Low-level laser therapy (808 nm) reduces inflammatory response and oxidative stress in rat tibialis anterior muscle after cryolesion

Abstract

Background and objective: Muscle regeneration is a complex phenomenon, involving coordinated activation of several cellular responses. During this process, oxidative stress and consequent tissue damage occur with a severity that may depend on the intensity and duration of the inflammatory response. Among the therapeutic approaches to attenuate inflammation and increase tissue repair, low-level laser therapy (LLLT) may be a safe and effective clinical procedure. The aim of this study was to evaluate the effects of LLLT on oxidative/nitrative stress and inflammatory mediators produced during a cryolesion of the tibialis anterior (TA) muscle in rats.

Material and methods: Sixty Wistar rats were randomly divided into three groups (n = 20): control (BC), injured TA muscle without LLLT (IC), injured TA muscle submitted to LLLT (IRI). The injured region was irradiated daily for 4 consecutive days, starting immediately after the lesion using a AlGaAs laser (continuous wave, 808 nm, tip area of 0.00785 cm(2) , power 30 mW, application time 47 seconds, fluence 180 J/cm(2) ; 3.8 mW/cm(2) ; and total energy 1.4 J). The animals were sacrificed on the fourth day after injury.

Results: LLLT reduced oxidative and nitrative stress in injured muscle, decreased lipid peroxidation, nitrotyrosine formation and NO production, probably due to reduction in iNOS protein expression. Moreover, LLLT increased SOD gene expression, and decreased the inflammatory response as measured by gene expression of NF-kβ and COX-2 and by TNF-α and IL-1β concentration.

Conclusion: These results suggest that LLLT could be an effective therapeutic approach to modulate oxidative and nitrative stress and to reduce inflammation in injured muscle.

Fig. 1

Freezing right tibialis anterior muscle injury (cryolesion) model. A: Dissection and muscle exposition; (B and C) Cryolesion procedure; and (D) Suture after surgical procedure. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/lsm]

Fig. 2

Diagram for the collection and storage of the right tibialis anterior muscle. A: Muscles from 10 animals for morphological analysis; (B) 10 muscles were cut transversely into two equal parts: the proximal fragment was used for RNA extraction protocol; the distal fragments were homogenate in RIPA buffer and used for de TBARs, Griess, ELISA, Western blot, and dot blot analyses. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/lsm]

Fig. 3

Serial transversal cross-sections in the medial region of right TA muscle. A: Toluidine blue staining; (B) acid phosphatase staining of: normal TA muscle—control (BC); injured TA muscle without LLLT (IC); injured TA muscle submitted to infrared laser irradiation (IRI); Bar: 100 μm (magnification: 200×). ●Normal tissue, ^*injured area; ■regenerating fiber; (−) negative reaction to acid phosphatase; and (+) positive reaction to acid phosphatase showing high concentration of lysosomes as a tissue necrosis marker. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/lsm]

Fig. 4

Lipid peroxidation. Normal TA muscle—control (BC); injured TA muscle without LLLT (IC); injured TA muscle submitted to infra-red laser irradiation (IRI); LLLT decreased lipid peroxidation levels (^†P < 0.05 vs. IC).

Fig. 5

Nitrotyrosine formation. Normal TA muscle—control (BC); injured TA muscle without LLLT (IC); injured TA muscle submitted to infrared laser irradiation (IRI); LLLT decreased nitrotyrosine formation (^†P < 0.01 vs. IC).

Fig. 6

Protein expression of iNOS and NO generation. A: iNOS expression and (B) NO production. Normal TA muscle—control (BC); Injured TA muscle without LLLT (IC); injured TA muscle submitted to infrared laser irradiation (IRI). LLLT decreased iNOS expression (^†P < 0.05 vs. IC) and NO production (^†P < 0.01 vs. IC).

Fig. 7

Cytokine levels. A: TNF-α and (B) IL-1β. Normal TA muscle—control (BC); injured TA muscle without LLLT (IC); injured TA muscle submitted to infrared laser irradiation (IRI). LLLT has decreased the concentration of inflammatory cytokines (^†P < 0.05 vs. IC).

Fig. 8

NF-kB (A), COX-2 (B), and SOD (C) gene expression. Normal TA muscle—control (BC); Injured TA muscle without LLLT (IC); injured TA muscle submitted to infrared laser irradiation (IRI). LLLT has decreased gene expression of NF-κB and COX-2, and enhanced SOD mRNA (^†P < 0.01 vs. IC).