John J. Lemasters

Protection by pentoxifylline against graft failure from storage injury after orthotopic rat liver transplantation with arterialization.

Destruction of the endothelial cell lining and activation of Kupffer cells after reperfusion limits the safe storage of livers for transplantation surgery. Tumor necrosis factor-alpha (TNF) release by activated Kupffer cells may contribute to graft failure from storage injury. Accordingly, we evaluated whether pentoxifylline, which suppresses macrophage TNF release, would improve graft survival after orthotopic rat liver transplantation with arterialization. Livers from syngeneic Lewis rats were stored for 12-24 h in cold UW solution. Prior to implantation, the livers were flushed with cold Ringers solution or warm Carolina rinse solution B. With either rinse, pentoxifylline treatment of graft recipients significantly improved graft survival. Combined use of pentoxifylline (50 mg/kg for 5 days) and Carolina rinse solution doubled the safe storage time to 24 h. Acidotic pH and antioxidants were essential components of Carolina rinse solution that acted synergistically with pentoxifylline. Pentoxifylline was also shown to suppress TNF release by lipopolysaccharide (LPS)-stimulated cultured rat Kupffer cells. Thus, pentoxifylline may protect against primary non-function and failure of grafts from storage injury by suppressing excessive TNF release by activated Kupffer cells. However, neutralization of TNF with excess anti-TNF antibody did not improve survival. This may mean that depletion of TNF is as deleterious as excess TNF production. Alternatively, other Kupffer cell secretions [e.g., interleukin-1 (IL-1), interleukin-6 (IL-6) and other cytokines] may be involved in the pathogenesis of graft failure. In conclusion, pentoxifylline could protect against graft failure from storage injury.

Reperfusion rather than storage injury predominates following long-term (48 h) cold storage of grafts in UW solution: studies with Carolina Rinse in transplanted rat liver.

Both storage injury and reperfusion injury have been reported in association with liver transplantation; however, which predominates is not clear. Therefore, these studies were designed to evaluate whether Carolina Rinse, which minimizes reperfusion injury following orthotopic liver transplantation in the rat, would be effective after long-term (48 h) storage of grafts in University of Wisconsin (UW) cold storage solution where sufficient time for development of storage injury exists. Livers were rinsed with either Ringers solution or Carolina Rinse solution immediately prior to completion of implantation surgery. In the Ringers group, 30-day survival was high following 24 h of cold storage (4/5) but was very low after 48 h (1/16). Importantly, survival was increased significantly (5/14) when grafts were rinsed with Carolina Rinse following 48 h of cold storage. In both groups, parenchymal cells appeared normal by scanning electron microscopy, excluded trypan blue, and released SGOT at values only slightly above the normal range immediately (i.e., less than 5 min) after 48 h of cold storage. However, SGOT values rose steadily during the 1st hour postoperatively following reperfusion in the Ringers rinse group and reached levels around 1,000 U/l. In addition, nonparenchymal cells were not labelled with trypan blue following storage, but significant labelling occurred within 1 h. Both SGOT release and nonparenchymal cell injury were reduced significantly when grafts were rinsed with Carolina Rinse prior to completion of surgery. Liver injury assessed histologically 24 h postoperatively was also reduced about 50% by Carolina Rinse. Oxidative stress appeared to be involved, since radical adducts, most likely of lipid origin, were trapped during the first 5 min after reperfusion with the spin trapping technique and detected by electron paramagnetic resonance spectroscopy. Lipid radical formation was reduced nearly completely on reperfusion by Carolina Rinse. Since Carolina Rinse improved survival of liver grafts following long periods of cold storage and reduced lipid radical formation and hepatocellular injury, we concluded that a reperfusion injury rather than a storage injury predominates following orthotopic transplantation of livers stored for long periods of time in cold UW solution.

The calcium channel blocker nisoldipine minimizes the release of tumor necrosis factor and interleukin-6 following rat liver transplantation

Kupffer cells, when activated, release toxic cytokines such as tumor necrosis factor (TNF), which can cause tissue injury. Takei et al. have reported that nisoldipine, a calcium channel blocker which decreases phagocytotic activity by Kupffer cells, also diminishes liver and lung injury and dramatically improves survival following liver transplantation. Therefore, we studied the effect of nisoldipine on the time course of TNF and interleukin-6 (IL-6) release following cold storage and liver transplantation in the rat. Livers were stored under survival and non-survival conditions in cold Euro-Collins solution in the presence or absence of nisoldipine (1.4 microM). After storage, the effluent was collected for determination of cytokines. The liver was then transplanted orthotopically and serum was collected at various time intervals for up to 5 h. In the effluent, TNF levels were very low in both the control and nisoldipine-treated groups and IL-6 was not measurable. Furthermore, when livers were stored under survival conditions and transplanted (liver stored in the cold for 4 h), serum TNF (2 U/ml) and IL-6 (350 U/ml) values were minimal in both the control and nisoldipine-treated groups. In contrast, when livers were stored under non-survival conditions and transplanted (liver stored in the cold for 10 h), TNF levels increased to 15 +/- 2 U/ml, 150 min after graft reperfusion, an increase which was prevented by nisoldipine (6.5 U/ml). Serum IL-6 levels were also elevated 300 min after transplantation in livers stored for 10 h. Nisoldipine also reduced the release of this cytokine. Serum transaminases (SGOT) were elevated to values around 2000 U/l 5 h following transplantation. In the nisoldipine-treated group, values were lower between 60 and 300 min. In the lung, interstitial and alveolar edema and cellular infiltration were detectable 5 h postoperatively and were diminished by nisoldipine. These data confirmed that TNF and IL-6 release were minimal following cold storage and transplantation of livers stored under survival conditions, but were elevated transiently after transplantation under non-survival conditions. Nisoldipine prevented cytokine release, most likely by blocking the activation of Kupffer cells, which may explain how it decreases liver and lung injury very early following liver transplantation.

SPC‐100270, a protein kinase C inhibitor, reduced hypoxic injury due to reperfusion following orthotopic liver transplantation in the rat

Abstract Recently, we reported that SPC‐100270, a sphingosine derivative and inhibitor of protein kinase C (50–90 μM) in mixed micelle assays, reduced reperfusion injury resulting from hypoxia in a low‐flow, reflow model of liver perfusion [8]. Here we report that SPC‐100270 has similar beneficial effects following liver transplantation in vivo. Rat liver transplantation was performed using nonarterial and rearterial techniques. Livers from syngenic rats were harvested surgically, prepared with vascular cuffs and a splint, and stored for 24 or 48 h in University of Wisconsin (UW) cold storage solution. Just prior to completion of vascular reconstruction, the organ was rinsed with 3 or 10 ml of Ringers solution, vehicle, or a solution containing SPC‐100270 (up to 500 μM). Following implantation surgery, low doses of SPC‐100270 were ineffective at reducing both parenchymal and nonparenchymal cell death, yet significant (P < 0.05) reductions were observed with 500 μM. Further, nonparechnymal cell viability was improved nearly four fold by the drug. SPC‐100270 (500 μM) tended to increase survival following 48 h cold storage in UW solution, but the improvement was not statistically significant. SPC‐100270 also did not diminish carbon‐centered free radical formation in transplanted livers from alcohol‐treated rats. Collectively, these data support the hypothesis that pretreatment of donor livers with an inhibitor of protein kinase C is effective in vivo at reducing reperfusion injury, particularly to nonparenchymal cells, following orthotopic liver transplantation in the rat.

The second generation of Carolina Rinse, solution II, improves graft survival following orthotopic liver transplantation in the rat by preventing reperfusion injury

Carolina Rinse solution was designed to minimize reperfusion injury following orthotopic liver transplantation. Carolina Rinse blocks reperfusion-induced endothelial cell killing, diminishes postoperative enzyme release and improves survival dramatically. Adenosine and mildly acidotic pH were identified as key components. Here we report results with a simplified formulation, Carolina Rinse II, which contains extracellular inorganic ions similar to Ringers solution, adenosine, as well as antioxidants and radical scavengers (allopurinol, glutathione and desferrioxamine). In this study, 44 rat livers were explanted and stored for 12 h in University of Wisconsin (UW) cold storage solution (non-survival conditions). Control livers were rinsed with 15 ml cold Ringers solution just prior to completion of implantation surgery. In this control group, average 30-day survival was poor (8%). However, survival was increased to around 60% when grafts were rinsed with Carolina Rinse II. Survival was not improved significantly by rinsing the graft with Ringers solution containing antioxidants and radical scavengers with adenosine omitted (about 30%). Peak SGOT values of nearly 3000 U/l, measured 1-3 days postoperatively in the Ringers rinse control group, were decreased 4- to 5-fold both by Carolina Rinse II and by Ringers solution containing antioxidants. On the other hand, the addition of adenosine to Ringers solution improved survival (around 60%) but did not decrease the postoperative elevation of serum enzymes significantly. Thus, it appears that adenosine was necessary for optimal survival whereas antioxidants and radical scavengers were needed to prevent injury to the transplanted graft. These data were consistent with the hypothesis that at least two mechanisms, one involving the liver and a second one non-hepatic, are responsible for post-transplant pathophysiology. Carolina Rinse II also reduced the postoperative elevation in serum enzymes 2- to 3-fold in livers stored under survival conditions (e.g., for 8 h in UW solution). This study demonstrated convincingly that a very simple rinse solution, Carolina Rinse II, improved survival significantly and minimized graft injury following orthotopic liver transplantation.

Endothelial cell damage and Kupffer cell activation in reperfusion injury to livers stored for transplantation

Liver transplantation is an accepted therapy that achieves good long-term survival and a return to productive life for children and adults with end-stage liver disease. Scarcity of donor organs and graft dysfunction or failure are major obstacles to more widespread and successful application of liver transplantation surgery. In the USA alone, more than 2000 patients await donor livers, and hundreds die each year because none can be found [1]. In addition, primary non-function of grafts leading to graft failure and retransplantation occurs in 5–15% of patients [2–4]. Initial poor function of transplanted liver grafts occurs in another 20% of patients [5,6]. Since the clinical incidence of primary non-function is strongly dependent on time of cold storage, its aetiology is related to injury associated with graft harvest, storage and reperfusion. Liver damage after cold ischaemic storage is a special instance of the more general phenomenon of ischaemia/reperfusion injury. Recent advances in the understanding of storage/reperfusion injury reveal the many facets of this injury, virtually all of which are applicable to reperfusion injury after warm ischaemia to liver and other organs.