Lasers in Medical Science

, Volume 31, Issue 5, pp 841–848 | Cite as

Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser

  • Larissa Alexsandra da Silva Neto Trajano
  • Camila Luna da Silva
  • Simone Nunes de Carvalho
  • Erika Cortez
  • André Luiz Mencalha
  • Adenilson de Souza da FonsecaEmail author
  • Ana Carolina Stumbo
Original Article


Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm2) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.


Apoptosis Cell viability Reactive oxygen species Low-level laser therapy Myoblast Necrosis 



This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).


  1. 1.
    Prisk V, Huard J (2003) Muscle injuries and repair: the role of prostaglandins and inflammation. Histol Histopathol 18:1243–1256PubMedGoogle Scholar
  2. 2.
    Filippin LI, Moreira AJ, Marroni NP, Xavier RM (2009) Nitric oxide and repair of skeletal muscle injury. Nitric Oxide 21:157–163CrossRefPubMedGoogle Scholar
  3. 3.
    Rola P, Doroszko A, Derkacz A (2014) The Use of Low-Level Energy Laser Radiation in Basic and Clinical Research. Adv Clin Exp Med 23:835–842CrossRefPubMedGoogle Scholar
  4. 4.
    de Freitas CE, Bertaglia RS, Vechetti Júnior IJ, Mareco EA, Salomão RA, de Paula TG, Nai GA, Carvalho RF, Pacagnelli FL, Dal-Pai-Silva M (2015) High Final Energy of Low-Level Gallium Arsenide Laser Therapy Enhances Skeletal Muscle Recovery without a Positive Effect on Collagen Remodeling. Photochem Photobiol 91(4):957–965CrossRefPubMedGoogle Scholar
  5. 5.
    Alves AN, Fernandes KP, Melo CA, Yamaguchi RY, França CM, Teixeira DF, Bussadori SK, Nunes FD, Mesquita-Ferrari RA (2013) Modulating effect of low level-laser therapy on fibrosis in the repair process of the tibialis anterior muscle in rats. Lasers Med Sci 29:813–821CrossRefPubMedGoogle Scholar
  6. 6.
    Vatansever F, Rodrigues NC, Assis LL, Peviani SS, Durigan JL, Moreira FM, Hamblin MR, Parizotto NA (2012) Low intensity laser therapy accelerates muscle regeneration in aged rats. Photonics Lasers Med 1:287–297PubMedPubMedCentralGoogle Scholar
  7. 7.
    Mesquita-Ferrari RA, Martins MD, Silva JA Jr, da Silva TD, Piovesan RF, Pavesi VC, Bussadori SK, Fernandes KP (2011) Effects of low-level laser therapy on expression of TNF-alpha and TGF-beta in skeletal muscle during the repair process. Lasers Med Sci 26:335–340CrossRefPubMedGoogle Scholar
  8. 8.
    Liu XG, Zhou YJ, Liu TC, Yuan JQ (2009) Effects of low-level laser irradiation on rat skeletal muscle injury after eccentric exercise. Photomed Laser Surg 27:863–869CrossRefPubMedGoogle Scholar
  9. 9.
    Burattini S, Ferri P, Battistelli M, Curci R, Luchetti F, Falcieri E (2004) C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem 48:223–233PubMedGoogle Scholar
  10. 10.
    Shefer G, Partridge TA, Heslop L, Gross JG, Oron U, Halevy O (2002) Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells. J Cell Sci 115:1461–1469PubMedGoogle Scholar
  11. 11.
    Ferreira MP, Ferrari RA, Gravalos ED, Martins MD, Bussadori SK, Gonzalez DA, Fernandes KP (2009) Effect of low-energy gallium-aluminum-arsenide and aluminium gallium indium phosphide laser irradiation on the viability of C2C12 myoblasts in a muscle injury model. Photomed Laser Surg 27:901–906CrossRefPubMedGoogle Scholar
  12. 12.
    Farivar S, Malekshahabi T, Shiari R (2014) Biological effects of low level laser therapy. J Lasers Med Sci 5:58–62PubMedPubMedCentralGoogle Scholar
  13. 13.
    Migliario M, Pittarella P, Fanuli M, Rizzi M, Renò F (2014) Laser-induced osteoblast proliferation is mediated by ROS production. Lasers Med Sci 29:1463–1467CrossRefPubMedGoogle Scholar
  14. 14.
    Gao X, Xing D (2009) Molecular mechanisms of cell proliferation induced by low power laser irradiation. J Biomed Sci 16:4–20CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Chen J, Zhao Y, Liu Y (2014) The role of nucleotides and purinergic signaling in apoptotic cell clearance - implications for chronic inflammatory diseases. Front Immunol 5:656–665PubMedPubMedCentralGoogle Scholar
  16. 16.
    Dai S, Xu C, Tian Y, Cheng W, Li B (2014) Stimulation of calcium overload and apoptosis by sonodynamic therapy combined with hematoporphyrin monomethyl ether in C6 glioma cells. Oncol Lett 8:1675–1681PubMedPubMedCentralGoogle Scholar
  17. 17.
    Miki Y, Akimoto J, Hiranuma M, Fujiwara Y (2014) Effect of talaporfin sodium-mediated photodynamic therapy on cell death modalities in human glioblastoma T98G cells. J Toxicol Sci 39:821–827CrossRefPubMedGoogle Scholar
  18. 18.
    Higuchi Y (2004) Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis. J Cell Mol Med 8:455–464CrossRefPubMedGoogle Scholar
  19. 19.
    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Festjens N, Vanden Berghe T, Vandenabeele P (2006) Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta 1757:1371–1387CrossRefPubMedGoogle Scholar
  21. 21.
    Circu ML, Aw TY (2010) Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 48:749–762CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Broekman MM, Roelofs HM, Wong DR, Kerstholt M, Leijten A, Hoentjen F, Peters WH, Wanten GJ, de Jong DJ (2015) Allopurinol and 5-aminosalicylic acid influence thiopurine-induced hepatotoxicity in vitro. Cell Biol Toxicol 31:161–71CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Palanki R, Arora S, Tyagi N, Rusu L, Singh AP, Palanki S, Carter JE, Singh S (2015) Size is an essential parameter in governing the UVB-protective efficacy of silver nanoparticles in human keratinocytes. BMC Cancer 15:636CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    El-Khatib M, Tepe C, Senger B, Dibué-Adjei M, Riemenschneider MJ, Stummer W, Steiger HJ, Cornelius JF (2015) Aminolevulinic acid-mediated photodynamic therapy of human meningioma: an in vitro study on primary cell lines. Int J Mol Sci 16:9936–48CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Maia ML, Bonjardim LR, Quintans Jde S, Ribeiro MA, Maia LG, Conti PC (2012) Effect of low-level laser therapy on pain levels in patients with temporomandibular disorders: a systematic review. J Appl Oral Sci 20:594–602CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Heu F, Forster C, Namer B, Dragu A, Lang W (2013) Effect of low-level laser therapy on blood flow and oxygen- hemoglobin saturation of the foot skin in healthy subjects: a pilot study. Laser Ther 22:21–30CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Rodrigues NC, Assis L, Fernandes KR, Magri A, Ribeiro DA, Brunelli R, Abreu DC, Renno AC (2013) Effects of 660 nm low-level laser therapy on muscle healing process after cryolesion. J Rehabil Res Dev 50:985–996CrossRefPubMedGoogle Scholar
  28. 28.
    Yonezu T, Kogure S (2013) The effect of low-level laser irradiation on muscle tension and hardness compared among three wavelengths. Laser Ther 22:201–207CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Eduardo FP, Mehnert DU, Monezi TA, Zezell DM, Schubert MM, Eduardo CP, Marques MM (2007) Cultured epithelial cells response to phototherapy with low intensity laser. Lasers Surg Med 34:365–372CrossRefGoogle Scholar
  30. 30.
    Souza NH, Ferrari RA, Silva DF, Nunes FD, Bussadori SK, Fernandes KP (2014) Effect of low-level laser therapy on the modulation of the mitochondrial activity of macrophages. Braz J Phys Ther 18:306–314Google Scholar
  31. 31.
    Huang YY, Sharma SK, Carroll J, Hamblin MR (2011) Biphasic dose response in low level light therapy - an update. Dose Response 9:602–618CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Rodrigues NC, Brunelli R, Abreu DC, Fernandes K, Parizotto NA, Renno AC (2014) Morphological aspects and Cox-2 expression after exposure to 780-nm laser therapy in injured skeletal muscle: an in vivo study. Braz J Phys Ther 18:395–401CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Hao D, Song Y, Che Z, Liu Q (2014) Calcium overload and in vitro apoptosis of the C6 glioma cells mediated by sonodynamic therapy (hematoporphyrin monomethyl ether and ultrasound). Cell Biochem Biophys 70:1445–1452CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Lee YH, Kim DH, Kim YS, Kim TJ (2013) Prevention of oxidative stress-induced apoptosis of C2C12 myoblasts by a Cichorium intybus root extract. Biosci Biotechnol Biochem 77:375–377CrossRefPubMedGoogle Scholar
  35. 35.
    Karu T (1989) Photobiology of low-power laser effects. Health Phys 56:691–704CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag London 2016

Authors and Affiliations

  • Larissa Alexsandra da Silva Neto Trajano
    • 1
    • 2
  • Camila Luna da Silva
    • 1
  • Simone Nunes de Carvalho
    • 1
  • Erika Cortez
    • 1
  • André Luiz Mencalha
    • 2
  • Adenilson de Souza da Fonseca
    • 2
    • 3
    • 4
    Email author
  • Ana Carolina Stumbo
    • 1
  1. 1.Laboratório de Pesquisa em Células-Tronco, Departamento de Histologia e Embriologia, Instituto de Biologia Roberto Alcantara GomesUniversidade do Estado do Rio de JaneiroRio de JaneiroBrazil
  2. 2.Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara GomesUniversidade do Estado do Rio de JaneiroRio de JaneiroBrazil
  3. 3.Departamento de Ciências Fisiológicas, Instituto BiomédicoUniversidade Federal do Estado do Rio de JaneiroRio de JaneiroBrazil
  4. 4.Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara GomesUniversidade do Estado do Rio de JaneiroRio de JaneiroBrazil

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