Pharmacologic Pre- and Post-Conditioning with Hydrogen Sulfide Protects Against Ischemia-Reperfusion Injury in Diabetic Tissue
Sophie Horbach, BA1, Natalia Jimenez, BA1, Peter W. Henderson, MD MBA1, John Ruffino, BA1, David D. Krijgh, BA1, Bennett Wechsler2, Jason A. Spector, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2Cornell University, Ithaca, NY, USA.
BACKGROUND: Diabetic tissue tolerates periods of ischemia and subsequent reperfusion poorly. This is likely due to a multiple factors, including altered complement activation, microangiopathy-related vessel wall changes and an elevated baseline production of reactive oxygen species. Previous studies in our lab and others have shown that the endogenous signaling molecule hydrogen sulfide (HS) has a cytoprotective effect against ischemia-reperfusion injury (IRI) in muscle both when delivered prior to the onset of ischemia (“pre-ischemic” delivery), as well as when delivered after the onset of ischemia (“post-ischemic” delivery). Research on the effects of HS to date, however, has only been performed on euglycemic models. The purpose of this study was to investigate the effects of pharmacologic pre- and post-conditioning with HS on the effects of IRI in a murine model of diabetes.
METHODS: In this study, 9 diabetic mice and 9 non-diabetic mice underwent 3 hours of tourniquet-induced hindlimb ischemia, followed by 3 hours of reperfusion. Within each group, 3 received an intravenous injection of HS sufficient to raise the bloodstream concentration to [10uM] 20 minutes prior to the onset of ischemia, 3 received the same injection 20 minutes prior to the onset of reperfusion, and 3 did not receive any HS. At the end of the ischemia-reperfusion cycle, the mice were sacrificed and the gastrocnemius and soleus muscles were harvested bilaterally and processed for histology. Slides were stained with hematoxylin and eosin (H&E) in order to evaluate the cellular architecture, and also underwent a TUNEL assay in order to determine the apoptotic index (AI) for each experimental group. Values are reported as AI ± standard error of the mean, and statistical significance was set at p<0.05.
RESULTS: Histologic examination showed that the ischemic muscle of the non-HS-treated diabetic mice displayed major architectural changes, such as extracellular edema and intracellular vacuolization, compared to non-ischemic muscle. Conversely, ischemic muscle of the HS-treated diabetic mice showed minimal structural changes (the results were comparable in the pre-ischemic and post-ischemic groups). Qualitatively, the results were similar to those seen in the non-diabetic group. The AI of the non-HS-treated tissues was similar in the diabetic (10.1% ± 0.9%) and the non-diabetic (9.0% ± 0.6%) groups. In both groups, both pre-ischemic (non-diabetic: 3.4% ± 0.4%, diabetic: 3.9% ± 0.9%) and post-ischemic delivery of HS (non-diabetic: 2.7% ± 0.4%, diabetic: 4.1% ± 0.7%) of HS resulted in statistically significant reduction in AI (p<0.001 for all groups, relative to the non-HS-treated ischemic control).
CONCLUSIONS: The results of this study indicate that pharmacologic pre- and post conditioning with HS is equally efficacious in conferring protection to skeletal muscle against the deleterious effects of IRI in diabetic organisms as it is non-diabetic organisms. These important findings significantly broaden the potential clinical applicability of HS, given the high incidence of diabetes among patients undergoing free tissue transfer, solid organ transplantation, and lower extremity revascularization.
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