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Diseases of the cornea are a frequent cause of blindness worldwide. Keratoplasty is an efficient method for treating severely damaged cornea. The functional competence of corneal endothelial cells is crucial for successful grafting, which requires improving the media for the hypothermic cornea preservation, as well as developing the methods for the evaluation of the corneal functional properties. The transport of water and ions by the corneal endothelium is important for the viability and optic properties of the cornea. We studied the impact of SkQ1 on the equilibrium sodium concentration in the endothelial cells after hypothermic preservation of pig cornea at 4°C for 1, 5, and 10 days in standard Eusol-C solution. The intracellular sodium concentration in the endothelial cells was assayed using the fluorescent dye Sodium Green; the images were analyzed with the custom-designed CytoDynamics computer program. The concentrations of sodium in the pig corneal endothelium significantly increased after 10 days of hypothermic preservation, while addition of 1.0 nM SkQ1 to the preservation medium decreased the equilibrium concentration of intracellular sodium (at 37°C). After 10 days of hypothermic preservation, the permeability of the plasma membrane for sodium decreased in the control cells, but not in the cells preserved in the presence of 1 nM SkQ1. Therefore, SkQ1 increased the ability of endothelial cells to restore the intracellular sodium concentration, which makes SkQ1 a promising agent for facilitating retention of the functional competence of endothelial cells during cold preservation.

Background Molecular hydrogen (H2) has protective effects against ischemia-reperfusion injury in various organs. Because they are easier to transport and safer to use than inhaled H2, H2-rich solutions are suitable for organ preservation. In this study, we examined the protective effects of an H2-rich solution for lung preservation in a canine left lung transplantation (LTx) model. Methods Ten beagles underwent orthotopic left LTx after 23 hours of cold ischemia followed by reperfusion for 4 hours. Forty-five minutes after reperfusion, the right main pulmonary artery was clamped to evaluate the function of the implanted graft. The beagles were divided into two groups: control group (n = 5), and H2 group (n = 5). In the control group, the donor lungs were flushed and immersed during cold preservation at 4°C using ET-Kyoto solution, and in the H2 group, these were flushed and immersed using H2-rich ET-Kyoto solution. Physiologic assessments were performed during reperfusion. After reperfusion, the wet-to-dry ratios were determined, and histology examinations were performed. Results Significantly higher partial pressure of arterial oxygen and significantly lower partial pressure of carbon dioxide were observed in the H2 group than in the control group (P = .045 and P Conclusions Our results demonstrated that the H2-rich preservation solution attenuated ischemia-reperfusion injury in a canine left LTx model.

Background Liver transplantation from donors after cardiac death (DCD) might increase the pool of available organs. Recently, some investigators reported the potential use of mesenchymal stem cells (MSCs) to improve the outcome of liver transplantation from DCD. The aim of this study was to evaluate the cytoprotective effects and safety of MSC transplantation on liver grafts from DCD. Methods Rats were divided into 4 groups (n = 5) as follows: 1. the heart-beating group, in which liver grafts were retrieved from heart-beating donors; 2. the DCD group, in which liver grafts were retrieved from DCD that had experienced apnea-induced agonal conditions; 3. the MSC-1 group, and 4. the MSC-2 group, in which liver grafts were retrieved as with the DCD group, but were infused MSCs (2.0 × 105 or 1.0 × 106, respectively). The retrieved livers were perfused with oxygenated Krebs-Henseleit bicarbonate buffer (37°C) through the portal vein for 2 hours after 6 hours of cold preservation. Perfusate, bile, and liver tissues were then investigated. Results Bile production in the MSC-2 group was significantly improved compared with that in the DCD group. Based on histologic findings, narrowing of the sinusoidal space in the both MSC groups was improved compared with that in the DCD group. Conclusions MSCs could protect the function of liver grafts from warm ischemia-reperfusion injury and improve the viability of DCD liver grafts. In addition, we found that the infusion of 1.0 × 106 MSCs does not obstruct the hepatic sinusoids of grafts from DCD.

The nutritional value of mutton chop rolls is gradually recognized by people, but it is easy to cause microbial contamination during storage, leading to spoilage and shortening of storage time. The bacterial diversity of mutton chop rolls in different cold preservation time was analyzed to explore the main pathogens of spoilage of mutton chop rolls. At the same time, the oxidative state of myoglobin and the change of mitochondrial Metmyoglobin (MMb) Reduction Ability (MRA) in different cold preservation were studied. It lays a foundation for further study on the mechanism of meat color stabilization of mutton chop rolls during cold preservation. A total of 10,123,180 effective Tags were obtained from three samples with different cold preservation time by high throughput sequencing. The relative abundance of Pseudomonas was the highest in the samples refrigerated for 8 days, Acinetobacter, Brochothrix and Lactobacillales showed the highest relative abundance in the samples refrigerated for 4 days, which were closely related to the deterioration of mutton chop rolls and color deterioration. With the increase of cold preservation time, Oxymyoglobin (OMb) content decreased and Metmyoglobin (MMb) content increased. MRA was negatively correlated with MMb. The content of NADH was extremely significant difference with OMb and MMb. At the same time, the content of NADH was a significant difference with MRA. This study provides theoretical basis for prolonging the shelf life, maintaining meat color stability, improving the quality of mutton chop rolls. And it also plays a certain role in promoting the production and consumption of chilled meat.

Organ transplantation is the treatment of choice against terminal and irreversible organ failure. Optimal preservation of the graft is crucial to counteract cold ischemia effects. As we developed an N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid–gluconate–polyethylene glycol (BGP)–based solution (hypothermic machine perfusion [HMP]), we aimed to analyze the use of this solution on static cold storage (SCS) of rat livers for transplantation as compared with the histidine tryptophan ketoglutarate (HTK) preservation solution. Livers procured from adult male Sprague Dawley rats were preserved with BGP-HMP or HTK solutions. Liver total water content and metabolites were measured during the SCS at 0°C for 24 hours. The function and viability of the preserved rat livers were first assessed ex vivo after rewarming (90 minutes at 37°C) and in vivo using the experimental model of reduced-size heterotopic liver transplantation. After SCS, the water and glycogen content in both groups remained unchanged as well as the tissue glutathione concentration. In the ex vivo studies, livers preserved with the BGP-HMP solution were hemodynamically more efficient and the O2 consumption rate was higher than in livers from the HTK group. Bile production and glycogen content after 90 minutes of normothermic reperfusion was diminished in both groups compared with the control group. Cellular integrity of the BGP-HMP group was better, and the histological damage was reversible. In the in vivo model, HTK-preserved livers showed a greater degree of histological injury and higher apoptosis compared with the BGP-HMP group. In conclusion, our results suggest a better role of the BGP-HMP solution compared with HTK in preventing ischemia/reperfusion injury in the rat liver model. Fil: Carnevale, Matías Emanuel. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lausada, Natalia Raquel. Universidad Nacional de Rosario; Argentina Fil: Juan de Paz, Leonardo. Universidad Nacional de Rosario; Argentina Fil: Stringa, Pablo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentina Fil: Machuca, Mariana Alejandra. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina Fil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentina Fil: Guibert, Edgardo Elvio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tiribelli, Claudio. Fondazione Italiana Fegato.; Italia Fil: Gondolesi, Gabriel Eduardo. Fundación Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rodriguez, Joaquin Valentin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Summary Organ preservation and re‐conditioning using machine perfusion technologies continue to generate promising results in terms of viability assessment, organ utilization and improved initial graft function. Here, we summarize the latest findings and study the results of ex‐vivo/ex‐situ hypothermic (HMP) and normothermic machine perfusion (NMP) in the area of abdominal organ transplantation (kidney, liver, pancreas and intestine). We also consider the potential role of normothermic regional perfusion (NRP) to re‐condition donors after circulatory death organs before retrieval. The findings from clinical studies reported to date suggest that machine perfusion will offer real benefits when compared with conventional cold preservation. Several randomized trials are expected to report their findings within the next 2 years which may shed light on the relative merits of different perfusion methods and could indicate which perfusion parameters may be most useful to predict organ quality and viability. Further work is needed to identify composite endpoints that are relevant for transplanted organs that have undergone machine preservation. Multi‐centre trials to compare and analyse the combinations of NRP followed by HMP and/or NMP, either directly after organ retrieval using transportable devices or when back‐to‐base, are needed. The potential applications of machine preservation technology beyond the field of solid organ transplantation are also considered.

Background Liver transplantation from donors after cardiac death (DCD) could increase the pool of organs. We previously reported that oxygenated subnormothermic (20°C-25°C) ex vivo liver perfusion (SELP) improved the graft viability in rats. This study aimed to compare the effectiveness of SELP and normothermic (37°C) ex vivo liver perfusion (NELP) after cold storage (CS) in DCD liver grafts. Methods Male Wistar rats were used, and grafts were retrieved 30 minutes after cardiac arrest. We performed oxygenated NELP and SELP with a Krebs-Henseleit buffer for different time points and durations: Group 0, donation performed from heart-beating donors (control); Group 1 (DCD group), donation performed from DCD donors with no treatments; Group 2, NELP performed before CS (30 minutes); Group 3, NELP performed after CS (30 minutes); Group 4, SELP performed after CS (30 minutes); Group 5, SELP performed after CS (60 minutes); and Group 6, SELP performed after CS (90 minutes). After 15 minutes of incubation at room temperature, the grafts were reperfused under normothermic conditions for 60 minutes as a model of liver transplantation. Results No significant differences in body and liver weight were observed between all groups. In the SELP after CS groups, even 30 minutes of perfusion improved bile production, tumor necrosis factor-α, and interleukin-1β significantly compared with the DCD group (P Conclusion SELP rescued DCD livers from ischemia-reperfusion injury the same as the normothermic perfusion before or after CS groups. SELP after CS is more convenient than normothermic perfusion; hence, this technique may increase the organ pool.

Purpose of review To summarize the history of organ preservation and place into this context the current trends in preservation. Recent findings Multiple large retrospective studies have analyzed cold preservation solutions in an attempt to determine superiority with largely negative results. Experimental and some clinical studies have examined machine perfusion of procured grafts, in both hypothermic and normothermic contexts with variable, but promising, results. Lastly, there are experimental efforts to evaluate mesenchymal stem cell therapy on rehabilitation of marginal donor organs. Summary New trends in organ preservation may soon translate into more efficient use of the limited donor pool.

We previously reported that short oxygenated warm perfusion before cold storage (CS) had improved the graft viability of rat livers from donors after circulatory death (DCD). In this study, we investigated the effectiveness of short-term oxygenated subnormothermic perfusion for different durations after CS in a rat DCD model.We used an isolated perfused rat liver system. In study 1: the grafts were retrieved from Wistar rats 30 minutes after cardiac arrest (thoracotomy), preserved in CS for 6 hours, and perfused with oxygenated subnormothermic (20-25°C) Krebs-Henseleit buffer for different durations (0, 15, 30, 60, and 90 minutes groups; n = 5 in each). In study 2: in addition to subnormothermic ex vivo liver perfusion (SELP), after 15-minute incubation at room temperature, the grafts were reperfused under normothermic condition for 60 minutes as a model of liver transplantation (0, 30, 60, and 90 minutes groups; n = 5 in each).In study 1, portal flow, bile production and tissue adenosine triphosphate increased with perfusion duration. In study 2, SELP significantly improved portal flow volume (P0.05), and bile production (P0.05), decreased liver enzymes (P0.05) and cytokines (P0.0001), and increased tissue adenosine triphosphate (P0.01). Histological examinations showed that additional SELP ameliorated tissue deterioration, preserved the parenchymal structure, and decreased apoptosis (P0.01). Furthermore, scanning electron microscopy revealed that additional SELP alleviated sinusoidal endothelial cells and hepatic microvasculature.Even 30 minutes of SELP after CS rescued DCD livers from ischemia-reperfusion injury, which may help the viability of the grafts.

Background A merit of subnormothermic perfusion has been reported to preserve grafts from ischemic injury in animal models. The split liver technique is commonly performed to solve the shortage of liver grafts. However, there has been no study showing the effect of a split liver graft on subnormothermic perfusion. We herein investigated the split liver protocol using a subnormothermic oxygenated circuit system (SOCS). Methods Auxiliary liver transplantation was performed in a porcine marginal donor model by using a SOCS. In the SOCS group, the portal vein and hepatic artery of the graft were cannulated, and the graft was perfused by SOCS. In the cold storage (CS) group, the graft was placed in cold preservation solution. In the preservation phase, the graft was split. Results There were no significant differences in the biochemical markers between the SOCS and CS groups. In terms of the histology, the sinusoidal spaces were widened in the CS group 12 hours after implantation. Conclusion We have demonstrated a possibility to use SOCS with the split liver protocol by using a porcine model. This split liver protocol using SOCS will extend the split liver criteria and rescue more patients from hepatic failure, including pediatric patients.

Allogenic osteochondral tissue (OCT) is used for the treatment of large cartilage defects. Typically, OCTs collected during the disease-screening period are preserved at 4°C; however, the gradual reduction in cell viability during cold preservation adversely affects transplantation outcomes. Therefore, improved storage methods that maintain the cell viability of OCTs are needed to increase the availability of high-quality OCTs and improve treatment outcomes. Here, we evaluated whether long-term hydrogen delivery to preservation solution improved the viability of rat OCTs during cold preservation. Hydrogen-supplemented Dulbecco’s Modified Eagles Medium (DMEM) and University of Wisconsin (UW) solution both significantly improved the cell viability of OCTs during preservation at 4°C for 21 days compared to nonsupplemented media. However, the long-term cold preservation of OCTs in DMEM containing hydrogen was associated with the most optimal maintenance of chondrocytes with respect to viability and morphology. Our findings demonstrate that OCTs preserved in DMEM supplemented with hydrogen are a promising material for the repair of large cartilage defects in the clinical setting.

OBJECTIVE: Transplantation of organs retrieved after cardiac arrest could increase the donor organ supply. However, the combination of warm ischemia and cold preservation is highly detrimental to the reperfused organ. Our objective was to maintain physiological temperature and organ function during preservation and thereby alleviate this injury and allow successful transplantation. BACKGROUND DATA: We have developed a liver perfusion device that maintains physiological temperature with provision of oxygen and nutrition. Reperfusion experiments suggested that this allows recovery of ischemic damage. METHODS: In a pig liver transplant model, we compared the outcome following either conventional cold preservation or warm preservation. Preservation periods of 5 and 20 hours and durations of warm ischemia of 40 and 60 minutes were tested. RESULTS: After 20 hours preservation without warm ischemia, post-transplant survival was improved (27%-86%, P = 0.026), with corresponding differences in transaminase levels and histological analysis. With the addition of 40 minutes warm ischemia, the differences were even more marked (cold vs. warm groups 0% vs. 83%, P = 0.001). However, with 60 minutes warm ischemia and 20 hours preservation, there were no survivors. Analysis of hemodynamic and liver function data during perfusion showed several factors to be predictive of posttransplant survival, including bile production, base excess, portal vein flow, and hepatocellular enzymes. CONCLUSIONS: Organ preservation by warm perfusion, maintaining physiological pressure and flow parameters, has enabled prolonged preservation and successful transplantation of both normal livers and those with substantial ischemic damage. This technique has the potential to address the shortage of organs for transplantation.

This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial., The urgent need to expand the donor pool in order to attend to the growing demand for liver transplantation has obliged physicians to consider the use of suboptimal liver grafts and also to redefine the preservation strategies. This review examines the different methods of liver graft preservation, focusing on the latest advances in both static cold storage and machine perfusion (MP). The new strategies for static cold storage are mainly designed to increase the fatty liver graft preservation via the supplementation of commercial organ preservation solutions with additives. In this paper we stress the importance of carrying out effective graft washout after static cold preservation, and present a detailed discussion of the future perspectives for dynamic graft preservation using MP at different temperatures (hypothermia at 4 °C, normothermia at 37 °C and subnormothermia at 20 °C-25 °C). Finally, we highlight some emerging applications of regenerative medicine in liver graft preservation. In conclusion, this review discusses the “state of the art” and future perspectives in static and dynamic liver graft preservation in order to improve graft viability., Supported by Grant from Fondo de Investigaciones Sanitarias, No. FIS PI12/00519; Eirini Pantazi is the recipient of a fellowship from Agència de Gestió d’Ajuts Universitaris i de Recerca, No. 2012FI_B00382, Generalitat de Catalunya, Barcelona, Spain.Fondo de Investigación Sanitaria (España)Generalidad de Cataluña

Although several types of culture medium have been used for preservation of osteochondral allografts, the viability of chondrocytes decreases with increasing storage duration. We previously showed the University of Wisconsin solution is more suitable for graft preservation than culture medium.We determined whether the addition of allogenic serum to University of Wisconsin solution increases chondrocyte survival during prolonged storage of osteochondral allografts.Osteochondral tissue samples harvested from the distal femora of rats were preserved in University of Wisconsin solution supplemented with 0%, 1%, 10%, and 50% allogenic serum at 4 °C for 14 days. Cell viability and chondrocyte degenerative changes of the samples then were assessed using a tetrazolium assay and histologic methods. We also evaluated time-dependent changes in cell viability and histologic findings of samples preserved for 7, 14, and 21 days in University of Wisconsin solution supplemented with or without 10% allogenic serum.After 14 days of preservation, osteochondral tissue samples maintained in University of Wisconsin solution containing 10% or greater allogenic serum exhibited the highest cell viability and lowest degenerative changes in chondrocytes. In the evaluation of time-dependent changes, we found the chondrocyte degenerative changes were greater in cartilage preserved in University of Wisconsin solution alone than in University of Wisconsin solution containing 10% allogenic serum after day 7 or later.Our results suggest the addition of 10% allogenic serum to University of Wisconsin solution enhances viability of osteochondral tissue samples.The use of allogenic serum-supplemented University of Wisconsin solution is expected to prolong the duration of osteochondral allograft storage and result in higher-quality grafts.

Background. Livers from donation after circulatory determination-of-death (DCD) donors suffer ischemic injury during a preextraction period of cardiac arrest and are infrequently used for transplantation; they have the potential, however, to considerably expand the donor pool. We aimed to determine whether hypothermic oxygenated machine perfusion would improve or further deteriorate the quality of these livers using a clinically relevant porcine model.Methods. Donor livers were subjected to 90 min of cardiac arrest and preserved at 4 degrees C with either static cold storage using University of Wisconsin solution (CS, n=6) or oxygenated machine perfusion using University of Wisconsin machine perfusion solution and 25% physiological perfusion pressures (HMP, n=5). After 4 hr of preservation, livers were transplanted into recipient pigs, which were followed intensively for up to 5 days.Results. Five-day survival was 0 in CS and 20% in HMP. Immediately after reperfusion, hepatocellular injury and function were improved in HMP versus CS. However, HMP grafts also demonstrated significant endothelial and Kupffer cell injury, and a progressive lesion developed 24 to 48 hr after reperfusion that led to death in all but one of the recipient animals.Conclusions. Although hypothermic oxygenated machine perfusion performed using subphysiological perfusion pressures seems to offer some advantages over cold storage in the preservation of ischemically damaged livers, it simultaneously conditions endothelial and Kupffer cell injury that may ultimately lead to the failure of these grafts.

Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution.Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0-4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis.The re-beating time was significantly shorter in hearts stored in the new solution (10.3±2.6 min vs. HTK 14.2±4.1 min; p0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3±0.4 vs. HTK 1.6±0.5; p0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8±0.2 vs. HTK 2.2±0.7), less interstitial fibrosis (4.8±1.9% vs. HTK 8.5±3.8%, p0.05), less vasculopathy (14.7±6.9% vs. 22.0±23.2%; p=0.06) and lower TGF-β1-expression (6.6±1.4% vs. HTK 12.0±4.6%).The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-L-histidine, glycine, alanine, arginine and sucrose.

Background: Although lung transplantation from donation after cardiac death (DCD), especially uncontrolled DCD, is limited by warm ischemic periods, the molecular mechanism of warm ischemia-reperfusion-injury (IRI) has not been well elucidated. The purpose of this study was to clarify the particular longitudinal mechanisms of molecular factors involved in warm IRI. Methods: Cold ischemic-time (CIT)-group lungs were retrieved and subjected to 3-h of cold preservation, whereas warm ischemic-time (WIT)-group lungs were retrieved after 3-h of warm ischemia. Orthotopic rat lung transplantation was performed and the grafts were reperfused for 1 or 4-h. The graft functions, gene expression, and activation of inflammatory molecules in the grafts were analyzed. Exhaled-carbon-monoxide-concentration (ExCO-C) was measured during reperfusion. Results: Only the WIT-group showed obvious primary graft dysfunction at 1-h reperfusion, but the graft function was recovered during 4-h reperfusion. Most of pro-inflammatory cytokines and stress-induced molecules showed different expression and activation patterns between CIT and WIT groups. In the WIT-group, the expressions of anti-inflammatory molecules, IL-10 and HO-1, were significantly increased at 1-h reperfusion compared to the CIT-group, and these high levels were maintained through 4-h reperfusion. Furthermore, ExCO-C levels in the WIT-group increased immediately after reperfusion compared to the CIT-group. Conclusions: This study indicates that warm IRI may involve a different mechanism than cold IRI and anti-inflammatory pathways may play important roles in the graft recovery after lung transplantation from uncontrolled DCD.

Background. Brain death and cold preservation are major alloantigen-independent risk factors for transplantation Outcome. The present study was conducted to assess the influence of these factors on transplantation-associated injury independently or in combination.Methods. Brain death was induced in F344 rats. Renal grafts were harvested after 6 hr and either directly transplanted in unilateral nephrectomized Lewis recipient or Subjected to 24 hr of cold preservation in University of Wisconsin solution before implantation. Allografts obtained from living donor rats were also subjected to cold preservation or not. DNA damage was assessed before implantation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Ten days after transplantation, renal histology was performed according to Banff '97 classification. The expressions of cytokines and adhesion molecules were analyzed by quantitative polymerase chain reaction.Results. Cold preservation significantly increased the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling positive cells in renal allografts. Ten days after transplantation, histology revealed a higher degree of tubulitis and vasculitis scores when the grafts were Subjected to cold storage. Vasculitis was aggravated when the graft was obtained from brain death (BD) donors. BD, but not cold preservation alone, was associated with papillary necrosis. This was more frequently observed after cold preservation. Immunohistology showed an increase in MHC class II+ cells after cold preservation. The combination of BD and cold preservation revealed a higher degree of VEGF and IL-10 expression.Conclusions. Our Study emphasizes that cold ischemia time should be limited when renal allografts from brain-dead donors are transplanted.

We compared Dulbecco's modified Eagle's medium (DMEM), saline, Euro-Collins (EC) solution and University of Wisconsin (UW) solution to determine which was best for cold preservation of rat osteochondral tissues (OCTs). After 7 days' cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of lactate dehydrogenase released from damaged cells. Histological evaluation revealed chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, particularly in the deeper layer of articular cartilage after preservation in saline and EC solution and predominantly in all layers if preserved in DMEM. In contrast, chondrocyte morphology in all layers of the articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. It is therefore concluded that UW solution is the most suitable for cold preservation of rat OCTs as well as solid organs.

31P-NMR spectroscopy was utilized to investigate rat and porcine pancreatic ATP:P(i) ratios to assess the efficacy of existing protocols for cold preservation (CP) in maintaining organ quality. Following sacrifice, rat pancreata were immediately excised or left enclosed in the body for 15 minutes of warm ischemia (WI). After excision, rat pancreata were stored at 6 degrees C to 8 degrees C using histidine-tryptophan-ketoglutarate solution (HTK) presaturated with air (S1), HTK presaturated with O2 (S2), or the HTK/perfluorodecalin two-layer method (TLM) with both liquids presaturated with O2 (S3). 31P-NMR spectra were sequentially collected at 3, 6, 9, 12, and 24 hours of CP from pancreata stored with each of the three protocols examined. The ATP:Pi ratio for rat pancreata exposed to 15 minutes of WI and stored with S3 increased during the first 9 hours of CP, approaching values observed for organs procured with no WI. A marked reduction in the ATP:Pi ratio was observed beyond 12 hours of CP with S3. After 6 hours of CP, the ATP:Pi ratio was highest for S3, substantially decreased for S2, and below detection for S1. In sharp contrast to the rat model, ATP was barely detectable in porcine pancreata exposed to minimal warm ischemia (15 minutes) stored with the TLM regardless of CP time. We conclude that 31P-NMR spectroscopy is a powerful tool that can be used to (1) noninvasively evaluate pancreata prior to islet isolation, (2) assess the efficacy of different preservation protocols, (3) precisely define the timing of reversible versus irreversible damage, and (4) assess whether intervention will extend this timing.

Protection of intestinal graft mucosa during cold preservation is still an unmet need in clinical practice, thus affecting the success of transplantation. The present study investigates the ability of two ischemic preconditioning (IPC) procedures to limit cold preservation injury. Three groups of Sprague-Dawley rats were recruited (n=11 each) as follows: the short IPC (SIPC) performed through 4 cycles of mesenteric ischemia of 4 min each followed by 10 min of reperfusion, the long IPC (LIPC) obtained by 2 ischemic cycles of 12 min each followed by 10 min of reperfusion, and the control group (C) without IPC. Grafts were then stored in cold histidine-tryptophan-ketoglutarate solution and samples were taken at 0, 3, 6 and 9 h lasting preservation. Both IPC groups showed an advanced degree of preservation with delayed development of graft mucosa damage, mainly in the crypt region. At the beginning of preservation, the graft mucosa in both IPC groups showed lower degree of mucosal injury index (MII) by 50% in comparison with C group. Specifically, a significant improvement of MII was observed after 3h of preservation in the LIPC group (p0.05) in comparison with untreated C grafts. Significant atrophy of the intestinal mucosa in C group was found after 3h of preservation (p0.01), in SIPC group the progress of atrophy was delayed to 6 h (p0.001), and in LIPC group only moderate decrease in that was found. A parallel increase of laminin expression with the MII rate after 6 and 9h of preservation in comparison with the level at time 0 was observed in all grafts (p0.001 and p0.01, respectively). In both IPC groups the apoptotic cell (AC) rate was significantly reduced at the beginning of cold preservation (p0.05 both). Moreover, in both the SIPC and C groups, the progressive increase in MII rate connected with AC rate decrease was due to a predominance of necrosis. By contrast in the LIPC group, after an increase of nearly 50% in the AC rate at the 3rd hour, its level remained fairly constant during the further 6 h of preservation, thus probably preventing necrosis and improving graft viability.

Background— Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Methods and Results— Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4°C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0±2.6% versus 47.7±7.0% in saline control and 48.6±6.1% in vector control, P P c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Conclusions— Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.

Defects in articular cartilage are often repaired with fresh osteochondral grafts. While fresh allografts provide viable chondrocytes, logistic limitations require surgical implantation within seven days of graft harvest. Here, we provide information on cold preservation of whole intact osteochondral materials that retains cartilage cell viability and function, and histologic and biochemical integrity for 28 days. Canine femoral condyles were obtained and stored at 4 degrees C for 14, 21 or 28 days. At the end of the storage period, cartilage was assessed for cell viability, 35S uptake, proteoglycan content and histologic parameters. The most noticeable histologic change was reduced Safranin-O near the cartilage surface with 14 days of cold preservation, but had recovered with 21 and 28 days. Cartilage thicknesses did not vary significantly. Cell viability was >95% at 14 days, 75-98% at 21 days and reduced to 65-90% at 28 days. Cell function measures showed that the level of 35SO4 incorporation was suppressed in samples stored at 4 degrees C. However, no significant differences were seen among groups at 14, 21 or 28 days of cold preservation. This data has implications for tissue banking protocols for osteochondral allograft material obtained for transplantation suggesting that cold preserved allograft material be implanted within 28 days.

This study investigated the effects of prolonged cold preservation and Schwann-cell injection on nerve regeneration through peripheral nerve allografts. Forty rats were randomized to the following groups: group I, isograft; group II, allograft; group III, isograft + Schwann cells; group IV, 6-week cold-preserved allograft; and group V, 6-week cold-preserved allograft with Schwann cells. Nerves from all animals were harvested at 4 weeks after surgery for histological and histomorphometric analysis. Untreated allograft recipients demonstrated poor nerve regeneration and histological evidence of rejection. The remaining four groups showed robust regeneration without evidence of rejection. In a short nerve allograft model, prolonged cold preservation of allografts supported robust nerve regeneration, but the addition of cultured Schwann cells conferred no additional benefit for nerve regeneration. Further work in large animals is needed to establish the role for exogenous Schwann cells in nerve allotransplantation.

Aim The aim of this study was to investigate the safe time limits of cold preservation in University of Wisconsin (UW) solution of liver grafts subjected to warm ischemia (WI) for 30 minutes from non–heart-beating donors (NHBDs). Methods The safe time limits were studied in a simple porcine orthotopic liver transplantation (LTx) model. In donors, livers were subjected to 30 minutes of WI and subsequent 6-hour (Group 1, n = 5), 10-hour (Group 2, n = 5), and 14-hour (Group 3, n = 3) cold preservation in UW solution. Results All 5 animals in Group 1 survived up to 7 days, the survey endpoint. In Group 2, only 2 animals survived to the same survey endpoint. All animals in Group 3 died within 12 hours. The 1-week survival rate of Group 1 was significantly higher than those of the other 2 groups. Group 1 showed a lower level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) after LTx, less pathological damage, higher concentration of adenosine triphosphate (ATP), and higher microcirculation blood flux in the grafted liver tissue at 1 hour after reperfusion than the other 2 groups. Conclusions It is concluded that about 6 hours is the safe time limit of cold preservation in UW solution for liver grafts from NHBDs subjected to WI for 30 minutes.

To define the protective effect of ischemic preconditioning on cold ischemia and reperfusion injury associated with intestinal transplantation, and the role of nitric oxide in this process.Ischemia/reperfusion injury continues to be a significant obstacle in small bowel transplantation. Preconditioning is a mechanism that protects against this injury.To study the capacity of preconditioning to prevent cold ischemia-associated injury and the inflammatory response associated with intestinal transplantation, the authors studied a control group of animals, cold ischemia groups with or without previous preconditioning and with or without previous administration of L-NAME or NONOS, and intestinal transplantation groups with or without previous preconditioning and with or without previous administration of L-NAME or NONOS.Histologic findings and the release of lactate dehydrogenase into the preservation solution showed that preconditioning protects against cold ischemic preservation-associated injury. Preconditioning also prevented the inflammatory response associated with intestinal transplantation, measured by the above parameters and by neutrophil recruitment in the intestine. Inhibition of nitric oxide eliminates the protective effect.Preconditioning protects the intestinal grafts from cold preservation and reperfusion injury in the rat intestinal transplantation model. Nitric oxide is involved in this protection.

Background: Dibutyryl cyclic adenosine monophosphate (db-cAMP) has been shown to protect vascular endothelial cells by increasing the level of intracellular cAMP, and we have previously reported its effectiveness in lung preservation. Here, the effects of db-cAMP in lung preservation were ultrastructurally investigated, and the ultrastructural changes before reperfusion were correlated with pulmonary function after reperfusion. Methods: The lungs of 17 Lewis rats were flushed with perfusate and prostaglandin E1, and were then divided into three groups. In the fresh group (n = 6), the lungs were flushed with extracellular-type trehalose-containing (ET-K) solution and were reperfused immediately. In the control group (n = 6) and db-cAMP group (n = 5), the lungs were flushed with ET-K solution and ET-K solution plus db-cAMP (2 mM), respectively, and were reperfused after cold preservation at 4°C for 15 h. Before reperfusion, tissue was sampled and ultrastructurally analyzed by transmission electron microscopy. Results: In the endothelial cells of pulmonary arterioles, the incidence of protrusion was significantly lower in the fresh and db-cAMP groups than in the control group (p < 0.05). The incidence of detachment and microvillus formation were significantly lower in the fresh and db-cAMP groups than in the control group (p < 0.01). The ultrastructure of the alveoli did not allow separation of the control and db-cAMP groups. The shunt fraction and wet to dry weight ratio of the lung tissue after reperfusion were significantly lower in the fresh and db-cAMP groups than in the control group (p < 0.01). Positive correlations were found between the incidence of these ultrastructural changes in the endothelial cells of the pulmonary arterioles and pulmonary function after reperfusion. Conclusion: These findings suggest that db-cAMP might attenuate the lung injury caused by cold preservation and ischemia-reperfusion, partly by suppressing the acceleration of the structural changes in the endothelial cells in the pulmonary arterioles.

Pyruvate has been shown to prevent intestinal mucosal injury after ischemia-reperfusion. The aim of the present study was to determine whether pyruvate can (1) prevent postreperfusion mucosal injury occurring after intestinal preservation and subsequent transplantation and (2) exert a protective effect on the intestinal graft mucosa during acute rejection. Preservation mucosal injury was evaluated, after 2 hours of reperfusion, by comparing grafts transplanted in a rat syngeneic combination (ACI to ACI) after 2 hours of cold preservation using pyruvate (n = 6) or placebo (n = 6). Mucosal parameters obtained during acute rejection (allogeneic combination: ACI to Lewis) were compared between placebo-treated (n = 6) and pyruvate-treated (nequals 6) animals. Tissue injury was evaluated by histopathologic examination, oxygen free radical production by luminol-enhanced chemiluminescence, and degree of neutrophil infiltration by myeloperoxidase staining. After reperfusion of the preserved grafts and during acute rejection, mucosal oxygen free radical levels and the number of infiltrating neutrophils were significantly (P0.05) increased in the untreated grafts, whereas there was a statistically significant inhibition of these parameters in those treated with pyruvate. Mucosal injury, seen after reperfusion of the preserved grafts, was prevented by pyruvate. The histopathologic abnormalities observed in the untreated grafts during rejection were also significantly reduced by pyruvate. Treatment with pyruvate before cold preservation of intestinal grafts, in this rat model, reduced reperfusion mucosal injury, neutrophil infiltration, and oxygen free radical production. Oxygen free radicals were produced in the mucosa of the graft during acute rejection and their production was reduced by pyruvate, which exerted a protective effect on the rejecting allograft mucosa.

The aim of the present study was to evaluate the potential of Celsior, a recently developed cardioplegic and heart storage solution, to protect the small bowel during ischemic storage. Small bowel segments were isolated from rats, flushed with either UW or Celsior solution, and cold-stored for 18 h at 4 degrees C in the respective solution. After ischemic storage, some preparations were freeze-clamped for analysis of tissue metabolites while other preparations were tested for structural and functional integrity by isolated perfusion in vitro using a previously validated model. After 18 h of ischemic storage no significant differences were seen between Celsior and UW with regard to the development of edema, energy charge, or creatine phosphate, but lactate accumulation was significantly reduced in the Celsior group, although glucose catabolism was not inhibited. Histological evaluation of the cold-stored organs showed no differences with regard to structural integrity between the two groups. Total vascular resistance upon reperfusion was significantly lower in the Celsior group (666 +/- 126 vs 827 +/- 88 MPa s m-3*), as was the intestinal release of LDH (9.7 +/- 4.4 vs 18.2 +/- 4.6 U/l*). Carbohydrate absorption from the intestinal lumen amounted to venous effluent concentrations of 0.58 +/- 0.24 vs 0.18 +/- 0.15 mg% * of galactose in the Celsior and UW groups, respectively. Within the limits of this in vitro pilot study, Celsior provided better postischemic recovery of the small bowel than UW in terms of vascular perfusion characteristics, enzyme release, and carbohydrate absorption and may, thus, be considered a suitable alternative for intestinal organ preservation.

Phosphate-buffered sucrose (PBSc) solution is effective for short-term hypothermic preservation of tissue during feline kidney transplantation. A high-molecular-weight polyethylene glycol (35,000 Da, PEG35) reportedly enhanced the protective effects against cold-induced tubular injuries in animal kidney transplantation models. We investigated the ability of PBSc solution containing PEG35 to preserve cultured feline kidney cells using in vitro WST-8 cell proliferation assays. PEG35 significantly improved cell viability during 24 hr of cold preservation. PBSc containing 20 g/l PEG35 achieved an effect almost equal to that of University of Wisconsin (UW) solution, the gold standard preservation solution used in human clinical kidney transplantation, for up to 24 hr of preservation. Our results suggest that PBSc containing PEG35 provides an excellent medium for graft cold storage during feline kidney transplantation.

International audience; BACKGROUND: In liver transplantation, the donor and recipient are in different locations most of the time, and longer preservation periods are inevitable. Hence, the choice of the preservation solution and the duration of the preservation period are critical for the success of the transplant surgery.OBJECTIVE: In this study, we examine the mechanical and histological properties of the bovine liver tissue stored in Lactated Ringer's (control), HTK and UW solutions as a function of preservation period.METHODS: The mechanical experiments are conducted with a shear rheometer on cylindrical tissue samples extracted from 3 bovine livers and the change in viscoelastic material properties of the bovine liver is characterized using the fractional derivative Kelvin-Voigt Model. Also, the histological examinations are performed on the same liver samples under a light microscope.RESULTS: The results show that the preservation solution and period have a significant effect on the mechanical and histological properties of the liver tissue. The storage and loss shear moduli, the number of the apoptotic cells, the collagen accumulation, and the sinusoidal dilatation increase, and the glycogen deposition decreases as the preservation period is longer.CONCLUSIONS: Based on the statistical analyses, we observe that the liver tissue is preserved well in all three solutions for up to 11 h. After then, UW solution provides a better preservation up to 29 h. However, for preservation periods longer than 29 h, HTK is a more effective preservation solution based on the least amount of change in mechanical properties. On the other hand, the highest correlation between the mechanical and histological properties is observed for the liver samples preserved in UW solution.

Background 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. Methods 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. Results 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. Conclusions Edaravone may improve the viability of liver grafts from NHBDs.

The purpose of this study was to compare University of Wisconsin (UW) solution with Euro-Collins (EC) solution in their cold preservation effects on rat limbs. Thirty-six Lewis rat limbs were preserved in EC solution (n=18) or UW solution (n=18) at 4 degrees C for 72 hr, and grafted orthotopically to a syngeneic rat using microsurgical techniques. The surgeon was blinded to the solution used. We evaluated the vascular patency rate and death rate of both groups at day 7 after surgery and performed histological evaluations of bone, muscle, growth plate, and articular cartilage for each specimen of successful grafts in both groups. The vascular patency rates of the EC and UW groups were 27.7% (5/18) and 11% (2/18), respectively, and showed no significant difference. The death rates of the EC and UW groups were 50% (9/18) and 60% (10/18), which were not significantly different. There were no clear differences in histological viability between both groups, in all tissues exclusive of bone marrow and muscle tissue. Our results showed that in comparing EC and UW solutions, one was not significantly superior to the other as a cold immersion storage medium after a 72 hr ischemia-induced reperfusion injury.

Serum-free preservation media such as University of Wisconsin (UW) may cause tissue damage through trophic factor (TF) deprivation. This study evaluated whether the addition of TFs to UW solution improves liver graft quality after extended cold preservation time in pigs. UW solution was supplemented with epidermal growth factor, insulin-like growth factor-1, nerve growth factor-beta, bactenecin, and substance P to create TF-supplemented (TFS) UW. Orthotopic liver transplantation was performed after 18 hr of static cold storage at 4 degrees C in UW (n=7) or TFS-UW (n=7) solution. Recipients of grafts preserved with TFS-UW demonstrated significantly better 5-day survival (57%) than those preserved with UW alone (14%) (P

1.Human and animal precision-cut organ slices are being widely used to obtain drug metabolism and toxicity profiles in vitro. These data are then used to predict what might be seen in human patients. The accuracy of this prediction and extrapolation of the findings based on human or animal in vitro systems to the findings that occur in vivo is dependent on both the quality of the tissue itself and the quality of the in vitro system. 2.The quality of human organs used in research is dependent on procurement methods, warm ischaemia time, preservation solutions, cold ischaemia time, and donor-specific factors. It is important to confirm that the organs being used are highly viable and fully functional before using them in scientific studies. 3.The optimal preparation and incubation of organ slices is also essential in maintaining slice viability and function. It is important to prepare the slices in a cold preservation solution, to prepare the slices at a correct thickness, and to incubate the slices in a system where the slice rotates in out of the oxygen atmosphere and medium. 4.Meeting the criteria outlined here will lead to successful organ slice cultures for investigating drug-induced mechanisms and organ-specific toxicity.

BACKGROUND The aim of the present study was to evaluate the effect of donor pre-treatment with atorvastatin in a model of brain death followed by prolonged cold preservation and allogeneic kidney transplantation in rats. MATERIAL/METHODS Donor rats were pre-treated with atorvastatin or vehicle 2 days prior to induction of brain death. After a brain death period of 6h kidneys were explanted and stored for 24 h at 4°C in UW solution and transplanted into allogeneic recipients. Non brain dead rats treated with vehicle were ventilated for 6h prior to explantation and served as controls. Grafts were harvested after 10 days. RESULTS Donor treatment of brain dead organ donors with atorvastatin had no influence on renal histology (Banff score) or renal inflammation compared to vehicle treated brain dead rats 10 days after cold preservation and allogeneic transplantation. Grafts from brain dead organ donors showed severe signs of vasculopathy compared to grafts from non brain dead organ donors 10 days after prolonged cold preservation and allogeneic kidney transplantation. Kidneys harvested after brain death before cold preservation and allogeneic transplantation showed an increased infiltration of ED1 and MHCII positive cells compared to kidneys of non-brain dead animals (NBD). CONCLUSIONS Atorvastatin donor pre-treatment of brain dead organ donors combined with 24h of cold preservation has no influence on graft rejection and infiltration with ED1 or MHCII positive cells in an allogeneic rat renal transplantation model. The detrimental combination of brain death and cold preservation might have overcome the possible protective effect of atorvastatin donor pre-treatment.

The relation between the level of cellular adenine nucleotides and graft viability after cold preservation and warm ischemia was studied by using a rat small-bowel transplantation model. The rat jejunum was preserved in lactated Ringer's solution at 4 degrees C for 120 min, and was then transplanted syngeneically under warm ischemia after between 60 and 180 min. The recovery levels of ATP and the energy charge at 30 min after reperfusion in the viable graft were significantly higher than those in the nonviable graft. None of the grafts were viable when the recovery level of ATP at 30 min after reperfusion was less than 2.8 mumol/g dry weight. These results suggest that the recovery level of ATP at 30 min after reperfusion is a reliable parameter for graft viability in small-bowel transplantation.

Cold preservation of isolated mammalian hearts attenuates cardiac contractility. We examined the effect of intracellular and extracellular Ca ion on the contractility of cold-preserved atria of rat. Rat hearts were isolated and stored in Collins' solution at 4 degrees C for either 0 or 12 h. Mechanical responses were then examined in electrically driven left and beating right atrial muscle preparations. When stimulus frequencies were increased stepwise from 0.1 to 3 Hz, the twitch contractions initially decreased (Woodworth phenomenon: WP) and then increased (Bowditch phenomenon: BP) in 0 hour-preserved (fresh) left atrial preparations. However, BP, but not WP, was abolished in 12 hour-preserved preparations. The absence of BP was also observed when fresh preparations were exposed to a low-Ca medium (0.9 mM). Ryanodine (3 x 10(-8) M) abolished the twitch contractions at low stimulus frequencies, but did not inhibit BP in fresh preparations. In 12 hour-preserved preparations, both ryanodine and low-Ca medium completely inhibited the twitch contraction. The positive inotropic effects of isoproterenol in right and left atria were markedly attenuated after 12-hour cold preservation. However, the rate of spontaneous beating and the positive chronotropic effect of isoproterenol in right atria were not changed in the cold-preserved preparations or in fresh preparations that had been treated with ryanodine or exposed to a low-Ca medium. These results clearly show that cold-preserved atrial muscle is more susceptible to changes in intracellular and extracellular Ca ion and suggest that the attenuation of cardiac contractility by cold preservation may be due to the impairment of Ca2+ influx which may result from membrane deterioration.

To determine the tolerance of old livers to prolonged preservation, livers from aged rats (corresponding to humans in the sixth to seventh decades of life) were transplanted after specific periods of cold preservation. Male BN/BiRij rats received orthotopic, arterialized liver grafts from either young (5 months) or old (25 to 28 month) donor rats after liver storage for 12 (n = 6), 24 (n = 6) or 30 (n = 10) hours in University of Wisconsin solution. Outcome was assessed by survival, liver enzymes after transplantation, and histology of the grafts. There were no significant differences in survival rates between recipients of old and young grafts. All rats survived after 12-hr and 24-hr preservation except one recipient of an old graft preserved for 24 hr. After 30-hr preservation recipients of old and young livers had identical survival rates (60%). There was a strong correlation between the highest postoperative AST and ALT and the duration of preservation in all groups (P < 0.0001), but only in the 24-hr preservation experiments was the ALT significantly higher in recipients of old grafts than in recipients of young livers (P = 0.025). Age of the donor did not significantly affect the peak AST, but there was a correlation between donor age and the highest postoperative ALT (P = 0.007). Although intracellular vacuolization was a prominent histological finding in more than half of the old livers at the end of preservation, it was not associated with an increase in mortality. It is concluded that under the ideal conditions provided in the experiments, old rat livers tolerate long preservation periods with satisfactory graft survival compared with young livers.

Background The ex vivo lung perfusion (EVLP) system has been used successfully to assess donor lungs. Perfadex (PX) is usually the flush and preservation solution in EVLP systems. We have used the extracellular-type–Kyoto (ET-K) solution containing 44 mEq/L potassium for clinical lung transplantation, investigating whether it rather than PX affects the EVLP system. Methods We used domestic slaughterhouse pigs to analyze the EVLP system. After 20-minute warm ischemia and 6-hour cold ischemia, EVLP was performed for 2 hours. Pig heart-lung blocks were divided into the PX (n = 5) and ET-K (n = 5) groups depending on the flush/cold preservation solution. At the beginning, we discarded the first 100 mL of effluent in the PX group and the first 200 mL in the ET-K group. We measured pulmonary physiological data and potassium levels. Results In both groups, perfusion for 2 hours showed no differences between the 2 groups with respect to the final flow, pulmonary arterial pressure, pulmonary vascular resistance, PaO2/FiO2, and shunt fraction. The potassium level in the perfusate was 4.4 mEq/L for the PX and 5.4 mEq/L for the ET-K group. Conclusion The pig EVLP system was not affected when ET-K was used instead of PX as the flush/preservation solution. The initial 200 mL of effluent should be discarded when using the ET-K to ensure that the potassium level does not increase.

Introduction Despite significant advances, widespread applicability of islet cell transplantation remains elusive. Refinement of current islet isolation protocols may improve transplant outcomes. Islet purification by magnetic separation has shown early promise. However, surgical protocols must be optimized to maximize the incorporation of paramagnetic microparticles (MP) within a greater number of islets. This study explores the impact of MP concentration and infusion method on optimizing MP incorporation within islets. Methods Five porcine pancreata were procured from donors after cardiac death. Splenic lobes were isolated and infused with varying concentrations of MP (8, 16, and 32 × 10 8 MP/L of cold preservation solution) and using one of two delivery techniques (hanging bag versus hand-syringe). After procurement and infusion, pancreata were stored at 0°C to 4°C during transportation (less than 1 hour), fixed in 10% buffered formalin, and examined by standard magnetic resonance imaging (MRI) and histopathology. Results T2*-weighted MRI showed homogeneous distribution of MP in all experimental splenic lobes. In addition, histologic analysis confirmed that MP were primarily located within the microvasculature of islets (82% to 85%), with few MP present in acinar tissue (15% to 18%), with an average of five to seven MP per islet (within a 5-μm thick section). The highest MP incorporation was achieved at a concentration of 16 × 10 8 MP/L using the hand-syringe technique. Conclusion This preliminary study suggests that optimization of a surgical protocol, MP concentrations, and applied infusion pressures may enable more uniform distribution of MP in the porcine pancreas and better control of MP incorporation within islets. These results may have implications in maximizing the efficacy of islet purification by magnetic separation.

Background Hepatic ischemia-reperfusion injury (I/Ri) is a serious complication occurring during liver surgery that may lead to liver failure. Hepatic I/Ri induces formation of reactive oxygen species, hepatocyte apoptosis, and release of pro-inflammatory cytokines, which together causes liver damage and organ dysfunction. A potential strategy to alleviate hepatic I/Ri is to exploit the potent anti-inflammatory and cytoprotective effects of carbon monoxide (CO) by application of so-called CO-releasing molecules (CORMs). Here, we assessed whether CO released from CORM-2 protects against hepatic I/Ri in a rat model. Methods Forty male Wistar rats were randomly assigned into four groups (n = 10). Sham group underwent a sham operation and received saline. I/R group underwent hepatic I/R procedure by partial clamping of portal structures to the left and median lobes with a microvascular clip for 60 minutes, yielding ~70% hepatic ischemia and subsequently received saline. CORM-2 group underwent the same procedure and received 8 mg/kg of CORM-2 at time of reperfusion. iCORM-2 group underwent the same procedure and received iCORM-2 (8 mg/kg), which does not release CO. Therapeutic effects of CORM-2 on hepatic I/Ri was assessed by measuring serum damage markers AST and ALT, liver histology score, TUNEL-scoring of apoptotic cells, NFkB-activity in nuclear liver extracts, serum levels of pro-inflammatory cytokines TNF-α and IL-6, and hepatic neutrophil infiltration. Results A single systemic infusion with CORM-2 protected the liver from I/Ri as evidenced by a reduction in serum AST/ALT levels and an improved liver histology score. Treatment with CORM-2 also up-regulated expression of the anti-apoptotic protein Bcl-2, down-regulated caspase-3 activation, and significantly reduced the levels of apoptosis after I/Ri. Furthermore, treatment with CORM-2 significantly inhibited the activity of the pro-inflammatory transcription factor NF-κB as measured in nuclear extracts of liver homogenates. Moreover, CORM-2 treatment resulted in reduced serum levels of pro-inflammatory cytokines TNF-α and IL-6 and down-regulation of the adhesion molecule ICAM-1 in the endothelial cells of liver. In line with these findings, CORM-2 treatment reduced the accumulation of neutrophils in the liver upon I/Ri. Similar treatment with an inactive variant of CORM-2 (iCORM-2) did not have any beneficial effect on the extent of liver I/Ri. Conclusions CORM-2 treatment at the time of reperfusion had several distinct beneficial effects on severity of hepatic I/Ri that may be of therapeutic value for the prevention of tissue damage as a result of I/Ri during hepatic surgery.

Evidence for in vivo antioxidative activity of reduced CoQ homologs has been presented. This came from studies with experimental endotoxemia in mice, reoxygenation of rat liver following ischemia, and reoxygenation of canine heart following 24-hour cold preservation. In radical-induced injury of hepatocytes, it has been first shown that reduced CoQ9 acts as a potential antioxidant regardless of its cellular concentration, whereas reduced CoQ10 acts in cells containing CoQ10 as the predominant homolog. The antioxidant activity of reduced CoQ homologs appears to be independent of that of alpha-tocopherol under the conditions employed.

Brain death (BD) is associated with tissue inflammation. As dopamine treatment of BD donor rats reduces renal monocyte infiltration, we tested if this treatment affects renal function and inflammation in recipients. BD was induced in F344 rats and was maintained for 6 h in all experiments. Dopamine was given for 6 (DA6) or 3 h (DA3) from the onset of BD. Ventilated non-BD (NBD) and BD animals served as controls. Kidneys were transplanted into bilaterally nephrectomized Lewis recipients. Serum creatinine (s-crea) was measured and leukocyte infiltration was assessed 10 days after transplantation. One day after transplantation, s-crea was significantly reduced in recipients who received a renal allograft from dopamine treated BD or from NBD rats compared to BD vehicle (P

Endothelial barrier dysfunction severely compromises organ function after reperfusion. Because dopamine pretreatment improves hypothermia mediated barrier dysfunction, we tested the hypothesis that dopamine treatment of lung allografts positively affects tissue damage associated with hypothermic preservation and reperfusion.Rats were treated for 1 hr with dopamine (5 microg/min/kg) or vehicle (NaCl). Thereafter lungs were explanted, flushed with Perfadex solution and stored at 4 degrees C for different time periods. Peak inspiratory pressure (PIP), pulmonary arterial pressure (PAP), and lung weight were measured online during reperfusion. Inflammatory mediators in the perfusate and the expression of adhesion molecules in situ were measured after perfusion.Lungs could tolerate a cold ischemia time of up to 6 hr with stable PIP, PAP, and no edema formation upon reperfusion. Cold ischemia time above 6 hr significantly increased PIP, PAP, and pulmonary edema in untreated but not in dopamine treated lungs (Por =0.001 dopamine treated vs. untreated). Perfusion and ventilation alone induced a strong up-regulation of cytokine-induced neutrophil chemoattractant-1 and adhesion molecules in untreated lungs, whereas in dopamine treated lungs significantly lower levels were found. Dopamine treatment also inhibited tissue damage associated with hypothermic preservation as measured by nicotinamide adenine dinucleotide staining.Our study suggests that donor dopamine treatment is a highly effective modality to maintain organ quality of lung allograft. These findings are of high clinical relevance because prevention of tissue damage might reduce complications associated with lung transplantation and hence improve graft survival in lung transplant recipients.

Cold preservation prior to small-bowel transplantation can moderate tissue injury, although it is unable to protect the bowel graft from acute reperfusion injury. One method to reduce oxidative stress is ischemic preconditioning (IPC). The limited data regarding IPC of the bowel encouraged us to investigate the key factor in this process, i.e., the activation of nuclear factor-kappa binding (NF-kB) in bowel tissue. The intestine was preconditioned by various cycles of IPC on rats. Activation of NF-kB was detected by a chemiluminescence-based ELISA method. Our findings showed that NF-kB level was elevated significantly 30 min after IPC. One hour following IPC, NF-kB decreased to control level; 2 h after IPC, the level significantly increased again. These changes were independent of the number of IPC cycles. Our experiments with various IPC cycles revealed that even a very short IPC cycle was able to activate the IPC cascade in small-bowel tissue.

During hypothermic storage of the cornea, corneal endothelial damage restricts storage times. We previously reported a new, iron-dependent mechanism of cold-induced injury to cultured liver cells. In this study, we sought to evaluate whether corneal endothelial cells incur a similar kind of injury.Cultured porcine corneal endothelial cells were exposed to 4 degrees C in either cell culture medium, Krebs-Henseleit buffer, Optisol-GS solution, or McCarey-Kaufman medium for 5 hours to 14 days and then rewarmed under cell culture conditions (3 hours). The cultures were assessed for lethal cell injury (LDH release); cellular, nuclear, and mitochondrial morphologic alterations; lipid peroxidation; and mitochondrial membrane potential.Corneal endothelial cells sustained substantial injury following cold storage and rewarming in cell culture medium (47% +/- 8% and 64% +/- 20% cell death after 2 and 5 days cold storage, respectively). The injury displayed some apoptotic features, and cells lost mitochondrial membrane potential before cell death occurred. The iron chelators deferoxamine, 1,10-phenanthroline, and 2,2'-dipyridyl and the antioxidant butylated hydroxytoluene completely inhibited this cell injury. Marked iron-dependent cell injury and lipid peroxidation also occurred during and after cold incubation in Krebs-Henseleit buffer and, most importantly, iron-dependent cell injury was also observed after cold incubation in Optisol solution and in McCarey-Kaufman medium.Cultured porcine corneal endothelial cells incur a strong iron-dependent injury elicited by hypothermia. This cold-induced injury might provide an explanation for the known corneal endothelial susceptibility to hypothermic preservation injury, which thus might be amenable to therapeutic interventions (ie, by iron chelators).