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Activated Kupffer cells (KC) have been implicated in the damage sustained by preserved liver grafts during ischemia and reperfusion. The aim of this study was to compare ischemia/reperfusion injury in preserved, KC-depleted rat livers and preserved control livers, with special regard to sinusoidal endothelial cell (SEC) injury. Wistar rats were injected with liposome-encapsulated dichloromethylene diphosphonate, 48 hr before hepatectomy, to eliminate KC, or were withheld this pretreatment (controls). Livers were flushed with cold University of Wisconsin solution and after 0, 8, 16, or 24 hr of storage at 4 degrees C, were reperfused in a recirculation system with 200 ml of oxygenated Krebs-Henseleit solution at 37 degrees C for 90 min. Damage to SEC was measured by the uptake of hyaluronic acid (HA) from the perfusate and release of purine nucleoside phosphorylase (PNP). Perfusate samples were, furthermore, analyzed for aspartate aminotransferase (AST) and tumor necrosis factor-alpha. Carbon particles were infused in the perfusate to determine the phagocytotic capacity of KC. Biopsies were taken for histological examination and sections were stained with ED2 monoclonal antibodies to confirm the absence of KC. After 90 min of reperfusion, immediately after cold flush (t0), the uptake of HA was 72.2+/-2.3% and 69.3+/-1.3% in KC-depleted livers and in control livers, respectively (n.s.). After 8 hr of storage, HA uptake was 21.6+/-4.5% and 34.6+/-8.0%, respectively (n.s.). After 16 and 24 hr of storage and reperfusion, no uptake of HA was found in either KC-depleted or control livers, indicating abolished SEC function. PNP activities in the perfusate were higher in control livers (after 8 and 24 hr of storage), presumably due to release from damaged KC. No difference was found in AST and no tumor necrosis factor-alpha was measured in the perfusates of normal and KC-depleted livers. Electron microscopic studies showed that after 8 and 24 hr of storage and reperfusion, KC were activated and were able to phagocytose colloidal carbon. Our conclusion was that the elimination of Kupffer cells did not result in reduction of ischemic and reperfusion damage in livers preserved up to 24 hr, as assessed in vitro by SEC uptake of HA, PNP release, and AST release.

The aim of this study was to investigate performance of preserved arterial allografts under the protection of a high-dose and a low-dose immunosuppressive regimen, with cyclosporine (CsA). Dog carotid arteries were harvested and stored for 14 days at 4 degrees C in University of Wisconsin organ preservation solution. Segments (6 cm) of carotid artery were orthotopically and bilaterally implanted in mongrel dogs (n = 18). CsA was given in two dosage regimens: 25 mg/kg/day (group I, n = 7) and 10 mg/kg/day (group II, n = 7). The control group received no CsA (group III, n=4). After 3 months of implantation, patency was assessed by angiography. The grafts were excised for investigation of vessel wall and endothelial function and morphology. For assessment of function in vitro, slices of arterial segments were connected as ring preparations to an isometric force transducer and immersed in a 5 ml organ bath (37 degrees C) containing Tyrode's solution. The contractile response was examined by adding 40 mM KCl and phenylephrine (100 microM) to the organ bath; endothelium-dependent relaxation was examined by adding methacholine (100 microM). Morphology was assessed semiquantitatively. The functional responses to KCl, phenylephrine (Phe) and methacho- line (Met) after 14 days of storage in UW, were 30.2 +/- 1.2 mN, 26.9 +/- 1.0 and 45 +/- 1.2% (means +/- SEM, n=9), respectively. Patency after three months of implantation for group I was 100% (14/14), for group II 50% (7/14), and for group III 75% (6/8). In vitro functional responses of preserved arteries, after 3 months of implantation in group I were 58.5 +/- 10.6 mN (KCl), 36.5 +/- 5.8 mN (Phe), and 57.4 +/- 9.7% (Met), respectively. Functions in group II were 1.2 +/- 0.1 mN (KCl, 0.0 mN (Phe), and 0.0% (Met). Grafts in group III showed no function. Measurement of medial thickness showed significant thinning (P <0.05) in groups II and III. Patency and function of arterial allografts under a therapeutic dose of CsA were superior to grafts implanted under low-dose CsA or no immunosuppressive treatment.

HTK (histidine-tryptophane-ketoglutarate) organ preservation solution has been shown to be effective in human kidney transplantation, but the efficacy of HTK for extended liver preservation has not been determined. In this study, canine livers were preserved in HTK and compared with livers preserved in University of Wisconsin solution. First, the right and left liver lobes in dogs were flushed separately with cold HTK and UW, respectively, according to a double-flush method. After splitting the liver, the right and left lobes were stored at 4 degrees C in either solution for 24 hr and 48 hr and assessed microscopically for parenchymal cell swelling, and enzyme histochemically for 5'-nucleotidase (5'-NT) as a marker of ischemic liver injury. Unlike livers preserved in UW (n = 5), HTK-preserved livers (n = 5) showed progressive parenchymal cell swelling after 24-hr and 48-hr storage. The 5'-NT scores in HTK livers were lower than in UW livers, indicating increased storage injury (0-5% and 66-85% in HTK- and UW-preserved livers, respectively, after 48-hr storage). Second, graft function was tested in an orthotopic liver transplantation model in the dog. Whole livers were flushed in situ with cold HTK or UW and stored at 4 degrees C for 24 hr or 48 hr. Liver grafts stored in HTK were not washed out prior to reflow in the recipient, in contrast to grafts stored in UW. Livers preserved for 24 hr using HTK showed life-supporting function after transplantation (n = 5, survival 12 hr-8 days). All grafts preserved for 48 hr in HTK did not function (n = 5, survival < 10 hr). UW-preserved grafts all functioned after 24-hr storage (n = 5, survival > 6 days), as well as after 48-hr storage (n = 6, survival > 6 days). Peak serum glutamic oxaloacetic transaminase (SGOT) values after transplantation of 24-hr and 48-hr HTK-preserved livers did not differ from peak SGOT values of UW-preserved livers after similar preservation times. In conclusion, UW solution is more effective than HTK solution in extended preservation of canine liver grafts: 24-hr storage of livers preserved with HTK solution is feasible, whereas 48-hr storage results in a nonfunctioning graft.

Glutathione spontaneously oxidizes in the UW solution for organ preservation and reduced glutathione (GSH) is converted to oxidized (GSSG) glutathione. To determine the effects of the oxidized or reduced forms of glutathione on liver preservation dog livers were preserved for 24 and 48 hr with the UW solution containing either GSH or GSSG. After 24 hr of preservation the form of glutathione did not affect survival or the postoperative course of the animals. All animals survived (three per group) with near-normal liver functions by the third to fifth postoperative day. When preservation was extended to 48 hr survival was 100% (6/6) with GSH and 29% (2/7) with GSSG. The dogs that died developed primary nonfunction of the liver. This study shows that GSH is an important component of the UW solution for 48-hr preservation of the dog liver. The presence of GSSG does not prevent successful 24-hr preservation of the liver, which has been confirmed in clinical studies. However, for 48-hr preservation GSH is required and GSSG is not suitable.