Lasers Med Sci DOI 10.1007/s10103-016-1908-9

ORIGINAL ARTICLE

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 Agnelo Neves Alves 1 & Beatriz Guimarães Ribeiro 1 & Kristianne Porta Santos Fernandes 2 & Nadhia Helena Costa Souza 1 & Lília Alves Rocha 3 & Fabio Daumas Nunes 4 & Sandra Kalil Bussadori 1,2 & Raquel Agnelli Mesquita-Ferrari 1,2

Received: 27 August 2015 / Accepted: 5 February 2016 # Springer-Verlag London 2016

Abstract 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; preinjury 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

* Raquel Agnelli Mesquita-Ferrari [email protected]

1

Postgraduate Program in Rehabilitation Sciences, Universidade Nove de Julho—UNINOVE, Rua Vergueiro, 235/249, Liberdade, CEP 01504-001 São Paulo, SP, Brazil

2

Postgraduate Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho—UNINOVE, São Paulo, SP, Brazil

3

Departament of Molecular Pathology, School of Dentistry, Universidade de São Paulo—FOUSP, São Paulo, SP, Brazil

4

Departament of Oral Pathology, School of Dentistry, Universidade de São Paulo—FOUSP, São Paulo, SP, Brazil

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. Keywords Low-level laser therapy . Muscle injury . Regeneration . Satellite cells . Myogenic factors . Cytokines

Introduction Muscle injuries are common among high-performance athletes and amateurs in different sports [1, 2]. Injury often results in the inability to participate in training and competitions, compromises athletic performance, and increases one’s susceptibility to recurrent injuries [3, 4]. The best treatment for muscle injuries has not yet been clearly defined. Thus, standardized therapies that can minimize muscle damage and enhance the repair process are important [5]. The muscle repair process is characterized mainly by muscle fiber necrosis, inflammatory infiltration, and a local increase in pro-inflammatory cytokines, growth factors, and proteolytic enzymes involved in the phagocytosis of cell debris [2, 6, 7]. Simultaneously, myogenic precursor (stem) cells, denominated satellite cells, are activated, proliferate, and differentiate into myoblasts, which fuse to repair damaged fibers or form a new functional muscle fiber [8–10]. This process ends with the maturation of new fibers, the contraction

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and reorganization of connective tissue, and the functional recovery of the injured muscle. The activation of satellite cells and subsequent differentiation in the myogenesis process are controlled by myogenic regulatory factors (MRFs): Myf5, MyoD, myogenin, and MRF4. Shortly after activation, Myf-5 and MyoD are rapidly expressed and play roles in myogenesis, proliferation, and the conversion of precursor cells into myoblasts, while myogenin and MRF4 are important to the terminal differentiation and formation of myotubes [8–10]. A number of different growth factors and cytokines have been implicated in the regulation of satellite cell responses [8]. Interleukin-6 (IL-6) is a muscle-derived cytokine known to be involved in the regulation of inflammation and the immune response [11, 12]. Recently, a number of studies have provided evidence that IL-6 may play a key role in muscle regeneration and growth by modulating the proliferation, differentiation, and fusion of skeletal muscle cells, indicating the importance of this cytokine to the muscle repair process [13, 14]. Low-level laser therapy (LLLT) has demonstrated positive effects on the inflammatory response and repair process when administered following a muscle injury [5]. However, the mechanisms of these effects are still under investigation. LLLT administered prior to exercise and injury has demonstrated the potential to improve performance, delay muscle fatigue, preserve muscle tissue against exercise-induced damage, and accelerate the recovery process [15, 16]. In addition, there are few reports in the literature on whether LLLT administered preemptively is able to minimize muscle injury. The aim of the present study was to analyze the effects of pre-injury and post-injury irradiation with LLLT on the mRNA expression of MyoD, myogenin, and IL-6 during the muscle repair process following cryoinjury.

Ethics Committee, process numbers AN12/2012 and AN16/ 2012. Experimental groups The animals were randomly divided into six groups: (1) control group—not submitted to any type of procedure (n = 7); (2) sham group—submitted only to exposure of the tibialis anterior (TA) muscle (n = 7); 3) LLLT group—submitted to LLLT irradiation over the TA muscle immediately prior to euthanasia (n = 7); (4) injury group—submitted to the injury procedure with no subsequent treatment (n = 28); (5) pre-injury LLLT group—submitted to LLLT irradiation over TA muscle immediately prior to injury (n = 28); and (6) post-injury LLLT—submitted to the injury procedure followed by LLLT (n = 28). Animals from the control, sham, and LLLT groups were euthanized on the first day. Animals from the injury, preinjury LLLT, and post-injury LLLT groups were euthanized on days 1, 3, 7, and 14 following the induction of injury. Cryoinjury procedure Cryoinjury procedure was performed as previously described by our research group [17–19]. The animals were anesthetized with ketamine (Dopalen, Vetbrands, São Paulo, Brazil) and xylazine (Anasedan, Vetbrands, São Paulo, Brazil) (80 and 10 mg/kg, respectively). The TA muscle was then surgically exposed and submitted to the cryoinjury procedure, which consisted of cooling the flat end of a metal rod (3 mm in diameter) in liquid nitrogen and applying it directly to the ventral surface of the exposed muscle for 10 s, followed by a second application to the same area for another 10 s and suturing of the incision. LLLT procedure

Materials and methods Male Wistar rats aged 12 weeks (body mass 220 ± 15 g) were acclimatized to the laboratory with free access to food and water. All procedures were performed in accordance with the guidelines of the Brazilian National Council for the Control of Animal Experimentation. This study received approval from the University Nove de Julho Animal Research

Table 1

An aluminum-gallium-arsenide (AlGaAs) diode laser (Twin Laser®, MM Optics, São Carlos, SP, Brazil) operating at a wavelength of 780 nm was employed. A power meter (Laser Check, MM Optics, São Carlos, Brazil) was used to determine the output power of the equipment. The dose and parameters were chosen based on a previous study by Alves et al. [19] and are summarized in Table 1.

LLLT parameters

Wavelength (nm)

Type of diode laser

Power output (mW)

Beam spot (cm2)

Power density (W/cm2)

Energy per point (J)

Energy density (J/cm2)

Time per point (s)

Irradiated points

Energy per treatment (J)

780

AlGaAs

40

0.04

1

0.4

10

10

8

3.2

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LLLT was administered in contact with the surface of the skin at an angle of 90° between the emitter and skin to prevent refraction. The pre-injury LLLT group was submitted to a single session immediately prior to muscle injury. Treatment in the post-injury LLLT group was initiated 2 hours following injury and was performed at 24-h intervals for a total of 1, 2, 6, and 13 sessions for the groups evaluated at 1, 3, 7, and 14 days, respectively. After the experimental period for each group, the animals were weighed and euthanized with an overdose of anesthesia (ketamine (240 mg/kg) and xylazine (30 mg/kg)), and the TA muscle was removed for analysis. Real-time quantitative polymerase chain reaction Total RNA was isolated from the TA muscle using cold Trizol Reagent (Invitrogen, CA, USA), following the manufacturer’s instructions. RNA quantity and integrity were assessed in a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and 1 % agarose gel electrophoresis stained with ethidium bromide. cDNA synthesis was performed with 1 μg of total RNA and a High-Capacity cDNA Reverse Transcription Kit (Invitrogen, CA, USA) using a Veriti ® Thermal Cycler (Applied Biosystems, USA). All samples received DNase I (Invitrogen, CA, USA) to avoid DNA contamination. RTqPCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, USA) and SYBR® Green PCR Master Mix (Applied Biosystems, USA). The thermal cycling conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Rat-specific primers for MyoD [20] (forward: 5′GGAGACATCCTCAAGCGATGC-3′; reverse: 5′AGCACCTGGTAAATCGGATTG-3′; GenBank accession number NM_176079.1), myogenin [20] (forward: 5′A C TA C C C A C C G T C C AT T C A C - 3 ′ ; r e v e r s e : 5 ′ TCGGGGCACTCACTGTCTCT-3′; GenBank accession number M24393.1), and IL-6 (forward: 5′TCCAGTTGCCTTCTTGGGAC-3′; reverse: 5′GTGTAATTAAGCCTCCGACTTG-3′; GenBank accession number NM_031168.1) were used for this procedure. A housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase; forward: 5′-GCATCCTGGGCTACACTGA-3′; reverse: 5′-CCACCACCCTGTTGCTGTA-3′; GenBank accession number NM_002046) was employed to normalize the data using the same amount of cDNA. Quantification was performed using the 2−ΔΔCT method [21].

confidence intervals and a 5 % level of significance (P < 0.05). All analyses were performed with the aid of the GraphPad Prism 5 program (GraphPad Software, CA, USA).

Results mRNA expression of MyoD Figure 1 displays the mRNA expression of MyoD. One day following injury, no differences were found among the experimental groups (P > 0.05). At 3 days, a significant increase in the expression of MyoD mRNA was found in the post-injury LLLT group in comparison to the control, sham, LLLT, and injury groups (P < 0.05). At 7 days, a significant increase in the expression of MyoD mRNA was found in the injury and pre-injury LLLT groups in comparison to the control group (P < 0.05), and a significant increase in MyoD mRNA was found in the post-injury LLLT group in comparison to all other groups (P < 0.01). At 14 days, no significant differences were found among the experimental groups (P > 0.05). mRNA expression of myogenin Figure 2 displays the mRNA expression of myogenin. No differences were found among the experimental groups on days 1, 3, and 7 (P > 0.05). At 14 days, a significant increase in the expression of myogenin mRNA was found in the postinjury LLLT group in comparison to all other experimental groups (P < 0.01). mRNA expression of IL-6 Figure 3 displays the mRNA expression of IL-6. One day following injury, no differences were found among the experimental groups (P > 0.05). However, at 3 and 7 days, a significant increase in the expression of IL-6 mRNA was found in the injury group in comparison to all other experimental groups (P < 0.01). Moreover, a significant reduction in the expression of IL-6 mRNA was found in the pre-injury LLLT (P < 0.05) and post-injury LLLT (P < 0.01) groups in comparison to the injury group. At 14 days, a significant increase in the expression of IL-6 mRNA was found in the post-injury LLLT group in comparison to all other experimental groups (P < 0.01).

Statistical analysis

Discussion Data on MyoD, myogenin, and IL-6 mRNAwere expressed as mean ± standard error of the mean (SEM). Values were tested for normality by the Kolmogorov-Smirnov test, being parametric data; the one-way ANOVA followed by Tukey’s test were used for comparisons among groups, with 95 %

To the best of our knowledge, this study is the first to compare the effects of LLLT prior to and following the occurrence of a muscle injury on mRNA expression of MRFs and cytokines involved directly in myogenic proliferation and

Lasers Med Sci Fig 1 mRNA expression of MyoD; data expressed as mean ± SEM; n = 7 animals per group (#P = 0.027 vs. control group; *P = 0.041 vs. injury group; **P = 0.008 vs. injury group)

differentiation. The findings suggest that both pre-injury and post-injury LLLT may contribute favorably to the muscle Fig 2 mRNA expression of myogenin; data expressed as mean ± SEM; n = 7 animals per group (**P = 0.006 vs. injury group)

repair process. However, the effects were more accentuated in the post-injury group.

Lasers Med Sci Fig 3 mRNA expression of IL-6; data expressed as mean ± SEM; n = 7 animals per group (#P = 0.002 vs. all groups)

MyoD is one of the key transcription factors responsible for the withdrawal of myoblasts to initiate myogenesis and the initial differentiation of muscle cells [8, 9]. Moreover, the deletion or inhibition of MyoD expression results in a reduced regenerative capacity and a decrease in regenerated myotubes [10, 22]. Myogenin plays a key role in the terminal differentiation and fusion of myoblasts [8–10]. The downregulation of myogenin causes the cleavage of myotubes through a mechanism that is independent on cell cycle re-entry [23]. In the present study, the daily administration of LLLT following cryoinjury led to a significant increase in MyoD mRNA at 3 and 7 days as well as an increase in myogenin mRNA at 14 days. These results are in agreement with findings reported by Rodrigues et al. [24] who administered LLLT (wavelength 660 nm; output power 40 mW; total energy 2 J) following cryoinjury to the TA muscle of rats and found an increase in the expression of MyoD mRNA after 7 days. Brunelli et al. [25] also found that LLLT (wavelength 780 nm; output power 20 and 40 mW; total energy 0.4 and 2.0 J, respectively) caused an increase in MyoD immunoexpression in rat muscle 7 days following injury. Interestingly, using the same dosimetric parameters employed in the present study, our research group has found an increase in the number of blood vessels after 3 and 7 days as well as an increase in the number of immature muscle fibers and matrix metalloproteinase-2 activity after 7 days [18].

Unlike the present investigation, two previous studies found no change in myogenin mRNA at 14 days following LLLT [24, 25]. This divergence may be explained by the different treatment and dosimetric parameters employed. The increase in myogenin mRNA in the present study was accompanied by an increase in IL-6 mRNA in the same group. IL-6 is a critical mediator of macrophage migration, myoblast proliferation, myotube fusion, and the progression of activated satellite cells to the early differentiation stage [6, 9, 26]. While IL-6 is produced by different cells, the main sources are monocytes/macrophages, fibroblasts, vascular endothelial cells, and skeletal muscle cells [27, 28]. Analyzing different cell types following acute muscle injury induced by crotoxin, Zhang et al. [26] found that monocytes/macrophages expressed the highest level of IL-6 mRNA, whereas muscle-resident cells (including myoblasts and fibroblasts) expressed very low levels. The authors also demonstrated the impairment of muscle regeneration along with the decreased expression of MyoD and myogenin mRNA and the inflammatory response as well as an increase in interstitial fibrosis in mice lacking IL-6. However, an elevated IL-6 level can also have negative effects on satellite cell functions and can cause muscle atrophy [14, 29]. The present findings revealed a significant increase in IL-6 mRNA level in the injury group at 3 and 7 days (acute phase) in comparison to the control group. In the same periods, both pre-injury and

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post-injury LLLT led to a reduction in IL-6 mRNA in comparison to the untreated injury group. This finding is in agreement with data described in previous studies, which report that LLLT modulates the inflammatory process [16, 17]. The increase in IL-6 mRNA in the post-injury LLLT group at 14 days may be directly related to the increase in myogenin mRNA, since the knockout of IL-6 mRNA in differentiating C2C12 myoblasts has been found to impair the expression of myogenin mRNA and myosin heavy chain IIb, with the subsequent impairment of myotube fusion [13]. Moreover, in a previous study, our research group demonstrated the absence of inflammatory infiltration and the moderate presence of immature fibers 14 days following cryoinjury, which lends support to the hypothesis that an increase in IL-6 mRNA is directly involved in the differentiation of myoblasts [20]. Thus, IL-6 may be of importance to the formation of muscle fibers following injury. Therefore, the two IL-6 peaks observed in this study are also consistent with LLLT helping in muscle repair, decreasing the inflammatory process and contributing to the differentiation of myoblasts. Muscle repair is a complex process that involves inflammatory cell infiltration, which promotes the proliferation and activation of satellite cells [6, 9]. The regulation of IL-6 may involve inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1). Using the same experimental cryoinjury model, our research group has demonstrated an increase in TNF-α and IL-1β mRNA 7 days following injury in comparison to a non-injury group [17, 18]. The studies cited demonstrated that LLLT (wavelength 660 nm; output power 20 mW; total energy 1.6 J) administered after cryoinjury led to a decrease in TNF-α and IL-1β mRNA in the same experimental period. TNF-α and IL-1 cause the translocation of NF-κB to the cell nucleus [6]. NF-κB activation induces an increase in the expression of other inflammatory mediators, such as IL-6 and CCL2, thereby amplifying the inflammatory process [6]. The reduction in the expression of these cytokines may explain the decrease in IL-6 mRNA found in the post-injury LLLT group. The reduction in mRNA IL-6 in the pre-injury LLLT group at 3 and 7 days can be directly related to the reduction in infiltration of inflammatory cells showed by Ribeiro et al. [16] after 1 and 3 days. This study also demonstrated that LLLT (wavelength 780 nm; output power 40 mW; total energy 3.2 J) applied prior to muscle injury led to an increase of blood vessels, immature fibers, and MMP-2 activity after 7 days. Moreover, recent studies report positive findings with regard to improvements in performance, delayed skeletal muscle fatigue, and the accelerated recovery when applied prior to exercise and tetanic contraction [15, 30]. LLLT has been found to stimulate mitochondrial activity and mitochondrial function [31], enhance ATP synthesis [31], and increase the amount of reactive oxygen species as well as the activity of cytochrome c oxidase [32]. These effects may explain the

findings of the present study, in which LLLT administered prior to muscle injury led to a reduction in the mRNA expression of IL-6 and therefore might exert a protective effect, minimizing tissue damage by attenuating the inflammatory process. In conclusion, the present findings demonstrate that LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin during the muscle repair process. Moreover, both forms of LLLT administration were able to modulate the mRNA expression of IL-6. Thus, LLLT administered prior to muscle injury may have considerable therapeutic value and further studies should be conducted to gain a better understanding of the effects of this resource on reducing tissue damage and accelerating the repair process. Acknowledgments The authors wish to thank UNINOVE and the Brazilian fostering agencies, Coordination for the Improvement of Higher Education Personnel—CAPES (grants 1182781; 1510536) and São Paulo Research Foundation—FAPESP (grants 2011/04452-8; 2011/17638-2; 2012/11461-6; 2013/21540-3; and 2014/12381-1) for financial support. Compliance with ethical standards All procedures were performed in accordance with the guidelines of the Brazilian National Council for the Control of Animal Experimentation. This study received approval from the University Nove de Julho Animal Research Ethics Committee, process numbers AN12/2012 and AN16/2012 Conflict of interest The authors declare that they have no competing interests.

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