raffinose and Ischemia

A safe and effective preservation solution is a precondition for liver transplantation, which is accepted as the radical treatment for patients with end-stage liver disease. The increasing use of marginal donors and non-heart beating donors as well as the establishment of a national organ allocation network call for better preservation. New preservation solutions like histidine-tryptophan-ketoglutarate (HTK) solution and Celsior solution have been introduced to liver preservation, and protective gene intervention and other modifications have also been investigated. In this article, we review recent advances in liver preservation solutions.. An English-language literature search was conducted using MEDLINE (1990-2005) on liver preservation solution, biliary complication, protective gene and other related subjects.. Although the high viscosity of the University of Wisconsin (UW) solution proved harmful to the hepatic microcirculation, three solutions showed equivalent preservation effects. When the cold ischemia time was short, there were no significant differences among the three solutions in the incidence of biliary complications. So far, modifications of preservation solutions have achieved great success. Several types of protective genes like A20, Bcl-2, Bcl-X(L) and HO-1 were reported to have definite liver protective effects. The addition of other substrates like TNF-alpha antibody, tacrolimus (FK506) and fructose-1,6-bisphosphate (FBP) can also improve the preservation effect. However, addition of insulin to UW solution is harmful to the graft.. In centers with highly-developed transplantation techniques, HTK and Celsior solutions are acceptable in liver preservation. Protective gene modification and addition of substrates like TNF-alpha antibody, FK506 and FBP are prominent approaches to improve liver preservation.

Topics: Adenosine; Allopurinol; Animals; Antibodies; Bile Duct Diseases; Disaccharides; Electrolytes; Fructosediphosphates; Glucose; Glutamates; Glutathione; Histidine; Humans; Insulin; Ischemia; Liver; Mannitol; Microcirculation; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Tacrolimus; Transformation, Genetic; Tumor Necrosis Factor-alpha

Reduced glutathione (GSH), a component of University of Wisconsin (UW) solution, is reported to oxidize during storage. Consequently the commercial manufacturer of UW recommends the supplemental addition of GSH to UW before utilization. We investigated the influence of supplemental GSH during cold ischemia on early renal allograft function.. One hundred kidneys were locally procured from heart-beating donors, preserved in our laboratory, and transplanted during an 18-month period. Selected donor, preservation, and outcome characteristics were collected and compared by presence of supplemental GSH and method of preservation. All kidneys were randomized to receive 3.0 mM supplemental GSH to perfusate or no supplementation (control) and were preserved by either cold storage (CS) in UW or machine perfused (MP) in UW-machine perfusate solution (MPS). During MP, perfusion characteristics (flow, resistance, perfusate electrolytes, and pH) were serially measured.. There were no significant differences among the groups when the donor characteristics of age, serum creatinine, and intra-operative urine output were compared. Preservation characteristics were similar among the groups with the exception of cold ischemia time, which was longer in the MP group compared to CS (26.1 h vs. 21.9 h, P=0.03). When compared with CS, kidneys preserved by MP exhibited a 33.4% increase in immediate function (93% vs. 62%, P=0.01), a corresponding 29.4% decrease in the incidence of delayed graft function (10% vs. 34%, P=0.02), and a 10% improvement in short-term (6-month) graft survival (98% vs. 88%, P=0.02). The addition of GSH supplementation to perfusate resulted in no significant differences in graft outcomes.. Despite recommendations by the manufacturer that UW solution be routinely supplemented with GSH, supplemental GSH does not influence early renal allograft function. Our data suggest that a far greater beneficial impact on early graft function is achieved by machine perfusion. We conclude that GSH supplementation of commercially available UW is not necessary.

Topics: Adenosine; Adult; Allopurinol; Cold Temperature; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Kidney Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reactive Oxygen Species; Tissue Donors; Transplantation, Homologous

The use of University of Wisconsin (UW) solution in liver transplantation (LTX) has significantly prolonged preservation times and facilitated semielective transplant procedures. Despite this advantage potential risk factors related to the donor, recipient, or cold storage method will persist in the UW era and detrimental effects will be reflected by primary dysfunction (PDF) after LTX. Concern has been voiced about the maximum period of UW preservation in LTX and various cold ischemia times (CIT) are mentioned. To evaluate the effect of UW solution in LTX, a prospective European multicenter study was initiated in 1988 and short-term results have been reported previously. This report focuses on the long-term effects and survival of prolonged preservation with UW solution and primary function after LTX. Three hundred and fifteen LTXs were performed in 288 patients in participating European centers. Complete follow up of at least 6 years was available for 296 grafts in 277 patients. Effects of donor, preservation, and recipient risk factors on PDF including primary non-function (PNF) and initial poor function (IPF) were evaluated. Next, the effect of risk factors on graft survival (GS) was analyzed including the long-term impact of PNF and IPF using multivariate analyses and the Kaplan-Meyer method. PDF occurred in 15.2% (45/296) with PNF in 7.8% and IPF in 7.4%. Patients with IPF had a 34% lower GS at 3 months those with immediate function (IF; 58% vs 91%; P < 0.001). This difference persisted up to 6 years for patients with IPF with a 39% GS vs 72% after IF (P < 0.001). Median CIT was significantly longer in grafts with PNF compared to IPF or IF (P = 0.03). Long-term GS, however, was significantly influenced at a lower CIT threshold with a 6-year GS for CIT < or = 16 h of 67%, compared to a CIT > 16 h of 51% (P = 0.02). Other independent risk factors for the 6-year survival rate were re-LTX, ABO incompatibility, and recipient diagnosis of acute hepatic failure. In conclusion, liver patients with PNF, but not with IPF, have a significantly lower CIT. IPF is associated with a significantly lower 3 month GS compared to IF, but this difference of 34% does not further increase during a 6-year follow up. Although a short term follow up (3 months) shows that with UW solution CIT up to 18 h has no adverse effect on GS, the 6-year data clearyl suggest that CIT should be kept to less than < 16 h to avoid tetrimental effects on lang-term GS after LTX.

Topics: Adenosine; Allopurinol; Cold Temperature; Follow-Up Studies; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Survival Rate; Time Factors

Topics: Adenosine; Allopurinol; Cadaver; Cold Temperature; Glucose; Glutathione; Graft Survival; Histocompatibility Testing; Humans; Hypertonic Solutions; Insulin; Ischemia; Kidney Transplantation; Mannitol; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Retrospective Studies; Time Factors

University of Wisconsin solution has become the most commonly used vascular perfusate during multiorgan donation world-wide. In the UK however, hyperosmolar citrate remains in common use. The purpose of this prospective randomized study was to compare the effect of systemic perfusion with UW or HOC on subsequent islet yield and purification for pancreata with short cold ischemic times. Seven pancreata were randomized to each group, with the donor age, pancreas weight, and period of cold ischemia being similar in both. Perfusion with UW was shown to inhibit collagenase digestion, and a higher concentration of this enzyme was needed to achieve comparable numbers of islets with good separation of exocrine and islet tissue after a similar period of digestion. There were no differences in the number, size, purity, or viability of islets between the two groups. In conclusion, UW solution offers no benefits over HOC for pancreata with short cold ischemic times, and because of its expense and need to use greater amounts of collagenase enzyme, we continue to use HOC.

Topics: Adenosine; Allopurinol; Cell Survival; Citrates; Citric Acid; Cold Temperature; Collagenases; Glutathione; Humans; Insulin; Ischemia; Islets of Langerhans; Organ Preservation Solutions; Osmolar Concentration; Pancreas; Perfusion; Prospective Studies; Raffinose; Random Allocation; Tissue Donors

Topics: Adenosine; Adult; Alanine Transaminase; Alkaline Phosphatase; Allopurinol; Aspartate Aminotransferases; Bilirubin; Child; Glutathione; Humans; Insulin; Ischemia; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Retrospective Studies; Serum Albumin; Solutions; Time Factors

The protective effects of carbon monoxide (CO) against ischemia/reperfusion (IR) injury during organ transplantation have been extensively investigated. Likewise, CO-releasing molecules (CORMs) are known to exert a variety of pharmacological activities via liberation of controlled amounts of CO in organs. Therefore, we hypothesized that intraluminal administration of water-soluble CORM-3 during cold storage of intestinal grafts would provide protective effects against IR injury.. Orthotopic syngeneic intestinal transplantation was performed in Lewis rats following 6 h of cold preservation in Ringer solution or University of Wisconsin solution. Saline containing CORM-3 (100 µmol/L) or its inactive counterpart (iCORM-3) was intraluminally introduced in the intestinal graft before cold preservation.. Histopathological analysis of untreated and iCORM-3-treated grafts revealed a similar erosion and blunting of the intestinal villi. These changes in the mucosa structure were significantly attenuated by intraluminal administration of CORM-3. Intestinal mucosa damage caused by IR injury led to considerable deterioration of gut barrier function 3 h postreperfusion. CORM-3 significantly inhibited upregulation of proinflammatory mRNA levels, ameliorated intestinal morphological changes, and improved graft blood flow and mucosal barrier function. Additionally, CORM-3-treated grafts increased recipient survival rates. Pharmacological blockade of soluble guanylyl cyclase activity significantly reversed the protective effects conferred by CORM-3, indicating that CO partially mediates its therapeutic actions via soluble guanylyl cyclase activation.. Our study demonstrates that luminally delivered CORM-3 provides beneficial effects in cold-stored rat small intestinal grafts and could be an attractive therapeutic application of CO in the clinical setting of organ preservation and transplantation.

Topics: Adenosine; Allopurinol; Animals; Carbon Monoxide; Glutathione; Humans; Insulin; Ischemia; Organ Preservation Solutions; Organometallic Compounds; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Soluble Guanylyl Cyclase; Water

Transplantation is lifesaving and the most effective treatment for end-stage organ failure. The transplantation success depends on the functional preservation of organs prior to transplantation. Currently, the University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) are the most commonly used preservation solutions. Despite intensive efforts, the functional preservation of solid organs prior to transplantation is limited to hours. In this study, we modified the UW solution containing components from both the UW and HTK solutions and analyzed their tissue-protective effect against ischemic injury. The composition of the UW solution was changed by reducing hydroxyethyl starch concentration and adding Histidine/Histidine-HCl which is the main component of HTK solution. Additionally, the preservation solutions were supplemented with melatonin and glucosamine. The protective effects of the preservation solutions were assessed by biochemical and microscopical analysis at 2, 10, 24, and 72 h after preserving the rat kidneys with static cold storage. Lactate dehydrogenase (LDH) activity in preservation solutions was measured at 2, 10, 24, and 72. It was not detectable at 2 h of preservation in all groups and 10 h of preservation in modified UW+melatonin (mUW-m) and modified UW+glucosamine (mUW-g) groups. At the 72nd hour, the lowest LDH activity (0.91 IU/g (0.63-1.17)) was measured in the mUW-m group. In comparison to the UW group, histopathological damage score was low in modified UW (mUW), mUW-m, and mUW-g groups at 10, 24, and 72 hours. The mUW-m solution at low temperature was an effective and suitable solution to protect renal tissue for up to 72 h.

Topics: Adenosine; Allopurinol; Animals; Glucosamine; Glucose; Glutathione; Histidine; Insulin; Ischemia; Kidney; Mannitol; Melatonin; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Raffinose; Rats

Appropriate hypothermic packaging techniques are an essential part of organ procurement. We present a case in which deviation from standard packaging practice may have caused sub-zero storage temperatures during transport, resulting in a clinical picture resembling PNF. An 18-month-old male with alpha-1-antitrypsin deficiency underwent liver transplant from a size-matched pediatric donor. Upon arrival at the recipient hospital, ice crystals were noted in the UW solution. The transplant proceeded uneventfully with short ischemia times. Surprisingly, transaminases, INR, and total bilirubin were markedly elevated in the postoperative period but returned to near normal by discharge. Follow-up of over five yr has demonstrated normal liver function. Upon review, it was discovered that organ packaging during recovery included storage in the first bag with only 400 mL of UW solution, and pure ice in the second bag instead of slush. This suggests that the postoperative delayed graft function was related to sub-zero storage of the graft during transport. This is the first report of sub-zero cold injury, or frostbite, following inappropriate packaging of an otherwise healthy donor liver. The clinical picture closely resembled PNF, perhaps implicating this mechanism in other unexpected cases of graft non-function.

Topics: Adenosine; Allopurinol; alpha 1-Antitrypsin Deficiency; Bilirubin; Cold Temperature; Delayed Graft Function; Frostbite; Glutathione; Humans; Ice; Infant; Insulin; International Normalized Ratio; Ischemia; Liver Transplantation; Male; Organ Preservation Solutions; Postoperative Period; Raffinose; Tissue and Organ Procurement; Treatment Outcome

Institut Georges Lopez-1 preservation solution (IGL-1) is an emerging extracellular-type electrolyte solution, low in viscosity, containing polyethylene glycol 35 as a colloid. Although IGL-1 has shown beneficial outcomes in kidney and liver preservation, this pilot study is the first to evaluate the efficacy of IGL-1 in pancreas transplantation (PT) compared with the University of Wisconsin solution (UW).. Sixteen Landrace pigs underwent allogeneic PT with 16 hr of cold ischemia. Grafts were preserved with IGL-1 (n=8) or UW (n=8). No immunosuppression was administered. We analyzed graft function, the acute-phase response, and oxidative stress in the pancreatic graft monitoring membrane fluidity and lipid peroxidation.. All eight grafts with IGL-1, but only six with UW, were functioning. Graft failures with UW resulted from graft thrombosis. There were no differences between the two solutions in the number of normoglycemic days (IGL-1: 11.5 ± 6.2 versus UW: 8.5 ± 4.4 days, P=0.1357), nor in lipid peroxidation during 16-hr cold ischemia (P=0.672), or reperfusion (P=0.185), but IGL-1 prevented changes in membrane fluidity after reperfusion when compared with UW (P=0.026).. IGL-1 offered the same degree of safety and effectiveness as UW in our model of pig PT with 16 hr of cold ischemia.

Topics: Adenosine; Allopurinol; Animals; Colloids; Electrolytes; Female; Glutathione; Immunosuppression Therapy; Insulin; Ischemia; Kidney; Lipid Peroxidation; Liver; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Pancreas; Pancreas Transplantation; Pilot Projects; Polyethylene Glycols; Raffinose; Swine; Time Factors; Viscosity

The differences and efficacy of standard preservation solutions in kidney transplantation, University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK), remain a topic of debate in recent clinical studies. P-selectins represent glycoproteins expressed on endothelial cells and platelets responsible for the earliest events in ischemia/reperfusion injury in kidney transplantation. This study aimed to compare the levels of P-selectin expression between cold preserved kidney tissues in UW and HTK solutions. Thirty kidneys were procured from male Lewis rats and stored in cold (4°C) solutions for periods of 4, 12, 16, 20, and 24h. Group 1 (n=15) kidneys were stored in UW solutions, and group 2 (n=15) kidneys were submerged in HTK solutions. At the end of each time point, the kidneys underwent preparation and levels of P-selectin expression in the tissues were measured using Immunoblot analyses and adjusted volumetric quantification of Western blot signals. For all periods of cold preservation, P-selectin expression was significantly down-regulated in kidney tissues stored in UW compared with HTK solutions (P<0.001). In summary, UW demonstrated a significant benefit over HTK solution in down-regulating P-selectin expression in cold preserved kidney grafts.

Topics: Adenosine; Allopurinol; Animals; Cryopreservation; Down-Regulation; Glucose; Glutathione; Insulin; Ischemia; Kidney; Male; Mannitol; Models, Animal; Necrosis; Organ Preservation Solutions; P-Selectin; Potassium Chloride; Procaine; Raffinose; Rats; Rats, Inbred Lew

Our study investigated the effect of ischemic postconditioning (IPO) in intestinal warm ischemia/reperfusion (I/R) and autotransplantation models.. Warm ischemia was performed by occlusion of superior mesenteric artery for 1, 3 and 6 hours in white domestic pigs (n = 15). Prior to 3 hours reperfusion the intestine was postconditioned by 3 cycles of 30-seconds ischemia and 30-seconds reperfusion (IPO protocol). In the cold ischemia group (n = 15) the bowel was preserved in University of Wisconsin solution for 1, 3, and 6 hours. Prior to 3 hours reperfusion IPO protocol was applied, too. Tissue samples were collected after laparotomy (control) and at the end of the reperfusion periods. As far as oxidative stress markers, malondialdehyde and reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity were determined. Tissue damage was evaluated by qualitative (Park-classification) and quantitative (Scion Image) methods.. As regards oxidative stress parameters, lipidperoxidation decreased and the protective effect of endogenous antioxidants (GSH, SOD) retained significantly by IPO procedure at the end of reperfusion. Tissue injury correlated significantly by the duration of warm ischemia and cold preservation. Quantitative analysis demonstrated that IPO ameliorated tissue injury in each group (p < 0.05).. IPO significantly attenuated intestinal oxidative stress and morphological damages in warm and cold I/R models.

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Disease Models, Animal; Glutathione; Insulin; Intestines; Ischemia; Ischemic Postconditioning; Laparotomy; Lipid Peroxidation; Malondialdehyde; Organ Preservation Solutions; Oxidative Stress; Raffinose; Reperfusion Injury; Superoxide Dismutase; Sus scrofa; Time Factors; Transplantation, Autologous; Warm Ischemia

Liver grafts preserved in cold storage undergo changes mainly manifested by morphological modifications of the sinusoidal endothelium that result in poor graft function upon reperfusion. The present studies aimed to determine if the addition of polyethylene glycol-albumin to University of Wisconsin (Peg-AlbUW) solution ameliorates the cold preservation injuries of liver grafts. Rat livers were preserved cold with various preservation solutions and evaluated for weight changes and endothelial morphology. Solutions that preserved graft weight and endothelial morphology were tested in the isolated perfused rat liver model to assess graft function. A rat hepatocyte cell line was evaluated for both viability and glutathione concentrations emulating cold preservation and reperfusion conditions. Liver grafts preserved with Peg-AlbUW maintained their initial weight and showed a conserved endothelial morphology compared with liver grafts preserved in UW for 30 h (P<0.05). Liver grafts preserved with Peg-AlbUW had improved portal blood flow and bile secretion compared with liver grafts preserved in UW for 30 h (P<0.05). In vitro we noted comparable hepatocyte viability when cells were preserved in Peg-AlbUW versus UW under similar preservation conditions (P>0.05); glutathione concentrations (reduced and total) were significantly increased in hepatocytes preserved in 3% Peg-AlbUW compared with other preservation solutions (P<0.05). The addition of Peg-Alb to UW preservation solution ameliorated the cold preservation injuries of rat liver grafts as shown by stable liver graft weight, a better preservation of the endothelial morphology, improved portal vein blood flow, and increased bile secretion. Peg-Alb-UW solution improved the integrity of the glutathione redox buffer system of a hepatocyte cell line after cold storage and reperfusion.

Topics: Adenosine; Albumins; Allopurinol; Animals; Apoptosis; Cell Line; Cryopreservation; Endothelial Cells; Glutathione; Graft Survival; Hepatocytes; Insulin; Ischemia; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Organ Size; Oxidative Stress; Polyethylene Glycols; Raffinose; Rats; Rats, Wistar

Ischemic-type biliary lesions (ITBL) account for a major part of patients' morbidity and mortality after orthotopic liver transplantation (OLT). The exact origin of this type of biliary complication remains unknown. This study retrospectively evaluated 1843 patients. Patients with primary sclerosing cholangitis were excluded from this study. The diagnosis of ITBL was established only when all other causes of destruction of the biliary tree were ruled out. Donor age (P = 0.028) and cold ischemic time (CIT) (P = 0.002) were found to be significant risk factors for the development of ITBL. Organs that were perfused with University of Wisconsin (UW) solution developed ITBL significantly more often than Histidine-Tryptophan-Ketoglutarate (HTK)-perfused organs (P = 0.036). The same applied to organs harvested externally and shipped to our center versus those that were procured locally by our harvest teams (P < 0.001). Pressure perfusion via the hepatic artery significantly reduced the risk of ITBL (P = 0.001). The only recipient factor that showed a significant influence was Child-Pugh score status C (P = 0.021). Immunologic factors had no significant impact on ITBL. The clinical consequences of this study for our institution have been the strict limitation of CIT to <10 h and the exclusive use of HTK solution. We further advocate that all organ procurement teams perform pressure perfusion on harvested organs.

Topics: Adenosine; Adult; Allopurinol; Berlin; Biliary Tract Diseases; Cold Ischemia; Female; Glucose; Glutathione; Humans; Incidence; Insulin; Ischemia; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Perfusion; Potassium Chloride; Pressure; Procaine; Raffinose; Retrospective Studies; Risk Factors; Tissue and Organ Harvesting; Tissue Donors

Donor hearts cannot be preserved beyond 6h using cold storage (CS). Improving preservation methods may permit prolonged storage of donor heart. We compared graft function in large animal model after prolonged preservation (8h) using continuous perfusion (CP) and CS method. Twenty-four miniature pigs were used as donors and recipients. Donor hearts were either stored in University of Wisconsin solution (UW solution) for 8h at 0-4°C (CS group, n=6) or were continuously perfused with oxygenated blood cardioplegia at 26°C for 8h (CP group, n=6). After preservation, hearts were transplanted into recipients and reperfused for 3h. Left ventricular (LV) function, cardiac output (CO), malondialdehyde (MDA) and adenosine triphosphate (ATP) levels, and water content were measured. Although water content of CP hearts was higher than that of CS, LV contractility and diastolic function of CP hearts were superior to those of CS. In addition, CP hearts performed better than CS hearts on CO in working heart state. ATP was better preserved and MDA levels were lower in CP hearts compared with those of CS (P<0.0001). Donor hearts can be preserved longer using continuous perfusion with oxygenated blood cardioplegia and this method prevents time-dependent ischemic injury.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cardiac Output; Glutathione; Heart Arrest, Induced; Heart Failure; Heart Transplantation; Insulin; Ischemia; Male; Malondialdehyde; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Swine; Swine, Miniature; Time Factors

The intestine is extremely sensitive to ischemic preservation and reoxygenation injury. Current vascular perfusion and cold storage with University of Wisconsin (UW) solution neglect the intestinal lumen and the ongoing mucosal metabolism during hypothermia. This study was designed to test the effects of luminal preservation with an alternative preservation solution in addition to the common vascular flush with UW solution on graft viability after preservation and ex vivo reoxygenation.. Rat intestine was preserved on ice for 6 hr in UW solution or Williams Medium E with additional buffering, impermeants, and a colloid (WMEplus) after being stapled or after flushing and filling the lumen with the respective preservation solution. Tissue slices were prepared from fresh and preserved intestines and were incubated with oxygen for 6 hr at 37°C to assess the viability after reoxygenation.. Directly after preservation, histologic damage was mild and unaffected by preservation strategy. Contrary to luminal preservation, closed preservation resulted in significantly decreased ATP levels compared with control. Reoxygenation aggravated damage and revealed differences between the strategies. Luminal preservation better maintained the ATP levels and histologic integrity (vs. closed preservation) for both solutions. Histomorphologic integrity was superior after preservation with WMEplus (vs. UW solution). Expression of stress responsive genes was least up-regulated in the slices from tissue preserved luminally with WMEplus.. In conclusion, preservation and reoxygenation injury can be attenuated by luminal preservation with WMEplus.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Gene Expression Regulation; Glutathione; Ice; Insulin; Intestinal Mucosa; Intestines; Ischemia; Microvilli; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; RNA, Messenger

Kidneys obtained from donors after cardiac death are damaged by the combination of warm and cold ischemia. Although the parenchymal damage of these kidneys is well studied, little is known about the functional effects of warm and cold ischemia on the renal vascular bed. We compared kidney preservation using the new extracellular-type cold storage solution from Institut Georges Lopez (IGL-1) with the University of Wisconsin solution (UW) and focused on vasomotor functions.. The influence of warm and cold ischemia on vasomotor functions was studied in an isolated perfused kidney model. Six groups of donation after cardiac death donor kidneys were studied with warm ischemia of 0, 15, and 30 min followed by 0 or 24 h cold storage preservation in IGL-1 or UW at 4 degrees C. Endothelial dependent vasodilation was studied using acetylcholine, smooth muscle cell (SMC) constriction was assessed using phenylephrine, and finally endothelial independent relaxation was tested using papaverine-sulfate.. SMCs were significantly affected by cold ischemia showing a 50% reduction of phenylephrine mediated constriction after preservation. Additional warm ischemia did not affect SMCs. After UW preservation endothelial dependent vasodilation was only significantly reduced when the combination of warm and cold ischemia was present. IGL-1 preserved kidneys showed a reduction in endothelial dependent vasodilation after isolated warm ischemia. Both preservation solutions rendered equal results after 24 h preservation.. Vasomotor functions are negatively influenced by the combination of warm and cold ischemia. Both IGL-1 and UW performed equally in preserving vasomotor functions. The interesting finding of the rapid decline of SMC function might point at the first step toward intimal hyperplasia as seen in late transplant dysfunction.

Topics: Adenosine; Allopurinol; Animals; Cadaver; Cold Temperature; Endothelium, Vascular; Glutathione; Insulin; Ischemia; Kidney; Male; Muscle, Smooth, Vascular; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred F344; Renal Circulation; Reperfusion; Tissue and Organ Harvesting; Tissue Donors; Vasoconstriction

The improvement of the ischaemic tolerance of myocutaneous flaps is of clinical importance and hence the subject of numerous investigations.. In an attempt to increase the ischaemic tolerance, 20 myocutaneous flaps (rectus abdominis muscle) in pigs were elevated and perfused with various, established solutions prior to the onset of ischaemia. The flaps were elevated, utilizing the superior epigastric vessels as the pedicle. Ten flaps were flushed with the University of Wisconsin solution, five with the Euro-Collins solution and the last five with a Ringer-Lactate solution, prior to the 6h long, normothermic ischaemia. On the day of operation, the first, third, fifth, seventh and tenth postoperative day clinical examinations and thermography were performed as well as biopsies. Additionally, on the tenth postoperative day, the rate of necrosis was determined morphometrically as the average of three measurements.. Ten days after surgery, the flaps pretreated with the University of Wisconsin solution displayed a vital surface area of 89%, the Euro-Collins solution 23% and the Ringer-Lactate solution 14%. Histologically, muscle tissue proved to be more susceptible to ischaemia than skin.. Regarding the rectus abdominis flap in a pig model, the University of Wisconsin solution proved superior in the prevention of ischaemic injury compared with the Euro-Collins solution and Ringer Lactate. In accordance with the literature, muscle tissue proved to be more susceptible to ischaemia than skin in our study.

Topics: Adenosine; Allopurinol; Animals; Biopsy; Disease Susceptibility; Epigastric Arteries; Glutathione; Graft Survival; Hypertonic Solutions; Insulin; Ischemia; Ischemic Preconditioning; Isotonic Solutions; Models, Animal; Necrosis; Organ Preservation Solutions; Raffinose; Rectus Abdominis; Reperfusion; Ringer's Lactate; Skin Transplantation; Surgical Flaps; Swine; Thermography; Time Factors; Tissue Preservation; Transplantation Conditioning; Warm Ischemia

We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Glutathione; Insulin; Ischemia; Islets of Langerhans; Male; Mitogen-Activated Protein Kinase Kinases; Organ Preservation Solutions; Pancreas; Phosphotransferases; Raffinose; Rats; Rats, Inbred Lew; Tissue and Organ Harvesting

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.. At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes.. 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.. 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.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glutathione; Hypoxanthine; Insulin; Ischemia; Kinetics; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Reticulin; Uric Acid; Uridine; Xanthine

Especially in damaged organs, adequate organ preservation is critically important to maintain viability. Institut Georges Lopez-1 (IGL-1) is a new preservation solution, with an extracellular sodium/potassium ratio and polyethylene glycol as a colloid. The influence of warm and cold ischemia was evaluated in a rat Lewis-Lewis transplant model with a follow up of 14 days. Eight groups of donation after cardiac death donor kidneys were studied with warm ischemia of 0 and 15 min followed by 0- or 24-h cold storage (CS) preservation in IGL-1 or UW-CSS. Blood was collected daily during the first week and at day 14. Recipients were placed in metabolic cages at day 4 and 14 after transplantation allowing urine collection and adequate measurement of glomerular filtration rate. Focussing on inflammation, reactive oxygen species production, proximal tubule damage, proteinuria, histology, and renal function after transplantation we could not show any relevant difference between IGL-1 and UW-CSS. Furthermore, the combination of 15-min warm ischemia and by 24-h cold ischemia did not result in life sustaining kidney function after transplantation, irrespective of the used solution. In the present experiment, static CS preservation of ischemically damaged rat kidneys in either IGL-1 or UW-CSS rendered equal results after transplantation.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Glomerular Filtration Rate; Glutathione; Insulin; Ischemia; Kidney; Kidney Transplantation; Kidney Tubules, Proximal; Male; Organ Preservation; Organ Preservation Solutions; Proteinuria; Raffinose; Rats; Rats, Inbred Lew; Reactive Oxygen Species

Cold ischemia time and preservation of organs are limited by I/R injury leading to primary nonfunction of the graft. In a rat heart transplant model, we compared cardioplegic St Thomas (ST) to histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin preservation solutions in terms of contractile function, and mitochondrial respiratory and enzymatic defects after prolonged cold ischemia and reperfusion. Contractile function was scored after transplantation and 24 h of reperfusion. Mitochondrial function was investigated by high-resolution respirometry of permeabilized myocardial fibers. Graft performance in terms of contractile function declined with the duration of cold storage. Recovery was significantly improved after 10 h of cold storage in HTK compared with ST (cardiac scores, 3.3+/-0.5 and 1.8+/-0.8, respectively). Tissue lactate dehydrogenase was better preserved in HTK than ST. Increase of tissue water content (edema) was less pronounced in HTK than ST (3.33+/-0.14 and 3.73+/-0.21 mg/mg dry weight, respectively). Similar cardiac scores (2.6+/-0.9 and 2.9+/-1.2, respectively) and mitochondrial respiratory parameters were obtained after preservation in HTK and University of Wisconsin. Decline in contractile function of individual grafts correlated well with loss of mitochondrial respiratory capacity, whereas citrate synthase activity remained largely preserved, indicating specific damage of respiratory complexes. Our data provide evidence for the superiority of preservation solutions versus a cardioplegic solution for prolonged cold storage of the heart. The correlation of graft performance and mitochondrial function indicates the potential of high-resolution respirometry for quantitative assessment of myocardial injury upon cold I/R, providing a basis for diagnostic approaches and evaluation of improved preservation solutions for heart transplantation.

Topics: Adenosine; Allopurinol; Animals; Cardioplegic Solutions; Citrate (si)-Synthase; Glutathione; Heart Transplantation; Insulin; Ischemia; Male; Mitochondria; Myocardial Contraction; Myocardium; Organ Preservation Solutions; Permeability; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

In kidney transplantation, cold storage is the dominant modality used to prolong organ viability ex vivo, but is inevitably followed by a period of warm ischemia. Preservation fluids limit tissue damage during the ischemic period, but there is little information on the influence of preservation fluids on the physiologic consequences of warm ischemia alone, or on the comparative ability of such preservation fluids to limit warm ischemic injury. In this study, warm ischemia was induced in rat kidneys by crossclamping the left renal pedicle for 45 min with contralateral nephrectomy. The ischemic kidneys were flushed with Euro-Collins (EC), hyper osmolar citrate (HOC), University of Wisconsin (UW), or phosphate buffered sucrose (PBS)140 solution. Over a period of 2 h after reperfusion, urine and blood samples were collected and physiological parameters related to the function of the postischemic kidneys were assessed. The data show that postischemic renal function can be influenced by the choice of preservation fluid. Essentially, the continued use of EC as a renal preservation solution finds little support in these data, and, while HOC and UW solutions were better able to limit the decline in renal function after warm ischemia than EC, the solution most able to limit functional impairment after warm ischemia was PBS140. This analysis compares the efficacies of the commonly used preservation solutions and could form the basis for future solid-organ transplant studies that may ultimately allow us to propose best-practice guidelines and an optimum platform for improved preservation solutions.

Topics: Adenosine; Allopurinol; Animals; Citric Acid; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Osmolar Concentration; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sugar Phosphates; Temperature

Ischemic type biliary lesions lead to considerable morbidity following orthotopic liver transplantation. The exact pathogenesis is unknown. One major hypothesis is that insufficient perfusion of the arterial vessels of the biliary tree, especially under perfusion with the high viscous University of Wisconsin solution, might be responsible for ischemic type biliary lesions. Due to low viscosity, HTK solution is reported to have a lower incidence of biliary complications. However, there is no data concerning ischemic type biliary lesions in HTK preserved livers. In this paper we report our results after orthotopic liver transplantation with special regard to ischemic type biliary lesions in liver grafts preserved with HTK solution. Between 09/1997 and 01/2005 300 liver transplantations were performed in our center. Thirty-two (10.7%) liver grafts were preserved with HTK solution, 268 (89.3%) were preserved with UW solution. Six and 43 grafts showed ischemic type biliary lesions after orthotopic liver transplantation in HTK- (18.8%) and UW- (16.0%) groups, respectively (p=0.696). There was no statistical significant difference between the two groups. Donor related factors, recipient age, indication for transplantation, transplantation technique, immunosuppression and ischemia time were comparable in both groups. Ischemic type biliary lesions occurred with the same frequency in HTK preserved livers compared to UW preserved organs. We suggest that low viscosity of the preservation fluid by itself does not guarantee reliable perfusion of the small arteries of a liver graft and a pressure perfusion might be beneficial even in HTK solution.

Topics: Adenosine; Allopurinol; Bile Ducts; Glucose; Glutathione; Humans; Insulin; Ischemia; Liver Transplantation; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Procaine; Prospective Studies; Raffinose

Redox-active iron, catalyzing the generation of reactive oxygen species, has been implicated in experimental renal ischemia-reperfusion injury. However, in clinical transplantation, it is unknown whether redox-active iron is involved in the pathophysiology of ischemic injury of non-heart-beating (NHB) donor kidneys. We measured redox-active iron concentrations in perfusate samples of 231 deceased donor kidneys that were preserved by machine pulsatile perfusion at our institution between May 1998 and November 2002 using the bleomycin detectable iron assay. During machine pulsatile perfusion, redox-active iron was released into the preservation solution. Ischemically injured NHB donor kidneys had significantly higher perfusate redox-active iron concentrations than heart-beating (HB) donor kidneys that were not subjected to warm ischemia (3.9 +/- 1.1 vs. 2.8 +/- 1.0 micromol/L, p = 0.001). Moreover, redox-active iron concentration was an independent predictor of post-transplant graft viability (odds ratio 1.68, p = 0.01) and added predictive value to currently available donor and graft characteristics. This was particularly evident in uncontrolled NHB donor kidneys for which there is the greatest uncertainty about transplant outcomes. Therefore, perfusate redox-active iron concentration shows promise as a novel viability marker of NHB donor kidneys.

Topics: Adenosine; Allopurinol; Cadaver; Cell Survival; Glutathione; Graft Survival; Heart Arrest; Humans; Hypothermia; Insulin; Iron; Ischemia; Kidney; Kidney Transplantation; Organ Preservation Solutions; Oxidation-Reduction; Perfusion; Predictive Value of Tests; Raffinose; Reactive Oxygen Species; Tissue Donors; Treatment Outcome

Transplantation of the uterus has been suggested as a possible future treatment of absolute uterine infertility. The tolerability of human uterine tissue to cold ischaemic storage was tested in the present study.. Small tissue samples of human uteri were subjected to cold (4 degrees C) ischaemia (6 and 24 h) in Ringer acetate (RIN), the intracellular-like University of Wisconsin solution (UW) or the extracellular-like Perfadex solution (PER). The ability of myometrial strips to contract, histology by light and electron microscopy as well as tissue concentrations of glutathione, ATP and protein were used as parameters to detect cold ischaemic injuries.. Contractile ability and response to prostaglandin F(2alpha) (PGF(2alpha)) was better preserved after 6 h cold ischaemia in UW and PER in comparison with the other groups. Histological examination did not reveal any major changes after 6 and 24 h cold ischaemic storage in UW and PER solutions, while specimens stored in RIN for 24 h displayed degenerative changes on the electron microscopy level. UW and PER preserved ATP concentrations significantly better than RIN. Myometrium stored in UW contained more total glutathione but also a larger proportion of oxidized glutathione than specimens stored in RIN and PER. Protein concentrations did not change with storage time in any of the solutions.. The results show that human uterine myometrial tissue is resistant towards cold ischaemia for at least 6 h if stored in UW and PER solutions.

Topics: Adenosine; Adenosine Triphosphate; Adult; Allopurinol; Citrates; Cold Temperature; Cryopreservation; Dinoprost; Female; Glutathione; Humans; Insulin; Ischemia; Isotonic Solutions; Microscopy, Electron; Middle Aged; Muscle Contraction; Myometrium; Organ Preservation; Organ Preservation Solutions; Organ Transplantation; Premenopause; Raffinose; Reperfusion; Specimen Handling; Temperature; Time Factors; Uterine Contraction; Uterus

Cold ischemia is the best known method to preserve kidneys for transplant. However, it produces several detrimental effects. First, cellular necrosis. Secondarily, during the hypothermic period a mitochondrial injury process develops which makes the cell entering a pre-apoptotic state. This apoptosis occurs definitively in the reperfusion. Preservation solutions currently available are not perfect and are not able to avoid cold-related cell injuries. The addition of certain substances to UW solution (desferrioxamine) has shown experimentally a reduction in mitochondrial cold-related lesions. Isolated hypothermic kidney perfusion reduces initial graft dysfunction about 20% in comparison to hypothermic storage. This fact relates to important either economical as functional consequences.

Topics: Adenosine; Allopurinol; Cold Temperature; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

The current models of liver ischemia/reperfusion injury (IRI) in mice are largely limited to a warm ischemic component. To investigate the mechanism of hepatic "cold" IRI, we developed and validated a new mouse model of prolonged cold preservation followed by syngeneic orthotopic liver transplantation (OLT). Two hundred and forty-three OLTs with or without rearterialization and preservation in University of Wisconsin solution at 4 degrees C were performed in Balb/c mice. The 14-day survivals in the nonarterialized OLT groups were 92% (11/12), 82% (9/11), and 8% (1/12) after 1-hour, 6-hour and 24-hour preservation, respectively. In contrast, hepatic artery reconstruction after 1-hour, 6-hour, and 24-hour preservation improved the outcome as evidenced by 2-week survival of 100% (12/12), 100% (10/10), and 33% (4/12), respectively, and diminished hepatocellular damage (serum alanine aminotransferase /histology). Moreover, 24-hour (but not 1-h) cold preservation of rearterialized OLTs increased hepatic CD4+ T-cell infiltration and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin 2, interferon-gamma) production, as well as enhanced local apoptosis, and Toll-like receptor 4/caspase 3 expression. These cardinal features of hepatic IRI validate the model. In conclusion, we have developed and validated a new mouse model of IRI in which hepatic artery reconstruction was mandatory for long-term animal survival after prolonged (24-h) OLT preservation. With the availability of genetically manipulated mouse strains, this model should provide important insights into the mechanism of antigen-independent hepatic IRI and help design much needed refined therapeutic means to combat hepatic IRI in the clinics.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; CD4 Lymphocyte Count; Disease Models, Animal; Glutathione; Immunohistochemistry; Inflammation; Insulin; Ischemia; Liver; Liver Transplantation; Male; Mice; Mice, Inbred BALB C; Organ Preservation Solutions; Postoperative Complications; Raffinose; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Treatment Outcome

Although the use of Celsior has been recently described for heart, lung, liver, and kidney transplantation, no data are available on its use for clinical pancreas preservation.. We herein describe the results of 112 pancreas transplants preserved with either University of Wisconsin (UW; (n = 56) or Celsior (n = 56) solution at two Italian transplant centers. The groups were comparable with regard to all donor and recipient characteristics.. Mean cold and warm ischemia times were 10.1 +/- 2.2 hours and 37.2 +/- 8.2 minutes for UW compared to 10.8 +/- 2.4 hours and 38.3 +/- 6.7 minutes for Celsior (P = NS). Delayed endocrine pancreas function was recorded in two UW-preserved grafts (3.6%). Actuarial 1-year patient survival was 94.6% for UW as compared with 100% for Celsior (P = NS). Equivalent graft survival figures were 91.0% for UW as compared with 96.4% for Celsior (P = NS).. Within the range of cold ischemia times reported in this study, UW and Celsior solutions have similar safety profiles for pancreas transplantation.

Topics: Adenosine; Adult; Allopurinol; Blood Glucose; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Graft Survival; Histidine; Histocompatibility Testing; Humans; Insulin; Ischemia; Italy; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Organ Transplantation; Pancreas; Pancreas Transplantation; Raffinose; Retrospective Studies; Survival Analysis; Tissue Donors

To evaluate the viability and energy metabolism of long warm ischemically damaged pancreas during preservation by the UW solution cold storage method.. The pancreas grafts subjected to 30-120 min warm ischemia were preserved by the UW solution cold storage method for 24 h. The tissue concentrations of adenine nucleotides (AN) and adenosine triphosphate (ATP) and total adenine nucleotides (TAN) were determined by using high performance liquid chromatography (HPLC) and the viability of the pancreas graft was tested in the canine model of segmental pancreas autotransplantation.. The functional success rates of pancreas grafts of groups after 30 min, 60 min, 90 min, 120 min of warm ischemia were 100%, 100%, 67.7%, 0%, respectively. There was an excellent correlation between the posttransplant viability and tissue concentration of ATP and TAN at the end of preservation.. The UW solution cold storage method was effective for functional recovery of the pancreas suffering 60-min warm ischemia. The tissue concentration of ATP and TAN at the end of 24 h preservation by the UW solution cold storage method would predict the posttransplant outcome of pancreas graft subjected to significant warm ischemia.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Dogs; Energy Metabolism; Female; Glutathione; Graft Survival; Hot Temperature; Insulin; Ischemia; Islets of Langerhans; Male; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose

Human pancreas preservation for islet transplantation holds additional challenges and considerations compared with whole pancreas transplantation. The purpose of this study was to clarify the limitations of the University of Wisconsin (UW) solution and the potentials of the two-layer method (TLM) for pancreas preservation before human islet isolation.. We retrospectively evaluated human islet isolation records between January 2001 and February 2003. One hundred forty-two human pancreata were procured from cadaveric donors and preserved by means of the UW solution (n=112) or TLM (n=30). Human islet isolations were performed using a standard protocol and assessed by islet recovery and in vitro function of islets.. Eight to ten hours of cold ischemia in the UW solution is a critical point for successful islet isolations. It is difficult to recover a sufficient number of viable islets for transplantation from human pancreata with more than 10 hours of cold storage in the UW solution. The overall islet recovery in the TLM group was significantly higher than in the UW group. With 10 to 16 hours of cold storage, the success rates of islet isolations remained at 62% in the TLM group but decreased to 22% in the UW group. Transplanted islets in the TLM group worked well in the recipients.. There are time limitations for using the UW solution for pancreas preservation before human islet isolation. The TLM is a potential method to prolong the optimal cold storage time for successful islet isolations.

Topics: Adenosine; Adult; Aged; Allopurinol; Cadaver; Fluorocarbons; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Islets of Langerhans Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Recovery of Function; Retrospective Studies

Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys.. Bactenecin (L- and D-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro.. Bactenecin (both L- and D- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin-Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics.. These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage.

Topics: Actins; Adenosine; Allopurinol; Animals; Antimicrobial Cationic Peptides; Cell Line; Cold Temperature; Cryopreservation; Culture Media, Serum-Free; Cytoskeleton; Dogs; Dose-Response Relationship, Drug; Female; Fluoresceins; Glutathione; Insulin; Ischemia; Kidney; Kidney Tubules; Liposomes; Membranes; Microtubules; Mitogens; Organ Preservation; Organ Preservation Solutions; Paclitaxel; Peptides, Cyclic; Permeability; Protein Binding; Raffinose; Reperfusion Injury; Stereoisomerism; Temperature; Time Factors

Topics: Adenosine; Allopurinol; Animals; Body Weight; Dogs; Female; Glutathione; Hepatectomy; Hypertonic Solutions; Insulin; Ischemia; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion; Time Factors; Tissue and Organ Harvesting; Transplantation, Autologous

The deleterious effect of steatosis on transplanted livers is mainly related to a microcirculation impairment. We investigated the effect of preservation duration on the recovery of isolated perfused rat steatotic livers and tested the effect of pentoxifylline (PTX), known to have a beneficial effect on hepatic microcirculation.. Fatty rat livers were obtained using a diet able to induce an 80% to 100% microvesicular steatosis within 7 days. We studied the effect of the duration of preservation (12 hr, 18 hr, and 24 hr) on fatty and normal isolated perfused rat liver. PTX was added to University of Wisconsin solution during cold storage (30 mM/kg of weight) and at reperfusion (3 mM) (n=5 livers in each group). Lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, bile production, and vascular resistance were evaluated. The liver injury at the end of perfusion was assessed by optical and electron microscopy.. For a 24-hr preservation period, fatty livers demonstrated increased enzymatic release (aspartate aminotransferase: 42+/-16 vs. 17+/-5 IU/L/g of liver, P<0.005; alanine aminotransferase: 32+/-13 vs. 13+/-3 IU/L/g of liver, P<0.005; lactate dehydrogenase: 1,207+/-497 vs. 291+/-195 IU/L/g of liver, P<0.001). Vascular resistance (0.32 vs. 0.15 cm H(2)O/min/mL, P<0.0005) and bile output (67+/-24 vs. 141+/-61 mg/g of liver, P<0.05) were decreased. Peliosis appeared after an 18-hr preservation period for fatty livers compared with a 24-hr preservation period for controls. All these negative effects were suppressed by PTX.. Diffuse microvesicular steatosis became deleterious only after long preservation times (24 hr). PTX prevented this effect.

Topics: Adenosine; Allopurinol; Animals; Fatty Liver; Free Radical Scavengers; Glutathione; Insulin; Ischemia; Liver; Liver Circulation; Liver Function Tests; Male; Organ Preservation; Organ Preservation Solutions; Pentoxifylline; Raffinose; Rats; Rats, Wistar; Time Factors

Ischemic biliary lesions are a threatening complication following orthotopic liver transplantation. Their exact pathophysiological mechanism is unknown so far, but insufficient perfusion of biliary arterial vessels might be responsible for the development of these lesions. This might be changed by improved perfusion techniques. We performed a controlled study of cases since February 2000.. We used arterial back table pressure perfusion to achieve reliable perfusion of the capillary system of the biliary tract, which may be impaired by the high viscosity of University of Wisconsin solution. In this study, 190 orthotopic liver transplantations performed between September 1997 and July 2002 were investigated with regard to ischemic biliary lesions.. One hundred thirty-one grafts were preserved by in situ standard perfusion including portal perfusion,whereas additional arterial back table pressure perfusion was performed in 59 cases. Donor-related factors, recipient age, indications for transplantation, transplantation techniques, and ischemia times were comparable between groups. Twenty-one (16%) of the patients in the standard perfusion group and only one of the those receiving arterial back table pressure perfusion developed ischemic biliary lesions. This difference was highly significant (P=0.004). Maximal aspartate aminotransferase and alanine aminotransferase levels in the first 3 days were significantly lower in the arterial back table pressure perfusion group (P>0.05).. Arterial back table pressure perfusion is an easy and reliable method for preventing ischemic biliary lesions in orthotopic liver transplantation. It should, therefore, be the standard technique in liver procurement.

Topics: Adenosine; Adult; Allopurinol; Angiography; Biliary Tract; Capillaries; Cholestasis; Female; Follow-Up Studies; Glutathione; Hepatic Artery; Humans; Hydrostatic Pressure; Insulin; Intraoperative Complications; Ischemia; Liver Function Tests; Liver Transplantation; Male; Middle Aged; Organ Preservation Solutions; Perfusion; Postoperative Complications; Raffinose; Reperfusion Injury

The purpose of this study is to examine the possibility of a long-term preservation of the ischemically damaged intestine by the cavitary two-layer method (TLM) in canine small intestinal transplantation.. The grafts were allotransplanted without preservation immediately (group 1) or after 30 minutes of warm ischemia (group 2). The ischemically damaged grafts were also allotransplanted after cold preservation for 24 hours in University of Wisconsin (UW) solution (group 3) or the cavitary TLM (group 4). Seven-day survivals, tissue adenosine triphosphate (ATP) concentrations, absorption tests, and histopathology were examined.. seven-day survivals in groups 1, 2, 3, and 4 were 8 of 8, 6 of 8, 0 of 8, and 6 of 8, respectively. In group 4, significant recovery of ATP tissue level was seen after preservation compared with group 3, and absorption function and regeneration of the graft mucosa recovered at day 14.. Ischemically damaged canine small intestine could be preserved for 24 hours by the cavitary TLM.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Dogs; Female; Glutathione; Graft Survival; Hot Temperature; Insulin; Intestine, Small; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Raffinose

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.

Topics: Adenosine; Allopurinol; Cadaver; Cell Separation; Glutathione; Humans; Insulin; Ischemia; Islets of Langerhans; Organ Preservation; Organ Preservation Solutions; Pancreas; Raffinose; Resuscitation; Tissue and Organ Harvesting; Tissue Donors

Topics: Adenosine; Allopurinol; Animals; Biological Transport; Cold Temperature; Dogs; Glutathione; Hepatocytes; Indocyanine Green; Insulin; Ischemia; Liver; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion

A two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method delivers sufficient oxygen to the pancreas during preservation and restores the ischemically damaged pancreas. In this study, we determined whether the additional preservation by the two-layer method could improve islet recovery from human pancreases with prolonged cold storage in UW.. Human pancreases were procured from cadaveric organ donors and preserved by the two-layer method (UW/PFC) for 2.9+/-0.7 hours (mean+/-SEM) at 4 degrees C after 11.8+/-1.5 hours of cold storage in UW (UW/PFC group, n=7), or by cold UW alone for 11.3+/-0.3 hours (UW group, n=14). The selected pancreases met the criteria of having at least 10 hours of cold storage in UW. All were processed by using a standard protocol of Liberase perfusion with Pefabloc by way of the duct, gentle mechanical dissociation, and Ficoll gradient purification. Transplanted islets were selected with the criteria of the Edmonton protocol (>5,000 islet equivalents [IE]/kg recipient body weight).. The islet recovery was significantly increased in the UW/PFC group compared with the UW group (349.2+/-44.1 x 10 and 214.0+/-31.0 x 10 IE, respectively; <0.05). This resulted in islet yields of 4.6+/-1.0 x 10 IE/g of pancreas in the UW/PFC group compared with 2.0+/-0.3 x 10 IE/g of pancreas in the UW group ( <0.05). Five of 7 cases (71%) in the UW/PFC group and 5 of 14 cases (36%) in the UW group were transplanted. The islet grafts in the UW/PFC group improved the ability of glycemic control and decreased exogenous insulin administration in all recipients.. Improvements in methods to preserve and recover ischemically damaged human pancreases before islet isolation and transplant could be extremely beneficial to the field of clinical islet transplantation. This preliminary study shows that additional short preservation by the two-layer (UW/PFC) cold-storage method can significantly improve islet recovery and increase opportunities of islet transplantation from human pancreases after prolonged cold ischemia.

Topics: Adenosine; Adult; Aged; Allopurinol; Cell Separation; Female; Fluorocarbons; Glutathione; Graft Survival; Humans; Hypothermia, Induced; Insulin; Insulin Secretion; Ischemia; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose

Topics: Adenosine; Allopurinol; Animals; Electron Spin Resonance Spectroscopy; Glutathione; Insulin; Iron; Ischemia; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Time Factors

Topics: Adenosine; Allopurinol; Animals; Blood Glucose; Glucose; Glutathione; Hyperinsulinism; Insulin; Ischemia; Mannitol; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Potassium Chloride; Procaine; Raffinose; Reperfusion; Swine; Temperature; Time Factors

Topics: Adenosine; Adult; Age Factors; Allopurinol; Body Mass Index; Cadaver; Cause of Death; Cell Separation; Female; Glutathione; Humans; Insulin; Ischemia; Islets of Langerhans; Male; Middle Aged; Organ Preservation Solutions; Raffinose; Retrospective Studies; Tissue and Organ Harvesting; Tissue Donors; Tissue Preservation

Topics: Adenosine; Allopurinol; Animals; Carrier Proteins; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Glutathione; Immunohistochemistry; Insulin; Intestine, Small; Ischemia; Jejunum; Male; Neoplasm Proteins; Nerve Tissue Proteins; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Time Factors

Hemodynamic disorders in brain dead organ donors induce hypoxia, warm ischemia and finally tissue damage. A cold preservation period also induces tissue and cellular lesions. The two major modes of preservation are cold storage (CS) and hypothermic pulsatile perfusion (HPP). We aimed to compare the influence of each mode of preservation and their combination on oxidative stress, perfusion characteristics and tissue damage, after a period of warm ischemia. Rat kidneys which had undergone ischemia (0, 30, 60 min) were preserved either by CS (12, 24 h), or by HPP (12 h), or by a combination of both (HPP+CS, CS+HPP), in University of Wisconsin cold storage solution (UWCSS) at + 4 degrees C. During HPP, renal vascular pressure decreased then increased to reach 90 mmHg after perfusion for 7 h. If HPP followed CS, the mean pressure reached 200 mmHg, showing successive high amplitude peaks. HPP had a deleterious effects on tissue structure with tubular necrosis, and induced an increase in catalase (Cat) and a decrease in manganese superoxide dismutase (Mn SOD) and gluthatione peroxidase (GPx) activity. Copper zinc superoxide dismutase (Cu/Zn SOD) activity was not reduced except with CS+HPP. During CS, we observed an increase in GPx, Cu/Zn SOD and Cat activity, a decrease in Mn SOD activity and no histological alterations in the kidney. CS induces a slight oxidative stress which is not important enough to induce major tissue damage. HPP with UWCSS induces a stronger stress, which overpowers the antioxidant defences, inducing tissue damage. The reperfusion of HPP with UWCSS emphasises the stress initiated by CS. In addition an increase in damage occurred in the CS + HPP group.

Topics: Adenosine; Allopurinol; Animals; Catalase; Cryopreservation; Glutathione; Glutathione Peroxidase; Hot Temperature; Insulin; Ischemia; Kidney; Male; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Perfusion; Pressure; Pulsatile Flow; Raffinose; Rats; Rats, Sprague-Dawley; Renal Circulation; Superoxide Dismutase

Human lung allografts can only be preserved for 6 hours. Experimental interventions that reduce ischemia-reperfusion (I/R) lung injury can be used to improve the properties of the preservation solution. The best solution for lung preservation is still a matter of controversy. The purpose of this study was to compare the protective effects of various solutions on I/R lung injury in Sprague-Dawley rats.. The following solutions were compared: a physiological salt solution; an intracellular preservation solution (the University of Wisconsin Solution, UW); an extracellular preservation solution (EP3); and the extracellular preservation solution with the addition of various protective agents--EP3 plus dexamethasone (Dex) (EP3-a), plus glutathione (GLU) and allopurinol (ALL) (EP3-b), and EP3 plus GLU, ALL, lactobionate (LACT), and raffinose (RAF) (EP3-c). I/R lung injury was induced by ischemia for either 45 or 60 minutes, followed by reperfusion for 60 minutes. Hemodynamic changes, lung weight gain (LWG), and capillary filtration coefficients were measured.. Both EP3 and UW preservation solutions had partial attenuation effects on I/R lung injury, but UW produced a better attenuation effect than EP3. Use of modified EP3 solutions containing either protective agents (GLU, ALL, or Dex) or impermeants (LACT and RAF) improved the ability of EP3 to reduce I/R lung injury. The LWG using the modified EP3-c solution was the lowest among all groups. UW induced pulmonary hypertension. After I/R challenge, pulmonary arterial pressure with EP3-c was lower than with UW. Based on a lower LWG and the changes in hemodynamics, EP3-c is a better lung preservation solution than UW and EP3.. Based on the attenuation of I/R injury, we conclude that there is no significant difference between intracellular UW and extracellular (EP3-a, EP3-b) preservation solutions in this rat model, but the addition of protective agents and impermeants to the solution are important. The findings suggest that EP3-c might be a better lung preservation solution than UW.

Topics: Adenosine; Allopurinol; Animals; Dextrans; Glucose; Glutathione; Hemodynamics; Heparin; Insulin; Ischemia; Lung; Lung Transplantation; Male; Organ Preservation Solutions; Organ Size; Phosphates; Prednisolone; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The development of biliary strictures (BSs) after liver transplantation (LT) continues to affect 10% to 30% of patients, causing substantial morbidity. The cause of BSs is multifactorial, including technical, immune, and, in particular, ischemic factors. The importance of adequate flushing of the peribiliary arterial tree has been stressed. We hypothesized that high-viscosity (HV) preservation solutions in the donor do not completely flush the small donor peribiliary plexus, leading to inadequate preservation of the bile ducts and posttransplant BSs. To test this hypothesis, we retrospectively compared the incidence of BSs in 2 groups of adults undergoing LT using different types of aortic preservation solution in the donor: group 1 (n = 24), low-viscosity (LV) Marshall solution; and group 2 (n = 27), HV University of Wisconsin (UW) solution. All donors in both groups received additional portal flushes with UW. All LTs were performed between November 1995 and August 1998 at 2 centers by the same surgeon, eliminating a technical bias. Terminal duct-to-duct anastomosis was performed in all recipients except 1 patient in group 1, who underwent a bile duct-to-jejunum anastomosis. BSs were first suspected on clinical and biochemical grounds and then confirmed by endoscopic retrograde cholangiopancreatography. Identical medical protocols were used in all patients. One-year patient survival rates in groups 1 and 2 were 92% and 100%, respectively (P =.9). One-year graft survival was identical to patient survival. The incidence of BSs in group 1 was 4.1% (1 of 24 patients), compared to 29.7% in group 2 (8 of 27 patients; P =.02). The BS in group 1 occurred 4 months post-LT and was anastomotic. BSs in group 2 occurred between 1 and 12 months post-LT and were anastomotic, extrahepatic, intrahepatic, or combined intrahepatic and extrahepatic. There were no significant differences in the following factors between groups 1 and 2: donor age, local versus imported liver, split-liver or full-liver transplantation, incidence of multiple vessels in the donor liver, indications for LT, recipient age, T-tube versus no T-tube, post-LT peak aspartate aminotransferase level, and treatment for rejection. There was no hepatic artery thrombosis or primary nonfunction in either group. Interestingly, cold ischemia time (CIT) was longer in group 1, which had the least incidence of BSs (692 +/- 190 v 535 +/- 129 minutes in group 2; P =.001). Follow-up was longer in group 1 (28.9 +/- 8.

Topics: Adenosine; Adult; Allopurinol; Aorta; Biliary Tract; Cold Temperature; Constriction, Pathologic; Glutathione; Graft Survival; Humans; Hypertonic Solutions; Insulin; Ischemia; Liver Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Retrospective Studies; Survival Rate; Time Factors; Tissue Donors; Viscosity

Prolonged cold ischemia has been shown to be an important factor in the development of posttransplant renal dysfunction. The exact mechanisms have not been completely defined. The expression of intercellular adhesion molecule-1 (ICAM-1) (CD 54) in rat kidneys stored in University of Wisconsin (UW) solution was studied in an attempt to correlate ischemia time with immunogenicity of the graft.. Kidneys from male Lewis rats were perfused with UW solution, removed, and bathed in UW solution at 4 degrees C for 4, 12, 24, and 48 h. For the evaluation of expression of ICAM-1, immunohistochemical staining, Western blotting, and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.. Immunohistochemical staining in normal, nonischemic kidneys revealed that glomerular capillaries expressed ICAM-1 but that tubular cells did not. The preserved kidneys were analyzed by immunohistochemistry, Western blotting, and semiquantitative RT-PCR and showed increased transcription and expression of ICAM-1 in the cortex of the kidney. Expression reached a maximum at 24 h and declined at 48 h. The ICAM-1 protein expression in the preserved kidney cortex relative to control kidneys was increased at 4 h (1.68 +/- 0.60-fold of control kidneys, P = 0.06), 12 h (2.38 +/- 0.90-fold, P = 0.02), 24 h (3.70 +/- 1.29-fold, P = 0.01), and 48 h (2.00 +/- 0.54-fold, P = 0.01). The messenger RNA expression (the ratio of ICAM-1 to glyceraldehyde-3-phosphate dehydrogenase) in preserved kidneys cortex relative to control kidneys was increased at 4 h (1.19 +/- 0.14-fold of control kidneys), 12 h (1.38 +/- 0.16-fold), 24 h (1.77 +/- 0.29-fold), and 48 h (1.19 +/- 0.12-fold) (P < 0.05 for all time points).. We conclude that cold preservation of rat kidneys in UW solution induces increasing levels of ICAM-1 cell surface expression and gene transcription. Further study is necessary to determine if this increase in adhesion molecule expression increases the immunogenicity of the allograft and contributes to the development of posttransplant renal dysfunction.

Topics: Adenosine; Allopurinol; Animals; Blotting, Western; Cryopreservation; Gene Expression; Glutathione; Immunohistochemistry; Insulin; Intercellular Adhesion Molecule-1; Ischemia; Kidney Cortex; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Despite the established preservation method for major organs of perfusion followed by immersion at hypothermia, a standard preservation technique for skeletal muscle is still a matter of controversy. The purpose of this study is to examine the effect of perfusion on the preservation of skeletal muscle in amputated limbs.. The rat hindlimb was amputated for perfusion with Euro-Collins (EC) or University of Wisconsin (UW) solution at different perfusion pressures (40 or 100 cm-gravity). After certain ischemic periods (4 or 5 h), the skeletal muscle viability was determined by measuring the tissue content of adenosine triphosphate (ATP).. The UW solution perfusion group maintained better ATP levels than the EC solution group when the ischemic period was extended to 5 h. The perfusion pressure of 100 cm-gravity was more effective for preserving muscle viability than 40 cm-gravity with both EC and UW solutions.. UW solution might be adequate to preserve muscle viability and perfusion pressure is recommended at 100 cm-gravity rather than at minimal pressure (40 cm-gravity), which washes out stagnant blood.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Hypertonic Solutions; Insulin; Ischemia; Male; Muscle, Skeletal; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Tissue Preservation

Topics: Adenosine; Allopurinol; Cold Temperature; Diuresis; Glomerular Filtration Rate; Glutathione; Humans; Insulin; Ischemia; Kidney; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Time Factors; Vascular Resistance

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glutathione; Insulin; Ischemia; Kidney; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion; Trimetazidine

With increasing time of cold preservation, levels of high-energy nucleotides in the liver are reducing. The authors hypothesized that cold preservation sensitizes hepatocyte function to ischemic injury occurring during graft rewarming and that the injury can be prevented by short-term reperfusion. Rat livers were cold-preserved in University of Wisconsin solution for 0 to 18 hours and ischemically rewarmed for 0 to 45 minutes to simulate the implantation stage of transplantation. Hepatobiliary function was assessed using a blood-free perfusion model. In comparison with controls, neither 18-hour preservation nor 45-minute ischemic rewarming significantly influenced hepatocyte function. Compared with livers subjected to 45-minute ischemic rewarming, livers subjected to 9-hour preservation and 45-minute rewarming, and livers subjected to 18-hour preservation and 45-minute rewarming exhibited, respectively: 3.8 and 24 times reduced bile production, 4.3- and 116-fold decreased taurocholate excretion, and 3.1 and 42 times depressed bromosulfophthalein excretion. Thirty-minute oxygenated warm reperfusion after 9- and 18-hour preservation nearly completely blunted sensitization of hepatocyte function to rewarming ischemia. The authors found that short-term oxygenated reperfusion restored adenine nucleotides in liver tissue to the values found before organ preservation and that reperfusion with energy substrate containing solutions increased tissue adenosine triphosphate concentration to a higher level than that found before preservation. In conclusion, sensitization of hepatocyte function to rewarming ischemia increases disproportionally with storage time, suggesting that this phenomenon may play a role in graft dysfunctions with increasing liver preservation time. Short-term oxygenated reperfusion of the liver may protect hepatocyte functions against warm ischemic insult, even after extended preservation.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Glutathione; Insulin; Ischemia; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion; Time Factors

A brief period of liver ischemia decreases sinusoidal endothelial cell killing after cold liver storage and improves graft survival after liver transplantation, a phenomenon called ischemic preconditioning. In this study, we investigated the mechanism of sinusoidal endothelial cell protection after ischemic preconditioning. Livers were preconditioned by 5 minutes of ischemia and 5 minutes of reperfusion. Subsequently, livers were stored for 30 hours in cold University of Wisconsin (UW) solution and reperfused briefly with physiological buffer containing Trypan blue. Ischemic preconditioning decreased sinusoidal endothelial cell killing after storage/reperfusion, as assessed by Trypan blue staining of nonparenchymal cells. Adenosine A(2) receptor blockade prevented the protective effect of ischemic preconditioning. By contrast, adenosine A(1) receptor blockade did not prevent protective ischemic preconditioning. Other rat livers were treated with adenosine A(1) and A(2) receptor agonists or dibutyryl-cyclic adenosine monophosphate (DB-cAMP) before storage. The adenosine A(2) receptor agonist, CGS-21680, and DB-cAMP decreased sinusoidal endothelial cell killing to the same extent as ischemic preconditioning, but the adenosine A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), had no effect. The adenosine A(2) agonist and prostaglandin E(2), another agent that preconditions sinusoidal endothelial cells against storage/reperfusion injury, but not the adenosine A(1) agonist, increased cAMP levels in cultured sinusoidal endothelial cells. In conclusion, an adenosine A(2) receptor pathway coupled to increased cAMP mediates sinusoidal endothelial cell protection by ischemic preconditioning.

Topics: Adenosine; Allopurinol; Animals; Cyclic AMP; Endothelium, Vascular; Glutathione; Insulin; Ischemia; Ischemic Preconditioning; Liver; Male; Organ Preservation Solutions; Purinergic P1 Receptor Agonists; Raffinose; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion Injury

Questions as to the critical stress factor and primary targets of cold ischemia/reperfusion (CIR) injury were addressed by comparing mitochondrial defects caused by (1) CIR injury and (2) intracellular Ca2+ overload. CIR was simulated in transformed human umbilical vein endothelial cell cultures (tEC) by 8 h cold anoxia in University of Wisconsin solution and reoxygenation at 37 degrees C. Intracellular Ca2+ concentrations were changed by permeabilization of suspended cells with digitonin in culture medium (RPMI, 0.4 mM Ca2+). Binding of free Ca2+ by ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid in RPMI or mitochondrial incubation medium served as controls. Extracellular Ca2+ protected the cell membrane against permeabilization. Mitochondrial functions were determined before and after permeabilization of the cell membrane. After CIR, mitochondrial respiratory capacity declined, but oxygen consumption remained coupled to adenosine triphosphate (ATP) production. In contrast, Ca2+ overload caused uncoupling of mitochondrial respiration. High intracellular Ca2+ overload, therefore, does not reproduce cold ischemia/reperfusion injury in endothelial cells.

Topics: Adenosine; Allopurinol; Calcium; Cell Hypoxia; Cell Line, Transformed; Cell Membrane Permeability; Cells, Cultured; Cold Temperature; Egtazic Acid; Endothelium, Vascular; Glutathione; Humans; Insulin; Ischemia; Mitochondria; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Reperfusion Injury; Umbilical Veins

Changes in cytosolic and mitochondrial calcium content were studied in an endothelial cell model after simulating cold ischemia reperfusion injury. Image analysis demonstrated that only a subpopulation of mitochondria in endothelial cells accumulate calcium. Observations led to the hypothesis that mitochondria which are in close contact with the plasma membrane are mainly affected by the Ca2+ efflux across that membrane, while those in other parts of the cell remain unaffected.

Topics: Adenosine; Allopurinol; Calcium; Cells, Cultured; Cytosol; Endothelium, Vascular; Glutathione; Humans; Insulin; Ischemia; Mitochondria; Organ Preservation Solutions; Raffinose; Reperfusion; Umbilical Veins

Topics: Adenosine; Allopurinol; Animals; Glutathione; Heart Arrest; Hepatic Veins; Insulin; Ischemia; Liver; Liver Circulation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Superoxide Dismutase; Tissue Donors

Topics: Adenosine; Allopurinol; Animals; Catalase; Glutathione; Glutathione Peroxidase; Insulin; Ischemia; Isoenzymes; Kidney; Kidney Tubular Necrosis, Acute; Models, Animal; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion; Superoxide Dismutase; Temperature

Applying the orthotopic rat liver transplantation (ORLT) model, postoperative survival has been shown to be mainly dependent on the portal vein clamping time (PVCT). It was hypothesized that prolonged intestinal congestion was responsible for the activation of Kupffer cells (KC) with overproduction of TNF, secondary to splanchnic endotoxin accumulation and release on reperfusion. The role of KCs was directly investigated in the context of long PVCTs by eliminating them (using liposome-encapsulated dichloromethylene diphosphonate), by preventing their activation (using a calcium channel blocker, nisoldipine) and by inhibiting TNF production (using thalidomide). Livers from different groups of rats were transplanted following 24-h cold preservation in the UW solution with long PVCTs (from 18-21 min). KCs depletion, preservation with nisoldipine and pretreatment with thalidomide significantly improved survival in conditions using long PVCTs. KC depletion and nisoldipine preservation had no effect on liver enzymes or pathological findings while lung injury was significantly improved. The present data confirm that, in the context of ORLT with long PVCTs, KCs are directly responsible for the systemic endotoxin-like shock syndrome and their effect is mediated through overproduction of TNF.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Allopurinol; Animals; Calcium Channel Blockers; Cell Count; Clodronic Acid; Cold Temperature; Constriction; Glutathione; Graft Survival; Hepatectomy; Immunosuppressive Agents; Insulin; Ischemia; Kupffer Cells; Liposomes; Liver; Liver Transplantation; Lung; Macrophage Activation; Male; Nisoldipine; Organ Preservation Solutions; Portal Vein; Postoperative Complications; Raffinose; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Shock; Thalidomide; Time Factors; Tissue and Organ Procurement; Tumor Necrosis Factor-alpha

A method is described for isolating human hepatocytes from tissue fragments after warm and cold ischemia as experienced during hepatic resections. Cells with a high trypan blue dye exclusion and good culture characteristics were isolated by employing an initial tissue perfusion with UW solution. The method could facilitate transfer of liver tissues between distant centers for cell isolation studies.

Topics: Adenosine; Adult; Aged; Allopurinol; Cell Separation; Cold Temperature; Female; Glutathione; Hepatectomy; Humans; In Vitro Techniques; Insulin; Ischemia; Liver; Male; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Temperature

Ischemia triggers secretion of proteins from the intestine, including type II secretory phospholipase A2 (sPLA2). This "secretory event" was studied in intestinal grafts during the first few hours of preservation by measuring total protein, sPLA2, and other enzymes in the UW preservation solution over time. The effect of PX-13, a PLA2 inhibitor, was also studied.. Twenty-five centimeter intestinal grafts were harvested from Lewis rats, flushed, and preserved in UW solution +/- PX-13 at 4 degrees C. UW samples from 0 to 48 h (n = 5 each) were analyzed for total protein, sPLA2, lactate dehydrogenase (LDH), N-acetylglucosamine (NAGA), and lysozyme. Nonpreserved grafts were homogenized in PBS as tissue controls. Standard biochemical methods were used for all assays.. Total protein increased rapidly by 5 min, continued to rise more slowly until 30 min, and then stabilized. The most significant increase in sPLA2 activity occurred between 90 and 180 min. NAGA increased most markedly between 30 and 180 min, while LDH increased in the first 30 min, although the level of both enzymes was negligible compared to tissue enzyme. Lysozyme levels were minimal at all times. PX-13 decreased sPLA2 activity markedly at all time points.. Total protein levels increased before sPLA2, suggesting that sPLA2 may be secreted in response to other proteins or enzymes released even earlier during preservation (e.g., cytokines). These elevations do not appear to be caused by cell death. Phospholipase A2 secretion may be blocked, and this may greatly improve the outcome of intestinal preservation.

Topics: Acetylglucosamine; Adenosine; Allopurinol; Animals; Cryopreservation; Glutathione; Insulin; Intestinal Mucosa; Intestines; Ischemia; L-Lactate Dehydrogenase; Male; Muramidase; Organ Preservation Solutions; Phospholipases A; Phospholipases A2; Proteins; Raffinose; Rats; Rats, Inbred Lew

Reperfusion injury following lung preservation has been associated with free radical formation and subsequent endothelial cell damage. Trolox is a water-soluble analogue of the free radical scavenger alpha-tocopherol. We hypothesized that addition of this form of vitamin E to University of Wisconsin (UW) solution would decrease reperfusion injury and improve lung function after cold ischemic preservation.. Bovine aortic endothelial cells were cultured and stored at 4 degrees C for 12, 24, and 48 h in UW or UW + Trolox (UWT). Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. An isolated rat perfused lung (IPL) model was used and lungs were flushed with the respective solutions with cold storage times of 6 and 12 h. Following storage, the lungs were reperfused with fresh blood and lung function was assessed by blood gas analysis, alveolar-arterial gradient, and compliance.. There was no difference in endothelial cell viability between UW and UWT after 12 or 24 h; however, UWT had higher endothelial cell viability than UW with 48 h of cold ischemic storage. Using the IPL model, the pO2 was higher with UWT than UW after 6 and 12 h of cold ischemia. The alveolar-arterial oxygen difference was significantly lower for UWT versus UW at 6 h. UWT provided increased compliance at 6 and 12 h of ischemia.. The addition of a water-soluble vitamin E analogue to UW solution resulted in increased endothelial cell viability after prolonged storage and improved whole lung preservation in the postreperfusion period as evidenced by higher oxygenation and increased compliance. These results are clinically relevant as the lung is extremely sensitive to reperfusion injury and UW solution is being increasingly used in lung transplantation and remains the predominant solution in abdominal organ transplantation.

Topics: Adenosine; Allopurinol; Animals; Cattle; Cells, Cultured; Child; Chromans; Endothelium, Vascular; Glutathione; Humans; In Vitro Techniques; Insulin; Ischemia; Lung; Organ Preservation Solutions; Pulmonary Circulation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solubility; Vitamin E

In order to investigate locally produced mediators during the process of organ storage in liver transplantation, we collected the liver preservation solution effluent of 15 transplanted livers and compared it with serum samples taken preoperatively from donor and recipient, as well as 60 min after reperfusion. The mean ischemia time +/- SEM was 10 h 10 min +/- 53 min. Mean concentrations in University of Wisconsin preservation solution effluent were: interleukin-(IL-)1beta 154 +/- 77 pg/ml; IL-1 receptor antagonist (IL-1 ra) 1281 +/- 309 pg/ml; IL-6 412 +/- 90 pg/ml; and for tumor necrosis factor-(TNF-)alpha 74 +/- 21 pg/ml. Cytokine levels in the donors were lower than those detected in the effluent. All measured cytokines showed higher concentrations in the effluent compared to those of the recipient prior to the operation. With respect to a comparison of donor and recipient values, no correlation is evident. Likewise, the ischemic time does not correlate with effluent values. Further development of liver preservation concepts requires information about the state of the graft before reperfusion. Data on cytokine liberation may serve as a helpful tool for the further development of preservation concepts because they enable an estimation of cell activation during preservation.

Topics: Adenosine; Adult; Allopurinol; Cytokines; Female; Glutathione; Humans; Insulin; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-6; Ischemia; Liver; Liver Transplantation; Male; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Sialoglycoproteins; Tissue Donors; Tumor Necrosis Factor-alpha

Topics: Adenosine; Allopurinol; Animals; Glutathione; Heart Arrest; Insulin; Ischemia; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Rabbits; Raffinose; Renal Artery; Renal Veins; Reperfusion; Time Factors; Tissue Donors

Pyruvate dehydrogenase has been thought to be involved in the improved recovery of livers, from fasted donors, reperfused with alanine after cold preservation. The aim of this work was to investigate the effect on perfused mouse liver of dichloroacetate, an activator of this enzyme. Livers from fed and fasted animals were perfused with oxygenated Krebs-Henseleit buffer for 30 min, then stored at 4 degrees C in University of Wisconsin solution for 48 h. Then reperfusion at 37 degrees C was performed with Krebs-Henseleit buffer containing 2 mM dichloroacetate for 1 h. (3-(13)C)Alanine (8 mM) was then added and perfusion was continued for a second hour. (31)P-NMR was used to measure nucleoside triphosphate recovery of the livers. At the end of reperfusion, (13)C-NMR spectra of perfusates were recorded. Dichloroacetate (DCA) was found to activate pyruvate dehydrogenase in all cases. However, it decreased the functional recovery of livers from both fed and fasted mice. In order to study the effect of alanine on this DCA deleterious effect, we reperfused the livers according to a modified protocol. The first hour of perfusion without alanine was omitted and the organs were reperfused directly for 1 h in the presence of 2 mM dichloroacetate and 8 mM (3-(13)C)alanine. In this protocol, the deleterious effect of DCA was completely suppressed for livers from fasted mice. These results led to the conclusion that the specific beneficial effect of alanine on livers from fasted livers persists in the presence of DCA and thus cannot be explained solely by the induction of a greater pyruvate dehydrogenase reaction rate.

Topics: Adenosine; Alanine; Allopurinol; Animals; Carbon Isotopes; Cold Temperature; Cryopreservation; Dichloroacetic Acid; Fasting; Glutamine; Glutathione; Insulin; Ischemia; Liver; Male; Mice; Nuclear Magnetic Resonance, Biomolecular; Organ Preservation; Organ Preservation Solutions; Phosphorus; Raffinose; Reperfusion Injury; Tissue Donors

The present study was devised to elucidate the influence of prolonged cold ischemia on the development of chronic transplant dysfunction (CTD) in kidney isografts (Brown Norway-->Brown Norway; BN-->BN) and in kidney allografts (BN-->Wistar Agouti/ Rij [WAG]) under temporary cyclosporine (CsA) therapy.. To induce ischemic injury, BN donor kidneys were preserved for 24 hr in 4 degrees C University of Wisconsin solution before transplantation. Renal function (proteinuria), histomorphology according to the BANFF criteria for CTD, and infiltrating cells were assessed. Grafts were examined both early at days 2, 3, 6, and 10, and late at week 26 (allografts) or at week 52 (isografts).. Nonischemic isografts preserved a normal function and morphology. Ischemic isografts developed a progressive proteinuria over time and demonstrated significantly more glomerulopathy with macrophage (Me) infiltration and intimal hyperplasia than nonischemic controls at week 52. During the initial 10 days, there was an increased infiltration of MHC class II+ cells, predominantly CD4+ cells and Mphi, coinciding with up-regulated intercellular adhesion molecule-1 expression. CsA treatment in ischemic isografts inhibited infiltration of MHC II+ cells in the early stage, which was accompanied by significantly less renal damage at week 52 compared with untreated controls (proteinuria: 59+/-8 vs. 134+/-19 mg/24 hr; BANFF score: 2.8+/-0.4 vs. 4.3+/-1.0). Under CsA therapy, 24-hr cold ischemia of the allograft affected neither the onset or progress of proteinuria, nor the histomorphology (BANFF score: 7.8+/-2.4 vs. 7.3+/-1.9). In both ischemic and nonischemic allografts, intercellular adhesion molecule-1 expression and mononuclear cell infiltration (CD4, CD8, Mphi was abundantly present during the first 10 days and function deteriorated rapidly.. Prolonged cold ischemia plays a role in the induction of CTD, but its deleterious effect can be successfully inhibited by CsA. Therefore, the alloantigeneic stimulus is the overriding component in the multifactorial pathogenesis of CTD.

Topics: Adenosine; Allopurinol; Animals; Cryopreservation; Cyclosporine; Glutathione; Graft Rejection; Immunohistochemistry; Immunosuppressive Agents; Insulin; Ischemia; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred BN; Rats, Wistar; Time Factors; Transplantation, Homologous; Transplantation, Isogeneic

Orthotopic rat liver transplantation (ORLT) following extended cold preservation in University of Wisconsin (UW) solution has been shown to induce alterations of the hepatic microcirculation, mainly characterized by areas of no-reflow. The present study was performed to determine whether these alterations were related to the portal vein clamping time (PVCT), shown to be the main determinant of survival after ORLT. The hepatic microcirculation was evaluated using the multiple-indicator dilution curve (MIDC) technique after ORLT following 24-hour cold ischemia in UW solution. Two groups of rats were studied: one with PVCTs of less than 14 min (survival conditions) and one with PVCTs of more than 18 min (nonsurvival conditions). Four hours after ORLT, only long PVCTs were associated with small, but significant, nonperfused areas, about 10% of the liver not being perfused by water; however, in both survival and nonsurvival conditions, the sinusoidal sieving function was well-maintained in perfused areas. In addition, liver viability parameters and hepatocyte function were similarly and minimally altered. The hepatic microcirculation is minimally altered 4 h after ORLT following extended cold preservation in UW solution, whatever the survival condition. Although only found after long PVCTs, the low magnitude of areas of no-reflow should not be associated with lethal injury of the transplanted liver, a finding further supporting the concept that survival after ORLT following 24-hour cold preservation in UW solution is mainly influenced by extrahepatic factors.

Topics: Adenosine; Allopurinol; Animals; Constriction; Cryopreservation; Glutathione; Graft Survival; Insulin; Ischemia; Liver; Liver Circulation; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Portal Vein; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Tissue and Organ Harvesting

The aim of this study was to compare the effects of Euro-Collins and University of Wisconsin solutions on pulmonary mitochondrial function after cold ischemia and subsequent warm reperfusion.. Seventeen pigs underwent lung harvesting after classical lung flush with either University of Wisconsin or Euro-Collins solutions. The mitochondria were isolated from fresh swine lungs, from swine lungs subjected to 24 hr of cold ischemia, and from swine lungs subjected to 24 hr of ischemia followed by 30 min of subsequent ex vivo reperfusion at 37 degrees C with Krebs-Henseleit buffer solution and air ventilation. Mitochondrial oxidative phosphorylation parameters were determined in isolated mitochondria by in vitro measurement of oxygen consumption rates. During reperfusion, the lung function was assessed by the pulmonary aerodynamic parameters and the pulmonary vascular resistance.. Relative to controls, mitochondria submitted to cold ischemia showed an alteration in the oxidoreductase activities of the respiratory chain. However, the yield of oxidative phosphorylation was conserved. After reperfusion, pulmonary mitochondria underwent a significant worsening in the oxidoreductase activities of the respiratory chain, and a decrease in the respiratory control and the efficiency of oxidative phosphorylation. Meanwhile, the reperfused lungs showed evidence of early dysfunction, assessed by the aerodynamic parameters and pulmonary vascular resistance. In this model, there was no advantage of University of Wisconsin solution over Euro-Collins solution.. The mild mitochondrial alterations after cold ischemia were not sufficient to explain the limited tolerance of lung to ischemia. After reperfusion, the mitochondrial damage was more severe and could be involved in the posttransplant lung dysfunction.

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Female; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Lung; Male; Mitochondria; Organ Preservation Solutions; Oxidation-Reduction; Oxygen Consumption; Phosphorylation; Raffinose; Reperfusion; Swine; Vascular Resistance

Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown.. We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired.. Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage.. We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.

Topics: Adenosine; Allopurinol; Animals; Bile; Bile Ducts; Cold Temperature; Glutathione; In Vitro Techniques; Insulin; Ischemia; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Regression Analysis; Reperfusion; Time Factors

Topics: Adenosine; Allopurinol; Amino Acids; Cold Temperature; Glutathione; Insulin; Ischemia; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Temperature; Time Factors

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Cold Temperature; Endothelium; Glutathione; Hyaluronic Acid; Insulin; Ischemia; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Swine; Temperature; Time Factors

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Intestine, Small; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Phospholipases A; Proteins; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors

The organ donor shortage has led to a reconsideration of the use of non-heart-beating donors (NHBDs). However, graft injury due to warm ischemia in NHBD livers strongly affects posttransplant outcome. The present study was aimed at investigating the role of the cellular cyclic (c)AMP second messenger signal with regard to hepatic viability after cold preservation of NHBD livers.. Cardiac arrest was induced in Wistar rats by frenotomy of the anesthetized nonheparinized animal. After 30 min, the livers were excised and flushed with 20 ml of heparinized saline solution, rinsed with 10 ml of University of Wisconsin (UW) solution, and stored submerged in UW solution at 4 degrees C for 24 hr. In half of the experiments, UW solution was supplemented with glucagon (0.5 microg/ml) to increase the cAMP signal in the liver. Reperfusion was carried out in vitro after all livers were incubated at 25 degrees C in saline solution to replicate the period of slow rewarming during surgical implantation in vivo.. Hepatic levels of cAMP (nmol/g dry weight) declined from 1.21+/-0.05 to 0.53+/-0.03 (P<0.01) at 30 min after cardiac arrest. Subsequent storage in UW solution resulted in a further decline to 0.35+/-0.04 after 24 hr in group A, whereas glucagon treatment enhanced cellular cAMP signal to 0.64+/-0.06 (P<0.01). Upon reperfusion, liver integrity was significantly improved after glucagon administration, with 66% reduction in alanine aminotransferase release and a threefold increase in hepatic bile production as compared with untreated livers. Moreover, liver ATP tissue levels were restored to only 2.19+/-0.51 micromol/g in the untreated group but reached 4.97+/-0.41 micromol/g (P<0.05) after treatment with glucagon.. Posthoc conditioning of predamaged livers by glucagon enhances cAMP tissue levels during ischemic preservation and improves hepatic integrity upon reperfusion. This may represent a promising approach for the use of livers from non-heart-beating donors in clinical transplantation.

Topics: Adenosine; Adenosine Triphosphate; Alanine Transaminase; Allopurinol; Animals; Bile; Cryopreservation; Cyclic AMP; Glucagon; Glutathione; Heart Arrest; Insulin; Ischemia; Liver; Liver Circulation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion; Signal Transduction; Tissue Donors

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Aspartate Aminotransferases; Cold Temperature; Glutathione; Humans; Insulin; Ischemia; Liver; Liver Circulation; Liver Transplantation; Microcirculation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Regression Analysis; Reperfusion Injury; Time Factors

Topics: Acetylcysteine; Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Choline Deficiency; Fatty Liver; Glutathione; Insulin; Intercellular Adhesion Molecule-1; Ischemia; Liver; Male; Methionine; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Topics: Adenosine; Aerobiosis; Allopurinol; Anaerobiosis; Animals; Glutathione; Hypoxia; Insulin; Ischemia; Liver; Male; Microscopy, Fluorescence; NAD; Organ Preservation Solutions; Oxidation-Reduction; Oxygen; Oxygen Consumption; Raffinose; Rats; Rats, Wistar

Triple therapy with S-adenosylmethionine (SAM) (given to the donor animal, included in University of Wisconsin solution [UW], and added to the reperfusing medium) has been shown to reduce the sequential cold and warm ischemia/reperfusion injuries characteristic of the liver transplantation procedure. To clarify the actions of SAM during different stages of ischemia/ reperfusion, we have compared its benefit in five dosage regimens, using perfused rat livers after sequential periods of 24 hr cold and 20 min rewarming ischemia. When added only to UW, the presence of SAM throughout ischemia improved hepatic blood flow by 26% after 15 min of reperfusion versus no treatment (2.32+/-0.18 vs. 1.84+/-0.11 ml/min/g liver, P<0.05). SAM also improved blood flow by 23% during the 3-hr perfusion overall (P<0.05). Oxygen consumption and the release of purine nucleoside phosphorylase (PNP) were decreased (both P<0.05). When added to both UW and the perfusate, SAM additionally increased bile production at 15 min (7.14+/-1.21 vs. 2.31+/-0.74 mg/h/g liver, P<0.01). By pretreating the liver donor with SAM in vivo, and including it in the preservation and reperfusing media, it was possible to prolong and amplify the benefits on blood flow (P<0.001) and bile production (P<0.05) and to sustain glucose uptake (P<0.01). An acute exposure to SAM, when used in saline to flush UW from the graft before reperfusion, increased blood flow at 15 min (by 68%) and over a 3-hr period (both P<0.001), but no indices of metabolic activity were improved. Oxygen consumption and PNP release were both decreased (P<0.05). When added to the perfusate (present throughout reperfusion), SAM increased blood flow at 15 min (58%) and over a 3-hr period (P<0.01 in both cases). Net glucose uptake was increased (P<0.05), whereas oxygen consumption (P<0.001) and PNP release fell (P<0.05). Actions of SAM achieved acutely and over the intermediate- and long-term all seem to underlie its benefits in reducing ischemia/reperfusion injuries.

Topics: Adenosine; Allopurinol; Animals; Bile; Glutathione; Insulin; Ischemia; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; S-Adenosylmethionine

A method to reduce ischemia-reperfusion (I/R) injury can be an important criterion to improve the preservation solution. Although University of Wisconsin solution (UW) works as a lung preservation solution, its attenuation effect on I/R injury has not been investigated. We attempted to determine whether, by adding various protective agents, modified UW solutions will enhance the I/R attenuation by UW. We examined the I/R injury in an isolated rat lung model. Various solutions, e.g., physiological salt solution (PSS), UW, and modified UW solutions containing various protective agents such as prostaglandin E1, dexamethasone, U-74389G, or dibutyryl adenosine 3',5'-cyclic monophosphate were perfused individually to evaluate the I/R injury. Isolated rat lung experiments, with ischemia for 45 min, then reperfusion for 60 min, were conducted in a closed circulating system. Hemodynamic changes, lung weight gain (LWG), capillary filtration coefficient (Kfc), protein content of lavage fluid, concentration of cytokines, and lung histopathology were analyzed. Results showed that the acute I/R lung injury with immediate permeability pulmonary edema was associated with an increase in tumor necrosis factor-alpha (TNF-alpha) production. A significant correlation existed between TNF-alpha and Kfc (r = 0.8, P < 0.0001) and TNF-alpha and LWG (r = 0. 9, P < 0.0001), indicating that TNF-alpha is an important cytokine modulating early I/R injury. Significantly lower levels of Kfc, LWG, TNF-alpha, and protein concentration of lung lavage (P < 0.05) were found in the UW-perfused group than in the control group perfused with PSS. Modified UW promoted the protective effect of UW to further decrease Kfc, LWG, and TNF-alpha (P < 0.05). Histopathological observations also substantiated this evidence. In the UW+U-74389G group, bronchial alveolar lavage fluid contained lowest protein concentration. We conclude that the UW solution attenuates I/R injury of rat lung and that the modified UW solutions further enhance the effect of UW in reducing I/R injury. Among modified solutions, UW+U-74389G is the best. Further investigation of the improved effects of the modified UW solutions would be beneficial in lung transplantation.

Topics: Adenosine; Allopurinol; Alprostadil; Animals; Bucladesine; Dexamethasone; Drug Synergism; Glutathione; Hemodynamics; In Vitro Techniques; Insulin; Interleukin-1; Ischemia; Lung; Male; Organ Preservation Solutions; Pregnatrienes; Pulmonary Circulation; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glutathione; Hypoxanthine; Insulin; Ischemia; Jejunum; Mesenteric Artery, Superior; Organ Preservation; Organ Preservation Solutions; Oxygen; Phosphocreatine; Portal Vein; Raffinose; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury

Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs.. The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion.. Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion.. The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Kidney; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Renal Circulation; Reperfusion Injury; Swine; Trimetazidine

Topics: Adenosine; Allopurinol; Animals; Buffers; Carnosine; Cold Temperature; Glutathione; Glycolysis; Histidine; Insulin; Ischemia; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley

The impact of storage solution composition on graft performance was evaluated following perfusion with either Euro-Collins (EC), low potassium Euro-Collins (rEC), low potassium dextran (LPD) or University of Wisconsin solution (UW) after brief (2 h) and extended ischemia (16 h) in an acute double lung transplantation model in the rat.. Following flush perfusion and ischemia the lungs were implanted in recipient rats allowing serial assessment of graft pulmonary vascular resistance (PVR) and alveolar arterial oxygen difference (AaDO2) during 120 min of reperfusion. Graft dynamic lung compliance (DLC) was determined by separate ventilation. Final evaluation included weight gain and histology.. After extended ischemia LPD provided superior graft function in respect to DLC (repeated measures ANOVA; LPD versus rEC P < 0.05; versus EC P < 0.03; versus UW P < 0.05) and AaDO2 (LPD versus rEC P < 0.04; versus EC P < 0.006). The PVR was significantly lower in LPD versus UW (P < 0.05). At the end of reperfusion the weight increase amounted to 229 +/- 49% in rEC, 207 +/- 22% in EC, 115 +/- 22% in UW and 87 + 17% in LPD (LPD versus rEC P < 0.01, LPD versus EC P < 0.001). The type of preservation solution used had little impact on graft function after 2 h ischemia.. Low potassium dextran provides superior graft function after extended ischemia. After short ischemia the type of preservation solution used in this study had little impact on global lung function.

Topics: Adenosine; Allopurinol; Animals; Bronchoalveolar Lavage Fluid; Dextrans; Glutathione; Hypertonic Solutions; Insulin; Ischemia; Lung; Lung Transplantation; Male; Organ Preservation Solutions; Phospholipids; Potassium; Proteins; Raffinose; Rats; Rats, Inbred Lew; Time Factors

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Anticoagulants; Bile; Cold Temperature; Gabexate; Glutathione; Insulin; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Portal System; Raffinose; Rats; Rats, Inbred Lew; Regional Blood Flow; Reperfusion; Reperfusion Injury; Time Factors

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Energy Metabolism; Glutathione; Heart; Heart Transplantation; Hemodynamics; Insulin; Ischemia; Myocardial Contraction; Organ Preservation; Organ Preservation Solutions; Phosphocreatine; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Time Factors; Transplantation, Heterotopic; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Animals; Blood; Blood Pressure; Glutathione; Hypertonic Solutions; Insulin; Ischemia; Lung; Lung Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Pneumonectomy; Pulmonary Artery; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Trehalose

Topics: Acetylcysteine; Adenosine; Allopurinol; Analysis of Variance; Animals; Bucladesine; Dogs; Endothelium, Vascular; Gluconates; Glutathione; Hydroxyethyl Starch Derivatives; Insulin; Ischemia; Lung; Lung Transplantation; Microscopy, Electron, Scanning; Nitroglycerin; Organ Preservation; Organ Preservation Solutions; Oxygen; Phosphates; Raffinose; Time Factors; Trehalose

Topics: Adenosine; Allopurinol; Animals; Bile Ducts; Bilirubin; Cardiopulmonary Bypass; Dogs; Glucose; Glutathione; Heart Arrest; Humans; Insulin; Ischemia; Liver; Liver Transplantation; Mannitol; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Swine; Temperature; Time Factors; Tissue Donors; Transplantation, Heterotopic

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Glutathione; Graft Survival; Heart Arrest; Hepatectomy; Humans; Insulin; Ischemia; Liver; Liver Circulation; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion; Tissue Donors

Ischemic preconditioning has not been assessed in an experimental model for myocardial preservation during heart transplantation. Using isolated working rat hearts, ischemic preconditioning was investigated as an adjunct to isolated hypothermic (group 1), crystalloid (group 2: University of Wisconsin solution; group 3: St. Thomas' Hospital cardioplegic solution II; group 4: Bretschneiders' cardioplegic solution), and noncrystalloid (group 5: cold blood cardioplegia) preservation during a 10-hr period of global ischemia at 4 degrees C. After acquisition of functional baseline data, ischemic preconditioning was induced with one cycle of 5 min of normothermic ischemia and 5 min of reperfusion before induction of global hypothermic ischemia (n= 10/group). Nonpreconditioned hearts (n= 10/group) were assessed for control. Ischemic preconditioning improved postischemic: functional recovery. Thus, aortic flow after 60 min of reperfusion recovered to 0%, 8%, 0%, 1% and 0% in control groups 1 to 5 without ischemic preconditioning and 21%, 25%, 10%, 8%, and 3% in groups 1 to 5 with ischemic preconditioning. The same pattern of recovery was observed in regard to postischemic maximum developed left ventricular pressure, which recovered to 21%, 56%, 30%, 36%, and 19% in groups 1 to 5 without preconditioning and 46%, 75%, 49%, 40%, and 47% in the corresponding groups with ischemic preconditioning. High-energy phosphate contents were not significantly different between preconditioned hearts and corresponding nonpreconditioned control hearts. Creatine kinase leakage during early reperfusion was found to be reduced with ischemic preconditioning. Thus, we have demonstrated that ischemic preconditioning can improve contractile function after global hypothermic ischemia in the isolated rat heart and we have shown that this protection is additive to that of hypothermia-induced protection during global ischemia at 4 degrees C. This endogenous mechanism of cardioprotection was effective regardless of whether preservation was accomplished using cardioplegic solution or topical hypothermia alone. This may have clinical implications in myocardial preservation for heart transplantation.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Coronary Circulation; Creatine Kinase; Energy Metabolism; Glutathione; Heart Transplantation; Insulin; Ischemia; Male; Myocardium; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar

Hypothermia and preservative perfusates have been used to decrease ischemic renal injury. This study was performed to identify the preservative function of perfusates independent of the effects of hypothermia. Rats underwent 45 minutes of renal ischemia. Rectal and renal parenchyma temperatures were monitored and maintained within 1 degree C of normal. Perfusates were University of Wisconsin solution (UW), Euro-Collins solution, normal saline solution, and Ringer's lactate solution. A nonperfused ischemic control and a nonischemic control group were also evaluated. Parameters evaluated included serum creatinine and blood urea nitrogen levels, renal ischemic injury grade, renal weight, and gross appearance of the injured kidney. Rats treated with UW solution were found to have a significantly lower creatinine, blood urea nitrogen, and injury grade than the other three perfused groups. The external gross appearance of the UW-treated kidneys was normal, whereas that of the other groups demonstrated moderate to severe injury. Although the mean right/left renal weight difference of the UW-treated group was lower than that of the other three groups, this was not statistically significant. Under normothermic conditions in rats, UW solution affords significant renal protection from ischemia. Euro-Collins, normal saline, and Ringer's lactate solutions display no significant protective effect.

Topics: Adenosine; Allopurinol; Animals; Blood Urea Nitrogen; Body Temperature; Cardioplegic Solutions; Creatinine; Glutathione; Hypertonic Solutions; Hypothermia, Induced; Insulin; Ischemia; Isotonic Solutions; Kidney; Male; Monitoring, Physiologic; Organ Preservation Solutions; Organ Size; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Rectum; Renal Artery; Ringer's Lactate; Sodium Chloride; Tissue Preservation

The University of Wisconsin solution has been proven to be effective for prolonged heart preservation. However, 24-hour heart preservation by simple cold immersion in University of Wisconsin solution has been disappointing. We have performed hypothermic low-pressure continuous coronary perfusion with oxygenated University of Wisconsin solution for experimental prolonged heart preservation. However, the high potassium concentration of University of Wisconsin solution combined with prolonged ischemia has detrimental effects on endothelial function, which increases coronary tone during preservation and after reperfusion. The severe vasoconstriction and tissue edema result in damage to the coronary microcirculation. The purpose of this study was to determine whether hypothermic low-pressure continuous coronary perfusion technique with oxygenated University of Wisconsin solution containing a selective endothelin-A receptor antagonist (FR139317) would increase the effectiveness of the perfusion technique and improve postischemic cardiac function, both minimizing tissue edema and suppressing vasoconstriction.. Preischemic and postischemic cardiac function of isolated rabbit hearts was evaluated with a Langendorff apparatus. The hearts were divided into three groups (n = 7 each): group I (hypothermic low-pressure continuous coronary perfusion with University of Wisconsin solution), group II (hypothermic low-pressure continuous coronary perfusion with oxygenated University of Wisconsin solution), and group III (hypothermic low-pressure continuous coronary perfusion with oxygenated University of Wisconsin solution containing 10 mg/L of FR139317). Preservation was performed for 24 hours. The initial perfusion pressure for continuous coronary perfusion was set at 5 mm Hg. Measurement of percentage of tissue water content and ultrastructural examination of the myocardium was then performed. In groups I, II, and III, the perfusion pressures at the end of the 24-hour preservation period increased from 5 mm Hg to 12.2 +/- 2.5, 8.1 +/- 1.3, and 5.4 +/- 0.8 mm Hg (p < 0.05), respectively. Percent recovery rate of cardiac output was 56.6 +/- 2.8, 82.3 +/- 8.2, and 93.3 +/- 6.0 (p < 0.05), respectively. And percent recovery rate of coronary flow was 55.5 +/- 8.1, 80.0 +/- 8.0, and 94.3 +/- 9.4 (p < 0.05), respectively. A significant inverse correlation was found between continuous coronary perfusion pressure at the end of preservation and the recovery rate of cardiac output (r = 0.85, p < 0.05). Tissue water content was significantly higher in group I than in groups II and III. These effects were inhibited by oxygenation of the University of Wisconsin solution (group II) and by the addition of the selective endothelin-A receptor antagonist (FR139317) (group III). Damage to coronary circulation was reduced by oxygenation and the addition of endothelin-A receptor antagonist during prolonged heart preservation.. We concluded that hypothermic low-pressure continuous coronary perfusion technique with oxygenated UW solution containing endothelin-A receptor antagonist (FR139317) maintained coronary circulation by suppressing tissue edema and vasoconstriction during preservation, which improved postischemic functional recovery.

Topics: Adenosine; Allopurinol; Animals; Azepines; Blood Pressure; Body Water; Cardiac Output; Cardioplegic Solutions; Coronary Circulation; Cryopreservation; Edema; Endothelin Receptor Antagonists; Glutathione; Heart Transplantation; Hypothermia, Induced; Indoles; Insulin; Ischemia; Microcirculation; Myocardial Contraction; Myocardium; Organ Preservation; Organ Preservation Solutions; Oxygen; Rabbits; Raffinose; Reperfusion; Time Factors; Vasoconstriction

Retrograde oxygen persufflation (ROP) has been reported to be beneficial to kidney preservation. The purpose of this study was to investigate whether use of ROP during cold storage (CS) with Universita of Wisconsin (UW) solution could ameliorate energy metabolism and functional recovery of ischemically injured rat kidneys and, moreover, to study the particular role of adenosine (ADO) in CS with ROP. Kidneys subjected to 30 min of warm ischemia (WI) were preserved for 24 h in 4 degrees C UW solution with or without ROP and with or without ADO. Measurements of tissue high-energy phosphate levels showed that reduced total adenine nucleotides (TAN) after 30 min of WI further declined during the subsequent CS. In ROP kidneys, however, TAN were less reduced, suggesting that even during CS, TAN can still be regenerated in the injured kidneys when ROP is combined with UW solution. When UW did not contain ADO, regeneration of TAN by ROP was slightly less than in the case of UW with ADO. This indicates that the supply of molecular oxygen is a significant factor in TAN resynthesis during CS. There was no statistically significant difference in survival rate between the ROP and CS groups, indicating that an improved energy status is not the sole determinant of functional recovery. We conclude that the gaseous oxygen supply provided by ROP during CS in UW solution ameliorates the energy state of ischemically injured rat kidneys and that exogenous ADO from the UW solution contributes to the improvement of energy metabolism to a limited extent.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Energy Metabolism; Glutathione; Graft Survival; Hypothermia, Induced; Insulin; Ischemia; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Inbred Lew; Solutions

The uptake of hyaluronic acid (HA) was used to assess preservation damage to sinusoidal endothelial cells (SEC) during cold storage and subsequent normothermic reperfusion of rat livers. After 8, 16, 24, and 48 h storage in University of Wisconsin (UW) solution, livers were gravity-flushed via the portal vein with a standard volume of cold UW solution containing 50 micrograms/l HA. The effluent was collected for analysis of HA, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The mean uptake of HA at 0 h was 59.1% +/- 4.6% (mean +/- SEM). After 8 h of storage, HA uptake was similar (55.5% +/- 7.3%), whereas after 16 h of storage it was reduced to 34.7% +/- 5.8%. At 24 and 48 h of storage, no uptake of HA was found. In a second series of experiments, livers were stored in UW solution and subsequently reperfused for 90 min with a Krebs-Henseleit solution (37 degrees C) in a recirculating system containing 150 micrograms/l HA. Following 8 h of storage, 34.6% +/- 8.0% of the initial HA concentration was taken up from the perfusate. After 16 and 24 h of storage, no uptake of HA was found. The results of this study indicate that damage to SEC occurs progressively during storage, leading to zero uptake of HA by the rat livers at 24 h of cold ischemia time. Additional reperfusion injury to the SEC was demonstrated by the reduced ability of the SEC to take up HA following normothermic reperfusion. The uptake of exogenous HA in preserved livers, used as a tool to assess SEC injury, enables the detection of early preservation damage.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Cold Temperature; Endothelium; Female; Glucose; Glutathione; Hyaluronic Acid; Insulin; Ischemia; Liver; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Temperature; Tromethamine

We investigated the role of adhesion molecules in the early phase of reperfusion following cold ischemia. Livers of male Lewis rats were preserved for 0 h (group A) or 24 h in University of Wisconsin (UW) solution without additives (group B) or in UW solution with anti-ICAM-1 antibody (group C) or anti-E-selectin-1, SLe(x) and SLe(a) antibodies (group D). The livers were then reperfused with diluted rat whole blood (DWB; groups A and B). DWB containing anti-ICAM-1 and LFA-1 antibodies (group C) or DWB containing anti-L-selectin, SLe(x) and SLe(a) antibodies (group D). The reperfusion was performed at 37 degrees C for 1 h at 5 cm H2O of perfusion pressure. During reperfusion, hepatic microcirculation was assessed by monitoring portal and peripheral tissue blood flow. Bile production was significantly reduced in group B livers compared with those in group A. Anti-ICAM-1 and LFA-1 antibodies failed to improve hepatic microcirculation, whereas anti-LECAM-1, SLe(x) and SLe(a) antibodies significantly improved the microcirculation. Bile production in group C and D livers was comparable to that in group B livers. Preservation for 24 h significantly increased the release of TNF-alpha from 0.207 to 43.7 pg/g per hour during reperfusion. Monoclonal antibodies to the adhesion molecules did not suppress the release of TNF-alpha in groups C and D. Histological examination demonstrated a lack of leukocyte infiltration or thrombus in hetapic microvessels. The extent of hepatocyte necrosis did not differ among groups B, C, and D. We conclude that the microcirculatory disturbance in the early phase of reperfusion occurs as a result of the tethering of leukocytes through the interaction of the selectin family and their ligands, and that the ICAM-1-LFA-1 pathway is not involved in this step. The lack of improvement in bile production with antibodies to the selectin family and their ligands strongly suggests that other mechanisms participate in the deterioration of hepatic function.

Topics: Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Antibody Specificity; Bile; CA-19-9 Antigen; Cell Adhesion; Cold Temperature; E-Selectin; Glutathione; Insulin; Intercellular Adhesion Molecule-1; Ischemia; L-Lactate Dehydrogenase; L-Selectin; Leukocytes; Liver; Lymphocyte Function-Associated Antigen-1; Male; Microcirculation; Necrosis; Oligosaccharides; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha

We have shown that 5-hr preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C allows ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. However, 8 hr of preservation using this method caused a disturbance of vascular microcirculation and did not resuscitate the grafts. The aim of this study was to examine the effect of thromboxane A2 synthesis inhibitor OKY046 on vascular endothelial cells and ATP tissue levels of canine pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C, and vascular microcirculation and pancreas viability after transplantation. Graft viability was judged by graft survival following autotransplantation. ATP tissue levels were measured by high-performance liquid chromatography at the end of preservation. Viability of the vascular endothelial cells was judged using nuclear trypan blue uptake of the graft after preservation. Pancreatic tissue perfusion was measured using an H2 clearance technique after reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). However, 5-hr preservation made it possible to recover the pancreas (5/5); 8-hr preservation was not successful (0/3). ATP tissue levels after 5-hr and 8-hr preservation were 9.40+/-2.09 and 7.37+/-1.06 micromol/g dry weight, respectively, and OKY046 did not affect ATP synthesis during 8-hr preservation (8.44+/-0.92 micromol/g dry weight). The percentage of nuclear trypan blue uptake of endothelial cells in 8-hr-preserved grafts was 37.6+/-11.6% and was significantly higher than the value in 5-hr-preserved grafts (5.0+/-3.0%; P<0.01). However, OKY046 significantly reduced trypan blue uptake in 8-hr-preserved grafts (8.2+/-3.6%; P<0.01). Pancreatic tissue perfusion in 8-hr-preserved grafts after 2 hr of reperfusion was 28.5+/-7.5 ml/min/100 g, and was significantly lower than the value in 5-hr-preserved grafts (57.1+/-4.4 ml/ min/100 g; P<0.01), but OKY046 dramatically improved pancreatic tissue perfusion (97.1+/-14.6 ml/min/100 g; P<0.01). As a consequence, 8-hr-preserved grafts were resuscitated (4/5). We conclude that OKY046 protects the vascular endothelium during preservation by the two-layer method at 20 degrees C and consequently improves vascular microcirculation on reperfusion. Together with ATP synthesis, which is essential for repairing damaged cells, the canine p

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Dogs; Endothelium, Vascular; Enzyme Inhibitors; Female; Glutathione; Graft Survival; Insulin; Ischemia; Male; Methacrylates; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Perfusion; Raffinose; Temperature; Thromboxane A2; Time Factors; Trypan Blue

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Hypertonic Solutions; Insulin; Intestinal Mucosa; Intestine, Small; Ischemia; Isotonic Solutions; Male; Mesenteric Artery, Superior; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Ringer's Lactate; Time Factors

Topics: Adenosine; Adult; Allopurinol; Glutathione; Graft Rejection; Heart; Heart Transplantation; Heart-Lung Transplantation; Humans; Hypertonic Solutions; Insulin; Ischemia; Length of Stay; Organ Preservation; Organ Preservation Solutions; Raffinose; Retrospective Studies; Time Factors; Tissue Donors

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Ischemia; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Rabbits; Raffinose; Reperfusion; Temperature; Transplantation, Autologous

Topics: Adenosine; Allopurinol; Animals; Cell Adhesion; Cold Temperature; Endothelium, Vascular; Glutathione; Graft Survival; Insulin; Ischemia; Leukocytes; Liver Circulation; Liver Transplantation; Microcirculation; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred ACI; Reperfusion Injury; Tacrolimus; Transplantation, Isogeneic

Taurine (2-aminoethane sulfonic acid) is a physiologic amino acid involved in cellular osmoregulation in various species including man. This study was intended to compare the respective effects of cold storage and consecutive ischemic rewarming of the liver postischemic hepatic flow and hepatocellular outcome upon reperfusion with or without the addition of taurine to the preservation medium. Livers from male Wistar rats were rinsed free of blood via the portal vein and stored ischemically at 4 degrees C in UW solution. Livers from group 1 were then rinsed again with 10 ml Ringer's solution and reperfused with Krebs-Henseleit buffer at a constant pressure of 10 mmHg for 45 min at 37 degrees C in a nonrecirculating manner. Livers from groups 2 and 3 were subjected to 30 min of warm ischemia subsequent to cold storage and prior to reperfusion with 10 mM taurine added to the UW solution in group 3. While there were only very few signs of hepatic injury in group 1, the additional period of warm ischemia (group 2) led to a significant reduction in early perfusate flow and enhanced enzyme leakage from the livers during postischemic rinse and reperfusion. Livers in group 3 exhibited an amelioration in hepatic circulation and significantly reduced enzyme release as compared to group 2. The results clearly demonstrate a remarkable impact of postischemic rewarming on graft viability. Furthermore, the addition of taurine to the preservation medium was shown to improve hepatic circulation and enhance viability of the liver upon reperfusion.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Ischemia; Liver; Male; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Wistar; Taurine; Temperature; Tissue Preservation

Topics: Adenosine; Adult; Allopurinol; Female; Glutathione; Humans; Insulin; Ischemia; Liver; Liver Transplantation; Male; Morbidity; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Raffinose; Retrospective Studies

Topics: Adenosine; Allopurinol; Elective Surgical Procedures; Emergencies; Female; Glutathione; Humans; Hypertonic Solutions; Insulin; Ischemia; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Probability; Prognosis; Raffinose; Renal Insufficiency; Retrospective Studies; Risk Factors; Time Factors

Topics: Adenosine; Allopurinol; Animals; Bacterial Physiological Phenomena; Glutathione; Insulin; Intestine, Small; Ischemia; Isotonic Solutions; Lipid Peroxidation; Male; Malondialdehyde; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Ringer's Lactate; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Blood Loss, Surgical; Constriction; Embolism, Air; Follow-Up Studies; Glutathione; Hepatectomy; Hepatic Veins; Humans; Hypothermia, Induced; Insulin; Ischemia; Ligation; Liver Circulation; Liver Diseases; Liver Failure; Liver Neoplasms; Organ Preservation Solutions; Perfusion; Portal Vein; Raffinose; Survival Rate; Time Factors; Tissue Preservation; Vena Cava, Inferior

The standard preservation technique in lung transplantation is cold single pulmonary artery flush (PAF) with Eurocollins solution (ECS). We compared ECS with University of Wisconsin (UW) solution, with and without added indomethacin, in single PAF preservation in an in vivo rabbit model of warm ischemia-reperfusion lung injury. Six groups of four New Zealand white rabbits each underwent isolation and hilar stripping of the left lung. In the four experimental groups, the left lung was flushed with (15 ml/kg) of cold ECS or UW solution, with or without added indomethacin, before warm ischemia for 120 minutes and before reperfusion for 60 minutes. The remaining two groups were the nonischemic and the ischemic "no flush" controls. Transcapillary flux of 99mTechnitium-labeled albumin and electron microscopy were used to demonstrate lung injury. Pulmonary vascular resistance (PVR) and thromboxane B2 (TXB2) concentrations were measured. There was a significant rise in PVR after ischemia/reperfusion in the ischemic control group (54.7 +/- 13.9 to 117.8 +/- 20.7 mm Hg/L.min-1, P < 0.05). The net rise in PVR after ischemia-reperfusion was significantly smaller in the two groups in which indomethacin was added (16.8 +/- 17.5 and 4.5 +/- 10.6 mm Hg/L.min-1 for UW and ECS, respectively) compared with the ischemic control (63.1 +/- 24.6 mm Hg/L.min-1, P < 0.05). Post-reperfusion TXB2 levels tended to be lower in the nonischemic control group and in the indomethacin-flush groups. We conclude that the increase in PVR produced by unilateral ischemia-reperfusion lung injury in this model was improved by single PAF perfusion. There was no significant difference between UW solution and ECS in this regard. The addition of indomethacin to the flush solution was associated with lower PVRs as well as morphologic improvement by electron microscopy. These findings may indicate a prominent role for the provision of PG synthesis inhibition during preservation for lung transplantation.

Topics: Adenosine; Allopurinol; Animals; Blood Pressure; Glutathione; Hypertonic Solutions; Indomethacin; Insulin; Ischemia; Lung; Organ Preservation; Organ Preservation Solutions; Oxygen; Partial Pressure; Pulmonary Artery; Rabbits; Raffinose; Reperfusion; Thromboxane B2; Vascular Resistance

Kupffer cell activation is hypothesized to play an etiopathogenic role in storage-related graft failure after liver transplantation. The aim of this study was to verify whether the elimination of Kupffer cells modifies the magnitude of cold ischemia/reperfusion injury of the liver.. Rat Kupffer cells were eliminated by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. Livers from control and treated rats were isolated and perfused before and after 24-hour cold ischemia in the University of Wisconsin solution (4 degrees C). Hepatocyte and sinusoidal endothelial cell functions were evaluated by taurocholate and hyaluronic acid elimination, respectively. Liver transplantation was also performed using control and treated donor livers stored under identical conditions.. Compared with baseline values, similar alterations were found in both groups after cold ischemia for hepatocyte function (intrahepatic resistance, bile secretion, lactate dehydrogenase release, oxygen consumption, and taurocholate intrinsic clearance) and for sinusoidal endothelial cell function (hyaluronic acid intrinsic clearance). The 10-day survival rate of animals undergoing transplantation was not different between the groups (6 of 15 vs. 4 of 15, control vs. treated donor livers, respectively).. The presence or absence of Kupffer cells does not modify the effect of 24-hour cold ischemia/reperfusion on the rat liver.

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Bile; Glutathione; Hyaluronic Acid; Hypothermia, Induced; In Vitro Techniques; Insulin; Ischemia; Kupffer Cells; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Macrophage Activation; Male; Metabolic Clearance Rate; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Rats; Reperfusion Injury; Taurocholic Acid; Tissue Survival

As adenine nucleotide content has been shown to correlate with post-transplant function of livers and hearts, it was the aim of our study to investigate the regeneration of rat small bowel tissue high-energy phosphates after 7 hr of cold storage followed by incubation of everted small bowel sacs in normothermic oxygenated KHB for 1 hr. We compared the University of Wisconsin (UW) and the Eurocollins (EC) solutions. Krebs-Henseleit-bicarbonate buffer (KHB) was used to point out the effect of simple cold ischemic storage. After 7 hr of cold storage only small bowel stored in UW and EC solutions retained the capacity for almost total regeneration of ATP necessary for optimal posttransplant function, whereas in the KHB group we found only minimal regeneration. A similar pattern was found for the energy charge. These data support the superiority of UW and EC solutions over simple cold storage in KHB for preservation of small bowel.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Glutathione; Hypertonic Solutions; Insulin; Intestine, Small; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Energy Metabolism; Glutathione; Hypertonic Solutions; Insulin; Intestinal Mucosa; Intestine, Small; Ischemia; Isotonic Solutions; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Ringer's Lactate; Time Factors

Topics: Adenosine; Adolescent; Adult; Allopurinol; Cadaver; Female; Glutathione; Graft Rejection; Humans; Insulin; Ischemia; Isotonic Solutions; Kidney; Kidney Transplantation; Male; Middle Aged; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Perfusion; Raffinose; Ringer's Solution; Therapeutic Irrigation; Thrombosis; Tissue and Organ Procurement; Tissue Donors

The role of true cold ischemia times (CIT) and rewarming ischemia times (WIT) in determining outcome after liver transplantation was investigated in 230 adult recipients. Using multivariate analysis, WIT (time from the start of implantation until restoration of arterial and portal blood supply) and donor intensive care stay (P = .04 and .0004, respectively) but not CIT (the time from donor portal vein flushing until the graft was removed from University of Wisconsin solution; P > .30) emerged as independent determinants of graft survival. In the small number of patients with a WIT of greater than 180 minutes, there were reductions in graft survival (58% v 80% for WIT greater than 180 minutes) but these just failed to reach significance (P = .055). CIT had no influence on graft survival using cut-offs of 12 or 18 hours. A WIT of greater than 180 minutes was associated with an increased median area under the curve of day 1 through 7 serum bilirubin (1,370 v 915 mumol/L.day; P = .048) and trends towards an increased incidence of primary graft nonfunction or dysfunction (22.2% v 6.2% for WIT of less than 180 minutes; P = .065) and the day 1 through 7 area under the curve of serum aspartate aminotransferase (3,310 v 1,440 IU/L.day; P = .092). A prolonged CIT (greater than 18 hours) led to a prolonged hospital stay (69 v 31 days; P = .03), an increased area under the curve of day 8 through 14 serum bilirubin (2,500 v 995 mumol/L.day; P = .003), and a trend towards an increased incidence of initial poor graft function (33.3% v 6.3% for less than 18 hours; P = .092). The incidence of acute rejection increased (to 64.3% from 53.4%; P = .04) in patients with preservation injury (serum aspartate aminotransferase greater than 1,500 IU/L during the first 2 postoperative days). True CIT and WIT are important determinants of outcome after liver transplantation.

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Cold Temperature; Female; Follow-Up Studies; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Liver; Liver Function Tests; Liver Transplantation; Male; Middle Aged; Multivariate Analysis; Organ Preservation; Organ Preservation Solutions; Raffinose; Retrospective Studies; Rewarming; Time Factors; Transplantation, Homologous; Treatment Outcome

Enzyme histochemical activity of catalase, a peroxisomal enzyme involved in cellular antioxidant systems, was studied in proximal tubular cells of human renal transplants as a marker of ischemia-reperfusion injury in the prediction of the evolution of renal transplants. A low enzymatic activity was observed in all renal biopsies performed at 30 min. reperfusion with no difference between the several evolution types of renal transplants. Reduced catalase activity due to ischemia-reperfusion injury could not be correlated with renal function or used as an index of renal function recovery.

Topics: Adenosine; Adolescent; Adult; Allopurinol; Biomarkers; Catalase; Creatinine; Follow-Up Studies; Glutathione; Histocytochemistry; Humans; Hypertonic Solutions; Insulin; Ischemia; Kidney; Kidney Transplantation; Kidney Tubules, Proximal; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion; Time Factors; Tissue Donors

The current shortage of transplantable organs has renewed interest in kidneys obtained from non-heart-beating donors. Kidneys from these donors have suffered warm ischemia (WI). The effectiveness of two preservation solutions, i.e., the University of Wisconsin (UW) and the histidine tryptophan ketoglutarate (HTK) solutions, for preservation of kidneys that have been subjected to WI was tested in dogs. The left kidney was autotransplanted after 30 min of WI, and subsequent 24-hr cold storage (CS) in either UW (n = 6) or HTK (n = 6), with immediate contralateral nephrectomy. Surgical biopsies from the cortex were taken before WI, after 30 min of WI, after 24 hr of CS, and after 1 hr of reperfusion for electron microscopy and for analysis of energy metabolites. At 2 weeks after transplantation in the UW group, 4 out of 6 and, in the HTK group, 1 out of 6 dogs survived. As from day 2, serum creatinine was lower in the UW group as compared with the HTK group (P < 0.05). After 24 hr of CS, in the HTK group the luminal membranes of proximal tubular cells were partly denuded of microvilli. Moreover, the tubular lumen was filled with blebs and debris. In the UW group, the brush borders remained intact, although microvilli were swollen. Energy metabolites were analyzed with HPLC. Thirty minutes of WI resulted in a +/- 45% reduction of total adenine nucleotide (TAN) content. During CS, TAN levels further decreased in both groups; however, after 24 hr of CS, the levels of adenosine, inosine, hypoxanthine, and xanthine were significantly higher in the UW group as compared with the HTK group (P < 0.05, P < 0.01, P < 0.01, P < 0.01). At 1 hr of reperfusion, TAN levels were higher in the UW group as compared with the HTK group (4.66 +/- 0.16 vs. 4.02 +/- 0.28, P < 0.05). Our results show that UW is a superior solution compared with HTK in the preservation of ischemically damaged kidneys, demonstrating better survival, better recovery of kidney function, better protection against ischemia-induced ultrastructural damage, and better preservation of energy metabolism indicated by (a faster) regeneration of TAN levels after reperfusion.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Cardioplegic Solutions; Cold Temperature; Creatinine; Dogs; Female; Glucose; Glutathione; Insulin; Ischemia; Kidney; Kidney Transplantation; Kidney Tubules; Mannitol; Organ Preservation; Organ Preservation Solutions; Organ Size; Potassium Chloride; Procaine; Raffinose; Reperfusion

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Cold Temperature; Edetic Acid; Glutathione; Graft Survival; Hypertonic Solutions; Insulin; Intestine, Small; Ischemia; Lipid Peroxidation; Male; Organ Preservation Solutions; Pregnatrienes; Raffinose; Rats; Rats, Inbred WF; Reperfusion Injury; Temperature; Tissue Preservation

Topics: Adenosine; Allopurinol; Cadaver; Cyclosporine; Glutathione; Graft Survival; Humans; Hypertonic Solutions; Insulin; Ischemia; Kidney; Kidney Transplantation; Kidney Tubular Necrosis, Acute; Organ Preservation; Organ Preservation Solutions; Raffinose; Time Factors; Tissue Donors

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Female; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Kidney; Kidney Transplantation; Microscopy, Electron; Mitochondria; Models, Biological; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

In a rat model, the left kidney was subjected to 60 min of normothermic ischemia followed by 15 min of reperfusion, whereas the right kidney, serving as a paired control, was not rendered ischemic. Both kidneys were then perfused in situ with either Euro-Collins (EC) solution (n = 12) or University of Wisconsin (UW) solution (n = 6) for 10 min. Each kidney was then harvested and stored at 4 degrees C in its respective solution. After 24 and 48 h of cold storage, the following vasoactive substances were measured in the preservation media: endothelin (ET), angiotensin II (A-II), thromboxane (B2) (TxB2), and prostaglandin I2 (PGI2). After 24 h in EC solution, left kidneys uniformly produced significantly higher concentrations of each vasoactive substance than right kidneys: ET 1.64 +/- 0.3 pg/ml vs 0.82 +/- 0.1 pg/ml (P < or = 0.009); A-II 20.8 +/- 6.2 pg/ml vs 7.75 + 2.3 pg/ml (P < or = 0.007); TxB2 100.8 +/- 17.7 pg/ml vs 40.1 +/- 11.7 pg/ml (P < or = 0.04); PGI2 638.3 +/- 41.1 pg/ml vs 318.3 +/- 36.4 pg/ml (P < or = 0.001), respectively. At 48 h, a similar pattern of results was obtained as the kidney continued to produce TxB2 and prostacyclins during the 24-48 h period. In the UW solution, basal levels of ET and A-II were lower than those in EC solution, but similarly increased after initial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Allopurinol; Angiotensin II; Animals; Cryopreservation; Endothelins; Glutathione; Humans; Hypertonic Solutions; Insulin; Ischemia; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Radioimmunoassay; Raffinose; Rats; Rats, Wistar; Thromboxane B2

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.

Topics: Adenosine; Allopurinol; Animals; Dogs; Female; Fluorocarbons; Glutathione; Graft Survival; Hot Temperature; Insulin; Ischemia; Male; Methods; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose

Topics: Adenosine; Allopurinol; Animals; Biopsy; Blood Glucose; Dogs; Female; Fluorocarbons; Glucose Tolerance Test; Glutathione; Insulin; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Time Factors; Transplantation, Autologous

Topics: Adenosine; Allopurinol; Animals; Dogs; Glutathione; Graft Survival; Insulin; Ischemia; Lung; Lung Transplantation; Organ Preservation; Organ Preservation Solutions; Pulmonary Circulation; Raffinose; Reperfusion; Time Factors; Transplantation, Homologous; Vascular Resistance

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Chromatography, High Pressure Liquid; Cryopreservation; Fluorocarbons; Glutathione; Humans; Insulin; Ischemia; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Temperature

In order to evaluate the protective effect of University of Wisconsin (UW) solution in heart transplantation, a retrospective comparative study with histidine-tryptophane-ketoglutarate (HTK) solution was initiated. In group I, we included 160 patients with HTK preservation, while group II consisted of 50 patients who had their transplant protected with UW solution. All patients received standard quadruple drug therapy for immunosuppression. The average ischaemic time of the donor hearts in group I was 142+/-44 min, ranging from 83 to 235 min. Acute immediate perioperative graft failure occurred in six cases (3.8%). Statistical analysis including the chi-square test, revealed a significant increase in the incidence of acute perioperative graft failure when compared with duration of ischaemic time (P < 0.01). Within the first 30 postoperative days, 24 patients died (15% early mortality). The same statistical correlation was evident between the incidence of early mortality and duration of graft ischaemic time. The 30-day and 6-month survival rates were 81% and 78%, respectively. The average ischemic time of the donor hearts in group II was 193+/-50 min ranging from 100 to 360 min, which was significantly longer in comparison with the group I (P < 0.05). Acute perioperative graft failure occurred once (2%); the patient was retransplanted successfully. Five patients died within the first 30 postoperative days (10% early mortality). There was no correlation between length of ischaemic time and incidence of acute graft failure or early mortality. The 30-day and 6-month survival rates were 90% and 88%, respectively and, thus, better when compared with group I. In both groups similar results were achieved with regard to postoperative NYHA status of the patients and incidence of cardiac arrhythmias. Myocardial preservation with HTK solution showed satisfying results as long as the ischaemic time did not exceed 4 h. The early functional results achieved with UW graft protection were excellent, even with ischaemic times longer than 4 h and not depending on lenght of ischaemic period.

Topics: Adenosine; Allopurinol; Arrhythmias, Cardiac; Chi-Square Distribution; Female; Glucose; Glutathione; Graft Survival; Heart; Heart Transplantation; Humans; Insulin; Ischemia; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Postoperative Complications; Postoperative Period; Potassium Chloride; Procaine; Raffinose; Reoperation; Retrospective Studies; Survival Rate; Time Factors; Treatment Failure

Frozen section examination was performed on 385 donor livers before transplantation. Exclusion criteria were applied to the donor livers examined to exclude potentially dysfunctional livers. The exclusion criteria included the following: severe macrovesicular steatosis, ischemic necrosis, prominent chronic portal inflammation, prominent periductular fibrosis, granulomatous inflammation, bridging fibrosis, and malignancy. Twenty-seven of the 385 donor livers examined were excluded before transplantation. The following histologic features were present in the excluded livers: severe steatosis (22), ischemic necrosis (2), portal inflammation (1), and periductular fibrosis (2). Steatosis was present in 51 of the 385 (13.25%) organs examined, including 22 of the donor organs excluded before transplantation. Twenty-nine livers with mild to moderate steatosis were implanted into size and blood type-matched recipients. Indicators of allograft function (prothrombin time and bilirubin) and damage (aspartate aminotransferase and alanine aminotransferase) were measured daily for the first 10 days after transplant. There was no statistically significant difference between the group of nonfat livers and donor livers containing mild steatosis. Statistically significant higher posttransplant serum alanine aminotransferase and prothrombin time levels were present in the patients with livers implanted with mild versus moderate steatosis. The 1-year survival rate for patients receiving fatty versus nonfatty donor livers was not statistically different (Kaplan-Meier, P = 0.592). No significant differences were found in the clinical and laboratory characteristics of donors whose organs were implanted compared with the clinical and laboratory characteristics of donors whose organs were excluded. The primary nonfunction rate after applying the exclusion criteria was 1.4%, which is a significant decrease compared with our primary nonfunction rate of 8.5% before using frozen section examination. Frozen section examination is useful in excluding donor organs which may become dysfunctional after transplantation.

Topics: Adenosine; Adult; Allopurinol; Azo Compounds; Child; Fatty Liver; Female; Fibrosis; Frozen Sections; Glutathione; Hepatitis; Humans; Hypertonic Solutions; Insulin; Ischemia; Liver; Liver Transplantation; Male; Necrosis; Organ Preservation; Organ Preservation Solutions; Raffinose; Staining and Labeling; Survival Rate; Tissue Donors

The enhancement of blood flow in experimental skin flaps following postischemic perfusion washout was investigated in rats. Unilateral island skin flaps based on the superficial epigastric vessels were raised and subjected to 6 hr of primary ischemia. Group 1 was designated as a control and did not undergo postischemic perfusion washout. In the remaining rats, postischemic washout was performed with one of five agents: Group 2--lactated Ringer's solution; Group 3--University of Wisconsin solution, an organ preservation medium; Group 4--verapamil, a calcium channel blocker; Group 5--urokinase, a thrombolytic agent; Group 6--iloprost, a stable prostacyclin analog. Two hours following perfusion washout, fluorometric analysis revealed a statistically significant enhancement of blood flow in Groups 4, 5, and 6, compared to Groups 2 and 3 (p < 0.05). Furthermore, a significant increase in skin surface fluorescence was demonstrated in all the flaps that underwent perfusion washout, compared to the control flaps (p < 0.05). By analyzing skin surface fluorescence, the enhancement of nutritive blood flow in flaps, following postischemic perfusion washout, was evaluated. This is the first study in which the above pharmacologic agents were compared in a quantitative manner.

Topics: Adenosine; Allopurinol; Animals; Female; Fluorescein; Fluoresceins; Glutathione; Iloprost; Insulin; Ischemia; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Skin; Surgical Flaps; Tissue Preservation; Urokinase-Type Plasminogen Activator; Verapamil

The two-layer cold storage method using the University of Wisconsin solution (UW) allows continuous tissue adenosine triphosphate (ATP) production during the preservation period. UW contains 5mM of adenosine which has several mechanisms of action, however, its efficacy is controversial. In this study, at first, we investigated the metabolism of exogenous adenosine during preservation by the two-layer method in the canine pancreas graft subjected to 60 min warm ischemia. High concentration (> 5 mM) of adenosine in Euro-Collins' solution (EC) remarkably increased ATP and total adenine nucleotide (TAN) levels during 24 hr preservation by the two-layer method using EC. Furthermore, experiments with 5 mM of [2-3H] adenosine demonstrated that adenosine was metabolized and incorporated into adenine nucleotides (ANs). However, neither 5 mM of inosine, hypoxanthine nor adenine substituted for adenosine. It was clear that adenosine was directly phosphorylated and converted into ANs. Secondly, the effect of adenosine on the viability of the ischemically damaged pancreas graft was evaluated by graft survival following autotransplantation. The ischemically damaged pancreas grafts were preserved for 24 hours by simple cold storage in EC (group 1), EC with 5 mM of adenosine (group 2), the two-layer method using EC (group 3), EC with 5 mM of adenosine (group 4). Graft survival rates were 0/5 (0%), 1/5 (20%), 0/3 (0%) and 4/5 (80%) in groups 1, 2, 3 and 4 respectively. Adenosine was clearly effective on graft survival via recovering high tissue ATP and TAN levels only in case of the preservation by the two-layer method. We conclude that exogenous adenosine works as a substrate for ANs synthesis and is directly phosphorylated to ANs during preservation by the two-layer method. This is essential to repair damaged cells and maintain cellular integrity, making it possible to preserve ischemically damaged pancreas.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Cryopreservation; Dogs; Female; Glutathione; Graft Survival; Insulin; Ischemia; Male; Organ Preservation Solutions; Pancreas; Raffinose

We have demonstrated that a two-layer (University of Wisconsin solution [UW]/perfluorochemical [PFC]) cold storage method restores the function of ischemically damaged pancreas during 24-hr preservation in canine autotransplantation model. The purpose of this study was to examine the possibility of a long-term preservation of the ischemically damaged pancreas by the two-layer (UW/PFC) method. After 60 or 90 min of warm ischemic time, pancreas grafts were preserved by the two-layer (UW/PFC) method or a simple cold storage in UW alone for up to 96 hr. A K value of i.v. glucose tolerance test more than 1.0 2 weeks after autotransplantation was considered successful preservation. After 60 min warm ischemia, limitation of preservation time by the simple cold storage in UW was 24 hr (5/5 100% and 0/5 0%; 24- and 48-hr preservation, respectively). However, the two-layer method made it possible to extend the preservation time up to 48 hr (5/5 100%, 5/5 100%, 2/5 40%, and 0/5 0%; 24-, 48-, 72-, and 96-hr preservation, respectively). After 90 min warm ischemia, the simple cold storage in UW was not effective even for 24-hr preservation (0/5 0%). However, 48-hr preservation was successful by the two-layer (UW/PFC) method (5/5 100%, 5/5 100%, and 0/5 0%; 24-, 48-, and 72-hr-preservation, respectively). After preservation by the two-layer (UW/PFC) method, ATP tissue concentrations of viable grafts were significantly higher compared with nonviable grafts (9.11 +/- 3.05 (n = 22) versus 5.22 +/- 1.02 (n = 13) mumol/g dry wt, P < 0.001). Based on analysis of individual ATP for each graft, if an ATP concentration of 6.0 mumol/g dry weight was determined as a critical value for doing the transplant, sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 84.6%, 91.7%, and 94.3%, respectively. This study clearly demonstrated that 48-hr preservation of the canine pancreas subjected to either 60 or 90 min warm ischemia was successfully achieved by the two-layer (UW/PFC) cold storage method, and ATP tissue concentration at the end of preservation by this method would predict the post-transplant outcome of the ischemically damaged pancreas just prior to transplantation.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Dogs; Female; Fluorocarbons; Glutathione; Insulin; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Time Factors

Topics: Adenosine; Allopurinol; Cold Temperature; Glutathione; Humans; Insulin; Ischemia; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Retrospective Studies; Time Factors

Topics: Adenosine; Allopurinol; Animals; Blood Urea Nitrogen; Creatinine; Glutathione; Hypertonic Solutions; Insulin; Ischemia; Kidney; Kidney Transplantation; Kidney Tubular Necrosis, Acute; Kidney Tubules, Proximal; Microscopy, Electron; Microvilli; Organ Preservation; Organ Preservation Solutions; Raffinose; Swine; Transplantation, Autologous

UW solution has been found to be an effective organ perfusate for transplantation. Initial studies in experimental pedicle skin flaps have also demonstrated its unique effectiveness in prolonged ischemia. To understand better the limits of its preservation properties without the influence of endothelial clamp damage, we have undertaken to study the properties of UW solution in experimental microvascular free flaps. Control, lactated Ringer's, and UW solutions were utilized in pedicle and microvascular free flaps in Sprague-Dawley rats over varying periods of ischemia. UW solution demonstrated a clear superiority over all other solutions in both flap models. In addition there also was a significant prolongation of critical ischemia time in UW-treated free flaps compared to pedicle flaps.

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Insulin; Ischemia; Isotonic Solutions; Microcirculation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Regression Analysis; Ringer's Lactate; Skin; Skin Transplantation; Surgical Flaps; Tissue Preservation

Hepatic artery thrombosis (HAT) after liver transplantation (LTx) usually mandates retransplantation. Prolonged preservation with Eurocollins solution has been associated with HAT. We reviewed our experience with 359 LTx patients to identify risk factors for HAT. All grafts were preserved in University of Wisconsin solution. HAT developed in 12 patients (3%) within 50 days. Seven patients were asymptomatic; four presented with biliary sepsis and 1 with poor graft function. Two patients had suffered acute rejection; another 2 had severe preservation injury. Technical problems accounted for 4 cases; in the remaining 8, no etiology was found. Diagnosis was at a mean 14.7 days after LTx. One patient maintains normal graft function 3 years after LTx without intervention. Eight underwent re-LTx, 3 of whom died. Routine surveillance via duplex enabled early diagnosis and revascularization in 3 patients; in all 3, no biliary complications occurred between 6 and 20 months. Overall graft and patient survival after HAT were 33.3% and 75%, respectively. Cold ischemic time (CIT) averaged 813 min in patients with HAT and 669 min in those without HAT (P < .05). HAT occurred in 7/165 patients with CIT > 12 hr, and in 3/234 patients with CIT < 12 hr (P = 0.0699). By avoiding CIT > 12 hr, we have recently avoided HAT in 78 consecutive patients. We conclude that CIT > 12 hr may increase the risk of HAT. When HAT is diagnosed before biliary sepsis develops, flow can often be restored and retransplantation averted.

Topics: Adenosine; Adult; Aged; Allopurinol; Child, Preschool; Cold Temperature; Glutathione; Hepatic Artery; Humans; Insulin; Ischemia; Liver; Liver Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Thrombosis; Time Factors; Ultrasonography

This study was designed to observe the effect of perfusion with University of Wisconsin (UW) preservation solution on skin flap survival following secondary ischaemia caused by venous obstruction in rats. An epigastric flap model was used. Saline-perfused flaps exhibited no significant improvement in survival compared to untreated animals (NS). Skin flaps perfused with UW solution, however, had a significant increase in survival to 40% (8/20) (p < 0.01) when perfused before the onset of primary ischaemia and 30% (p < 0.05) when given before the onset of secondary ischaemia. These results show that UW solution improves skin flap survival, presumably through preservation of the microvasculature.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Survival; Insulin; Ischemia; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Skin; Skin Transplantation; Solutions; Surgical Flaps; Time Factors; Tissue Preservation

Topics: Adenosine; Allopurinol; Cold Temperature; Endothelins; Glutathione; Humans; Insulin; Ischemia; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Bile; Glutathione; Hepatic Artery; Insulin; Ischemia; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Organ Size; Pressure; Raffinose; Reperfusion Injury; Swine

Topics: Adenosine; Allopurinol; Animals; Blood Glucose; Dogs; Female; Fluorocarbons; Glutathione; Hyperglycemia; Insulin; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Solutions; Temperature; Transplantation, Autologous

A myriad of investigations have been published on the pharmacologic manipulation of flaps to enhance tolerance to ischemia. We recently reported a threefold increase in ischemic tolerance of the rat abdominal skin flap pedicle after 6 hours of primary ischemia and 12 hours of reperfusion. Flaps underwent normothermic perfusion washout with lactated Ringer's or U.W. solution, a newly developed organ preservation medium. Perfusion washouts were performed at one of three different points in the protocol: (1) onset of primary ischemia; (2) onset of secondary ischemia; or (3) 2 hours after onset of secondary ischemia. The last group was used to simulate the clinical situation in which flaps are discovered and salvage procedures instituted at a delayed time interval. This is the longest normothermic ischemic interval reported. We undertook the present study to determine the utility of the U.W solution in prolonging the tolerance of the flap to a second ischemic insult after a period of reperfusion. Seventy-five unilateral rat abdominal skin flaps were raised. Secondary ischemia was produced by placing a microvascular clamp across the inferior epigastric pedicle. Flap survival was assessed at 1 week postoperatively. While none of the nonperfused flaps survived 8 hours of secondary ischemia, at least 50% of the U.W. perfused flaps survived an average of 14 hours of secondary ischemia. Lactated Ringer's perfusion washout only modestly increased the ischemic tolerance. Perfusion washout in the secondary ischemic phase improved the ischemic tolerance to a significantly greater degree than in the primary ischemic interval.

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Graft Survival; Insulin; Ischemia; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion; Skin; Solutions; Surgical Flaps; Time Factors; Tissue Preservation

The activity and localization of the plasma membrane-bound enzyme 5'-nucleotidase (5'-NT) in liver tissue are sensitive parameters of ischemic damage. The value of 5'-NT as a marker of liver graft viability was studied in relation to liver preservation. In six mongrel dogs, the main right and left branches of the portal vein were cannulated and flushed separately in situ with cold University of Wisconsin (UW) solution and Euro-Collins (EC) solution, respectively. After hepatectomy, the right and left liver lobes were split and stored at 5 degrees C in either of the two solutions. 5'-NT activity was demonstrated in cryostat sections of liver tissue using the lead salt method. After 48 h of storage in EC solution, the 5'-NT score had decreased to 31% +/- 16% (n = 6), whereas in UW solution the 5'-NT score was 76% +/- 10% (n = 6). Significantly (P < 0.05) higher 5'-NT scores were also found after 24-h and 72-h preservation times in UW versus EC solutions. This result is in keeping with the higher preservation tolerance of liver grafts preserved in UW solution. The 5'-NT assay was studied in relation to graft function in orthotopic liver transplantation experiments in dogs. All dogs with liver grafts preserved in UW solution for 24 h (n = 4) and 48 h (n = 3) survived (> 5 days). Pretransplant 5'-NT scores ranged from 61% to 100%. The 72-h-preserved livers (n = 5) did not show life-supporting function.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 5'-Nucleotidase; Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Dogs; Female; Glutathione; Graft Survival; Hypertonic Solutions; Insulin; Ischemia; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Solutions

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Blood Glucose; Dogs; Female; Fluorocarbons; Furans; Glutathione; Graft Survival; Hot Temperature; Insulin; Ischemia; Male; Neck; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Solutions; Transplantation, Autologous; Transplantation, Heterotopic

Topics: Adenosine; Allopurinol; Animals; Body Water; Cold Temperature; Energy Metabolism; Glutathione; Hot Temperature; Hydrogen; Insulin; Ischemia; Liver; Magnetic Resonance Spectroscopy; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Solutions

Topics: Adenosine; Adult; Allopurinol; Brain Death; Glutathione; Humans; Indocyanine Green; Insulin; Ischemia; Liver; Liver Function Tests; Liver Transplantation; Metabolic Clearance Rate; Organ Preservation; Organ Preservation Solutions; Raffinose; Regression Analysis; Solutions; Tissue Donors

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glucose; Glutathione; Insulin; Ischemia; Liver; Male; Mannitol; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Rats; Rats, Sprague-Dawley; Solutions; Temperature

Successful liver transplantation is dependent upon many factors, one of which is the quality of the donor organ. Previous studies have suggested that the donor nutritional status may affect the outcome of liver transplantation and starvation, due to prolonged stay in the intensive care unit, may adversely affect the liver. In this study we have used the orthotopic rat liver transplant model to measure how fasting the donor affects the outcome of liver transplantation. Rat livers were preserved with UW solution either at 37 degrees C (warm ischemia for 45-60 min) or at 4 degrees C (cold ischemia for 30 or 44 hr). After preservation the livers were orthotopically transplanted and survival (for 7 days) was measured, as well as liver functions 6 hr after transplantation. After 45 min of warm ischemia 50% (3 of 6) animals survived when the liver was obtained from a fed donor about 80% (4 of 5) survived when the liver was obtained from a three-day-fasted donor. After 60 min warm ischemia no animal survived (0 of 8, fed group). However, if the donor was fasted for 3 days 89% (8 of 9) of the animals survived for 7 days. Livers cold-stored for 30 hr were 50% viable (3 of 6) and fasting for 1-3 days did not affect this outcome. However, if the donor was fasted for 4 days 100% (9 of 9) survival was obtained. After 44-hr preservation only 29% (2/7) of the recipients survived for 7 days. If the donor was fasted for 4 days, survival increased to 83% (5/6). Liver functions, bile production, and serum enzymes were better in livers from the fasted rats than from the fed rats. Fasting caused a 95% decrease in liver glycogen content. Even with this low concentration of glycogen, liver viability (animal survival) after warm or cold ischemia was not affected, and livers with a low glycogen content were fully viable. Thus liver glycogen does not appear to be important in liver preservation. This study shows that fasting the donor does not cause injury to the liver after warm or cold ischemia. In fact, the livers appeared to be better able to tolerate ischemia when obtained from fasted rats. Thus donor nutritional status may be an important factor for outcome of liver transplantation. Livers from fasted donors may be capable of tolerating long-term preservation better than livers from fed donors.

Topics: Adenosine; Allopurinol; Animals; Bile; Body Water; Cold Temperature; Fasting; Glutathione; Graft Survival; Hot Temperature; Immunity, Innate; Insulin; Ischemia; Liver; Liver Glycogen; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Organ Size; Raffinose; Rats; Rats, Inbred BN; Solutions; Time Factors; Tissue Donors

Application of the University of Wisconsin cold storage solution has rapidly expanded to include medium-term to long-term preservation of virtually all intraabdominal organs. Its use in intrathoracic organ transplantation has also been suggested. We therefore examined the efficacy of the University of Wisconsin solution in a primate allotransplantation model for preservation of hearts, and as a simple single-solution system for static preservation of heart-lung blocks, for periods of ischemia ranging from 6 to 24 hours. For comparison, we employed the histidine-tryptophane-ketoglutarate cardioplegic solution of Bretschneider. University of Wisconsin solution provided superior results with regard to clinical outcome and hemodynamic recovery of hearts after ischemic periods of up to 16 hours. This was in contrast to Bretschneider's solution, which allowed storage of hearts for periods of only up to 10 hours. Heart-lung blocks were equally well preserved with either University of Wisconsin or Bretschneider's solution after 6 to 12 hours, although the University of Wisconsin solution group exhibited a more notable increase in pulmonary water content. This was in accordance with histological data, which suggested that, although hemodynamic recovery of hearts stored for periods longer than 10 hours was poor, preservation of pulmonary ultrastructure was far superior using Bretschneider's solution as compared with University of Wisconsin solution after an ischemic period of up to 16 hours.

Topics: Adenosine; Allopurinol; Animals; Body Water; Cardiac Output; Cardioplegic Solutions; Cardiopulmonary Bypass; Catecholamines; Glucose; Glutathione; Heart Arrest, Induced; Heart Transplantation; Heart-Lung Transplantation; Hypertonic Solutions; Insulin; Ischemia; Lung; Mannitol; Monitoring, Physiologic; Myocardium; Organ Preservation Solutions; Papio; Positive-Pressure Respiration; Potassium Chloride; Procaine; Raffinose; Solutions; Stroke Volume; Survival Rate; Time Factors; Tissue Preservation; Ventricular Function, Left

Studies in animals on the use of UW solution in liver transplantation have shown an inverse relation between cold ischaemia time (CIT) and graft function. There are few clinical data on this relation in human beings. We have investigated the effect of extended cold ischaemia in a prospective study. We assessed early graft function and subsequent outcome for 306 consecutive elective liver transplantations; for analyses, grafts were grouped according to CIT (< 12 h group A, > or = 12 h group B), since a preliminary study identified 12 h as a significant cut-off point. Initial graft function was better in group A than group B, as shown by maximum alanine aminotransferase activity (mean 623 [805] vs 946 [1148], p = 0.02), bile production on days 1-3 (p < 0.05), maximum serum bilirubin by day 10 (206 [166] vs 244 [163] mumol/l, p = 0.04), and frequencies of primary non-function (1 [0.4%] vs 4 [7%], p = 0.006) and hepatocyte necrosis on routine biopsy sample after reperfusion (18% vs 31%, p = 0.04). Long-term outcome was also better in group A than group B; graft and patient survival rates were higher and fewer retransplantations were needed. These findings suggest that cold ischaemia in UW solution for longer than 12 h is a risk factor for graft function and patient survival. We recommend that the limit of the safe CIT be reconsidered.

Topics: Adenosine; Adult; Alanine Transaminase; Allopurinol; Bile; Bilirubin; Cold Temperature; Female; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Prospective Studies; Raffinose; Reoperation; Risk Factors; Solutions

Lazaroids are a class of novel 21 aminosteroids. They have been reported to be potent inhibitors of lipid peroxidation, which is a major contributing factor to ischemia-reperfusion injury in the lung. A Lewis rat orthotopic left lung isotransplant model was used to investigate the effects of the lazaroid U74500A on pulmonary preservation. The heart-lung blocks of donor rats were flushed with and then stored in either standard University of Wisconsin solution or University of Wisconsin solution with 30 mumol/L of U74500A substituted for the dexamethasone. After 6 or 12 hours of cold storage at 0 degrees C, the left lungs were transplanted into recipient rats and reperfused for 1 hour. Pulmonary function was assessed by measuring oxygen and carbon dioxide tensions in arterial blood after removal of the right lung. Lipid peroxide concentrations were measured as a thiobarbituric acid-reactive substance. Although arterial oxygen and carbon dioxide pressures and water content after 6 hours of preservation followed by reperfusion were similar in both the lazaroid and dexamethasone groups, lipid peroxide concentration was significantly higher in the dexamethasone group (0.88 +/- 0.07 mumol/gm) than in the lazaroid group (0.54 +/- 0.07 mumol/gm) (p < 0.01). After 12 hours of preservation, there were significant differences between the lazaroid and dexamethasone groups in arterial oxygen pressure (339 +/- 70 vs 27 +/- 3 mm Hg, p < 0.01), arterial carbon dioxide pressure (24.3 +/- 2.7 vs 47.7 +/- 7.0 mm Hg, p < 0.001), and lipid peroxide concentrations (0.69 +/- 0.07 vs 1.30 +/- 0.09 mumol/gm, p < 0.001). We conclude that addition of U74500A to the flush and storage solution enhances the preservation of the pulmonary graft in this transplant model.

Topics: Adenosine; Allopurinol; Animals; Body Water; Carbon Dioxide; Cardioplegic Solutions; Cold Temperature; Dexamethasone; Glutathione; Insulin; Ischemia; Lipid Peroxides; Lung; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Pregnatrienes; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions

This study compared the function of reduced grafts prepared in situ or ex vivo and transplanted immediately or after 4 hr of cold storage. Measurements of acid/base balance, plasma electrolytes, albumin, and urea showed no differences between groups. There was no difference between the increase and decline of plasma AST in recipients of grafts transplanted immediately after either ex vivo or in situ reduction; the increase in plasma AST of recipients of stored grafts was up to 10-fold and persisted until the end of the study at 7 days, with some decline. Plasma fibrinogen decreased intraoperatively but levels were restored within 24 hr in all groups; plasma prothrombin and partial thromboplastin times were not significantly disturbed. The patterns of decline and return of tissue adenine nucleotides were similar in all groups. While the regenerative response measured by tissue thymidine kinase and mitotic figures was not different between the groups, comparison with results from a group of partially hepatectomized animals showed a 3-4-fold depression in response in reduced liver grafts. The contributions of the effects of ischemia, flushing, and preservation to the depressed regenerative response of reduced liver grafts need to be determined. The present studies suggest however, that with regard to functional assessment, results are not affected either by ex vivo or in situ reduction of the graft, or by cold storage for 4 hr.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Blood Coagulation; Cryopreservation; Glutathione; Graft Survival; Insulin; Ischemia; Liver; Liver Regeneration; Liver Transplantation; Organ Preservation Solutions; Raffinose; Solutions; Swine; Time Factors

Nonanastomotic biliary strictures that involve only the biliary tree of the graft occur after orthotopic liver transplantation in patients with hepatic artery thrombosis, chronic ductopenic rejection and ABO blood group incompatibility. This complication may also occur in the absence of these known risk factors. The major focus of our study was to evaluate the risk factors for nonanastomotic biliary stricturing of unknown cause after orthotopic liver transplantation. Results demonstrate that the development of biliary strictures is strongly associated with the duration of cold ischemic storage of allografts in both Euro-Collins solution and University of Wisconsin solution. Results also demonstrate that strictures are not associated with the type of biliary reconstruction, the primary liver disease, cytomegalovirus infection, allograft rejection or the presence of a positive lymphocytotoxic crossmatch. More recently, we have markedly reduced the occurrence of nonanastomotic biliary stricturing by decreasing the ischemia time of our allografts. Thus nonanastomotic biliary strictures appear to be the result of the ischemia/reperfusion-induced tissue injury associated with the harvest and implantation of allografts.

Topics: Adenosine; Adult; Allopurinol; Child; Cholangiography; Gallbladder; Glutathione; Humans; Hypertonic Solutions; Insulin; Ischemia; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Raffinose; Retrospective Studies; Risk Factors; Solutions

The benefit of perfusion washout in both experimental and clinical skin flaps has long been debated. By perfusing ischemic rat pedicled flaps with UW solution, a recently developed, high-molecular-weight, organ-preservation medium, a 170 percent increase in the critical ischemia time of treated versus untreated control flaps was demonstrated. Sixty rats were used in this study. A 3- x 6-cm unilateral abdominal skin flap based on the superficial inferior epigastric artery and vein was raised. The flaps were divided into three groups: Group 1 (control--no perfusion washout (n = 15); Group 2 (LR)--perfusion washout with lactated Ringer's solution (n = 15); Group 3 (UW)--perfusion washout with UW solution (n = 30). Flaps were subjected to varying periods of ischemia, ranging between 8 and 30 hr. The primary ischemia time at which 50 percent of the flaps survived clinically was 10 hr for Group 1, 15 hr for Group 2, and 27 hr for Group 3. The differences between the survival rates for flaps in Groups 1, 2, and 3 were statistically significant (p less than .0005). By bathing the vascular and parenchymal cells in an impermeant preservation solution, it was hypothesized that cellular swelling would be inhibited, thereby significantly improving a skin flap's tolerance to warm ischemia. Furthermore, after reviewing the pertinent literature, it is evident that the primary critical ischemia time of 27 hr is the highest reported to date for the normothermic experimental rat pedicled flap. Clinical application of these findings, as well as the need for further studies, are discussed.

Topics: Abdomen; Adenosine; Allopurinol; Animals; Female; Glutathione; Graft Survival; Insulin; Ischemia; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred Strains; Skin; Solutions; Surgical Flaps; Tissue Preservation

Topics: Adenosine; Alanine Transaminase; Allopurinol; Aspartate Aminotransferases; Cold Temperature; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Prothrombin Time; Raffinose; Retrospective Studies; Solutions; Survival Analysis; Time Factors

Five hundred ninety-three cadaveric livers were used for primary liver transplantation between October 24, 1987, and May 19, 1989. The grafts were procured with a combined method, using in situ cooling with cold electrolyte solution and backtable flushing with UW solution. The mean cold-ischemia time was 12.8 (range 2.4-34.7) hr. The cases were divided into 5 groups according to the cold-ischemia time: group 1: less than 10 hr (n = 223); group 2: 10-14 hr (n = 188); group 3: 15-19 hr (n = 101); group 4: 20-24 hr (n = 52); and group 5: greater than or equal to 25 hr (n = 29). There was no difference between the 5 groups in 1-year patient survival, highest SGOT in first week after operation, and SGOT and total bilirubin during the first month after operation. However, with a logistic regression model, the retransplantation rate (P = 0.001) and primary nonfunction rate (P = 0.006) significantly rose as cold-ischemia time increased, meaning that the equivalency of patient survival was increasingly dependent on aggressive retransplantation.

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Child; Child, Preschool; Cold Temperature; Glutathione; Graft Survival; Humans; Infant; Infant, Newborn; Insulin; Ischemia; Liver; Liver Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Reoperation; Solutions; Time Factors; Transplantation, Homologous

Cardiac transplantation remains constrained by poor graft tolerance of prolonged cold ischemia. University of Wisconsin solution has remarkably extended ischemic preservation in pancreas, kidney, and liver transplantation. To assess its efficacy in cardiac preservation, modified University of Wisconsin solution flush and storage were tested against St. Thomas' cardioplegia flush and normal saline solution storage after six hours of ischemia at 0 degrees C in 46 isolated rat hearts. After ischemia, groups were compared before and after reperfusion. After ischemia but before reperfusion, University of Wisconsin solution hearts had significantly less tissue water (3.8%), superior tissue sodium, potassium, calcium, and magnesium profiles, and elevated adenosine and inosine levels, and tended toward better histological preservation. After reperfusion, University of Wisconsin solution more effectively preserved left ventricular compliance (75% versus 35% of baseline), developed pressure (71% versus 45% of baseline), histological integrity, and tissue potassium and calcium profiles than St. Thomas' solution. The University of Wisconsin solution provided superior preservation of systolic and diastolic ventricular function, tissue histology, tissue water, and tissue electrolytes than did St. Thomas' cardioplegia and normal saline solution storage in this experimental model, and might result in improved graft tolerance of ischemia in clinical cardiac transplantation.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Bicarbonates; Blood Pressure; Body Water; Calcium; Calcium Chloride; Cardioplegic Solutions; Cryopreservation; Glutathione; Heart; Heart Transplantation; Insulin; Ischemia; Magnesium; Myocardial Contraction; Myocardium; Organ Preservation Solutions; Potassium; Potassium Chloride; Raffinose; Rats; Rats, Inbred Strains; Sodium Chloride; Solutions; Spectrophotometry, Atomic; Tissue Preservation

The role of recipient hemodilution for postischemic renal medullary red cell trapping was investigated after different periods of cold storage in a conventional cold storage solution (Sacks'). At all cold storage times investigated (4, 12, 24, and 48 hr) medullary red cell trapping was reduced by isovolemic hemodilution, with about 50% reduction of recipient hematocrit. Trapping was also reduced when a modification of a new preservation solution (University of Wisconsin solution [UW]) was used and compared with flush-out and storage in a standard preservation solution (Sacks'). The combination of hemodilution and preservation in modified UW solution had additional capacity to reduce medullary red cell trapping. Thus, even after 48 hr of cold storage, only a moderate trapping was observed. The results also indicate that measurements of medullary red cell trapping offers an accurate method of grading postischemic renal damage.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Erythrocytes; Glutathione; Hemodilution; Insulin; Ischemia; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Solutions