J.P.A. Lodge

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.

The Impact of Postoperative Infection on Long-Term Outcomes in Liver Transplantation

INTRODUCTION Postoperative infection (POI) prolongs inpatient stay, delays return to normal activity, and may be detrimental to long-term survival after cancer resections. This study sought to identify the impact of postoperative infection on liver transplantation outcomes. METHODS We analyzed our prospective database of 910 adult patients who underwent liver transplantation between 2000 and 2010 in a single UK center. POI was defined as pyrexia plus positive cultures from blood, sputum, urine, wound, or ascitic fluid. Patient demographic features and perioperative variables were analyzed for their effects on POI. The impacts of POI on overall survival (OS) and graft survival were analyzed using Kaplan-Meier curves with log-rank tests for significance, before entry into a multivariate regression analysis. We analyzed the effects of POI on the length of hospital stay (LOS) and the incidence of acute rejection episodes and readmissions within 1 year as secondary outcomes. RESULTS Patients who developed a postoperative chest or wound infection showed poorer OS at a mean of 7.0 versus 8.8 years (P = .009) and 7.0 versus 8.8 years (P = .003), respectively. Infection in blood, ascitic fluid, or urine showed no significant impact on survival. LOS was significantly increased among patients with a wound (median 21 vs 17 days, P = .011), a sputum (median 24 vs 17 days, P < .001), or a blood infection (median 32 vs 17 days, P < .001). Higher rates of intraoperative blood transfusion were observed among subjects who developed a chest or a wound infection. There was no difference in other variables between those who did versus did not develop an infection. Upon multivariate analysis, wound infection was the strongest independent predictor of OS (P = .007). CONCLUSION We demonstrated that wound or chest infections were associated with poorer OS. More aggressive prophylactic and/or therapeutic interventions targeting specific sites of infection may represent a simple and cost-effective measure to reduce hospital stay and improve OS.

Dopexamine and microcirculatory flow in transplanted small bowel: the Leeds experience.

Abstract Intestinal mucosa is extremely vulnerable to ischemic injury. In transplanted small bowel, this may represent preservation, ischemia-reperfusion injury, technical errors, postimplantation ischemia, or rejection. Such injury may compromise the mucosal barrier. Barrier dysfunction can lead to bacterial translocation and endotoxemia, a trigger for multisystem organ failure (MSOF). Prevention of further ischemic injury in the postoperative period by maintaining optimal graft perfusion may have a major role to play in ensuring graft viability. Previous methods of monitoring graft function have included clinical assessment, serum procoagulant levels, serial endoscopy and mucosal biopsy, and intestinal permeability studies. These methods are not ideal for monitoring early graft function. A number of noninvasive measures of graft perfusion are now available. These include laser Doppler flowmetry (LDF) and gut tonometry. Neither of these techniques has been applied to the transplanted small bowel. LDF is easy to use, is noninvasive, allows continuous monitoring, and its use has been validated in orthotopic liver transplantation (OLT) and in postoperative monitoring of new grafts. Gut tonometry was designed to allow measurement of bowel intramucosal pH (pHi). Low intestinal pH indicates splanchnic hypoperfusion or anaerobic metabolism, is common in critical illness, and is associated with increased morbidity and mortality after major surgery and trauma. Gastric tonometry has been used to monitor early graft function in OLT. Both techniques enable evaluation both of graft function and of therapies aimed at improving perfusion. The pharmacologic profile of dopexamine suggests it would prove a valuable agent in improving splanchnic blood flow. It is a dopamine (DA) analog with action at β2 adrenoceptors and DA1 receptors and only moderate activity at β1 and DA2 receptors. Dopexamine possesses no direct α-adrenoceptor activity, but norepinephrine reuptake is inhibited. Dopexamine improves cardiac performance by vasodilatation and mild isotropic activity. These hemodynamic effects are achieved without increased myocardial oxygen consumption. Dopexamine has been shown to improve splanchnic oxygenation, an effect which appears to be independent of its systemic effects. Boyd et al have shown that deliberately increasing oxygen delivery perioperatively significantly reduces mortality and morbidity in high-risk surgical patients. We have therefore studied the effects of dopexamine on gut mucosal blood flow following small bowel transplantation, using LDF and gastric tonometry.

Influence on energy kinetics and histology of different preservation solutions seen during cold ischemia in the liver.

BACKGROUND AND PURPOSE Cold flush preservation prolongs tissue viability during ischemia. However, there is little understanding of the effects of various preservation fluids on events during this period. A study of cold ischemia in rat livers was undertaken to compare biochemical and histological changes over time, using three preservation solutions: University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), and Leeds solution (LS) under development at our institution. Leeds solution is a phosphate-based sucrose solution that like UW contains the impermeant lactobionate and the metabolite allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) which acts as a competitive inhibitor of xanthine oxidase, stopping the breakdown of hypoxanthine to xanthine by oxidizing it to alloxanthine, inhibiting both the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid. MATERIALS AND METHODS At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes. RESULTS In all livers, ATP, ADP, and AMP degraded over 4 hours. In UW and LS groups, degradation beyond hypoxanthine was halted, and it continued in the HTK group. This blockade led to a significant reduction in the accumulation of xanthine and uric acid. Histological analysis showed protected architecture and maintenance of reticulin scaffolds in the UW and LS groups, whereas tissue breakdown was seen from earlier time points in the HTK group. Additionally, throughout ischemia, signs of pathological injury were more pronounced with UW- than with LS-preserved tissue. CONCLUSIONS These results implied that cold ischemia in the liver is characterized by dynamic biochemical changes coincident with pathological injury which are initiated from the time of organ perfusion and influenced by the choice of the perfusion fluid. Allopurinol in UW and LS appears to be critical. We hypothesized that it may also affect the degree of subsequent reperfusion injury. The data supported the assertion that LS offerred improved preservation over UW, adding to the impetus to shorten ischemic times in clinical transplantation.

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).

Functional and Histological Comparison of Rat Liver Preserved in University of Wisconsin Solution Compared with Tissue Preserved in a Novel Solution

An isolated perfused rat liver model was used to investigate biochemical and histologic changes during 2 hours of reperfusion after 24 hours of cold storage to compare Leeds solution (LS) with University of Wisconsin solution (UW). Compared with livers stored in UW, those perfused with LS showed significantly higher bile flow and lower enzyme production (P < .05 by 1-way analysis of variance). For example, after 120 minutes, alanine aminotransferase results were: LS 38.9 U/L vs UW 66.8 U/L and bile flows were LS 10.3 μg/15 min/g liver vs UW 9.2 μg/15 min/g liver. Histologically the reticulin breakdown was greater and its reformation slower in UW-preserved livers. Liver tissue was viable in both groups, as shown by the increased glycogen content after reperfusion in both groups, but seen at a higher rate among LS, perfused livers. In conclusion, LS compared favorably with UW to prevent ischemic damage and so could offer an alternative perfusion medium to UW.

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.

Alternative techniques for arterialization in multivisceral grafting.

Abstract For a small group of carefully selected patients, multivisceral grafting is a viable treatment. While early data suggest that the 3-year survival is less than 50%, 1 it is unlikely that any of these patients would have survived without transplantation. While improving results rely on advances in management, particularly in technique and immunosuppression, it is essential that the acquisition of the multivisceral graft does not compromise the procurement of the donor kidneys for transplantation. Described techniques generally employ the use of an aortic conduit or large aortic patch, 2,3 but these techniques have a number of drawbacks. For example, the use of the aortic conduit compromises the aortic patch around the orifices of the donor renal arteries which is a particular problem if there are multiple renal arteries. In addition, these techniques require mobilization of a long length of recipient aorta, often appear cumbersome, and can be difficult to orientate satisfactorily. In addition, the thoracic aorta needs to have the multiple intercostal arteries oversewn. If an aortic patch bearing the origins of the celiac and superior mesenteric arteries is used, the short length of vessels can make the anastomosis of the patch to recipient aorta difficult and hazardous.