Red/Near-infrared irradiation therapy (R/NIR IT) LASER pretreatment promotes neuroprotection of retinal neurons in a model of retinal metabolic dysfunction in vitro.

Abstract

Purpose : R/NIR IT using light-emitting diodes (LEDs) has been shown to promote protection, survival and healing of retinal cells. However, there remains scepticism regarding its efficacy due to a lack of clarity regarding the mechanism of action (MOA) and the use of varying treatment parameters. We believe that the use of LEDs is central to this scepticism due to the fact that they emit divergent light, which is unable to deliver a well-defined irradiance to a target. We have therefore formed a collaboration with Ellex Medical Lasers Ltd to develop a R/NIR IT LASER capable of delivering precise irradiances to its target in the hope of clarifying treatment parameters, MOA and safety of R/NIR IT, as well as its neuroprotective efficacy in a model of retinal metabolic dysfunction in vitro using sodium azide (NaN₃) - an inhibitor of complex IV (cytochrome C oxidase) of the mitochondrial electron transport chain.

Methods : One week old mixed rat retinal cultures derived from Sprague-Dawley rat pups were treated with R/NIR IT LASER (670nm for 90 seconds at irradiances of 25, 100 or 500 mW/cm²) or sham (no LASER). Cells were then either (a) left untreated to provide a control and also investigate the safety of the different LASER irradiances, or (b) treated immediately with NaN₃ at 500 μM. All cells were fixed at 16 hours with 10% neutral buffered formalin and immunocytochemistry (calretinin-labelling) performed to quantify retinal neuron survival in cell cultures. Mean cell counts were obtained using Image J, with safety data analysed using an equivalency test, and neuroprotection data with a one-way ANOVA and Dunnett's Post-hoc test.

Results : R/NIR IT LASER treatment alone did not decrease retinal neuron survival (Equivalency test, δ = 49.8, P<0.05, n=6 [25, 100mW/cm2], n=4 [500mW/cm2]). R/NIR IT LASER pretreatment at all irradiances provided neuroprotection of retinal neurons in a model of NaN₃-induced metabolic compromise (P<0.05, one-way ANOVA and Dunnett’s Post-hoc tests, n=3).

Conclusions : Our data suggest that R/NIR IT LASER alone does not affect retinal neuron survival in vitro. We have also shown that R/NIR IT LASER provides neuroprotection to retinal neurons in an in vitro model of metabolic injury using NaN₃, further supporting evidence in the literature that cytochrome C oxidase is the primary target of R/NIR IT.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.