Experimental Assessment of Intestinal Damage in Controlled Donation After Circulatory Death for Visceral Transplantation

Abstract

There is an urgent need to address the shortage of potential multivisceral grafts in order to reduce the average time in waiting list. Since donation after circulatory death (DCD) has been successfully employed for other solid organs, a thorough evaluation of the use of intestinal grafts from DCD is warranted. Here, we have generated a model of Maastricht III DCD in rodents, focusing on the viability of intestinal and multivisceral grafts at five (DCD5) and twenty (DCD20) minutes of cardiac arrest compared to living and brain death donors. DCD groups exhibited time-dependent damage. DCD20 generated substantial intestinal mucosal injury and decreased number of Goblet cells whereas grafts from DCD5 closely resemble those of brain death and living donors groups in terms intestinal morphology, expression of tight junction proteins and number of Paneth and Globet cells. Upon transplantation, intestines from DCD5 showed increased ischemia/reperfusion damage compared to living donor grafts, however mucosal integrity was recovered 48 h after transplantation. No differences in terms of graft rejection, gene expression and absorptive function between DCD5 and living donor were observed at 7 post-transplant days. Collectively, our results highlight DCD as a possible strategy to increase multivisceral donation and transplantation procedures.

Keywords: donation after cardiac death; experimental transplantation; intestinal transplantation; organ procurement; solid organ transplant.

FIGURE 1

Scheme of the experimental design in the different donation models and ITx. The study encompassed several types of organ donors in rats, representing the different stages and component organs of intestinal and multivisceral grafts (A). Experimental design of allogeneic-heterotopic ITx model in rats. DCD5 and LD were considered to ITx. Transplanted intestines were sampled after transplantation to evaluate long-term outcome of the graft (B) LD, living donors; LVD, ventilated living donors; BDD, brain death donors; BDI, brain death induction; DCD, donation after circulatory death with two different times of CA (5 and 20 min); LLST, limitation of life support therapy; CA, cardiac arrest. This figure was generated with BioRender program (biorender.com).

FIGURE 2

Donor and graft monitoring during DCD. (A) MAP in living donor (LD), brain death (BD), and donation after circulatory death (DCD) groups exhibited normal values throughout the experimental procedure. No significant differences were observed between groups at any time-points. (B) Elapsed period (seconds) between limitation of life support therapy and CA diagnosis in the DCD groups. (C) After “no-touch period” cannulation time was measured from skin incision to the start of cold perfusion (black and white circles correspond to donation after 5 min of circulatory death [DCD5] and donation after 20 min of circulatory death [DCD20] groups, respectively). (D) Representative macroscopic image of an intestine from a DCD20 donor at the end of the experimental procedure. The pale appearance denoted correct washing of the graft.

FIGURE 3

Warm ischemia time impacts intestinal graft histology in donation after circulatory death (DCD). (A) Architecture in DCD5-group small bowel was similar to that of LD. Groups with the longest ischemia time presented with more severe damage based on Chiu/Park scores compared to the other donors, with significant differences vs. LD and DCD5 groups (p < 0.05). (B,C) Despite a worsening trend in the crypt villus index and intestinal mucosa thickness in the donation after 20 min of no-touch period (DCD20) group, no differences between groups in these morphometric studies were observed. Representative microphotographs from DCD5 (D) and DCD20 (E) group.

FIGURE 4

Cell population study and principal component analysis of the intestinal graft in the different donation scenarios. (A) Goblet cell count using Alcian Blue staining. Significant differences between DCD20 and LD groups were observed (*p < 0.05; **p < 0.01). (B) Examples of high and low numbers of goblet cells in intestinal samples corresponding to LD and DCD20 groups, respectively. (C) All experimental groups exhibited similar numbers of Paneth cells. (D) Biplot visualizing correlations among variables. The DCD5 group closely resembled BDD (grafts used in human settings). Samples from the DCD20 group were the most distinct compared to the other experimental groups and were associated with histological damage variables based on Chiu/Park scores. Claudin-3 staining in different scenarios of intestinal procurement (E).

FIGURE 5

Histological analysis of the remaining organs comprising multivisceral grafts in the different donation scenarios. Liver (A), stomach (B), and pancreas (C) analyses are presented. In all cases, significant differences were observed between the donation after 20 min of CA (DCD20) and LD groups (*p < 0.05; **p < 0.01). Representative microscopic images of low and high damage of the three organs are presented.

FIGURE 6

Intestinal Graft monitoring from DCD after ITx. Macroscopic appearance of transplanted intestines during intra-surgical graft reperfusion (DCD5 and LD) (A). Survival analysis of DCD and LD ITx recipients (B). Histological analysis of Ischemia-Reperfusion injury (C) and acute cellular rejection (D) of the DCD5 and LD graft after ITx did not show significant differences between groups. Intestinal graft absorption (E) and gene expression of CLDN3, TJP1, TNF and IL-6 after transplantation were similar in DCD5 and LD animals (F).