raffinose and Reperfusion-Injury

To update the reader on the recent literature in liver, kidney, pancreas, and intestine static cold preservation, and to identify which solutions are most advantageous for each organ.. The comparison of randomized trials of histidine-tryptophan-ketoglutarate (HTK), Celsior, and University of Wisconsin solutions has shown equivalent risk of delayed graft function after kidney transplantation. Similar outcomes have been observed after pancreas preservation with University of Wisconsin, HTK, and Celsior solution. In live-donor liver transplantation, University of Wisconsin and HTK solution have shown equivalent results, whereas in the recent trials of deceased-donor liver transplantation, University of Wisconsin, HTK, and Celsior solutions have shown equivalence. Contrary to the most clinical trials, national registry data in kidney, pancreas, and liver transplantation demonstrate more detrimental effects and earlier graft loss after preservation with HTK versus University of Wisconsin solution. Early outcomes after intestinal transplantation with University of Wisconsin or HTK solution have shown no significant difference and animal studies indicate intraluminal preservation may be beneficial.. The University of Wisconsin solution is the standard criterion static cold preservation for the procurement of liver, kidney, pancreas, and intestine. University of Wisconsin, HTK, and Celsior solutions all provide similar allograft outcomes in most clinical trials, but subtle differences have become more apparent in the recent studies and registry reports.

Topics: Abdomen; Adenosine; Allografts; Allopurinol; Cryopreservation; Disaccharides; Electrolytes; Glucose; Glutamates; Glutathione; Histidine; Humans; Insulin; Intestines; Kidney; Liver; Mannitol; Organ Preservation Solutions; Organ Transplantation; Pancreas; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Tissue Donors

Topics: Acute Disease; Adenosine; Allopurinol; Animals; Chronic Disease; Glutathione; Graft Rejection; Humans; Insulin; Intestine, Small; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Transplantation, Homologous

Topics: Adenosine; Allopurinol; Free Radicals; Glutathione; Humans; Insulin; Liver; Liver Circulation; Organ Preservation Solutions; Oxygen; Preservation, Biological; Preservatives, Pharmaceutical; Raffinose; Reperfusion Injury; Solutions; Specimen Handling; Tissue Preservation

Ischemia reperfusion injury (IRI) is the main cause of early allograft dysfunction (EAD) and subsequent primary allograft failure (PAF).. The purpose of this study is to compare IRI, EAD, and PAF in liver transplantation in a cohort of patients perfused with histidine-tryptophan-ketoglutarate (HTK) solution and University of Wisconsin (UW) solution versus HTK alone.. A randomized trial was performed to compare outcomes in liver recipients who underwent transplantation surgery in the University Regional Hospital of Malaga, Spain. Forty patients were randomized to two groups. Primary endpoints included IRI, EAD, PAF, re-intervention, acute cellular rejection, retransplantation, arterial complications, and biliary complications at postoperative day 90.. Postoperative glutamic oxaloacetic transaminase (1869.15 ± 1559.75 UI/L vs. 953.15 ± 777.27 UI/L; P = .004) and glutamic pyruvic transaminase (1333.60 ± 1115.49 U/L vs. 721.70 ± 725.02 U/L; P = .023) were significantly higher in patients perfused with HTK alone. A clear tendency was observed in recipients perfused with HTK alone to present moderate to severe IRI (7 patients in the HTK + UW solution group vs. 15 patients in the HTK-alone solution group; P = .06), EAD (0 patients in the HTK + UW solution group vs. 0 patients in the HTK-alone solution group; P = .76), and PAF (3 patients in the HTK + UW solution group vs. 8 patients in the HTK-alone solution group; P = .15).. Initial perfusion with HTK solution followed by UW solution in liver transplantation improves early liver function as compared to perfusion with HTK alone.

Topics: Adenosine; Adult; Alanine Transaminase; Allopurinol; Aspartate Aminotransferases; Cohort Studies; Drug Therapy, Combination; Female; Glucose; Glutathione; Graft Rejection; Humans; Insulin; Liver; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Perfusion; Postoperative Period; Potassium Chloride; Procaine; Raffinose; Reoperation; Reperfusion Injury; Spain; Treatment Outcome

One of the main concerns in liver transplant is the prolonged ischemia time, which may lead to primary graft nonfunction or delayed function. N-acetylcysteine is known as a hepato-protective agent in different studies, which may improve human hepatocyte viability in steatotic donor livers. This study investigated whether N-acetylcysteine can decrease the rate of ischemia-reperfusion syndrome and improve short-term outcome in liver transplant recipients.. This was a double-blind, randomized, control clinical trial of 115 patients. Between April 2012 and January 2013, patients with orthotopic liver transplant were randomly divided into 2 groups; in 49 cases N-acetylcysteine was added to University of Wisconsin solution as the preservative liquid (experimental group), and in 66 cases standard University of Wisconsin solution was used (control group). We compared postreperfusion hypotension, inotrope requirement before and after portal reperfusion, intermittent arterial blood gas analysis and potassium measurement, pathological review of transplanted liver, in-hospital complications, morbidity, and mortality.. There was no significant difference between the groups regarding time to hepatic artery reperfusion, hospital stay, vascular complications, inotrope requirement before and after portal declamping, and blood gas analysis. Hypotension after portal reperfusion was significantly more common in experimental group compared with control group (P = .005). Retransplant and in-hospital mortality were comparable between the groups.. Preservation of the liver inside Univer-sity of Wisconsin solution plus N-acetylcysteine did not change the rate of ischemia reperfusion injury and short-term outcome in liver transplant recipients.

Topics: Acetylcysteine; Adenosine; Allopurinol; Cold Ischemia; Double-Blind Method; Female; Glutathione; Hospital Mortality; Humans; Hypotension; Insulin; Iran; Length of Stay; Liver Transplantation; Male; Middle Aged; Operative Time; Organ Preservation Solutions; Perfusion; Protective Agents; Raffinose; Reperfusion Injury; Risk Factors; Time Factors; Treatment Outcome; Warm Ischemia

Celsior solution (CS) is a high-sodium, low-potassium, low-viscosity extracellular solution that has been used for liver graft preservation in recent years, although experience with it is still limited. We performed an open-label randomized active-controlled trial comparing CS with the University of Wisconsin solution (UW) for liver transplantation (LT), with a follow-up period of 5 years.. Adult transplant recipients (n=102) were prospectively randomized to receive either CS (n=51) or UW (n=51). The two groups were comparable with respect to donor and recipient characteristics. The primary outcome measure was the incidence of postreperfusion syndrome (PRS). Secondary outcome measures included primary nonfunction (PNF) or primary dysfunction (PDF), liver retransplantation, and graft and patient survival. Other secondary outcome measures were days in the intensive care unit (ICU) and the rates of acute rejection, chronic rejection, infectious complications, postoperative reoperations, and vascular and biliary complications.. In all, 14 posttransplant variables revealed no significant differences between the groups. There were no cases of PNF or PDF. The incidence of PRS was 5.9% in the CS group and 21.6% in the UW group (P=0.041). After reperfusion, CS revealed greater control of serum potassium (P=0.015), magnesium levels (P=0.005), and plasma glucose (P=0.042) than UW. Respective patient survivals at 3, 12, and 60 months were 95.7, 87.2, and 82.0% for the CS group and 95.7, 83.3, and 66.6% for the UW group (P=0.123).. While retaining the same degree of safety and effectiveness as UW for LT, CS may yield postliver graft reperfusion benefits, as shown in this study by a significant reduction in the incidence of PRS and greater metabolic control.

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Disaccharides; Electrolytes; Female; Follow-Up Studies; Glutamates; Glutathione; Histidine; Humans; Insulin; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion Injury; Syndrome; Time Factors; Young Adult

Celsior solution (CS), which has recently become available, that might theoretically offer a new means for improving graft preservation quality. The present prospective, randomized study was designed to evaluate the efficacy of CS compared with University of Wisconsin (UW) for pancreas allografts. Between January 2001 and January 2007, 88 patients underwent pancreatic transplantation, including the last 30 consecutive simultaneous pancreas kidney patients who were randomly assigned to either CS or UW. There was no case of graft thrombosis in either group. There were 2 cases of pancreatitis in the UW group compared with 1 in the CS group. No case of primary nonfunction occurred in either group. There were 2 cases of early duodenal stump fistulae in the CS group that required transplantectomy, whereas this complication was not observed in the UW group. Relaparotomy in the UW group was required in 3 cases due to infection and treated by close drainage that which, progressed to fatal sepsis in 1 patient. In the UW group with 6 months of follow-up, there were 12 patients insulin free. In the CS group, 6 patients underwent relaparotomy, 3 for transplantectomy and the others for intra-abdominal infection, which was fatal in 2 cases. In the CS group with 6 months of follow-up, there were 10 patients insulin free. Two patients died with functioning grafts. These results provided indirect evidence that CS solution is at least as safe as UW to mitigate postreperfusion graft edema and pancreatitis, as well as graft thrombosis.

Topics: Adenosine; Adult; Allopurinol; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Histidine; Humans; Insulin; Kidney Transplantation; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury; Safety; Young Adult

We studied the evolution of the liver graft with preservation in Celsior (CS) compared with University of Wisconsin solution (UW).. A randomized prospective clinical study in 80 liver transplants (OLTs) from May 2001 to October 2003, compared CS (group I; n = 40) and UW (group II; n = 40). The characteristics of the donors were homogeneous, with no significant differences in 15 variables. CS was perfused with 4 L through the aorta, 2 L through the portal vein, and 1 L, through the portal vein on the back table; and the UW, as 3 L, 2 L, and 1 L, respectively. All OLTs were performed using the piggyback technique.. Group I experienced reperfusion syndrome (n = 2; 5.9%), primary graft nonfunction (n = 0); vascular complications (n = 0); biliary anastomosis stenosis (n = 8; 22.9%), intensive care unit (ICU) days (n = 4.1 +/- 1), death within 30 days (n = 1; 3.1%). The patient and graft survivals at 1, 3, 6, 12, and 24 months were 93.7%, 93.7%, 90.2%, 85.7%, 85.7%, and 94.3%, 88.5%, 85.2%, 78%, 78%, respectively. For group II; the reperfusion syndrome occured in 6 patients (17.6%); primary graft nonfunction (n = 0); vascular complications (n = 0), biliary anastomosis stenosis (n = 3; 8.6%), ICU days (n = 4.9 +/- 2.4) and death within 30 days (n = 1; 3.1%); The patient and graft survival at 1, 3, 6, 12, and 24 months were 96.9%, 93.5%, 89.8%, 79.8%, 79.8% and 94.3%, 88.3%, 84.9%, 75.5%, 66.1%, respectively.. CS offers the similar safety to UW for preservation of liver grafts within these ischemia times.

Topics: Adenosine; Adult; Aged; Allopurinol; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Histidine; Humans; Insulin; Liver; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Portal Vein; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion; Reperfusion Injury; Treatment Outcome; Vascular Diseases

Topics: Adenosine; Adult; Allopurinol; Cholagogues and Choleretics; Glutathione; Humans; Insulin; Liver; Liver Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Taurochenodeoxycholic Acid; Transplantation, Homologous

High-sodium as opposed to high-potassium lactobionate/raffinose preservation solution offers potential advantages in improving the quality of liver storage by reducing potassium-induced vasoconstriction and preventing hyperkalaemia on reperfusion. In our study we evaluated in a prospective trial (encompassing 40 consecutive cadaver donor hepatic retrievals and subsequent transplants) the efficacy of a high-sodium formulation versus the standard high-potassium solution. Quality of preservation was assessed by clinical indices of liver function in the intraoperative and early postoperative phases, including measurements of requirements for blood and blood products and potassium, circulating liver enzymes and bilirubin. Frequencies of acute rejection episodes and primary non-function were also recorded. No significant differences were evident in any of the measured parameters. Thus a sodium-based solution can be used for hepatic preservation, advancing the possibility that it may be possible to develop a single storage solution for clinical multi-organ donor operations.

Topics: Adult; Cadaver; Disaccharides; Female; Graft Rejection; Humans; Intraoperative Period; Liver; Liver Function Tests; Liver Transplantation; Male; Middle Aged; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Postoperative Period; Potassium; Prospective Studies; Raffinose; Reperfusion Injury; Tissue Donors

Topics: Adenosine; Allopurinol; Biopsy; Glutathione; Graft Rejection; Humans; Hypertonic Solutions; Insulin; Liver Transplantation; Muromonab-CD3; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Retrospective Studies; Solutions; Transplantation, Homologous

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H

Topics: Animals; Deuterium Oxide; Glutathione; Hydrogen; Insulin; Liver; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion; Reperfusion Injury; Reproducibility of Results

The University of Wisconsin (UW) solution is the gold standard for preserving the liver, kidneys, and pancreas. For renal preservation, the addition of the flavonoid, quercetin (QE), to the preservation solution reduces damage to renal tubular cells, and the addition of sucrose (Suc) is also beneficial for preservation. The aim of this study was to investigate the protective effects of QE and Suc on porcine livers in terms of warm and cold injury and to evaluate whether their use improves ischemia-reperfusion (I/R) injury after simple cold storage (CS).. We tested porcine livers procured after 30 minutes of warm ischemia followed by preservation for 6 hours under the following 2 conditions: group 1, preserved with the CS/UW solution (n = 4); group 2, preserved with the CS/UW solution containing Que 33.1 μM and Suc 0.1 M (n = 6). All livers were evaluated using an ex vivo isolated liver reperfusion model with saline-diluted autologous blood.. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase levels in group 2 were significantly lower at 30 minutes of reperfusion than in group 1. Furthermore, histologic evaluation by hematoxylin and eosin staining showed significantly fewer morphologic changes in group 2 than in group 1, as indicated by the total Suzuki score. Group 2 also had significantly better scores for sinusoidal congestion and hepatocyte cytoplasmic vacuolization.. Adding Que and Suc to the UW solution can effectively prevent cold injury in livers donated after circulatory death.

Topics: Allopurinol; Animals; Cold Injury; Glutathione; Humans; Insulin; Liver; Organ Preservation; Organ Preservation Solutions; Quercetin; Raffinose; Reperfusion Injury; Swine

Cold ischemia-reperfusion injury (IRI) is an inevitable event that increases post-transplant complications. We have previously demonstrated that supplementation of University of Wisconsin (UW) solution with non-FDA-approved hydrogen sulfide (H. Thirty Lewis rats underwent bilateral nephrectomy followed by syngeneic orthotopic transplantation of the left kidney after 24-hour preservation in either UW or UW+STS solution at 4 °C. Rats were monitored to post-transplant day 14 and sacrificed to assess renal function (urine output, serum creatinine and blood urea nitrogen). Kidney sections were stained with H&E, TUNEL, CD68, and myeloperoxidase (MPO) to detect acute tubular necrosis (ATN), apoptosis, macrophage infiltration, and neutrophil infiltration.. UW+STS grafts showed significantly improved graft function immediately after transplantation, with improved recipient survival compared to UW grafts (p < 0.05). Histopathological examination revealed significantly reduced ATN, apoptosis, macrophage and neutrophil infiltration and downregulation of pro-inflammatory and pro-apoptotic genes in UW+STS grafts compared to UW grafts (p < 0.05).. We show for the first time that preservation of renal grafts in STS-supplemented UW solution protects against prolonged cold IRI by suppressing apoptotic and inflammatory pathways, and thereby improving graft function and prolonging recipient survival. This could represent a novel clinically applicable therapeutic strategy to minimize the detrimental clinical outcome of prolonged cold IRI in kidney transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Blood Urea Nitrogen; Cold Ischemia; Creatinine; Glutathione; Insulin; Kidney Function Tests; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate; Thiosulfates

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

The possible reno-protective effect of a controlled brief heat-shock treatment during isolated ex vivo machine perfusion of donor grafts prior to reperfusion should be investigated in a primary in vitro study. Porcine kidneys (n = 14) were retrieved after 20 minutes of cardiac standstill of the donor and subjected to 20 hours of static cold storage in University of Wisconsin solution. Prior to reperfusion, kidneys were subjected to 2 hours of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35°C. In half of the kidneys (n = 7), a brief hyperthermic impulse (10 minutes perfusion at 42°C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Hyperthermic treatment resulted in a 50% increase of heat shock protein (HSP) 70 and HSP 27 mRNA and was accompanied by ~ 50% improvement of tubular re-absorption of sodium and glucose upon reperfusion, compared with the controls. Furthermore, renal loss of aspartate aminotransferase was significantly reduced to one-third of the controls as was urinary protein loss, evaluated by the albumin to creatinine ratio. It is concluded that ex vivo heat-shock treatment seems to be an easily implementable and promising option to enhance renal self-defense machinery against reperfusion injury after preservation that merits further investigation in preclinical models.

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Insulin; Kidney; Kidney Transplantation; Models, Animal; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Rewarming; Sus scrofa; Tissue and Organ Harvesting

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Glutathione; Humans; Insulin; Mice; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

The eIF5A hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) was shown to protect from ischemic injuries. We hypothesized that GC7 could be useful for preconditioning kidneys from donors before transplantation. Using a preclinical porcine brain death (BD) donation model, we carried out in vivo evaluation of GC7 pretreatment (3 mg/kg iv, 5 minutes after BD) at the beginning of the 4h-donor management, after which kidneys were collected and cold-stored (18h in University of Wisconsin solution) and 1 was allotransplanted. Groups were defined as following (n = 6 per group): healthy (CTL), untreated BD (Vehicle), and GC7-treated BD (Vehicle + GC7). At the end of 4h-management, GC7 treatment decreased BD-induced markers, as radical oxygen species markers. In addition, GC7 increased expression of mitochondrial protective peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC1α) and antioxidant proteins (superoxyde-dismutase-2, heme oxygenase-1, nuclear factor [erythroid-derived 2]-like 2 [NRF2], and sirtuins). At the end of cold storage, GC7 treatment induced an increase of NRF2 and PGC1α mRNA and a better mitochondrial integrity/homeostasis with a decrease of dynamin- related protein-1 activation and increase of mitofusin-2. Moreover, GC7 treatment significantly improved kidney outcome during 90 days follow-up after transplantation (fewer creatininemia and fibrosis). Overall, GC7 treatment was shown to be protective for kidneys against BD-induced injuries during donor management and subsequently appeared to preserve antioxidant defenses and mitochondria homeostasis; these protective effects being accompanied by a better transplantation outcome.

Topics: Adenosine; Allopurinol; Animals; Brain Death; Glutathione; Insulin; Kidney; Kidney Transplantation; Organ Preservation Solutions; Peptide Initiation Factors; Raffinose; Reperfusion Injury; RNA-Binding Proteins; Swine

Ex vivo machine perfusion of the liver after cold storage has found to be most effective if combined with controlled oxygenated rewarming up to (sub)-normothermia. On disconnection of the warm graft from the machine, most surgeons usually perform a cold flush of the organ as protection against the second warm ischemia incurred upon implantation. Experimental evidence, however, is lacking and protective effect of deep hypothermia has been challenged for limited periods of liver ischemia in other models. A first systematic test was carried out on porcine livers, excised 30 min after cardiac arrest, subjected to 18 h of cold storage in UW and then machine perfused for 90 min with Aqix-RSI solution. During machine perfusion, livers were gradually rewarmed up to 20 °C. One group (n = 6) was then reflushed with 4 °C cold Belzer UW solution whereas the second group (n = 6) remained without cold flush. All livers were exposed to 45 min warm ischemia at room temperature to simulate the surgical implantation period. Organ function was evaluated in an established reperfusion model using diluted autologous blood. Cold reflush after disconnection from the machine resulted in a significant increase in bile production upon blood reperfusion, along with a significant reduction in transaminases release alanine aminotransferase and of the intramitochondrial enzyme glutamate dehydrogenase. Interestingly, free radical-mediated lipid peroxidation was also found significantly lower after cold reflush. No differences between the groups could be evidenced concerning histological injury and recovery of hepatic energy metabolism (tissue content of adenosine triphosphate). Post-machine preservation cold reflush seems to be beneficial in this particular setting, even if the organs are warmed up only to 20 °C, without notion of adverse effects, and should therefore be implemented in the protocol.

Topics: Adenosine; Allopurinol; Animals; Bile; Cold Temperature; Female; Glutathione; Insulin; Lipid Peroxidation; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion; Reperfusion Injury; Rewarming; Swine; Time Factors

Liver transplantation (LT) is considered the standard treatment for end-stage liver disease, but ideal donors remain in limited supply, resulting in an unavoidable increase in the need to use grafts from marginal donors. The attenuation of ischemia-reperfusion injury (IRI) in such marginal donors is therefore crucial for reducing the possibility of the primary non-function of grafts and graft loss. Some reports have found that molecular-hydrogen showed antioxidant and anti-inflammatory effects in preventing IRI in some non-hepatic transplant models. Therefore, we investigated whether or not molecular-hydrogen could attenuate IRI in LT model rats.. We used a hydrogen-rich water bath to dissolve hydrogen into solution and graft tissues and performed isogenic and orthotopic LT in Lewis rats with University of Wisconsin (UW) solution. Blood and tissue samples were collected 6 h after the reperfusion. Hepatic enzymes in serum were measured. Pathological findings including the expressions of cytokines and heme oxygenase (HO)-1 in liver tissues were evaluated.. The concentration of hydrogen inside the graft tissues increased depending on the storage time, plateauing after 1 h. Serum liver enzyme levels were significantly lower and the histology score of liver damage markedly attenuated in the group given grafts preserved in hydrogen-rich UW solution than in the control group. The hydrogen-rich UW solution group also showed less oxidative damage and hepatocyte apoptosis than the control group, and the expression of proinflammatory cytokines tended to be lower while the protein levels of HO-1 were significantly increased (n = 3-12 per group, P < 0.05).. Storage of liver grafts in hydrogen-rich UW solution resulted in superior functional and morphologic protection against IRI via the up-regulation of HO-1 expression.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cold Temperature; Glutathione; Hepatocytes; Hydrogen; Hydrogen-Ion Concentration; Inflammation; Insulin; Kidney; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; RNA, Messenger

Hypothermic machine perfusion (HMP) has become standard care in many center's to preserve kidneys donated after circulatory death (DCD). Despite a significant reduction in metabolism at low temperatures, the remaining cellular activity requires oxygen. Because of the role and safety of oxygen during HMP has not been fully clarified, its supply during HMP is not standard yet. This study investigates the effect of administering oxygen during HMP on renal function in a porcine DCD model.. After 30 minutes of warm ischemia, porcine slaughterhouse kidneys were preserved for 24 hours by means of cold storage (CS), or HMP with Belzer Machine Perfusion Solution supplemented with no oxygen, 21% or 100% oxygen. Next, kidneys were reperfused for 4 hours in a normothermic machine perfusion setup.. HMP resulted in significantly better kidney function during normothermic machine perfusion. Thiobarbituric acid-reactive substances, markers of oxidative stress, were significantly lower in HMP preserved kidneys. HMP preserved kidneys showed significantly lower aspartate aminotransferase and lactate dehydrogenase levels compared with kidneys preserved by CS. No differences were found between the HMP groups subjected to different oxygen concentrations. Adenosine triphosphate levels significantly improved during HMP when active oxygenation was applied.. This study showed that preservation of DCD kidneys with HMP is superior to CS. Although the addition of oxygen to HMP did not result in significantly improved renal function, beneficial effects were found in terms of reduced oxidative stress and energy status. Oxygen addition proofed to be safe and did not show detrimental effects.

Topics: Adenosine; Allografts; Allopurinol; Animals; Biopsy; Disease Models, Animal; Glutathione; Humans; Hypothermia, Induced; Insulin; Kidney; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Oxygen; Perfusion; Raffinose; Reperfusion; Reperfusion Injury; Swine; Tissue and Organ Harvesting; Warm Ischemia

Ischemia‑reperfusion injury (IRI) is a notable cause of tissue damage during surgical procedures and a major risk factor in graft dysfunction in liver transplantation. Livers obtained from donors after circulatory death (DCD) are prone to IRI and toll‑like receptor 4 (TLR4) serves a prominent role in the inflammatory response associated with DCD liver IRI. The present study was designed to investigate whether TAK242, a specific TLR4 inhibitor, improves hepatic IRI following a DCD graft and to investigate its underlying protective mechanisms. Male Sprague‑Dawley rats were randomized into 4 groups: Control, TAK242, DCD and DCD+TAK242 groups. Rats were pretreated with TAK242 or its vehicle for 30 min, then the livers were harvested without warm ischemia (control group and TAK242 group) or with warm ischemia in situ for 30 min. The livers were stored in cold University of Wisconsin solution for 24 h and subsequently perfused for 60 min with an isolated perfused rat liver system. Rat liver injury was evaluated thereafter. When compared with the DCD group, DCD livers with TAK242 pretreatment displayed significantly improved hepatic tissue injury and less tissue necrosis (P<0.05). Compared with DCD livers, mechanistic experiments revealed that TAK242 pretreatment alleviated mitochondrial dysfunction, reduced reactive oxygen species and malondialdehyde levels and inhibited apoptosis. Additionally, TAK242 significantly inhibited the IRI‑associated inflammatory response, indicated by the decreased expression of TLR4, interleukin (IL)‑1β, IL‑6 and cyclooxygenase 2 at the mRNA and protein levels (P<0.05). TAK242 ameliorates DCD liver IRI via suppressing the TLR4 signaling pathway in rats. The results of the present study have revealed that TAK242 pretreatment harbors a potential benefit for liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sulfonamides; Toll-Like Receptor 4; Warm Ischemia

storage is the most prevalent method for graft preservation in kidney transplantation (KTX). The protective effects of various preservation solutions have been studied extensively in both clinical trials and experimental animal models. However, a paucity of studies have examined the effect of different preservation solutions on graft function in mouse KTX; in addition, the tolerance of the transplanted grafts to further insult has not been evaluated, which was the objective of the present study. We performed mouse KTX in three groups, with the donor kidneys preserved in different solutions for 60 min: saline, mouse serum, and University of Wisconsin (UW) solution. The graft functions were assessed by kidney injury markers and glomerular filtration rate (GFR). The grafts that were preserved in UW solution exhibited better functions, reflected by 50 and 70% lower plasma creatinine levels as well as 30 and 55% higher plasma creatinine levels in GFR than serum and saline groups, respectively, during the first week after transplants. To examine the graft function in response to additional insult, we induced ischemia-reperfusion injury (IRI) by clamping the renal pedicle for 18 min at 4 wk after KTX. We found that the grafts preserved in UW solution exhibited ~30 and 20% less injury assessed by kidney injury markers and histology than in other two preservation solutions. Taken together, our results demonstrated that UW solution exhibited a better protective effect in transplanted renal grafts in mice. UW solution is recommended for use in mouse KTX for reducing confounding factors such as IRI during surgery.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Creatinine; Cytoprotection; Glomerular Filtration Rate; Glutathione; Hepatitis A Virus Cellular Receptor 1; Insulin; Kidney; Kidney Transplantation; Lipocalin-2; Male; Mice, Inbred C57BL; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Saline Solution; Time Factors

University of Wisconsin (UW) solution is not optimal for preservation of marginal organs. Polyethylene glycol (PEG) could improve protection. Similarly formulated solutions containing either 15 or 20 g/L PEG 20 kDa or 5, 15 and 30 g/L PEG 35 kDa were tested in vitro on kidney endothelial cells, ex vivo on preserved kidneys, and in vivo in a pig kidney autograft model. In vitro, all PEGs provided superior preservation than UW in terms of cell survival, adenosine triphosphate (ATP) production, and activation of survival pathways. Ex vivo, tissue injury was lower with PEG 20 kDa compared to UW or PEG 35 kDa. In vivo, function recovery was identical between UW and PEG 35 kDa groups, while PEG 20 kDa displayed swifter recovery. At three months, PEG 35 kDa 15 and 30 g/L animals had worse outcomes than UW, while 5 g/L PEG 35 kDa was similar. PEG 20 kDa was superior to both UW and PEG 35 kDa in terms of function and fibrosis development, with low activation of damage pathways. PEG 20 kDa at 15 g/L was superior to 20 g/L. While in vitro models did not discriminate between PEGs, in large animal models of transplantation we showed that PEG 20 kDa offers a higher level of protection than UW and that longer chains such as PEG 35 kDa must be used at low doses, such as found in Institut George Lopez (IGL1, 1g/L).

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cell Hypoxia; Disease Models, Animal; Endothelial Cells; Glutathione; Insulin; Kidney; Kidney Function Tests; Kidney Transplantation; Male; Molecular Weight; Organ Preservation; Organ Preservation Solutions; Polyethylene Glycols; Primary Cell Culture; Raffinose; Recovery of Function; Reperfusion Injury; Swine; Transplantation, Autologous

Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation.. To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 μM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion.. We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6.. Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.

Topics: Adenosine; Allografts; Allopurinol; Animals; Carbon Monoxide; Cold Ischemia; Glutathione; Insulin; Kidney; Kidney Transplantation; Male; Manganese; Models, Animal; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Sus scrofa

Cold ischemia-reperfusion injury is unavoidable during organ transplantation, and prolonged preservation is associated with poorer function recovery. Cardiotrophin-1 (CT-1) is an IL-6 family cytokine with cytoprotective properties. This preclinical study in rats tested whether CT-1 mitigates cold renal ischemia-reperfusion injury in the context of the transplantation of long-time preserved kidneys.. Kidneys were flushed with cold (4°C) University of Wisconsin solution containing 0.2 μg/mL CT-1 and stored for several periods of time at 4°C in the same solution. In a second approach, kidneys were first cold-preserved for 6 hours and then were perfused with University of Wisconsin solution containing CT-1 (0, 16, 32, or 64 μg/mL) and further cold-preserved. Organ damage markers were measured in the kidneys at the end of the storage period. For renal transplantation, recipient consanguineous Fischer rats underwent bilateral nephrectomy and received a previously cold-preserved (24 hours) kidney as described above. Survival and creatinine clearance were monitored over 30 days.. Cardiotrophin-1 in perfusion and preservation fluids reduced oxidative stress markers (superoxide anion and inducible nitric oxide synthase), inflammation markers (NF-κB and tumor necrosis factor-α), and vascular damage (vascular cell adhesion molecule-1) and activated leukemia inhibitory factor receptor and STAT-3 survival signaling. Transplantation of kidneys cold-preserved with CT-1 increased rat survival and renal function (ie, lower plasma creatinine and higher creatinine clearance) and improved kidney damage markers after transplantation (ie, lower superoxide anion, tumor necrosis factor-α, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 and higher NF-κB).. Cardiotrophin-1 represents a novel therapeutic strategy to reduce ischemia-reperfusion and cold preservation injury to rescue suboptimal kidneys and, consequently, to improve the clinical outcomes of renal transplantation.

Topics: Adenosine; Allografts; Allopurinol; Animals; Cold Ischemia; Cytokines; Disease Models, Animal; Glutathione; Graft Survival; Humans; Insulin; Kidney; Kidney Function Tests; Kidney Transplantation; Male; Nephrectomy; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred F344; Reperfusion Injury; Tissue and Organ Harvesting

Renal transplantation is the preferred treatment for end-stage renal disease. Currently, there is a large gap between the supply and demand for transplantable kidneys. The use of sub-optimal grafts obtained via donation after cardiac death (DCD) is on the rise. While static cold storage (SCS) in University of Wisconsin (UW) solution on ice (4°C) is the clinical standard of care for renal graft preservation, cold storage has been associated with negative graft outcomes. The alternative, normothermic machine perfusion, involves mechanical perfusion of the organ at physiological or normothermic temperature (37°C) and this technique is expensive, complicated and globally inaccessible. As such, simpler alternatives are of interest. Preliminary results revealed that UW solution is more protective at 21°C than 37°C and subnormothermic preservation is of interest because it may facilitate the use of existing solutions while preventing cold injury. We have previously shown that SCS in UW solution supplemented with mitochondria-targeted H

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cell Line; Cell Survival; Cytoprotection; Epithelial Cells; Glutathione; Hydrogen Sulfide; Insulin; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Organophosphorus Compounds; Perfusion; Raffinose; Reperfusion Injury; Swine; Temperature; Thiones

Deceased donor kidneys are exposed to cold ischemic insult which makes them particularly susceptible to the effects of cold ischemic injury during hypothermic preservation resulting in high rates of delayed graft function. Bone morphogenetic protein-7 (BMP-7) is a valuable reagent in the field of tissue regeneration and preservation under ischemic conditions. Following these insights, we investigated the effect of recombinant human BMP-7 (rhBMP-7) on graft preservation during cold ischemia. The study was conducted on an experimental model of kidney cold ischemia in rats. Kidneys were perfused with University of Wisconsin (UW) saline solution, rhBMP-7, or rhBMP-7 + UW, and exposed to cold ischemia for 6, 12, and 24 hours. In tubular epithelial cells of kidneys perfused with rhBMP-7 and rhBMP-7+UW solution, the expression of BMP-7 and E-cadherin was observed after 24 hours of cold ischemia. In kidneys not perfused with rhBMP-7, high expression of transforming growth factor-β and α-smooth muscle actin was found. Also, in kidneys perfused with rhBMP-7 solution, statistically higher levels of Smad1, Smad5, and Smad8 messenger RNA expressions were proven. BMP-7 maintains the morphology of kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells.

Topics: Adenosine; Allopurinol; Animals; Bone Morphogenetic Protein 7; Cold Ischemia; Delayed Graft Function; Glutathione; Insulin; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury

During liver transplantation, graft ischemia-reperfusion injury leads to a systemic inflammatory response producing postoperative organ dysfunctions. The aim of this observational and prospective study was to compare the impact of Solution de conservation des organes et tissus (SCOT) 15 and University of Wisconsin (UW) preservation solutions on early cytokine release, postreperfusion syndrome and postoperative organ dysfunctions.. Thirty-seven liver transplantations were included: 21 in UW Group and 16 in SCOT 15 group. Five cytokines were measured in systemic blood after anesthetic induction, 30minutes after unclamping portal vein and on postoperative day 1.. Following unclamping portal vein, cytokines were released in systemic circulation. Systemic cytokine concentrations were higher in UW than in SCOT 15 group: Interleukin-10, Interleukine-6. In SCOT 15 group, significant reduction of postreperfusion syndrome incidence and acute kidney injury were observed. Alanine and aspartate aminotransferase peak concentrations were higher in SCOT 15 group than in UW group. However, from postoperative day 1 to day 10, aminotransferase returned to normal values and did not differ between groups.. Compared to UW, SCOT 15 decreases systemic cytokine release resulting from graft ischemia-reperfusion injury and reduces incidence of postreperfusion syndrome and postoperative renal failure.

Topics: Adenosine; Allopurinol; Cytokines; Female; Glutathione; Humans; Insulin; Liver Transplantation; Male; Middle Aged; Multiple Organ Failure; Organ Preservation Solutions; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion Injury; Time Factors

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC-/MSC-derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold-perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT-PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC-/EV-treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up-regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Energy Metabolism; Extracellular Vesicles; Gene Expression; Gene Expression Profiling; Glucose; Glutathione; Insulin; Ion Transport; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Lactic Acid; Malondialdehyde; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Organ Preservation; Organ Preservation Solutions; Perfusion; Pyruvic Acid; Raffinose; Rats; Rats, Inbred F344; Rats, Transgenic; Reperfusion Injury

The outcomes of liver transplantation (LT) from donation after cardiac death (DCD) donors remain poor due to severe warm ischemia injury. Perfluorocarbon (PFC) is a novel compound with high oxygen carrying capacity. In the present study, a rat model simulating DCD LT was used, and the impact of improved graft oxygenation provided by PFC addition on liver ischemia/reperfusion injury (IRI) and survival after DCD LT was investigated. Orthotopic liver transplants were performed in male Lewis rats, using DCD liver grafts preserved with cold University of Wisconsin (UW) solution in the control group and preserved with cold oxygenated UW solution with addition of 20% PFC in the PFC group. For experiment I, in a 30-minute donor warm ischemia model, postoperative graft injury was analyzed at 3 and 6 hours after transplantation. For experiment II, in a 50-minute donor warm ischemia model, the postoperative survival was assessed. For experiment I, the levels of serum aspartate aminotransferase, alanine aminotransferase, hyaluronic acid, malondialdehyde, and several inflammatory cytokines were significantly lower in the PFC group. The hepatic expression levels of tumor necrosis factor α and interleukin 6 were significantly lower, and the expression level of heme oxygenase 1 was significantly higher in the PFC group. Histological analysis showed significantly less necrosis and apoptosis in the PFC group. Sinusoidal endothelial cells and microvilli of the bile canaliculi were well preserved in the PFC group. For experiment II, the postoperative survival rate was significantly improved in the PFC group. In conclusion, graft preservation with PFC attenuated liver IRI and improved postoperative survival. This graft preservation protocol might be a new therapeutic option to improve the outcomes of DCD LT. Liver Transplantation 23 1171-1185 2017 AASLD.

Topics: Adenosine; Allografts; Allopurinol; Animals; Disease Models, Animal; Fluorocarbons; Glutathione; Graft Survival; Humans; Insulin; Liver; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Postoperative Period; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate; Time Factors; Treatment Outcome; Warm Ischemia

To compare the effect of University of Wisconsin (UW) solution with or without metformin, an AMP-activated protein kinase (AMPK) activator, for preserving standard and marginal liver grafts of young and aged rats. Eighteen young (4 mo old) and 18 aged (17 mo old) healthy male SD rats were selected and randomly divided into three groups: control group, UW solution perfusion group (UWP), and UW solution with metformin perfusion group (MUWP). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), interleukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-α) in the perfused liquid were tested. The expression levels of AMPK and endothelial nitric oxide synthase (eNOS) in liver sinusoidal endothelial cells were also examined. Additionally, microscopic evaluation of the harvested perfused liver tissue samples was done.. AST, ALT, LDH, IL-18 and TNF-α levels in the young and aged liver-perfused liquid were, respectively, significantly lower in the MUWP group than in the UWP group (. The addition of metformin to the UW preservative solution for

Topics: Adenosine; Alanine Transaminase; Allopurinol; AMP-Activated Protein Kinases; Animals; Aspartate Aminotransferases; Cold Ischemia; Glutathione; Hepatocytes; Humans; Infusion Pumps; Insulin; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Metformin; Microscopy, Electron, Transmission; Models, Animal; Nitric Oxide Synthase Type III; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue and Organ Harvesting

The purpose of this study is to explore whether normothermic machine perfusion (NMP) preservation is superior to cold preservation during reduced-size liver transplantation (RSLT) in pigs. Twenty-four healthy Ba-Ma mini pigs were used (aged >13 months; weight 25-35 kg; regardless of sex). The animals were randomized into 2 groups. In group A (NMP), donor livers were harvested without warm ischemia time and heartbeats and then were connected to the NMP system to reduce the livers' size under the normothermic condition. In group B (University of Wisconsin [UW] solution), donor livers were harvested without warm ischemia time and heartbeats after being perfused by UW solution and were then preserved in 0°C-4°C UW solution to reduce the livers' size under cold conditions. After that, liver transplantation without venovenous bypass was performed. General RSLT information of the pigs from the 2 groups was recorded; the serological indices were measured; and routine pathological examination of liver tissue was observed. A significant difference was observed in the intraoperative bleeding between the 2 groups (P < 0.05), whereas no significant difference was found in the other indices (all P > 0.05). Significant differences of alanine aminotransferase levels, aspartate aminotransferase levels, and lactate dehydrogenase levels between the 2 groups were observed between postoperative days 3 and 5 (P < 0.05). Significant differences of lactic acid levels between the 2 groups were observed between postoperative days 2 and 5 (P < 0.05). Compared with the cold preservation group, the liver tissues of the NMP preservation group only rarely experienced liver cell necrosis and maintained integrities in the hepatic sinusoid spaces and endothelial cells. In conclusion, NMP preservation is superior to cold preservation during RSLT in pigs. Liver Transplantation 22 968-978 2016 AASLD.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Cold Ischemia; Glutathione; Hepatocytes; Humans; Insulin; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Necrosis; Organ Preservation; Organ Preservation Solutions; Perfusion; Postoperative Period; Raffinose; Reperfusion Injury; Swine; Swine, Miniature; Temperature

Deceased after cardiac death donors represent an important source of organs to reduce organ shortage in transplantation. However, these organs are subjected to more ischaemia-reperfusion injury (IRI). Reducing IRI by targeting coagulation is studied here in an experimental model.. The effect of an anti-Xa compound (fondaparinux) was evaluated using an autotransplanted kidney model in pigs. Kidneys were clamped for 60 min (warm ischaemia) and then preserved for 24 h at 4 °C in University of Wisconsin solution (UW). The anti-Xa compound was injected intravenously before warm ischaemia and used during cold storage, and its effects were compared with those of intravenous injection of unfractionated heparin (UFH) before warm ischaemia and use during cold storage, or use of UW alone during cold storage.. At 3 months after transplantation, anti-Xa treatment improved recovery of renal function and chronic serum creatinine levels compared with UW and UFH (mean(s.e.m.) 89(4), 250(4) and 217(8) µmol/l respectively). The anti-Xa treatment also reduced fibrosis, and decreased tissue expression of markers of the epithelial-mesenchymal transition compared with UW and UFH. Cleaved protease-activated receptor 2 was overexpressed in the UW group compared with the anti-Xa and UFH groups. Leucocyte infiltrates were decreased in the anti-Xa group compared with the UW and UFH groups. Macrophage invasion was also decreased by anticoagulation treatment.. Peritransplant anticoagulation therapy was beneficial to graft outcome, in both the acute and chronic phases. Moreover, specific inhibition of coagulation Xa protease further protected kidney grafts, with better recovery and decreased expression of chronic lesion markers. Surgical relevance The increasing use of marginal donors highlights the importance of organ quality in transplantation. Renal ischaemia-reperfusion injury (IRI), which includes a deleterious activation of coagulation, plays a central role in determining graft quality and outcome. Using an established porcine renal autotransplantation preclinical model with high clinical relevance, the benefits of anticoagulation therapy using an antifactor Xa molecule were evaluated. Peritransplantion anticoagulation treatment, specifically with an anti-Xa compound, protected marginal kidney grafts, improving functional recovery and reducing chronic lesions. This study demonstrates the benefits of anticoagulation therapy at the time of organ collection, particularly for marginal organs, encountered in cases of extended criteria and deceased after circulatory death donors. This anticoagulation strategy could be an important addition to current donor and organ management protocols in order to limit IRI and improve outcome.

Topics: Adenosine; Allopurinol; Animals; Anticoagulants; Constriction; Cytokines; Fondaparinux; Glutathione; Insulin; Kidney; Kidney Transplantation; Leukocytes; Nephritis; Organ Preservation Solutions; Polysaccharides; Raffinose; Reperfusion Injury; Swine; Transplantation, Autologous; Warm Ischemia

By binding to T cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 co-stimulation may rescue IR-stressed orthotopic liver transplants from innate immunity-driven inflammation.. We used wild type (WT) and TIM-3 transgenic (Tg) mice (C57BL/6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20 h at 4°C in UW solution) and syngeneic orthotopic liver transplantation (OLT).. Orthotopic liver transplants in WT or TIM-3Tg→TIM-3Tg groups were resistant against IR-stress, evidenced by preserved hepatocellular function (serum ALT levels) and liver architecture (Suzuki's score). In contrast, orthotopic liver transplants in WT or TIM-3Tg→WT groups were susceptible to IRI. TIM-3 induction in circulating CD4+ T cells of the recipient: (1) depressed T-bet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in orthotopic liver transplants; (2) promoted T cell exhaustion (PD-1, LAG-3) phenotype; and (3) depressed neutrophil and macrophage infiltration/function in orthotopic liver transplants. In parallel studies, we documented for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in vivo and in vitro. Moreover, exogenous recombinant Gal-9 (rGal-9) potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells.. Harnessing TIM-3/Gal-9 signalling at the T cell-hepatocyte interface facilitates homeostasis in IR-stressed orthotopic liver transplants. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cell Differentiation; Disease Models, Animal; Galectins; Glutathione; Hepatitis A Virus Cellular Receptor 2; Hepatocytes; Immunity, Innate; In Vitro Techniques; Insulin; Liver Transplantation; Lymphocyte Activation; Macrophages; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neutrophils; Organ Preservation; Organ Preservation Solutions; Raffinose; Receptors, Virus; Reperfusion Injury; Signal Transduction; T-Lymphocytes

BACKGROUND Results in small bowel transplantation are inferior compared to other solid organ transplantations, among other reasons, due to a specific vulnerability to ischemia/reperfusion injury. New strategies are needed to improve organ storage. Here we compare static cold storage in University of Wisconsin solution to storage supplemented with molecular oxygen gas insufflation. MATERIAL AND METHODS Rat small bowel was retrieved and either stored unoxygenated (UW) or oxygenated (UW+O2) for 18 h at 4°C. Biochemical parameters, mucosal function, Toll-like receptor upregulation, and parameters of structural integrity were evaluated following isolated reperfusion in vitro for 30 min at 37°C. RESULTS Oxygenation showed: ATP concentration was 82 times higher; lactate dehydrogenase release was continuously lower over 30 min; malondialdehyde, a final product of lipid peroxidation (UW+O2 vs. UW; 2.7±0.92 nmol/mL vs. 17.22±10.1 nmol/mL; P<0.05) and nitric oxide concentration (0.87±0.27 µmol/L vs. 2.17±0.29 µmol/L; P<0.001) were significantly lower; whereas mucosal functional integrity (galactose uptake) was better preserved (0.47±0.18 mg/dL vs. 0.35±0.05 mg/dL). Amelioration of tissue damage could be demonstrated by reduced apoptosis (3.3±1.2 AU vs. 28.4±10 AU; P>0.05), and preserved subcellular integrity. Toll-like receptors were significantly less upregulated (TLR2 0.32±0.1 vs. 2.1±1.5-fold and TLR4 1.53±1.14 vs. 11.79±5.4-fold; P<0.05). CONCLUSIONS Oxygenated storage is superior to standard storage in University of Wisconsin solution in terms of energetics, tissue damage, and mucosal integrity.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insufflation; Insulin; Intestine, Small; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cells, Cultured; Chemistry, Pharmaceutical; Curcumin; Cyclodextrins; Disease Models, Animal; Fibrosis; Flow Cytometry; Glutathione; Graft Rejection; Humans; Inflammation; Insulin; Kidney Transplantation; Kidney Tubules; Male; Organ Preservation Solutions; Oxidative Stress; Prostate; Raffinose; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine

Although liver transplantation from non-heart-beating donors (NHBDs) is an effective way to overcome shortage of donors, primary graft nonfunction is often noted in these grafts. We have previously reported that edaravone, a free radical scavenger, has a cytoprotective effect on warm ischemia-reperfusion injury and improves the function of liver grafts from NHBDs in a rat model of ischemia-reperfusion. The purpose of this study was to investigate the effects of edaravone on liver transplantations from NHBDs.. Pigs were divided into three groups: (1) a heart-beating (HB) group (n = 5), in which liver grafts were retrieved from HB donors; (2) a non-heart-beating (NHB) group (n = 4), in which liver grafts were retrieved under apnea-induced NHB conditions; and (3) an edaravone-treated (ED) group (n = 5), in which liver grafts were retrieved in the same manner as the NHB group and treated with edaravone at the time of perfusion (3 mg/L in University of Wisconsin [UW] solution), cold preservation (1 mg/L in UW solution), and after surgery (1 mg/kg/d). The grafts from all groups were transplanted after 4 hours of cold preservation.. In the ED group, the 7-day survival rate was significantly higher than that in the NHB group (80% versus 0%, P = .0042, Kaplan-Meier log-rank test). Furthermore, on histologic examination, the structure of sinusoids in the ED group was well preserved and similar to that in the HB group.. Edaravone may improve the viability of liver grafts from NHBDs.

Topics: Adenosine; Allopurinol; Animals; Antipyrine; Biomarkers; Cold Ischemia; Cytoprotection; Edaravone; Free Radical Scavengers; Glutathione; Graft Survival; Insulin; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine; Time Factors; Tissue and Organ Harvesting; Tissue Survival; Warm Ischemia

Use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, this requires the development of novel preservation methods to recover the organ from changes due to warm ischemia time (WIT).. Porcine livers were perfused with a newly developed machine perfusion (MP) system. The livers were perfused with modified University of Wisconsin solution (UW) - gluconate. All grafts were procured after acute hemorrhagic shock with the ventilator off. For group 1 (n = 6), grafts were procured after WIT of 60 minutes and preserved by hypothermic MP (HMP) for 3 hours. For group 2 (n = 5), grafts were preserved with 2 hours of simple cold storage (SCS) and HMP for 2 hours. For group 3 (n = 6), grafts were preserved with 2 hours of SCS and rewarming up to 25°C by MP for 2 hours (RMP). The preserved liver grafts were transplanted orthotopically.. The alanine aminotransferase level in perfusate in RMP during perfusion preservation was maintained at less than that of HMP. The levels of aspartate aminotransferase and lactate dehydrogenase in the 2 hours after reperfusion were significantly lower in group 3. Histologically, the necrosis of hepatocytes was less severe in group 3. The survival rate in group 3 was 2/4, but 0/4 in the other group.. RMP is expected to facilitate the recovery of the DCD liver grafts.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Cold Ischemia; Disease Models, Animal; Female; Glutathione; Graft Survival; Heart Arrest; Hepatectomy; Insulin; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Necrosis; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Rewarming; Sus scrofa; Time Factors; Tissue and Organ Harvesting; Warm Ischemia

The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model.. Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy.. Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr.. Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.

Topics: Adenosine; Allopurinol; Animals; Cold Ischemia; Dose-Response Relationship, Drug; Extremities; Glucose; Glutathione; Inflammation; Insulin; Male; Mannitol; Microscopy, Confocal; Microscopy, Electron, Transmission; Muscle, Skeletal; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Sciatic Nerve; Time Factors

Biolasol solution (Pharmaceutical Research and Production Plant "Biochefa," Sosnowiec, Poland) is a novel extracellular perfusion and ex vivo hypothermic kidney preservation solution. It ensures maintenance of homeostasis, reduces tissue edema, has low viscosity, and allows the graft to preserve structural and functional integrity. It minimizes ischemia-reperfusion damage.. Perfundates from control and transplanted kidneys flushed with Biolasol or ViaSpan solutions (Arkas, Warszawa, Poland) were analyzed. Parameters of serum and urine collected from 12 pigs after auto-transplantation were also analyzed. Renal medulla was investigated for structural alterations by analyzing hematoxylin and eosin-stained slides. The mean survival time of pigs after the auto-transplantation procedure was the measure for the novel Biolasol solution effectiveness.. We observed a statistically significant decrease in marker enzyme levels alanine transaminase, aspartate transaminase, lactic dehydrogenase, and ions (Na and K) in pigs with grafts flushed with Biolasol. Histopathologic examination revealed that the renal cortex structure was not damaged after the use of Biolasol solution.. Biolasol solution protects kidneys against ischemia damage and does not differ significantly from the "golden standard" ViaSpan solution.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Creatinine; Glutathione; Insulin; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Organ Preservation Solutions; Poland; Raffinose; Reperfusion Injury; Swine; Transplantation, Autologous

To test whether a new rinse solution containing polyethylene glycol 35 (PEG-35) could prevent ischemia-reperfusion injury (IRI) in liver grafts.. Sprague-Dawley rat livers were stored in University of Wisconsin preservation solution and then washed with different rinse solutions (Ringer's lactate solution and a new rinse solution enriched with PEG-35 at either 1 or 5 g/L) before ex vivo perfusion with Krebs-Heinseleit buffer solution. We assessed the following: liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity), liver function (bile output and vascular resistance), oxidative stress (malondialdehyde), nitric oxide, liver autophagy (Beclin-1 and LCB3) and cytoskeleton integrity (filament and globular actin fraction); as well as levels of metalloproteinases (MMP2 and MMP9), adenosine monophosphate-activated protein kinase (AMPK), heat shock protein 70 (HSP70) and heme oxygenase 1 (HO-1).. When we used the PEG-35 rinse solution, reduced hepatic injury and improved liver function were noted after reperfusion. The PEG-35 rinse solution prevented oxidative stress, mitochondrial damage, and liver autophagy. Further, it increased the expression of cytoprotective heat shock proteins such as HO-1 and HSP70, activated AMPK, and contributed to the restoration of cytoskeleton integrity after IRI.. Using the rinse solution containing PEG-35 was effective for decreasing liver graft vulnerability to IRI.

Topics: Adenosine; Allopurinol; Animals; Autophagy; Biomarkers; Cold Ischemia; Cytoprotection; Cytoskeleton; Disease Models, Animal; Glutathione; Hepatectomy; Insulin; Liver; Liver Function Tests; Male; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Polyethylene Glycols; Raffinose; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Time Factors

S-Nitrosated human serum albumin (SNO-HSA) is useful in preventing liver ischemia/reperfusion injury, and SNO-HSA should thus be able to prevent cell injury during liver transplantation. However, the potential protective effect of SNO-HSA on a combination of cold and warm ischemia, which is obligatory when performing liver transplantation, has not been examined. Therefore, we evaluated the protective effect of SNO-HSA added to University of Wisconsin (UW) solution during cold or/and warm ischemia in situ and in vitro. First, we observed that apoptotic and necrotic cell death were increased during cold and warm ischemia, respectively. SNO-HSA, which possesses anti-apoptosis activity at low NO concentrations, can inhibit cold ischemia injury both in situ and in vitro. In contrast, SNO-HSA had no significant effect on warm liver ischemia injury which, however, can be reduced by UW solution. We also demonstrated that the cellular uptake of NO from SNO-HSA can occur during cold ischemia resulting in induction of heme oxygenase-1 within 3h of cold ischemia. Our results indicate that treatment with SNO-HSA or UW solution alone is not sufficient to inhibit liver injury during a period of both cold and warm ischemia. However, a combination of SNO-HSA and UW solution can be used to prevent the two types of ischemia. SNO-HSA-added UW solution could be very useful in transplantation, because the previously imposed constraints on preservation time can be removed. This is a great advantage in a situation as the present one with increased utilization of scarce donor organs for more recipients.

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Apoptosis; Glutathione; Hep G2 Cells; Humans; Insulin; Liver; Liver Diseases; Liver Transplantation; Male; Necrosis; Nitric Oxide Donors; Nitroso Compounds; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Serum Albumin; Serum Albumin, Human

The small intestine is one of the most sensitive organs to ischemia-reperfusion injury during transplantation. Cytoprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) is well known. The aim of our study was to measure changes of PACAP-38-like immunoreactivities and cytokine levels in intestinal grafts stored PACAP-38 containing preservation solution.. Small-bowel autotransplantation was performed on male Wistar rats (n = 56). Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 (GI), 3 (GII), and 6 hours (GIII); and in PACAP-38 containing UW solution for 1 (GIV), 3 (GV), and 6 hours (GVI). Reperfusion lasted 3 hours in each group. Intestinal PACAP-38 immunoreactivities were measured by radioimmunoassay. To measure cytokine from tissue homogenates we used rat cytokine array and Luminex Multiplex Immunoassay.. Levels of PACAP-38-like and PACAP-27-like immunoreactivities decreased by preservation time compared to control. This decrease was significant following 6 hours cold storage (p < 0.05). Values remained significantly higher in grafts stored in PACAP-38 containing UW. Expressions of sICAM-1, L-selectin, tissue inhibitor of metalloproteinase-1 were increased in GIII and were decreased in GVI.. PACAP-38 increased tissue levels of PACAP-38 and PACAP-27, and decreased cytokine expression. This indicates that PACAP-38 has anti-inflammatory and cytoprotective effects in intestinal autotransplantation model.. Bevezetés: A vékonybél ischaemia-reperfusióval szembeni fokozott érzékenysége a szerv transzplantációjakor is jelen lévő probléma. Ismert a hypophysis adenilát-cikláz aktiváló polipeptid (PACAP) sejtvédő hatása. Munkánkban azt vizsgáltuk, hogy PACAP-38-at tartalmazó University of Wisconsin (UW) oldatban történő konzerválás hogyan befolyásolja a szöveti PACAP- és citokinszinteket. Anyag és módszer: Wistar-patkányokon (n = 56) vékonybél-autotranszplantációt végeztünk. A graftokat 4 °C-os UW oldatban tároltuk 1 (I. csoport), 3 (II.) és 6 órán (III.), illetve 100 μg PACAP-38-at tartalmazó UW oldatban 1 (IV.), 3 (V.) és 6 órán (VI.) át. A reperfusio 3 óra volt. Bélmintákból a PACAP-38- és PACAP-27-szinteket radioimmunassayjel határoztuk meg, míg a citokinexpressiót kemilumineszcens módszerrel és Luminex Multiplex Immunoassayjel mértük. Eredmények: A szöveti PACAP-38-szint a kontrollhoz (57,32 ± 3,5 fmol/mg) képest a konzerválás idejével csökkent, és 6 óra után szignifikáns volt (III.: 32,6 ± 3,9 fmol/mg, p < 0,05), míg a IV–VI. csoportoknál szignifikánsan nőtt. A PACAP-27 szöveti értéke is hasonló tendenciával változott. Az sICAM-1, L-selectin és a metalloproteáz-1 szöveti inhibitorának emelkedett expressióját mértük a III. csoportban, és jelentős csökkenés volt a VI. csoportban. Következtetés: UW oldathoz adott PACAP-38 növelte a szöveti PACAP-38- és PACAP-27-szinteket, és csökkentette a citokinexpressiót. Mindez a PACAP-38 citoprotektív és anti-inflammatórikus hatását jelzi bél-autotranszplantációs modellben. Támogatta: OTKA (PD77474, 104984, CNK78480), MTA Bolyai-ösztöndíj és Lendület program.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents; Antioxidants; Cytokines; Cytoprotection; Down-Regulation; Glutathione; Graft Survival; Growth Substances; Insulin; Intercellular Adhesion Molecule-1; Intestine, Small; L-Selectin; Male; Organ Preservation; Organ Preservation Solutions; Pituitary Adenylate Cyclase-Activating Polypeptide; Radioimmunoassay; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transplantation, Autologous

Organ cold storage and subsequent transplantation are associated with significant ischemia-reperfusion injury, leading to cell death, graft inflammation and decreased graft function.. CORM-3s reduce oxidative stress and prevent inflammation in kidneys stored at 4C and subsequently transplanted. Graft survival and function are markedly improved compared to kidneys preserved and stored in University of Wisconsin solution alone. We determined whether CORM-3 has direct antiapoptotic effects on in vitro preparations of human HUVECs exposed to anoxic conditions. We also determined whether direct administration of CORM-3 to renal grafts before and/or after cold storage would prevent renal damage during the transplantation process.. CORM-3 supplementation led to a significantly increased frequency of live cells (mean ± SD 72.3% ± 1.9%, p <0.01), reduced apoptosis (14.9% ± 6.1%, p <0.01) and decreased mitochondrial transmembrane potential (40.2% ± 7.2%, p <0.05) in HUVECs exposed to 20 hours of cold storage compared to controls (11.6% ± 3.5%, 82.2% ± 2.3% and 78.2% ± 3.2%, respectively). In keeping with this antiapoptotic effect CORM-3 supplementation led to a mean 7.4 ± 2.1-fold up-regulation in Bcl-2 gene expression. CORM-3 supplementation in standard preservation solution was most beneficial at initial ischemic injury and before cold storage exposure. However, additional reflushing before vascular reperfusion showed an additive benefit to graft survival and function after transplantation. This was confirmed by decreased glomerular and tubular necrosis, and apoptosis in double flushed grafts.. CORM-3 supplementation in standard University of Wisconsin solution has a significant impact on decreasing cellular and graft injury, and improving survival through its antiapoptotic effects, which are likely mediated through mitochondrial membrane stabilization.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Glutathione; Graft Survival; Inflammation; Insulin; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Organometallic Compounds; Oxidative Stress; Raffinose; Rats, Inbred Lew; Reperfusion Injury

Cyclosporin-A (CsA) has been reported to protect livers from warm ischemia/reperfusion (I/R) injury. To study if CsA has also a protective effect on cold I/R injury, two models were used: the isolated perfused rat liver (IPRL) and the orthotopic rat liver transplantation (ORLT). (1) IPRL: Livers were preserved for 24 h (5°C) in University of Wisconsin (UW) solution alone (group 1), with CsA (400 nM) dissolved in dimethylsulfoxide (50 μM) (group 2), and with dimethylsulfoxide (DMSO) alone (group 3). Livers were reperfused for 60 min (37°C) (n = 8/group). Cell necrosis was evaluated by trypan blue uptake and apoptosis by laddering and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and by caspase-3 activation. Marked and similar sinusoidal endothelial cell necrosis was found in the three groups while apoptosis was found similarly deceased in groups 2 and 3 compared with group 1. (2) ORLT: Donors received either CsA (5 mg/kg) or corn oil 24 h before transplantation. Recipients were sacrificed after 240 min; cell necrosis and apoptosis were then evaluated. No difference was found between treated and control groups. The current data strongly suggest that CsA has no protective effect on hepatic cold I/R injury. Hepatocyte apoptosis can be reduced by antioxidants, as occurred with DMSO, but introduction of CsA does not provide additional protective effect.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Cold Ischemia; Cyclosporine; Dimethyl Sulfoxide; Endothelium; Glutathione; Hepatocytes; Insulin; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Regional Blood Flow; Reperfusion Injury

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia-induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO-UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase-3 and PARP expression. In contrast, grafts in CO-UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO-UW with less TUNEL(+) cells and reduced mRNA upregulation for proinflammatory mediators (IL-6, TNF-α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO-UW for 24 h, graft PARP expression, TUNEL(+) cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia-induced apoptosis and significantly improved kidney graft function and outcomes of KTx.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Carbon Monoxide; Cold Temperature; Cytosol; Death; Glutathione; Humans; Inflammation; Insulin; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; RNA, Messenger; Treatment Outcome

Pathophysiological alterations in the endothelial phenotype result in endothelial dysfunction. Flow cessation, occurring during organ procurement for transplantation, triggers the endothelial dysfunction characteristic of ischemia/reperfusion injury, partly due to a reduction in the expression of the vasoprotective transcription factor Kruppel-like Factor 2 (KLF2). We aimed at (1) characterizing the effects of flow cessation and cold storage on hepatic endothelial phenotype, and (2) ascertaining if the consequences of cold stasis on the hepatic endothelium can be pharmacologically modulated, improving liver graft function. Expression of KLF2 and its vasoprotective programs was determined in (i) hepatic endothelial cells (HEC) incubated under cold storage conditions with or without the KLF2-inducer simvastatin, and (ii) rat livers not cold stored or preserved in cold University of Wisconsin solution (UWS) supplemented with simvastatin or its vehicle. In addition, upon warm reperfusion hepatic vascular resistance, endothelial function, nitric oxide vasodilator pathway, apoptosis, inflammation, and liver injury were evaluated in not cold stored livers or livers preserved in cold UWS supplemented with simvastatin or vehicle. Expression of KLF2 and its vasoprotective programs decrease in HEC incubated under cold storage conditions. Cold-stored rat livers exhibit a time-dependent decrease in KLF2 and its target genes, liver injury, increased hepatic vascular resistance, and endothelial dysfunction. The addition of simvastatin to the storage solution, maintained KLF2-dependent vasoprotective programs, prevented liver damage, inflammation, and oxidative stress and improved endothelial dysfunction.. Our results provide a rationale to evaluate the beneficial effects of a vasoprotective preservation solution on human liver procurement for transplantation.

Topics: Adenosine; Allopurinol; Animals; Cells, Cultured; Cryoprotective Agents; Endothelium, Vascular; Glutathione; Insulin; Kruppel-Like Transcription Factors; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation; Organ Preservation Solutions; Phenotype; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Simvastatin

We evaluated whether IGL-1, a graft preservation solution containing polyethylene glycol, improves the outcome of small bowel grafts in comparison to the University of Wisconsin (UW) solution in a pig allotransplantation model.. Seventeen pigs were randomly allocated to group 1 (n = 10; intestinal allotransplantation with IGL-1) and group 2 (n = 7; allotransplantation with UW). Pigs received no immunosuppression and were sacrificed on postoperative d (POD) 8. Intestinal specimens were obtained from the animal immediately before cold flushing (T0), 2 h after graft reperfusion (T1), and at sacrifice (T2).. Survival rate to POD 8 was 50% in group 1 compared with 16% in group 2 (P < 0.05); 62% of pigs in group 1 did not present any acute cellular rejection (ACR) compared to 16% in group 2 (P < 0.05). Severe ACR rate was 25% in group 1 and 66% in group 2 (P < 0.05). iNOS activity and intestinal caspase 3 levels increased significantly between T0 and T1 in group 1 compared to group 2 (P < 0.05). Cell necrosis increased significantly between TO and T1 in group 2 compared with group 1 (P < 0.05) whereas cell apoptosis was significantly higher at T1 compared with T0 in group 1 in comparison to group 2.. Our results show that IGL-1 improves intestinal graft viability as compared to UW solution, possibly by reducing graft immunogenicity and by favoring intestinal epithelial repair.

Topics: Acute Disease; Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Female; Glutathione; Graft Rejection; Graft Survival; Immunosuppression Therapy; Insulin; Intestinal Mucosa; Intestine, Small; Organ Preservation Solutions; Polyethylene Glycols; Raffinose; Reperfusion Injury; Sus scrofa; Transplantation, Homologous

Matrix metalloproteinases and tissue inhibitor of metalloproteinases play an important role in the regulation of mesangial cell proliferation and may be involved in ischemia-reperfusion injuries. Preservation solutions are thought to diminish the ischemic injury and appropriate choice of the solution should guarantee a better graft function and good prognosis for graft survival. The aim of the study was to examine the effect of preservation solutions UW and EC on the expression of matrix metalloproteinase II and tissue inhibitor of metalloproteinase II genes in rat kidney. The study was carried out on Wistar rat kidneys divided into 3 groups: kidneys perfused with 0.9% NaCl (control group), with UW, and with EC preservation solution. The results show an enhancement of MMP-2 and TIMP-2 gene expression after 12 min of cold ischemia. This increase was more expressed in kidneys preserved with UW solution in comparison with kidneys perfused with EC solution and 0.9% NaCl. After 24 h of cold ischemia the expression of MMP-2 and TIMP-2 genes in kidney perfused with UW solution decreased, while in kidneys perfused with EC it was increased. After warm ischemia the MMP-2 and TIMP-2 gene expression increased, whereas it was significantly lower in kidneys perfused with EC solution.

Topics: Adenosine; Allopurinol; Animals; Gene Expression; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Male; Matrix Metalloproteinase 2; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Inhibitor of Metalloproteinase-2

Injury due to cold ischemia reperfusion (I/R) is a major cause of primary graft non-function following liver transplantation. We postulated that I/R-induced cellular damage during liver transplantation might affect the secretory pathway, particularly at the endoplasmic reticulum (ER). We examined the involvement of ER stress in organ preservation, and compared cold storage in University of Wisconsin (UW) solution and in Institute Georges Lopez-1 (IGL-1) solution. In one group of rats, livers were preserved in UW solution for 8 h at 4 °C, and then orthotopic liver transplantation was performed according to Kamada's cuff technique. In another group, livers were preserved in IGL-1 solution. The effect of each preservation solution on the induction of ER stress, hepatic injury, mitochondrial damage and cell death was evaluated. As expected, we found increased ER stress after liver transplantation. IGL-1 solution significantly attenuated ER damage by reducing the activation of three pathways of unfolded protein response and their effector molecules caspase-12, C/EBP homologous protein-10, X-box-binding protein 1, tumor necrosis factor-associated factor 2 and eukaryotic translation initiation factor 2. This attenuation of ER stress was associated with a reduction in hepatic injury and cell death. Our results show that IGL-1 solution may be a useful means to circumvent excessive ER stress reactions associated with liver transplantation, and may optimize graft quality.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 12; Cold Ischemia; Cold Temperature; DNA-Binding Proteins; Endoplasmic Reticulum Stress; Gene Expression; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Regulatory Factor X Transcription Factors; Reperfusion Injury; Signal Transduction; TNF Receptor-Associated Factor 2; Transcription Factors; Unfolded Protein Response

Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation and frequently influences both short- and long-term allograft survival rates. One of the major events in I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to display antioxidant properties and protective effects against organ dysfunction induced by various I/R injuries. We investigated whether hydrogen-rich University of Wisconsin (HRUW) solution attenuates renal cold I/R injury.. We prepared HRUW solution by a novel method involving immersion of centrifuge tubes containing UW solution into hydrogen-saturated water. Hydrogen readily permeates through the centrifuge tubes, and thus, the hydrogen concentration of the UW solution gradually increases in a time-dependent manner. Syngeneic rat kidney transplantation was performed, and the animals were divided into three groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution for 24 to 48 hr (UW group), and recipients with grafts preserved in HRUW solution for 24 to 48 hr (HRUW group).. In the early phases, HRUW solution decreased oxidative stress, tubular apoptosis, and interstitial macrophage infiltration in the kidney grafts. Consequently, HRUW solution improved renal function and prolonged recipient survival rate compared with simple cold storage using UW solution. Histopathologically, HRUW treatment alleviated tubular injury and suppressed development of interstitial fibrosis.. HRUW solution improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cold Temperature; Glutathione; Graft Survival; Hydrogen; Insulin; Kidney; Kidney Transplantation; Kidney Tubules; Macrophages; Male; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate

Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca²⁺-dependent and Ca²⁺-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation.. Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry.. Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal-regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action.. The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Biomarkers; Blotting, Western; Cell Proliferation; Cold Temperature; Creatinine; Cytokines; Cytoprotection; Delayed Graft Function; Glutathione; Immunohistochemistry; Inflammation Mediators; Insulin; Kidney; Kidney Transplantation; Male; Mycophenolic Acid; Organ Preservation; Organ Preservation Solutions; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Quinazolines; Raffinose; Rats; Rats, Inbred Lew; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Signal Transduction; Time Factors

The initial flush of an organ is important to remove any cellular components from the microcirculation before storage. The aim of this study was to assess graft function after an ex vivo warm flush with a novel non-phosphate buffered preservation solution AQIX RS-I (AQIX) compared with a traditional cold flush.. Porcine kidneys were either warm-flushed with AQIX RS-I at 30°C, or cold-flushed at 4°C with University of Wisconsin solution (UW) or hyperosmolar citrate (HOC) preservation solution at a pressure of 100 cmH2O (n = 6). Renal function was measured ex vivo by perfusing the organs with autologous blood at 37°C on an isolated organ perfusion system.. The AQIX group flushed significantly quicker than the cold stored groups (22 ± 1.8 versus UW 4.9 ± 1.6 versus HOC 10 ± 1.6 mL/min/100g; P = 0.001) and gained less weight than the UW group (19 ± 2.9 versus UW 30 ± 3.4 versus HOC 21% ± 7.7%; P = 0.025). The AQIX group also had superior acid-base homeostasis. Functional results, histologic analysis, and ADP: ATP levels were comparable between the groups.. Flushing kidneys with AQIX at 30°C cleared the renal microcirculation of blood more rapidly without any detrimental effects when compared to traditional cold flushing with UW or HOC at 4°C. Warm initial flushing has potential to be developed as part of normothermic renal preservation techniques.

Topics: Acid-Base Equilibrium; Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Allopurinol; Animals; Citrates; Glutathione; Hypothermia, Induced; Insulin; Kidney; Kidney Transplantation; Microcirculation; Organ Preservation Solutions; Phosphates; Raffinose; Reperfusion Injury; Spouses; Temperature

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H₂S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H₂S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4 °C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h-4 °C) in UW; and UWS: IPRL was performed in livers preserved (48 h-4 °C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H₂S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H₂S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H₂S donor showed an enhanced capacity for BSP uptake (k(A) CON = 0.29 min⁻¹; k(A) UW = 0.29 min⁻¹ ; k(A) UWS = 0.36 min ⁻¹). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H₂S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.

Topics: Adenosine; Allopurinol; Allyl Compounds; Animals; Bile; Cold Ischemia; Disulfides; Gases; Glutathione; Glycogen; Hydrogen Sulfide; Insulin; L-Lactate Dehydrogenase; Liver; Liver Circulation; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sulfobromophthalein; Time Factors; Vascular Resistance

The rapid uniform delivery of University of Wisconsin solution (UW) to the microcirculation may be compromised by its vasoactivity.. In 2 different rodent models, we tested whether UW-mediated vasoconstriction could be reversed with nicardipine.. In the perfused, splanchnic circulation, intravascular control solutions (lactated Ringers [LR], Hextend [HEX], histidine-tryptophan-ketoglutarate [HTK]) or UW (± nicardipine) evoked pressure changes in 3 protocols (series 1; n = 35). In the cremaster muscle, topical control solutions or UW (± nicardipine) evoked vascular responses measured by video microscopy in 4 protocols (series 2; n = 47). In series 1A, 37°C UW increased perfusion pressure, but there was no change caused by LR, HEX, or HTK. In series 1B, 4°C UW caused a similar, albeit transient, increase. In series 1C, nicardipine reversed 37°C UW-mediated vasoconstriction in a dose-related manner. In series 2A, UW caused a 30%-59% constriction that varied with arteriolar branching order. In series 2B, the recovery from UW-induced vasoconstriction varied with duration of exposure, but nicardipine fully reversed residual vasoconstriction. In series 2C, cold and warm UW were equipotent, near maximal, vasoconstrictors. In series 2D, UW potentiated no-reflow.. UW causes a potent temperature-independent vasoconstriction by a calcium-mediated mechanism and this effect can be mitigated with nicardipine.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Male; Microcirculation; Nicardipine; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Temperature; Vasodilator Agents

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

Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis.. Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW+SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor.. AST was significantly lower during cold storage with UW than with the older media (P=.001); ALT was lower in the FBP group (P=.023) and LDH was lower in the FBP and SNAC groups (P=.007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P=.008 and P=.006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P=.004). Renin levels were significantly lower in the FBP group (P=.022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor.. In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Topics: Acetylcysteine; Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Catalase; Disease Models, Animal; Fructosediphosphates; Glutathione; Insulin; L-Lactate Dehydrogenase; Liver; Liver Cirrhosis; Liver Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Renin; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Time Factors; Transforming Growth Factor beta1

There is increasing support for the use of hypothermic machine perfusion (HMP) in an attempt to reduce preservation injury. However, experimental evidence is needed to further examine the effects of HMP on renal ischemia reperfusion injury.. Porcine kidneys were subjected to 10 min of warm ischemia followed by 18 hr of static cold storage with hyperosomolar citrate (HOC), histidine-tryptophan-ketoglutarate (HTK), or University of Wisconsin (UW) solutions or 18 hr HMP with Kidney Perfusion Solution using the Lifeport perfusion system. Renal function, oxidative damage, and morphology were assessed during 3 hr of reperfusion with autologous blood using an isolated organ perfusion system.. During reperfusion, intrarenal resistance was significantly lower in the HMP group compared with HOC and UW (area under the curve; HMP 3.8+/-1.7, HOC 9.1+/-4.3, UW 7.7+/-2.2, HTK 5.6+/-1.9 mm Hg/min; P=0.006), and creatinine clearance was significantly higher compared with the UW group (area under the curve creatinine clearance; HMP 9.8+/-7.3, HOC 2.2+/-1.7, UW 1.8+/-1.0, HTK 2.1+/-1.8 mL/min/100 g; P=0.004). Tubular function was significantly improved in the HMP group (P<0.05); however, levels of lipid peroxidation were significantly higher (P=0.005).. HMP demonstrated a reduced level of preservation injury compared with the static techniques resulting in improved renal and tubular function and less tubular cell inflammation during reperfusion.

Topics: Acid-Base Equilibrium; Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Citrates; Cold Ischemia; Creatinine; Disease Models, Animal; Equipment Design; Glucose; Glutathione; Hypothermia, Induced; Insulin; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Lipid Peroxidation; Mannitol; Organ Preservation Solutions; Oxidative Stress; Perfusion; Peroxidase; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Swine; Time Factors

Here we examine the effect of adding carvedilol (CVD) to University of Wisconsin (UW) solution on the preservation of steatotic and nonsteatotic livers during cold ischemia and after normothermic reperfusion. We used an isolated perfused rat liver model. The following protocols were evaluated. Protocol 1 concerned the effect of CVD after cold ischemia. Steatotic and nonsteatotic livers were preserved for 24 hours in UW solution alone or with CVD. Livers without cold ischemia were used as controls. Transaminases were evaluated in the flushing effluent. Protocol 2 involved the effect of CVD after reperfusion. Both liver types were preserved for 24 hours in UW solution alone or with CVD and then perfused ex vivo for 2 hours at 37 degrees C. Livers flushed and perfused without ischemia were used as controls. Hepatic injury and functionality [transaminases, bile production, and hepatic clearance of sulfobromophthalein (BSP)] were evaluated after reperfusion. In addition, factors potentially involved in hepatic ischemia-reperfusion injury, including oxidative stress (malondialdehyde and superoxide anion levels), mitochondrial damage (glutamate dehydrogenase activity), microcirculatory disorders (flow rate and vascular resistance), and adenosine triphosphate (ATP) depletion, were evaluated after reperfusion. After cold ischemia, steatotic livers preserved in UW solution showed higher transaminase levels than nonsteatotic livers. After reperfusion, steatotic livers preserved in UW solution showed higher transaminase levels and lower bile production and BSP clearance than nonsteatotic livers. Alterations in the perfusion flow rate and vascular resistance, mitochondrial damage, and reduced ATP content were more evident in steatotic livers preserved in UW solution. The addition of CVD to UW solution reduced hepatic injury, obstructed its mechanisms, and improved hepatic functionality in both liver types. We conclude that CVD is a useful additive for UW solution that improves the preservation of steatotic and nonsteatotic livers subjected to prolonged cold ischemia.

Topics: Adenosine; Adenosine Triphosphate; Adenylate Kinase; Adrenergic beta-Antagonists; Allopurinol; Animals; Bile; Carbazoles; Carvedilol; Cold Temperature; Fatty Liver; Glutathione; Insulin; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Propanolamines; Raffinose; Rats; Rats, Zucker; Reperfusion Injury; Vascular Resistance

Fingolimod (FTY720) is a potent agonist of sphingosine 1 phosphate receptors and thereby interferes with lymphocyte trafficking. We previously showed that FTY720 protects from mild preservation reperfusion injury induced by 4 hr of cold ischemia. The purpose of this study was to explore the role of FTY720 in ischemic injury and regeneration using a clinically relevant rat renal transplant model with 24 hr of cold ischemia.. Donor kidneys were cold stored in the University of Wisconsin solution for 24 hr before transplantation into bilaterally nephrectomized syngeneic recipients (n=6 per group), which received 0.5 mg/kg/d FTY720 or vehicle through oral gavage. Grafts were harvested 2 or 7 days posttransplantation. Renal tissue was examined histologically, stained for apoptosis, proliferation, inflammatory cell infiltrates, and studied for transforming growth factor-beta, and tumor necrosis factor-alpha expression. Rat proximal tubular cells were incubated with 0.1 to 30 micromol/L of phosphorylated FTY720 to test for in vitro cytopathic effects.. FTY720 induced peripheral lymphopenia and significantly reduced intragraft CD3 and ED1 infiltrates. Acute tubular damage scores and graft function were not influenced by FTY720. Tubular apoptosis was significantly reduced, whereas the number of proliferating cell nuclear antigen-positive tubular cells were markedly increased. FTY720 attenuated renal tumor necrosis factor-alpha and transforming growth factor-beta expression. In vitro, pharmacologic concentrations up to 1 micromol/L of phosphorylated FTY720 did not affect tubular cell viability.. FTY720 confers tubular epithelial protection in the presence of severe preservation reperfusion injury. Beneficial effects may in part be due to reduction in cell-mediated immune mechanisms. Furthermore, FTY720 could be helpful in patients with delayed graft function.

Topics: Adenosine; Allopurinol; Animals; Cell Culture Techniques; Cell Division; Cell Survival; Fingolimod Hydrochloride; Flow Cytometry; Glutathione; Immunohistochemistry; Immunosuppressive Agents; Inflammation; Insulin; Kidney Transplantation; Male; Organ Preservation Solutions; Propylene Glycols; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Sphingosine

Orthotopic liver transplantation (OLT) is today the gold standard treatment of the end-stage liver disease. Different solutions are used for graft preservation. Our objective was to compare the results of cadaveric donor OLT, preserved with the University of Wisconsin (UW) or Celsior solutions in the portal vein and Euro-Collins in the aorta.. We evaluated retrospectively 72 OLT recipients, including 36 with UW solution (group UW) and 36 with Celsior (group CS). Donors were perfused in situ with 1000 mL UW or Celsior in the portal vein of and 3000 mL of Euro-Collins in the aortia and on the back table managed with 500 mL UW or Celsior in the portal vein, 250 mL in the hepatic artery, and 250 mL in the biliary duct. We evaluated the following variables: donor characteristics, recipient features, intraoperative details, reperfusion injury, and steatosis via a biopsy after reperfusion. We noted grafts with primary nonfunction (PNF), initial poor function (IPF), rejection episodes, biliary duct complications, hepatic artery complications, re-OLT, and recipient death in the first year after OLT.. The average age was 33.6 years in the UW group versus 41 years in the CS group (P = .048). There was a longer duration of surgery in the UW group (P = .001). The other recipient characteristics, ischemia-reperfusion injury, steatosis, PNF, IPF, rejection, re-OLT, and recipient survival were not different. Stenosis of the biliary duct occured in 3 (8.3%) cases in the UW group and 8 (22.2%) in the CS (P = .19) with hepatic artery thrombosis in 4 (11.1%) CS versus none in the UW group (P = .11).. Cadaveric donor OLT showed similar results with organs preserved with UW or Celsior in the portal vein and Euro-Collins in the aorta.

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Aorta, Abdominal; Cadaver; Child; Child, Preschool; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Histidine; Humans; Hypertonic Solutions; Immunosuppressive Agents; Insulin; Liver Failure; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Portal Vein; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion Injury; Retrospective Studies; Tissue Donors

The two-layer organ preservation method (TLM) based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution has been successfully used in clinical islet and experimental heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve kidney function in a model of syngeneic kidney transplantation with prolonged ischemia time.. Kidneys were stored for 24 h either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation.. One-month survival rates in the UW, TLM and control groups were 12.5, 62.5 and 100%, respectively (UW vs. TLM, p < 0.01). Median creatinine levels 24 h after transplantation were 381, 299 and 121 microM, respectively (UW vs. TLM, p < 0.02). Histological scoring showed more severe tissue damage in the UW group than in the TLM group (p < 0.05). Apoptosis was more frequent in the UW group than in the TLM group (p < 0.05).. We demonstrated for the first time that conservation with TLM significantly improves the outcome of kidney transplantation in a rat model and should therefore be further studied in larger animals.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Blood Substitutes; Cold Ischemia; Creatinine; Fluorocarbons; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred BN; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

This study examined the effects of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) supplementation to University of Wisconsin solution (UW) in steatotic and nonsteatotic livers during cold storage. Hepatic injury and function were evaluated in livers preserved for 24 hours at 4 degrees C in UW and in UW with EGF and IGF-I (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. AKT was inhibited pharmacologically. In addition, hepatic injury and survival were evaluated in recipients who underwent transplantation with steatotic and nonsteatotic livers preserved for 6 hours in UW and UW with EGF and IGF-I (separately or in combination). The results, based on isolated perfused liver, indicated that the addition of EGF and IGF-I (separately or in combination) to UW reduced hepatic injury and improved function in both liver types. A combination of EGF and IGF-I resulted in hepatic injury and function parameters in both liver types similar to those obtained by EGF and IGF-I separately. EGF increased IGF-I, and both additives up-regulated AKT in both liver types. This was associated with glycogen synthase kinase-3beta (GSK3(beta)) inhibition in nonsteatotic livers and PPAR gamma overexpression in steatotic livers. When AKT was inhibited, the effects of EGF and IGF-I on GSK3(beta), PPAR gamma, hepatic injury and function disappeared. The benefits of EGF and IGF-I as additives in UW solution were also clearly seen in the liver transplantation model, because the presence of EGF and IGF-I (separately or in combination) in UW solution reduced hepatic injury and improved survival in recipients who underwent transplantation with steatotic and nonsteatotic liver grafts. In conclusion, EGF and IGF-I may constitute new additives to UW solution in steatotic and nonsteatotic liver preservation, whereas a combination of both seems unnecessary.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Cold Ischemia; Disease Models, Animal; Epidermal Growth Factor; Fatty Liver; Glutathione; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Insulin; Insulin-Like Growth Factor I; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Perfusion; PPAR gamma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Raffinose; Rats; Rats, Zucker; Recombinant Proteins; Reperfusion Injury; Time Factors

Improved kidney preservation methods are needed to reduce ischemia-reperfusion (IR) injury in kidney allografts. Lifor is an artificial preservation solution comprised of nutrients, growth factors, and a non-protein oxygen and nutrient carrier. The current study compared the effectiveness of Lifor to University of Wisconsin solution (UW) in protecting rat kidneys from warm IR and cold storage injury.. In a warm IR model, rat kidneys were perfused in situ with either saline, UW, or Lifor for 45 min. Renal function and histology were assessed 24 h later. In a cold IR model, kidney slices were cold-stored in saline, UW, or Lifor at 4°C. Kidney injury was assessed by the release of lactate dehydrogenase (LDH) and immunoblot analysis for cleaved caspase-3.. Lifor perfusion significantly mitigated renal dysfunction and tubular injury at 24 h compared with saline or UW. Lifor and UW prevented LDH release in hypoxic kidney slices in vitro, however activation of caspase-3 following hypoxia-reoxygenation was attenuated only with Lifor. Cold storage with Lifor or UW significantly decreased LDH release from kidney slices or normal rat kidney cells in comparison to storage in saline or culture media. After 24 h of cold storage there was a significant decrease in cleaved caspase-3 in Lifor stored slices compared that seen following cold storage in saline or UW solution.. Lifor solution mitigates both warm and cold renal IR and appears to provide greater protection from apoptosis compared with UW solution.

Topics: Adenosine; Allopurinol; Animals; Caspase 3; Glutathione; In Situ Nick-End Labeling; Insulin; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Male; Nephrectomy; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Transplantation, Homologous

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

Topics: Adenosine; Alanine Transaminase; Allopurinol; Analysis of Variance; Animals; Aspartate Aminotransferases; Bile; Cold Ischemia; Glutathione; Glycogen; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Reticulin; Time Factors; Tissue Survival

To investigate the effects of dextrans of various molecular weights (Mw) during a 12 h cold storage time-course on energetics, histology and mucosal infiltration of fluorescein isothiocyanate (FITC)-dextran.. Rodent intestines were isolated and received a standard University of Wisconsin vascular flush followed by intraluminal administration of a nutrient-rich preservation solution containing dextrans of varying Mw: Group D1, 73 kdal; Group D2, 276 kdal; Group D3, 534 kdal; Group D4, 1185 kdal; Group D5, 2400 kdal.. Using FITC-labeled dextrans, fluorescent micrographs demonstrated varying degrees of mucosal infiltration; lower Mw (groups D1-D3: 73-534 kdal) dextrans penetrated the mucosa as early as 2 h, whereas the largest dextran (D5: 2400 kdal) remained captive within the lumen and exhibited no permeability even after 12 h. After 12 h, median injury grades ranged from 6.5 to 7.5 in groups D1-D4 (73-1185 kdal) representing injury of the regenerative cryptal regions and submucosa; this was in contrast to group D5 (2400 kdal) which exhibited villus denudation (with intact crypts) corresponding to a median injury grade of 4 (P < 0.05). Analysis of tissue energetics reflected a strong positive correlation between Mw and adenosine triphosphate (r(2) = 0.809), total adenylates (r(2) = 0.865) and energy charge (r(2) = 0.667).. Our data indicate that dextrans of Mw > 2400 kdal act as true impermeant agents during 12 h ischemic storage when incorporated into an intraluminal preservation solution.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Ischemia; Dextrans; Energy Metabolism; Fluorescein-5-isothiocyanate; Glutathione; Insulin; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Molecular Weight; Organ Preservation Solutions; Osmosis; Oxidative Stress; Permeability; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors

We compared the susceptibility of liver grafts from lean and obese Zucker rats to preservation injury, using two organ-preservation techniques: conventional static preservation (SP) and machine perfusion (MP) preservation. SP: livers preserved by UW solution at 4, 8 or 20 degrees C for 6-h. MP: livers perfused for 6-h with an improved oxygenated Krebs-Henseleit solution (KH) at 4, 8 or 20 degrees C. Reperfusion with KH (2-h) was performed either with the SP or MP preserved livers. Fatty livers tolerate SP poorly at 4, 8 and 20 degrees C as compared with MP at the same temperatures. SP induced a decrease in the ATP/ADP ratio both at 8 and 20 degrees C in obese rats while an increase in energy status was found with MP at 8 and 20 degrees C. Nitrate/nitrite (NOx) concentration was higher and bile flow lower in livers preserved with SP than MP. In lean rats, no differences were observed between MP and SP as regards enzyme release, bile production and NOx levels except for SP at 20 degrees C in which high enzyme release and low bile flow were observed. In lean rats ATP/ADP was higher and NOx was lower with MP at 20 degrees C than with SP at 20 degrees C. To optimize steatotic liver preservation SP should be avoided because it is particularly detrimental as compared with MP.

Topics: Adenosine; Allopurinol; Animals; Cryopreservation; Fatty Liver; Glucose; Glutathione; Insulin; Liver Transplantation; Male; Obesity; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Zucker; Reperfusion Injury; Tromethamine

Hepatic ischemia/reperfusion (I/R) injury significantly influences short-term and long-term outcomes after liver transplantation (LTx). The critical step initiating the injury is known to include sinusoidal endothelial cell (SEC) alteration during the cold preservation period. As carbon monoxide (CO) has potent cytoprotective functions on vascular endothelial cells, this study examined if CO treatment of excised liver grafts during cold storage could protect SECs and ameliorate hepatic I/R injury. Rat liver grafts were preserved in University of Wisconsin (UW) solution containing 5% CO (CO-UW solution) for 18 to 24 hours and were transplanted into syngeneic Lewis rats. After 18 hours of cold preservation, SEC damage was evident with propidium iodide (PI) nuclear staining on SECs, and the frequency of PI(+) SECs was significantly lower in grafts stored in CO-UW solution versus those stored in control UW solution. SEC protection with CO was associated with decreased intercellular cell adhesion molecule translocation and less matrix metalloproteinase release during cold preservation. After LTx with 18 hours of cold preservation, serum alanine aminotransferase levels and hepatic necrosis were significantly less in the CO-UW group than in the control UW group. With 24 hours of cold storage, 35% (7/20) survived with control UW solution, whereas the survival with CO-UW solution improved to 80% (8/10). These beneficial effects of CO-UW solution were associated with a significant reduction of neutrophil extravasation, down-regulation of hepatic messenger RNA for tumor necrosis factor alpha and intercellular cell adhesion molecule 1, and less hepatic extracellular signal-regulated kinase activation. Liver grafts from Kupffer cell-depleted donors or pseudogerm-free donors showed less SEC death during cold preservation, and CO-UW solution further reduced SEC death. In conclusion, CO delivery to excised liver grafts during cold preservation efficiently ameliorates SEC damage and hepatic I/R injury.

Topics: Adenosine; Allopurinol; Animals; Carbon Monoxide; Cryopreservation; Cryoprotective Agents; Endothelial Cells; Glutathione; Graft Survival; Hepatitis; Insulin; Kupffer Cells; Liver Transplantation; Male; MAP Kinase Signaling System; Matrix Metalloproteinases; Neutrophils; Organ Preservation; Organ Preservation Solutions; Primary Graft Dysfunction; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Several studies have been carried out investigating different preservation methods and preservation solutions for the pancreata of various species. Attention has to be drawn to the extreme vulnerability of porcine pancreata (PP) to oxidative stress due to the lack of endogenous antioxidants. This study sought to evaluate the influence of cannulation and infusion of different volumes of University of Wisconsin (UW) solution immediately after organ retrieval on PP organ quality.. PP from 24 slaughterhouse pigs were harvested with immediate cannulation of the pancreatic duct for infusion of 10 mL, 20 mL, 50 mL, or 100 mL UW solution. The organs were stored in cold UW solution. Control organs were only stored in UW. After 6 hours of cold ischemia, tissue and supernate samples were analyzed for markers of oxidative cell damage, adenosine triphosphate (ATP) levels, and occurrence of apoptosis.. The fewest apoptotic cells were detected in the PP infused with 50 mL UW via the pancreatic duct (PP 50) as compared with all other groups. Oxidative cell damage was lowest and ATP levels were highest in the PP 50 group.. Because PP 50 showed significantly better results when compared with all other groups, we suggest that infusion of 50 mL UW via the pancreatic duct immediately after organ retrieval may be useful to minimize oxidative cell damage and cell death in PP.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Lipase; Models, Animal; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury; Swine; 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

The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation.. Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism.. p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134+/-297 vs. 1477+/-145 IEQ/g or 65,012+/-9385 vs. 45,700+/-5103 IEQ/pancreas; P<0.05). Apoptotic beta-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%+/-9.4% vs. 61.6%+/-4.8%, P<0.05). Tumor necrosis factor-alpha expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40+/-0.78 vs. 0.21+/-0.05 ng/mL, P<0.05).. Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents beta-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cytokines; Dogs; Enzyme Activation; Enzyme Inhibitors; Glutathione; Insulin; Insulin-Secreting Cells; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Organ Preservation; Organ Preservation Solutions; p38 Mitogen-Activated Protein Kinases; Raffinose; Reperfusion Injury

To determine if blockade of P-selectin in the isolated blood-perfused cold ex vivo rat liver model protects the liver from ischemia-reperfusion injury.. The effect of P-selectin blockade was assessed by employing an isolated blood-perfused cold ex vivo rat liver with or without P-selectin antibody treatment before and after 6 h of cold storage in University of Wisconsin solution.. In our isolated blood-perfused rat liver model, pre-treatment with P-selectin antibody failed to protect the liver from ischemia-reperfusion injury, as judged by the elevated aspartate aminotransferase activity. In addition, P-selectin antibody treatment did not significantly reduced hepatic polymorphonuclear leukocyte accumulation after 120 min of perfusion. Histological evaluation of liver sections obtained at 120 min of perfusion showed significant oncotic necrosis in liver sections of both ischemic control and P-selectin antibody-treated groups. However, total bile production after 120 min of perfusion was significantly greater in P-selectin antibody-treated livers, compared to control livers. No significant difference in P-selectin and ICAM-1 mRNAs and proteins, GSH, GSSG, and nuclear NF-kappaB was found between control and P-selectin antibody-treated livers.. In conclusion, we have shown that blockade of P-selectin alone failed to reduced polymorphonuclear leukocyte accumulation in the liver and protect hepatocytes from ischemia-reperfusion injury in the isolated blood-perfused cold-ex vivo rat liver model.

Topics: Adenosine; Allopurinol; Animals; Antibodies; Aspartate Aminotransferases; Bile; Cold Ischemia; Glutathione; Glutathione Disulfide; In Vitro Techniques; Insulin; Intercellular Adhesion Molecule-1; Liver; Male; Necrosis; Neutrophils; NF-kappa B; Organ Preservation Solutions; P-Selectin; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Time Factors

University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions are the 2 most commonly used liver preservation solutions. The aim of this study was to compare cardiovascular stability, acid-base status, and potassium concentrations between patients who received grafts preserved in either UW or HTK solution in orthotopic liver transplantation (OLT).. In this retrospective study, 87 patients who underwent living donor OLT were divided into 2 groups: UW (n = 28) and HTK (n = 59). Group HTK was subdivided into group NF-HTK (n = 31; nonflushed before reperfusion) and group F-HTK (n = 28; flushed before reperfusion). We determined mean arterial pressure (MAP) and heart rate every minute for 5 minutes after reperfusion and the maximum change in these values and incidence of postreperfusion syndrome (PRS). Body temperature, cardiovascular and acid-base parameters, as well as potassium concentrations were compared at 5 minutes before and 5 and 30 minutes after reperfusion.. The maximum decreases in MAP within 5 minutes after reperfusion were significantly greater in both the NF-HTK and the F-HTK groups. The rate of PRS was significantly greater in the NF-HTK compared with the UW group. Flushing with HTK solution decreased the rate of PRS; there was no significant difference between the F-HTK and UW groups. All serial changes in body temperature, cardiovascular and acid-base parameters, as well as potassium concentrations were similar among the 3 groups.. The incidence of PRS was greater using HTK compared with UW solution during the reperfusion period. Therefore, careful hemodynamic management is advised when using HTK solution.

Topics: Acid-Base Equilibrium; Adenosine; Adult; Allopurinol; Blood Pressure; Carcinoma, Hepatocellular; Female; Glucose; Glutathione; Hemodynamics; Humans; Insulin; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Living Donors; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Portal Vein; Potassium; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Retrospective Studies

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

This study investigated the role of a novel nutrient-rich preservation solution in alleviating intestinal ischemia-reperfusion (IR) injury in a large animal model.. Porcine intestines were treated in vivo with the following intraluminal flush solutions: group 1, none; group 2, University of Wisconsin solution; group 3, an amino acid-based solution, previously shown to be effective in reducing IR injury in rodent models. Intestinal ischemia was induced in vivo for 60 min, followed by 180 min reperfusion. Key metabolic aspects were assessed in relation to two fundamental kinase mechanisms that govern cell fate, AMP kinase, and Jun kinase.. After 180 min reperfusion, groups 1 and 2 exhibited clefting, denudation, and mucosal hemorrhage, whereas injury was markedly reduced in group 3 (median grades 4.5 and 5 vs. 0; P<0.05). In contrast to groups 1 and 2, group 3 tissues exhibited a full recovery of adenylates (ATP, total adenylates) and an effective control of oxidative stress throughout reperfusion. Neutrophil-mediated inflammation was abrogated in group 3. An up-regulation of two key enzymes (glutaminase and alanine aminotransferase) provided a mechanism for the superior recovery of energetics and the preservation of mucosal integrity in group 3. A strong activation of AMP-activated protein kinase resulting in the up-regulation of a primary proapoptotic kinase mechanism, Jun kinase, was evident in groups 1 and 2.. A strategy of intraluminal administration of a nutrient-rich solution represents a potential therapy for alleviating intestinal IR injury; these findings suggest a more effective method for the ischemic storage of intestine.

Topics: Adenosine; Adenylate Kinase; Alanine Transaminase; Allopurinol; Animals; Glutaminase; Glutathione; Hemorrhage; Insulin; Intestinal Mucosa; Intestine, Small; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine

ET-Kyoto solution (ET-K) is an extracellular-type organ preservation solution containing the cytoprotective disaccharide, trehalose. A previous study reported the supplement of dibutyryl cyclic adenosine monophosphate (db-cAMP) in conventional ET-K to attenuate lung ischemia-reperfusion injury. In this study, the efficacy of this modified ET-K for liver preservation was investigated by comparison with University of Wisconsin solution (UW). ET-K was supplemented with db-cAMP (2 mmol/L). Lewis rats were randomly assigned to two groups, and liver grafts were flushed and stored at 40C for 24 h with ET-K or UW before syngeneic liver transplantation. The graft function and histological changes at 4 h posttransplant as well as 7-day survival were evaluated. Recipient rat survival rate was significantly higher in the ET-K group than in the UW group. Preservation in ET-K resulted in a significant reduction in serum parenchymal transaminase level and promotion of bile production in comparison with UW. The serum hyaluronic acid level, an indicator of sinusoidal endothelial cell injury, was significantly lower after ET-K preservation than that in UW. Histologically, at 4 h after transplantation, the liver grafts preserved in UW solution demonstrated a greater degree of injury than those in ET-K, which appeared to be apoptosis, rather than necrosis. The continuity of the sinusoidal lining was better preserved in ET-K than in UW. In conclusion, ET-K supplemented with db-cAMP is superior to UW in rat liver preservation. This modified ET-K might therefore be a novel candidate for the procurement and preservation of multiple organs.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Aspartate Aminotransferases; Bucladesine; Gluconates; Glutathione; Graft Survival; Hydroxyethyl Starch Derivatives; Insulin; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Phosphates; Raffinose; Rats; Reperfusion Injury; Trehalose

This study investigates how the addition of trimetazidine (TMZ) and aminoimidazole-4-carboxamide ribonucleoside (AICAR) to University of Wisconsin (UW) solution protects steatotic livers. Steatotic and nonsteatotic livers were preserved for 24 hours at 4 degrees C in UW and UW with TMZ and AICAR (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. Adenosine monophosphate-activated protein kinase (AMPK) or nitric oxide (NO) synthesis inhibition in livers preserved in UW with TMZ was also investigated. Hepatic injury and function (transaminases, bile production, and sulfobromophthalein clearance) and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion (I/R), including vascular resistance, mitochondrial damage, adenosine triphosphate depletion, and oxidative stress were evaluated. AMPK, NO synthase (NOS), nitrate, and nitrite levels were also determined. The addition of TMZ and AICAR (separately or in combination) to UW reduced hepatic injury, improved functionality, and protected against the mechanisms responsible for the vulnerability of steatotic livers to I/R. Like AICAR, TMZ increased AMPK, constitutive NOS, and nitrates and nitrites, and conversely, AMPK or NO synthesis inhibition abolished the benefits of TMZ. In conclusion, TMZ, by means of AMPK, increased NO, thus protecting steatotic livers against their vulnerability to I/R injury. TMZ and AICAR may constitute new additives to UW solution in steatotic liver preservation, whereas a combination of both seems unnecessary.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Enzyme Activators; Fatty Liver; Glutathione; Insulin; Liver; Mitochondria, Liver; Multienzyme Complexes; Nitric Oxide; Nitric Oxide Synthase; Organ Preservation Solutions; Oxidative Stress; Protein Serine-Threonine Kinases; Raffinose; Rats; Rats, Zucker; Reperfusion Injury; Ribonucleotides; Trimetazidine; Vascular Resistance

Topics: Adenosine; Allopurinol; Animals; Cold Ischemia; Glutathione; Graft Survival; Humans; Insulin; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Polyethylene Glycols; Raffinose; Rats; Rats, Sprague-Dawley; 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

Cold ischemia and reperfusion during renal transplantation result in release of reactive oxygen species. The aim of this study is to examine whether cold storage induced cell injury can be ameliorated by adding flavonoids directly to preservation solutions.. Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin (UW) or Euro-Collins (EC) solution at 4 degrees C for 20 hours. Preservation solutions were supplemented with various flavonoids. After rewarming, structural and metabolic cell integrity was measured by lactate dehydrogenase (LDH) release and MTT-test, and lipid peroxidation was assessed from generation of thiobarbituric acid-reactive substances (TBARS).. Twenty hours of cold storage resulted in a substantial loss of cell viability in both preservation solutions (in EC: LDH release 92.4+/-2.7%; MTT-test 0.5+/-0.7%). Addition of luteolin, quercetin, kempferol, fisetin, myricetin, morin, catechin, and silibinin significantly reduced cell injury (for luteolin in EC: LDH release 2.4+/-1.6%; MTT-test 110.3+/-10.4%, P<0.01; TBARS-production (related to cold stored control cells) 8.9+/-2.6%). No cytoprotection was found for apigenin, naringenin, and rutin. Protective potency of flavonoids depends on number of hydroxyl-substituents and lipophilicity of the diphenylpyran compounds.. Cold storage induced injury of renal tubular cells was substantially ameliorated by adding selected flavonoids directly to preservation solutions.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Cold Temperature; Cryoprotective Agents; Flavonoids; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Kidney Transplantation; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Lipid Peroxidation; LLC-PK1 Cells; Luteolin; Molecular Structure; Organ Preservation; Organ Preservation Solutions; Quercetin; Raffinose; Reperfusion Injury; Swine

Preconditioning treatments hold significant potential for improving outcomes in solid organ transplantation. Protective phenotypes can be induced using certain drugs. Curcumin is a biologically active component of turmeric and has been reported to induce stress proteins in certain cell lines, leading to cell protection. This study investigates in detail the effect of curcumin on the stress-response in human hepatocytes, in particular its effect on heme oxygenase 1 (HO-1) and its cytoprotective effect. Pretreatment with curcumin protected hepatocytes in a model of oxidative injury and this protection was mediated through HO-1. In a model of cold preservation injury, curcumin pretreatment resulted in elevation of HO-1 throughout the cold storage and rewarming period, and was cytoprotective against oxidative injury. This is the first study to demonstrate that curcumin induces HO-1 in human hepatocytes, and that the protective effects of curcumin pretreatment may have clinical potential in hepatic transplantation.

Topics: Adenosine; Allopurinol; Curcumin; Gene Expression Regulation, Enzymologic; Glutathione; Heme Oxygenase-1; Hepatocytes; HSP70 Heat-Shock Proteins; Humans; Insulin; Liver Circulation; Liver Transplantation; Organ Preservation Solutions; Polymerase Chain Reaction; Raffinose; Reperfusion Injury; Tissue Preservation; Transplantation Conditioning

During ischemia-reperfusion, free oxygen radicals which directly affect renal cells may cause delayed graft function. We investigated whether there was a difference regarding antioxidant enzyme content between use of Ringer's lactate (RL) versus University of Wisconsin (UW) perfusion solutions in kidney transplantation. Ischemia was achieved by clamping the renal pedicle for 20 minutes followed by perfusion with either solution for 20 minutes and reperfusion for another 20 minutes. A parenchymal biopsy was taken before and after the ischemia, perfusion, and reperfusion (IPR) process. The levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) were investigated in the biopsy specimens. We used paired t tests within groups and t tests for comparisons between groups. The results were expressed as mean values +/- SEM with P < .05 accepted as statistically significant. After IPR, SOD, GPx, and MDA were decreased in all groups: only GPx (P = .001) and MDA (P = .04) for the RL group and SOD (P = .001) and MDA (P = .05) for the UW group were statistically significant. In the control group, we did not observe any difference (P > .05). Comparisons between groups did not reveal differences (P > .05). In our study, no difference was observed between RL and UW regarding their effects on antioxidant enzymes following renal I/R injury in pigs. More investigations are needed to evaluate graft function in this setting.

Topics: Adenosine; Allopurinol; Animals; Disease Models, Animal; Glutathione; Glutathione Peroxidase; Insulin; Isotonic Solutions; Kidney Transplantation; Male; Malondialdehyde; Organ Preservation Solutions; Perfusion; Raffinose; Renal Circulation; Reperfusion Injury; Ringer's Lactate; Solutions; Swine; Thiobarbituric Acid Reactive Substances

In pancreas transplantation (PTx), ischemia/reperfusion-induced deterioration of graft-microcirculation is accompanied by alterations of intermittent capillary perfusion (IP; alternating cessation and resumption of capillary blood flow) is known to counteract malperfusion. Incidence and effectiveness of IP following short- versus long-term preservation of pancreas grafts with University of Wisconsin (UW) solution has not been examined so far. PTx was performed in Lewis rats following 2-h or 18-h preservation in UW solution. Using intravital fluorescence microscopy, functional capillary density (FCD), red blood cell (RBC) velocity, IP-incidence and -frequency were analyzed. Laser Doppler flowmetry allowed for the determination of erythrocyte flux and velocity. Measurements were performed at 30, 60 and 120 min after reperfusion. Nontransplanted animals served as controls. FCD, RBC-velocity and -flux remained unchanged in the 2-h group. IP was encountered in 87% of all observation areas at 120 min. After 18-h ischemia, FCD was significantly reduced, which was paralleled by a 50% incidence of IP at 120 min. Tissue edema and leukocyte infiltration in pancreas grafts following 18-h preservation were significantly enhanced. Therefore, IP is an important mechanism aimed at improving microcirculation and UW solution is suitable to preserve vasomotion-activities enabling long-term preservation in a pancreas graft.

Topics: Adenosine; Allopurinol; Animals; Blood Flow Velocity; Capillaries; Glutathione; In Vitro Techniques; Insulin; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.

Topics: Adenosine; Allopurinol; Animals; Antimetabolites; Carbon Monoxide; Disease Models, Animal; Glutathione; Graft Survival; Insulin; Intestinal Mucosa; Intestine, Small; Male; Microscopy, Electron, Transmission; Organ Preservation; Organ Preservation Solutions; Organ Transplantation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Treatment Outcome

To investigate whether tanshinone IIA could improve the effect of UW solution for skeletal muscle preservation and to determine the dose range of tanshinone IIA providing optimal protection during ischemia and reperfusion.. Ischemic rat limbs were perfused with UW solution or UW plus tanshinone IIA (UW+T, 0.05, 0.1, or 0.2 mg/mL) for 0.5 h before reperfusion; controls (I/R) received no perfusion. Serum creatine phosphokinase (CPK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured pre-ischemia and after reperfusion (2-h, 4-h, and 6-h). Muscle water content, superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphatase (ATPase) were assessed pre-reperfusion and after 6-h reperfusion. Intercellular adhesion molecule-1 (ICAM-1) and apoptosis were detected after 6-h reperfusion. Reperfusion blood flow was monitored during reperfusion period.. UW and UW+T prevented luxury perfusion during reperfusion and inhibited ICAM-1 expression and apoptosis after 6-h reperfusion. Serum CPK, AST, and LDH levels in UW rats were significantly less than those in controls after 2-h reperfusion (no difference, 4-h or 6-h reperfusion). After 4-h ischemia, there were significant differences in water content, MDA, SOD, and ATPase between UW and controls, but no difference after 6-h reperfusion. All tests with UW+T rats were significantly different from control results at corresponding durations. Higher tanshinone doses improved results.. UW plus tanshinone IIA increased protection against I/R injury, suggesting that tanshinone IIA has clinical value.

Topics: Abietanes; Adenosine; Adenosine Triphosphatases; Allopurinol; Animals; Apoptosis; Aspartate Aminotransferases; Creatine Kinase; Dose-Response Relationship, Drug; Glutathione; Hindlimb; Insulin; Intercellular Adhesion Molecule-1; L-Lactate Dehydrogenase; Male; Malondialdehyde; Muscle, Skeletal; Organ Preservation Solutions; Phenanthrenes; Raffinose; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

Cold ischemia--warm reperfusion (CI/WR) injury of liver transplantation involves hepatocyte cell death, the nature and underlying mechanisms of which remain unclear. Isolated hepatocytes and isolated perfused livers were used to determine the prevalence of necrosis and apoptosis as well as mitochondrial dysfunction. In isolated cells, propidium iodide and Hoechst 33342 staining showed a cold-storage, time-dependent increase in necrosis, whereas apoptosis was minimal even after 48 h of hypothermia. Nonetheless, a progressive loss of mitochondrial membrane potential was observed. Translocation of mitochondrial cytochrome c toward microsomes occurred within 24 h of CI/WR, with cytochrome c reaching the cytosol later. Mitochondria isolated from whole livers subjected to CI/WR also display reduced metabolic parameters and increased susceptibility to swelling. These events are associated with increased activity of major initiator (caspase 9) and effector (caspase 3) caspases. The results demonstrate that CI/WR induces mitochondrial dysfunction in isolated cells and in the whole organ; only in the latter is that sufficient to trigger the classical mitochondrial pathway of apoptosis. Our study also provides evidence for the involvement of endoplasmic reticulum stress in CI/WR hepatocyte injury. Combined protection of mitochondria and endoplasmic reticulum may thus represent an innovative therapeutic avenue to enhance liver graft viability and functional integrity.

Topics: Adenosine; Allopurinol; Animals; Caspase 3; Caspase 9; Caspases; Cell Death; Cold Temperature; Cytochromes c; Glutathione; Hepatocytes; In Vitro Techniques; Insulin; Liver; Liver Transplantation; Male; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

The transplanted lung suffers ischemia-reperfusion injury and many efforts have been made to improve preservation. The aim of this study was to compare the effectiveness of intracellular high-potassium Belzer solution versus extracellular Celsior in 24-hour lung preservation.. Sixteen pigs weighing 25.1-28.1 kg underwent single, left-lung allotransplantation. Sixteen other pigs were donors of the left lung. Heart and lungs were retrieved from the donors using single-flush perfusion by a randomly selected solution that was also used for cold storage (4 degrees C) for 24 hrs. Biopsies were taken from the right lung before storage, after the preservation, and finally three hours following transplantation. The observation period lasted three hours following transplantation.. Pulmonary artery pressure and pulmonary vascular resistance were significantly higher in the Belzer group (p<0.05), while the pO2/FiO2 ratio was much higher in the Celsior group (p<0.05). Compliance was reduced approximately equally in both groups. Histology showed less edema, atelectasis, and hemorrhagic infiltration in the Celsior group than in the Belzer group, which developed more interstitial thickening and presented more leukocyte infiltration and desquamation of alveoli cells.. According to hemodynamic, respiratory, and histological data, the extra-cellular solution Celsior offers improved prolonged preservation compared with Belzer.

Topics: Adenosine; Allopurinol; Anesthesia; Animals; Blood Pressure; Disaccharides; Electrolytes; Glutamates; Glutathione; Graft Survival; Histidine; Insulin; Lung; Lung Transplantation; Mannitol; Organ Preservation Solutions; Potassium; Pulmonary Artery; Raffinose; Reperfusion Injury; Swine; Temperature; Time Factors

Graft pancreatitis (GP) is one the main technical problems associated with pancreas transplant (PT). It occurs in 20% of patients representing a risk factor for thrombosis and cause of graft loss. GP is related to oxidative effects from oxygen-derived free radicals (OFR) in ischemia-reperfusion injury. We evaluated lipid peroxidation by the OFR in the PT of pig organs preserved with either Celsior or Wisconsin solutions.. In Landrace pigs we performed 24 pancreas allografts, which were preserved 18 or 24 hours: 12 with Celsior solution (CS) and 12 with Wisconsin solution (UW). No immunosuppression was administered. The oxidative effects were determined by quantification of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) and of the carbonyl groups of proteins in our pancreatic tissue samples and measured at different times: (A) baseline in the donor, (B) after perfusion of the graft, (C) after the ischemia period, and (D) 30 minutes after ischemia-reperfusion of the graft.. The MDA and 4-HDA values were similar in conditions A, B, and C, but showed an extraordinary increase after ischemia-reperfusion in D, among both the 18- or 24-hour preserved grafts and in the same proportion with CS and UW. The carbonyl groups of the proteins rose in conditions B and C (cold ischemia), but less so in state D (reperfusion).. The oxidative injury of a pancreatic graft preserved for 18 or 24 hours occurs during reperfusion, with an extraordinary intensity, but similarly with CS and UW, an observation that may help to explain graft pancreatitis.

Topics: Adenosine; Allopurinol; Animals; Disaccharides; Electrolytes; Glutamates; Glutathione; Histidine; Insulin; Mannitol; Models, Animal; Organ Preservation Solutions; Oxidative Stress; Pancreas; Pancreas Transplantation; Postoperative Complications; Raffinose; Reperfusion Injury; Swine

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

In organ transplantation, ischemia-reperfusion injury (IRI) has been implicated in delayed graft function (DGF) as well as in short- and long-term complications. Using an autotransplant pig kidney model, changes in renal function and morphology were determined after different periods of cold ischemia in kidneys preserved in the University of Wisconsin solution (UW), high-Na(+) version of UW (HEH) or Celsior (CEL) a newly developed high-Na(+) solution, with or without trimetazidine (TMZ). Kidney function was better preserved in CEL, UW and particularly HEH in combination with TMZ, particularly after 48 and 72 h. Mitochondria integrity was improved in TMZ-preserved groups. This study indicates that TMZ is efficiently protective against IRI even after prolonged preservation and in different preservation solutions.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Cryopreservation; Disaccharides; Disease Models, Animal; Drug Evaluation, Preclinical; Electrolytes; Glutamates; Glutathione; Histidine; Insulin; Kidney Function Tests; Kidney Transplantation; Mannitol; Mitochondria; Organ Preservation; Organ Preservation Solutions; Raffinose; Receptors, GABA-A; Reperfusion Injury; Swine; Time Factors; Transplantation, Autologous; Trimetazidine

We assessed the effect of adding exogenous fructose-1,6-biphosphate (F16BP) to the preservation solution (University of Wisconsin storage solution) used during an experimental procedure of small bowel transplantation in rats.. We studied levels of the nucleotides hypoxanthine/xanthine and adenosine in tissue after cold ischemia, as well as histologic changes and associated deleterious processes such as bacterial translocation produced by the reperfusion associated with the transplantation.. The groups of rats treated with F16BP showed the lowest levels of hypoxanthine/xanthine and uric acid, the highest levels of adenosine, and the lowest levels of histologic damage and lactate dehydrogenase release to the bloodstream. Consumption of intestinal hypoxanthine during reperfusion was lowest in the groups treated with F16BP, as was the incidence of bacterial translocation.. This study shows a protective effect of exogenous F16BP added to University of Wisconsin solution during experimental intestinal transplantation in rats. This protective effect, reflected by decreased intestinal damage and bacterial translocation, was related to a decrease in adenosine triphosphate depletion during cold ischemia before intestinal transplantation, and to the reduced availability of xanthine oxidase substrates for free radical generation during reperfusion.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Bacterial Translocation; Cryopreservation; Cytoprotection; Fructosediphosphates; Glutathione; Hypoxanthine; Insulin; Intestine, Small; Intestines; L-Lactate Dehydrogenase; Male; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Uric Acid; Xanthine

The mechanisms underlying liver graft dysfunction are not completely defined, although much of the injury derives from oxidative stress in organ reperfusion. The antioxidant glutathione in its reduced form (GSH) is an important agent to detoxify oxygen species after reperfusion. However, this effect might be limited by low concentrations at the end of cold storage. The objective of this study was to evaluate GSH and glutathione oxidized (GSSG) hepatic levels pre- and postreperfusion and correlate with hepatocellular injury and liver function in the 5 subsequent days after transplantation.. Liver biopsies were taken immediately before implant and 2 hours after venous reperfusion in 34 grafts, determining GSH, GSSG levels, and GSSG/GSH ratio. Aminotransferases (ALT, AST) and PT were measured for 5 days.. There was a strong decrease in GSH concentration (P <.0001), increase of GSSG levels (P <.01), and increase of the GSSG/GSH ratio (P <.0001). No correlations were found between GSH, GSSG, or GSH/GSSH levels and AST, ALT, and PT.. Glutathione levels showed significant changes after 2 hours of reperfusion, due to intense oxidative stress. Therapies to replenish GSH should be considered as a protective measure to avoid liver graft dysfunction after transplantation.

Topics: Adenosine; Adult; Allopurinol; Cause of Death; Female; Glutathione; Glutathione Disulfide; Hepatocytes; Humans; Insulin; Liver; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Raffinose; Reperfusion Injury; Retrospective Studies

This study investigated the effects of pyrrolidine dithiocarbamate (PDTC), a novel NF-kappaB inhibitor, on the expression of multiple inflammatory mediators and on neutrophilic inflammation of the graft in rats following liver transplantation. Orthotopic liver transplantation (OLT) was performed after 24 hr of cold storage using University of Wisconsin (UW) solution that contained various concentrations of PDTC. We determined the time course of NF-kappaB activation and of the expression of multiple inflammatory signals: tumor necrosis factor-alpha (TNF-alpha), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM)-1. Serum alanine aminotransferase (ALT), intrahepatic myeloperoxidase (MPO/WBC ratio, a measure of neutrophil accumulation), and Mac-1 expression (CD11b/CD18, a measure of circulating neutrophil activity) were also evaluated. The results showed that PDTC decreased OLT-induced NF-kappaB activation in a dose-dependent manner (from 20 mmol/L to 60 mmol/L), diminished TNF-alpha, CINC, and ICAM-1 protein levels in the graft, and reduced the OLT-induced increase of serum TNF-alpha level. Pretreatment with PDTC significantly suppressed OLT-induced neutrophilic inflammation of the graft. The PDTC-exposed livers (PDTC, 40 mmol/L), in comparison with the control livers, had a significant reduction of MPO/WBC ratio (7.04+/-0.97 vs 14.07+/-1.31) and Mac-1 expression (181+/-11.3% vs 281+/-13.2%) at 6 hr after reperfusion. Furthermore, PDTC inhibited the increase of serum ALT levels after liver transplantation. In conclusion, PDTC inhibited NF-kappaB activation and the expression of the inflammatory mediators. These effects were associated with improved graft viability through inhibited intrahepatic neutrophilic inflammation. A therapeutic strategy directed at inhibition of NF-kappaB activation within the transplanted liver might be effective in reducing intrahepatic neutrophilic inflammation, and be beneficial to prolonged graft storage.

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Chemokines, CXC; Glutathione; Insulin; Intercellular Signaling Peptides and Proteins; Liver Transplantation; Neutrophils; NF-kappa B; Organ Preservation Solutions; Pyrrolidines; Raffinose; Rats; Reperfusion Injury; Thiocarbamates; Time Factors; Tumor Necrosis Factor-alpha

Ischemia / reperfusion (I / R) injury is related to tissue graft energy status. Insulin, which is currently used in the University of Wisconsin (UW) preservation solution with insulin (UWI), is an anabolic hormone and was shown to exacerbate the hepatic I / R injury in our previous study. In this study, the energy status and regulation of metabolism genes by insulin were investigated in liver grafts preserved by UW solution. Insulin could significantly decrease adenosine triphosphate (ATP) level after 3 hours of preservation, as well as total adenine nucleotides (TANs) and energy charge (EC) levels. Energy regeneration deteriorated in the grafts preserved by insulin in terms of ATP and EC levels at 24 hours after transplantation. The insulin signal was transduced through the insulin receptor substrate-2 (IRS-2) pathway and the activity of IRS-2 was decreased gradually at the messenger ribonucleic acid (mRNA) level during cold preservation. Downstream targeting genes such as sterol regulatory element-binding protein-1c (SREBP-1c), glucokinase (GKC), and fatty acid synthase (FAS) genes, as well as phospho-glycogen synthase kinase-3beta (GSK-3beta) were activated and they showed the similar expression profiles during cold preservation. Lipoprotein metabolism was accelerated by insulin through upregulation of the activity of apolipoprotein C-III (Apo C-III) during cold preservation. The insulin-like growth factor-binding protein-1 pathway was inhibited during cold preservation. In conclusion, insulin in UW solution exacerbates hepatic I / R injury by energy depletion as the graft maintains its anabolic activity. The key enzyme activities of the energy-consuming process of glycogen and fatty acid synthesis as well as lipoprotein metabolism were accelerated by insulin through the IRS-2 / SREBP-1c pathway.

Topics: Adenosine; Allopurinol; Animals; Apolipoprotein C-III; Apolipoproteins C; Blotting, Western; CCAAT-Enhancer-Binding Proteins; DNA-Binding Proteins; Energy Metabolism; Glutathione; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hypoglycemic Agents; Insulin; Insulin Receptor Substrate Proteins; Intracellular Signaling Peptides and Proteins; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Phosphoproteins; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Transcription Factors; Up-Regulation

This study examined the potential therapeutic effects of a combination therapy consisting of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and N-acetyl cysteine (NAC) to attenuate ischemia-reperfusion (I/R) injury in a canine model of autologous renal transplantation.. Male mongrel dogs (15-20 kg) underwent left nephrectomy followed by flushing and static preservation of the kidney in University of Wisconsin (UW) solution for 48 hr. The treatment group received AICAR (50 mg/kg) plus NAC (100 mg/kg) intravenously before the left nephrectomy. The compounds were added to the UW solution as well. All dogs underwent right nephrectomy 48 hr later followed by autotransplantation of the preserved left kidney. Treated dogs received a second dose of AICAR and NAC before implantation of the renal autograft.. The treated dogs had excellent urine output posttransplant, with peak serum creatinine of 7.26 mg/dL on postoperative day (POD) 3 that normalized after 14 days. The control group were anuric and developed clinical symptoms of uremia on POD 1. Morphologic evaluation supported the protective effects of combination therapy. Immunohistochemical analysis revealed decrease of tumor necrosis factor-alpha, interferon-gamma, and inducible nitric oxide synthase; and TUNEL assay showed decreased apoptosis in the treated group.. Combination therapy with AICAR and NAC attenuates renal I/R injury and improves the outcome of the transplanted kidney after prolonged cold preservation.

Topics: Acetylcysteine; Adenosine; Allopurinol; Aminoimidazole Carboxamide; Animals; Apoptosis; Creatinine; Disease Models, Animal; Dogs; Glutathione; Graft Survival; Immunohistochemistry; Insulin; Kidney; Kidney Transplantation; Male; Nephrectomy; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Ribonucleotides; Transplantation, Autologous; Tumor Necrosis Factor-alpha

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

Increases in intracellular calcium ion (Ca(2+)) levels of sinusoidal endothelial cell (SEC) may have a crucial role in mediating the expression of adhesion molecules and thus contribute to the microcirculatory disturbances observed in primary graft dysfunction. The effect of changes in the composition and/or temperature of the reperfusion solution on cytosolic Ca(2+) was studied in isolated rat SECs. Cells were preserved in cold University of Wisconsin (UW) solution for 0, 12, or 24 hours and loaded with Fura-2AM dye (Cedarlane, Eugene, OR) at 20 degrees C in N-2-hydroxyethylpiperazine-propanesulfonic acid (HEPES)-buffered physiological solution (HEPES 20 degrees C) or UW solution (UW 20 degrees C). SEC Ca(2+) levels were measured by cytofluorimetry. Basal steady-state Ca(2+) levels were much lower when SECs were loaded in UW 20 degrees C (37 +/- 2 nmol/L) than in HEPES 20 degrees C (114 +/- 32 nmol/L). In unstored controls (0 hour), going from UW 20 degrees C to HEPES 37 degrees C induced a large transient increase (185 +/- 31 nmol/L) in SEC Ca(2+) levels, which was greatly inhibited (43 +/- 13 nmol/L) in Ca(2+)-free HEPES 37 degrees C. A similar large transient increase was observed going from UW 20 degrees C to HEPES 20 degrees C (163 +/- 22 nmol/L). Changing temperature only (20 degrees C to 37 degrees C) in UW or HEPES solution had a much smaller effect on SEC Ca(2+) levels (14 +/- 2 and 60 +/- 18 nmol/L, respectively). These changes were similar in cold-preserved cells. In unstored controls, solution changes greatly attenuated the intensity of subsequent Ca(2+) responses to the purinergic agonist adenosine triphosphate (ATP). Cold preservation (CP) greatly attenuated both the frequency of appearance and intensity of ATP-induced Ca(2+) responses. Hence, changing reperfusion solution composition has a greater impact on SEC steady-state Ca(2+) levels than changing temperature. Cold preservation does not significantly affect changes in SEC steady-state Ca(2+) levels, but greatly impairs the capacity of SECs to subsequently respond to Ca(2+)-mobilizing agonists.

Topics: Adenosine; Allopurinol; Animals; Calcium; Cells, Cultured; Cryopreservation; Cytosol; Endothelium, Vascular; Glutathione; HEPES; Homeostasis; Hot Temperature; Insulin; Liver Circulation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Temperature

Using in situ fluorescence microscopy with Sprague Dawley rats, we studied the hypothesis of compromised microvascular kidney perfusion on organ harvest in non-heart-beating donors (NHBDs), and we evaluated the potential benefit of an additional preflush with saline solution containing streptokinase. Aortal flush of NHBD kidneys solely with University of Wisconsin solution resulted in a significantly (P <0.05) reduced functional capillary density (FCD) with increased perfusion heterogeneity compared with kidneys of heart-beating controls. This was associated with an increased lactate dehydrogenase (LDH) release on 24 hr postpreservation rinse of the grafts (76.7+/-18.9 U/L). Warm preflush with low-viscosity Ringer's lactate (RL) solution alone did not influence the decreased renal FCD and the postpreservation LDH release (76.2+/-29.1 U/L). In contrast, the addition of streptokinase to the RL preflush solution resulted in a significant (P <0.05) improvement of FCD with values not statistically different from those of heart-beating controls. This was associated with an attenuation of perfusion heterogeneity and a significantly lowered postpreservation LDH release (17.0+/-2.5 U/L). Furthermore, in transplanted and reperfused NHBD kidney grafts, the use of streptokinase-supplemented RL for preflush during organ harvest significantly (P <0.05) reduced early manifestation of tubular necrosis (29%+/-8%) when compared with kidneys preflushed exclusively with University of Wisconsin solution (56%+/-4%). Thus, we conclude that kidney harvest from NHBDs is prone to severe microvascular perfusion deficits, which are likely to preclude successful preservation of organ integrity during cold storage. Temporary fibrinolytic preflush with streptokinase may represent a feasible tool to improve microvascular graft equilibration, which effectively protects the renal integrity during both cold storage and posttransplant reperfusion.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Fibrinolytic Agents; Glutathione; Graft Survival; Heart Arrest; Insulin; Kidney; Kidney Transplantation; Male; Microcirculation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Streptokinase

Cold ischemia (CI)-warm reperfusion (WR) liver injury remains a problem in liver transplantation. CI-WR initially causes sinusoidal endothelial cell (SEC) apoptosis through a caspase-dependent mechanism. We previously showed that the caspase inhibitor IDN-1965 prevents CI-WR-induced SEC apoptosis. However, this agent required to be administered to the donor, preservation solution, and recipient for efficacy. Here, we show that a second-generation caspase inhibitor, IDN-6556, effectively prevents CI-WR-induced SEC injury when added only to University of Wisconsin (UW) cold storage media. Rat livers were stored in UW solution for 24 hours at 4 degrees C and reperfused for 1 hour at 37 degrees C. Apoptosis was quantitated using terminal deoxynucleotide transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and caspase 3 activation determined by biochemical measurement and immunohistochemical analysis. Pan-caspase inhibitors (IDN-8066, IDN-7503, IDN-7436, IDN-1965, and IDN-6556) were applied at preischemic, cold preservation, or reperfusion periods. TUNEL-positive SEC and caspase 3-like activity in the liver was increased by CI-WR. Three caspase inhibitors (IDN-8066, IDN-1965, and IDN-6556) effectively attenuated SEC apoptosis and caspase 3 activation. The most potent inhibitor, IDN-6556, reduced SEC apoptosis and caspase 3 activity by 55% and 94%, respectively. Prevention of SEC apoptosis by IDN-6556 was not reduced when this agent was administered only during the cold preservation period. When added to the preservation solution, the caspase inhibitor IDN-6556 appears to be a feasible therapeutic agent against ischemia-reperfusion injury in liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Endothelium; Enzyme Inhibitors; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Ischemic-type biliary lesions (ITBLs) lead to considerable morbidity after orthotopic liver transplantation (OLT). The exact pathogenesis is unknown. We tested the hypothesis that insufficient perfusion of biliary arterial vessels might be responsible for ITBLs. This could be prevented by improved perfusion techniques. Since February 2000, we performed a controlled study using arterial back-table pressure perfusion (AP) to achieve reliable perfusion of the biliary-tract capillary system, which may be impaired by the high viscosity of University of Wisconsin solution. We retrospectively analyzed 190 OLTs performed between September 1997 and July 2002 with regard to ITBLs. One hundred thirty-one grafts were preserved by in situ standard perfusion (SP), including portal perfusion, whereas in 59 cases, additional AP was performed. Donor-related factors, recipient age, indication for OLT, OLT technique, immunosuppression, and ischemia time were similar in both groups. In the SP group, 21 of 131 patients (16%) developed ITBLs. Only 1 of 59 patients with grafts receiving AP developed ITBLs. This difference was highly significant (P =.004). Peak aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels within the first 3 days were significantly lower in the AP group (AST, P =.016; ALT, P =.007). Multivariate analysis showed a significant influence of AP (P =.010) and donor age (P =.003) on the development of ITBLs. AP is an easy and reliable method to prevent ITBLs in OLT. It therefore should be used as the standard technique in liver procurement.

Topics: Adenosine; Adult; Aged; Allopurinol; Bile Ducts; Glutathione; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Insulin; Liver Diseases; Liver Transplantation; Middle Aged; Organ Preservation Solutions; Perfusion; Postoperative Complications; Pressure; Raffinose; Reperfusion Injury; Retrospective Studies; Survival Rate; Viscosity

The effect of University of Wisconsin (UW) solution perfusion for extremity preservation is still unknown although it is widely used. The purpose of this study is to examine the effect of UW solution perfusion on skeletal muscle preservation in a rat model.. Rat hindlimbs were amputated and either preserved with UW solution perfusion (UW perfusion group) or given no perfusion (no-perfusion group) for 5 h at 25 degrees C. They were then transplanted to other isogeneic rats. ATP in the muscle and serum creatine phosphokinase were measured after 24 h of reperfusion. The vascular endothelial function of the femoral artery rings was measured before and after 24 h of reperfusion in the presence or absence of indomethacin (cyclooxygenase inhibitor) and L-NMMA (nitric oxide synthase inhibitor). TEA (calcium-activated potassium channel inhibitor) was also used to verify the vasodilator function. Reperfusion blood flow was monitored during the first 2 h of reperfusion.. ATP in the UW perfusion group was significantly decreased after 24 h of reperfusion, while that in the no-perfusion group recovered. Reperfusion blood flow in the UW solution perfusion group was significantly lower than that in the no-perfusion group. Acetylcholine-induced relaxation in the UW perfusion group was significantly reduced before and after 24 h of reperfusion compared to that in the no-perfusion group and was mostly diminished by indomethacin and L-NMMA administration.. Skeletal muscle injury is augmented by UW solution perfusion, probably due to deterioration of the vascular endothelial function resulting in blood supply diminution.

Topics: Adenosine; Allopurinol; Animals; Endothelium, Vascular; Exocrine Glands; Glutathione; Hindlimb; In Vitro Techniques; Insulin; Male; Muscle, Skeletal; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Cells, Cultured; Cryopreservation; Disaccharides; Electrolytes; Glucose; Glutamates; Glutathione; Hepatocytes; Histidine; Humans; Insulin; L-Lactate Dehydrogenase; Mannitol; Organ Preservation Solutions; Oxidation-Reduction; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Tetrazolium Salts; Thiazoles

University of Wisconsin (UW) solution (Viaspan) is currently used to preserve organs from nonheartbeating donors. Histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is of proven efficacy in experimental pancreas preservation, but its efficacy in combined warm ischemia (WI) and cold ischemia (CI) is unknown. The viability of HTK-preserved porcine pancreatic grafts was assessed after various periods of WI and compared with grafts flushed and preserved with UW solution.. A total of 14 pigs were used: G1 (n=4, UW) and G2 (n=4, HTK) with 15-min WI and 16-hr cold storage; G3 (n=3, UW) and G4 (n=3, HTK) with 30-min WI and 16-hr cold storage.. All animals in G1 and G2 were normoglycemic, whereas only 66% of pancreases were functioning in G3 and G4. HTK perfusion was associated with increased wet weight. Transient hyperinsulinemia was noted in all the groups on postoperative day 1 (mean range: 8.9-12.4 microU/L). Postoperative serum amylase and lipase were more pronounced in G3 and G4. However, HTK-stored grafts exhibited less evidence of biochemical pancreatitis as compared with UW-stored grafts on the first postoperative day in the group with 15-min WI. Mean K values of intravenous glucose tolerance tests on postoperative day 14 were similar in both groups. Vascular congestion was uniformly observed and was considered a typical feature of WI.. Porcine pancreatic grafts are viable after 16-hr CI following 15-min WI in this experimental nonheartbeating donor model. HTK solution seems to provide reliable graft function in this setting and to be equivalent to UW.

Topics: Adenosine; Allopurinol; Amylases; Animals; Cold Temperature; Edema; Glutathione; Graft Survival; Hot Temperature; Hyperinsulinism; Hypoglycemia; Insulin; Lipase; Models, Animal; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury; Swine

Ischemia-reperfusion injury has been associated with both early and late effects on allografts in the form of delayed graft function and decreased graft survival. Recent studies demonstrated that functional parameters were influenced by cold storage conditions and particularly the ratio of Na+:K+ of the preservation solution.. We have extended this study to examine whether the high-Na+ low-K+ formulation of Belzer's solution (HEH) was efficient in an autotransplanted pig kidney model when compared with the classical low-Na+ high-K+ University of Wisconsin solution and the new high-Na+ low-K+ Celsior solution. Kidneys were harvested, cold flushed, and preserved for 24, 48, or 72 hr with HEH, Celsior solution, or University of Wisconsin solution and autotransplanted. Renal function was determined on days 1, 3, 7, and 14, and at 4 to 16 weeks after autotransplantation. Histologic changes and cell infiltration were assessed on kidney biopsy specimens taken after reperfusion (30-40 min), at days 5 and 14, and at 4 to 5 and 10 to 12 weeks after surgery. Peripheral benzodiazepine receptor (PBR), a structural mitochondrial protein, was also studied.. Cold storage in HEH resulted in reduction of delayed graft function and renal damage, with a decrease in interstitial inflammation. HEH reduced interstitial fibrosis, tubular atrophy, and improved PBR expression.. This study suggests that cold preservation in HEH has a beneficial action in in vivo renal preservation and reduces tubular necrosis, interstitial inflammation, and fibrosis in these groups. In addition, PBR detection was correlated to the level of preservation integrity.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Male; Models, Animal; Organ Preservation; Organ Preservation Solutions; Potassium; Raffinose; Reperfusion Injury; Sodium; Swine; Time Factors; Transplantation, Autologous

Insulin keeps the liver in a metabolically vigorous state. However, organ preservation aims to decrease the metabolic rate. The objective of this study was to clarify the effect of insulin used in University of Wisconsin (UW) preservation solution on the liver graft.. The liver grafts were preserved by UW solution with or without insulin for 7, 9, and 24 hr, respectively. The influence of insulin was studied by 7-day survival rate, liver function, morphology, and intragraft gene expression 24 hr after transplantation. Morphology was studied on the preserved grafts.. The morphology of the graft in the insulin group showed more severe ischemia-reperfusion injury. The 7-day graft survival rates of the 7-hr subgroups with and without insulin were 55% and 93%, respectively (P=0.02). In the 9-hr subgroups, the survival rates were 0% and 78%, respectively (P=0.002). The serum levels of aspartate aminotransferase (AST) (P=0.008) and alanine aminotransferase (ALT) (P=0.032) were higher in the 7-hr subgroup with insulin. The same trend was found in the 9-hr subgroups (AST, P=0.016; ALT, P=0.016). The expression level of 215 genes were much lower at 24 hr after transplantation in the grafts preserved with insulin than in those preserved without insulin, and most of the genes were related to metabolic activities.. Insulin in UW solution may exacerbate graft ischemic injury and decrease the graft survival rate in rat liver transplantation. Insulin, in the absence of glucose in UW solution, may exhaust the metabolic activity of the liver graft. It is harmful rather than helpful for isolated rat liver grafts preserved in UW solution.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Gene Expression Regulation; Glutathione; Graft Survival; Insulin; Liver; Liver Circulation; Liver Function Tests; Liver Transplantation; Oligonucleotide Array Sequence Analysis; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

Although classic ischemia-reperfusion injury is mediated by reactive oxygen intermediaries, increasing evidence implicates a role for immune-mediated apoptosis during ischemic injury in transplantation. Herein, we report the effects of polyclonal rabbit anti-thymocyte globulin (rATG) on mediators of hepatic apoptosis during cold storage.. Three-month-old male Lewis rats were placed under halothane anesthesia and the portal vein cannulated. University of Wisconsin (UW) solution (35 ml) with (n = 5) and without (n = 5) 20 mg/kg anti-rat rATG was infused before hepatectomy. The liver was stored in UW solution +/- rATG (143 ng/ml) at 4 degrees C for various times up to 24 h. Specimens were terminal deoxyuridine nick end labeling-stained for apoptosis. Tissue lysates were analyzed by Western blotting and densitometry.. Compared to UW alone, significantly fewer apoptotic cells were present in UW + rATG perfused and stored livers. There were early and sustained significant increases in Bcl-X(L) and decreases in Bcl-X(S) with rATG. There was an initial, but not sustained, significant decrease in Bax with rATG. Moreover, there was a significant one-third decrease in caspase-9 production with rATG at 0, 6, 12, and 18 h.. Decreased proapoptotic Bcl-X(S) and increased antiapoptotic Bcl-X(L), as well as decreased downstream proapoptotic caspase-9 expression, during liver ischemia after treatment with rATG, all favor cell survival. Because apoptotic ischemic injury results in allograft dysfunction, preservation strategies that ameliorate such immunological effects may improve organ function.

Topics: Adenosine; Adjuvants, Immunologic; Allopurinol; Animals; Apoptosis; bcl-X Protein; Caspase 9; Caspases; Cold Temperature; Glutathione; Immunoglobulins; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Proto-Oncogene Proteins c-bcl-2; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Heme oxygenase (HO)-1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO-1 could be substituted by a simple treatment with heme-degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO-1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4 degrees C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO-1-preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin-IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short-term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO-1 preconditioning. The protective effects of HO-1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO-1-mediated preconditioning.

Topics: Adenosine; Allopurinol; Animals; Bile; Bilirubin; Carbon Monoxide; Cholestasis, Intrahepatic; Glutathione; Graft Survival; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemin; Insulin; Ischemic Preconditioning; Liver Transplantation; Male; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

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

Sirolimus (SRL) seems to impair renal recovery from ischemic injury in animal models and delayed graft function after clinical renal transplantation. This study determined the impact of SRL on renal recovery after ischemia-reperfusion injury in a rat kidney transplant model.. Syngeneic kidneys were preserved in University of Wisconsin solution before transplantation into bilaterally nephrectomized rats. Recipients received vehicle or SRL targeting for whole-blood trough levels of 10 to 20 ng/mL as measured by high-performance liquid chromatography. Renal function was assessed by animal survival or daily serum creatinine. Tissue samples were collected for histologic examination.. Median SRL whole-blood concentrations were 16.6 +/- 1.6 ng/mL on postoperative day (POD) 1 and 12.0 +/- 0.9 ng/mL on POD 3. Transplantation of kidneys after 39 hr of cold storage resulted in 30% survival in the SRL-treated group compared with 100% survival in the control group (P=0.002). Transplantation of kidneys after 24 hr of cold storage resulted in no survival differences, but there were significant differences in renal function. Daily serum creatinine (PODs 1-4) was higher in the SRL-treated group compared with the control group (P<0.05 at all time points). Grafts from SRL-treated animals showed more severe tubular necrosis compared with control animals.. When given at therapeutic immunosuppressive doses, SRL compromises renal function after ischemia-reperfusion injury in a rat kidney transplant model. The antiproliferative effect of SRL may translate into impairment of tubular repair and regeneration necessary for recovery after such injury.

Topics: Adenosine; Allopurinol; Animals; Creatinine; Glutathione; Immunosuppressive Agents; Insulin; Kidney; Kidney Transplantation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Sirolimus; Time Factors; Transplantation, Isogeneic; Treatment Outcome

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Hepatocytes; In Vitro Techniques; Insulin; Ischemic Preconditioning; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Protein Biosynthesis; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Temperature

Topics: Adenosine; Adult; Alanine Transaminase; Allopurinol; Aspartate Aminotransferases; Brain Death; Glutathione; Glycine; Hepatectomy; Humans; Infusions, Intravenous; Insulin; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue and Organ Harvesting; Tissue Donors

Topics: Adenosine; Allopurinol; Animals; Endoplasmic Reticulum; Glutathione; Insulin; Liver; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Polyethylene Glycols; Raffinose; Rats; Reperfusion Injury

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

University of Wisconsin (UW) solution has been proven able to prevent liver injury during cold ischemia. During rewarming ischemia, however, the efficacy of this solution in preserving hepatocyte function is unclear. The aim of the present study was to investigate to what extent UW solution protects rat liver during rewarming ischemia.. Livers were washed out with cool physiologic saline or with UW solution and subjected to rewarming ischemia for periods of 20 min or 45 min followed by reperfusion using a blood-free perfusion model.. In comparison with controls, ischemia for 20 min in saline-treated livers led to mild depression of hepatocyte function, while UW solution afforded complete protection of the liver. In UW-treated livers, compared with saline-treated livers exposed to ischemia for 45 min, portal flow was slightly but significantly higher, bile production was increased by 62%, and lactate dehydrogenase leakage into the perfusate was reduced by 61%. In an attempt to explain mechanisms of liver protection by UW solution, we found that UW solution inhibited conversion of hypoxanthine into uric acid, but this effect was not associated with decreased degradation of adenine nucleotides in the liver during ischemia. Following 30 min reperfusion, UW solution increased tissue levels of adenosine triphosphate (not significantly) and adenosine diphosphate (significantly). Further, UW solution markedly reduced tumor necrosis factor-alpha release by the liver both after ischemia and after reperfusion.. These results create the hypothesis that UW solution may protect liver tissue during ischemia in liver surgery as well as during the implantation stage of liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Hot Temperature; Insulin; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Our objective was to evaluate graft equilibration with high viscosity (University of Wisconsin solution [UW]) or low viscosity (Bretschneider's histidine-tryptophan-ketoglutarate [HTK]) during liver procurement from non-heart beating donors (NHBD) and the potential impact of a preceding fibrinolysis with streptokinase on postpreservation viability.. After 60 min of cardiac arrest, rat livers were perfused by gravity (60 cm H2O) via the portal vein with either 60 ml of HTK, 20 ml of UW, or 20 ml of Ringer's solution (22 degrees C including 7500U of streptokinase) and, subsequently, 20 ml of UW. After 24 h of storage at 4 degrees C, viability of the livers was assessed upon isolated reperfusion in vitro.. Magnetic resonance imaging revealed severe perfusion deficits, which were mildly attenuated with HTK, upon flush-out with UW. After preflush with streptokinase, a mostly homogenous distribution of the preservation solution was observed throughout the liver tissue. The choice of the flush-out solution (UW or HTK) had no influence on parenchymal enzyme leakage, hepatic bile production, or tissue levels of ATP after reperfusion of the livers. Fibrinolytic preflush, however, resulted in a relevant and significant improvement of structural integrity as well as functional and metabolic recovery.. Compromised vascular tissue perfusion upon organ harvest in NHBD triggers graft dysfunction after cold storage and can easily be circumvented by temporary fibrinolysis before graft retrieval.

Topics: Adenosine; Allopurinol; Animals; Energy Metabolism; Fibrinolytic Agents; Glucose; Glutathione; Heart Arrest; Insulin; Liver; Magnetic Resonance Imaging; Male; Mannitol; Organ Preservation Solutions; Potassium Chloride; Preoperative Care; Procaine; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Streptokinase; Tissue and Organ Harvesting; Tissue Donors; Tissue Survival

Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the development of ischemia/reperfusion injury in nonhepatic organs, such as the heart. However, the role of p38 MAPK activation in the liver is unclear. We examined the effects of FR167653, a novel p38 MAPK inhibitor, as an additive to University of Wisconsin (UW) solution in rat liver transplantation.. Rat orthotopic liver transplantation was performed after 30 hr of cold storage using UW solution with or without FR167653. Ten-day survival rates, serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels, liver tissue blood flow, histological findings, and activities of p38 MAPK and p46/p54 c-Jun N-terminal kinase (JNK) in liver grafts were evaluated.. The addition of FR167653 significantly increased animal survival rates. FR167653 significantly suppressed serum ALT and LDH levels and improved liver tissue blood flow after transplantation. FR167653 also ameliorated histological damage to the liver graft. Neither p38 MAPK nor p46/p54 JNKs was activated during cold storage, whereas both were markedly activated within 30 min of reperfusion and remained activated until 60 min after reperfusion. FR167653 inhibited the activation of p38 MAPK both 30 and 60 min after reperfusion, but it did not affect the activation of p46/p54 JNKs.. The addition of FR167653 to UW solution improved liver graft viability and animal survival rates associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate ischemia/reperfusion injury in liver transplantation.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Cryopreservation; Enzyme Inhibitors; Glutathione; Insulin; JNK Mitogen-Activated Protein Kinases; L-Lactate Dehydrogenase; Liver; Liver Circulation; Liver Transplantation; Male; Mitogen-Activated Protein Kinases; Organ Preservation Solutions; p38 Mitogen-Activated Protein Kinases; Protein Isoforms; Pyrazoles; Pyridines; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Analysis

Ischaemia/reperfusion (I/R) lung injury using University of Wisconsin solution (UW) as perfusate has not been well studied. Isolated rat lungs were challenged with various periods of ischaemia and/or reperfusion. Haemodynamics, lung weight gain (LWG), capillary filtration coefficient (K(fc)), tissue pathology, the concentrations of cytokines in the perfusate, and mRNAs for the various cytokines in the lung tissues were measured. I/R induced a permeability type of pulmonary oedema, as reflected by increases in LWG and K(fc). LWG and K(fc) in the I(45)R(60)(UW) group (45 min of ischaemia followed by 60 min of reperfusion with UW) were only 2% and 5% respectively of those in the I(45)R(60)(NS) group (where NS is normal saline). LWG and K(fc) in the UW group had both increased by 180 min, to values similar to those in the I(45)R(60)(NS) group. However, these findings show that UW was remarkably effective at preventing LWG after 60 min of reperfusion, and was more than 3-fold more effective than NS in delaying LWG. For longer ischaemic times only, or the same period of ischaemia followed by longer reperfusion periods, greater lung injury occurred. I/R lung injury also induced increased concentrations of tumour necrosis factor-alpha (TNF-alpha), interleukin 1 and interleukin 6 in the perfusate, and increased the mRNAs for these cytokines in lung tissue. A significant correlation was obtained between TNF-alpha concentration and LWG. TNF-alpha production in the I(45)R(60)(UW) group was only 7% of that in the I(45)R(60)(NS) group. However, TNF-alpha mRNA expression in the I(45)R(60)(UW) group was 80% of that in the I(45)R(60)(NS) group. This indicates that transcription/translation do not correlate well with cytokine production, and also suggests that one reason for the effectiveness of UW in delaying LWG may be because it delays TNF-alpha production. In summary, ischaemia or I/R caused a permeability-type pulmonary oedema that was associated with leucocyte infiltration and the up-regulation of various cytokines, regardless of the perfusion fluid. Except for pulmonary hypertension, less severe I/R lung injury and delayed cytokine production in lungs perfused with UW, the pattern of injury associated with I/R challenge was similar to that in lungs perfused with NS. We propose that more or long-acting protective agents are required as additives in order to modify UW to produce an optimal preservation solution.

Topics: Adenosine; Allopurinol; Animals; Cytokines; Glutathione; Hemodynamics; Insulin; Lung; Male; Organ Preservation Solutions; Organ Size; Polymerase Chain Reaction; Pulmonary Edema; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Up-Regulation

A recent clinical study demonstrated that in renal allografts preserved in the cold apoptosis occurred soon after reperfusion. The mode of cell death during cold storage is generally considered necrotic. Whether apoptosis occurs as a part of cold storage is uncertain. The objective was to determine in human renal tubular cells whether apoptosis is specific for rewarming or it also occurs during cold storage and whether it could be modified.. Cold storage (4 degrees C) of primary human renal proximal tubular epithelial (RPTE) in University of Wisconsin (UW) solution up to 48 hr caused a time-dependent increase in cell death measured by lactic dehydrogenase (LDH) release and vital dye exclusion methods. Transmission electron microscopy (TEM) demonstrated that cell death in the cold was necrotic, involving considerable mitochondrial disruption, and was not apoptotic. The TUNEL assay that provides a specific, quantitative measure for apoptosis showed no increase in TUNEL-positivity during flow cytometry of cells stored in cold: 37 degrees C, 0.23+/-0.14%; 24 hr cold, 0.23+/-0.1%; 48 hr cold, 1.79+/-0.58%. Annexin-V staining, a sensitive method for detecting early apoptosis, similarly showed no increase in positively stained cells during cold storage. Addition of antioxidants 2-methyl aminochroman and deferoxamine to UW solution inhibited necrotic cell death and preserved mitochondrial structure. In contrast to cold storage alone, rewarming (37 degrees C for 24 hr) of cold stored cells, however, resulted in significant apoptosis (TUNEL positive: 48 hr cold: 2+/-0.6%, 48 hr cold and 24 hr rewarming: 54+/-17%), which was confirmed by the TEM based on typical apoptotic features. Addition of 2-MAC and DFO significantly inhibited rewarming-induced apoptotic cell death (plus 2-MAC: 3+/-1%, plus DFO: 3+/-2%).. Our study in human tubular cells provides evidence that cold storage per se does not result in apoptosis, but is primarily necrotic. However, rewarming is associated with significant apoptosis in the presence of ongoing necrosis, speculatively due to the activation of the apoptotic enzymic process of sublethally injured cells. Inclusion of antioxidants in the storage solution confers protection against both cold storage and rewarming-induced necrosis and apoptosis.

Topics: Adenosine; Allopurinol; Annexin A5; Apoptosis; Cells, Cultured; Cold Temperature; Epithelial Cells; Glutathione; Hot Temperature; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Insulin; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Microscopy, Electron; Necrosis; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Preservation; Trypan Blue

The effects of three hemoglobin solutions were compared with those of iso-oncotic human serum albumin in rats with ischemic renal failure and sham-operated controls. Unmodified and alpha-alpha cross-linked hemoglobins both increase mean arterial pressure and systemic vascular resistance and reduce cardiac output substantially and to a comparable extent. In contrast, omicron-raffinose cross-linked hemoglobin has no deleterious effect on any of these parameters. In sham-operated rats unmodified hemoglobin reduces the glomerular filtration rate (GFR) by approximately 30%, whereas neither of the two cross-linked hemoglobins has any adverse effect on GFR in this group. None of the three hemoglobin solutions exacerbated the degree to which GFR was reduced by ischemia-reperfusion injury. Also, the degree of tubular necrosis induced by ischemia-reperfusion injury was also comparable in all groups. We conclude the following: (1) omicron-raffinose cross-linking, but not alpha-alpha cross-linking, ameliorates the effects of unmodified hemoglobin on vascular resistance and cardiac output; (2) both forms of cross-linking reduce the nephrotoxicity exhibited by unmodified hemoglobin in sham-operated rats; and (3) none of the hemoglobin solutions exacerbate renal injury induced by ischemia-reperfusion.

Topics: Acute Kidney Injury; Animals; Aspirin; Blood Pressure; Glomerular Filtration Rate; Heart Rate; Hemoglobins; Humans; Kidney Tubules; Male; Necrosis; Oxygen; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Serum Albumin; Sodium; Vasoconstriction

In clinical transplantation, exposure of donors to gut-derived endotoxin occurs frequently and may adversely affect liver transplantation therapy. The aim of this study was to investigate: 1) whether brief exposure of rats to endotoxin before liver procurement aggravates the early phase of reperfusion injury of hepatic explants; and if so 2) whether Kupffer cell activation is a contributing factor to liver injury; and 3) whether heparin and pentoxifylline could minimize this effect.. Male Wistar rats were injected with 0.2-4.0 mg/kg of Escherichia coli lipopolysaccharide 2 h prior to liver harvest. After preservation in University of Wisconsin cold-storage solution, the livers were reperfused using a blood-free perfusion model. To inactivate Kupffer cells, some rats were pretreated with gadolinium chloride or liposome-encapsulated dichloromethylene-diphosphonate before lipopolysaccharide administration. The other rats received lipopolysaccharide with heparin or pentoxifylline.. In a dose-independent fashion, lipopolysaccharide impaired portal flow during graft reperfusion. In a dose-dependent way, lipopolysaccharide increased lactate dehydrogenase release into the perfusate and decreased bile flow and bromosulfophthalein excretion. Gadolinium chloride, liposomal dichloromethylene-diphosphonate, heparin, and pentoxifylline reduced lactate dehydrogenase release by 34%, 43%, 59%, and 64%, respectively, and improved functional parameters of the liver. A 52-fold increased neutrophil infiltration in the liver sinusoids after lipopolysaccharide exposure was not affected significantly by the drugs studied; however, heparin reduced markedly neutrophil activation.. The results of this investigation provide direct evidence that aggravation of preservation-reperfusion injury of rat liver by endotoxin is mediated by Kupffer cell-dependent mechanism(s) and it can be minimized by heparin and pentoxifylline.

Topics: Adenosine; Allopurinol; Animals; Bile; Clodronic Acid; Cryopreservation; Dose-Response Relationship, Drug; Escherichia coli; Glutathione; Heparin; Insulin; Kupffer Cells; L-Lactate Dehydrogenase; Lipopolysaccharides; Liver; Male; Neutrophils; Organ Preservation; Organ Preservation Solutions; Pentoxifylline; Perfusion; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Hepatocyte membrane potential differences (PDs) may be altered by the preservation solutions used in liver transplantation. Such alterations could impact on the survival of the donor liver, extent of biochemical injury, and flux of important ionic compounds. The purpose of the present study was to document these outcomes in the presence of four different preservation solutions.. Livers of adult male Sprague-Dawley rats (N = 3 to 4 per group) were impaled with intracellular microelectrodes prior to and at various time periods for 6 hours following complete hepatic resection. Just prior to resection, each liver was perfused with preservation solutions associated with high (normal saline [NS]), moderate (Euro-Collins [EC]), and low (University of Wisconsin solution [UW]) risks of reperfusion injury.. Baseline (in situ) PDs were similar in all groups (-37 +/- 4 mV, mean +/- SD). Ten minutes postresection, hepatic PDs were as follows: NS, -23.8 +/- 3.5 mV; EC, -11.4 +/- 0.4 mV; and UW, -8.7 +/- 0.3 mV (P <0.01 for all groups). Maximum depolarization occurred at 6 hours postresection (NS, -8.1 +/- 1.1 mV; EC, -7.7 +/- 1.3 mV; and UW, -8.6 +/- 1.0 mV). To determine whether these changes are of pathophysiologic importance, the NS solution was modified (addition of 0.1% ethanol) to achieve similar PD changes as those observed with UW. Liver transplants were then performed where the donor livers had been perfused and preserved for 6 hours with either NS or the modified NS (MNS) solution. Posttransplant (10 day) survival was 1 of 6 (17%) in the NS group and 4 of 6 (67%) in the MNS group (P <0.05). Regarding the effects of PD changes on ionic flux, intracellular calcium levels were documented for up to 4 hours by fluorescence video microscopy using Fura-2 in isolated hepatocytes exposed to NS, UW, and MNS solutions. Intracellular calcium levels were similar in all solutions at each time point studied.. The results of this study indicate that hepatocytes undergo prompt and marked depolarization following hepatic resection, and the extent of the depolarization correlates with survival following transplantation.

Topics: Adenosine; Allopurinol; Animals; Calcium; Calcium Signaling; Ethanol; Fluorescent Dyes; Fura-2; Glutathione; Graft Survival; Hepatectomy; Hypertonic Solutions; Insulin; Liver; Liver Transplantation; Male; Membrane Potentials; Microscopy, Fluorescence; Microscopy, Video; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Statistics, Nonparametric; Treatment Outcome

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Chemokine CCL2; Glutathione; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Insulin; Kidney; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction

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

Delayed graft function (DGF) has remained an important complication after renal transplantation. The exact causes of DGF remain to be clarified, particularly the impact of retrieval conditions and preservation factors. In the present investigation, (1)HNMR spectroscopy of urine was assessed in order to detect the influence of retrieval condition on renal medulla damage.. The isolated perfused pig kidney (IPK) was used to assess initial renal function from multiorgan donors (MOD) or single organ donors (SOD) after in situ cold flush and 24-h cold storage (CS) preservation with two standard preservation solutions: Euro-Collins (EC) and University of Wisconsin (UW) solutions. Kidneys flushed with cold heparinized saline and immediately perfused were used as the control group. Kidneys were perfused for 90 min at 37.5 degrees C for functional evaluation. During reperfusion, renal perfusion flow rate (PF) was measured. Glomerular filtration rate (GFR), tubular reabsorption of Na(+), and lactate dehydrogenase (LDH) and N-acetyl-beta-d-glucosaminidase (NAG) excretions were determined. Ischemia-reperfusion impairment was also determined by histological techniques and (1)HNMR spectroscopy.. PF, GFR, and tubular reabsorption of Na(+) were significantly decreased in experimental groups when compared to the control group but there was no significant difference between experimental SOD groups. GFR was significantly greater in UW-MOD than in EC-MOD and tubular reabsorption of Na(+) was significantly greater in UW-MOD than in EC-MOD after 45 min of reperfusion. The release of LDH in the effluent and the urinary excretion of NAG were not significantly different after 24-h CS in the various experimental groups. The most relevant resonances determined by (1)HNMR spectroscopy were citrate, trimethylamine-N-oxide, lactate, acetate, and amino acids. Excretion of these markers was significantly different when compared to biochemical markers. A resonance (P) detected particularly in EC-MOD after 24-h CS was identified and well correlated to renal dysfunction. Histological study showed that ultrastructural damage and mitochondrial injury were more pronounced in the EC-MOD group.. These results show that retrieval condition influences renal medullary damage. NMR spectroscopy, which is a noninvasive and nondestructive technique, is more efficient in assessing renal damage than conventional histology and biochemical analysis.

Topics: Acetylglucosaminidase; Adenosine; Allopurinol; Animals; Glutathione; Insulin; Kidney Medulla; Kidney Transplantation; L-Lactate Dehydrogenase; Magnetic Resonance Spectroscopy; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Swine; Tissue Donors

Ischemia-reperfusion injury involves free radical production, polymorphonuclear neutrophil chemotaxis/degranulation, and production of proteolytic enzymes, complement components, coagulation factors, and cytokines. Activated polymorphonuclear neutrophils, endothelial cells, and macrophages produce platelet activating factor, which further promotes these inflammatory reactions. The recently cloned plasma form of platelet activating factor-acetylhydrolase (PAF-AH) demonstrates antiinflammatory effects by degrading platelet activating factor. We evaluated the effects of PAF-AH in an isolated perfused rat lung model by adding it to the flush solutions or to the reperfusion blood.. Rat lungs were isolated, flushed with EuroCollins (EC) or University of Wisconsin (UW) solution, stored at 4 degrees C for 6 or 12 hours, and reperfused using a cross-circulating syngeneic support rat. During reperfusion, oxygenation, compliance, and capillary filtration coefficient were calculated. There were four groups in the study; group I (control) had no PAF-AH added, group II had PAF-AH added to the flush solution, group III had PAF-AH added to reperfusion blood, and group IV had PAF-AH added to both flush solution and reperfusion blood.. After 6 hours of storage, oxygenation, compliance, and capillary filtration coefficient significantly improved for EC in group IV. For UW, oxygenation improved in group IV whereas compliance improved in groups II, III, and IV. After 12 hours of storage, compliance improved for EC in group IV and capillary filtration coefficient improved in groups III and IV. For UW, oxygenation and compliance improved in groups II and IV, whereas capillary filtration coefficient improved in group IV.. Addition of PAF-AH to intracellular organ preservation solutions and to the blood reperfusate significantly improves postreperfusion oxygenation and compliance, and reduces lung capillary permeability.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Adenosine; Allopurinol; Animals; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Lung; Male; Organ Preservation Solutions; Oxidative Stress; Phospholipases A; Platelet Activating Factor; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Topics: Adenosine; Allopurinol; Blood Pressure; Glutathione; Hemodynamics; Humans; Insulin; Intestines; Lactates; Mannitol; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Short Bowel Syndrome; Transplantation, Homologous

Reperfusion injury with pulmonary edema continues to be a major complication after lung transplantation. Alveolar fluid homeostasis is regulated by Na+/K+-ATPase activity on the basolateral surface of alveolar epithelial cells. Intact Na+/K+-ATPase is essential to the resolution of pulmonary edema. We characterized the effects of cold ischemia and reperfusion on expression of Na+/K+-ATPase mRNA and protein.. Baseline values for Na+/K+-ATPase mRNA and protein were determined from freshly harvested lungs with no cold storage time or reperfusion (group I). Group II lungs were analyzed after cold storage times of 12 or 24 hr without subsequent reperfusion. Group III lungs were analyzed after cold storage times of 12 or 24 hr with subsequent reperfusion. Lungs were flushed with either Euro-Collins (EC) or University of Wisconsin (UW) solution in each group. All samples were quantified for Na+/K+-ATPase mRNA and Na+/K+-ATPase protein. Physiological parameters including oxygenation and compliance were also measured.. There were no significant differences in the level of mRNA and protein for samples that were cold stored without reperfusion (group II). With reperfusion (group III) there was a significant increase in the level of the Na+/K+-ATPase mRNA after 12 hr of storage for both EC and UW. After 24 hr of storage and subsequent reperfusion, lungs flushed with EC had significantly decreased Na+/K+-ATPase protein and mRNA, although lungs preserved with UW maintained their increased levels of Na+/K+-ATPase protein and mRNA.. Our data suggest that ischemia-reperfusion injury results in an initial up-regulation of Na+/K+-ATPase mRNA. With prolonged injury in lungs preserved with EC, the level of the mRNA decreased with a corresponding decrease in the Na+/K+-ATPase protein. The different response seen in EC versus UW may be explained by better preservation of pump function with UW than EC and correlates with improved physiological function in lungs preserved with UW solution.

Topics: Adenosine; Allopurinol; Animals; Blotting, Western; Cryopreservation; Gene Expression; Glutathione; Insulin; Lung; Lung Compliance; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ribonucleases; RNA, Messenger; Sodium-Potassium-Exchanging ATPase; Tissue Preservation

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

Topics: Adenosine; Allopurinol; Animals; Cell Line; Glutathione; Hot Temperature; HSP70 Heat-Shock Proteins; Insulin; Intestinal Mucosa; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Tissue Preservation

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Glutathione; In Vitro Techniques; Insulin; Isotonic Solutions; Liver; Organ Preservation Solutions; Oxygen; Partial Pressure; Raffinose; Rats; Reperfusion Injury; Ringer's Lactate

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

Injury during the transplant process affects the alloantigen-dependent factors and the alloantigen-independent processes of "chronic" rejection. Consequently, the determination of reliable parameters for the assessment of ischemic damage is essential for the prediction of renal changes after ischemia/reperfusion injury. The aim of this study was to assess the ability of (1)H NMR spectroscopy to predict the early graft dysfunction in an ischemia/reperfusion model after preservation in two standard preservation solutions, Euro-Collins (EC) and University of Wisconsin (UW). The second aim was to specify the role of the UW solution in preventing renal medullary injury. Urine and plasma samples from three experimental groups were examined during 2 weeks: control group (n = 5), EC group (cold flushed and 48-h cold storage of kidney in EC and autotransplantation, n = 12), and UW group (cold flushed and 48-h cold storage of kidney in UW and autotransplantation; n = 12). We also examined these kidneys 30-40 min after implantation and on the sacrifice day. Creatinine clearance was significantly reduced in the EC group during the second week. Fractional excretion of sodium and urine N-acetyl-beta-d-glucosaminidase activity were improved but not significantly different in the preserved groups. Urinary concentrations of the alpha-class glutathione S-transferase were significantly greater in the EC group during the first week after transplantation. The most relevant resonances for evaluating renal function after transplantation determined by (1)H NMR spectroscopy were those arising from citrate, dimethylamine (DMA), lactate, and acetate in urine and trimethylamine-N-oxide (TMAO) in urine and plasma. These findings suggest that graft dysfunction is associated with damage to the renal medulla determined by TMAO release in urine and plasma associated with DMA and acetate excretion. Citrate is also a urinary marker that can discriminate kidneys with a favorable evolution. Our results suggest that (1)H NMR spectroscopy is an efficient technique for detecting ischemic damage when accurate and precise data on graft injury is required. In addition, this study outlines the specific impact of the UW solution against injury to the renal medulla.

Topics: Acetic Acid; Acetylglucosaminidase; Acute Kidney Injury; Adenosine; Allopurinol; Animals; Biomarkers; Citric Acid; Cold Temperature; Creatinine; Dimethylamines; Glutathione; Glutathione Transferase; Hypertonic Solutions; Insulin; Isoenzymes; Kidney; Kidney Medulla; Kidney Transplantation; Lactic Acid; Magnetic Resonance Spectroscopy; Male; Methylamines; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine; Transplantation, Autologous

Experimental interventions that reduce ischaemia/reperfusion (I/R) lung injury can be used to improve the properties of preservation solutions. We attempted to increase the attenuation of I/R injury by University of Wisconsin solution (UW) by adding an antibody against tumour necrosis factor-alpha (TNF-alpha), to neutralize TNF-alpha, and/or by adding 3-deaza-adenosine (c3-Ado), to inhibit leucocyte adhesion and the biosynthesis of ICAM-1 (intercellular cell-adhesion molecule 1). We examined I/R injury using an isolated rat lung model. Six different solutions were perfused individually, followed by evaluation of I/R injury: (1) 0.9% NaCl (normal saline; NS), (2) NS+anti-TNF-alpha antibody, (3) UW alone, (4) UW+anti-TNF-alpha, (5) UW+c3-Ado and (6) UW+anti-TNF-alpha+c3-Ado. Haemodynamic changes, lung weight gain, capillary filtration coefficient, TNF-alpha levels and lung pathology were analysed in order to evaluate I/R injury. Compared with lungs perfused with NS, lungs treated with NS+anti-TNF-alpha showed less I/R injury. The addition of anti-TNF-alpha and/or c3-Ado to UW reduced I/R injury compared with unmodified UW. Among the six solutions tested, UW containing anti-TNF-alpha antibody reduced I/R injury to the greatest extent. We conclude that addition of anti-TNF-alpha antibody or c3-Ado protects against I/R lung injury when using UW. Further investigation of the improved properties of modified UWs would be beneficial with regard to lung transplantation research.

Topics: Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Glutathione; Hemodynamics; Insulin; Lung; Lung Transplantation; Male; Organ Culture Techniques; Organ Preservation; Organ Preservation Solutions; Organ Size; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tubercidin; Tumor Necrosis Factor-alpha

The lung is particularly susceptible to reperfusion injury, both experimentally and clinically after transplantation. The extracellular-type preservation solution Celsior, which has been predominantly studied in cardiac preservation, has components designed to prevent cell swelling, free radical injury, energy depletion, and calcium overload. Using an isolated blood-perfused rat lung model, we investigated whether Celsior would decrease preservation injury and improve lung function after cold ischemic storage and reperfusion compared to Euro-Collins (EC) and University of Wisconsin (UW) solutions.. Lewis rat lungs were isolated, flushed with the respective cold preservation solution, and then stored at 4 degrees C for 6 or 12 hr. After ischemic storage, the lung block was suspended from a force transducer, ventilated with 100% O2, and reperfused for 90 min with fresh blood via a cannula in the pulmonary artery. Lung compliance, alveolar-arterial oxygen difference, and outflow oxygen tension were all measured. The capillary filtration coefficient (Kf), a sensitive measure of changes in microvascular permeability, was determined.. For 6 hr of cold storage, lungs stored in Celsior had lower Kf values than those stored in EC, indicating decreased microvascular permeability. No other significant differences were noted between Celsior and EC or UW. For 12 hr of cold storage, Celsior provided increased oxygenation, decreased alveolar-arterial O2 differences, increased compliance, and decreased Kf values as compared to both EC and UW.. Celsior provides better lung preservation than EC or UW as demonstrated by increased oxygenation, decreased capillary permeability, and improved lung compliance, particularly at 12-hr storage times. These results are highly relevant, inasmuch as EC and UW are the most common clinically used lung preservation solutions. Further studies of Celsior in experimental and clinical lung transplantation, as well as in other solid organs, are indicated.

Topics: Adenosine; Allopurinol; Animals; Arteries; Capillary Permeability; Disaccharides; Electrolytes; Glutamates; Glutathione; Histidine; Hypertonic Solutions; Insulin; Lung; Lung Compliance; Male; Mannitol; Organ Preservation Solutions; Oxygen; Partial Pressure; Pulmonary Alveoli; Pulmonary Circulation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

The objective of this study was to determine if allopurinol (AL) and/or trifluoperazine (TFP) added to the Belzer machine preservation solution (MPS) improves the function of non-heart-beating donor (NHBD) canine kidneys.. Anesthetized canines underwent bilateral dissection of the renal vessels, obtaining baseline flow. After removing one kidney (heart-beating donor [HBD]), the dog was exsanguinated. After remaining in situ for 120 min (30-min warm ischemia time, 90-min cold ischemia time), the second kidney was removed (NHBD), flushed, biopsied, and weighed. The kidneys were machine-perfused separately for 20 hr, and pressure, flow, and resistance were measured serially. The kidneys were randomly assigned to a perfusate group (G): G1=MPS, G2=MPS+TFP, G3=MPS+AL, and G4=MPS+TFP+AL. Kidneys were implanted separately into a single recipient dog. Flow, resistance, and urine output were measured serially for 4 hr. Blood and urine samples and kidney biopsies were then obtained. All measurements were standardized to 100 g of kidney weight.. HBD kidneys functioned better than NHBD kidneys in all groups, as expected. Although perfusate G1 was the most effective solution for HBD kidneys, the TFP additive (perfusate G2) more effectively reversed the vasospastic effects of ischemia/reperfusion for NHBD than the MPS solution (G1) with or without other additives. In HBD kidneys, the addition of AL resulted in the best creatinine clearance; however, AL was less effective than MPS alone in NHBD kidneys. TFP+AL together were completely ineffective in preserving renal function, regardless of whether the kidneys were from HBD or NHBD.. MPS+TFP more effectively protected renal function against reperfusion injury in the NHBD than MPS alone, AL, or AL+TFP. AL exerts a salutary effect on creatinine clearance in HBD but not in the NHBD. The TFP and AL combination should not be used together with the MPS in machine preservation of kidneys.

Topics: Adenosine; Allopurinol; Animals; Diuresis; Dogs; Female; Glutathione; Heart Arrest; Insulin; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Renal Circulation; Reperfusion Injury; Trifluoperazine

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Female; Gadolinium; Glutathione; Glycine; Graft Survival; Hepatectomy; Infusions, Intravenous; Insulin; Kupffer Cells; Liver; Liver Transplantation; Microcirculation; Necrosis; Nitrites; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Tumor Necrosis Factor-alpha; Valine

A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated.. Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine).. There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis.. The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Chromans; Cryopreservation; Cytosol; Deferoxamine; Dithiothreitol; Glutathione; Insulin; Leucine; Liver; Organ Preservation Solutions; Protein Biosynthesis; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tritium; Vitamin E

Initial ischemia-reperfusion injury is associated with organ retrieval, storage, and transplantation adversely affects early graft function and influences the development of chronic graft dysfunction. We have recently shown that the protective agent trimetazidine (TMZ) added to preservation solutions: Euro-collins (EC) and University of Wisconsin (UW) was efficient to protect kidneys from ischemia-reperfusion injury in an isolated perfused kidney model. We extended these observations to investigate the role of this drug in the development and progression of organ dysfunction in the autotransplant pig kidney model.. Five experimental groups were studied. After 48-hr cold preservation, autotransplantation and immediate controlateral nephrectomy was then performed in group EC (EC+placebo (n=8), EC+TMZ (n=8), UW+placebo (n=7), and (UW+TMZ) (n=7) and compared with control group (uninephrectomized, n=4) during 14 days. Blood and urine samples were collected for the measurement of creatinine and blood urea nitrogen on postoperative days 1, 3, 5, 7, 11, and 14. Histological analysis was performed after reperfusion and at day 14.. Survivals were 100% in group B and D versus 42% in group A and 57% in group C. Urine production occurred earlier after autotransplantation from TMZ preserved kidneys than in placebo preserved groups. Peak creat and blood urea nitrogen was significantly greater in groups B and D than in groups A and C. TMZ was also efficient both to reduce ischemia-reperfusion injury and to decrease cellular infiltration.. These results support the beneficial effect of TMZ against ischemia-reperfusion injury and its early effects on grafts in the form of delayed graft function and decreased graft survival. In addition, TMZ reduces inflammatory cellular infiltration in the renal parenchyma.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Cryoprotective Agents; Glutathione; Graft Survival; Hypertonic Solutions; In Vitro Techniques; Insulin; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine; Trimetazidine

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

Current methods of preserving lung tissue for transplantation are inadequate. In this study, we tested whether the combination of hypothermia plus prostaglandin E(1) (PGE(1)) treatment would have synergistic attenuation on ischemia-reperfusion (I/R) lung injury. Isolated rat lung experiments with ischemia for 1 h then reperfusion for 1 h, were conducted using six different perfusates: (1) University of Wisconsin solution (UW) at 30 degrees C (n = 5), (2) UW at 22 degrees C (n = 5), (3) UW at 10 degrees C (n = 4), (4) UW+PGE(1) at 30 degrees C (n = 4), (5) UW+PGE(1) at 22 degrees C (n = 4), and (6) UW+PGE(1) at 10 degrees C (n = 4). Hemodynamic changes, lung weight gain, capillary filtration coefficients, and lung pathology were analyzed to evaluate the I/R injury. Compared with 30 degrees C UW, animals treated with 22 degrees C UW and 10 degrees C UW had less I/R lung injury, with the groups receiving 22 degrees C UW showing superior results to group receiving 10 degrees C UW. The addition of PGE(1) to UW solution produced more attenuation of I/R injury than did UW alone. Among the six groups, 10 degrees C UW+PGE(1) produced the most reduction of I/R injury. This study has shown that hypothermia can attenuate I/R injury with the optimal flushing temperature being near 22 degrees C. PGE(1) also has a protective effect on I/R. Furthermore, hypothermia and PGE(1) have synergistic attenuation of I/R lung injury. We propose that pulmonary artery flushed with cooling UW+PGE(1) might improve lung preservation and improve results in lung transplantation.

Topics: Adenosine; Allopurinol; Alprostadil; Animals; Capillary Permeability; Glutathione; Hypothermia, Induced; Insulin; Lung; Male; Organ Preservation; Organ Preservation Solutions; Organ Size; Pulmonary Circulation; Pulmonary Wedge Pressure; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Vascular Resistance; Vasodilator Agents

The inclusion of the colon in the intestinal graft resulted in worsening patient and graft outcome and increased the incidence of infection and rejection. In this study, we examine the role of ischemia on the barrier function of the epithelium during cold ischemia. Samples were collected from 15 harvested and transplanted human donor grafts (colon, 10; ileum, 6), which were immersed in University of Wisconsin (UW) solution. Ischemia (6, 12, 24, and 48 h) and reoxygenation were performed to evaluate the mucosal electrical status using the Ussing chamber technique. The functions of enterocytes and crypt cells were tested by glucose and theophylline challenge. Modified Park's classification was applied to evaluate the severity of mucosal damage under light microscopy. The colon had higher levels of baseline potential difference, short-circuit current, and resistance than the ileum during 6 48 h of ischemia. Colonic epithelial cells responded well to theophylline stimulation at 24 h of ischemia, while there was no ileal response. The colonic mucosa was histopathologically well preserved in UW solution for 48 h, and mucosal damage induced by reoxygenation was less than in the ileum. In conclusion, electrophysiologically and histopathologically, the colon is less susceptible to cold preservation damage than the ileum during storage with UW solution.

Topics: Adenosine; Allopurinol; Cold Temperature; Colon; Electrophysiology; Glutathione; Humans; Ileum; In Vitro Techniques; Insulin; Intestinal Mucosa; Organ Preservation Solutions; Raffinose; Reperfusion Injury

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

Topics: Adenosine; Allopurinol; Anastomosis, Surgical; Animals; Cell Survival; Gadolinium; Glutathione; Hepatic Artery; Insulin; Kupffer Cells; Liver; Liver Circulation; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Portal Vein; Raffinose; Regional Blood Flow; Reperfusion Injury; Swine; Vena Cava, Inferior

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

Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4 degrees C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8, 16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 degrees C, 20 degrees C, or 37 degrees C and reperfused for 90 min (37 degrees C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme hystochemistry (5'-nucleotidase and LDH). After 8-h CIT and a flush at 4 degrees C, a release of endogenous HA (-7%) was observed, whereas uptake of exogenous HA occurred after the 20 degrees C flush (2%, P = NS) and after the 37 degrees C flush (24%, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 degrees C (-10%) that at 4 degrees C and 20 degrees C (-64% and -17%, respectively, p = 0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 degrees C and 20 degrees C preflush groups, but not in the 37 degrees C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 degrees C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4 degrees C or 37 degrees C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 degrees C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 degrees C before reperfusion, SEC and hepatocellular damage is attenuated.

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Bile; Cold Temperature; Female; Glutathione; Hot Temperature; Hyaluronic Acid; Insulin; L-Lactate Dehydrogenase; Liver; Organ Preservation Solutions; Purine-Nucleoside Phosphorylase; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Preservation

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K+ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na+ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na+ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K+ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na+ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na+ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K+ UW solution. Liver preservation in the high-Na+ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Creatine Kinase; Cryopreservation; Glutathione; Indocyanine Green; Insulin; Isoenzymes; Liver; Liver Circulation; Liver Transplantation; Male; Microcirculation; Organ Preservation Solutions; Potassium; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sodium

Organ preservation injury is associated with endothelial cell damage, destabilization of mitochondrial and cell membranes, and the release of proteolytic enzymes. In addition to its well-known clinical effect of reducing perioperative blood loss, aprotinin has antiproteolytic and membrane-stabilizing properties. We hypothesized that adding aprotinin to Euro-Collins (EC) and University of Wisconsin (UW) solutions would decrease preservation injury in cultured endothelial cells and a whole organ rat lung model.. Bovine aortic endothelial cells were cultured and stored in the respective solution at 4 degrees C for 12 or 48 hours. Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. In the whole organ model, rat lungs were isolated, flushed with the respective solution, and stored at 4 degrees C for 6 or 12 hours. The lungs were ventilated with 100% O2 and reperfused with fresh blood. Alveolar-arterial O2 difference, O2 tension, capillary filtration coefficient, and compliance were determined.. Endothelial cell viability was optimized with the addition of aprotinin to EC and UW at a dose of 150 KIU/mL (0.02 mg/mL). In the isolated perfused lung model, after 6 hours of ischemic storage, aprotinin-enhanced (100 KIU/mL [0.014 mg/mL]) EC and UW decreased alveolar-arterial O2 difference, increased O2 tension, and decreased capillary filtration coefficient compared with EC and UW alone. After 12 hours of ischemic storage, aprotinin-enhanced EC and UW decreased alveolar-arterial O2 difference, increased O2 tension, decreased capillary filtration coefficient, and increased compliance compared with EC and UW alone.. The addition of aprotinin to EC and UW solutions increases endothelial cell viability in hypoxic cold storage conditions. In terms of whole organ function, aprotinin improves lung preservation as demonstrated by increased oxygenation and compliance, and decreased capillary permeability. This study is clinically applicable as there is already extensive experience with the use of aprotinin in heart and lung transplant recipients, in addition to its routine use in conventional cardiac operations.

Topics: Adenosine; Allopurinol; Animals; Aprotinin; Capillaries; Cattle; Cell Membrane; Cell Survival; Cells, Cultured; Cryopreservation; Endopeptidases; Endothelium, Vascular; Glutathione; Hemostatics; Hypertonic Solutions; Insulin; Lung Compliance; Lung Diseases; Lung Transplantation; Male; Mitochondria; Organ Preservation Solutions; Oxygen; Oxygen Consumption; Protease Inhibitors; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Ventilation-Perfusion Ratio

1. An intervention to reduce ischaemia-reperfusion lung injury will be an important advance in transplant medicine. Although the mechanisms associated with producing ischaemia-reperfusion endothelial injury have not been completely elucidated, many of the injury mediators have been studied in detail. While no single pharmacological therapy is likely to be totally effective in eliminating this complex injury, we have developed a mixture of agents that are known to block pathways involved in producing ischaemia-reperfusion-associated lung vascular injury.2. The present study modified University of Wisconsin solution (UW) by adding one of the protective agents prostaglandin E1 (PGE1), dexamethasone (Dex) or dibutyryl cAMP (Bt2-cAMP), or a combination of these, to the perfusate of rat lungs exposed to 4 h of cold ischaemia followed by 1 h of reperfusion. Nine modified UW solutions were studied: (1) UW+Dex, (2) UW+PGE1, (3) UW+Bt2-cAMP, (4) UW+Dexx3, (5) UW+PGE1x3, (6) UW+Bt2-cAMPx3, (7) UW+Dex+PGE1, (8) UW+Dex+Bt2-cAMP, (9) UW+PGE1+Bt2-cAMP. These solutions were utilized in individual experiments to assess haemodynamic changes, lung weight gain, the capillary filtration coefficient (Kfc) and pathology in all lungs.3. The results indicate that lung weight gain and Kfc values were significantly lower than with UW alone in groups 1, 2 and 3, which contained only one additional protective agent. In groups 4, 5 and 6, which contain three times the concentration of each protective agent, both Kfc and lung weight gain were similar to those measured in groups 1, 2 and 3, i.e. lungs were protected but the protection was not dose dependent. In groups 7, 8 and 9, which contained two protective agents, lung weight gain and Kfc were greatly reduced compared with UW alone. Histopathological studies showed similar decreases in the injury profiles of lungs.4. Although UW contains several antioxidant protective agents such as allopurinol and glutathione, it did not provide effective protection in our ischaemia-reperfusion lung injury model. UW modified with an additive of PGE1, Dex or Bt2-cAMP attenuated ischaemia-reperfusion injury. Furthermore, UW containing two of these protective agents augmented the protection. Among the modified solutions, it appears that UW+PGE1+Bt2-cAMP protects the lungs to a greater extent than all other solutions used in our study. We suggest that preservation solutions containing PGE1-Bt2-cAMP will provide additional protective effects to organs s

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Anti-Inflammatory Agents; Bucladesine; Dexamethasone; Glutathione; Insulin; Lung; Lung Transplantation; Male; Organ Preservation Solutions; Organ Size; Prostaglandins E; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue Preservation; Vascular Resistance

Topics: Adenosine; Alkanesulfonates; Allopurinol; Animals; Enzyme Inhibitors; Glutathione; Insulin; Intestinal Mucosa; Intestines; Microvilli; Organ Preservation; Organ Preservation Solutions; Phospholipases A; Phospholipases A2; Raffinose; Rats; Reperfusion Injury

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

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Graft Rejection; Insulin; Intestinal Mucosa; Intestine, Small; Muscle, Smooth; Myenteric Plexus; Neurons; Organ Preservation; Organ Preservation Solutions; Phosphopyruvate Hydratase; Raffinose; Reperfusion Injury; S100 Proteins; Swine; Transplantation, Homologous

Nonspecific injury in cadaveric renal transplants adversely affects early graft function and influences long-term graft survival after organ transplantation. Trimetazidine (TMZ) has been reported to exert a protective action against normothermic ischemia and reperfusion injury in several experimental and clinical studies. In an isolated perfused pig kidney model, we investigated the effects of TMZ added to University of Wisconsin solution (UW) during 48 or 72 h of cold storage (CS) and the consequence during reperfusion. Under all conditions tested renal perfusate flow rate (PFR), renal functions, and tubular injury markers were determined during a 120-min perfusion period. Lipid peroxidation and histological examination (optical and electron microscopy) were also determined after CS and reperfusion. The addition of TMZ (10(-6) M) to the UW solution improved dramatically the quality of preserved kidneys and consequently the functional recovery during reperfusion. TMZ + UW also significantly had a protecting role against reperfusion injury and lipid peroxidation when compared to UW alone. These results were correlated with both a better preservation of the proximal brush border membrane and reduced cellular and mitochondrial swelling. These results also suggested that the TMZ-induced renoprotection correlated well with the observed decrease membrane lipid peroxidation. Therefore, trimetazidine may be useful for clinical kidney graft preservation.

Topics: Adenosine; Allopurinol; Animals; Citric Acid; Cold Temperature; Glomerular Filtration Rate; Glutathione; Humans; Insulin; Kidney; Kidney Transplantation; Kidney Tubules; Lactic Acid; Lipid Peroxidation; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Swine; Trimetazidine

Cyclic nucleotides mediate intracellular signal transduction of several vasodilators. In addition to its vascular relaxant effects, cAMP is known to protect endothelial cells and to suppress Kupffer cell activation. On the other hand, cGMP potently ameliorates adhesion of leukocytes and platelets. We tested the effects of two analogs of cyclic nucleotides (8bromo cyclic adenosine monophosphate [8br-cAMP] and 8bromo cyclic guanosine monophosphate [8br-cGMP]) in rat liver preservation.. In experiment 1, either analog (0.1-1.0 mM) alone was added to University of Wisconsin (UW) solution in a survival study. In experiment 2, donors and recipients were also treated with 8br-cAMP or 8br-cGMP, with the following three groups tested: group 1=control; group 2=administration of 8br-cAMP to donors, UW solution, and recipients; group 3=administration of 8br-cGMP to donors, UW solution, and recipients. Experiment 3 tested combined treatments: group 4=administration of 8br-cGMP to donors and UW solution, and cAMP to recipients; group 5=administration of 8br-cAMP to donors and UW solution, and 8br-cGMP to recipients. To elucidate the roles of each nucleotide, two further groups were tested: group 6=administration of 8br-cAMP to donors and UW solution; group 7=administration of 8br-cGMP to recipients. In experiment 4, rats in groups 1, 5, 6, and 7 were killed at several time points after reperfusion, and percent graft blood flow (%BF), number of accumulated neutrophils, plasma levels of tumor necrosis factor-alpha and interleukin-1, and serum alanine aminotransferase levels were examined.. In experiments 1 and 2, no significant effect was observed on animal survival. In experiment 3, a significant increase in animal survival was observed only in group 5 (100%, 7/7, P=0.0004 vs. group 1: 16.7%, 2/12). In group 5, no improvement of %BF was observed during the early phase of reperfusion (15 and 30 min) compared with that in group 1. On the other hand, the %BF of group 5 was significantly higher in the later phase (6 hr), consistent with the decrease in accumulation of neutrophils observed then. Production of tumor necrosis factor-alpha and serum alanine aminotransferase levels were also reduced with this treatment. Histologically, the bleeding and segmental necrosis, observed in group 1, were completely prevented in group 5.. We conclude that restoration of grafts with cAMP and administration of cGMP to recipients led to successful transplantation, and that the two analogs acted synergistically in opposing preservation and reperfusion injury without improvement of graft blood flow during the early phase of reperfusion. The effect was due to their regulation of neutrophil activation and sequestration.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Allopurinol; Animals; Cyclic GMP; Drug Combinations; Drug Synergism; Glutathione; Insulin; Interleukin-1; Liver; Liver Circulation; Male; Organ Preservation; Organ Preservation Solutions; Postoperative Period; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Tissue Donors; Tumor Necrosis Factor-alpha

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: Adenosine; Allopurinol; Animals; Antioxidants; Cold Temperature; Creatinine; Diuresis; Dogs; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Kidney; Kidney Transplantation; Male; Niacinamide; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transplantation, Autologous

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

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

The effects of hypothermic injury to the liver were investigated on an isolated perfusion circuit by comparing porcine livers with varying degrees of preservation injury.. A group of unstored livers (n = 5) were compared to livers stored in University of Wisconsin (UW) solution for 18 h (n = 5), and a group of livers stored in Hartmann's solution for 18 h (n = 5).. We observed that the degree of platelet sequestration was directly related to the severity of the preservation injury. After 2 h of isolated liver perfusion, the perfusate platelet count fell from 148 +/- 14 x 10(9)/L to 84 +/- 13 x 10(9)/L for control livers. In comparison for livers stored in UW solution, the platelet count fell from 173 +/- 43 x 10(9)/L to 61 +/- 14 x 10(9)/L representing a 64.8% fall, while for those stored in Hartmann's solution, an even more profound fall from 152 +/- 36 x 10(9)/L to 19 +/- 9 x 10(9)/L (87.5% fall) was observed. The difference between the UW-stored and Hartmann's-stored livers was significant (P < 0.05). However, using this model, the degree of leukocyte sequestration did not differentiate the groups. Both histological and ultrastructural examination of liver biopsies taken immediately following revascularization demonstrated that for mild degrees of preservation injury following hypothermic storage, changes occur to the sinusoidal lining cells well before changes to the parenchymal elements.. These findings substantiate the hypothesis that the primary injury associated with hypothermia involves the sinusoidal lining cells (non-parenchymal elements), that it is predominantly a reperfusion phenomenon and that efforts at improving preservation should therefore be targeted primarily at these cells and not the hepatocytes.

Topics: Adenosine; Allopurinol; Animals; Biopsy; Cryopreservation; Disease Models, Animal; Glutathione; Insulin; Isotonic Solutions; Liver; Liver Function Tests; Organ Preservation; Organ Preservation Solutions; Platelet Count; Raffinose; Reperfusion Injury; Ringer's Lactate; Swine

Proteases as well as alterations in intracellular calcium have important roles in hepatic preservation-reperfusion injury, and increased calpain activity recently has been demonstrated in liver allografts. Experiments were designed to evaluate (i) hepatic cytosolic calpain activity during different periods of cold ischemia (CI), rewarming, or reperfusion, and (ii) effects of inhibition of calpain on liver graft function using the isolated perfused rat liver and arterialized orthotopic liver transplantation models. Calpain activity was assayed using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (AMC) and expressed as mean +/- SD pmol AMC released/min per mg of cytosolic protein. Calpain activity rose significantly after 24 hr of CI in University of Wisconsin solution and further increased with longer preservation. Activity also increased within 30 min of rewarming, peaking at 120 min. Increased durations of CI preceding rewarming resulted in significantly higher activity (P < 0.01). Calpain activity increased rapidly upon reperfusion and was significantly enhanced by previous CI (P < 0.01). Calpain inhibition with Cbz-Val-Phe methyl ester significantly decreased aspartate aminotransferase released in the isolated perfused rat liver perfusate (P < 0.05). Duration of survival after orthotopic liver transplantation using livers cold-preserved for 40 hr was also significantly increased (P < 0.05) with calpain inhibitor. In conclusion, calpain proteases are activated during each phase of transplantation and are likely to play an important role in the mechanisms of preservation-reperfusion injury.

Topics: Adenosine; Allopurinol; Animals; Calpain; Female; Glutathione; Insulin; Liver; Liver Transplantation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Rats, Wistar; Reperfusion Injury; Tissue Preservation

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

Little is known about the possible contribution of apoptosis to ischemia-reperfusion injury in human liver transplantation. Therefore, we studied postreperfusion surgical biopsy specimens of 16 human liver allografts using the TUNEL assay for in situ demonstration of apoptotic cells. In all patients, a variable proportion of hepatocytes and sinusoidal endothelial cells presented labeled nuclei. The mean +/- standard deviation percentages of positive hepatocytes were 18.7% +/- 12.2% in the whole section, 30.4% +/- 18.7% in the subcapsular region, 14.5% +/- 13.5% in the centrilobular zones, and 10.3% +/- 9.5% in the periportal zones. The percentage of positive hepatocytes were not correlated with the duration of cold ischemia but was higher in grafts harvested from donors with elevated preoperative aspartate aminotransferase (AST) levels. The percentage of positive hepatocytes was correlated with postoperative serum levels of AST (P = .015) and inversely correlated with postoperative serum levels of factor V (P = .019). Apoptotic biliary epithelial cells were detected in only 3 cases. In conclusion, apoptosis is a frequent event in postreperfusion biopsy specimens of liver allografts and probably contributes to preservation injury of hepatocytes.

Topics: Adenosine; Allopurinol; Apoptosis; Biopsy; Cryopreservation; Deoxyuracil Nucleotides; DNA Nucleotidyltransferases; Female; Glutathione; Humans; Immunoenzyme Techniques; Insulin; Liver; Liver Diseases; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Random Allocation; Reperfusion Injury; Transplantation, Homologous

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; Bile; Enalapril; Epoprostenol; Glutathione; Insulin; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Digitonin; Glucose; Glutathione; Humans; Insulin; Lung Neoplasms; Mannitol; Mitochondria; Organ Preservation Solutions; Oxygen Consumption; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Rotenone; Tumor Cells, Cultured; Umbilical Veins

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Calcium; Glutathione; In Vitro Techniques; Insulin; Kidney Cortex; Kinetics; Male; Mitochondria; Organ Preservation Solutions; Permeability; Quinacrine; Rabbits; Raffinose; Reperfusion Injury; Temperature

Intestinal ischaemia-reperfusion (IR) injury has largely been attributed to cellular necrosis. Apoptosis, a distinct form of cell death has been observed following IR to the brain, heart, adrenals and the kidneys. In order to characterize the role of apoptosis in intestinal IR, small bowel grafts were stored in saline (n = 6) or modified University of Wisconsin solution (n = 6) at 4 degrees C for 12 h and reperfused for 6 h in syngeneic rats. Samples of normal, stored and reperfused intestines at 1, 3 and 6 h were analysed by light and electron microscopy. Following reperfusion, there was crypt and villous epithelial apoptosis, loss of crypt and villous structures, and an increase in mucosal inflammatory cell infiltration. Ongoing apoptosis was maximum at 1 h, its degree decreasing with increasing reperfusion intervals. Large numbers of apoptotic bodies dominated the picture from 3 h of reperfusion. This study has demonstrated the induction of apoptosis by intestinal IR injury, which begins within an hour of reperfusion and is probably responsible for the observed crypt and villous loss. This has potential therapeutic implications as, opposed to necrosis, apoptosis is an active process with genetic regulators and biochemical effectors, which can be specifically targeted to prevent or alleviate IR injury.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Glutathione; Insulin; Intestinal Mucosa; Intestine, Small; Male; Microscopy, Electron; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Sodium Chloride; Tissue Preservation

This study focuses upon two discrete components of posttransplant hepatic reticuloendothelial system (RES) function-phagocytosis and killing of bacteria-under various conditions of ischemic preservation. We had previously reported that, following intravenous injection of rats with 51Cr and 125I double-labeled Escherichia coli, hepatic 51Cr levels can be used to reliably quantify hepatic phagocytic clearance of the bacteria from the blood (HPC), while the subsequent release of 125I from the liver accurately parallels hepatic bacterial killing. Here, Wistar rats were transplanted with syngeneic livers perfused with either normal saline (NS) or University of Wisconsin solution (UW) and stored at 4 degrees C for 1, 2, or 3 hr prior to implantation. Control rats underwent laparotomy and hepatic artery ligation. Using the double-labeled E coli assay, HPC was decreased in all transplanted animals when compared with controls, reaching a nadir on the third postoperative day (P < 0.05). In rats transplanted with livers preserved in NS, the fraction of phagocytosed organisms that were subsequently killed (hepatic killing efficiency=HKE) was increased to 142%, 129%, or 112% of normal following 1, 2, or 3 hr of cold ischemia, respectively; P < 0.05). Conversely, preservation of donor allografts in UW was associated with marked depression of HKE. Moreover, rats receiving NS- or UW-preserved livers tolerated an intravenous challenge with Streptococcus pneumoniae poorly (50% mortality) compared with hepatic artery ligated controls (12% mortality) at 7 days. Ischemic preservation of rat livers in NS resulted in a dose (of ischemia)-dependent reduction of hepatic phagocytosis coupled with a potentiation of HKE. Preservation in UW, however, produced a striking suppression of both components of hepatic RES function. Following a septic challenge survival was reduced in both groups of transplanted rats.

Topics: Adenosine; Allopurinol; Animals; Escherichia coli; Escherichia coli Infections; Glutathione; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Preservation

Topics: Adenosine; Allopurinol; Animals; Cells, Cultured; Free Radical Scavengers; Glutathione; Glutathione Peroxidase; Insulin; Lipid Peroxidation; Liver; Liver Transplantation; Male; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Phosphatidylcholines; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Swine; 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: Adenosine; Alanine Transaminase; Allopurinol; Animals; Anti-Ulcer Agents; Biomarkers; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Pentoxifylline; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Tumor Necrosis Factor-alpha; Vasodilator Agents; Xanthines

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Cold Temperature; Glutathione; Hyaluronic Acid; Insulin; Kinetics; L-Lactate Dehydrogenase; Liver; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Time Factors

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: Adenosine; Allopurinol; Animals; Benzamidines; Blood Glucose; Glutathione; Graft Survival; Guanidines; Insulin; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Protease Inhibitors; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Arginine; Cyclic GMP; Enzyme Inhibitors; Glutathione; Insulin; Kidney; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Temperature

Extracellular yeast glycoproteins (YG) produced by Rhodosporidium toruloides have been shown to increase the survival rate of different yeast species after storage in liquid nitrogen. The purpose of this study was to investigate the effect of YG on cold-stored rat livers. Water-soluble YG produced either by Phaffia rhodozyma (G3) or by Leucosporidium antarcticum (G4) were added to a modified University of Wisconsin solution (mUW) and used for cold storage (1 degree C) of isolated livers. The functional status of each liver was then assessed under conditions of 90-min normothermic reperfusion. The 46-h cold storage in mUW without G3 and G4 resulted in serious preservation-reperfusion injury of the liver. The addition of G3 to mUW for 46-h preservation of the liver resulted in significantly higher bile flow (4.32 +/- 0.35 vs 2.35 +/- 0.49 microliters/min/10 g at 75-90 min), higher portal blood flow (10.99 +/- 0.2 vs 4.78 +/- 1.07 ml/min/g at 90 min), lower liver weight after reperfusion (102.4 +/- 1.5 vs 116.7 +/- 6.6% of weight before preservation), and lower total tissue water after reperfusion (2.49 +/- 0.05 vs 2.92 +/- 0.13 g water/g dry weight). However, the activity of ALT, AST, and LDH in perfusate was not changed. The beneficial effect of G4 was less pronounced. The 24-h storage in mUW resulted in a significant increase of AST and LDH activity in perfusate; the addition of G3 to mUW for 24-h preservation did not affect these parameters. In conclusion, the addition of 0.05% G3 or G4 to mUW was only partially beneficial in improving rat liver preservation.

Topics: Adenosine; Allopurinol; Animals; Basidiomycota; Cold Temperature; Evaluation Studies as Topic; Female; Fungal Proteins; Glutathione; Glycoproteins; In Vitro Techniques; Insulin; Liver; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

Rat livers were flushed with different preservation solutions and stored at 4 degrees C for 24 h before being reperfused with a synthetic air-equilibrated, water-based solution. Four solutions were tested using this isolated rat liver model: Marshall's hypertonic citrate (HC); modified University of Wisconsin solution (Mod UW); a histidine-based solution (HIS); and a histidine-lactobionate-raffinose-based solution (HLR). After storage, livers were perfused at 4 degrees C for a period of 2 h and biopsy samples taken, at different time points, to investigate energy metabolism. Livers stored in HLR and HIS had higher 24-h storage levels of ATP (0.41 and 0.24 mumol/g respectively; P < 0.05) than those stored in Mod UW and HC. On reperfusion, all groups regenerated ATP by 1-2 h. However, significantly greater levels of ATP regeneration occurred in livers stored in the HLR (1.6 +/- 0.08 mumol/g) and Mod UW (1.3 +/- 0.18) than HC (0.58 +/- 0.19) and HIS (0.96 +/- 0.12); P < 0.05. Energy charge (1) (EC) recovered in all groups but was significantly higher in HLR and Mod UW (0.79 and 0.68, respectively; P < 0.05) than HC and HIS. These represent 95% (HLR) and 80% (Mod UW) of values observed in FIL. During the reperfusion period, total adenine nucleotide levels (TA) did not vary significantly within each storage group, except in the HIS solution. However, TAs were greater with livers stored for 24 h in HLR (2.5 +/- 0.25) and Mod UW (2.7 +/- 0.20) than those the other two storage groups (P < 0.05 in each case). This study has demonstrated that it was possible to resuscitate liver energetics after prolonged hypothermic ischemia by a period of cold reperfusion, and the method can differentiate between preservation solutions. The livers stored in each solution showed varying degrees of success in regeneration of ATP and EC, demonstrating that oxygen was not a limiting factor when using an air-equilibrated perfusate. The solutions providing the better preservation conditions gave the greater resuscitation of liver energetics (Mod UW and HLR). Overall, livers stored in HLR had the greatest resuscitation of energy metabolism, which correlates with survival data from other studies (29-31, 33).

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Disaccharides; Energy Metabolism; Evaluation Studies as Topic; Glutathione; Histidine; Hypertonic Solutions; In Vitro Techniques; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Solutions

Simple models are needed that effectively test the variables that may be important in liver preservation. Two such models are isolated hepatocytes and tissue slices. In this study the effects of hypothermic preservation on the viability of hepatocytes (HC) and tissue slices (TS) from rat livers were measured by LDH leakage after cold storage and rewarming. We compared how glycine, calcium, and fasting, shown previously to affect preservation injury in hepatocytes, affected both HC and TS viability. Hepatocytes were cold-stored in University of Wisconsin organ preservation solution for up to 48 h and rewarmed in Krebs-Henseleit Bicarbonate (KHB) for 120 min. Tissue slices were studied in two ways. Either livers were cold-stored intact and then tissue slices (TS-A) prepared and rewarmed in KHB, or tissue slices were prepared from the fresh liver, cold-stored, and then rewarmed (TS-B). The latter method may be similar to cold storage of HC. Freshly prepared samples (HC, TS-A, or TS-B) showed < 15% LDH leakage during the rewarming phase. Cold storage for 24 h resulted in < 30% LDH leakage in all preparations. After 48 h cold storage there was a significant increase in LDH leakage (HC, 65.1 +/- 5.1%; TS-A, 52.9 +/- 0.8%, TS-B, 53.6 +/- 2.6%). Glycine (3 mM) or calcium (1.5 mM) included in the KHB significantly reduced LDH leakage from 48 h cold-stored HC to 20.7 +/- 1.8 and 26.3 +/- 2.4%, respectively. These agents caused a smaller decrease in LDH release from tissue slices (around 40%). Hepatocytes appear more susceptible to preservation/reperfusion damage than the more structurally intact tissue slices as suggested by the greater release of LDH. Another difference was that the agents which improved preservation quality of HC were not as effective in TS. Hepatocytes may be more vulnerable to preservation/reperfusion damage because of the harsh methods used in their preparation. The damage induced during preparation appears amenable to suppression by glycine or calcium. Tissue slices, which are intact pieces of liver tissue, may be more suitable for studies related to development of better methods for liver preservation. The intact cells in TS have not been exposed to harsh conditions and maintain a more natural cell-cell relationship.

Topics: Adenosine; Allopurinol; Animals; Calcium; Cell Separation; Cold Temperature; Fasting; Glutathione; Glycine; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Liver; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue Preservation

We have shown that 24-hour preservation by a two-layer [University of Wisconsin solution (UW)/perfluorochemical (PFC)] cold storage method at 4 degrees C allowed tissue ATP synthesis and resuscitated canine pancreases subjected to 90 minutes of warm ischemia. The purpose of this study was to examine whether the two-layer (UW/PFC) mild hypothermic storage method at 20 degrees C could shorten a preservation period for recovery of ischemically damaged pancreas and clarify changes of tissue adenine nucleotide metabolism and tissue perfusions. After 90 minutes of warm ischemia, canine pancreas grafts were preserved by the two-layer method and then autotransplanted. Tissue adenine nucleotide levels at the end of preservation and tissue perfusions after reperfusion were measured. Pancreas grafts subjected to 90 minutes of warm ischemia did not survive (0 of 5), without preservation. During a 5-hour preservation by the two-layer cold storage method the grafts did not synthesize enough ATP to repair damaged cell, although tissue perfusions were maintained after reperfusion. Consequently, ischemically damaged pancreases were not resuscitated (0 of 3). However, during 5-hour preservation by the two-layer mild hypothermic storage method, the grafts supplied enough ATP for processes that repair damaged cells, and tissue perfusions were maintained after reperfusion. As a result, ischemically damaged grafts were resuscitated (5 of 5). We conclude that 5-hour preservation by the two-layer mild hypothermic storage method accelerates ATP synthesis, which is essential for repairing damaged cells and protects the vascular microcirculation. This method can resuscitate ischemically damaged pancreas faster and holds promise for pancreas-kidney transplantation from cardiac arrest donors.

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cryopreservation; Dogs; Female; Fluorocarbons; Glutathione; Graft Survival; Insulin; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury

Altered cellular calcium (Ca) homeostasis may be important in mediating hypothermic injury in preserved kidneys. In this study the effect of hypothermic (5 degrees C) storage on ionized intracellular Ca concentration ([Ca]i) in rabbit tubules was examined using Indo-1. Tubules were stored up to 250 min in UW-gluconate solution containing either 0.0, 0.5, 1.5, or 5.0 mM Ca (yielding about 3.6, 62, 371, and 1,010 microM ionized solution Ca (Ca2+) at 5 degrees C, respectively). [Ca]i increased to about 1,600 nM within 1 min after suspension in UW solution followed by a decrease in [Ca]i during the subsequent 60 min in all groups, suggesting mitochondrial Ca sequestration. Thereafter, [Ca]i either 1) increased in tubules incubated with 1.5 and 5.0 mM Ca to levels greater than 2,500 nM; 2) decreased to about 800 nM in tubules incubated with 0.5 mM Ca and then remained stable; or 3) continued to decrease in tubules incubated with 0.0 mM added Ca to reach an apparent steady-state concentration of about 175 nM after 180 min of incubation. The early spike in [Ca]i was unaffected by adding EGTA (solution Ca2+ = 50 nM). Ryanodine eliminated the [Ca]i spike, indicating that cooling in UW-gluconate solution caused release of endoplasmic reticulum Ca. This study shows that [Ca]i initially increases after exposure to UW-gluconate solution and appears to be transiently buffered through intracellular, probably mitochondrial, sequestration. Saturation of cellular buffer mechanisms resulted in a sustained dependence of [Ca]i on extracellular Ca2+. These results support the hypothesis that the effect of Ca on kidney viability is related to solution-induced alterations in [Ca]i.

Topics: Adenosine; Allopurinol; Animals; Calcium; Cattle; Cold Temperature; Gluconates; Glutathione; Homeostasis; Hypothermia; In Vitro Techniques; Insulin; Intracellular Fluid; Kidney Tubules; Kinetics; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Preservation

Despite suggestions of a connection between endothelial damage and permeability alterations after ischemia and reperfusion in pulmonary tissue undergoing transplantation, no direct correlation between vascular endothelial discontinuity and parenchymal edema has yet been shown.. Forty-two rat lungs were harvested and stored for 48 or 72 hours under hypothermic and ischemic conditions. Stored pulmonary tissue was studied before transplantation and 5 minutes or 24 hours after transplantation by light microscopy and scanning electron microscopy of arterial vascular endothelium.. Stored lungs not subjected to revascularization showed moderate perivascular edema, with small intercellular gaps in endothelial monolayers. Five minutes after transplantation, pulmonary tissue appeared congested, with perivascular and alveolar edema. Examination of vascular endothelium by scanning electron microscopy showed detachment of endothelial cells. Twenty-four hours after transplantation, edema, hemorrhage, and vascular congestion were found in all specimens. Arterial vascular endothelium showed weak intercellular connections, numerous intercellular gaps, and widespread cell detachment. Bronchial epithelial cells appeared damaged after storage, with loss of cilia, blebbing of apical cytoplasm, and cellular rounding. These changes were maintained 5 minutes after transplantation but appeared totally reversed after 24 hours in specimens stored 48 hours, whereas bronchial denudation was observed in 72-hour stored lungs. Statistically significant positive correlations (Kendall p < 0.001) between revascularization time and alveolar edema and hemorrhage were found for both storage periods.. The results from this study demonstrate correlation between loss of endothelial monolayer continuity and histologic evidence of vascular permeability increases in pulmonary tissue before and after lung transplantation.

Topics: Adenosine; Allopurinol; Animals; Bronchi; Capillary Permeability; Cold Temperature; Endothelium; Endothelium, Vascular; Female; Glutathione; Insulin; Lung; Lung Transplantation; Microscopy, Electron, Scanning; Organ Preservation; Organ Preservation Solutions; Pulmonary Edema; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

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

The University of Wisconsin solution is widely used for organ preservation. We show that this fluid is often contaminated by traces of iron catalytic for free radical reactions, as demonstrated by the bleomycin assay and the ability of the iron to stimulate lipid peroxidation. Reduced glutathione (GSH) added to University of Wisconsin solution during its commercial preparation had oxidized to the disulfide in all samples examined. Addition of GSH to UW solution showed its half-life to be about one day. The metal ion chelators desferrioxamine and phenanthroline did not substantially delay the loss of GSH, although desferrioxamine was able to suppress iron-mediated lipid peroxidation induced by the contaminant iron in the preservation solution. Additional iron was released from rat liver after a period of cold storage/rewarming.

Topics: Adenosine; Allopurinol; Animals; Catalysis; Copper; Drug Stability; Free Radicals; Glutathione; Insulin; Ions; Iron; Lipid Peroxidation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Tissue Preservation

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Glutathione; Insulin; Intestinal Mucosa; Jejunum; L-Lactate Dehydrogenase; Organ Preservation; Organ Preservation Solutions; Phospholipases A; Phospholipases A2; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Time Factors

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Glutaminase; Glutamine; Glutathione; Insulin; Intestinal Mucosa; Intestine, Small; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Glutathione; Humans; Insulin; Intestinal Mucosa; Intestine, Small; Jejunum; Microvilli; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Donors

Topics: Adenine Nucleotides; Adenosine; Allopurinol; alpha-Glucosidases; Animals; Dogs; Glutathione; Hypertonic Solutions; Insulin; Intestinal Mucosa; Intestine, Small; Isotonic Solutions; Organ Preservation; Organ Preservation Solutions; Peroxidase; Raffinose; Reperfusion; Reperfusion Injury; Ringer's Lactate

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; Allopurinol; Biomarkers; Colon; Glutathione; Humans; Ileum; Insulin; Intestinal Mucosa; Membrane Potentials; Muscle, Smooth; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Sodium-Potassium-Exchanging ATPase; Theophylline; Transplantation, Homologous

Livers of male Wistar rats (250-300 g) were isolated and flushed with 10 ml of Ringer's solution and 10 ml of UW preservation solution. Then the organs were stored for 24 h at 4 degrees C in UW solution. Livers of Group 1 were rinsed with 10 ml of Ringer's solution and reperfused after hypothermic storage with oxygenated Krebs-Henseleit solution (95% O2; 5% CO2) in a nonrecirculating system at constant pressure (10 mmHg) and 37 degrees C. Livers of Group 2 were incubated for 30 min at 37 degrees C prior to reperfusion, in order to simulate rewarming of the organ upon surgical implantation. Livers of Group 3 were treated like Group 2, but taurine was admixed to the UW solution (1 mM). Livers of Group 1 showed little signs of a preservation/reperfusion injury, with low enzyme activities of the parenchymal ALT and endothelial purine nucleoside phosphorylase (PNP) in the postischemic rinse solution (ALT: 19.9 +/- 12.4; PNP: 3.3 +/- 0.4 U/liter), adequate portal flow values about 3 ml/g/min and high O2 uptake at the end of the experiment (VO2: 3.2 +/- 0.4 ml/100g/min). Livers of Group 2 exhibited nearly tenfold higher enzyme activities in the rinse solution (ALT: 247.0 +/- 94.7*; PNP: 29.5 +/- 17.0* U/l) and disturbed tissue perfusion with significantly reduced flow values of about 2 ml/g/min during the first 10 min of reperfusion. As a result, the recovery of O2 uptake was only 2.2 +/- 0.3 ml/100 g/min*. Addition of taurine (Group 3) resulted in a significant reduction of the enzyme loss (ALT: 96.2 +/- 50.0#; PNP:12.4 +/- 7.0# U/liter) and improved portal flow values and O2 uptake at the end of reperfusion (2.7 +/- 0.3 ml/100 g/min#). The results give evidence for the importance of the rewarming period after hypothermic storage, which is inevitable during implantation of the organ in vivo. Taurine seems to exert a protective effect, affecting both the vascular endothelium and parenchymal tissue (*p < 0.05 vs Group 1; # p < 0.05 vs Group 2).

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Biomarkers; Cold Temperature; Glutamate Dehydrogenase; Glutathione; In Vitro Techniques; Insulin; Liver; Male; Organ Preservation; Organ Preservation Solutions; Purine-Nucleoside Phosphorylase; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Taurine

Hepatic vascular exclusion allows the performance of major hepatic resections with minimal intraoperative blood loss. We have previously shown that normothermic ischemia can be tolerated by a healthy liver for up to 90 minutes, and this period is increased to 4 hours if the liver is cooled to 4 degrees C using University of Wisconsin solution.. This study assessed whether these techniques could be successfully applied for patients requiring resection of a diseased liver, which is more sensitive to ischemic damage. Between July 1990 and May 1994, 12 patients (6 men, 6 women; mean age, 57.8 years) in whom the planned hepatic resection was believed to require hepatic vascular exclusion for more than 1 hour were treated with perfusion with the University of Wisconsin solution. The surgical procedures were right hepatectomy (one patient), extended right hepatectomy (seven patients), and extended left hepatectomy (four patients). The underlying hepatic disease was cirrhosis or severe fibrosis with hepatocellular carcinoma (four patients), cholestasis (due to cholangiocarcinoma and biliary stricture, one patient each), and more than 30 percent steatosis after treatment of hepatic metastases with chemotherapy (six patients). The University of Wisconsin solution that had been cooled to 4 degrees C was perfused through a cannula placed in the portal vein or the hepatic arterial branch of the segment to be resected, but with flow directed toward the liver that should be retained and effluent fluid drained through a cavotomy. Before reperfusion, the liver was rinsed with Ringer's lactate solution, which was also 4 degrees C.. The mean duration of hepatic ischemia was 121 minutes (range, 65 to 250 minutes), and venovenous bypass was used in three cases. The mean amount of blood transfused intraoperatively was 4.3 +/- 4 U; four cases required no transfusion. One patient died on postoperative day seven of portal vein thrombosis. The median hospital stay was 21 days (range, 12 to 56 days). Postoperative complications consisted of pneumonia (one patient), liver insufficiency (one patient, who recovered spontaneously), and subphrenic abscess (one patient). The postoperative tests of hepatic function were altered to the same degree as that seen after hepatic vascular exclusion of less than 1-hour duration in healthy livers. All patients who left the hospital were alive at 1 year.. Cooling of the hepatic parenchyma allowed us to perform major hepatic resection in patients with diseased livers using hepatic vascular exclusion for longer than 1 hour without increased morbidity or mortality. However, because of particular difficulties due to the size or location of the lesions, the application of these new techniques should only be considered for the largest and most complex hepatic resections for which hepatic vascular exclusions longer than 1 hour are foreseen.

Topics: Adenosine; Adult; Aged; Allopurinol; Blood Loss, Surgical; Cryopreservation; Female; Follow-Up Studies; Glutathione; Hepatectomy; Hepatic Artery; Humans; Hypothermia, Induced; Insulin; Liver; Liver Circulation; Liver Diseases; Male; Middle Aged; Organ Preservation Solutions; Portal Vein; Raffinose; Reperfusion Injury; Tissue Preservation

Preservation with University of Wisconsin (UW) solution can maintain liver graft function and produces survival rates of recipients higher than that with Euro Collins (EC) solution. To explore the underlying mechanisms, we transplanted rat livers following cold preservation with EC or UW solution for 18 hr, and measured hepatic adenine nucleotide levels, the percentage of water content, lactate levels, and endogenous antioxidant levels (alpha-tocopherol [alpha-Toc], reduced coenzyme Q9 [CoQ9H2], reduced coenzyme Q10, [CoQ1OH2] and reduced glutathione [GSH] during preservation and after transplantation. The adenosine triphosphate levels of the liver grafts preserved with UW solution recovered after reperfusion more rapidly and reached a higher level than those preserved with EC solution. UW solution caused a reduction in hepatic water content during preservation. Conversely, EC solution induced remarkable tissue edema. In addition, UW solution reduced the rate of hepatic lactate production both during preservation and after reperfusion. The concentrations of hepatic GSH, alpha-Toc, CoQ9H2, and CoQ1OH2 immediately after the graftectomy, and after the 18 hr of preservation with both EC and UW solutions, did not differ from those in the normal liver, and decreased only after transplantation. However, UW solution suppressed significantly the reduction in hepatic GSH, alpha-Toc, and CoQ9H2 after reperfusion, compared with EC solution. These results suggest that long-term cold storage induces tissue edema, reflecting a disturbance of the microcirculation during preservation, followed by parenchymal cell damage mediated by free radicals after reperfusion. The protective effects of UW solution could be attributable to the inhibition of free radical production after reperfusion.

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Antioxidants; Cold Temperature; Glutathione; Insulin; Lactates; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Survival Rate; Tissue Preservation; Ubiquinone; Vitamin E; Water

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

We examined the cytology of the exudate in preserved intestinal grafts on reperfusion and compared it with the histological findings in rat small intestinal transplantation. The jejunal graft was harvested from the Lewis rat and was preserved in University of Wisconsin solution for 6, 12, 24 and 48 h at 4 degrees C (n = 6, in each group) and was then syngeneically transplanted. On reperfusion, the exudate was collected and studied cytologically. Full thickness biopsies were performed at the end of the preservation and at 30 min after reperfusion for histological examination. Histological examination after reperfusion showed that the crypt layer was preserved until 24 h. However, it was destroyed by 48 h preservation. The cytological findings correlated with the depth of tissue injury shown histologically. The degeneration of villus epithelial cells, the decrease in the content of mucin in both the goblet cells as well as villus cells, and the appearance of crypt cells are all considered to be signs of poor graft viability. Cytological examination is therefore recommended as an effective, non-invasive and real-time method for evaluating graft viability just after reperfusion in small intestinal transplantation.

Topics: Adenosine; Allopurinol; Animals; Cell Death; Glutathione; Graft Survival; Insulin; Intestine, Small; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Tissue Preservation

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

Free radicals are important mediators of reperfusion injury; however, the mechanism(s) of oxyradical production after liver reimplantation are not well understood. A model of cold storage and reperfusion using low-level chemiluminescence to directly measure oxyradical production during reperfusion was developed.. Rat livers were harvested and stored at 4 degrees C in University of Wisconsin cold-storage solution or Euro-Collins solution for 0-48 hours and then flushed and reperfused with warm oxygenated (37 degrees C) Krebs-Henseleit buffer. Liver chemiluminescence was measured using a sensitive photomultiplier tube, and hepatocellular injury was assessed by measuring aspartate aminotransferase release into the perfusate.. Chemiluminescence reached a maximum within 5 minutes of reperfusion and then decreased to a baseline within 30 minutes. There was a marked increase in chemiluminescence after only a short period of storage in University of Wisconsin cold-storage solution. Chemiluminescence decreased with longer periods of storage but steadily increased again after 16 hours of storage. Chemiluminescence after 22 hours of storage, but not after 3 hours of storage, was decreased by pretreatment with the Kupffer cell inactivator gadolinium chloride.. The data suggest two mechanisms of oxyradical production during cold storage and reperfusion of the rat liver. The later phase seems to be Kupffer cell dependent.

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Cold Temperature; Free Radicals; Glutathione; Hypertonic Solutions; Insulin; Kupffer Cells; Least-Squares Analysis; Liver; Luminescent Measurements; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury

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

To investigate the role of platelet-activating factor (PAF) in the preservation/reperfusion injury of the liver graft, the effect of treatment with a potent PAF antagonist (E5880) was evaluated in a pig orthotopic liver transplantation model. The graft liver was flushed out and preserved for 8 hr at 4 degrees C using a simplified University of Wisconsin solution. The PAF antagonist was administered into the University of Wisconsin solution (1 mg/L), into the rinsing solution (1 mg/L), and to a recipient pig (0.3 mg/kg d.i.v.) in group 1. The PAF antagonist was not given in the control group (group 2). Postoperative survival of more than 12 hr was 100% (9/9) in group 1 and 56% (5/9) in group 2 (P < 0.05). At 12 hr after reperfusion of the graft (RPF), the arterial ketone body ratio (acetoacetate to 3-hydroxybutyrate) increased to 1.54 +/- 0.15 (mean +/- SEM) in group 1, compared with 0.95 +/- 0.09 (P < 0.05) in group 2. In group 2, blood leukocyte count decreased to 8.3 +/- 0.9 (x 10(3)/microliters) at 2 hr after RPF, in contrast to a slight increase in group 1 (14.3 +/- 1.8 x 10(3)/microliter, P < 0.01). At 4 hr after RPF, glutamic oxaloacetic transaminase (461 +/- 59 vs. 712 +/- 97 U/L, P < 0.05), glutamic pyruvic transaminase (65 +/- 4 vs. 82 +/- 5 U/L, P < 0.05), and the lactate level (6.2 +/- 1.1 vs. 9.4 +/- 1.0 mmol/L, P < 0.05) in arterial blood were significantly lower in group 1 than in group 2. Light and electron microscopic study at 1 hr after RPF showed neutrophil sludging in the sinusoids and sinusoidal endothelial cell damage in group 2, while these findings were attenuated in group 1. It is suggested that PAF plays a key role in microcirculatory disturbance of the liver graft manifested on reperfusion, and that the treatment with E5880 has a protective effect against preservation/reperfusion injury of the graft in liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Rejection; Insulin; Leukocyte Count; Liver; Liver Transplantation; Microscopy, Electron; Mitochondria, Liver; Organ Preservation Solutions; Piperidines; Platelet Activating Factor; Pyridinium Compounds; Raffinose; Reperfusion Injury; Swine; Tissue Preservation; Transaminases

The role of eicosanoid metabolism and its relationship with nitric oxide production in the ischemia-reperfusion associated with pancreas transplantation in the rat is explored in this study. Twenty-six male Sprague-Dawley rats were randomized into 3 groups, as follows: group 1, control animals not surgically manipulated; group 2, pancreas transplantation, after 12 hr of organ preservation in University of Wisconsin solution; group 3, same as group 2 but with administration of NG-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor) (10 mg/kg) before organ revascularization. The results show posttransplantation increases in edema and in 6-keto-prostaglandin F1 alpha (x1.9), thromboxane B2 (x4), and prostaglandin E2 (x5) levels in pancreatic tissue. Nitric oxide synthase inhibition reversed the increases in edema and eicosanoid production, which suggests that eicosanoid generation in the recipient rat would be mediated, in part, through a nitric oxide-dependent mechanism.

Topics: Adenosine; Allopurinol; Animals; Arginine; Dinoprostone; Edema; Glutathione; Insulin; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Organ Preservation; Organ Preservation Solutions; Pancreas Transplantation; Prostaglandins F; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thromboxane B2

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Glutathione; Insulin; Kupffer Cells; Liver; Liver Circulation; Liver Transplantation; Microcirculation; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Tissue Preservation; Transplantation, Isogeneic

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Glucose; Glutathione; Glycine; Graft Survival; Insulin; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Reperfusion Injury; Solutions; Time Factors; Transplantation, Homologous; Tromethamine

Topics: Adenosine; Allopurinol; Animals; Benzamidines; Glutathione; Graft Survival; Guanidines; Heart; Heart Transplantation; Insulin; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions; Transplantation, Isogeneic

Using isolated rat lungs, we compared prevention of ischemia-reperfusion injury provided by flushing the lungs with modified Euro-Collins solution (EC), University of Wisconsin solution (UW), low-potassium-dextran solution (LPD), or Wallwork solution (WA). After 4 hours' and 6 hours' cold ischemia, reperfusion injury was assessed on the basis of changes in filtration coefficients (Kfc) and pressure-flow curves, characterized by the slope of the curves (incremental resistance) and the extrapolation of this slope to zero flow (pulmonary pressure intercept [Ppi]). After 4 hours, Kfc and Ppi were higher with EC than with UW, LPD, and WA, and the incremental resistance was higher with EC and UW. After 6 hours, Kfc and incremental resistance Ppi were higher with LPD than with WA. Because ischemia-reperfusion injury is associated with decreased endothelial synthesis of prostacyclin and nitric oxide, we tested whether the addition of prostacyclin or the nitric oxide precursor L-arginine to WA would improve preservation. The Kfc and Ppi were lower with both treatments. In conclusion, ischemia-reperfusion injury was best prevented by using WA. The favorable effect of prostacyclin or L-arginine emphasizes the role played by endothelial dysfunction in ischemia-reperfusion injury.

Topics: Adenosine; Albumins; Allopurinol; Animals; Blood Pressure; Cardioplegic Solutions; Chlorides; Epoprostenol; Glutathione; Hypertonic Solutions; Hypothermia, Induced; In Vitro Techniques; Insulin; Lung; Lung Compliance; Male; Mannitol; Organ Preservation; Organ Preservation Solutions; Organ Size; Potassium Compounds; Propionates; Pulmonary Artery; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Vascular Resistance

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; Adenosine Diphosphate; Adenosine Triphosphate; Allopurinol; Animals; Biomarkers; Cats; Cold Temperature; Energy Metabolism; Enzymes; Female; Glutathione; Insulin; Intestinal Mucosa; Intestine, Small; Male; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Preservation; Transplantation, Autologous

Glycine has been shown to decrease membrane injury in isolated cells due to hypoxia or cold ischemia. The mechanisms of action of glycine are not known, but glycine may be useful in organ preservation solutions or in treating recipients of liver transplantation. In this study the isolated, perfused rabbit liver was used to measure how glycine affected liver performance after 48-h preservation in University of Wisconsin (UW) solution without added glutathione. UW solution is less effective for 48-h liver preservation when glutathione is omitted. Rabbit livers stored for 48 h without glutathione show a large increase in enzyme release (LDH and AST) from the liver and a reduction in bile production. The addition of 15 mM glycine to UW solution, in place of glutathione, did not improve bile production or reduce enzyme release. However, infusion of 10 mM glycine into the reperfused liver lowered LDH release significantly (from 2383 +/- 562 units/100 g to 1426 +/- 286 units/100 g) during the initial reperfusion of the 48-h preserved liver. Hepatamine, a parenteral nutrition solution containing glycine, as well as other amino acids, was also effective in lowering LDH release from the preserved liver. Although glycine reduced LDH release, it did not decrease the amount of AST released from the liver, nor did it improve bile production. Thus, we conclude that glycine, either in UW solution or given to the liver upon reperfusion, has no significantly beneficial effect as tested in this model. Further testing of glycine, however, should be conducted in an orthotopic transplant model in the rat or dog.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Glycine; Insulin; Liver; Liver Function Tests; Organ Preservation; Organ Preservation Solutions; Perfusion; Rabbits; Raffinose; Reperfusion Injury

Liver endothelial cells appear to be particularly vulnerable to cold ischemia reperfusion. However, their function has not yet been evaluated, except using electron microscopic changes and trypan blue exclusion (an index of cell death). Hyaluronic acid is a polysaccharide highly extracted by normal liver endothelial cells. We thus evaluated liver endothelial cell function by measuring hyaluronic acid elimination in a model of ischemia-reperfusion injury using isolated perfused Wistar rat livers. We compared the effects of two preservation solutions during cold ischemia (4 degrees C): normal saline with 2 mM CaCl2 (4 h and 8 h ischemia) and the University of Wisconsin solution (8 h and 24 h ischemia). Eliminations were measured during two 40-min periods before and after ischemia; during each period, hyaluronic acid (150 ng/ml) and also, to evaluate hepatocyte function, propranolol (100 ng/ml) were infused into the reservoir. We show that, whatever the preservation solution or time used, liver endothelial cell function is altered to a larger extent than hepatocyte function. University of Wisconsin solution does not appear to protect liver endothelial cells during preservation, particularly after 24 h of cold ischemia. Hyaluronic acid elimination can be a useful tool in the investigation of an ideal preservation solution to protect liver endothelial cells from ischemia-reperfusion damage.

Topics: Adenosine; Allopurinol; Animals; Endothelium; Glutathione; Hyaluronic Acid; In Vitro Techniques; Insulin; Liver; Liver Circulation; Male; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sodium Chloride; Tissue Preservation; Trypan Blue

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

The purpose of this study was to clarify the role of adenosine in preservation of ischemically damaged pancreas by the two-layer (Euro-Collins solution [EC]/perfluorochemical [PFC]) method using a canine model. Twenty-four-hour preservation of the pancreas graft subjected to 60-min warm ischemia was successful by the two-layer (EC with adenosine/PFC) method (4/5, 80%), but neither simple cold storage in EC (0/5, 0%), nor EC with adenosine (1/5, 20%), nor the two-layer (EC/PFC) method (0/3, 0%) was successful. Tissue ATP concentrations at the end of preservation by the two-layer (EC with adenosine/PFC) method were significantly higher compared with the two-layer (EC/PFC) method (7.23 +/- 2.17 vs. 1.56 +/- 0.40 mumol/g dry weight, P < 0.01). Studies with [2-3H]adenosine demonstrated that only part of adenosine was converted to inosine, hypoxanthine, and adenine, whereas the remainder was incorporated into adenine nucleotides in the pancreas graft. In addition, hypoxanthine, inosine, and adenine did not substitute for adenosine. We conclude that provision of adenosine to ischemically damaged pancreas during preservation by the two-layer (EC/PFC) method allows ATP synthesis within the graft via direct phosphorylation of adenosine. Metabolic processes vital to repair damaged cells and maintain cellular integrity can be maintained, which makes it possible to preserve ischemically damaged pancreas.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Dogs; Female; Fluorocarbons; Glutathione; Graft Survival; Hot Temperature; Hypertonic Solutions; Insulin; Male; Nucleosides; Nucleotides; Organ Preservation; Organ Preservation Solutions; Pancreas; Raffinose; Reperfusion Injury

Biopsies taken from 13 human liver grafts at different stages of the transplantation process were used for study of the morphology of sinusoidal cells prior to harvesting (5 biopsies), after preservation in UW solution (10 biopsies), and after complete revascularization (13 biopsies). The mean cold ischemic period was 12 h 30. Immediate follow up was uneventful and the mean peak of post-operative transaminases below 1300 IU/l. Biopsies were perfusion-fixed by the transparenchymal route to ensure satisfactory ultrastructural results. There were no loose sinusoidal endothelial cells in the lumen and no signs of cellular death. Some endothelial cells presented signs of activation at the end of the preservation period, and even more after revascularization, with numerous lucent vacuoles resembling endosomes in the cytoplasm. Kupffer cells also presented signs of activation, particularly after reperfusion. The retraction of endothelial cell processes which formed large gaps during cold ischemia proved to be partly reversible after reperfusion. Signs of endothelial cell damage with gaps and partial rupture of the plasmic membrane were also observed, particularly after revascularization, in areas which contained numerous inflammatory cells adhering to the wall. The Disse space was not generally enlarged and contained no inflammatory cells. The sinusoidal pole of hepatocytes was occasionally damaged with the formation of blebs. These results strongly suggest that any drug or preservation solution that will inhibit endothelial and Kupffer cell activation could be beneficial in the prevention of preservation and reperfusion injury.

Topics: Adenosine; Adult; Allopurinol; Biopsy; Endothelium; Female; Glutathione; Humans; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Microscopy, Electron; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

Recently, it has been demonstrated that the use of both cold Carolina rinse (CR, 4 degrees C) as well as warm Ringer's lactate (RL, 37 degrees C) attenuates microvascular perfusion failure and leukocyte (WBC) accumulation in liver grafts. The aim of this study was to analyse in vivo whether warming of CR can also lead to a reduction in microvascular reperfusion injury in rat liver transplantation. Syngeneic orthotopic liver transplantation, including arterial reconstruction, was performed in male Lewis rats (180-300 g). Livers were stored in University of Wisconsin (UW) solution for 24 h and rinsed with 15 ml CR which was either cold 4 degrees C (n = 7) or warm 37 degrees C (n = 8) prior to reperfusion. Hepatic microcirculation and WBC accumulation were assessed by intravital fluorescence microscopy, and graft function was determined by analysis of bile flow during the 90-min reperfusion period. Warm CR yielded significantly (P < 0.01) improved sinusoidal perfusion when compared with cold CR; however, the extent of WBC adherence in both sinusoids and postsinusoidal venules did not vary between the groups. In addition, bile flow was slightly increased after warm CR. We conclude that after 24 h of cold storage in UW solution, warming of CR may offer additional benefit in the prevention of microcirculatory reperfusion injury without affecting WBC accumulation.

Topics: Adenosine; Allopurinol; Animals; Bile; Glutathione; Hemodynamics; Insulin; Isotonic Solutions; Liver; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Ringer's Lactate; Solutions; Transplantation, Isogeneic

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

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Enzyme Inhibitors; Fatty Liver; Female; Glutathione; Graft Survival; Insulin; Liver Transplantation; Organ Preservation Solutions; Protein Kinase C; Raffinose; Rats; Reperfusion Injury; Sphingosine; Transplantation, Isogeneic

This study investigated whether prostaglandin E1 (PGE1) could reduce hepatic injury to the liver graft caused by harvesting and 24-h preservation in University of Wisconsin (UW) solution in a canine model. The PGE1-treated group was intravenously administered 0.5 microgram/kg per minute of PGE1 for 30 min before harvesting, as well as a concentration of 1 mg/l PGE1 in the washout and UW solutions. In both the PGE1-treated and the control group, all recipients survived for 1 week or more after transplantation. Arterial ketone body ratio (AKBR) remained over 1.0 in the early postoperative period. The PGE1 group showed significant reductions in guanase, GOT, and LDH during the early postoperative period compared to the untreated control group. Histological examination disclosed partial mitochondrial swelling, hepatocyte vacuolation, and necrosis in the control group, while such abnormalities were rarely seen in the PGE1 group. These results suggest that PGE1 can effectively reduce hepatic injury to liver grafts preserved in UW solution prior to transplantation.

Topics: Adenosine; Allopurinol; Alprostadil; Animals; Aspartate Aminotransferases; Dogs; Glutathione; Guanine Deaminase; Insulin; Ketone Bodies; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Time Factors

The effect of adding a 21-aminosteroid, U74500A, and a Ca2+ antagonist, lidoflazine, alone and together to UW solution was assessed in a rat liver preservation model. Following preservation, the livers were reperfused using a closed circuit, and the release of hepatocellular enzymes (ASAT, ALAT, and LDH) into the perfusate was determined with increasing time. Both drugs reduced the amount of enzymes lost from the liver. The combination of the two drugs was better than either drug alone. These data suggest that both agents may be of value in organ preservation for clinical liver transplantation.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Glutathione; Insulin; L-Lactate Dehydrogenase; Lidoflazine; Lipid Peroxides; Liver; Male; Organ Preservation; Organ Preservation Solutions; Pregnatrienes; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

This quantitative in vivo fluorescence microscopy study investigated the relative impact of an optimized rinse solution (warm Carolina rinse) and that of an established storage solution (University of Wisconsin solution) on various pathomechanisms of hepatic reperfusion injury after cold storage. Syngeneic orthotopic, arterialized liver transplantation was performed in male Lewis rats after 24 hr of cold ischemia (n = 24). The four experimental groups differed according to the type of preservation/rinse solution used: University of Wisconsin solution/albumin rinse (group 1), autologous blood (just external cooling)/albumin rinse (group 2), blood/Carolina rinse (group 3) and University of Wisconsin solution/Carolina rinse (group 4). Hepatic microvascular perfusion, leukocyte accumulation and phagocytic activity of Kupffer cells were assessed by means of intravital fluorescence microscopy 30 to 90 min after reperfusion. Disturbances of microvascular perfusion were most pronounced in group 2, markedly reduced by University of Wisconsin solution (group 1) and Carolina rinse (group 3) and minimized by combined use of University of Wisconsin solution and Carolina rinse in group 4. Intrahepatic leukocyte-endothelium interaction in sinusoids and postsinusoidal venules was found to depend on the application of Carolina rinse before reperfusion rather than the use of University of Wisconsin solution during cold storage. Activation of phagocytosis by Kupffer cells was most pronounced in group 1, intermediate in groups 2 and 3 and not noticeable in group 4. Hepatocellular excretory function as assessed on the basis of total bile flow and excretion of bile acids during the first 90 min after reperfusion was found to be improved by application of Carolina rinse, both after storage in blood or in University of Wisconsin solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Allopurinol; Animals; Bile; Bile Acids and Salts; Cell Adhesion; Cryopreservation; Glutathione; Insulin; Kupffer Cells; Leukocytes; Liver; Liver Circulation; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions

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

Lactobionic acid, [4-beta-(galactosido)-D-gluconic acid] = LBA, is the major component of the Wisconsin organ transplantation preservant fluid and may suppress oxygen radical-induced tissue damage upon reperfusion by the control of FeII autoxidation. FeII and FeIII complexes of LBA and the related gluconic acid (GLC) have been studied herein by titrimetric, infrared, and electrochemical methods (CV; DPP). FeII(GLC) forms quickly at pH 7, but FeII(LBA) reacts in two steps, the second requiring 4 hr. The initial complex lacks coordination of the LBA carboxylate (C-1) and is bound by the "2,3,5" hydroxyl groups. The slow rearrangement forms a "1,2,3,6" chelate which FeII(LBA) shares in common with the donor set of the FeIII(LBA) complex. Titration data shows the removal of three protons from LBA through pH 5 and an additional proton from pH 6 to 9 which is indicative of the [FeIII(LBA)(OH)(H2O)]- formulation with LBA donating at the "1,2,3,6" positions. The more stable, second form of FeII(LBA) has been investigated in its oxidation mechanisms with H2O2 and O2 using selected trapping agents for HO. and ferryl intermediates. Eighty-six percent of the oxidation events of FeII(LBA)/H2O2 occurs in steps involving formation and reduction of freely diffusible HO.. These pathways are altered by the known HO. traps t-butanol, dmso, ethanol, and methanol in the manner predictable for beta-oxidizing radicals (from t-butanol or dmso) and alpha-reducing radicals (from ethanol and methanol). Fourteen percent of the FeII(LBA)/H2O2 reaction occurs via FeIVO intermediates not trapped by t-butanol or dmso, but intercepted by primary and secondary alcohols. The HO. generating pathways are responsible for a competitive LBA ligand oxidation at the C-2 position via HO., formed from FeII(LBA) and H2O2 within the original reaction cage. Competitive ligand oxidation at C-2 is absent for the FeII(LBA)/O2 autoxidation, indicative of a different redox mechanism. The FeII(LBA)/O2 reaction rate is first-order in each component and is insensitive to the presence of t-butanol as an HO. trap. These observations support a ferryl intermediate in the autoxidation pathway and the absence of HO. or free H2O2 during autoxidation. Although chelation of FeII by hard ligand donors such as edta4-, Cl-, or HPO4(2-) accelerate the rate of autoxidation of FeII, chelation of carboxylate, alkoxy, and hydroxyl donors of LBA does not accelerate autoxidation. The implications of these findings, and the abse

Topics: Adenosine; Allopurinol; Disaccharides; Electrochemistry; Free Radical Scavengers; Gluconates; Glutathione; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Insulin; Iron; Organ Preservation; Organ Preservation Solutions; Oxidation-Reduction; Raffinose; Reactive Oxygen Species; Reperfusion Injury; Solutions; Spectrophotometry, Infrared

Topics: Adenosine; Allopurinol; Animals; Dogs; Glutathione; Insulin; Lipid Peroxides; Lung; Lung Compliance; Organ Preservation; Organ Preservation Solutions; Pulmonary Circulation; Pulmonary Edema; Raffinose; Reperfusion Injury; Solutions; Time Factors; Vascular Resistance

Topics: Adenosine; Allopurinol; Animals; Free Radicals; Glucose; Glutathione; Graft Survival; Hypertonic Solutions; Insulin; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions; Superoxide Dismutase; Transplantation, Isogeneic

The critical injury to liver during cold preservation is believed to occur to the sinusoidal endothelium. In this study the viability of cultured sinusoidal endothelial cells from rat liver was assessed during storage in University of Wisconsin solution at 4 degrees C. The vast majority of cells (83 +/- 12%) died within 24 hr of storage. Addition of KCN (1 mM) to the solution to simulate hypoxia markedly increased survival: only 3 +/- 2% of cells had lost viability after 24 hr in the presence of cyanide. Further experiments showed that other inhibitors of mitochondrial ATP formation (antimycin A 1 microM, rotenone 1 microM, oligomycin 10 microM, carbonyl cyanide m-chlorophenylhydrazone 1 microM) were protective as well, whereas glucose (10 mM) greatly diminished the protective effect of cyanide (loss of viability 38 +/- 7% after 24 hr). ATP measurements confirmed the correlation between the energy state of the cells and cell death: ATP levels after 6 hr of incubation were 19.9 +/- 4.0 nmol/10(6) cells in UW solution, 13.7 +/- 2.9 nmol/10(6) cells in UW + glucose, 6.9 +/- 1.9 nmol/10(6) cells in UW + KCN + glucose and 1.9 +/- 1.5 nmol/10(6) cells in UW + KCN. In contrast to the protective effect observed in UW solution, addition of KCN to Krebs-Henseleit buffer led to increased endothelial cell damage upon cold storage. We therefore conclude that in UW solution damage to the sinusoidal endothelium is energy-dependent.

Topics: Adenosine; Adenosine Triphosphate; Aerobiosis; Allopurinol; Animals; Antimycin A; Cell Survival; Cryopreservation; Cyanides; Endothelium; Glucose; Glutathione; Hypoxia; Insulin; Liver; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Rotenone; Solutions; Time Factors; Tissue Preservation

Flushing hepatic grafts immediately before revascularization with a specially designed rinse solution such as "Carolina rinse" has been reported to improve survival after liver transplantation in the rat. This study investigated the influence of Carolina rinse and adenosine rinse on early graft function, microcirculation, and leukocyte (WBC)-endothelial cell interaction of arterialized syngeneic orthotopic liver transplants in Lewis rats. Livers were preserved for 24 hr in University of Wisconsin solution and flushed immediately before reperfusion with either Ringer's lactate (group A: n = 7), Ringer's lactate + 0.2 mmol/liter adenosine (group B: n = 6), or Carolina rinse (group C: n = 7). Microvascular perfusion and WBC accumulation were assessed by intravital fluorescence microscopy. In group C, acinar perfusion was significantly improved, accompanied by a lower percentage of nonperfused sinusoids 1 hr after reperfusion (mean +/- SEM: 26 +/- 2% [group A], 21 +/- 2% [B], 11 +/- 1% [C], P < 0.01 for C vs. A or B). In addition, Carolina rinse and, to a lesser extent, adenosine rinse reduced the number of WBC sticking in sinusoids and postsinusoidal venules. Better graft function in group C was indicated by increased bile flow during the observation period of 90 min after reperfusion (0.5 +/- 0.3 ml/100 g liver [group A], 1.5 +/- 0.7 [B], 3.7 +/- 0.6 [C], P < 0.01 for C vs. A or B). We conclude that Carolina rinse is able to improve early excretory hepatocellular function, microvascular perfusion, and intrahepatic WBC accumulation after prolonged cold ischemia and reperfusion, but adenosine is unlikely to be the key component of this rinse solution.

Topics: Adenosine; Allopurinol; Animals; Bile; Cell Adhesion; Cell Communication; Endothelium, Vascular; Glutathione; Hemodynamics; Insulin; Leukocytes; Liver; Liver Transplantation; Male; Microcirculation; Microscopy, Fluorescence; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions

Pharmacologic manipulation of free flaps to enhance tolerance to ischemia has become a subject of great interest in the research literature. In an effort to improve survival, perfusion washout of experimental free flaps was performed following an episode of primary ischemia. The perfusates utilized were lactated Ringer's solution (LR), University of Wisconsin solution (UW), a high-molecular-weight medium used in organ preservation, and urokinase, a thrombolytic agent. Seventy-five rats were used in this study and divided into groups of 5 each. A 3 x 6-cm abdominal free flap based on the superficial inferior epigastric vessels was raised in each rat. The free flaps were subjected to either 12 or 18 hr of primary ischemia. Following the period of ischemia, perfusion washout was performed with either LR, UW solution, or urokinase at increasing concentrations alone or in combination with UW solution. Urokinase was first evaluated as a perfusate alone at increasing concentrations. In the 12-hr ischemia group, free-flap survival was shown to increase from 0 percent in the LR-perfused flaps to 20 percent, 60 percent, and 80 percent in flaps perfused with 12,500, 25,000, and 100,000 U of urokinase, respectively (p < 0.05). A similar increase in survival was demonstrated in the 18-hr ischemia group, where 0 percent, 20 percent, and 40 percent of flaps survived following perfusion with 12,500, 25,000, and 100,000 U of urokinase, respectively (p < 0.05). Urokinase was then perfused along with UW solution to evaluate the combined effect on flap survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Allopurinol; Animals; Drug Combinations; Glutathione; Graft Survival; Insulin; Isotonic Solutions; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ringer's Solution; Solutions; Surgical Flaps; Time Factors; Tissue Preservation; Urokinase-Type Plasminogen Activator

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

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Survival; Insulin; Liver; Liver Transplantation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion; Reperfusion Injury; 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

The purpose of this study was to investigate the possibility of improving the organ preservation properties of the University of Wisconsin (UW) solution by adding the calcium entry blocker lidoflazine. We also investigated the possibility of decreasing the cold ischemia and reperfusion damage by pretreatment with lidoflazine of the donor and/or recipient. The protective effects of lidoflazine treatment were estimated by measuring the amount of trapped erythrocytes in the rat renal medulla after 48 h of cold storage, subsequent transplantation, and 20 min of reperfusion. Lidoflazine (20 mg/liter) added to the UW solution decreased the amount of erythrocyte trapping from 14.8 +/- 3.1% in controls to 8.6 +/- 1.7% (P less than 0.01). The flow rate of the flush-out solution during the harvesting procedure was also significantly (P less than 0.01) increased when lidoflazine was included in the UW solution (1.10 +/- 0.21 ml/min vs 0.75 +/- 0.22 ml/min). Administration of lidoflazine (0.28 mg/kg body wt) to the donor and/or the recipient did not further reduce the postischemia/reperfusion damage as estimated by the degree of erythrocyte trapping. In conclusion, the results indicate that the preservation properties of the UW solution can be significantly improved by adding lidoflazine to the solution.

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

Optimal techniques for lung preservation are yet to be defined. Platelet activating factor is a phospholipid released by a variety of cells and produces pulmonary abnormalities similar to posttransplantation pulmonary dysfunction. We investigated the strength of the effect of the platelet activating factor antagonist BN 52021 as compared with that of deferoxamine, an iron chelator previously shown to improve lung preservation. Differential lung function and pulmonary hemodynamics were used to assess preservation after a 6-hour period of cold ischemic storage in a modified canine model of left lung allotransplantation. Thirty size- and weight-matched mongrel male dogs were used for 15 transplant procedures randomized to one of three preservation techniques. The University of Wisconsin solution was used as the basic flush solution for all experimental animals. BN 52021 was added to the flush solution in one group (10 mg/kg, n = 5) and deferoxime in another group (10 mg/kg, n = 5). No additives were used for the control animals (n = 5). BN 52021 and deferoxime were administered to respective donor animals 30 minutes before organ harvesting (10 mg/kg) and to recipient animals 30 minutes before reperfusion (10 mg/kg). The pulmonary artery flush solution was administered (40 ml/kg) over 4 minutes. Recipient animals received double-lumen endotracheal tubes and were monitored with balloon-tipped, flow-directed catheters in both pulmonary arteries and dual-angle ultrasonic flow probes around each pulmonary artery. Solid-state high-fidelity micromanometers were used to measure pressures in the pulmonary artery, the left atrium, and the left ventricle. Systemic arterial, right and left pulmonary venous, and mixed venous blood samples were analyzed at 1, 2, 4, and 6 hours after transplantation. Pulmonary venous oxygen tension of the transplanted lung for the control group was 202 +/- 81 mm Hg versus 282 +/- 53 mm Hg for the BN 52021 group 6 hours after transplantation (p less than 0.05). Pulmonary vascular resistance of the transplanted lung for the control group was 319 +/- 54 dynes.sec.cm-5 versus 149 +/- 71 dynes.sec.cm-5 for the BN 52021 group (p less than 0.05). Proton magnetic resonance spectroscopy was performed on segments of upper and lower lobes of the native and transplanted lung from recipient animals to determine total lung water content. The BN 52021 group had a total lung water content of 57.3% as compared with 51.9% for the deferoxime group (p = not s

Topics: Adenosine; Allopurinol; Animals; Deferoxamine; Diterpenes; Dogs; Ginkgolides; Glutathione; Insulin; Lactones; Lung; Lung Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Platelet Activating Factor; Raffinose; Reperfusion Injury; Solutions; Tissue Preservation

Recently, we described a new solution, Carolina rinse, that prevents nonparenchymal cell injury in vitro after reperfusion of livers stored in University of Wisconsin cold solution (Currin RT, Toole JG, Thurman RG, Lemasters JJ. Transplantation 1990; 50: 1076). The present study was designed to examine the effect of Carolina rinse on graft survival in vivo. Unlike UW cold storage solution, which is high in potassium, Carolina rinse contains extracellular inorganic ions at levels similar to blood, a calcium channel blocker and a radical scavenger. Carolina rinse also contains fructose and mildly acidotic pH to reduce hypoxic cell death. Livers from Lewis rats were explanted, stored in UW cold storage solution under nonsurvival conditions, and rinsed with either 15 ml of Ringer's, UW solution, Carolina rinse, or Carolina rinse saturated with nitrogen prior to completion of implantation surgery. In the Ringer's rinse group, only 4% of recipients survived 30 days postoperatively. In this group, SGOT levels reached maximal values of about 5000 U/L. Survival was also poor (25%) when grafts were rinsed with UW solution. In the Carolina rinse group, however, 9 of 16 rats (56%) survived indefinitely, and maximal postoperative SGOT levels were reduced 3-fold. Liver injury indexed histologically was also decreased about 3-fold by Carolina rinse compared with the control group rinsed with Ringer's solution. Carolina rinse diminished postoperative sinusoidal endothelial cell damage assessed by electron microscopy and reduced carbon particle phagocytosis due to Kupffer cells significantly. Moreover, Carolina rinse diminished graft swelling and improved postoperative hepatic microcirculation compared with the Ringer's rinse group. Taken together, these results indicate that Carolina rinse is a superior alternative to Ringer's solution in vivo to protect liver grafts from reperfusion injury when removing high-potassium-containing cold storage solutions clinically prior to implantation.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Endothelium; Female; Glutathione; Graft Survival; Insulin; Liver; Liver Circulation; Liver Transplantation; Organ Preservation Solutions; Postoperative Complications; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions; Tissue Preservation

Topics: Adenosine; Allopurinol; Amino Acids; Animals; Dogs; Glutathione; Glycine; Insulin; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Rabbits; Raffinose; Reperfusion Injury; Solutions

Reperfusion injury characterized by loss of endothelial cell viability occurs after cold ischemic storage of livers for transplantation surgery. Here, ultrastructural changes in stored rat livers were examined by scanning and transmission electron microscopy. With increasing times of storage in Euro-Collins solution (4 to 24 hr) followed by 15 min of reperfusion at 37 degrees C, a sequence of structural alterations was observed involving endothelial and Kupffer cells. Widening of endothelial fenestrations occurred after 4 hr and progressed over 8 to 24 hr to retraction of cellular processes, ball-like rounding, sinusoidal denudation and ultrastructural derangements consistent with loss of cell viability. Kupffer cells exhibited progressive rounding, ruffling of the cell surface, polarization, appearance of wormlike densities, vacuolization and degranulation over a similar time course. By contrast, the structures of parenchymal and fat-storing cells were relatively undisturbed by cold storage and reperfusion. Alterations to endothelial and Kupffer cells were also studied as a function of time of reperfusion. After 24 hr of storage, endothelial cells showed retraction of cytoplasm before reperfusion that progressed quickly to loss of viability and denudation during reperfusion. Kupffer cell activation (ruffling, degranulation) during reperfusion was slower and occurred after deterioration of endothelial cells. Livers stored in Euro-Collins solution were also compared with livers stored in University of Wisconsin cold storage solution, an improved preservation medium for transplantation. University of Wisconsin solution provided better preservation of endothelial structure and markedly reduced parenchymal cell blebbing and swelling before reperfusion. University of Wisconsin solution also reduced Kupffer cell activation and release of lysosomal enzymes. In conclusion, endothelial cell deterioration followed by Kupffer cell activation occurred after increasing times of cold ischemic storage and reperfusion of rat livers. Both changes may contribute to the pathophysiology of graft failure caused by reperfusion-mediated storage injury.

Topics: Acid Phosphatase; Adenosine; Allopurinol; Animals; Endothelium; Glutathione; Hypertonic Solutions; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Lipid Peroxides; Organ Preservation; Organ Preservation Solutions; Organ Transplantation; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions; Time Factors

The present studies show clearly that both dexamethasone and insulin can be omitted without altering the efficacy of UW. Adenosine and glutathione are both helpful additives, as is allopurinol. These findings suggest an important role of reperfusion injury after preservation, and confirm the benefits of adding pharmacological agents likely to reduce this injury. Cold ischemic damage was significantly ameliorated by UW solution in this stringent model of rat kidney preservation for 48 hr. A substantially simplified modification of UW solution has been shown to give equally effective kidney preservation, after removal of hydroxyethyl starch, dexamethasone, and insulin. Adenosine, glutathione, and allopurinol have been confirmed as helpful pharmacological additives. These findings have defined some of the mechanisms of effectiveness of UW solution and suggest avenues of further exploration to improve simple hypothermic storage and to prevent reperfusion injury.

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Insulin; Kidney Diseases; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions; Tissue Preservation