Background:
A period of ischemia followed by reperfusion is an inevitable aspect of free tissue transfer (FTT). This cycle results in oxidative and inflammatory processes that lead to ischemia-reperfusion injury (IRI), which is a source of significant morbidity and mortality. In the cardiac, central nervous, and hepatobiliary systems, low doses of hydrogen sulfide (H₂S) have been shown to decrease cellular metabolism in a reversible, non-toxic manner, thereby protecting against IRI. This study aimed to determine whether H2S ameliorates cellular injury caused by IRI in an in vitro model of a commonly performed form of FTT, the muscle free flap.
Methods:
Human umbilical vein endothelial cells (HUVECs) and myotubes were chosen as the cell lines that together constitute an in vitro model of muscle flaps. The cell lines were plated in separate chamber slides and treated with media containing aqueous NaHS (0, 1μM, 10μM, 100μM, or 1mM). Chamber slides were then exposed to anoxia (0%) for 5 hours, followed by normoxia for 3 hours. Control slides were maintained in normoxia throughout. A TUNEL assay was performed on all cells in order to determine the proportion of cells undergoing apoptosis, and a series of Fisher's exact tests with pooled variances and controlled family error rate of α = 0.05. Results were expressed as mean percent apoptosis, and the level for statistical significance was set at p < 0.05.
Results:
Five hours of ischemia and 3 hours of reperfusion in the absence of exogenous H2S resulted in 32% and 38% apoptosis in HUVECs and myotubes, respectively (Figure). Treatment with increasing doses of H₂S resulted in a dose-dependent decrease in apoptosis in both HUVECs and myotubes, with maximal protection at 10uM (3.8% and 0.3% apoptosis, respectively); at this dose there was no statistical difference between cells exposed to anoxia and those that remained in normoxia throughout. At higher doses there is a loss of protective effect. No evidence of toxicity was observed in normoxic cells treated with any dose of H₂S.
Conclusion:
This is the first study to investigate the potential cytoprotective effect of H₂S on the most important cellular constituents of muscle flaps. Our results demonstrate that H₂S significantly decreases apoptosis in vitro in the setting of IRI. These data suggest H₂S has potential as a therapy for improving tissue survivability in clinical scenarios.