Adenosine A2a Receptor Stimulation Attenuates Ischemia-Reperfusion Injury and Improves Survival in A Porcine Model of DCD Liver Transplantation

Abstract

Orthotopic liver transplantation (OLT) using allografts from donation after circulatory death (DCD) is potentially associated with compromised clinical outcomes due to ischemia-reperfusion injury (IRI)-induced organ damage and graft-related complications. The aim of this study was to provide in vivo data on the effects of adenosine A_2a receptor stimulation in a clinically relevant large animal model of DCD liver transplantation. Cardiac arrest was induced in German Landrace pigs (n = 10; 20-25 kg). After 30 min of warm ischemia, the donor liver was retrieved following a cold flush with 3 L of histidine-tryptophan-ketoglutarate-HTK solution. Animals of the treatment group (n = 5/group) received a standard dose of the selective adenosine receptor agonist CGS 21680 added to the cold flush. All grafts were stored for 4.5 h at 4 °C in HTK-solution before OLT. Hepatocellular injury, apoptosis, protein kinase A-PKA activity, graft microcirculation, liver function, and animal survival were assessed. Compared to untreated livers, adenosine A_2a receptor stimulation resulted in improved tissue microcirculation (103% ± 5% vs. 38% ± 4% compared to baseline; p < 0.05), accelerated functional recovery of the graft (indocyanine green-plasma disappearance rate (ICG-PDR) of 75% ± 18% vs. 40% ± 30% after 3 h), increased PKA activity ratio (56% ± 3% vs. 32% ± 3%; p < 0.001 after 1 h), and consequently reduced tissue necrosis and apoptosis. The potent protective effects were clinically manifested in significantly improved survival in the treatment group after 72 h (100% vs. 40%; p = 0.04). The ex vivo administration of adenosine A_2a receptor agonist during the back-table flush mitigates IRI-mediated tissue damage and improves functional graft recovery and survival in a large animal model of DCD liver transplantation.

Keywords: adenosine A2a agonist; donation after circulatory death; hepatoprotection; liver transplantation; microcirculation; porcine model; reperfusion injury; survival.

Figure 1

Hepatic microcirculation. (A) Graft microcirculation was evaluated one hour after reperfusion at three standard locations on the surface of the liver using a laser Doppler monitor and corresponding surface probe. The mean value of the measurement points was used to characterize graft microcirculation. (B) Due to the large individual variations between animals, the data were expressed as a percentage of the baseline value (mean ± standard error of the mean (s.e.m.), * p < 0.05 donation after circulatory death (DCD) + A2 vs. DCD, Student’s t-test, n = 5/group).

Figure 2

Graft functional recovery. (A) Bile production is presented as total amount of bile collected over the 1st hour of reperfusion in the DCD and DCD + A2 groups. Prothrombin time (PT)/Quick values at post-operative day 3 (POD3) following orthotopic liver transplantation (OLT) in the DCD and DCD + A2 groups. (B) Indocyanine green (ICG) plasma disappearance rate was measured in the DCD and DCD + A2 groups at 1 h and 3 h after reperfusion and presented as a percentage of ICG eliminated compared to baseline. (mean ± s.e.m., * p < 0.05 DCD + A2 vs. DCD, Student’s t-test, n = 5/group).

Figure 3

Hepatocellular damage and histopathology. (A) Time course of serum aspartate-aminotransferase (AST) levels expressed in IU/L in the DCD and DCD + A2 groups (mean ± s.e.m., * p < 0.05 DCD + A2 vs. DCD, Student’s t-test, n = 5/group) (B) Time course of serum glutamate dehydrogenase (GLDH) levels expressed in IU/L in the DCD and DCD + A2 groups (mean ± s.e.m., n = 5/group (C1–3) Representative microscopic images of the histological specimens at sacrifice in the DCD group (stained with hematoxylin-eosin; original magnification 100×, 200×, 400×, respectively). (D1–3) Representative microscopic images of the histological specimens at sacrifice in the DCD + A2 group (stained with hematoxylin-eosin; original magnification 100×, 200×, 400×, respectively).

Figure 4

PKA activation and corresponding DNA fragmentation and apoptosis. (A) Serum activity of protein kinase A (PKA) after 1 hour of reperfusion shown in activity ratio in the DCD and DCD + A2 groups. (mean ± s.e.m., *** p < 0.001 DCD + A2 vs. DCD, Student’s t-test, n = 5/group). (B) Serum levels of histone-associated DNA fragments after 1 hour of reperfusion are shown in arbitrary units (AU) of absorption in the DCD and DCD + A2 groups. (mean ± s.e.m., ** p < 0.01 DCD + A2 vs. DCD, Student’s t-test, n = 5/group).

Figure 5

Animal survival. Kaplan–Meier analysis and log-rank test for 3-day animal survival (p = 0.04 log-rank test, n = 5/group).

Figure 6

Study flowchart and surgical protocols. Animals were randomized into two experimental groups (DCD and DCD + A2). Following a 7-day acclimatization period in our housing facility, a liver graft from the donor animal was retrieved and implanted into the recipient animal following 30 min of circulatory death/in situ warm ischemia and 4.5 hours of cold preservation. Liver grafts in the DCD + A2 group were flushed with a standard dose of CGS 21680 adenosine A2a receptor agonist added to the HTK solution. Animals were monitored closely and sacrificed after 3 days of reperfusion for sample collection and further analysis (n = 5/group). Abbreviations used: DCD—donation after circulatory death; HTK—histidine-tryptophan-ketoglutarate solution; POD—postoperative day; CS—cold storage; WIT—warm ischemia time.