The Effects of Photobiomodulation on Inflammatory Infiltrate During Muscle Repair in Advanced-Age Rats.

Photobiomodulation (PBM) enhances muscle repair in aged animals, but its effect on the modulation of the phenotype of immune cells has not yet been determined. Rats (20-month-old) were submitted to cryoinjury of the tibialis anterior muscle and were treated with PBM. After 1, 3, and 7 days, the muscles were submitted to immunohistochemical analysis for the determination of neutrophils and macrophage phenotypes. The muscles treated with PBM exhibited a smaller number of neutrophils after 1 day of treatment and a greater number of both M1 and M2 macrophages after 3 days of treatment. The irradiated tissues exhibited a smaller amount of both macrophage phenotypes after 7 days of treatment. PBM produced temporal alterations in the phenotype of the inflammatory cells during muscle repair process in advanced-age animals, indicating that these mechanisms may contribute to the beneficial effects of this therapy in the treatment of muscle injuries.

Effect of prior application with and without post-injury treatment with low-level laser on the modulation of key proteins in the muscle repair process.

The aim of the present study was to evaluate the effects of LLLT prior to muscle injury with and without post-injury irradiation on the expression of isoforms of myosin heavy chain (MyHC), calcineurin (CaN), and myostatin during the repair process. Wistar rats were divided into five groups: control (n = 7); injury (n = 21); LLLT + injury (n = 21); injury + LLLT (n = 21), and LLLT + injury + LLLT (n = 21). Cryoinjury was performed on the tibialis anterior (TA) muscle. The injured groups were euthanized at 3, 7, and 14 days after injury. LLLT was performed using an infrared laser (780 nm) with the following parameters: 10 J/cm2, 40 mW, 10 s per point, 8 points, and 3.2 J of total energy. At the end of each period, the TA muscle was removed for the analysis of MyHC, CaN, and myostatin gene expression using real-time PCR. The data were tested statistically by Kruskal-Wallis with Dunn's post hoc test (p < 0.05). The results demonstrated that prior irradiation reduced the mRNA expression of all proteins at 3 days. Post irradiation reduced the mRNA expression of MyHC-1, MyHC-2a, MyHC-2b, and CaN at 7 days. Prior irradiation combined with post-injury irradiation reduced the mRNA expression of MyHC-2x and CaN at 14 days and increased the mRNA expression of myostatin in the same period. In conclusion, different protocols of photobiomodulation can modulate the expression of the different isoforms of MyHC, CaN, and myostatin during the repair process. It is noteworthy that the combination of the prior and post-injury irradiation was the protocol that most promoted changes in the final phase of the repair process.

Effect of photobiomodulation on connective tissue remodeling and regeneration of skeletal muscle in elderly rats.

The purpose of this study was to evaluate the effects of low-level laser therapy (LLLT) on morphological aspects, IL-6 and IL-1ß expressions, as well as the distribution and organization of collagen in the tibialis anterior (TA) muscle of elderly rats submitted to cryoinjury. Histological photomicrographs were taken of TA muscles stained with HE and picrosirius red. Immunohistochemistry was used for the evaluation of IL-6 and IL-1ß. Male Wistar rats, aged 20 months, were distributed into three groups: (1) control animals not injured or treated with LLLT (n = 5), (2) cryoinjury without LLLT treatment (n = 15), and (3) cryoinjury treated with infrared LLLT (n = 15). LLLT was applied to the TA 2 h after of the injury induction and consisted of daily applications until the sacrifice (1, 3, and 7 days). The following parameters were used: λ = 780 nm, power density 1 W/cm2, output power 40 mW, 10 s per point, 8 points, and 3.2 J of total energy. In the histomorphological analysis, the treated group exhibited a significant decrease in inflammatory infiltrate (p < 0.001) as well as an increase immature fibers and new blood vessels at 7 days compared to the untreated group (p < 0.05). Furthermore, treatment induced a better collagen distribution and organization at 7 days in comparison to the untreated group (p < 0.05). In conclusion, LLLT demonstrated a modulatory effect on the muscle repair process in elderly animals with regard to the collagen remodeling and morphological aspects of muscle tissue.

Red and Infrared Low-Level Laser Therapy Prior to Injury with or without Administration after Injury Modulate Oxidative Stress during the Muscle Repair Process.

INTRODUCTION: Muscle injury is common among athletes and amateur practitioners of sports. Following an injury, the production of reactive oxygen species (ROS) occurs, which can harm healthy muscle fibers (secondary damage) and delay the repair process. Low-level laser therapy (LLLT) administered prior to or following an injury has demonstrated positive and protective effects on muscle repair, but the combination of both administration times together has not been clarified. AIM: To evaluate the effect of LLLT (660 nm and 780 nm, 10 J/cm², 40 mW, 3.2 J) prior to injury with or without the administration after injury on oxidative stress during the muscle repair process. METHODS: Wistar rats were divided into following groups: control; muscle injury alone; LLLT 660 nm + injury; LLLT 780 nm + injury; LLLT 660 nm before and after injury; and LLLT 780 nm before and after injury. The rats were euthanized on days 1, 3 and 7 following cryoinjury of the tibialis anterior (TA) muscle, which was then removed for analysis. RESULTS: Lipid peroxidation decreased in the 660+injury group after one day. Moreover, red and infrared LLLT employed at both administration times induced a decrease in lipid peroxidation after seven days. CAT activity was altered by LLLT in all periods evaluated, with a decrease after one day in the 780+injury+780 group and after seven days in the 780+injury group as well as an increase in the 780+injury and 780+injury+780 groups after three days. Furthermore, increases in GPx and SOD activity were found after seven days in the 780+injury+780 group. CONCLUSION: The administration of red and infrared laser therapy at different times positively modulates the activity of antioxidant enzymes and reduces stress markers during the muscle repair process.

Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

Light-emitting diode therapy increases collagen deposition during the repair process of skeletal muscle.

This study analyzed the effects of light-emitting diode (LED) therapy on the morphology of muscle tissue as well as collagen remodeling and matrix metalloproteinase 2 (MMP-2) activity in the skeletal muscle of rats following acute injury. Wistar rats were divided into four groups: (1) control, (2) sham, (3) untreated cryoinjury, and (4) cryoinjury treated with LED. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior muscle. For treatment, the LED equipment (wavelength 850 nm, output power 30 mW, and total energy 3.2 J) was used daily. The study periods were 1, 3, and 7 days after cryoinjury. Morphological aspects were evaluated through hematoxylin-eosin staining. The amount of collagen fibers was evaluated using Picro Sirius Red staining under polarized light. The gelatinase activity of MMP-2 was evaluated using zymography. The results showed significant reductions in inflammatory infiltrate after 3 days and an increased number of immature muscle fibers after 7 days. Furthermore, treatment induced a reduction in the gelatinolytic activity of MMP-2 after 1, 3, and 7 days in comparison to the untreated injury groups and increased the collagen deposition after 3 and 7 days in the treated groups. LED therapy at 850 nm induced a significant reduction in inflammation, decreased MMP-2 activity, and increased the amount of immature muscle and collagen fibers during the muscle repair process following acute injury.

Photobiomodulation with 660-nm and 780-nm laser on activated J774 macrophage-like cells: Effect on M1 inflammatory markers.

M1 profile macrophages exert a major influence on initial tissue repair process. Few days after the occurrence of injury, macrophages in the injured region exhibit a M2 profile, attenuate the effects of the M1 population, and stimulate the reconstruction of the damaged tissue. The different effects of macrophages in the healing process suggest that these cells could be the target of therapeutic interventions. Photobiomodulation has been used to accelerate tissue repair, but little is known regarding its effect on macrophages. In the present study, J774 macrophages were activated to simulate the M1 profile and irradiated with two different sets of laser parameters (780 nm, 70 mW, 2.6J/cm(2), 1.5s and 660 nm, 15 mW, 7.5 J/cm(2), 20s). IL-6, TNF-α, iNOS and COX-2 gene and protein expression were analyzed by RT-qPCR and ELISA. Both lasers were able to reduce TNF-α and iNOS expression, and TNF-α and COX-2 production, although the parameters used for 780 nm laser provided an additional decrease. 660 nm laser parameters resulted in an up-regulation of IL-6 expression and production. These findings imply a distinct, time-dependent modulation by the two different sets of laser parameters, suggesting that the best modulation may involve more than one combination of parameters.

The effect of low-level laser therapy (LLLT) applied prior to muscle injury.

AIM: To evaluate the effect of LLLT (780 nm; 10 J/cm2 , 40 mW, 3.2 J) prior to injury on the morphological analysis, collagen deposition, and activity of matrix metalloproteinase-2 (MMP-2). METHODS: Wistar rats were divided into groups: control; sham; only LLLT; only muscle injury and LLLT + injury. The rats were euthanized at 1, 3, and 7 days following cryoinjury to muscle that was removed for analysis. RESULTS: LLLT applied prior to muscle injury led to a reduction in myonecrosis and inflammatory cells, an increase of blood vessels and immature muscle fibers. An increase in MMP-2 activity and a decrease in collagen deposition were also found, with a better collagen organization and distribution. CONCLUSION: LLLT applied immediately prior to injury had positive effects during the muscle regeneration process. Therefore, this resource may have considerable therapeutic value, especially for athletes who practice sports in which there is a constant risk of muscle injury. Lasers Surg. Med. 47:571-578, 2015. © 2015 Wiley Periodicals, Inc.

Low-level laser irradiation modulates cell viability and creatine kinase activity in C2C12 muscle cells during the differentiation process.

Low-level laser irradiation (LLLI) is increasingly used to treat musculoskeletal disorders, with satisfactory results described in the literature. Skeletal muscle satellite cells play a key role in muscle regeneration. The aim of the present study was to evaluate the effect of LLLI on cell viability, creatine kinase (CK) activity, and the expression of myogenic regulatory factors in C2C12 myoblasts during the differentiation process. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum and submitted to irradiation with GaAlAs diode laser (wavelength, 780 nm; output power, 10 mW; energy density, 5 J/cm2). Cell viability and the expression of myogenic regulatory factors were assessed 24, 48, and 72 h after irradiation by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. CK activity was analyzed at 24 and 72 h. An increase in cell viability was found in the laser group in comparison to the control group at all evaluation times. CK activity was significantly increased in the laser group at 72 h. Myogenin messenger RNA (mRNA) demonstrated a tendency toward an increase in the laser group, but the difference in comparison to the control group was non-significant. In conclusion, LLLI was able to modulate cell viability and CK activity in C2C12 myoblasts during the differentiation process.

Effects of low-level laser therapy on skeletal muscle repair: a systematic review.

A review of the literature was performed to demonstrate the most current applicability of low-level laser therapy (LLLT) for the treatment of skeletal muscle injuries, addressing different lasers, irradiation parameters, and treatment results in animal models. Searches were performed in the PubMed/MEDLINE, SCOPUS, and SPIE Digital Library databases for studies published from January 2006 to August 2013 on the use of LLLT for the repair of skeletal muscle in any animal model. All selected articles were critically appraised by two independent raters. Seventeen of the 36 original articles on LLLT and muscle injuries met the inclusion criteria and were critically evaluated. The main effects of LLLT were a reduction in the inflammatory process, the modulation of growth factors and myogenic regulatory factors, and increased angiogenesis. The studies analyzed demonstrate the positive effects of LLLT on the muscle repair process, which are dependent on irradiation and treatment parameters. The findings suggest that LLLT is an excellent therapeutic resource for the treatment of skeletal muscle injuries in the short-term.

Effect of laser therapy on skeletal muscle repair process in diabetic rats.

Skeletal muscle myopathy is a common source of disability in diabetic patients. This study evaluated whether low-level laser therapy (LLLT) influences the healing morphology of injured skeletal muscle. Sixty-five male Wistar rats were divided as follows: (1) sham; (2) control; (3) diabetic; (4) diabetic sham; (5) nondiabetic cryoinjured submitted to LLLT (LLLT); (6) diabetic cryoinjured submitted to LLLT (D-LLLT); and (7) diabetic cryoinjured non-treated (D). Diabetes was induced with streptozotocin. Anterior tibialis muscle was cryoinjured and received LLLT daily (780 nm, 5 J/cm(2), 10 s per point; 0.2 J; total treatment, 1.6 J). Euthanasia occurred on day 1 in groups 1, 2, 3, and 4 and on days 1, 7, and 14 in groups 5, 6, and 7. Muscle samples were processed for H&E and Picrosirius Red and photographed. Leukocytes, myonecrosis, fibrosis, and immature fibers were manually quantified using the ImageJ software. On day 1, all cryoinjured groups were in the inflammatory phase. The D group exhibited more myonecrosis than LLLT group (p < 0.05). On day 14, the LLLT group was in the remodeling phase; the D group was still in the proliferative phase, with fibrosis, chronic inflammation, and granulation tissue; and the D-LLLT group was in an intermediary state in relation to the two previous groups. Under polarized light, on day 14, the LLLT and D-LLLT groups had organized collagen bundles in the perimysium, whereas the diabetic groups exhibited fibrosis. LLLT can have a positive effect on the morphology of skeletal muscle during the tissue repair process by enhancing the reorganization of myofibers and the perimysium, reducing fibrosis.

Effect of photobiomodulation on expression of IL-1ß in skeletal muscle following acute injury.

Muscle repair is regulated by growth factors and cytokines. Low-level laser therapy (LLLT) seems to influence acute inflammation and accelerate skeletal muscle repair. This study verifies the effect of LLLT on the expression of IL-1ß in the tibialis anterior (TA) muscle of rats following acute injury. Wistar rats (n=35) were allocated into three groups: control (without lesion and LLLT, n=5), injury group (n=15), and injury + LLLT group (n=15). The acute injury was induced by the contact with a cooled metal probe (3 mm in diameter) during 10 s, twice, in the same muscle area. LLLT was used three times a week using the InGaAlP laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2) during 10 s). The animals were analyzed at 1, 7, and 14 days following injury. TA muscles samples were used for obtaining total RNA and performing cDNA synthesis. Real-time polymerase chain reactions were realized using IL-1ß primer. There was a decrease in IL-1ß expression after 7 days in LLLT group in comparison with the no treated group. In conclusion, LLLT was able to decrease IL-1ß expression during the skeletal muscle repair following an acute injury.

Efeito da natação na expressão de fatores regulatórios miogênicos durante o reparo do musculoesquelético de rato / Swimming influence on the expression of myogenic regulatory factors during skeletal muscle repair of rats

CONTEXTUALIZAÇÃO: O músculo esquelético tem a capacidade de adaptação frente a estímulos variados, tais como atividade contrátil, danos diretos e indiretos. Uma das modalidades terapêuticas utilizadas na reabilitação de disfunções musculoesqueléticas que vem demonstrando resultados positivos no tratamento e na prevenção de várias patologias é a terapia aquática. OBJETIVO: Analisar o efeito da natação na expressão dos fatores regulatórios miogênicos MyoD e miogenina durante o reparo do músculo esquelético de rato após criolesão. MÉTODOS: Foram utilizados 40 ratos Wistar, divididos em 04 grupos: (1) Controle; (2) "Sham" (sem lesão, submetido a exposição do músculo tibial anterior (TA); (3) Criolesionado e (4) Criolesionado e submetido à natação, analisados em 7, 14 e 21 dias. A criolesão foi realizada por meio de duas aplicações, utilizando um bastão metálico de extremidade plana, resfriado em nitrogênio líquido diretamente no ventre muscular. O protocolo consistiu de sessões de natação com duração de 90 minutos, realizadas 6 vezes por semana. Ao término do protocolo os animais foram eutanasiados, os músculos TA foram removidos e o RNA total foi extraído. Em seguida, foi obtido o cDNA para a realização do PCR em tempo real utilizando primers específicos para MyoD e miogenina. RESULTADOS: Os resultados evidenciaram uma redução na expressão de miogenina após 7 dias nos grupos criolesionado com (p<0.01) e sem (p<0.01) natação e após 14 no grupo criolesionado com natação (p<0.05) com relação aos grupos controle e "sham", respectivamente. Não encontramos diferenças entre os grupos criolesionados com (p>0.05) e sem natação (p>0.05). Com relação à expressão de MyoD não houve diferença entre os grupos avaliados. CONCLUSÃO: A natação não influenciou a expressão dos fatores regulatórios miogênicos durante o processo de reparo de músculo esquelético de rato após criolesão.

BACKGROUND: Skeletal muscle has the ability to adapt to several stimuli, such as contractile activity as well as direct and indirect damage. Aquatic therapy has been used in the rehabilitation of musculoskeletal disorders. In addition, it has demonstrated positive results in the therapeutic process and preventing several diseases.. OBJECTIVE: The aim of the present study was to analyze the effect of swimming on the expression of the myogenic regulatory factors MyoD and myogenin during the skeletal muscle repair process in rats following cryoinjury. METHODS: Forty Wistar rats were randomly divided into four groups: 1) Control; 2) Sham - non-muscle damaged, submitted to procedure for exposure of the tibialis anterior (TA) muscle; 3) Cryoinjured; and 4) Cryoinjured and submitted to swimming. Analyses were carried out at 7, 14 and 21 days. Cryoinjury was performed with two applications of the flat end of a metal rod previously cooled in liquid nitrogen directly to the belly of the TA muscle. The protocol consisted of 90-minute swimming sessions six times a week. At the end of the protocol, the animals were euthanized and the TA muscles were removed. Total RNA was extracted using the TRIzol reagent. Next, cDNA was obtained to perform real-time PCR using specific primers for MyoD and myogenin. RESULTS: The results showed a reduction in the expression of myogenin in the groups cryoinjury with p<0.01and without p <0.01 swimming after 7 days, and in group cryoinjury with swimming (p<0.05) after 14 days respect to the control groups and "sham", respectively. There were no differences between groups cryoinjury with (p> 0.05) and without (p>0.05) swimming. Regarding the expression of MyoD there was no difference between the groups. CONCLUSION: Swimming did not affect the expression of myogenic regulatory factors during the skeletal muscle repair process in rats following cryoinjury.