Warm HTK donor pretreatment reduces liver injury during static cold storage in experimental rat liver transplantation.

BACKGROUND: Organ shortage has led to an increased number of transplantations from extended criteria donors. These organs are more vulnerable to ischemia-reperfusion injury. Thus, improvement of organ preservation is needed. HTK is a widely used preservation solution for static cold storage in liver transplantation. The present study was to investigate the beneficial effect of warm HTK donor pretreatment on liver preservation. METHODS: Male inbred Wistar rats (weighing 230-260 g) served as donors and recipients (n=6/group). Donors of treatment groups received i.v. 0.01 mL/g body weight (BW) warm (21 degree centigrade) HTK systemically 15 minutes prior to cold perfusion. Control groups received 0.01 mL/g BW warm (21 degree centigrade) NaCl 0.9%. Following pretreatment, donors were flushed with 4 degree centigrade cold HTK, livers were explanted and stored in 4 degree centigrade HTK for six hours. Thereafter orthotopic liver transplantation was performed. Recipients were harvested four hours, two and five days after reperfusion and blood and liver tissue samples were obtained. Blood samples were analyzed for AST, ALT, lactate dehydrogenase and bilirubin. Liver histological analysis as well as tissue analysis for pro-MMP2, MMP2 and pro-MMP9 using zymography was conducted. RESULTS: Treatment groups showed significantly lower ALT and lactate dehydrogenase levels as well as significantly lower activities of pro-MMP2, MMP2 and pro-MMP9. Histological analysis revealed only minor damage in all groups. CONCLUSIONS: The new concept of warm HTK pretreatment significantly reduced ischemia-reperfusion injury. The reduced ischemia-reperfusion injury was due to MMP inhibition. Warm HTK donor pretreatment is easy to handle and could further improve HTK's potency in liver preservation.

The influence of storage temperature during machine perfusion on preservation quality of marginal donor livers.

BACKGROUND: Although non-heart-beating donors have the potential to increase the number of available organs, the livers are used very seldom because of the risk of primary non-function. There is evidence that machine perfusion is able to improve the preservation of marginal organs, and therefore we evaluated in our study the influence of the perfusate temperature during oxygenated machine perfusion on the graft quality. METHODS: Livers from male Wistar rats were harvested after 60-min warm ischemia induced by cardiac arrest. The portal vein was cannulated and the liver flushed with Lifor (Lifeblood Medical, Inc.) organ preservation solution for oxygenated machine perfusion (MP) at 4, 12 or 21 degrees C. Other livers were flushed with HTK and stored at 4 degrees C by conventional cold storage (4 degrees C-CS). Furthermore two groups with either warm ischemic damage only or without any ischemic damage serve as control groups. After 6h of either machine perfusion or cold storage all livers were normothermic reperfused with Krebs-Henseleit buffer, and functional as well as structural data were analyzed. RESULTS: Contrary to livers stored by static cold storage, machine perfused livers showed independently of the perfusate temperature a significantly decreased enzyme release of hepatic transaminases (ALT) during isolated reperfusion. Increasing the machine perfusion temperature to 21 degrees C resulted in a marked reduction of portal venous resistance and an increased bile production. CONCLUSIONS: Oxygenated machine perfusion improves viability of livers after prolonged warm ischemic damage. Elevated perfusion temperature of 21 degrees C reconstitutes the hepatic functional capacity better than perfusion at 4 or 12 degrees C.

Improvement of microsurgical techniques in orthotopic rat liver transplantation.

BACKGROUND: Because of the limited tolerance to portal venous clamping, the model of liver transplantation in rats represents a difficult task, which requires a great proportion of experience. Since techniques that include the introduction of an artificial stent increase the risk of thrombosis, it was our goal to modify the classical vascular hand-sewn venous anastomosis technique by using a modified end-to-end knotless procedure. MATERIALS AND METHODS: Seventy-two animals were randomly assigned to 1 of 2 experimental groups, which differed by the technique for the 3 venous anastomoses (supra- and infrahepatic vena cava, portal vein). Group 1 comprised the established suturing technique for rat liver transplantation, whereas all venous anastomosis of the second group were performed using our modified technique. RESULTS: With our method, average anhepatic time could be significantly reduced from 14 min 10 s (+/-100 s) to 11 min 40 s (+/-60 s) (P < 0.001). Kaplan-Meier survival rates demonstrated a better 7-d survival for the knotless (94%) compared to the classic technique (83%) (not significant, P = 0.137). Biliary complications were low in both groups but tended to be higher in the classical group. CONCLUSIONS: Our modified knotless anastomosis proves to be equally safe in regard to complications, improves timing, and provides excellent results in the model of orthotopic rat liver transplantation.

Endoscopic management of biliary obstruction after successful reuse of a liver graft.

The widening gap between donated organs and potential recipients waiting for liver transplantation leads towards intensive efforts to increase the availability of liver grafts. This aim can be accomplished by using organs with extended criteria for acceptance like advanced age, steatosis or donation after cardiac death with prolonged warm ischemic time. Far less frequently, but also worthwhile considering as a valuable source for donor organs is the possibility to reuse organs in case of brain death after orthotopic liver transplantation (OLT). In this case the first organ recipient has to be evaluated to become a potential organ donor for a second recipient.
In our study we report on a case in which we reused a liver graft 24 hours after the first transplantation and present the successful endoscopic management of biliary obstruction.

Iloprost donor treatment reduces ischemia-reperfusion injury in an isolated extracorporeal pig liver perfusion model.

OBJECTIVES: Iloprost has the potential to protect the liver transplant graft before and during cold ischemia. We studied iloprost administration during organ procurement and reperfusion in an extracorporeal pig liver perfusion model. MATERIALS AND METHODS: German Landrace pigs (n = 7/group; 22-26 kg each) were used as donors. Preservation was performed by aortic perfusion with 2 L Bretschneiders' Histidine-Tryptophan-Ketoglutarate solution HTK and cold ischemia time (4°C) 20 hours followed by normothermic extracorporeal perfusion for 8 hours. Untreated controls (1) were compared to iloprost (2) donor bolus-treatment (1 µg/kg body weight), (3) addition of iloprost to Bretschneiders' Histidine-Tryptophan-Ketoglutarate solution HTK (0.0125 µg/mL), (4) continuous infusion during reperfusion (2 ng/kg/min), and (5) combined treatment (2) and (4). RESULTS: Iloprost donor treatment led to significantly higher bile production. Addition of iloprost to the preservation solution significantly improved hepatic artery perfusion and was accompanied by improvements of microcirculation and bile production. Iloprost reperfusion treatment alone significantly improved bile production. Enzyme levels were positively affected by all treatment regimens. Combined use of iloprost before and after ischemia improved hepatic artery flow and microcirculation and showed significantly lower hypoxia staining versus controls. CONCLUSIONS: Iloprost donor treatment and use of iloprost in the preservation solution significantly improved graft perfusion and function. The effects of graft treatment seemed greater before than after reperfusion. Combined treatment did not reveal a synergistic advantage.

Improved microcirculation by low-viscosity histidine- tryptophan-ketoglutarate graft flush and subsequent cold storage in University of Wisconsin solution: results of an orthotopic rat liver transplantation model.

As previously shown in a model of isolated rat liver perfusion, the combined use of an initial graft flush with low-viscosity histidine-tryptophan-ketoglutarate (HTK) solution followed by cold storage in University of Wisconsin (UW) solution markedly improved the preservation during an extended cold storage period. In this study, we aimed to transfer our results into an in vivo model of orthotopic rat liver transplantation, and to elucidate the potential mechanism of the improved preservation by focusing on the hepatic microcirculation. Livers were harvested from male Wistar rats. Aortic perfusion with a pressure of 100 cm H(2)O was performed with either UW (group UW) or HTK (groups UW and HTK_UW), followed by additional back-table perfusion with UW (group HTK_UW). After 20-h cold storage at 4 degrees C, livers were orthotopically transplanted with reconstructing the hepatic artery. As measured by bile flow and liver enzymes, HTK flush followed by UW storage was superior compared to single use of either UW or HTK solution. The hepatic microcirculation was significantly improved, as shown by the increased percentage of reperfused sinusoids and reduced sinusoidal leucostasis. HTK and UW effectively reduce ischaemia-reperfusion injury after liver transplantation. By combining the comparative advantages of both solutions, a cumulative effect resulting in an improved preservation was shown. Thus, this mechanism improves microcirculatory reperfusion.

24-h storage of pig livers in UW, HTK, hydroxyethyl starch, and saline solution: is microdialysis an appropriate method for the continuous graft monitoring during preservation?

Recent studies demonstrate the feasibility of microdialysis to monitor metabolism in ischemic livers. Whether these parameters correlate with markers of liver cell integrity in an experimental model using pig livers and different preservation solutions was an aim of this study. Pig livers were flushed with either 4 degrees C Histidine-Typtophan-Ketoglutarate solution (HTK) (Custodiol), University of Wisconsin solution (ViaSpan), and hydroxyethyl starch, or 12 degrees C saline solution. After 24-h storage, the livers were rinsed with saline to measure liver enzymes and lactate from the effluate. Utilizing microdialysis, intraparenchymal lactate, pyruvate, glucose, and glycerol was monitored. Tissue biopsies were taken for histological examinations. Cold preservation resulted in a decrease of metabolic activity measured by intrahepatic glucose, lactate, and pyruvate levels, as well as lactate in the effluate, independently of the solution used. Of particular interest, glycerol levels partially reflected the extent of hepatocellular damage and liver enzyme release. Glycerol levels partially discriminated preservation of different quality and were in accordance to histological findings and liver enzyme release. Lactate, pyruvate, and glucose levels were not appropriate as markers during cold storage. Whether or not glycerol monitoring could represent an additional and rational complementation to the current practice of macroscopic, microscopic and donor evaluation has to be clarified by further studies.

Low viscosity histidine-tryptophan-ketoglutarate graft flush improves subsequent extended cold storage in University of Wisconsin solution in an extracorporeal rat liver perfusion and rat liver transplantation model.

Adequate flushing for liver donation requires large fluid volumes delivered at a high flow. This can be achieved more effectively with crystalloid solutions than with colloid-based solutions. This study examined the combination of initial histidine-tryptophan-ketoglutarate solution (HTK) graft flush and subsequent storage in University of Wisconsin solution (UW) to that of the single use of each solution. Livers from inbred Wistar rats were procured using aortic perfusion with UW or HTK for initial perfusion and reflushed after 30 minutes using either solution. In a third group, after perfusion with HTK, organs were reflushed with UW. A 60-minute in-vitro recirculating perfusion was performed after 24 hours of cold storage in the subsequent solution, as well as allotransplantation after 18 and 24 hours of cold storage. In extracorporeal perfusion, the HTK flush followed by UW storage was superior compared to the single use of either UW or HTK solution, as measured by portal venous pressure, bile flow, liver enzymes released into the effluent perfusate, glycerol leakage, and histological examinations. These data were consistent with the transplantation study. Histological damage and enzyme release after 5-day survival were lowest in the HTK flush and subsequent UW storage groups following 18 hours of cold storage; likewise, the 5-day survival was superior following 24 hours of cold storage. In conclusion, the combined use of HTK solution for initial graft rinse and subsequent storage in UW solution resulted in a cumulative protection. Choosing low-viscosity HTK solution for the initial organ flush may represent a feasible improvement in liver preservation, which also further reduces the required amount of UW solution.

Oxygenated machine perfusion mitigates surface antigen expression and improves preservation of predamaged donor livers.

The aim of the present study was to evaluate the potential benefit of machine preservation with the Belzer MPS or HTK solution, compared to standard cold storage, after procurement of marginal livers from non-heart beating donors in an experimental pilot study. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein and cold stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to oxygenated machine perfusion with HTK or Belzer's MPS at 5 ml/min at 4 degrees C. Cold perfusion of livers with the non-colloidal HTK was not compromised by the lack of oncotic agents and there was no rise in vascular resistance during the 24 h of machine preservation with HTK or the colloidal Belzer MPS. Viability of the livers was assessed after the cold preservation period by warm reperfusion in vitro. Oxygenated machine perfusion was found to significantly increase viability of the livers vs simple cold storage with respect to portal vascular resistance upon reperfusion, enzyme release as well as functional recovery of oxygen utilization or bile production. Moreover, tissue antigen expression of ICAM-1 or histocompatibility antigen class II could be markedly reduced by oxygenated perfusion preservation as compared to cold storage. It is concluded that predamaged organs should preferably be preserved by oxygenated machine perfusion thus minimizing functional alterations and immunogenicity of the graft. In this setup HTK appeared equally effective as Belzer's MPS for machine preservation.

Liver preservation with HTK: salutary effect of hypothermic aerobiosis by either gaseous oxygen or machine perfusion.

The aim of the present study was to improve the viability of marginal livers from non-heart beating donors upon cold preservation using two different techniques for the provision of tissue aerobiosis. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein with 20 mL of heparinized Ringer's solution and 60 mL of histidine-tryptophan-ketoglutarate (HTK) preservation solution. Control livers were then stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to aerobic conditions by either insufflation of gaseous oxygen via the venous vascular system of the cold stored organ (VSOP) or pulsatile machine perfusion (MP) with oxygenated HTK at 5 mL/min at 4 degrees C. Superoxide dismutase (SOD) (7500 IU) was added to the last 10 mL of HTK in order to prevent adverse effects of high oxygen tensions at hypothermia. Viability of the livers was assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer at constant flow. VSOP or MP, both significantly improved vascular conductivity upon reperfusion as evaluated by portal venous pressure, reduced hepatic enzyme release and led to a rise in hepatic bile production upon reperfusion. Induction of apoptosis was also looked for in tissue homogenates by Western analysis for cleavage of poly(ADP-ribose)polymerase (PARP). Expression of cleaved PARP fragment could be found in reperfused control livers but also, though to a lesser extend, after VSOP or MP. In conclusion, provision of oxygen during cold preservation significantly contributes to improve organ viability upon reperfusion and must be regarded as a useful adjunct for marginal or pre-damaged livers. HTK has been shown for the first time to be also suitable for long-term MP preservation of the liver, but, as inferred from these data, simple insufflation of gaseous O2 may be considered a feasible alternative.

Renal transplants from non-heart beating paracetamol overdose donors.

INTRODUCTION: Non-heart beating donors (NHBD) are widely encouraged to avert the critical shortage in the kidney donor pool. Ischaemic injury at the time of cardiac arrest in the NHBD is more pronounced and therefore the kidneys resulting are considered marginal. This review describes our experience with four kidneys from two controlled NHBDs who were exposed to paracetamol intoxication and subsequently were treated with mannitol prior to organ donation. MATERIALS AND METHOD: Two patients with fulminant liver failure following paracetamol overdose were referred as 'withdrawal of treatment' NHBD. As the two patients had developed hepatic encephalopathy they were treated with mannitol to reduce intra-cerebral oedema. The two donors were oligoanuric for at least 24 h prior to cardiac arrest. Following cardiac arrest, in situ perfusion was carried out and the kidneys were removed. One pair of kidneys were machine perfused while the second pair of kidneys were cold stored prior to transplantation. RESULTS: Pre-transplant assessment of NHBD kidneys resulted in three of four kidneys being transplanted. The NHBD kidneys exhibited a period of delayed graft function (DGF). The early transplant biopsies showed evidence of diffuse cytoplasmic vacuolation. These histological features disappeared with time and the renal function improved until the time of discharge. DISCUSSION: Non-heart beating donor kidneys are considered marginal and the effect of mannitol and paracetamol drug intoxication will induce reversible sub-lethal injury. A period of dialysis is inevitable in clearing the reactive intermediates of mannitol and paracetamol. The kidneys behaved as traditional controlled NHBD at time of discharge.