Vijay Nagineni

Therapeutic Metabolic Inhibition: Hydrogen Sulfide Significantly Mitigates Skeletal Muscle Ischemia Reperfusion Injury In Vitro and In Vivo

Background: Recent evidence suggests that hydrogen sulfide is capable of mitigating the degree of cellular damage associated with ischemia-reperfusion injury. The purpose of this study was to determine whether it is protective in skeletal muscle. Methods: This study used both in vitro (cultured myotubes subjected to sequential anoxia and normoxia) and in vivo (mouse hind-limb ischemia followed by reperfusion) models in which hydrogen sulfide (0 to 1000 &mgr;M) was delivered before the onset of oxygen deficiency. Injury score and apoptotic index were determined by analysis of specimens stained with hematoxylin and eosin and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, respectively. Results: In vitro, hydrogen sulfide reduced the apoptotic index by as much as 99 percent (p = 0.001), with optimal protection conferred by raising intravascular hydrogen sulfide to 10 &mgr;M. In vivo, 10 &mgr;M hydrogen sulfide delivered before 3 hours of hind-limb ischemia followed by 3 hours of reperfusion resulted in protection against ischemia-reperfusion injury–induced cellular changes, as evidenced by significant decreases in injury score and apoptotic index (by as much as 91 percent; p = 0.001). These findings were consistent at 4 weeks after injury and reperfusion. Conclusion: These findings confirm that the preischemic delivery of hydrogen sulfide limits ischemia-reperfusion injury–induced cellular damage in myotubes and skeletal muscle and suggests that, when given in the appropriate dose, this molecule may have significant therapeutic applications in multiple clinical scenarios.

Hydrogen sulfide attenuates ischemia-reperfusion injury in in vitro and in vivo models of intestine free tissue transfer.

Background: Ischemia-reperfusion injury is the propagation of injury following reintroduction of oxygen to previously ischemic tissue. The purpose of this study was to evaluate whether hydrogen sulfide provides protection against ischemia-reperfusion injury in enteric tissue. Methods: In vitro (enterocyte anoxia-normoxia) and in vivo (rat intestinal ischemia-reperfusion) models of ischemia-reperfusion injury were tested with or without the addition of hydrogen sulfide. Apoptotic index was determined in vitro, and gross appearance, histology, and villus height (a measure of mucosal integrity) were assessed in vivo. Statistical analysis was performed, and significance was defined as p < 0.05. Results: In vitro, cells treated with 10 &mgr;M hydrogen sulfide after 1-hour anoxia experienced a significant decrease in apoptotic index compared with untreated control (0.5 ± 0.3 percent versus 2.8 ± 0.7 percent); after 3 hours of anoxia, cells treated with 1 &mgr;M, 10 &mgr;M, and 100 &mgr;M hydrogen sulfide experienced significant decreases in apoptotic index versus untreated control (1.6 ± 0.8 percent, 1.8 ± 0.9 percent, and 2.8 ± 0.7 percent versus 8.6 ± 1.7 percent). In vivo, intestine treated with [10 &mgr;M] or [100 &mgr;M] hydrogen sulfide retained normal coloration and villus architecture after 1-hour ischemia; after 2 hours of ischemia, only intestine treated with [10 &mgr;M] hydrogen sulfide appeared uninjured. After 1, 2, or 3 hours of ischemia, villus heights of intestine treated with [10 &mgr;M] or [100 &mgr;M] hydrogen sulfide were significantly higher than heights of non–hydrogen sulfide-treated villi. Conclusions: Hydrogen sulfide significantly attenuates ischemia-reperfusion injury in intestinal tissue in vitro and in vivo. These results have significant implications for enteric free tissue transfers and other gastrointestinal procedures in which ischemic intervals may be anticipated.

Development of an acellular bioengineered matrix with a dominant vascular pedicle.

BACKGROUND This study assessed the feasibility of creating a tissue engineering platform by decellularization of fasciocutaneous tissue. MATERIALS AND METHODS A fasciocutaneous flap based upon the superficial inferior epigastric artery was harvested from the abdominal wall of 8-wk-old male Sprague-Dawley rats. All cellular components were removed by sequential treatment with sodium azide, DNAse, and sodium deoxycholate. The degree of decellularization was qualitatively assessed by histology and quantitatively assessed by spectrophotometry. Persistence of relevant extracellular matrix proteins was shown following staining with orcein and hematoxylin. The duration of circuit patency was determined by continuous perfusion with a peristaltic perfusion pump. RESULTS Gross and histologic examination demonstrated removal of cellular constituents with preservation of tissue matrix architecture, including macrochannels and microchannels. This was confirmed by the application of spectrophotometry to DNA isolates, which showed that the decellularized flap retained 4.04 ng/μL DNA, compared with the non-processed control, which retained 37.03 ng/μL DNA, and the acellular control, which was read as having 0.65 ng/μL DNA. The extracellular matrix of vessel walls was shown to remain intact. Peristaltic perfusion of the cannulated pedicle inflow channel with phosphate buffered saline at a rate of 200 μL/min confirmed circuit patency for 6 h. CONCLUSION Fasciocutaneous flaps harvested with an intact vascular pedicle and associated tissue vascular network can be successfully decellularized and perfused ex vivo. This methodology, which is scalable to human size tissues, provides promise as a technique for the production of customizable engineered tissues.

164A: OPTIMIZING NEOVASCULARIZATION OF TISSUE REGENERATION TEMPLATES BY RATIONAL DESIGN AND MICROFABRICATION

Objective: Tissue engineering has long sought to design constructs that rapidly achieve functionality upon implantation. Current techniques, however, cannot produce pre-fabricated tissues with an intact microvascular network that is connected to macrovascular inflow and outflow vessels. Therefore, we assessed the feasibility of decellularizing tissue with an intact vascular pedicle that contains a dominant artery and vein.