THE HISTOLOGIC AND MOLECULAR ALTERATIONS DURING THE COLD STORAGE OF HUMAN RIGHT COLON

Abstract

Introduction: Colon transplantation is being increasingly performed together with the small intestine and other viscera due to increased water absorption and improved renal function. The small intestine is particularly sensitive to ischemia/preservation-reperfusion injury to ischemia yet the colon alterations incurred by cold storage (CS) are basically unknown. We set out to study the histological and molecular changes developing during the preservation of the human colon. Materials and methods: Following aortic perfusion with IGL-1 solution, the right colon was obtained from nine multiorgan deceased brain-dead donors. No evacuation of the luminal content was performed. Samples were taken after 8h, 14h and 24 hours of cold preservation and histology (Hematoxillin-eosin), goblet cells (Alcian blue staining) and apoptosis (active caspase-3 staining) were evaluated. Tight junction protein zonula-occludens (ZO)-1 and claudin-1 (cldn-1) were also studied using immunofluorescence and Western blot. Results and Discussion: During the first 8h of CS the colonic mucosa revealed minimal alterations, with the epithelium tightly adherent to the underlying lamina propria. The Goblet cells were abundant and fully repleted with mucins. After 14h the overall mucosal architecture was well maintained but a discrete detachment (edema) was noted extending under the epithelium in the luminal half of the crypts. Both the epithelial continuity and Goblet cells were well maintained. After 24h the epithelial layer detached further from the lamina propria. Enterocyte apoptosis (active caspase-3) was not observed. Immediately after retrieval the colon revealed strong, reticular staining around the apical pole (ZO-1) and basolateral membrane (cldn-1) from the lumen down to the crypt bottom. Eight hours of CS induced minimal molecular changes but after 14h we found maintained but weaker ZO-1 staining at the mucosal surface and broader cldn-1 staining pattern along the crypts. After 24h, ZO-1 was maintained as a continuous, although weaker line along the apex of the colonic cells (possibly degradation) whereas cldn-1 sizeable cytoplasmic staining suggesting internalization. Taken together, these findings suggest a certain ischemic resilience of the human colon and should partly relieve the concerns over the mucosal barrier injury when this intestinal segment is transplanted as part of a composite intestinal graft. However, the molecular alterations may imply that fluid and electrolyte transport may be impaired. Conclusions: The human colon tolerates very well up to 14h of CS, with minimal cellular and molecular changes. The small intestine remains the most sensitive part of a visceral graft.