D.J Potts

Use of isolated perfused rat liver model for testing liver preservation solutions

THE HISTORY of liver perfusion is one of continuous progress toward simulation in vitro of conditions, which exist for the normal organ inside the body. The perfused liver has been used successfully for more than 145 years. The isolated perfused liver maintains lobular architecture and microcirculation stays intact. It allows direct access to vascular inflow and also to mixed hepatic venous blood outflow for experimental measurements. The model is particularly valuable for studying the response of normal systems to experimental manipulation and eliminates the possible influence of extraneous systemic factors within the whole animal. The perfusion model closely mimics physiologic conditions, and the data are simple to collect and are reproducible. It is also cost-effective and reliable to use. It is easier to run than in vivo models, including transplantation models. Its only disadvantage is that function deteriorates with time and some functions are lost after 2 to 5 hours. In view of these considerations, IPRL offers a unique opportunity for the study of hepatic function for simulating in vivo conditions, with the advantage of being able to control precisely experimental conditions. This model was used to test the effectiveness of liver transplant preservation solutions in our laboratory. The livers were stored in an experimental preservation solution for 24 hours followed by assessment of liver function and hemodynamics using isolated perfused rat liver model (IPRL). A new preservation solution (PBSL) was compared with other solutions used clinically.

Histidine-Tryptophan-Ketoglutarate and Delayed Graft Function After Prolonged Cold Ischemia

BACKGROUND Several articles have compared histidine-tryptophan-ketoglutarate solution (HTK) with other preservation solutions in both liver and kidney transplantation, and the results suggest that HTK is as good or better than the criterion standard University of Wisconsin solution (UW) for short periods of cold ischemia, such as in live donation, but that it is not so efficient for longer periods of cold ischemia, causing a higher incidence of delayed graft function. OBJECTIVE To evaluate energy levels, metabolites, and histologic findings to determine why HTK is inefficient for longer periods of cold ischemia. METHODS Rat livers were perfused with either HTK or UW, and at various times, tissue samples were obtained for analysis of adenine triphosphate and metabolites using high-performance liquid chromatography or for histologic analysis. RESULTS The high energy charge observed with HTK-perfused livers plateaued after 5 minutes, and by 60 minutes began to decrease, following the same trend as other samples. The plateau is due to excess available glucose; however, after 1 hour, it is beginning to be consumed. Low levels of uridine, required for glycogen synthesis, are found in HTK-perfused livers, which suggests that at reperfusion, there is none available, whereas the higher concentrations found in UW-perfused livers may be advantageous after reperfusion. This will be especially detrimental to use of HTK because glycogen is used up rapidly because of the presence of alpha-ketoglutarate in the solution, enabling continuation of the tricarboxylic acid cycle. CONCLUSIONS Overall, HTK seems to do well for the first 2 hours, after which any advantage observed initially starts to disappear. A liver perfused in HTK and transplanted after more than 1 hour reacts like an organ from an individual who has been starved, because of the low energy charge and absence of a glycogen store or ability to synthesis glycogen because of lack of uridine. Livers perfused with UW demonstrate higher levels of uridine and do not lose their glycogen content to the same extent as HTK-perfused livers. These findings explain in part why HTK sometimes causes delayed graft function after longer periods of cold ischemia.

Comparison of two preservation solutions in the protection of the pH regulation mechanism of perfused rat livers after 24 hours of cold storage.

LIVER has a pH stabilizing mechanism that can be impaired by ischaemic damage such as occurs during organ preservation for transplantation. We compared the effectiveness of University of Wisconsin solution (UW) and phosphate-buffered sucrose for liver (PBSL) on pH regulation and pH stabilization following flush and storage at 0 to 4°C for 24 hours, and reperfusion at 37°C. During both warm and cold ischemia hypoxia leads to the accumulation of metabolically generated intracellular acid. In hepatocytes, the pH regulatory system has been partly characterized. Roles for the Na and H exchanger and the Na and HCO3 2 cotransporter have been suggested. Upon reperfusion, hepatocytes have a requirement to eliminate acid to restore their intracellular pH to a normal physiologic level. It has been shown that intracellular pH buffering power of the hepatocytes remains intact even after 48 hours of storage in the preservation solutions. This correction of pH is of vital importance to the liver, as impairment will affect cell membrane potential; energy supply; and the intracellular concentration of ions, including Ca, K, and Na. Hepatocytes have two major mechanisms for eliminating H from the cell. These are the electroneutral Na/H exchanger and electrogenic Na/HCO3 2 transporter. Both of these transporters increase their activity with an increase of intracellular H ion concentration. As the hepatocytes eliminate intracellular acid, the extracellular fluid tends to become more acidic. During reperfusion hepatocytes normally correct the pH to the physiologic level. It is important that the cells of a liver graft retain effective function of this mechanism, particularly in the early stages of reperfusion. This may depend on the effectiveness of the preservation fluid. We compared the effects of UW on control of pH with those of a (combined) series of phosphate-buffered sucrose-based preservation solutions developed for use on the liver (PBSL).

Effective protection against prolonged warm ischemia of rat kidney using a simple preservation solution

HYPOTHERMIC preservation in conjunction with intravascular flush is the established routine for solid organ preservation in transplantation. Normothermic protection (conventionally called “warm” ischemia protection) is, however, also crucially important. It has been shown that 30 minutes of warm ischemia is potentially more damaging than 24 hours of cold storage. It has also been demonstrated that the warm ischemic damage is an essential component in transplant kidney dysfunction. The present experiment evaluates the protective role of the current preservation solutions in the prevention of warm ischemia and reperfusion injury in a rat kidney model. Three clinically established solutions, Euro-Collins (EC), hyperosmolar citrate (HOC), and University of Wisconsin (UW) solution were compared against phosphate-buffered sucrose (PBS140).

Addition of Adenosine to University of Wisconsin Solution: Does It Help?

Adenosine Triphosphate (ATP) precursors are sometimes added to preservation solutions in the belief that once the organ is reperfused, these precursors will build up ATP rapidly, returning it to its original metabolic state. This work studied ATP and metabolites during preservation of the rat liver using University of Wisconsin solution (UW), which contains adenosine, versus histidine tryptophan ketaglutarate solution a new phosphate-based preservation solution, or leeds solution (LS), which is under development at our institution (neither of the latter 2 contains adenosine). Tissue samples of perfused livers were analyzed for ATP and metabolites by high-performance liquid chromatography. UW did initially show the expected significant difference in overall adenosine levels, but the advantage had disappeared by 4 hours. At no time did UW show significantly higher levels of ATP; this was not seen following adenosine addition to LS. Only in living donor transplants where the cold ischemic time is short may there be some advantage to the addition of adenosine.

PBSH: A New Improved Cardiac Preservation Solution in Comparison With Three Clinically Proven Solutions

INTRODUCTION A solution under development at our institute, based on phosphate-buffered sucrose, has shown good preservation for kidneys and livers. This work used a refined version of this solution (PBSH-phosphate-buffered sucrose for the heart) in heart preservation, comparing it to solutions already widely used (University of Wisconsin, St. Thomass, and Celsior solutions). METHODS Following an initial washout phase and control working mode on a Langendorff system, hearts were flushed with preservation solution and after 6 hours at 4 degrees C were then reperfused for 15 minutes followed by working heart mode for a further 30 minutes. Hemodynamic parameters were measured and compared with their preischemic values and expressed as percentage recovery. Enzyme measurement came from the collection of the initial 1.5 mL of the coronary effluent after the storage period. This was to test for creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). Hearts were then placed immediately into liquid nitrogen for adenosine triphosphate (ATP), lactate, and creatine phosphate (CP) testing. Spectrophotometric analysis was used to assess both the release of CPK and LDH into the coronary effluent, and the level of ATP, lactate, and CP in the frozen heart tissue. RESULTS These results show that hearts that are preserved in PBSH are hemodynamically as well preserved as the hearts preserved in other solutions tested and their enzyme and lactate content is lower while having higher levels of energy compounds in these hearts. CONCLUSION Overall these results show that PBSH is at least as effective in cardiac preservation in the rat model of 6 hours of cold ischemia as these other widely used solutions tested.

Protective Effects of Polyethylene Glycol (20 mol/L) in Phosphate-Buffered Sucrose for Rat Liver Preservation

THE USE of polyethylene glycol (PEG) in preservation solutions has been associated with a decreased incidence of rejection in clinical and experimental transplantation. Using the isolated perfused rat liver model, we found that PEG, when added to phosphate-buffered sucrose solution (PBSL), provided improved liver preservation. A direct comparison of three different molecular configurations of PEG in phosphate-buffered sucrose (PBSL) was carried out. The following solutions were studied in our isolated perfused rat liver (IPRL) model: ● PBSL-I (without PEG); ● PBSL-II (containing polyethylene glycol; MW 3350); ● PBSL-III (containing polyethylene glycol; MW 8000); and ● PBSL-IV (containing polyethylene glycol; MW 20,000).