SLR - October 2015 - Sasha Valdizan

Hydrogen-Rich Saline Attenuates Skin Ischemia/Reperfusion Induced Apoptosis via Regulating Bax/Bcl-2 Ratio and Ask-1/Jnk Pathway

Reference: Liu YQ, Liu YF, Ma XM, Xiao YD, Wang YB, Zhang MZ, Cheng AX, Wang TT, Li JL, Zhao PX, Xie F, Zhang X. Hydrogen-rich Saline Attenuates Skin Ischemia/Reperfusion Induced Apoptosis Via Regulating Bax/Bcl-2 ratio and ASK-1/JNK Pathway. J Plast Reconstr Aesthet Surg. 2015 Jul;68(7):e147-56.

Scientific Literature Review

Reviewed By: Sasha Valdizan, DPM
Residency Program: Mount Sinai Hospital Manhattan

Podiatric Relevance: Skin flaps are frequently used in podiatric medicine for wound closure and ulcerations. Ischemia/reperfusion injury still occurs in spite of technical advancements resulting in partial or complete loss of the skin flap. Damage to the skin flap is due to reactive oxygen and nitrogen species that develop through the reperfusion process. The formation of the reactive species enhances chemotaxis resulting in the influx of neutrophils to the skin flap site and subsequent activation of the inflammatory process. The use of hydrogen-rich saline has been shown to increase anti-apoptosis factors bcl-2, lower the apoptosis factors pASK-1, pJNK and Bax as well as decrease inflammatory infiltration to the skin flap with an overall decrease in cellular apoptosis. This study hypothesized that hydrogen-rich saline therapy to a skin flap would decrease skin flap necrosis by decreasing ischemia/reperfusion injury through regulation of the expression of pASK-1, pJNK, Bax, Caspase-3 and anti-apoptotic proteins Bcl-2 in skin tissue.

Methods: Thirty-six adult male Sprague-Dawley rats were randomly divided into three groups: Group 1: sham surgery group where a 6cm x 9cm skin flap was marked along the abdomen, Group 2: Ischemia/reperfusion: six hours ischemia of skin flap with ischemia induced by clamping superficial epigastric artery followed by physiological saline reperfusion Group 3: ischemia/reperfusion: six-hour ischemia of skin flap by clamping superficial epigastric artery followed by hydrogen-rich saline therapy. Vascularization was avoided in all three groups by placement of a silicone sheet along the borders of the skin flap site. Blood perfusion of flap and evaluation of the flap microcirculation were measured by laser Doppler flowmeters and laser speckle contrast analysis. Total flap tissue survival was analyzed on post-op day three using Hematoxylin and eosin staining to observe histological changes of recovered tissue. Apoptosis assays were completed and results were observed through TUNEL staining and presented as the percentage of TUNEL-positive cells of the total number of nuclei per field. pASK-1, pJNK, Bcl-2 and Bax were examined through western blot analysis. In addition Bcl-2, Bax and caspase-3 were detected by qPCR. Caspase-3 was also measured using a Fluorometric Assay Kit.

Results: Results showed that Group 2 tissues had lower survival (23.65±9.36 percent) as compared to Group 3 (35.36±15.32 percent), which had received hydrogen-rich saline therapy. Flap perfusion for Group 3 was increased ((56.66±9.14) PU) as compared to Group 2 ((28.644±8.29) PU). Tissues from Group 3 were found with a higher expression of Bcl-2 and a low expression of pASK-1, pJNK and Bax indicating that Hydrogen-rich saline decreases the expression of factors associated with cellular apoptosis.
The results indicate that hydrogen-rich saline therapy could increase flap survival rate in humans by preventing ischemia-reperfusion injury. Hydrogen-rich saline therapy suppresses the expression of apoptosis factors in vivo by regulating the expression of pASK-1, pJNK, Bax and increasing anti-apoptotic factor Bcl-2 expression. Clinical studies should be designed to assess this possibility for future flap surgery to increase viability.

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