Hypothermic Machine Preservation Research Articles

Role of perfusion medium, oxygen and rheology for endoplasmic reticulum stress-induced cell death after hypothermic machine preservation of the liver

Recently, the endoplasmic reticulum (ER) has been disclosed as subcellular target reactive to ischaemia/reperfusion and possibly influenced by hypothermic machine preservation. Here, the respective role of perfusate, perfusion itself, and the effect of continuous oxygenation to trigger ER-stress in the graft should be investigated. Livers were retrieved 30 min after cardiac arrest of male Wistar rats and preserved by cold storage (CS) in histidine-tryptophan-ketoglutarate (HTK) for 18 h at 4 degrees C. Other organs were subjected to aerobic conditions either by oxygenated machine perfusion with HTK (MP-HTK) or Belzer solution (MP-Belzer) at 4 degrees C or by venous insufflation of gaseous oxygen during cold storage (VSOP). Viability of livers was evaluated upon reperfusion in vitro according to previously validated techniques for 120 min at 37 degrees C. Oxygenation during preservation (MP-HTK, MP-Belzer or VSOP) concordantly improved functional recovery (bile flow, ammonia clearance), reduced parenchymal enzyme leakage and histological signs of necrosis and significantly attenuated mitochondrial induction of apoptosis (cleavage of caspase 9) compared to CS. However, MP with either medium produced about 500% elevated protein expression of CHOP/GADD153, suggesting pro-apoptotic ER-stress responses, paralleled by a significant elevation of caspase-12 enzyme activity compared to CS or VSOP. Although MP also promoted a slight (20%) induction of the cytoprotective ER-protein Bax inhibitor protein (BI-1), prevailing of proapoptotic reactions was seen by increased cleavage of caspase-3 and poly (ADP-Ribase)-polymerase (PARP) in both MP-groups. Endoplasmic stress activation is conjectured a specific side effect of long-term machine preservation irrespective of the medium, actually promoting cellular apoptosis via activation of caspase-12. The simple insufflation of gaseous O2 may be considered a feasible alternative, apparently indifferent to the endoplasmic reticulum.

How to make steatotic livers suitable for transplantation

Improvements in the surgical and nonsurgical care of patients with end-stage liver disease have led to an expansion of the indications for liver transplantation, resulting in an increasing shortage of donor organs. Despite many attempts to expand the donor pool, such as utilizing split or living donor liver transplants, the amount of available organs is insufficient to meet the demand. Alternatives to human organs, such as bioartificial organs, xenotransplantation, or stem-cell technology do not appear to have widespread clinical applicability in the foreseeable future. It seems logical, therefore, to focus attention on organs from compromised donors—organs that are at a higher risk of postoperative complications, such as initial poor graft function (or even primary nonfunction) or biliary complications. Although there is no general consensus on the definition of a compromised, or extended-criteria, donor, most would include donors who are older ( 60 years), and who have diabetes mellitus, hypertension, renal insufficiency, or hepatic steatosis in this category. Obesity, diabetes mellitus, the metabolic syndrome, and associated nonalcoholic fatty liver disease are reaching epidemic proportions throughout the world. A recent survey has indicated that up to 30% of the population in Western societies has a certain degree of hepatic steatosis. In case of advanced degrees of steatosis, this condition may preclude certain individuals as liver donors. Although some controversy about whether all steatotic livers should be excluded from transplantation remains, and some groups have reported reasonable outcomes utilizing livers with a certain degree of steatosis, livers that are deemed steatotic are frequently discarded. The reason not to utilize a potential liver is frequently based on subjective findings of hepatic steatosis during the procurement of postmortem organs, and in most programs, potential living donors are excluded when hepatic steatosis is detected during routine preoperative evaluation. There is a growing body of evidence to argue that steatotic livers are more susceptible to ischemia-reperfusion injury and, following transplantation, have less favorable outcomes than nonsteatotic livers. Hence, if manipulations, in vivo or ex vivo, could increase the quality of these organs and improve their outcome following transplantation, the number of livers available for transplantation would increase and, thereby, reduce current waiting lists for liver transplantation. These manipulations can be divided into 2 groups, the first focusing on reversing hepatic steatosis. If reversal cannot be achieved, the other group can focus on protecting the liver from ischemia-reperfusion injury associated with steatosis. In this issue of Liver Transplantation, Bessems et al. present their data on hypothermic machine preservation of fatty livers in a rodent model of hepatic steatosis. On the basis of an elegant and well-documented study, the authors conclude that less liver damage is observed following machine preservation of steatotic livers in comparison to conventional storage and that hepatobiliary function is superior in livers preserved with machine perfusion. Although the experiments

Evaluation of eight preservation solutions for endothelial in situ preservation.

Non-heart-beating donors (NHBDs) have the potential to reduce the increasing numbers of patients on kidney and liver graft waiting lists. One problem observed with kidneys obtained from NHBDs is the endothelial injury seen on protocol core biopsies after implantation. We postulate that this is caused by a combination of warm ischemia, cold ischemia, and hypertonic citrate during in situ preservation (ISP) rather than hypothermic machine preservation. Our aim was to optimize ISP methods to preserve endothelial structure and function. An animal model of ISP was used to compare the ability of eight different preservation solutions to protect mammalian vascular tissue exposed to a combination of warm and cold ischemia. Smooth muscle contractile function and endothelial dependent relaxation (nitric oxide production) were determined using an organ bath method. Bretchneider's HTK solution preserved the ability of endothelial tissue to relax vascular tissue in response to acetylcholine (91% relaxation vs. 17% saline control; ANOVA, P<0.001); in stark contrast, Marshall's solution performed no better than saline (15% relaxation vs. 17% saline control, P=NS). UW solution (80%) and a derivative lacking the starch colloid (70%) were comparable with HTK. Belzer-MPS (55%), Celsior (57%), and Perfadex (44%) showed a roughly equivalent level of endothelial preservation. Electron microscopy confirmed an anatomical loss of structure correlating with loss of function. ISP requires a large volume of fluid to be pumped at high flow rates. In this model, HTK retained a powerful ability to preserve endothelial structure and function during warm ischemia.

INTRA ARTERIAL DONOR COOLING: THE OPTIMAL PERFUSION SOLUTION

A16 Aims: Non-heart-beating donors (NHBD) have the potential to reduce the increasing numbers of patients on kidney and liver graft waiting lists. One problem observed with kidneys obtained from NHBDs is the vascular endothelial injury seen on protocol core biopsies after implantation. We postulate this is caused by a combination of warm ischaemia, cold ischaemia and Marshall’s hypertonic citrate during in situ preservation (ISP) rather than hypothermic machine preservation. Our aim was to optimise ISP methods in order to preserve endothelial structure and function. Methods: An animal model of ISP was used to compare the ability of seven different preservation solutions to protect mammalian vascular tissue exposed to a combination of warm and cold ischaemia. Smooth muscle contractile function and relaxation (endothelial nitric oxide production) was determined using an organ bath method. Results: Bretchneider’s HTK solution preserved the ability of endothelial tissue to relax vascular tissue in response to acetylcholine (91% relaxation vs. 17% control; ANOVA, p<0.001); in stark contrast Marshall’s solution performed no better than saline (15% relaxation vs. 17% control, p=NS) (Figure 1). UW solution (80%), and a derivative lacking the starch colloid (70%), were comparable to HTK. Belzer-MPS (55%), Celsior (57%) and Perfadex (44%) showed a roughly equivalent level of endothelial preservation. Electron microscopy confirmed an anatomical loss of structure correlating with loss of function. Figure 1: Vascular endothelial relaxation comparison of seven preservation solutions. * ANOVA p<0.05 different to saline 0.9% control.FigureConclusions: ISP requires a large volume of fluid to be pumped at high flow rates. HTK retains a powerful ability to preserve endothelial structure and function during human NHBD retrieval.

Hypothermic machine preservation in liver transplantation revisited: concepts and criteria in the new millennium.

To overcome the present shortage of liver donors by expansion of the existing donor pool and possibly lengthening of the storage time, hypothermic machine perfusion of the liver as a dynamic preservation method is revisited. The three most important aspects are defined to be the type of preservation solution, the characteristics of perfusion dynamics, and the oxygen supply. Reviewing hypothermic liver machine perfusion experiments, the University of Wisconsin machine preservation solution is the solution most used. It is also found that nothing conclusive can be said about the optimal perfusion characteristics, since either perfusion pressure or perfusion flow is reported. The best estimation is perfusion of the liver in a physiological manner, i.e. pulsatile arterial perfusion and continuous portal venous perfusion. The applied pressures could be chosen to be somewhat lower than physiological pressures to prevent possible endothelial cell damage. Oxygen supply is necessary to achieve optimal preservation of the liver. The minimal amount of partial oxygen pressure required is inversely related to the normalized flow. Incorporating these features in a system based on existing standard surgical and organ sharing procedures and which is able to work stand-alone for 24 h, weighing less than 23 kg, could successfully implement this technique into every day clinical practise.

Comparison of Glutathione S-Transferase and Alanyl Aminopeptidase as Viability Markers in a Porcine NHBD Model

A critical shortage in the kidney donor pool has led to a renewed interest in non-heart beating donors (NHBDs). As warm ischaemic tissue injury is a characteristic of NHBD, it is therefore crucial to screen such kidneys for viability to disqualify kidneys that will never work, i.e. develop primary non-function. Viability screening is somewhat time-consuming, usually involving a combination of donor history, visual assessment of the donor kidney at procurement and monitoring during hypothermic machine preservation. Machine perfusion of NHBD kidneys enables physical characteristics to be evaluated by perfusion flow rate and pressure. Glutathione S-transferase (GST) is an established kidney injury biomarker, but its prolonged assay time may result in delays in any potential transplantation. Alanyl aminopeptidase (Ala-AP) has been developed as an alternative novel biomarker of ischaemic renal injury. A porcine NHBD model with differential ischaemic renal injury, using heparin ± streptokinase in the initial in situ flush solution, was developed to evaluate the two biomarker enzymes, GST and Ala-AP. Two groups of 16 porcine NHBD kidneys (heparin alone or heparin + streptokinase) were machine perfused for 4 h, with documentation of pressure, flow, resistance and temperature and samples of kidney perfusate were taken at hourly intrevals. Wedge sections of the kidneys were analysed at termination of perfusion. Histopathology of renal wedge sections, machine perfusion physical parameters and biomarkers demonstrated the differential renal ischaemic insult. The two biomarkers, GST and Ala-AP appear to be correlate with the degree of ischaemic injury and with each other. Mild ischaemic injury occurs in the streptokinase-treated group, with lower efflux of GST and Ala-AP. Assays for Ala-AP activity in humans are significantly shorter than the comparative assays for GST, enabling early decision making on viability. This can, therefore, facilitate transplantation of the potential NHBD kidney within the recommended 24-hour period of cardiac arrest of donor.

Successful 96- and 144-hour experimental kidney preservation: A combination of standard machine preservation and newly developed normothermic ex vivo perfusion

In order to enable time-consuming matching and selection procedures (PLT, MLC) in kidney transplantation, a new approach to prolonged kidney preservation was studied. In a canine autotransplantation and contralateral nephrectomy model hypothermic machine preservation was interrupted by a short period of ex vivo perfusion on the donor animal. In a 96-hr experiment post-transplantation serum creatinines were significantly lower in the ex vivo perfused group versus the control group. All animals survived. In a 144-hr study five out of six survived in the ex vivo perfused group as opposed to one out of six in the control group. The beneficial effect of this ex vivo perfusion in the course of hypothermic machine preservation may be due either to a washout of metabolic waste products or to a restoration of exhausted enzyme systems.