Mechanism Underlying Hyperbaric Oxygen's Effect on Nitric Oxide in an in Vitro Model of Traumatic Brain Injury

Abstract

Background: Hyperbaric oxygen (HBO) therapy functions as a possible therapeutic option for traumatic brain injury (TBI). The aim of this study is to detect the mechanism of HBO on TBI.

Methods: Neurons and astrocytes isolated from healthy neonatal rat cortices were co-cultured, a TBI model was established, and cells were cultured under HBO conditions. Neuron/astrocyte viability and glutamate transporter-1 (GLT-1) expression in neuron/astrocyte co-cultures were assessed by immunofluorescence. Tumor necrosis factor (TNF)-α/tumor necrosis factor receptor 1 (TNFR1)/nitric oxide (NO)/neuronal nitric oxide synthase (nNOS)/interleukin (IL)-1β/inducible nitric oxide synthase (iNOS)/GLT-1 levels in neuron/astrocyte co-cultures were detected using quantitative real-time polymerase chain reaction (qRT-PCR), colorimetry, and western blotting. To identify the key role of the target gene TNF receptor 1 (TNFR1) in HBO therapy, TNFR1 was silenced or overexpressed. After transfection, the cellular functions and the levels of related factors were re-examined.

Results: HBO (2 atmospheric absolute (ATA) for 30/60 min) attenuated the effect of TBI-induced on decrease of neuronal viability, increase of astrocyte viability, up-regulation of TNF-α, IL-1β, NO, nNOS, iNOS, and TNFR1 levels, down-regulation of GLT-1 levels, and reduce of GLT-1-positive astrocytes in neuron/astrocyte co-cultures (p < 0.05). TNFR1 knockdown and HBO (2 ATA for 60 min) enhanced neuronal viability, decreased astrocyte viability, and down-regulated TNF-α, IL-1β, NO, nNOS, iNOS, and TNFR1 levels in TBI-induced neuron/astrocyte co-cultures (p < 0.01). TNFR1 overexpression reversed the above role of HBO in TBI-induced neuron/astrocyte co-cultures. HBO (2 ATA for 60 min) up-regulated GLT-1 levels in TBI-induced neuron/astrocyte co-cultures (p < 0.05).

Conclusions: HBO inhibits TNFR1 expression to down-regulate NO content in TBI in an in vitro model.

Keywords: TNFR1; glutamate transporter-1; hyperbaric oxygen; nitric oxide; traumatic brain injury.