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Hydrogen Sulfide Mitigates Reperfusion Injury in a Porcine Model of Vascularized Composite Autotransplantation

Villamaria, Carole Y. MD*†; Fries, C. Anton MA, MRCS; Spencer, Jerry R. BS; Roth, Mark MD§; Davis, Michael R. MD, FACS

doi: 10.1097/SAP.0000000000000021
Transplantation Surgery and Research

Background: Devastating extremity injuries are prevalent but often survivable on the modern battlefield. These complex injuries require advanced methods of reconstruction, involving prolonged ischemic periods and reperfusion injury. Using our group’s validated porcine model of gracilis myocutaneous flap transplantation, this study demonstrates that an interim perfusion of hydrogen sulfide (H2S) mitigates the effects of reperfusion injury in the setting of delayed restoration of blood flow.

Methods: A gracilis myocutaneous flap (200–400 g; surface area, 250 cm2) was procured from the hind limb of a Yorkshire swine (70–90 kg, n = 16). The right external carotid artery and the internal jugular vein are the recipient axis. Group 1 (control, n = 6) underwent delayed anastomosis with a 3-hour ischemic period. Group 2 (n = 10) underwent a similar delayed anastomosis with an interim perfusion of H2S during the ischemic period. The animals survived for 14 days. Systemic biomarker assays for skeletal muscle tissue injury (creatine kinase, lactate dehydrogenase, and aspartate transaminase) and proinflammatory markers (tumor necrosis factor α and interleukin 6) provide assessment of reperfusion injury at the cellular level.

Results: The control animals (3 hours of ischemia with an interim perfusion of heparinized saline) demonstrated increased levels of injury biomarkers and proinflammatory cytokines compared with the animals receiving H2S infusion and identical ischemic interval. The control flaps had a mean creatine kinase level of 280 × 103 U/L (±80 × 103), compared with the H2S group, which had a mean of 99 × 103 U/L (±14 × 103; P = 0.0007 at postoperative day 2). lactate dehydrogenase levels (mean) were 26 × 103 U/L (±8 × 103) versus 9 × 103 U/L (±3 × 103; P = 0.0004) and aspartate transaminase levels (mean) were 1651 U/L (±324) versus (873 U/L [±279]; P = 0.0013) for the control and treatment groups, respectively. Similarly, an intergroup difference in IL-6 was found, although not statistically significant. Tumor necrosis factor α levels (mean) were 93 pg/mL (±14) versus 39 pg/mL (±4; P = 0.0013) for the control and treatment groups, respectively.

Conclusions: This study demonstrated the mitigating properties of H2S on reperfusion injury. Interim perfusion with H2S resulted in diminution of ischemia-dependent biomarkers after 3 hours of ischemia. Follow-up studies will translate these findings as an evolving method for reconstructing previously unreconstructable injuries.

From the *Department of Surgery, University of Texas Health Science Center at San Antonio, San Antonio, TX; †59MDW/Science and Technology, Joint Base San Antonio, San Antonio, TX; ‡United States Army Institute of Surgical Research, Fort Sam Houston, TX; and §Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA.

Received April 12, 2013, and accepted for publication, after revision, September 24, 2013.

Conflicts of interest and sources of funding: none declared.

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

Reprints: C. Anton Fries, MA, MRCS, United States Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston, TX 78234. E-mail:

© 2014 by Lippincott Williams & Wilkins