Salvage of the Acutely Ischemic Flap: Pharmacologic Post-Conditioning with Hydrogen Sulfide Prevents Ischemia-Reperfusion Injury in Muscle When Delivered After the Onset of Ischemia
Natalia Jimenez, B.A., Peter Henderson, M.D., M.B.A, Sophie Horbach, B.S., John Ruffino, B.S., Jason Spector, M.D..
Weill Cornell Medical College, New York, NY, USA.
Ischemia-reperfusion injury (IRI) is an unavoidable consequence of revascularization following acute vascular occlusion. Recent in vitro and in vivo work from our group has shown that hydrogen sulfide (HS) mitigates IRI in muscle cells when delivered prior to the onset of ischemia (“pre-ischemic”). In the clinical setting, however, most ischemic events are unanticipated; in these cases, pre-ischemic delivery of HS is impossible. Therefore, the purpose of this study was to determine whether HS would confer a similar protective effect against IRI when delivered after the onset of an ischemic event (“post-ischemic”).
In vitro, myotubes were placed in a hypoxic chamber (1% O2) for 1, 3, or 5 hours. Twenty minutes prior to returning cells to normoxic (21% O2) conditions, the myotubes were treated with media containing either 0, 1μM, 10μM, or 100μM HS. At the completion of 3 hours of normoxia, the cells were subjected to a TUNEL assay in order to determine the apoptotic index (AI) for each time point and dose. In vivo, 9 C57/BL6 mice underwent 3 hours of unilateral tourniquet-induced hindlimb ischemia, and received intravenous saline or HS sufficient to raise the bloodstream concentration to 10μM, 20 minutes prior to reperfusion. After 3 hours of reperfusion, the gastrocnemius and soleus muscles were harvested bilaterally, stained with H&E, and subjected to a TUNEL assay to determine the AI.
In vitro, the dose response curve for myotubes exposed to 1 hour of hypoxia shows that HS is significantly protective at the 10μM (1.0%±0.3%, p=0.005) and 100μM (1.4%±0.5%, p=0.008) doses. Cells exposed to 3 hours of hypoxia were significantly protected by 10μM (1.7%±0.04, p=0.011). Cells exposed to 5 hours of hypoxia were significantly protected by the 1μM (1.5%±0.3, p<0.001), 10μM (1.7%±0.4%, p<0.001), and 100μM (3.0%±0.3%, p=0.001) doses of HS. In vivo, ischemic tissue from non-HS-treated mice demonstrated significant architectural disruption. Ischemic tissue from mice treated with HS 20 minutes prior to reperfusion showed minimal architectural disruption. Non-ischemic tissue treated with HS was not different in appearance from non-ischemic, non-HS-treated tissue. Compared to the AI of non-HS-treated mice (18.1% ± 11.0%), there was a significant reduction in AI in mice treated with HS 20 minutes prior to reperfusion (1.8% ± 1.4%, p=0.027).
The results of this study suggest that, similar to pre-ischemic delivery, post-ischemic delivery of HS provides significant protection in muscle against IRI. In vitro, the 10μM dose of HS provided the most consistent protection against IRI when administered 20 minutes prior to simulated reperfusion. Further strengthening this finding were the in vivo results, which similarly showed that post-ischemic HS delivery conferred protection against IRI. Importantly, the absence of injury in non-ischemic HS-treated tissue suggests that HS is non-toxic at the tested doses. These data highlight the benefits of the therapeutic delivery of HS in clinical situations where tissue has become acutely ischemic. This has obvious clinical implications in the setting of free tissue transfer as well as to the fields of transplant and vascular surgery.
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