Histomorphometric evaluation of skin wounds in rats submitted to biomodulatory therapies – research protocol
Keywords:Wound healing, Low-Level Light Therapy, Ozone, Rats, Wistar
INTRODUCTION: The use of biomodulatory therapies in order to help tissue repair has been increasingly common in different areas of health. OBJECTIVE: This study aims to comparatively evaluate the effects of 660 nm laser photobiomodulation, ozone therapy, and ozonated oil on repair through histomorphometric analysis in skin wounds in rats. Forty Wistar rats will be divided into 4 groups of 10 animals each, Control Group (GC), Laser Group (LG), Ozone Gas Group (OGG), and Ozonated Oil Group (OOG). MATERIALS AND METHODS: Standard skin wounds will be made on the back of the animals, and the different experimental groups will be treated with the biomodulatory therapies described for three consecutive days. Five and ten days after surgery, five rats from each group will be euthanized. Skin fragments, including the wound area, will be removed for histological processing and subsequent staining of histological sections with Hematoxylin-eosin and Sirius red. Micrographs of the histological sections will be obtained and ten standard images will be captured for quantitative evaluation of the variables collagen area, number of blood vessels, and epithelium thickness. The variables infiltrate of polymorphonuclear and monomorphonuclear inflammatory cells, as well as edema, will be analyzed semiquantitatively. Statistical analysis of the study variables will be performed, with a significance level of p<0.05. CONCLUSION: It is expected to verify which of the biomodulatory therapies used can favor the resolution of tissue repair, in particular, by promoting collagen biosynthesis.
(1) Müller KP, Rodrigues CRMD, Núñez SC, Rocha R, Jorge AOC, Ribeiro MS. Effects of low power red laser on induced-dental caries in rats. Arch Oral Biol. 2007;52(7):648-54. https://doi.org/10.1016/j.archoralbio.2006.12.018
(2) Fiório FB, Albertini R, Leal-Junior ECP, Carvalho PTC. Effect of low-level laser therapy on types I and III collagen and inflammatory cells in rats with induced third-degree burns. Lasers Med Sci. 2014;29(1):313-9. https://doi.org/10.1007/s10103-013-1341-2
(3) Tabakoglu HO, Sani MM, Uba AI, Abdullahi UA. Assessment of circular wound healing in rats after exposure to 808-nm laser pulses during specific healing phases. Lasers Surg Med. 2016;48(4):409-15. https://doi.org/10.1002/lsm.22462
(4) Colombo F, Valença Neto AAP, Sousa APC, Marchionni AMT, Pinheiro ALB, Reis SRA. Effect of low-level laser therapy (l660 nm) on angiogenesis in wound healing: A immunohistochemical study in a rodent model. Braz Dent J. 2013;24(4):308-12. https://doi.org/10.1590/0103-6440201301867
(5) Nesi-Reis V, Lera-Nonose DSSL, Oyama J, Lalucci MPPS, Demarchi IG, Aristides SMA, et al. Contribution of photodynamic therapy in wound healing: a systematic review. Photodiagnosis Photodyn Ther. 2018;21:294-305. https://doi.org/10.1016/j.pdpdt.2017.12.015
(6) Dortbudak O, Hass R, Malath-Pokorny G. Biostimulation of bone marrow cells with a diode soft laser. Clin Oral Implants Res. 2000;11(6):540-45. https://doi.org/10.1034/j.1600-0501.2000.011006540.x
(7) Moreira SH, Pazzini JM, Álvarez JLG, Cassino PC, Bustamante CC, Bernardes FJL, et al. Evaluation of angiogenesis, inflammation, and healing on irradiated skin graft with low-level laser therapy in rats (Rattus norvegicus albinus wistar). Lasers Med Sci. 2020;35:(5)1103-9. https://doi.org/10.1007/s10103-019-02917-y
(8) Gonçalves RV, Mezêncio JMS, Benevides GP, Matta SLP, Neves CA, Sarandy MM, et al. Effect of gallium-arsenide laser, gallium-aluminum arsenide laser and healing ointment on cutaneous wound healing in Wistar rats. Braz J Med Biol Res. 2010;43(4):350-55. https://doi.org/10.1590/S0100-879X2010007500022
(9) Bocci V, Zanardi I, Huijberts MSP, Travagli V. Diabetes and chronic oxidative stress. A perspective based on the possible usefulness of ozone therapy. Diabetes Metab Syndr. 2011;5(1):45-49. https://doi.org/10.1016/j.dsx.2010.05.014
(10) Bocci VA. Scientific and medical aspects of ozone therapy. Arch Med Res. 2006;37(4):425-435. https://doi.org/10.1016/j.arcmed.2005.08.006
(11) Rowen RJ. Ozone therapy as a primary and sole treatment for acute bacterial infection: case report. Med Gas Res. 2018;8(3):121-24. https://doi.org/10.4103/2045-9912.241078
(12) Rodrigues KL, Cardoso CC, Caputo LR, Carvalho JCT, Fiorini JE, Schneedorf JM. Cicatrizing and antimicrobial properties of an ozonised oil from sunflower seeds. Inflammopharmacology. 2004;12(3):261-70. https://doi.org/10.1163/1568560042342275
(13) Schulz S. The role of ozone/oxygen in clindamycin-associated enterocolitis in the Djungarian hamster (Phodopus sungorus sungorus). Lab Anim. 1986;20(1):41-8. https://doi.org/10.1258/002367786781062160
(14) Kazancioglu HO, Erisen M. Comparison of low-level laser therapy versus ozone therapy in the treatment of oral lichen planus. Ann Dermatol. 2015;27(5):485-91. https://doi.org/10.5021/ad.2015.27.5.485
(15) Grootveld M, Aylin B, Siddiqui N, Sim J, Silwood C, Lynch E. History of the clinical applications of ozone. In: Lynch E, organizator. Ozone: the revolution in dentistry. London: Quintessence Publishing Co; 2004. p. 23-31.
(16) Alan H, Vardi N, Özgür C, Acar AH, Yolcu Ü, Doğan DO. Comparison of the Effects of Low-Level Laser Therapy and Ozone Therapy on Bone Healing. J Craniofac Surg. 2015;26(5):e396-400. https://doi.org/10.1097/scs.0000000000001871
(17) Orsted HL, Keast D, Forest-Lalande L, Mégie MF. Basic principles of wound healing: An understanding of the basic physiology of wound healing provides the clinician with the framework necessary to implement the basic principles of chronic wound care. Wound Care Canada [Internet]. 2004;9(2):4-13. Available from: https://www.woundscanada.ca/docman/public/wound-care-canada-magazine/2011-vol-9-no-2/424-wcc-spring-2011-v9n2-principles-wound-healing/file
(18) Medrado AP, Soares AP, Santos ET, Reis SRA, Andrade, ZA. Influence of laser photobiomodulation upon connective tissue remodeling during wound healing. J Photochem Photobiol B. 2008;92(3):144-52. https://doi.org/10.1016/j.jphotobiol.2008.05.008
(19) Silva, DCGG, Plapler H, Costa MM, Silva SRG, Sá MCA, Silva BSL. Low level laser therapy (AlGaInP) applied at 5J/cm² reduces the proliferation of Staphylococcus aureus MRSA in infected wounds and intact skin of rats. An Bras Dermatol. 2013;88(1):50-5. https://doi.org/10.1590/s0365-05962013000100005
(20) Damy SB, Camargo RS, Chammas R, Figueiredo LFP. The fundamentals of experiments with animals - applications in experimental surgery. Rev Assoc Med Bras. 2010;56(1):103-11. https://doi.org/10.1590/S0104-42302010000100024
(21) Sampaio LAS, Costa JS, Freire TFC, Reis SRA, Deiró TCBJ, Medrado ARAP. Influence of protein malnutrition on cutaneous wound healing in rats. Rev Nutr. 2018;31(5):433-42. https://doi.org/10.1590/1678-98652018000500001
(22) Tannenbaum J, Bennett BT. Russell and Burch's 3Rs then and now: the need for clarity in definition and purpose. J Am Assoc Lab Anim Sci. 2015; 54(2):120-32. Cited: PMID: 25836957.
(23) Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying the number of animals for each experimente. Nucl Med and Biol. 2007;34(3):229-32. https://doi.org/10.1016/j.nucmedbio.2007.01.005
(24) Scheibe PO. Number of samples - hypothesis testing. Nucl Med Biol. 2008;35(1):3-9. https://doi.org/10.1016/j.nucmedbio.2007.10.006
(25) Fortuna T, Gonzalez AC, Sá MF, Andrade ZA, Reis SRA, Medrado ARAP. Effect of 670 nm laser photobiomodulation on vascular density and fibroplasia in late stages of tissue repair. Int Wound J. 2018;15(2):274-82. https://doi.org/10.1111/iwj.12861
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