Objective

The inevitable, anticipated period of ischemia and subsequent reperfusion during free tissue transfer (FTT) can potentially lead to ischemia-reperfusion injury (IRI).  The degree of IRI is frequently more severe, however, during episodes of unanticipated ischemia that occur as a result of acute post-operative vascular insufficiency.  Our recent in vitro and in vivo work has shown that pharmacologic pre-conditioning with hydrogen sulfide (HS) mitigates muscle IRI. It is less well-known, however, whether HS has a similar effect in those scenarios in which HS could only be delivered following the onset of ischemia.  Therefore, this study sought to determine whether HS would confer a similar protective effect against IRI when delivered after the onset of ischemia.

Methods

Nine C57/BL6 mice underwent 3h tourniquet-induced hindlimb ischemia and were randomized to 3 groups: no HS, 10µM intravenous HS delivered 1min prior to reperfusion, and 10µM HS delivered 20min prior to reperfusion. After 3h reperfusion, the gastrocnemius and soleus muscles were harvested bilaterally, sectioned, stained with H&E, and subjected to a TUNEL assay in order to determine the apoptotic index (AI), a measure of apoptosis.

Results

Upon histologic examination, ischemic tissue from non-HS-treated mice demonstrated architectural changes consistent with apoptosis. Tissue from mice treated with HS 1min prior to reperfusion appeared similar.  Ischemic tissue from mice treated with HS 20min prior to reperfusion showed no architectural disruption. Non-ischemic tissue treated with HS was not different in appearance from non-ischemic non-treated tissue.  Compared to the AI of non-HS-treated mice (18.1% ± 11.0%), there was a significant reduction in the AI of mice treated with HS 20min prior to reperfusion (1.8% ± 1.4%, p=0.027).  The AI of mice treated with HS 1min prior to reperfusion (16.0% ± 7.7%) was not significantly different than that of non-HS-treated mice (p=0.771)

Conclusions

After 3h ischemia, HS confers significant protection against IRI when administered 20min prior to reperfusion, but not when administered 1min prior to reperfusion, thereby suggesting that HS protects via temporally sensitive mechanisms. Importantly, the absence of injury in non-ischemic, HS-treated tissue suggests that HS is non-toxic at the tested dose.  We believe these results support the use of HS even after the onset of ischemia because of its ability to significantly ameliorate the consequences of IRI.