Dose-dependent effects of hyperbaric oxygen on PC12 cell viability and neurite outgrowth with and without NGF stimulation

Hyperbaric oxygen therapy (HBOT) is a promising approach to promote peripheral nerve regeneration. However, the underlying mechanisms of action are poorly understood resulting in inconsistent treatment protocols and unclear indications. The present study evaluated the effects of single or repeated HBOT at different atmospheric pressures, with or without nerve growth factor (NGF) on PC12-cells. Cell proliferation, survival, and neurite outgrowth were analyzed. PC12-cells were exposed to a single session of HBOT or daily sessions over 4 consecutive days. Four groups were studied with or without NGF stimulation: control (21% oxygen at 1ATA (atmosphere absolute)) and 100% oxygen at 2.0ATA, 2.5ATA, or 3.0ATA. Brightfield imaging, live/dead assay, Alamar blue reagent, and tubulin-β-III immunostaining were performed. A single dose of HBOT enhanced proliferation of unstimulated PC12-cells under all experimental conditions compared to the control (p<0.05). In contrast, repeated HBOT increased dead cell count at 2.0ATA, 2.5ATA, and 3.0ATA compared to the control (p<0.01). Notably, repeated HBOT in NGF-stimulated PC12 cells resulted in maintained stable cell numbers and significant increase in axonal outgrowth (p<0.05). However, 3.0ATA impeded axonal outgrowth and induced cell death with or without NGF. Repeated HBOT decreased cell count and induced cell death in non-stimulated PC12-cells. NGF-stimulated cells showed stable cell numbers and no significant increase in cell death at 2.0 and 2.5ATA, whereas 2.5ATA significantly stimulated axonal outgrowth. Notably, 2.5ATA was the best pressure setting for neurite outgrowth stimulation in vitro. The data for nerve regeneration applications are promising but further research is needed to confirm the data in vivo and to investigate underlying mechanisms.