The effect of hyperbaric oxygen on nitric oxide synthase activity and expression in ischemia-reperfusion injury

Hyperbaric oxygen (HBO) mitigates ischemia-reperfusion (IR) injury via a nitric oxide mechanism that is nitric oxide synthase (NOS) dependent. The purpose of this study was to investigate this NOS-dependent mechanism by examining isoform-specific, tissue-specific, and time-specific upregulation of NOS mRNA, protein, and enzymatic activity.

We raised a gracilis flap in Wistar rats that were separated into early and late phases. Treatment groups included nonischemic control, IR, HBO-treated ischemia-reperfusion (IR-HBO), and nonischemic HBO control. We harvested tissue-specific samples from gracilis, rectus femoris, aorta, and pulmonary tissues and processed them by reverse transcription polymerase chain reaction and Western blot to determine upregulation of isoform-specific NOS mRNA and protein. We also harvested tissue for NOS activity to investigate upregulation of enzymatic activity. Data are presented as mean ± standard error of the mean with statistics performed by analysis of variance. P ≤ 0.05 was considered significant.

There was no increase in NOS mRNA in the early phase. In the late phase, there was a significant increase in endothelial-derived NOS (eNOS) mRNA in IR-HBO compared with IR in gracilis muscle (79.4 ± 22.3 versus 36.1 ± 4.5; P < 0.05) and pulmonary tissues (91.0 ± 31.2 versus 30.2 ± 3.1; P < 0.01). There was a significant increase in the late-phase eNOS pulmonary protein IR-HBO group compared with IR (235.5 ± 46.8 versus 125.2 ± 14.7; P < 0.05). Early-phase NOS activity was significantly increased in IR-HBO compared with IR in pulmonary tissue only (0.049 ± 0.009 versus 0.023 ± 0.003; P < 0.05).

The NOS-dependent effects of HBO on IR injury may result from a systemic effect involving an early increase in eNOS enzymatic activity followed by a late-phase increase in eNOS protein expression within the pulmonary tissues.