Characterizing Autophagy in the Cold Ischemic Injury of Small Bowel Grafts: Evidence from Rat Jejunum

Cold ischemic injury to the intestine during preservation remains an unresolved issue in transplantation medicine. Autophagy, a cytoplasmic protein degradation pathway, is essential for metabolic adaptation to starvation, hypoxia, and ischemia. It has been implicated in the cold ischemia (CI) of other transplantable organs. This study determines the changes in intestinal autophagy evoked by cold storage and explores the effects of autophagy on ischemic grafts. Cold preservation was simulated by placing the small intestines of Wistar rats in an IGL-1 (Institute George Lopez) solution at 4 °C for varying periods (3, 6, 9, and 12 h). The extent of graft preservation injury (mucosal and cellular injury) and changes in autophagy were measured after each CI time. Subsequently, we determined the differences in apoptosis and preservation injury after activating autophagy with rapamycin or inhibiting it with 3-methyladenine. The results revealed that ischemic injury and autophagy were induced by cold storage. Autophagy peaked at 3 h and subsequently declined. After 12 h of storage, autophagic expression was reduced significantly. Additionally, enhanced intestinal autophagy by rapamycin was associated with less tissue, cellular, and apoptotic damage during and after the 12-h long preservation. After reperfusion, grafts with enhanced autophagy still presented with less injury. Inhibiting autophagy exhibited the opposite trend. These findings demonstrate intestinal autophagy changes in cold preservation. Furthermore, enhanced autophagy was protective against cold ischemia–reperfusion damage of the small bowels.

Intestinal Preservation Injury: A Comparison Between Rat, Porcine and Human Intestines

Advanced preservation injury (PI) after intestinal transplantation has deleterious short- and long-term effects and constitutes a major research topic. Logistics and costs favor rodent studies, whereas clinical translation mandates studies in larger animals or using human material. Despite diverging reports, no direct comparison between the development of intestinal PI in rats, pigs, and humans is available. We compared the development of PI in rat, porcine, and human intestines. Intestinal procurement and cold storage (CS) using histidine–tryptophan–ketoglutarate solution was performed in rats, pigs, and humans. Tissue samples were obtained after 8, 14, and 24 h of CS), and PI was assessed morphologically and at the molecular level (cleaved caspase-3, zonula occludens, claudin-3 and 4, tricellulin, occludin, cytokeratin-8) using immunohistochemistry and Western blot. Intestinal PI developed slower in pigs compared to rats and humans. Tissue injury and apoptosis were significantly higher in rats. Tight junction proteins showed quantitative and qualitative changes differing between species. Significant interspecies differences exist between rats, pigs, and humans regarding intestinal PI progression at tissue and molecular levels. These differences should be taken into account both with regards to study design and the interpretation of findings when relating them to the clinical setting.