How long after laser irradiation should cellular responses be measured to determine the laser effect?

Background: Low level laser therapy (LLLT) is a type of photomodulation, which uses photons to modulate biological activity. This photobiological effect depends on the wavelength, dose, intensity, as well as cell culture conditions. One problem with most of the published data on low level laser irradiation is that most studies do not record the duration between the exposure and the evaluation. It has been established that a single dose of 5 J/cm² stimulates cell proliferation and mitochondrial activity to normalize cell function and accelerate wound healing. This study investigates the cellular responses of irradiated human skin fibroblasts to establish if the duration after laser irradiation could influence the results. Methods: Normal and wounded human skin fibroblasts were exposed to helium-neon (632.8 nm) laser irradiation using a single dose of 5 J/cm² on one day or 5 J/cm² on two consecutive days. The cellular responses were measured 15 min, 1, or 3 h postirradiation. Responses evaluated included the effect on cell migration, changes in mitochondrial activity (cell viability), cell proliferation, and damage or stress caused by the irradiation. Results: The results indicate that a duration of between 1 and 3 h postirradiation is sufficient when measuring the direct effect of laser radiation on cells (cell viability, ATP mitochondrial activity, IL-6 activity, membrane integrity, and DNA damage), however, a longer duration of 24 h is required to demonstrate the indirect effect (cell proliferation and protein expression). Conclusion: This study indicates that the duration of effect should be included as one of the main parameters when reporting on the parameters used in LLLT.