Hypothermic Oxygenated Machine Perfusion Prevents Arteriolonecrosis of the Peribiliary Plexus in Pig Livers Donated after Circulatory Death

BACKGROUND: Livers derived from donation after circulatory death (DCD) are increasingly accepted for transplantation. However, DCD livers suffer additional donor warm ischemia, leading to biliary injury and more biliary complications after transplantation. It is unknown whether oxygenated machine perfusion results in better preservation of biliary epithelium and the peribiliary vasculature. We compared oxygenated hypothermic machine perfusion (HMP) with static cold storage (SCS) in a porcine DCD model. METHODS: After 30 min of cardiac arrest, livers were perfused in situ with HTK solution (4°C) and preserved for 4 h by either SCS (n = 9) or oxygenated HMP (10°C; n = 9), using pressure-controlled arterial and portal venous perfusion. To simulate transplantation, livers were reperfused ex vivo at 37°C with oxygenated autologous blood. Bile duct injury and function were determined by biochemical and molecular markers, and a systematic histological scoring system. RESULTS: After reperfusion, arterial flow was higher in the HMP group, compared to SCS (251±28 vs 166±28 mL/min, respectively, after 1 hour of reperfusion; p = 0.003). Release of hepatocellular enzymes was significantly higher in the SCS group. Markers of biliary epithelial injury (biliary LDH, gamma-GT) and function (biliary pH and bicarbonate, and biliary transporter expression) were similar in the two groups. However, histology of bile ducts revealed significantly less arteriolonecrosis of the peribiliary vascular plexus in HMP preserved livers (>50% arteriolonecrosis was observed in 7 bile ducts of the SCS preserved livers versus only 1 bile duct of the HMP preserved livers; p = 0.024). CONCLUSIONS: Oxygenated HMP prevents arteriolonecrosis of the peribiliary vascular plexus of the bile ducts of DCD pig livers and results in higher arterial flow after reperfusion. Together this may contribute to better perfusion of the bile ducts, providing a potential advantage in the post-ischemic recovery of bile ducts

Criteria for Viability Assessment of Discarded Human Donor Livers during Ex Vivo Normothermic Machine Perfusion

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial

Changes in cholangiocyte bile salt transporter expression and bile duct injury after orthotopic liver transplantation

Background Biliary bile salts have been shown to contribute to bile duct injury after orthotopic liver transplantation (OLT). While cholangiocytes modify bile composition by reabsorption of bile salts (cholehepatic shunt) and contribute to bile flow by active secretion of sodium and water via cystic fibrosis transmembrane conductance regulator (CFTR), there is no formation on the expression of cholangiocyte transporters after OLT. We, therefore, examined the expression of cholangiocyte transporters in liver grafts and correlated this with the development of bile duct injury. Methods In 37 adult liver transplant recipients, biopsies were taken from the grafted liver: at the end of cold storage, 3hr after graft reperfusion and at one week after transplantation. Changes in the apical sodium-dependent bile salt transporter (ASBT), the organic solute transporters (OST-alpha and beta), and CFTR were assessed using real time RT-PCR and immunofluorescence staining. Gene expressions were correlated with biliary bile salt secretion as well as with the development of bile duct injury, as assessed by histology. Results Compared to normal controls, OST-alpha expression was significantly downregulated before transplantation and 3hr after graft reperfusion, but levels normalized at one week after OLT. OST-beta expression was more than 8-fold up-regulated at the time of transplantation and levels increased further during the first postoperative week. There were no major changes in the expression of CFTR. Expression of OST-alpha/ beta correlated with changes in biliary bile salt secretion. However, OST-alpha/beta and CFTR expression were not correlated with the development of microscopic bile duct injury after OLT. Conclusions Liver transplantation is associated with marked differential changes in the expression of the bile salt transporters OST-alpha and OST-beta, while CFTR expression remains stable. Although changes in OST-alpha/beta correlate with biliary bile salt secretion, OST-alpha/beta and CFTR do not correlate with the histological degree of bile duct injury, suggesting that the cholehepatic shunt does not play a major role in the development of bile salt induced bile duct injury after OLT.nrpages: 20status: publishe

Changes in cholangiocyte bile salt transporter expression and bile duct injury after orthotopic liver transplantation

Background: Bile salts have been shown to contribute to bile duct injury after orthotopic liver transplantation (OLT). Cholangiocytes modify bile composition by reabsorption of bile salts (cholehepatic shunt) and contribute to bile flow by active secretion of sodium and water via cystic fibrosis transmembrane conductance regulator (CFTR). We hypothesized that changes in these transporters may be associated with the development of bile duct injury after OLT. Aim: To examine the expression of cholangiocyte transporters in liver grafts and to correlate this with the development of bile duct injury. Methods: In 37 adult liver transplant recipients, liver biopsies were taken during and after OLT. Changes in the apical sodium-dependent bile salt transporter (ASBT), the organic solute transporters (OSTalpha and beta), and CFTR were assessed using real-time RT-PCR and immunofluorescence staining. Gene expressions were correlated with bile salt concentration as well as with the histological degree of bile duct injury. Results: Compared to normal controls, OSTalpha expression was significantly down-regulated at the end of cold storage and 3 hours after graft reperfusion, but levels normalized at one week after OLT. OSTbeta expression was more than 8-fold up-regulated at the time of transplantation and levels increased further during the first week. There were no major changes in the expression of CFTR. Expression of these transporters did not correlate with changes in bile salt concentration or with the development of bile duct injury after OLT. Conclusion: Liver transplantation is associated with changes in the expression of the bile salt transporters OST alpha and OST beta, while CFTR expression remains stable. Changes in OST alpha/beta do not correlate with bile salt concentration or the degree of bile duct injury, suggesting that the cholehepatic shunt does not play a major role in the development of bile salt induced bile duct injury after OLT

Regeneration of human extrahepatic biliary epithelium:the peribiliary glands as progenitor cell compartment

Background & Aims: Although regeneration of intrahepatic bile ducts has been extensively studied and intrahepatic progenitor cells have been identified, few studies have focussed on the extrahepatic bile duct (EHBD). We hypothesized that local progenitor cells are present within the EHBD of humans. Human EHBD specimens (n = 17) were included in this study. Methods: Specimens of normal EHBD tissue were obtained from healthy donor livers (n = 6), mildly injured EHBD from patients with cholangitis (n = 6) and severely injured EHBD from patients with ischaemic type biliary lesions (n = 5). Double immunostaining for K19 and the proliferation marker Ki-67 was performed to identify and localize proliferating cells. In addition, immunofluorescent doublestaining using antibodies against K19 and c-Kit was performed to identify and localize cholangiocytes co-expressing putative progenitor cell markers. Results: In normal EHBD, few Ki-67 + cells were detected, whereas large numbers of Ki-67 + were found in the diseased EHBD. In EHBD affected by cholangitis, Ki-67 + cells were mainly located in the basal layer of the lumen. EHBD specimens from patients with ischaemic type biliary lesions displayed histological signs of epithelial cell loss and large numbers of Ki-67 + cells were observed in the peribiliary glands. C-Kit expression was localized throughout the EHBD wall and immunofluorescent doublestaining identified a few K19 +/c-Kit + cells in the luminal epithelium of the EHBD as well as in the peribiliary glands. Conclusions: These findings support the hypothesis that progenitor cells exist in the EHBD and that the peribiliary glands can be considered a local progenitor cell niche in the human EHBD