Therapeutic Delivery of Hydrogen Sulfide to Profoundly Ischemic Muscle: Timing Is Everything

Purpose: Research performed in our laboratory has shown that the gasotransmitter hydrogen sulfide (HS) has a powerful protective effect against the detrimental effects of ischemia-reperfusion injury (IRI) when delivered either before or after an ischemic event has occurred. The optimal timing of treatment, however, remains undefined. In order to better understand the potential clinical application of pharmacologic pre- and post-conditioning with HS, we sought to define the therapeutic window during which delivery of HS provides protection against IRI.

Methods: Twenty-four C57BL/6 mice were randomized to 8 groups: 1 non-ischemic control group and 7 groups that underwent 3 hours of tourniquet-induced hindlimb ischemia, followed by 3 hours of reperfusion. HS was administered by an intravenous NaHS injection sufficient to raise the circulating concentration of HS to [10µM] at different time points: 5 hr prior to reperfusion (-5hr), 20 min prior to ischemia (-3.3hr), at the time of ischemia (-3hr), 20 min prior to reperfusion (-0.3hr), at the start of reperfusion (0hr), and 1 hr after reperfusion (+1hr). After the reperfusion period, the gastrocnemius and soleus muscles were harvested bilaterally, and the specimens were subjected to a TUNEL assay in order to calculate an apoptotic index (AI) for each time point. Statistical significance was set at p<0.05.

Results: The AI for ischemic, non-HS-treated group was 17.2%. A significant reduction in AI was seen for HS administration at -3.3hr (2.6%, P=0.015) and at -0.3hr (1.8%, P=0.022). No protection was seen at -5hr, -3hr, 0hr, and +1hr.

Conclusion: The two time points for delivery of HS that resulted in significant cytoprotection were either 20min before ischemia or 20min prior to reperfusion. The results of this time-course study demonstrate a bi-modal distribution for the protective effect of HS in the setting of IRI, and suggest that the mechanism by which HS confers cytoprotection works through multiple pathways. Taken together, these data provide crucial insight into the “golden periods” within which HS treatment provides protection against IRI, either before or after the ischemic interval, and may serve to guide future clinical application of this novel therapy.