly expressed on Day 2, as described previously. 1 Four days after injury, reductions were found TNF-α is a proinflammatory cytokine that stimulates myogenic cell proliferation, but also inhibits its differentiation and fusion. 3,24 Therefore regions. [9][10][11] The absorption of light by cytochrome c-oxidase promotes the dissociation of its inhibitory nitric oxide, which leads to an increase in electron transport, mitochondrial membrane potential, ATP generation and the activation of other signalling pathways. [9][10][11] The results of these secondary effects include the activation of many transcription factors, which could explain the effects of PBM on cell proliferation and survival, protein synthesis and the activation of anti-inflammatory and antioxidant pathways, although the mechanism of action of PBM is yet to be fully described. [9][10][11] An important concept regards the therapeutic window for PBM dosimetry. Dosimetric parameters, such as wavelength, power density, energy density, frequency of irradiation, operation regime and interval between consecutive irradiations, are fundamental to achieving the desired results. In the present study, red and NIR light with the same dosimetric parameters were compared and the results were better for NIR light (780 nm laser). The explanation for this difference could reside in the amount of light that actually reaches the cells, which is subject to light wavelength as well as the scattering and reflection properties of tissues. [9][10][11]29 The effects of PBM on macrophage phenotypes are beginning to be described in cell cultures studies, 10,[30][31][32][33][34] evidencing that this therapeutic modality, especially using the NIR wavelength, is capable of altering the polarization of these cells in vitro. In vivo experiments have demonstrated that PBM (808 nm) can shift the phenotype of brain microglial polarization from the pro-inflammatory phenotype (M1) to the anti-inflammatory (M2) phenotype after ischemic stroke, promoting cortical neurogenesis. 35 There are also reports of the ability of red and NIR PBM to modulate the macrophage/microglia phenotype, leading to a predominance of M2 macrophages associated with better recovery of the spinal cord and peripheral nerves after a spinal cord injury 36,37 and spared nerve injury, 38 respectively.
In the present study, treatment with PBM at both wavelengths was able to decrease the amount of M1 macrophages 2 days after injury and increase the number of CD163 + (M2) macrophages 7 days after injury. However, only treatment with NIR PBM was able to increase the number of CD206 + M2 macrophages after 2 days.
These results demonstrate that the treatment with PBM can modulate the inflammatory phase, optimize the transition from the inflammatory to regenerative phase (mainly with NIR light) and improve the final step of regeneration, thereby enhancing the tissue repair processes. Moreover, all these events could be correlated with the presence of distinct macrophage phenotypes.
Although the data described above strongly suggest that PBM can modulate macrophage phenotypes and muscle regeneration after an acute injury, further analyses involving other injury methods and, especially, humans are essential to gaining a better understanding of the role of this therapeutic tool in muscle regeneration.

| CONCLUSION
As macrophage phenotype and function have been suggested to be critical and determinant in downstream outcomes in regenerative medicine 6,7 and muscle therapies, 1,3,4 the evidence presented herein that PBM is a useful tool for accelerating the skeletal muscle repair process by modulating macrophage phenotypes can improve our understanding of the mechanism of action of this therapeutic modality and also indicates new possibilities for macrophage-based therapies.