Photobiomodulation assay of muscle cells C2C12 after irradiation with LED device
Keywords:Photobiomodulation; Cell culture; Muscle cells.
Introduction: One of the ways that have been observed to reduce musculoskeletal fatigue is the use of protocols for the application of light sources (photobiomodulation) such as low-intensity laser and LED (Light Emitting Diode). Work involving photobiomodulation has shown promising results in strength performance or reduction of muscle fatigue. At the cellular level, photobiomodulation can modulate fibroblasts proliferation, the fixation and synthesis of collagen and procollagen, promote angiogenesis and improving energy metabolism in mitochondria. Compared with laser devices, LED has several advantages, such as being smaller, lighter, lower cost, and easier for operation. Objective: The present work objective is to verify if irradiation with LED device (650 nm and 860 nm) in muscle cells C2C12 modify the viability, morphology and cytoskeleton components. Methodology: C2C12 cells line (ATCC CRL - 1772) were cultured in 25 cm2 bottles at 37ºC under 5% CO2 in DMEM. The cells were irradiated with the light-emitting diodes (LED) device, Sportllux Ultra that consists of 84 LEDs, each individual LED has 8 mW of power, emitting in 660±20 nm (42 LEDs) and 850±20 nm (42 LEDs), and covering an area (A) of 120 cm2. The power density of delivered light was 5,6 mW/cm2, and the exposure time was 10 minutes, totalizing the fluence of 3,4 J/cm2. Viability assay was performed where the cells were incubated with 100 µL of Crystal Violet (CV) solution and mitochondrial activity assay was evaluated by the colorimetric MTT assay. Nucleus (DAPI) and Cytoskeleton (Rhodamine Phalloidin) fluorescence assay was performed to study the cytoskeleton based on the change in the actin filaments. Results: Our results demonstrate that the synergism of LED irradiation (660nm and 850nm) induced the proliferation of C2C12 cells. The light-emitting diode (LED) device, Sportllux Ultra has a significant effect on C2C12 cell culture. Mitochondrial activity and cell viability showed a significative increase in their activities after irradiation. The microscopy fluorescence observations showed an alignment of cytoskeletal components of C2C12 cells after irradiation. Conclusion: The application of irradiation with the Sportlux Ultra LED device stimulated an increase of energy by mitochondrial activity assay, number of cells by cell viability assay and alignment of cytoskeleton components by fluorescence assay in C2C12 line cells. Our results suggest that organizated cytoskeletal actin filaments normally contribute to cell survival and that induced major cell changes in the cytoskeleton that result in cell shape change. These results suggest that the Sport Lux Ultra LED device can help in the repair of tissue injuries and to collaborate to increase of performance in athletes in a faster way.
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., (2008). Molecular Biology of the Cell (5th ed.). New York: Garland Science. ISBN 978-0-8153-4105-5.
Al-Ghamdi, K. M., Kumar, A., Moussa, N. A., (2012) Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells. Lasers Med Sci 27, 237–249.
Al-Watban, F. A., Andres, B. L., (2006). Polychromatic LED in oval full-thickness wound healing in nondiabetic and diabetic rats,Photomed. Laser Surg. 24, 10–16.
Ates, G. B., Can, A. A., Gülsoy, M., (2017). Investigation of photobiomodulation potentiality by 635 and 809 nm lasers on human osteoblastos. Lasers Med Sci. 32, 591–599
Avni, D., Levkovitz, S., Maltz, L., Oron, U., (2005). Protection of skeletal muscles from ischemic injury: low-level laser therapy increases antioxidant activity. Photomed Laser Surg. 23, 273-277.
Barolet, D., (2008). Light-emitting diodes (LEDs) in dermatology, Semin. Cutan. Med. Surg. 27, 227–238.
Borsa, P. A., Larkin, K. A., True, J. M., (2013). Does phototherapy enhance skeletal muscle contractile function and postexercise recovery? A systematic review. J Athl Train. 48, 57–67.
Cooper, G. M., (2000). "Actin, Myosin, and Cell Movement". The Cell: A Molecular Approach. 2nd Edition.
Desmet, K. D., Paz, D. A., Corry, J. J., Eells, J. T., Wong-Riley, M. T., Henry, M. M., Buchmann, E. V., Connelly, M. P., Dovi, J. V., Liang, H. L., Henshel, D. S., Yeager, R. L., Millsap, D. S., Lim, J., Gould, L. J., Das, R., Jett, M., Hodgson, B. D., Margolis, D., Whelan, H. T., (2006). Clinical and experimental applications of NIR-LED photobiomodulation. Photomed. Laser Surg. 24(2), 121-128
Dima, V. F., Suzuko, K., Liu, Q., (1997). Effects of GaALAs Diode Laser on Serum Opsonic Activity Assessed by Neutrophil- associated Chemiluminescence. Laser Therapy. 9, 153–158
Ferraresi, C., de Brito Oliveira, T., de Oliveira Zafalon, L., de Menezes Reiff, R. B., Baldissera, V., de Andrade Perez, S. E., Matheucci Júnior, E., Parizotto, N. A., (2011). Effects of low level laser therapy (808 nm) on physical strength training in humans. Lasers Med Sci. 26, 349–358.
Ferraresi, C., Hamblin, M. R., Parizotto, N. A., (2012). Low-level laser (light) therapy (LLLT) on muscle tissue: performance, fatigue and repair benefited by the power of light. Photonics Lasers Med. 1, 267–286.
Frangez, I., Cankar, K., Ban Frangez, H., Smrke, D. M., (2017). The effect of LED on blood microcirculation during chronic wound healing in diabetic and non-diabetic patients-a prospective, double-blind randomized study. Lasers Med. Sci. 32, 887–894.
Gao, X., Xing, D., (2009). Molecular mechanisms of cell proliferation induced by low power laser irradiation. J Biomed Sci 16:409-415.
Hamblin, M. R., (2017). Mechanisms and applications of the anti-inflammatory effects of photobiomodulation. AIMS Biophys. 3, 337–361.
Hardin, J., Bertoni, G., Kleinsmith, L. J., (2015). Becker's World of the Cell (8th ed.). New York: Pearson. pp. 422–446. ISBN 978013399939-6.
Herrmann, H., Bär, H., Kreplak, L., Strelkov, S. V., Aebi, U. (2007). "Intermediate filaments: from cell architecture to nanomechanics". Nature Reviews. Molecular Cell Biology. 8, 562–573.
Hopkins, S. L., Siewert, B., Askes, S. H. C., Veldhuizen, P., Zwier, R., Hegerc, M., Bonnet, S., (2016). An in vitro cell irradiation protocol for testing
photopharmaceuticals and the effect of blue, green, and red light on human cancer cell lines. Photochem. Photobiol. Sci. 15, 644–653.
Huang, Y. Y., Sharma, S. K., Carroll, J., Hamblin, M. R., (2011). Biphasic dose response in low level light therapy - an update. Dose Response, 9, 602-618.
Kury, M., Wada, E. E., Da Silva, D.P., Tabchoury, C. P. M., Giannini, M., Cavalli, V., (2020). Effect of violet LED light on in-office bleaching protocols: a randomized controlled clinical trial. Journal of Applied Oral Science. 28, 3-11.
Lam, T. S., Abergel, R. P., Meeker, C. A., Castel, J. C., Dwyer, R. M., Uitto, J., (1986). Laser Stimulation of Collagen Synthesis in Human Skin Fibroblast Cultures. Lasers in Life Science. 1, 61–77.
Li, D. Y., Zheng, Z., Yu, T. T., Tang, B. Z., Fei, P., Qian, J., Zhu, D., (2020). Visible-near infrared-II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation, J. Biophoton. 13, e202000142.
Li, W. T., Leu, Y. C., Wu, J. L., (2010). Red-light light-emitting diode irradiation increases the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells. Photomed Laser Surg. 28, S157-165.
Manabe, Y., Miyatake, S., Takagi, M., Nakamura, M., Okeda, A., Nakano, T., Hirshman, M. F., Goodyear, L. J., Fujii, N. L., (2012). Characterization of an Acute Muscle Contraction Model Using Cultured C2C12 Myotubes. PLoS ONE 7, e52592.
Mangnall, D., Bruce, C., Fraser, R. B., (1993). Insulin-stimulated glucose uptake in C2C12 myoblasts. Biochem Soc. Trans. 21, 438S.
McKinley, M.; Dean O'Loughlin, V., Pennefather-O'Brien, E., Harris, R., (2015). Human Anatomy (4th ed.). New York: McGraw Hill Education. p. 29. ISBN 978-0-07-352573-0.
Mester, E., Mester, A. F., Mester, A., (1985) The biomedical effects of laser application. Lasers Surg Med 5,31–39
Pereira, A. S., Shitsuka, D. M., Parreira, F. J., Shitsuka, R. (2018). Metodologia da pesquisa científica. Ed. Santa Maria, RS: UFSM, NTE.
Osanai, T., Shiroto, C., Mikami, Y., (1990). Measurement of Ga ALA Diode Laser Action on Phagocytic Activity of Human Neutrophils as a Possible Therapeutic Dosimetry Determinant. Laser Therapy. 2, 123–134.
Rastelli, A. N., Dias, H. B., Carrera, E. T., Barros, A. C., Santos, D. D., Panhóca, C. H., Bagnato, V. S., (2018). Violet LED with low concentration carbamide peroxide for dental bleaching: a case report. Photodiagnosis Photodyn Ther.23, 270-272.
Ricci, R., Pazos, M. C., Borges, R. E., Pacheco-Soares, C., (2009). Biomodulation with low-level laser radiation induces changes in endothelial cell actin flaments and cytoskeletal organization. Journal of Photochemistry and Photobiology B: Biology 95, 6–8.
Rohringer, S., Holnthoner, W., Chaudary, S., Slezak, P., Priglinger, E., Strassl, M., Pill, K., Mühleder, S., Redl, H., Dungel, P., (2017). The impact of wavelengths of LED light-therapy on endothelial cells. Sci Rep 7, 10700.
Russell, B. A., Kellett, N., Reilly, L. R., (2005). A study to determine the efficacy of combination LED light therapy (633 nm and 830 nm) in facial skin rejuvenation. J Cosmet Laser Ther. 7, 196-200.
Silveira, P. C., Ferreira, K. B., da Rocha, F. R., Pieri, B. L., Pedroso, G. S., De Souza, C. T., Nesi, R. T., Pinho, R. A., (2016). Effect of low-power laser (LPL) and light-emitting diode (LED) on inflammatory response in burn wound healing, Inflammation 39, 1395–1404.
Sommer, A. P., (2019). Revisiting the photon/cell interaction mechanism in low-level light therapy. Photobiomodul Photomed Laser Surg., 37, 336-341.
Teuschl, A., Balmayor, E. R., Redl, H., van Griensven, M., Dungel, P., (2015). Phototherapy with LED light modulates healing processes in an in vitro scratch-wound model using 3 different cell types. Dermatol Surg. 41, 261-268.
Turrioni, A. P. S., Montoro, L. A., Basso, F. G., Almeida, L. F. D., Costa, C. A. S., Hebling, J., (2015). Dose-responses of Stem Cells from Human Exfoliated Teeth to Infrared LED Irradiation. Brazilian Dental Journal, 26, 409-415.
Vistica, V. T., Skehan, P., Scudiero, D., Monks, A., Pittman, A., Boyd, M. R., (1991). Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res. 51, 2515-2520.
Wong, C. Y., Al-Salami, H., Dass, C. R., (2020). C2C12 cell model: its role in understanding of insulin resistance at the molecular level and pharmaceutical development at the preclinical stage. Journal of Pharmacy and Pharmacology. 72, 1667–1693.
Young, S., Bolton, P., Dyson, M., Harvey, W., Diamantopoulos, C., (1989). Macrophage Responsiveness to Light Therapy. Lasers Surg Med. 9. 497–505.
Yu, W., Naim, J. O., Lanzafame, R. J. (1994). The effect of laser irradiation on the release of bFGF from 3T3 fibroblasts. Photochemistry and Photobiology, 59, 167–170.
Zabeu, A. M. C., Carvalho, I. C. S., Pacheco-Soares C., Da Silva, N. S., (2021). Biomodulatory effect of low intensity laser (830 nm.) in neural model 9L/lacZ. Research Society and Development, 10, e11310817025.
Zhao, H., Ji, T., Sun, T., Liu, H., Liu, Y., Chen, D., Wang, Y., Tan, Y., Zeng, J., Qiu, H., Gu, Y., (2022). Comparative study on Photobiomodulation between 630 nm and 810 nm LED in diabetic wound healing both in vitro and in vivo. Journal of Innovative Optical Health Sciences 15, 2250010, 1 - 10.
How to Cite
Copyright (c) 2022 Elessandro Váguino de Lima; Cristina Pacheco-Soares; Newton Soares da Silva
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
1) Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
2) Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
3) Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.