Preservation Of Normothermic Machine Perfusion Of Pig Small Intestine And Its Effect On Intestinal Mucosal Barrier

Objective: Mechanical perfusion(MP)can transport metabolic substrates and remove metabolic products from isolated organs,and reducing organ damage,which is one of the methods to improve the function of isolated organs.Normothermic machine perfusion(NMP)provides oxygen and nutrients to isolated organs at physiological temperatures while expelling metabolic waste.Therefore,NMP not only restores the normal metabolic activity of the tissue,but also reduces the ischemia-reperfusion injury of the tissue,which is an ideal way of organ preservation.NMP also beneficial for liver,kidney,lung and heart transplantation,and its preservation effect is significantly better than traditional SCS.However,MP is use in the transplantation of solid organs only.There are few studies on the transplantation of hollow organs in the small intestine.At present,this technique is still in the preclinical study in small bowel transplantation.At present,there is no research on the protective effect of pig small intestine on intestinal mechanical barrier.In this study,we compared the effects of conventional static cold storage and NMP preservation on the intestinal barrier,and evaluated whether NMP is superior to conventional static preservation in preserving the intestinal barrier,thus providing a better solution for alleviating ischemia-reperfusion injury in small bowel transplantation.Methods: In this study,the pig small intestine NMP model was established,and the small intestine jejunum segment are preserve by mechanical perfusion at room temperature and cold preservation.The pathological changes of small intestine mucosa at various time points are measure.The intestinal mucosal barrier was evaluated by HE staining,immunohistochemistry and immunofluorescence staining.Investigate the protective effect of NMP on intestinal mucosal barrier of porcine DCD.Results: 1.NMP perfusion device are establish successfully and inject the intestine into the experimental group to maintain stable temperature,pressure and flow.2.The intestinal mucosa of normal small intestine.The intestinal mucosa remains normal after warm ischemia and Chiu’s score is 0 ～1 point.Mild mucosal injury was found in 2h and 4h of UW group;NMP group also showed mild mucosal damage at 2h and 4h and 6h.In the 6h of UW group,the epithelial layer of the small intestine was separated from the lamina propria(2 ～4 points).It was found that the capillaries were obviously dilated and congested with a small amount of villus damage.3.There is a large amount of GCs on the villus in the normal small intestine mucosa;and there is still a large amount of GCs on the mucosa of the small intestine after warm ischemia.There were still more GCs on the intestinal mucosa in the UW group at 2h,4h and the NMP group at 2h,2h,4h and 6h,while the small intestine mucosal GCs were significantly reduced in the UW group at 6h.4.Bcl-2 and Bax expression were normal in normal intestinal mucosa,and immunohistochemical score(IHS)of Bcl-2 and Bax was weaker immune response(1～3 points).The IHS of Bcl-2 and Bax in the small intestinal mucosa after warm ischemia was a weakly immune response(2～4 points).The expression of Bcl-2 in the small intestine 0f 4h and 6h of NMP group was significantly higher than that of other groups(P<0.05),and IHS was 6-9 points.The expression of Bax in the small intestine of 4h and 6h of UW group was significantly higher than that of other groups(P<0.05),the IHS showed moderate-intensity immune response(8-9 points).5.In normal small intestine tissue,ZO-1 is widely expressed in the small intestine villi.After warm ischemia,the ZO-1 protein in the small intestine can still maintain a similar distribution with normal small intestine.ZO-1 in the NMP group remained at a state of continuous distribution at 2h and 4h.After 6h,ZO-1 appeared partially discontinuous at the small intestine villi.The ZO-1 of the UW group maintained a substantially continuous state for 2 hours,and some discontinuities appeared after 4 hours.After 6 hours,the ZO-1 of the villi disappeared extensively.Conclusion: This study show that NMP can significantly reduce intestinal mucosal tissue damage,reduce apoptosis,and effectively reduce ZO-1 down-regulation,has a better protective effect on the small intestinal mucosal barrier,compared with the use of UW liquid SCS has obvious advantages.