24-hour Preservation Research Articles

24-hour supercooling preservation of vascularized composite allografts in rats: A proof-of-concept study

24-hour supercooling preservation of vascularized composite allografts in rats: A proof-of-concept study

Antagonist of sphingosine 1-phosphate receptor 3 reduces cold injury of rat donor hearts for transplantation

Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-β-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.

421.2: One Organ Procurement Organization’s 26-Year Experience With Uncontrolled DCD Donation

Introduction: Uncontrolled DCD donation is an underutilized method of procurement in the U.S., with 44% of all uncontrolled DCD donors to date being procured by one of 57 OPOs. The purpose of this study is to demonstrate that uncontrolled DCD organ donation can lead to the successful retrieval of transplantable organs and to evaluate kidney and liver transplant outcomes for recipients of organs from uncontrolled DCD donors. Methods: This is a retrospective single OPO, multi-center study. As a standard of practice the OPO routinely pursued uncontrolled DCD donors in the setting of hemodynamic instability and/or sudden cardiac arrest. The aggressive pursuit of these types of donors required the OPO to establish the following: an organizational expectation, training, active AOC oversight, a system to rapidly deploy resources, a communication plan with care-giving team members, and 24-hour organ preservation and surgical recovery support. Donor warm ischemic time was determined by the time from patient arrest to cross-clamp with most donors receiving CPR during that interval. Graft survival was determined by the Kaplan-Meier method. WIT, age, kidney PNF and DGF were further evaluated by donor Maastricht category. Results: Between 1996 and 2021, 334 kidneys, 15 livers, 2 pancreata and 1 lung was transplanted from 262 uncontrolled DCD donors. The mean donor age was 33 years (r = 0.5 – 76, sd=15). The median warm ischemic time was 44 minutes (IQR = 22 - 83). Kidney graft survival was 84% at 6 months, 78% at 1 year and 71% at 3 years. Of the 15 livers transplanted, 9 grafts implanted prior to 2010 failed within 3 months, but all 3 of the grafts implanted in 2021 are functioning at 6 months post-transplant. Two of the liver grafts are functioning at 12 years post-transplant and one is functioning at 19 years post-transplant. The lung graft is functioning at 8 years post-transplant.Conclusions: Uncontrolled DCDs provide acceptable kidney graft survival outcomes and represent a pool of organs that should be pursued for transplantation. OPOs should develop protocols to effectively recover organs from uncontrolled DCDs, particularly in light of promising new advancements in organ preservation.

Arterial graft preservation with an endothelial damage inhibitor reduces vascular dysfunction in a rat model of in vitro ischemia/reperfusion injury

Coronary artery bypass grafting (CABG) with autologous conduits is a common surgical operation to redirect blood flow to the ischemic myocardium. Vascular ischemia/reperfusion injury (IR) occurs during graft harvest and implantation, and can lead to endothelial dysfunction. DuraGraft®, a novel one-time intraoperative vascular graft treatment, has been shown to protect integrity and function in saphenous vein grafts against IRI. We compared the effect of DuraGraft® to saline solution on arterial grafts submitted to IRI. Rat thoracic aorta was harvested, prepared, and immediately mounted in organ bath chambers (control, n = 7 rats) or underwent 24-hour cold ischemic preservation (4 °C) either in physiological saline (IR, n = 9 rats) or DuraGraft® (IR + Dura, n = 9 rats). Reperfusion-associated endothelial injury was simulated by hypochlorite. Vascular function was measured ex vivo and immunohistochemistry was performed. Impaired maximal vasorelaxation (Rmax) to acetylcholine (ACh) in the IR-group compared to controls was ameliorated by DuraGraft®, indicating an improvement in endothelial function (Rmax to ACh (%): IR + Dura 73 ± 2 vs. IR 48 ± 3, P < 0.05). Additionally, decreased aortic ring sensitivity to ACh (pD2-value: -log 50% maximum response) seen after IR in the saline group was increased by DuraGraft® (pD2 to ACh: IR + Dura 7.1 ± 0.1 vs. IR 6.3 ± 0.2, P < 0.05). Impaired maximum contractile response to phenylephrine and high K+-induced depolarization in the IR-group compared to controls was improved by DuraGraft®. Immunohistochemical analysis for ICAM-1 showed positivity confined to the endothelial layer in the IR group compared to controls, which was decreased by the preservation of aortic rings with DuraGraft®. Furthermore, DuraGraft® decreased nitrotyrosine immunoreactivity in the IR + DuraGraft® compared to both control and IR groups. Also, endothelial PECAM-1 immunoreactivity was significantly reduced in the IR rings compared to controls, whereas a similar pattern was observed between control and IR + Duragraft® groups. DuraGraft® alleviates vascular dysfunction following in vitro IR injury, in part, by reducing nitro-oxidative stress and by lowering inflammatory response through ICAM-1.

Proof of concept study for a closed ex vivo limb perfusion system for 24-hour subnormothermic preservation using acellular perfusate.

The two approaches to vascularized tissue machine perfusion use either the open (nonpressurized) or closed (pressurized) perfusion system. Most studies describing isolated limb perfusion preservation rely on open perfusion systems and report tissue edema exceeding 40% after 12 to 14 hours of preservation. A variant of machine perfusion places the limb and perfusate into a reservoir closed to atmosphere. It is hypothesized that the reservoir pressure, acting as a transmural pressure, has the advantage of reducing edema formation by counteracting the hydrostatic pressure gradient from the perfusion pressure. This proof-of-concept study aim was to demonstrate feasibility of the Universal Limb Stasis System for Extended Storage (ULiSSES) device (closed, vertical perfusion system) to preserve forelimbs of Sus scrofa swine for 24 hours of subnormothermic perfusion compared with an open, horizontal perfusion system. The ULiSSES is a compact, practical device that applies pulsatile, pressurized perfusion through the novel use of a diaphragm pump powered by compressed oxygen. Forelimbs from swine were preserved in ULiSSES device (closed perfusion system) (n = 9) and in an open perfusion system (n = 4) using subnormothermic modified Krebs-Henseleit solution. Physiological parameters were measured at the start and every 3 hours for 24 hours. Limbs were weighed before and after perfusion to compare weight gain. Edema and cellular integrity were evaluated using histopathology pre and post perfusion. Closed perfusion system showed superiority compared with the open perfusion system in terms of oxygen consumption, reduction in vascular resistance, and overall tissue integrity. The closed perfusion system demonstrated a 21% reduction in weight gain compared with the open perfusion system and significantly reduced intracellular edema. The ULiSSES closed, pressurized perfusion technology has translatable military applications with the potential to preserve porcine limbs for 24 hours with improved results compared with an open perfusion system.

Concentration of Tumor Necrosis Factor–α and Interleukin-1β in Isolated Porcine Liver Depending on Type of Transgenesis

BackgroundTransgenic animals may serve as organ donors in human organ transplantation. However, the number of the studies addressing all doubts related to this issue is currently insufficient for the clinical application of this approach. The aim of this study was to analyze the hepatic tumor necrosis factor–α (TNF-α) and interleukin-1β (IL-1β) synthesis during a 24-hour cold preservation of the transgenic pig liver, depending on the type of transgenesis. Materials and MethodsThe study was carried out on wild-type and transgenic pig livers with transferred human α1,2-fucosyltransferase (FUT) and/or α-galactosidase (GAL) gene (four groups; n = 6). Harvested livers were perfused for 30 minutes and stored for 24 hours in Biolasol (Biochefa) solution at 4°C with a subsequent 30-minute reperfusion (reflush). TNF-α and IL-1β concentrations were analyzed with an enzyme-linked immunosorbent assay. Perfusates were collected during the initial perfusion as well as after 24 hours of preservation and during the reperfusion. Tissue samples were harvested just after animal sacrifice, and after organ perfusion and reperfusion. ResultsA decrease in TNF-α concentration in homogenates was noted after both perfusion and reperfusion in all experimental groups. In contrast, there was a significant decrease in IL-1β concentration in the group with combined human FUT and GAL transgenes. Concurrently, increases in TNF-α and IL-1β concentrations were observed in the reperfusion perfusates in all groups. ConclusionThis study shows that IL-1β is synthesized in the ischemic livers of the transgenic animals with both human α1,2-fucosyltransferase and α-galactosidase transgenes. Further analysis is required to determine the importance of this observation.

5-HT Receptor Antagonism Attenuates the Ischemia-Reperfusion Injury After Rabbit Lung Preservation

The success of lung transplantation is threatened by the appearance of ischemia-reperfusion injury, which is characterized by increased vascular permeability. 5-Hydroxytryptamine (5-HT; serotonin) is known to produce microvascular leakage in the systemic circulation, but its possible role in ischemia-reperfusion injury after lung preservation has not been reported. In this work we measured the release of 5-HT during a 24-hour rabbit lung preservation, and the effect of methiothepin (antagonist of the majority of 5-HT receptors) and SB204741 (antagonist of 5-HT2B/2C receptors) on the modified capillary filtration coefficient (mKf,c) was evaluated at the end of this period. Our results showed that the highest release rate of 5-HT occurred during the first 15 minutes after the lung harvesting and progressively decreased in the following time intervals. The baseline mKf,c greatly increased after 24 hours of lung preservation, and this increment was partially reduced by methiothepin and even more by SB204741. We concluded that 5-HT may play an important role in the ischemia-reperfusion process after lung preservation.

Cytochrome P450 3 A Expression in Pigs Livers After 24-Hour Preservation in Biolasol Solution Depending on the Type of Transgenesis

BackgroundAn insufficient number of organs for transplantation shows the need for the development of new technologies. Xenotransplantation might be the answer. ObjectiveTo determine if the type of transgenesis influences the level of CYP3A4, which takes an active part in xenobiotics metabolism in livers after 24-hour storage, depending on the kind of solution used for preservation. Materials and methodsThe experiment was carried out on 30 livers of Polish White Landrace divided into 5 groups depending on transgene type. The following human genes were transferred: α1,2-fucosyltransferase (groups I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's solution (group I) or Biolasol solution (II-V). Reperfusion/reflush was performed. CYP3A29 isomer concentration was analyzed in liver specimens collected twice: 30 minutes after perfusion and 30 minutes after reperfusion/reflush. Expression of mRNA CYP3A29 was marked using RT-PCR analysis and of protein CYP3A29 using Western blotting technique. ResultsThe most significant decrease in protein CYP3A29 expression after 24-hour preservation was observed in group I (55.88% decrease), while the least significant was observed in group IV (10.44% decrease). mRNA expression evaluation was similar: the most significant decrease was observed in group I (87.8% decrease) and the least significant in group III (4.6% decrease). Conclusionα1,2-Fcosyltransferase transgene seems to influence mRNA and protein CYP3A expression in case of liver grafting and preservation for transplantation. CYP3A expression was also influenced by the kind of preservation solution used.

Interleukin-6 Concentration in the Transgenic Pig's Liver Preserved for 24 Hours in Biolasol Solution

IntroductionIncreasing the human lifespan contributes to a higher number of patients with end-stage organ failure, which in turn stimulates the search for alternative sources. Xenotransplantation seems to be a promising approach in this respect. ObjectiveAnalysis of changes in interleukin (IL)-6 concentration during 24-hour preservation of transgenic swine livers, depending on the kind of transgenesis and preservation solution used. Materials and methodsThe experiment was carried out in swine livers with transferred human genes that were divided into 5 groups. The following human genes were transferred: α1,2-fucosyltransferase (group I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's (group I) or Biolasol solutions (II–V). Reflush was then performed. IL-6 concentration was analyzed in the solution samples collected at the beginning and end of perfusion, and after 24 hours of preservation. ELISA was used to evaluate IL-6 concentration. ResultsIn liver homogenates from group I, IL-6 concentration after 24 hours of preservation increased by 8.24% compared to the levels observed after perfusion, whereas in the other groups IL-6 concentration decreased. The most significant decrease, 49.51%, was observed in group II; the least significant in group IV, 10.72%. In case of supernatants, a statistically significant increase of AUC0–30min level in relation to perfusion was observed in every group after 24-hour preservation and reperfusion. The highest values of AUC0–30min were observed in group I (α1,2-fucosyltransferase, Ringer's solution). ConclusionThe study indicates the hepatoprotective action of Biolasol solution.

Prolactin (PRL) reduces the release of transaminases from the isolated pig liver

This study investigated methods of increasing the effectiveness of preservation solutions. Recently, it has been reported that solution effectiveness can be improved by the addition of prolactin (PRL). This study determines the effect of prolactin (PRL) on the amount and release rate of alanine aminotransferase (ALAT) and aspartate aminotransferase (AST) from liver during its 24-hour preservation period. Isolated porcine liver was kept in the HTK (histidine-tryptophan-ketoglutaric acid) solution with and without PRL. Once infusion and the 24-hour preservation period had been finished, samples of the preservation solution were taken and the amounts of released indicator enzymes were determined. Aminotransferases were released from hepatocytes to the preservation solution at various rates. ALT was released much faster into the solution without PRL (k=-0.1230 [U/l/h-1]) and slower with PRL (k=-0.0895 [U/l/h-1]). The enzyme was released into the solution with PRL at a 27% slower rate. Similar results were obtained when the release rate of AST was analyzed. AST was more quickly released into the solution without PRL (k=-0.0642 [U/l/h-1]), and more slowly with PRL (k=-0.0205 [U/l/h-1]). The enzyme was released into the PRL-containing solution at a 68% slower rate. The obtained results indicate that the addition of PRL significantly lowers the amount of aminotransferases released from hepatocytes and also lessens their release rate, as it significantly affects the time of efficient in vitro storage of the liver.

New Stepwise Cooling System for Short-Term Porcine Islet Preservation

Porcine islets are the most suitable for xeno-islet transplantation. However, it is necessary to establish an effective preservation method against its fragility. Recently, we developed a new cooling and preservation (Keep and Fresh [KFC]; FUJIYA Co, Tokushima, Japan) system, which can maintain viability of hepatocyte. In this study, we examined the KFC for porcine islet preservation. Isolated porcine islets were preserved in CMRL 1066 culture media with bovine serum at 37°C, 22°C, and 4°C and KFC for 24, 48, and 72 hours. Islet recovery rate, purity, and viability were evaluated. After 24-hour preservation, the recovery rate was the highest in the KFC, but no significant difference was found. After 48-hour preservation, the recovery rate by the KFC was 73.9% ± 17.3%, which was significantly higher than the other groups (48.7% ± 28.6% at 37°C, P < 0.01; 46.6% ± 15.5% at 22°C, P < 0.01; 61.5% ± 20.0% at 4°C, P < 0.05). After 72-hour preservation, the difference of recovery rate was clearer. In the KFC group, purities and viabilities were the highest among the groups after 24-, 48-, and 72-hour preservation. The KFC system significantly improved porcine islet preservation; therefore, the KFC might be useful for porcine islet preservation.

Actin Cytoskeletal and Functional Studies of the Proximal Convoluted Tubules After Preservation

Background Proximal tubule cells have specialized apical membranes with microvilli that provide an extensive surface area for unidirectional transport of solute from lumen to blood. The major structural solute component is F-actin, which interacts with transmembrane proteins, including ion transport molecules related to normal absorptive and secretory functions. Our study was to evaluate F-actin and fluid absorption (Jv) in proximal tubules after exposure to preservation solutions. Methods In vitro microperfusion technique and immunohistochemistry analysis. Results 1. Absorptions were similar in 1- and 24-hour–preserved tubules, as well as in fresh tubules. The exception was tubules for 24 hours in Euro-Collins solution, which did not show absorption, suggesting that it was affected. 2. Fluorescence intensity of actin tubules preserved for 1 hour in both solutions showed similar values to each other and to the control group; tubules preserved for 24 hours in both solutions were similar to each other, although statistically different than the control group and those preserved for 1 hour in Belzer (UW) solution. Conclusion There were differences among groups in the distribution of F-actin; Jv values were different for 24-hour preservation in each solution, whereas fluorescence intensity was similar in both 24-hour solutions. Thus, actin cytoskeleton was not responsible for it, because 24-hour preservation in UW showed Jv results comparable to the control group.

A Rho-kinase inhibitor improves cardiac function after 24-hour heart preservation

The Rho-kinase signaling pathway is associated with coronary vasculopathy and myocardial dysfunction after cardiac transplantation. This study evaluated whether using a Rho-kinase inhibitor during allograft storage could limit early endothelial dysfunction and improve myocardial performance after reperfusion. This experiment was performed with an isolated working rabbit heart model and a support rabbit. Donor hearts (control group, n = 8) were arrested with an extracellular type of cardioplegia, preserved with University of Wisconsin solution, and then immersed in University of Wisconsin solution for 24 hours (1 degrees C). The Rho-kinase inhibitor (Rho-kinase inhibitor group, n = 8) was administrated in the cardioplegic solution, the preservation University of Wisconsin solution, and the storage University of Wisconsin solution. Left ventricular performance was evaluated from the modified Frank-Starling curve in the working mode. Coronary blood flow and donor heart rate were measured in Langendorff mode. Effective evaluation of the Rho-kinase inhibitor was inferred from phosphorylated myosin light chain. The expression of endothelial nitric oxide synthase mRNA was analyzed to assess endothelial function. The Frank-Starling curve showed a significant left and upward shift in the Rho-kinase inhibitor group compared with the control group (P < .05). The coronary blood flow and heart rate in the Rho-kinase inhibitor group at 120 minutes was significantly higher than in the control group (P < .05). Phosphorylated myosin light chain was significantly suppressed in the Rho-kinase inhibitor group (P < .05). Endothelial nitric oxide synthase mRNA levels in the Rho-kinase inhibitor group increased 4-fold relative to those seen in the control group. Treatment with Rho-kinase inhibitor during allograft harvest and storage enhanced coronary blood flow and ventricular recovery through nitric oxide-dependent endothelial protection after reperfusion. Rho-kinase inhibitor could help prevent early myocardial dysfunction after transplantation.

Endothelial Dysfunction After Long-term Cold Storage in HTK Organ Preservation Solutions: Effects of Iron Chelators and N-α-acetyl- l-histidine

To improve the rate of successful heart transplantations, organ preservation should be optimized in cardiac transplantation surgery. Iron-dependent oxidative damage and iron-independent, chloride-dependent injury of the endothelium have been described after cold ischemic storage and reperfusion, leading to an enhanced rate of complications and unfavorable outcomes. This screening study tested the effects of iron chelator supplementation in different histidine-tryptophan-ketoglutarate (HTK) organ preservation solutions on endothelial function in a long-term storage model of the isolated rat aorta. Isolated rat aortic rings underwent a 24-hour cold ischemic preservation in different HTK solutions supplemented with iron chelators of low (deferoxamine) and high (LK-614) membrane permeability. In vascular reactivity measurements we investigated the phenyleprine-induced contraction and both endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside with and without an additional external oxidant injury during re-oxygenation. Traditional HTK solution, Custodiol, failed to prevent endothelial dysfunction in our experiments. Use of chloride-poor HTK solutions containing N-alpha-acetyl-l-histidine with and without supplementation with LK-614, but not with deferoxamine, resulted in significant improvement of impaired endothelial function; moreover, complete protection of the endothelium was feasible after 24-hour cold storage. Endothelium-independent functions of vascular smooth muscle were not affected in any of the groups. Our results demonstrate the important pathophysiologic role of iron-dependent oxidative injury in the development of endothelial dysfunction after cold storage, which can be prevented by cell-permeable iron chelators.

Successful 40-hour preservation of the canine small intestine with the cavitary 2-layer method with glutamine supplementation

We recently reported that the cavitary 2-layer method (cTLM) allowed stable 24-hour preservation of canine intestine. The aim of this study was to examine the possibility of the 40-hour preservation by cTLM that is supplemented with glutamine. Canine jejunal segments (40 cm) were allotransplanted heterotopically without storage (group 1), after 40-hour cold storage with University of Wisconsin solution (group 2), cTLM (group 3), UW with 2% glutamine (group 4), or cTLM with 2% glutamine (group 5). Mucosal glutamine, histidine, tryptophan, glutathione, and adenosine triphosphate concentrations were determined immediately after preservation. At day 7 after the transplantation, maltose absorption test and histopathologic analysis were performed. Mucosal glutamine concentrations increased significantly with glutamine supplementation during preservation in groups 4 and 5 (P < .01). Mucosal adenosine triphosphate levels in cTLM groups (groups 3 and 5) were similar to those in group 1; group 4 showed very low levels after preservation, despite glutamine supplementation. Mucosal glutathione did not differ among groups 2, 3, 4, and 5 and were significantly lower than in group 1. In the absorption test, serum glucose curves showed a peak level by 30 minutes in groups 1 and 5; delayed peaks were seen in groups 2, 3, and 4 compared with group 1. The villous heights were 879, 555, 685, 688, and 773 microm in groups 1, 2, 3, 4, and 5, respectively (group 1 vs group 2, 3, and 4: P < .05). cTLM that was supplemented with glutamine extended a safe preservation period up to 40 hours in canine segmental small intestinal transplantation, although this study failed to elucidate a precise mechanism of the glutamine beneficial effect on the graft mucosa.

Oxygenated University of Wisconsin solution for 24-hour pancreas preservation: Equivalent to two-layer method?

Oxygenated University of Wisconsin solution for 24-hour pancreas preservation: Equivalent to two-layer method?

Kidney preservation by hypothermic perfusion with albumin versus plasma and with pulsatile versus non-pulsatile flow.

Abstract Four groups of canine kidneys were preserved by hypothermic perfusion for 24 hours in an attempt to compare the relative merits of using cryoprecipitated plasma or albumin as perfusates and of using pulsatile or non-pulsatile flow. All the kidneys were subsequently autotransplanted At 0, 3 and 24 hours during preservation the following parameters were measured: perfusate and urine flow, renal vascular resistance, perfusate serum glutamic oxaloacetic transamianase, lactic dehydrogenase, acid and alkaline phosphatases, ammonia, free fatty acids, clearances of creatinine, 131I hippuran, Na+ and K+. After reimplantation similar renal clearance studies were carried out. After transplantation overall kidney viability was assessed by recipient survival and by serum creatinine levels. The results obtained were subjected to statistical analysis and compared between the groups. It was found that perfusion with albumin solution gave a significantly higher perfusate flow rate (P&amp;lt;0·02) and lower vascular resistance (P&amp;lt; 0·001) than perfusion with plasma. With albumin as the perfusate there was no significant difference in perfusion characteristics or in the post-implantation renal clearances regardless of whether pulsatile or non-pulsatile flow was used. With plasma as the perfusate there was significantly higher perfusate flow (P&amp;lt;0·01), lower vascular resistance and higher post-implantation creatinine and hippuran clearances (P&amp;lt;0·02, P&amp;lt;0·001) with pulsatile than with non-pulsatile flow. In 24-hour renal preservation overall recipient survival and graft function after transplantation were similar regardless of the nature of the perfusate or the type of perfusion.

Cryoprecipitated plasma perfusion preservation and cold storage preservation of duct-ligated pancreatic allografts

It has been shown previously that, at least in dogs, a vascularized pancreatic allograft with ligation of the pancreatic duct can maintain normal beta-cell function for at least 5 years. In this study common organ preservation techniques, as used in human cadaveric kidney transplantation, were applied to canine pancreatic allografts to determine the influence of 24-hour preservation on beta-cell function, graft survival and histological appearances. Three groups of dogs were compared: group A consisted of 9 dogs with fresh grafts; group B consisted of 5 dogs who received grafts preserved for 24 hours by pulsatile hypothermic cryoprecipitated plasma perfusion; and group C was composed of 5 animals who received grafts that had been flushed with Collins' solution followed by hypothermic storage for 24 hours. It appeared that both preservation methods were equally effective and that preservation did not alter either the graft's function or its histological appearance. No significant differences after transplantation were observed in the endocrine function tests of the three groups when compared with the preoperative values; neither was there a significant difference in the mean graft survival time between the groups.

Angiotensin II Type 1 Receptor Antagonist Protects Ventricular and Coronary Endothelial Function After 24-hour Heart Preservation

Angiotensin II type 1 (AT1) receptor antagonists may enhance the cyclic guanosine monophosphate-nitric oxide system and thereby attenuate ventricular and coronary endothelial dysfunction after heart preservation. We used an isolated rabbit heart preparation perfused with blood from a support rabbit. The rabbit heart was excised, stored for 24 hours, and then perfused with blood from a support rabbit that was treated with an AT1 receptor antagonist (telmisartan; 5 mg/kg) or solvent. We evaluated the cardiac output with the working preparation, and coronary blood flow and coronary endothelial function with the Langendorff preparation. In addition, we measured the serum nitric oxide level in the coronary effluent. The Telmisartan Group showed higher plasma angiotensin II levels (928.6 +/- 136.2 vs 271.6 +/- 81.6 pg/ml, p < 0.01), better cardiac output (116.2 +/- 5.4 vs 88.8 +/- 7.1 ml/min, p < 0.05), and higher coronary blood flow (25.0 +/- 2.2 vs 14.9 +/- 1.3 ml/min, p < 0.01). The coronary blood flow in response to acetylcholine was higher in the Telmisartan Group (47.8 +/- 3.9 vs 28.0 +/- 2.1 ml/min, p < 0.01), but there was no difference in response to sodium nitroprusside. The Telmisartan Group showed higher serum nitric oxide levels in the coronary effluent (33.9 +/- 4.6 vs 20.6 +/- 3.3 mumol/liter, p < 0.05). Treatment with the AT1 receptor antagonist improved ventricular and endothelial function after 24-hour heart preservation. These data imply that AT1 activation plays a critical role in reperfusion injury. AT1 receptor blockade may be a promising strategy for long-term heart preservation.

Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation

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