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An in vitro Method to Test the Safety and Efficacy of Low-Level Laser Therapy (LLLT) in the Healing of a Canine Skin Model

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Title: An in vitro Method to Test the Safety and Efficacy of Low-Level Laser Therapy (LLLT) in the Healing of a Canine Skin Model
Author: Gagnon, Dominique
Department: Department of Clinical Studies
Program: Veterinary Science
Advisor: Gibson, Thomas
Abstract: Low-level laser therapy (LLLT) aims to photoactivate cellular mechanisms that have the potential to improve healing of the treated regions, and reduce local edema, inflammation and pain. Low-level laser therapy has been used clinically as a treatment modality in humans for a variety of medical conditions, including the improvement of wound-healing processes. The results obtained appear to be closely related to parameters such as dose, time of exposure and wavelength. Although LLLT is gaining popularity, a universally accepted theory that explains the cellular effects and beneficial biological processes seen in wound healing has not been described. This research was designed to primarily evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK), in an in vitro wound healing model. A secondary objective was to perform a pilot study with this model in order to evaluate the effect of LLLT on the expression of microRNA-21 (miR-21), a microRNA associated with wound healing, using quantitative real time polymerase chain reaction (qRT-PCR). Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. The proliferation assay was performed using water-soluble tetrazolium (WST-1), a proliferation reagent. Canine keratinocytes were cultured in tissue culture flasks and then seeded in 6-well tissue culture plates at different cellular concentrations. Cells between the third- and fifth-passage were used for all experiments. Laser treatment of monolayer cultures was performed using a Helium-Neon Class IV laser system. Sham irradiation and different energy doses of 0.1, 0.2, 1.2 and 10 J/cm2 were evaluated. In this in vitro wound healing model, keratinocytes exposed to doses of 0.1, 0.2, and 1.2 J/cm2 migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < 0.0001) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm2. Irradiation with 10 J/cm2 was characterized by decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior and high doses may be detrimental to wound healing.
Date: 2015-07
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