Xenogeneic Heterotopic Auxiliary Liver transplantation (XHALT) promotes native liver regeneration in a Post-Hepatectomy Liver failure model

Abstract

The liver's regenerative capacity is unique, but too small a segment can overwhelm its ability to simultaneously regenerate and support the host, resulting in liver dysfunction and death. Here we tested a temporary Xenogeneic Heterotopic Auxiliary Liver Transplant (XHALT) from Gal-KO miniature swine in a baboon model of Post-Hepatectomy Liver Failure (PHLF) by 90%- hepatectomy. Immunosuppression consisted of CVF, ATG, FK 506 and steroids. 90%-hepatectomized animals died within 4-5 days with the clinical picture of PHLF, (high LFTs and bilirubin, ascites, encephalopathy and coagulopathy). The 10% remnants had macroscopic and histological evidence of severe steatosis and absence of hepatocyte replication. In contrast, the addition of XHALT prolonged survival up to 11 days, with the cause of death being sepsis, rather than liver failure. The remnant liver appeared grossly normal, and on histology, there was no evidence of fatty infiltration, but there was pronounced Ki-67 staining. In conclusion, temporary auxiliary xenografts have the potential to support a small for size liver graft while it grows to adequate size or provide an opportunity for organ recovery in acute liver failure.

Fig 1. Auxiliary liver transplantation in 90% hepatectomized baboons.

(A) Table depicting the procedures performed to each animal, the medications administered and the survival length. (B) Blood and (C) Fresh Frozen Plasma requirements throughout the survival of the animals. (red bars indicate animals undergoing 90% Hepatectomy alone; black bars, 90%Hx plus the addition of an auxiliary pig liver xenotransplantation (XHALT). (D) Survival of the animals.

Fig 2. Liver function test.

(A) Animal’s liver function tests were evaluated in peripheral blood: ALT, AST, Bilirubin as well as platelets until the animal’s demise. (B) blood coagulations tests were also evaluated by means of INR, PTT, Fibrinogen, and PT. Black bars indicate 90% Hepatectomy alone group. White bars indicate 90% hepatectomy plus Xenogeneic Heterotopic Auxiliary Liver Transplantation (XHALT).

Fig 3. Native liver evaluation.

Macroscopic and histological appearance of the native liver from animal B347 undergoing 90%Hx+ XHALT at the time of exploratory laparotomy (A-C) and on autopsy (D-F). Both time points demonstrating normal healthy-looking livers and with a large number of Ki67+ cells.

Fig 4. Xenogeneic auxiliary liver evaluation.

(A) Serum IL-6 levels in both groups. (B,C) Macroscopic and histological appearance of the xenogeneic auxiliary liver from animals undergoing 90%Hx+ XHALT at the time of exploratory laparotomy and at graftectomy. Both animals at laparotomy demonstrated a large number of Ki67+ cells (B-C).

Fig 5. Macroscopic and microscopic analysis of remnant livers.

(A) macroscopic image of one representative animal from the 90%Hx alone group at the time of autopsy with yellow appearance indicating steatosis, which was confirmed histologically (B). (C) Ki67 staining depicting very few replicating hepatocytes positive. Representative macroscopic (D), and histological images of the livers of animals undergoing 90%Hx +XHALT (E,F). Note the normal appearance of the remnant liver, no presence of steatosis and good amount of Ki67+ cells. Bars = 100μm.

Fig 6. Rejection monitoring in xenogeneic auxiliary liver recipients.

(A) Histological appearance of xenogeneic auxiliary liver from animals undergoing 90%Hx+ XHALT at the time of exploratory laparotomy and at graftectomy indicating absence of rejection signs. (B) representative Flow Cytometry data from peripheral blood CD3 and CD20 in from animals undergoing 90%Hx+ XHALT at different time points.