Background: A potentially significant source of morbidity in free tissue transfer results from the obligatory period of ischemia required after the flap is removed from the source vessel. Even the most expeditious revascularization produces ischemia-reperfusion injury (IRI), manifested by flap edema and poor blood flow within the microvasculature, potentially leading to partial or even total flap failure. Though traditionally considered a toxic species, hydrogen sulfide (HS^-) has recently been recognized as an important endogenous signaling molecule, much like NO and CO, with cytoprotective properties through reversible inhibition of cellular metabolism.  As such, we hypothesized that HS^- could be utilized to decrease IRI in muscle, a tissue that is well-known to be the among the most IRI-sensitive tissue types.

Methods: For in vitro trials, murine myoblasts were pretreated with aqueous HS^- at varying concentrations (0µM(PBS), 0.1µM, 1.0µM, 10µM, 100µM, and 1mM) and then exposed to 5 hours of anoxia followed by 3 hours of reperfusion.  A TUNEL assay was used to determine extent of apoptosis.  In vivo trials included 2 trials that utilized 36 C57/b6 mice.  Eighteen were treated with the previously described concentrations of HS^- delivered via tail vein injection 20 minutes prior to ischemia. IRI was induced in the hind limb by tourniquet application above the greater trochanter for 3 hours followed by 3 hours of reperfusion.  An additional 18 mice underwent a similar experiment to that described above, but were allowed to recover for 4 weeks post-IRI.  In both in vivo trials gastrocnemius and soleus muscles were harvested bilaterally, and the contralateral leg served as a control.  Muscle sections were stained with H&E and TUNEL assay and examined for signs of disruption of muscle architecture (vacuole formation, edema, fiber separation) and apoptosis, respectively.  Statistical significance was determined by ANOVA with Tukey-Kramer post-hoc pair testing.

Results: HS^--treated anoxic myotubes showed a statistically significant decrease in apoptosis versus non-treated anoxic controls.  Overall, there was a dose-dependent decrease in the number of apoptotic cells with increasing concentrations of HS^- (Figure 1) with maximum protection observed at 10µM.  HS^- treated ischemic hindlimb muscle tissue showed preservation of normal architecture versus untreated ischemic limbs as evidenced by lack of vacuole formation, increased edema, and fiber separation.  These findings were noted to still be apparent at 4 weeks post-ischemia. Furthermore, IRI-induced apoptosis was significantly decreased in HS^- treated ischemic limbs, as measured by TUNEL-positive nuclei (Figure 2).  There was a dose-dependent decrease of apoptosis in HS^- treated ischemic limbs versus untreated ischemic controls, and maximal protective effect was observed at 10µM.  No increase in the degree of apoptosis was observed in non-ischemic myotubes or hindlimb muscle at any concentration of HS^- (data not shown).

Conclusions: Our results demonstrate that treatment with HS^- results in significant, dose-dependent mitigation of cellular injury without evidence of toxicity up to 10µM, in both in vitro and in vivo models of IRI. These data validate use of this molecule as a cytoprotectant with significant therapeutic potential in free tissue transfer and other conditions (e.g. solid organ transplantation) commonly associated with IRI.

Figure 1:

Figure 2: