Photobiomodulation with IR1072 nm protects cultured neurons from H2O2- or A β -induced toxicity, independent of proliferation

NA Duggett, PL Chazot. University of Durham, Durham, County Durham, UK

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), is a natural by-product of oxidative phosphorylation and at low and high levels can act as signalling molecules for a variety of cellular processes. However, high levels of ROS are also known to be detrimental and Aβ oligomers have been reported to cause oxidative damage via production of H2O2. Many studies have reported the use of low-intensity light therapy (LILT) as a means to modulate a considerable number of biological processes, termed photobiomodulation.

Previously, we have reported beneficial effects following LILT at 1072 nm, including improved working memory in ageing mice (Michalikova et al., 2008), protection of lymphocytes against UVA insults (Bradford et al., 2005) protection of primary cortical neurons from glutamate-induced cytotoxicity (unpublished) and reduction of Aβ levels in an AD mouse in vivo model (submitted). The primary aims of this study was to investigate what effect H2O2 (50-250 μM) or synthetic Aβ fibrils (range 1-25 μM) had on cell viability using a Cath a. Differentiated (CAD) neuroblastoma cell line, and if IR1072 exposure altered the degree of H2O2- or Aβ-induced cell toxicity. Furthermore, the effect of IR1072 on proliferation was assessed using the Ki67 marker. For H2O2 insults, CAD cells were grown for 72 hours before IR preconditioning. Media was then changed to that containing H2O2 (150 μM) or PBS, cells were then subjected to five sets of 3 minute IR1072 exposures, thirty minutes apart. Four hours after this time, an MTT assay was performed. For Aβ insults, CAD cells were passaged and grown for 24 hours. CAD cells were subjected to five sets of 3 minute IR1072 exposures for two consecutive days. At this point media was changed to that containing Aβ (1-25 μM); cells were then maintained for a further 2 days, with IR exposures on both days. After this time, a lactate dehydrogenase (LDH) release assay (CytoTox 96®, Promega) was performed. Data were compared using a paired two tailed Student’s T-test, n=3-4 replicates for Aβ and n=48 for H2O2 experiments (3 different cultures) (GraphPad Prism 5).

H2O2 and Aβ elicited up to 40 and 70% neurotoxicity in a concentration-dependent manner, respectively. IR1072 exposure was found to completely prevent cell death caused by H2O2, at 150 μM (***p<0.001) (Sham + H202 82.8+/- 3.0%, IR + H202 104.0 +/- 4.1% of sham control viability (100%). or reduced cytotoxicity up to 24% caused by Aβ,at 25 μM (***p<0.001). Furthermore, IR1072 treatment did not elicit any significant change in proliferation (p>0.05), suggesting a neuroprotective mechanism. This study provides further new evidence for the potential of IR1072 as an effective non-invasive CNS therapeutic strategy. A pilot clinical study with Alzheimer’s patients recently conducted in the US is showing early promise.

Bradford, A. et al, (2005). J Photochem Photobiol B, 81, 9-14.

Michalikova, S. et al (2008). Neurobiol Learn Mem, 89, 480-8.

This study was funded by BBSRC (UK), Durham BSI and Virulite Ltd. (UK)