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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.

In liver transplantation primary graft nonfunction (PGN) occurs in 10–15% of all cases. The cause of PGN is likely associated with free radical and lipid peroxidation at reperfusion, however, the mechanism of PGN is still unknown. We investigated in rats the electron leakage of mitochondria, i.e., free radical generation, and lipid peroxidation of mitochondria after cold ischemia to clarify the mechanism of PGN. After cold ischemia, generation of free radical was recognized, but decreased in proportion to cold ischemic time. The chemiluminescence value increased slightly after 8 h of cold ischemia, however, thereafter gradually decreased. Mitochondrial ATP synthesis also decreased in proportion to cold ischemic time. From these results we concluded that free radical generation occurs while the mitochondria maintain energy synthesis and the generation of free radical decreases with cold preservation. Although mitochondria with cold ischemia suffer lipid peroxidation with ease, leaked electrons from the mitochondria are not a main cause of the lipid peroxidation of mitochondria at reperfusion.

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.

The beneficial effects of (-)-epigallocatechin-3- O-gallate (EGCG) on the nonfrozen preservation of mammalian cells and tissues are generally not well understood. A storage solution containing EGCG was employed to test the hypothesis that EGCG is capable of extending the storage duration for the cold preservation of articular cartilages. Human articular cartilages were preserved in a storage solution composed of serum-free RPMI-1640 medium with 1% antibiotic-antimycotic solution and 1 mM EGCG at 4°C for 1, 2, and 4 weeks. The chondrocyte viability (CCK-8 assay), biochemical and immunohistochemical composition [glycosaminoglycans (GAG) and (type II) collagen], and biomechanical property (compressive elastic modulus) were assessed. The chondrocyte viability of the cartilages preserved with EGCG was significantly well maintained for at least 2 weeks with high content of GAG and total collagen. These beneficial effects of EGCG were confirmed by the immunohistochemical observations of well-preserved cartilaginous structures and delayed denaturation of the extracellular matrix in preserved cartilages. There was no significant difference in the compressive elastic modulus (MPa) between the cartilages preserved with and without EGCG. These results suggest that EGCG may play an effective role in preserving osteochondral allografts, which can be exploited in devising strategies for the long-term preservation of other tissues under cold storage conditions.

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.

Coskun, Abdurrahman/0000-0002-1273-0604; WOS: 000280566800019 Aim: To investigate the effect of melatonin added to preservation solution on liver cold ischemia Materials and methods: Twenty male Wistar albino rats were divided equally into groups treated with University of Wisconsin (UW) solution with and without melatonin The LDH, AST, and ACP activities of the preservation solutions were determined in samples collected at 2, 24, 36, and 48 h Tissue malondialdehyde and heat shock protein (HSP 70) levels were also measured and a histological examination was performed at 48 h Results: Melatonin prevented enzyme elevation, induced heat shock protein (HSP 70), and decreased lipid peroxidation effectively in comparison to the control group (P < 0.05) The histological examination revealed that UW solution containing melatonin prevented Kupffer cell activation and inflammation significantly (P < 0.05) Conclusion: Melatonin in preservation solutions such as UW solution may protect tissue effectively from damage during cold ischemia for up to 48 h Abant Izzet Baysal University Department of Scientific and Research Projects, Bolu, TurkeyAbant Izzet Baysal University [2005-07-01-236] This study was supported by Abant Izzet Baysal University Department of Scientific and Research Projects (2005-07-01-236), Bolu, Turkey.

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.

Introducción: Los resultados de los trasplantes efectuados con donantes con criterios expandidos (DCE) son inferiores a los obtenidos con donantes con criterios estándar (DCS). Para optimizar su evolución, se podría reducir su tiempo de isquemia fría (TIF) reduciendo su daño de preservación. Comparamos los resultados obtenidos al aplicar TIF < 15 horas tanto a DCE como a DCS. Material y métodos: Realizamos un estudio unicéntrico, de cohortes, prospectivo, de casos incidentes de trasplante renal de cadáver entre junio de 2003 y diciembre de 2007. El tiempo mínimo de seguimiento fue de 12 meses. Comparamos los datos de los donantes, de los receptores y de la evolución de los trasplantes efectuados con DCE frente a los de los DCS. Resultados: El TIF para los DCE (N = 24) y para los DCS (N = 50) fue, respectivamente, de 9,3 ± 2,5 y 8,3 ± 3,3 horas (p = 0,18). No encontramos diferencias significativas entre los receptores de DCE y DCS en cuanto a: no función primaria del injerto 4,2 vs. 4%, retardo en la función del injerto 16,7 vs. 10%, complicaciones quirúrgicas 25 vs. 16% y rechazos agudos 8,3 vs. 2%. El filtrado glomerular estimado al año para los DCS fue de 65,8 ± 14,9 ml/min y para los DCE de 49,4 ± 12,5 ml/min (p < 0,0001). La supervivencia renal al año fue del 95,8% para los receptores de DCE y del 94% para los DCS (p = 0,75). Conclusiones: La aplicación de TIF cortos a los DCE permite conseguir una evolución similar a la de los DCS, aunque su función renal sea en todo momento inferior. Introduction: Outcome of renal transplant from expanded criteria donors (ECD) is usually inferior than those from standard criteria donors (SCD) and may be improved decreasing cold ischemia time (CIT) and minimizing preservation injury. We compare the results obtained with CIT < 15 hours in kidney transplants from ECD vs. SCD. Subjects and Methods: Prospective, single center study of kidney transplants performed since June 2003 to December 2007. Minimum follow-up period was 12 months. Data of donors, receptors and transplant outcome from ECD and SCD are compared. Results: CIT (mean ± SD) was 9.3 ± 2.5 hours in transplants from ECD (n = 24) and 8.3 ± 3.3 hours in those from SCD (N = 50), p = 0.18. We did not find significant differences among recipients of grafts from ECD and those from SCD regarding: primary non-function (4.2% vs. 2%, respectively), delayed graft function (16.7% vs. 10%), surgical complications (25% vs. 16%) or acute rejection episodes (8.3% vs. 2%). Glomerular filtration rate at one year follow-up was 65.8 ± 14.9 ml/min in ECD recipients and 49.4 ± 12.5 ml/min (p < 0.0001). One year graft survival was 95.8% in ECD recipients and 94% in SCD recipients (p = 0.75). Conclusions: Short CIT in kidney transplant from ECD leads to similar outcome than that obtained from SCD, although renal function is inferior in ECD grafts.

Chilling temperatures (0–15°C) disrupt the metabolism of plants, resulting in abnormal growth and development and, often, in the death of the plant. Sensitivity to chilling is a major constraint in the production and cold preservation of crops, but the metabolic origin of sensitivity to chilling is not clear and, consequently, the phenomenon is not easily controlled. However, recent data suggest that chilling may destabilize the assembly and function of biopolymers by disrupting macromolecule-water interactions. A similar explanation may account for the altered properties and quality of food crops that have been chilled.

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.

Background: After thoracic and abdominal organ dissection, a preservation solution is chosen and preparation for organ perfusion is started. Cold preservation solution is taken from the transport box or refrigerator and afterwards, the surgeon, together with the transplant coordinator and the scrub nurse, prepares the abdominal aorta perfusion system and also, if necessary, the inferior vena cava (IVC) decompression system. At the same time, the thorax team performs similar preparations. The IVC decompression system is not necessary in every case of multi-organ procurement, because the IVC could be also vented into the thorax.

Donors after cardiac death (DCD) suffer irreversible cardiac arrest prior to donation. We describe our liver transplant experience with DCD whose cardiac arrest is unexpected, not following the removal of ventilatory support, whom we maintain with normothermic extracorporeal membrane oxygenation (NECMO). A potential donor goes into cardiac arrest outside the hospital and is brought to the hospital under continuous cardiopulmonary resuscitation (CPR). The donor is declared dead and placed on a cardiocompressor. Femoral vessels are cannulated and connected to cardiopulmonary bypass (CPB) to establish NECMO. Blood parameters and CPB pump flow are monitored throughout NECMO, which is continued until cold preservation. From April 2002 to May 2006, 10 of 40 potential DCD livers were transplanted. Only one graft was lost to primary nonfunction (PNF) and another to hepatic artery thrombosis. Posttransplant hepatic function was good. Certain parameters, such as CPR and NECMO times, hepatic transaminases during NECMO, and donor age, determined the viability of DCD liver grafts and were used to establish criteria for their acceptance. Though considered marginal, unexpected DCD can represent an important source of viable livers for transplant if strict acceptance criteria are employed and they are maintained with NECMO prior to recovery.

A microbial survival curve is constructed by plotting the number of recoverable cells or their logarithm vs. the exposure time to the hostile agent, be it high or low temperature, a chemical preservative or disinfectant, etc. Since the recovery is usually done in a medium and under conditions that favor growth, the result is insensitive to whether the counted survivors are intact or injured. If or when both the total number of survivors and those remaining intact follow a Weibullian decay pattern (with different parameters), then the momentary number of injured cells will be the momentary difference between the two. Such a scenario can be easily modeled mathematically and the resulting model enables to simulate a variety of survived-injury patterns in thermal and non-thermal food preservation processes. Under certain conditions according to this model, almost all the survivors would be injured to at least some extent and hence may perish during the food's storage and transportation. Isothermal survival-injury curves generated with the Weibullian model based on the above considerations were in general agreement with published experimental data. In principle, the methodology can be extended to simulate mortality-injury patterns under dynamic conditions, i.e., when the temperature or chemical agent's concentration vary with time. Whether a cell is considered injured depends on the recovery method, e.g., on whether it can or cannot grow in a saline medium. Thus recovery in different media may yield somewhat different quantitative results but very unlikely a qualitatively different pattern. Although the model used was based on that microbial mortality and injury both follow the Weibullian model, very similar results would have been obtained had other survival modes been assumed.

The scientific basis of bone transplantation was established during the mid-nineteenth century, when the osteogenic properties of the bone and periosteum, beneficial influence of cold preservation of bones, and first conventional attempt to store bones for elective use, were described. A bone bank has several advantages, such as the immediate availability of grafts in large quantities, and different shapes and sizes, and maintenance of osteoinductive activity in grafts. In addition, it reduces patient blood loss, surgical time, and quantity of anesthetics required. Clinical applications include the correction of comminuted fractures, treatment of non-union bones, and replacement of bone loss due to infections or malignancies. The success of these procedures depends on the preservation and integrity of the graft. This study aimed to evaluate the feasibility of maintaining a canine bone bank (Canis familiaris) by quantifying the bacterial contamination of bone grafts preserved at –24°C. The samples were evaluated through a monthly assessment of bacteriological cultures over a period of 6 months. The harvest method was efficient and sterile, reducing the risk of contamination. We conclude that the techniques chosen for the implementation of a bone bank were effective and feasible (use of a common freezer reaching a temperature below –24°C). The viability of the bones was attested during a 6-month period, and the samples demonstrated a 100% sterility rate.

Mammalian cells appear to be naturally tolerant to cold temperatures, but the formation of ice when cells are cooled leads to a variety of damaging effects. The study of cryo-injury, therefore, becomes the study of when and how ice is formed both inside and outside the cell during cooling. Protectant chemicals are used to control or prevent ice formation in many preservation protocols, but these chemical themselves tend to be damaging. Cooling and warming rates also strongly affect the amount and location of ice that is formed. Through careful modification of these parameters successful cold preservation techniques for many cell types have been developed, but there are many more cell types that have defied preservation techniques, and the extension of cell-based techniques to tissues and whole organs has been very limited. There are many aspects to the damaging effects of ice in cells that are still poorly understood. In this brief article we review our current understanding of cellular injury and highlight the aspects of cellular injury during cryopreservation that are still poorly understood.

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).

In May 2003, at Indiana University, the standard cold preservation solution University of Wisconsin (UW) solution was replaced by histidine-tryptophan ketogluatarate (HTK) solution. Earlier, we presented our initial experience with HTK in pancreas preservation with an analysis of the first 10 pancreas transplants. Here we report updated results with HTK in pancreas transplantation over the past 18 months. Between May 2003 and March 2005, a total of 87 pancreas transplants were performed with 78 of these organs utilizing HTK. Seventy five patients received 78 organ transplants. Surgical procedures performed were: simultaneous kidney pancreas transplantation (n = 50, 64%), pancreas after kidney transplantation (n = 19, 24%), solitary pancreas transplantation (n = 9, 12%). Donor and recipient data were collected with primary outcomes as primary nonfunction and 30-day graft and patient survivals, and compared to the UW cohort from our original report. Donor and recipient demographics were similar. Mean follow-up time is 12 +/- 6 months. The mean cold ischemia time was 9 +/- 3 hours. There were no cases of primary graft nonfunction. Thirty-day and 1-year patient survivals were 99% and 93%. The 30-day and 1-year graft survivals were 96% and 93%. There were five grafts lost, including three within the first month (two venous and one arterial thrombosis). There was one case of chronic rejection and one noncompliance. All other patients were insulin-independent by discharge. Serum fasting blood glucose and serial amylase remained comparable at all intervals posttransplantation. Within this range of cold ischemia time, HTK appears to provide effective pancreas preservation.

In the case of living tissues, the spectral width of the electrical bioimpedance dispersions (closely related with the alpha parameter in the Cole equation) evolves during the ischemic periods. This parameter is often ignored in favor of other bioimpedance parameters such as the central frequency or the resistivity at low frequencies. The object of this paper is to analyze the significance of this parameter through computer simulations (in the alpha and beta dispersion regions) and to demonstrate its practical importance through experimental studies performed in rat kidneys during cold preservation. The simulations indicate that the dispersion width could be determined by the morphology of the extra-cellular spaces. The experimental studies show that it is a unique parameter able to detect certain conditions such as a warm ischemia period prior to cold preservation or the effect of a drug (Swinholide A) able to disrupt the cytoskeleton. The main conclusion is that, thanks to the alpha parameter in the Cole equation, the bioimpedance is not only useful to monitor the intra/extra-cellular volume imbalances or the inter-cellular junctions resistance but also to detect tissue structural alterations.

Non-heart-beating donors sustain an ischemic insult of unknown severity and duration, which can compromise the viability of the graft. This preliminary study aimed to assess whether electrical bioimpedance monitoring of cold preserved organs could be useful to identify kidneys that have suffered previous warm ischemia (WI). Two rat groups were studied during 24 h of preservation in University of Wisconsin solution (UW): a control cold ischemia group and another group subjected previously to 45 min of WI. Multi-frequency bioimpedance was monitored during preservation by means of a miniaturized silicon probe and the results were modeled according to the Cole equation. Tissular ATP content, lactate dehydrogenase in UW solution and histological injury were assessed. Renal function and cell injury, evaluated during 3 h of ex vivo reperfusion using the isolated perfused rat kidney model, demonstrated differences between groups. Bioimpedance results showed that the time constant and the high frequency resistivity parameters derived from the Cole equation were able to discriminate between groups at the beginning of the preservation (Δτ ∼ 78%, ΔR∞ ∼ 36%), but these differences tended to converge as preservation time advanced. Nevertheless, another of the Cole parameters, α, showed increasing significant differences until 24 h of preservation (Δα ∼ 15%). © 2004 Elsevier B.V. All rights reserved., This work was supported by the European Commission through projects ESPPRIT-LTR-23485, IST-1999-13047 and QLK6-CT-2000-00064 and by the Spanish government through projects TIC98-1634-CE, TIC2000-2486-CE, FISS 01/1691 and SAF 2000-3090-CE, SAF 2003-04225 CE. Meritxell Genescà was supported by a grant from IDIBAPS

Decreasing numbers of organ donors lead to the use of critical organs in transplantation surgery with the consequence of an increased risk of primary graft failure or dysfunction. The improvement of organ preservation could result in amelioration of graft quality. As endotoxin pretreatment was reported to protect against hepatic warm ischemia-reperfusion injury, the aim of the present study was the to evaluate the effect of low dose LPS on liver cold preservation-reperfusion injury. Male Sprague-Dawley rats were pretreated with 0.5 mg/kg bw LPS; after 24 h livers were harvested and subjected to 24 h of cold preservation in histidine-tryptophan-ketoglutarate (HTK) solution and in vitro reperfusion with Krebs-Henseleit buffer at 37 °C for 2 h in a non-recirculating fashion. Apoptotic cell death was evaluated by means of intravital fluorescence microscopy. Hepatocellular function, integrity and metabolism were assessed by determination of bile flow, oxygen consumption, carbon dioxide production, K+-efflux and enzyme release. Trypan blue perfusion allowed for assessment of cell viability.

Cold preservation of the liver before transplantation may change uptake and excretory functions of hepatocytes. We hypothesized that an increase in the duration of preservation would result in a progressive decrease in the hepatic uptake and/or biliary excretion of indocyanine green (ICG), which would be attenuated by pharmacologic interventions.Donor rats (n = 40) were administered saline (control) or single 5 mg/kg doses of methylprednisolone (MP) or its liver-targeted prodrug (DMP) 2 h prior to liver harvest. Following preservation in cold University of Wisconsin solution for 0, 24, 48, or 72 h, livers were reperfused in a single-pass manner for 30 min in the presence of ICG (approximately 4 microg/ml), followed by 60 min of ICG-free perfusion. The inlet, outlet, and bile concentrations of ICG were measured periodically by high performance liquid chromatography (HPLC), and kinetic parameters were estimated.Effects of duration of preservation: In unpreserved livers, a significant portion of ICG dose (16%) was effluxed from the liver during the washout period. Cold preservation for 24-72 h progressively increased (p0.05) the efflux of ICG (2-fold at 72 h). Similarly, average extraction ratio showed a modest (30-40%) decrease with increasing preservation time (p0.05). However, biliary excretion of ICG showed the most sensitivity to the preservation time (14 to800-fold decline). Effects of pretreatment: DMP caused significant (p0.05) increases in biliary ICG levels (12-fold) and bile flow rates (6-15-fold) of preserved livers. Although MP pretreatment significantly (p0.05) increased (6-fold) bile flow rates in 48-h preserved livers, its effects on biliary ICG levels were not significant (p0.05).Biliary excretion of ICG is the most sensitive kinetic parameter to prolonged cold ischemia-reperfusion injury in a rat liver perfusion model. The injury may be significantly attenuated by pharmacologic pretreatment of the liver donors.

Publisher Summary Orthotopic liver transplantation (OLT) requires replacement of the liver by a donor organ. In this case, the transplanted liver is placed in the space made available following removal of the native liver. Trimming of the donor organ may be necessary if the recipient individual is of small body size. The most common sources of donor livers are brain-dead individuals on life-support. Organ transplantation would no doubt flourish were it possible to identify universal organ donors. When an organ is removed from a donor, it is necessary to preserve it in the best possible condition until transplantation. A guiding principle in organ preservation is avoidance of warm ischemia-reperfusion to prevent tissue injury. Another principle is to decrease the metabolic activity and oxidative damage in the organ. Blood is flushed out of the organ to prevent vascular occlusion by thrombus formation, followed by infusion of cold preservation solution. Surgery for OLT proceeds in three distinct stages: the dissection phase, anhepatic phase, and the reperfusion phase. Briefly, it is during the dissection phase, that the host liver is mobilized and vascular structures are prepared for resection and reanastomosis with the donor liver.

To control the sugar content in fish fillets, the permeability of various sugar compounds into fillets by soaking together with their dispersing ability inside the soaked fillets were investigated. Irrespective of the kind of sugars, an increase in the osmotic pressure given by sugars in the soaking solution caused the increase in the content of sugars and the decrease in the moisture of the soaked fillets. Comparing the permeability and the dewatering effect of sugars in connection with their molecular weights, lowering the molecular weights of sugars caused an increase in the permeability and a decrease in the dewatering effect. Furthermore, the content of sugars in the external parts were several times higher than those in the internal parts, even when the fillets were soaked for more than 100 h. In contrast, the moisture remained higher in the internal parts than in the external parts of the soaked fillets. Following preservation of the soaked fillets at 4°C for over 50 h was necessary to disperse sugars and moisture in the fillets. Moreover, lowering the molecular weights of sugars also caused an increase in their dispersing ability.

In order to clarify the effect of cold preservation using the UW solution for vascularized composite limb tissue, the authors performed a comparative study between the Euro-Collins and UW solutions for assessment of simple cold preservation of rat limbs in reimplantation.Thirty Lewis rat limbs were preserved at 4°C in EC solution (n=18) or UW solution (n=12) for 72 hours, and grafted orthotopically to a syngeneic rat using microsurgical techniques. We evaluated the vascular patency rate and death rate a week after surgery for both groups (EC group and UW group), and performed histological evaluation of bone, muscle, growth plate, and articular cartilage for each specimen of successful grafts in both groups.Both the vascular patency rate and the death rate between the EC group and the UW group showed no significant differences. There were no clear differences in histological viability between both groups, in all tissues.In conclusion, we could not demonstrate a protective effect of the UW solution as a cold preservation medium, as compared to the EC solution.

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

THE USE OF A FATTY LIVER for transplantation particularly after prolonged cold preservation often leads to postoperative graft dysfunction, whereas a mildly to moderately fatty liver with less prolonged preservation shows a function similar to that of a normal liver. In this study, we investigated the function of transplanted organs showing a moderate fatty content in the setting of reducedsize-graft liver transplantation after limited cold preservation.

Purpose: Monitoring ischemic liver injury is important in liver transplantation. We previously reported that dielectric properties were correlated with tissue adenosine triphosphate during liver preservation; however, it is unknown what the changes in dielectric properties are based on. Using rats, we studied the relationship between dielectric parameters and microstructural changes in liver tissues during preservation. Methods: We serially measured the percent decrease in conductivity and the percent increase in relative permittivity in rat livers, and determined the areas of cellular and extracellular components using a charge-coupled device microscope and an NIH image. Thereafter, we evaluated the relationship between dielectric parameters and microstructural changes in the liver tissues. Results: A positive linear and exponential correlation was found between the percent decrease in conductivity and the percent decrease in extracellular components (P < 0.01). A positive linear and exponential correlation was also found between the percent increase in relative permittivity and the percent increase in cellular components (P < 0.01). Conclusions: Our results suggest that the serial changes in tissue conductivity and the relative permittivity reflect changes in liver tissue microstructures during cold preservation.

The aim of this study was to determine whether the minimum necessary volume of a moderate fatty liver graft was similar to the normal liver volume and to elucidate means for improving the function of the transplanted fatty liver if it were inferior in volume to a normal liver under conditions of permissible cold preservation. Nine-week-old male Wistar rats were used. Normal rat chow was fed to the normal liver group, and fat-enriched rat chow was fed to the fatty liver group for 4 weeks to induce a moderately fatty liver. Liver transplantation with various volumes of reduced-size grafts, including whole liver graft (100%LT), 70% volume graft (70%LT), and 30% volume graft (30%LT), was performed with both groups of rats as donors. All procedures were performed under the conditions of 2-hour cold preservation. All rats with an implanted normal liver were surviving at 7 days after the operation regardless of the graft volume (100%LT, 5 of 5; 70%LT, 5 of 5; 30%LT, 5/5). In contrast, the survival rates decreased according to the graft volume in rats implanted with fatty livers (100%LT, 8 of 8; 70%LT, 5 of 8; 30%LT, 2/8). To improve the survival of 30%LT with fatty liver, we employed two potent inhibitors of ischemia-reperfusion injury: FK506 and prostaglandin E1. Though FK506 had no advantageous effect, prostaglandin E1 significantly improved the survival rate and diminished serum levels of alanine aminotransferase and hyaluronic acid. In conclusion, the volume of graft necessary for successful transplantation is larger in fatty livers than in normal livers in permissible cold preservation. Also, prostaglandin E1 protects grafts against ischemia-reperfusion injury and improves the functioning of a transplanted fatty liver.

The shortage of donor organs for liver transplantation requires investigations into expanding the donor pool. Non-heart beating donor (NHBD) livers are an untapped source of organs. NHBD organs are those that have had a period of warm ischemia prior to cold preservation. A main concern of NHBD livers is that there is a depletion of energy stores during the warm ischemia and subsequent cold storage. This study is to investigate the use of hypothermic machine perfusion to regenerate energy stores in the liver during 5hr storage and after 1hr rewarming in a rat transplantation model. Results will be compared with control. (no warm ischemia) and simple cold stored tissues.Copyright © 2003 by ASME

The aim of this study was to establish a liver transplant model with a reduced warm ischemic period between cold preservation and completed anastomosis. Male brown Norway rats were used. A cuff was fixed in the portal vein, stents were inserted in the A. hepatica, the suprahepatic and infrahepatic v. cava and in the bile duct. After cross clamping of the recipient liver, another stent, occluded by a small pin, was introduced in the recipient suprahepatic v. cava. After ligation of this recipient suprahepatic caval stent the recipient liver was removed and the cold prepared donor liver was pushed forward along the pin and both suprahepatic stents of recipient and donor were connected. Then cuff anastomosis of the portal vein was performed as well as anastomosis of the infrahepatic v. cava and of the A. hepatica and the bile duct. The median clamping time of the portal vein was 6.5 minutes with a warm ischemic period for the liver of 2.7 minutes. All transplanted rats survived 7 days, histologic examination after reperfusion showed normal hepatocytes and endothelial cells. The described method allows fast and safe anastomosis of the suprahepatic v. cava. Studies concerning the role of the warm ischemic period between cold preservation and reperfusion are thus feasible.

A solução de preservação da Universidade de Wisconsin (UW) é considerada a solução padrão para preservação de fígados, rins e pâncreas. A frutose-1,6-bisfosfato (FBP) é uma substância que apresenta efeito protetor do fígado contra injúrias provocadas por agentes químicos e ocorridas durante o período de isquemia-reperfusão. O objetivo deste estudo foi avaliar os efeitos da FBP na composição de soluções para preservação de fígados para transplante. Neste estudo experimental, a perfusão e a preservação dos fígados foram realizadas em cada grupo com as soluções de UW, UW contendo 10 mmol/L de FBP (UWM) e FBP 10 mmol/L (FBPS), respectivamente. Os fígados foram armazenados em um recipiente plástico contendo solução de preservação a 4oC por 24 horas. As mensurações bioquímicas de AST, ALT, LDH e TBARS foram realizadas em amostras das soluções de preservação nos tempos de 0, 12, 18 e 24 horas. A análise histológica foi realizada em fígados preservados por 24 horas. O grau de preservação observado com as soluções de UW e FBPS foi similar até 18 horas de armazenamento. A adição de 10 mmol/L de FBP à solução de UW provocou um aumento das injúrias ocorridas e uma pior preservação quando comparado ao grupo armazenado em UW. A FBP protegeu os fígados contra danos causados pelos radicais livres por tempos de preservação inferiores a 18 horas. Não houve diferença significativa entre os grupos na análise histológica dos fígados preservados no tempo de 24 horas. A FBP utilizada em solução foi eficaz na preservação de fígados, podendo ser um importante constituinte para outras formulações. University of Wisconsin (UW) solution is considered the standard preservation solution for livers, kidneys and pancreas. Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect in liver injury caused by chemical agents and ischemic-reperfusion damages. The aim of this study was to evaluate the effects of FBP in storage solution for cold liver preservation. In this experimental study, we divided the rats in three experimental groups. The perfusion and preservation of the liver were performed on each group with UW, UW containing 10 mmol/L of FBP (UWM) and FBP 10mmol/L (FBPS) solutions, respectively. The livers were stored in a plastic bag containing storage solution at 4oC for 24 hours. Biochemical measurements of AST, ALT, LDH e TBARS were realized in cold storage solution samples at 0, 12, 18 and 24 hours of preservation. Histological evaluation was performed in tissue samples obtained after 24 hors of cold storage. The preservation grade was similar in FBPS and UW solutions during 18 hours. Addition of 10 mmol/L of FBP in UW solution induced liver injury and a poor preservation when compared to group of livers preserved in UW solution. FBP protects the liver from injury caused by free radicals for preservation times bellow 18 hours. There were no significant histological differences between groups after 24 hours of preservation. FBP could be an important component of cold storage solutions for liver preservation.

Fatty infiltration of the liver is common in the brain-dead donor population and has a strong correlation with primary nonfunction after cold preservation, a condition that is catastrophic to liver transplant recipients. This literature review examines factors associated with the development, diagnosis, quantification, and clinical management of this difficult condition.

solation of endothelial cells (EC) from large humanblood vessels obtained from multi-organ donorscannot be performed as described for human umbili-cal cords (Jaffe et al, 1973) because of the availabilityof only small segments with side branches and inci-sions. We isolated EC from segments of human iliacarteries and veins with collagenase while the adventi-tia side of the vessels was attached to the culture dishwith fibrin glue. This prevented the collagenase frommaking contact with the adventitia side, resulting in ahigh yield of EC with minimized contamination of cellsfrom nonendothelial origin.Iliac arteries and veins from multiorgan donors werestored in cold preservation solution (UW) until 7 daysafter liver transplantation. Before isolation of EC, atissue sample of 100 mm