University Of Wisconsin Solution Research Articles

Effectiveness of metformin for the reversal of cold-ischemia-induced damage in hepatosteatosis

BackgroundPrimary dysfunction and rejection are more common in donor liver tissues with steatosis. AMP-activated protein kinase (AMPK) assumes organ-protective functions during ischemia. Metformin was used for the activation of AMPK in hepatocytes. The aim of this study is to investigate the effectiveness of metformin administration for the reversal of cold-ischemia-induced damage in hepatosteatosis. Material and methodsSeven-week-old C7BL56 male-mice (n = 109) were separated into four groups depending on diet type and metformin use. A specific diet model was followed for 10 weeks to induce hepatosteatosis. A group of the animals was administered with metformin for the last four weeks via oral gavage. After resection, the liver tissues were perfused and kept for 0–6–12–24 h in the UW solution. Histopathological examinations were performed, and Western blot was utilized to analyze p-AMPK and AMPK expression levels. ResultsHepatosteatosis decreased significantly with metformin. The steatotic liver group had more prominent pericentral inflammation, necrosis as well as showing a decreased and more delayed AMPK response than the non-fat group. All these alterations could be corrected using metformin. ConclusionMetformin can increase the resistance of livers with hepatosteatosis to cold-ischemia-induced damage, which in turn may pave the way for successful transplantation of fatty living-donor livers.

Effect of PERLA®, a new cold-storage solution, on oxidative stress injury and early graft function in rat kidney transplantation model

BackgroundThe composition of organ preservation solutions is crucial for maintaining graft integrity and early graft function after transplantation. The aim of this study is to compare new organ preservation solution PERLA® with the gold standard preservation solution University of Wisconsin (UW) regarding oxidative stress and early graft injury.MethodsIn order to assess oxidative stress after cold storage, kidney grafts have been preserved for 18 h at 4° C in either UW solution or PERLA® solution and then assessed for oxidative stress injury (protocol 1). To assess kidney injuries and oxidative stress after reperfusion, rat kidneys were harvested, stored in cold UW or in PERLA® solutions for 18 h at 4 °C and then transplanted heterotopically for 6 h (protocol 2). PERLA® is a high Na+/low K+ solution including PEG-35 (1 g/L), trimetazidine (1 µM), carvedilol (10 µM) and tacrolimus (5 µM).ResultsOur results showed that preservation of kidneys in PERLA® solution significantly attenuates oxidative stress parameters after cold storage and reperfusion. We found a significant decrease in oxidative damage indicators (MDA, CD and CP) and a significant increase in antioxidant indicators (GPx, GSH, CAT, SOD and PSH). Moreover, PERLA® solution decreased kidney injury after reperfusion (creatinine, LDH and uric acid).ConclusionPERLA® solution was more effective than UW storage solution in preserving rat’s kidney grafts.

Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Association of cardiac preservation solution with short-term outcomes after heart transplantation.

There is wide variability in the practice of cardiac preservation for heart transplantation. Prior reports suggest that the type of solution may be linked with a reduced incidence of posttransplantation complications. Adult (≥18 years old) heart recipients who underwent transplantation between 2015 and 2021 in the United States were examined. Recipients were stratified by solution utilized for their grafts at the time of recovery: University of Wisconsin, histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. The primary endpoint was a composite of 30-day mortality, primary graft dysfunction, or re-transplantation. Risk adjustment was performed for the recipient, donor, and procedural characteristics using regression modeling. Among 16 884 recipients, the group distribution was University of Wisconsin solution 53%, HTK 22%, Celsior solution 15%, and other 10%. The observed incidence of the composite endpoint (University of Wisconsin solution = 3.6%, HTK = 4.0%, Celsior solution = 3.7%, P = 0.301) and 1-year survival (University of Wisconsin solution = 91.7%, HTK = 91.3%, Celsior solution = 91.7%, log-rank P = 0.777) were similar between groups. After adjustment, HTK was associated with a higher risk of the composite endpoint [odds ratio (OR) 1.249, 95% confidence interval (CI) 1.019-1.525, P = 0.030] in reference to University of Wisconsin solution. This association was substantially increased among recipients with ischemic periods of greater than 4 h (OR 1.817, 95% CI 1.188-2.730, P = 0.005). The risks were similar between University of Wisconsin solution and Celsior solution (P = 0.454). The use of the histidine-tryptophan-ketoglutarate solution during cold static storage for cardiac preservation is associated with increased rates of early mortality or primary graft dysfunction. Clinician discretion should guide its use, especially when prolonged ischemic times (>4 h) are anticipated.

Preservation Efficacy of a Quercetin and Sucrose Solution for Warm Ischemically Damaged Porcine Liver Grafts

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

Static Cold Storage with Mitochondria-Targeted Hydrogen Sulfide Donor Improves Renal Graft Function in an Ex Vivo Porcine Model of Controlled Donation-after-Cardiac-Death Kidney Transplantation.

The global donor kidney shortage crisis has necessitated the use of suboptimal kidneys from donors-after-cardiac-death (DCD). Using an ex vivo porcine model of DCD kidney transplantation, the present study investigates whether the addition of hydrogen sulfide donor, AP39, to University of Wisconsin (UW) solution improves graft quality. Renal pedicles of male pigs were clamped in situ for 30 min and the ureters and arteries were cannulated to mimic DCD. Next, both donor kidneys were nephrectomized and preserved by static cold storage in UW solution with or without AP39 (200 nM) at 4 °C for 4 h followed by reperfusion with stressed autologous blood for 4 h at 37 °C using ex vivo pulsatile perfusion apparatus. Urine and arterial blood samples were collected hourly during reperfusion. After 4 h of reperfusion, kidneys were collected for histopathological analysis. Compared to the UW-only group, UW+AP39 group showed significantly higher pO2 (p < 0.01) and tissue oxygenation (p < 0.05). Also, there were significant increases in urine production and blood flow rate, and reduced levels of urine protein, serum creatinine, blood urea nitrogen, plasma Na+ and K+, as well as reduced intrarenal resistance in the UW+AP39 group compared to the UW-only group. Histologically, AP39 preserved renal structure by reducing the apoptosis of renal tubular cells and immune cell infiltration. Our finding could lay the foundation for improved graft preservation and reduce the increasingly poor outcomes associated with DCD kidney transplantation.

Ethyl Pyruvate; from Liver Preservation Solutions University of Wisconsin (UW) Increases the Effectiveness of the Solution

The most important factor affecting the success rate of liver transplants is the preservation of the normal histologic and biochemical properties of the cells in the tissue taken. The study aimed to identify the possible increase in efficacy of ethyl pyruvate, which has a hepatoprotective effect, on the University of Wisconsin (UW) solution. Rats were randomly selected and divided into 4 groups. After a laparotomy, the small intestines were removed from the abdomen and the portal pedicle was identified. Arterial and venous circulation of the liver was interrupted. After the portal vein was cannulated (and the distal of the portal pedicle was ligated, the liver was perfused with a solution. Perfusion solution was selected as Ringer Lactate in Group 1. In group 2, UW solution was chosen as the perfusion solution. In Group 3, the perfusion solution was chosen as the UW solution, but ethyl pyruvate at a dose of 40 mg/kg was administered intraperitoneally to the experimental animals 30 minutes before hepatectomy. In Group 4, as a perfusion solution, a UW solution with 40 mg/kg dose of ethyl pyruvate added to it was used. With TUNEL and Caspase-3 staining, a significant decrease was found in the apoptosis rates of Groups 2, 3, and 4 at the 12th hour post hepatectomy when compared with Group 1. When the morphometric liver sinusoid/parenchyma ratios and vena centralis diameters of the groups were examined, it was found that all preservation solutions containing the UW solution were more protective than the RL solution. Ethyl pyruvate is regarded as a promising agent that can increase the effect of the UW solution on organ preservation solutions. Because this study is the first in literature to apply ethyl pyruvate in preservation solutions, additional studies with larger series and different doses are needed.

Comparative cryopreservation of bovine and porcine primary hepatocytes.

The isolation of primary hepatocytes from liver tissue of farm animals yields a very high number of cells, and a part of them can be stored by cryopreservation for future experiments. As no experience exists with the cryopreservation of hepatocytes from cattle, our study aimed at the cryopreservation of bovine hepatocytes by use of different protocols compared with the cryopreservation of hepatocytes from pig. We tested different freezing media (William's Medium E vs. University of Wisconsin solution), cryoprotectants (dimethyl sulfoxide with vs. without trehalose as additional additive), freezing systems (standard freezing container vs. controlled-rate freezer) and freezing times (4 vs. 28 d). These tests identified a general influence of species and freezing systems, whereas the influence of freezing media, trehalose additive and freezing time was less or not obvious. In this regard, we determined a mean recovery of 30% of bovine hepatocytes and 55% of porcine hepatocytes cryopreserved in a controlled-rate freezer, whereas the rates were about 10% less when hepatocytes were frozen in a standard freezing container. In accordance with this observation, the cultivation of cryopreserved hepatocytes from cattle was less effective than that of porcine hepatocytes. Hepatocytes from cattle can be successfully cryopreserved and partially cultured after cryopreservation but with lower percentage than porcine hepatocytes.

Protective Effects of Different Hypothermal Preservation Solutions on Structure and Function of Isolated Rat Arteries.

With the increasing application of vascular reconstruction in surgical procedures, allogeneic vessels are becoming more popular in clinical practice due to their abundant sources, precise diameter matching, improved histocompatibility, and higher long-term patency rate. This study aimed to investigate the protective effect of various preservation solutions on the function and structure of the isolated rat abdominal aorta preserved under hypothermal conditions. The study utilized a total of 150 Sprague-Dawley (SD) rats, with 144 rats allocated to the experimental groups and 6 rats allocated to the control groups. The abdominal aorta of the rats was chosen as the subject of our research. The aorta in the experimental groups were randomly assigned to 4 groups: University of Wisconsin (UW) solution group, histidine-tryptophan-ketoglutarate (HTK) solution group, normal saline (NS) group, and sodium lactate Ringer's solution (RS) group. Samples were subjected to examination after preservation periods of 1 day, 3 days, 5 days, 7 days, 14 days, 30 days, and 90 days. Evaluation of vascular physiological function involved detecting and assessing vasoconstriction ability and measuring cell viability through the MTT test. Evaluation of the vascular wall structure involved tension tolerance tests and pathological staining. The pathogen-positive rate in the HTK group and NS group at 1 month was 16.7%. Regarding the vascular skeleton structure, both the UW group and HTK group exhibited intact structures after 2 weeks of preservation, with slightly edematous collagen and elastic fibers, which was significantly better than that of the NS group and RS group. In terms of cell activity and contractile function, all preservation groups showed similar effects within 2 weeks. However, after 2 weeks, the UW group showed the most favorable preservation effect (P<0.05). In terms of vascular tension, different groups exhibited similar effects within 1 week. However, after 2 weeks, the UW group showed the best preservation effect (P<0.05). All 4 types of preservation solution had a preservation effect on the structure and function of isolated blood vessels during short-term hypothermal preservation. However, after 2-week preservation, the UW solution was found to be the most suitable solution for the preservation of blood vessels.

Effect of Melatonin on Increasing the Effectiveness of Liver Preservation Solution.

Various tissue preservation solutions are used during the removal of the organ and during transplantation to protect the normal histological and biochemical characteristics of tissue while performing a successful liver transplant. In our study, it was aimed to investigate the effects of intraperitoneal melatonin administration on liver preservation damage before transplantation. In our study, the histological and biochemical characteristics of University of Wisconsin+melatonin group rats treated with melatonin 45 minutes before hepatectomy were compared between serum physiologic group and University of Wisconsin group. When hematoxylin and eosin staining was evaluated in terms of hydropic degeneration, sinusoidal dilatation, and hepatocyte necrosis, there was no statistically significant difference. Caspase 3 immunohistochemical staining showed a significant increase in Caspase 3 immunoreactivity positivity at the 12th-hour University of Wisconsin group compared to University of Wisconsin+melatonin group. As a result of biochemical analysis, the malondialdehyde and total oxidant status levels in the University of Wisconsin+melatonin group decreased significantly compared to the University of Wisconsin group. When the reduced glutathione activity and total antioxidant capacity level were compared, a significant increase was observed in the University of Wisconsin+melatonin group compared to the University of Wisconsin group at the 12th hour. It was also found that aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels decreased significantly in the University of Wisconsin+melatonin 12th-hour group compared to the University of Wisconsin 12th hour and control group. When the findings were evaluated, intraperitoneal administration of melatonin, a cytoprotective antioxidant, was found to play an effective role in preserving immunohistochemical and biochemical properties of liver tissue integrity and hepatocytes in University of Wisconsin solution.

An Examination of Packing Methods for Grafts to Prevent Freezing Injury during Transportation for Liver Transplantation.

University of Wisconsin solution (UW) may freeze at temperatures below -0.7 °C, damaging the graft. The present study assessed the effectiveness of the liver graft package protocol, which recommends filling a package with sufficient liquid to prevent grafts from sustaining freezing injury. We filled ice cubes at two temperatures (-80 and -20 °C) around packages and performed a comparative study with four groups based on the temperature and filling of the second layer with lactated Ringer's solution (LR) (A: -80 °C, LR-; B: -80 °C, LR+; C: -20 °C, LR-; D: -20 °C, LR+). The bovine liver was used as a graft and preserved for 6 h in the first isolation bag filled with UW. While temperatures dropped below -0.7 °C at some points for 6 h in groups A, B, C, they never dropped to -0.7 °C in group D. The macroscopic findings in groups A, B, C showed freezing of the UW and grafts, but no such results in group D. A pathological study including electron microscopy showed freezing injury in groups A, B, and C but no significant changes in group D. The graft package protocol prevents freezing of the UW and liver grafts.

Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death.

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Does an Additional Bile Duct Flush With Low-viscosity Preservation Solution Reduce Bile Duct Injury? A Single-blinded Randomized Clinical Trial.

A randomized trial was conducted using 64 liver grafts from brain dead donors. The control group received a bile duct flush with University of Wisconsin (UW) solution after donor hepatectomy. The intervention group received a bile duct flush using low-viscosity Marshall solution immediately after the onset of cold ischemia and a bile duct flush with University of Wisconsin solution after donor hepatectomy. The primary outcomes were the degree of histological bile duct injury, assessed using the bile duct injury score, and biliary complications within 24 mo of transplant. Bile duct injury scores were not different between the 2 groups. Similar rates of biliary complications occurred in the intervention group (31% [n = 9]) and controls (23% [n = 8]) (P = 0.573). No difference between groups was observed for anastomotic strictures (24% versus 20%, P = 0.766) or nonanastomotic strictures (7% versus 6%, P = 1.00). This is the first randomized trial to investigate an additional bile duct flush using low-viscosity preservation solution during organ procurement. The findings from this study suggest that performing an earlier additional bile duct flush with Marshall solution does not prevent biliary complications and bile duct injury.

Isolation and Purification of Human Pancreatic Islets.

Successful islet isolation is the key to islet transplantation in diabetic patients. However, islet isolation is a technically complex and time-consuming manual process. Optimizing the islet isolation process can improve islet yield and quality, reduce operators, and thus reduce costs.The isolation and purification of human islets include pancreas acquisition and preservation, pancreas digestion, islet purification, islet culture, and islet quality identification. Briefly, after the duodenum was removed, the pancreas was trimmed, the main pancreatic duct was intubated at the distal end of the pancreatic head, collagenase was injected into the pancreatic duct, and the perfused pancreatic tissue was cut and then digested in a Ricordi chamber. A digestion temperature of 37°C was continuously used to assess the number of samples and the integrity of the lysed and released islets. At the end of the digestion process, collect the digested tissue in a 500 mL centrifuge tube prefilled with 25mL of cold (4°C) human serum albumin and centrifuge twice at 150g for 3min. After mixing with UW solution as islet storage solution, put it on ice (shake occasionally to prevent clumping) after 30min. Digested pancreatic tissue was centrifuged at 2200rpm for 5min in a COBE 2991 cell processor to isolate islets from exocrine tissue using a continuous density gradient. The purified islet fractions were washed twice in HBSS supplemented with 10% human serum albumin and finally collected in CMRL1066 medium supplemented with the corresponding liquid. The purity of purified islets was calculated by DTZ staining, the survival rate of islets was calculated by FDA/PI staining, and islet function was determined by in vitro glucose-stimulated insulin secretion test.

Application of polymerized porcine hemoglobin in the ex vivo normothermic machine perfusion of rat livers.

Background: In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion (NMP) may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. The polymerized porcine hemoglobin is a kind of hemoglobin oxygen carrier prepared by crosslinking porcine hemoglobin by glutaraldehyde to form a polymer. The pPolyHb has been proved to have the ability of transporting oxygen which could repair the organ ischemia-reperfusion injury in rats. Objective: In order to evaluate the effectiveness of rat liver perfusion in vitro based on pPolyHb, we established the NMP system, optimized the perfusate basic formula and explored the optimal proportion of pPolyHb and basal perfusate. Methods: The liver was removed and perfused for 6h at 37°C. We compared the efficacy of liver perfusion with different ratios of pPolyHb. Subsequently, compared the perfusion effect using Krebs Henseleit solution and pPolyHb perfusate of the optimal proportion, and compared with the liver preserved with UW solution. At 0h, 1h, 3h and 6h after perfusion, appropriate samples were collected for blood gas analysis and liver injury indexes detection. Some tissue samples were collected for H&E staining and TUNEL staining to observe the morphology and detect the apoptosis rate of liver cells. And we used Western Blot test to detect the expression of Bcl-2 and Bax in the tissues. Results: According to the final results, the optimal addition ratio of pPolyHb was 24%. By comparing the values of Bcl-2/Bax, the apoptosis rate of pPolyHb group was significantly reduced. Under this ratio, the results of H&E staining and TUNEL staining showed that the liver morphology was well preserved without additional signs of hepatocyte ischemia, biliary tract injury, or hepatic sinusoid injury, and hepatocyte apoptosis was relatively mild. Conclusion: Through the above-mentioned study we show that within 6h of perfusion based on pPolyHb, liver physiological and biochemical activities may essentially be maintained in vitro. This study demonstrates that a pPolyHb-based perfusate is feasible for NMP of rat livers. This opens up a prospect for further research on NMP.

Application of a novel artificial perfusate based on oxygen-carrying nanoparticles in normothermic machine perfusion for porcine liver preservation after cardiac death.

To investigate the efficacy of a novel artificial perfusate based on oxygen-carrying perfluoronaphthalene-albumin nanoparticles in normothermic machine perfusion (NMP) for preservation of porcine liver donation after cardiac death. Artificial perfusate with perfluoronaphthalene-albumin nanoparticles was prepared at 5% albumin (w/v) and its oxygen carrying capacity was calculated. The livers of 16 Landrace pigs were isolated after 1 h of warm ischemia, and then they were divided into 4 groups and preserved continuously for 24 h with different preservation methods: cold preservation with UW solution (SCS group), NMP preservation by whole blood (blood NMP group), NMP preservation by artificial perfusate without nanoparticles (non-nanoparticles NMP group) and NMP preservation by artificial perfusate containing nanoparticles (nanoparticles NMP group). Hemodynamics, tissue metabolism, biochemical indices of perfusate and bile were monitored every 4 h after the beginning of NMP. Liver tissue samples were collected for histological examination (HE and TUNEL staining) before preservation, 12 h and 24 h after preservation. The oxygen carrying capacity of nanoparticles in 100 mL artificial perfusate was 6.94 μL/mmHg (1 mmHg=0.133 kPa). The hepatic artery and portal vein resistance of nanoparticles NMP group and blood NMP group remained stable during perfusion, and the vascular resistance of nanoparticles NMP group was lower than that of blood NMP group. The concentration of lactic acid in the perfusate decreased to the normal range within 8 h in both nanoparticles NMP group and blood NMP group. There were no significant differences in accumulated bile production, alanine aminotransferase and aspartate aminotransferase in perfusate between nanoparticles NMP group and blood NMP group (all P>0.05). After 24 h perfusion, the histological Suzuki score in blood NMP group and nanoparticles NMP group was lower than that in SCS group and non-nanoparticles NMP group (all P<0.05), and the quantities of TUNEL staining positive cells in blood NMP group and non-nanoparticles NMP group was higher than those in nanoparticles NMP group and SCS group 12 h and 24 h after preservation (all P<0.05). Artificial perfusate based on oxygen-carrying nanoparticles can meet the oxygen supply requirements of porcine livers donation after cardiac death during NMP preservation, and it may has superiorities in improving tissue microcirculation and alleviating ischemia-reperfusion injury.

Cellular Cytosolic Energy Replenishment Increases Vascularized Composite Tissue Tolerance to Extended Cold Ischemia Time.

Vascularized composite allotransplantation (VCA) is a restorative surgical procedure to treat whole or partially disfiguring craniofacial or limb injuries. The routine clinical use of this VCA surgery is limited using compromised allografts from deceased donors and by the failure of the current hypothermic preservation protocols to extend the allograft's cold ischemia time beyond 4 h. We hypothesized that the active replenishment of the cellular cytosolic adenosine-5`-triphosphate (ATP) stores by means of energy delivery vehicles (ATPv) encapsulating high-energy ATP is a better strategy to improve allograft's tolerance to extended cold ischemia times. We utilized established rat model of isolated bilateral in-situ non-cycled perfusions of both hind limbs. Ipsilateral and contralateral limbs in the anesthetized animal were randomized for simultaneous perfusions with either the University of Wisconsin (UW) solution, with/without O2 supplementation (control), or with the UW solution supplemented with the ATPv, with/without O2 supplementation (experimental). Following perfusion, the hind limbs were surgically removed and stored at 4°C for 12, 16, or 24 hours as extended cold ischemia times. At the end of each respective storage time, samples of skin, and soleus, extensor digitalis longus, and tibialis anterior muscles were recovered for assessment using tissue histology and tissue lysate studies. Control muscle sections showed remarkable microvascular and muscle damage associated with loss of myocyte transverse striation and marked decrease in myocyte nucleus density. A total of 1,496 nuclei were counted in 179 sections of UW-perfused control muscles in contrast to 1,783 counted in 130 sections of paired experimental muscles perfused with the ATPv-enhanced perfusate. This yielded 8 and 13 nuclei/field for the control and experimental muscles, respectively (P < .004). Oxygenation of the perfusion solutions before use did not improve the nucleus density of either the control or experimental muscles (n = 7 animals, P > .05). Total protein isolated from the muscle lysates was similar in magnitude regardless of muscle type, perfusion protocol, or duration of cold ischemia time. Prolonged static cold preservation of the hind limbs completely degraded the composite tissue's Ribonucleic acid (RNA). This supplementary result confirms the notion that that reverse transcription-Polymerase Chain Reaction, enzyme-linked immunosorbent assay, or the respiratory complex II enzyme activity techniques should not be used as indices of graft quality after prolonged static cold storage. In conclusion, this study demonstrates that active cellular cytosolic ATP replenishment increases hind limb composite tissue tolerance to extended cold ischemia times. Quality indicators and clinically relevant biomarkers that define composite tissue viability and function during static cold storage are warranted.

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival.

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic β-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary β-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

Luminal Preservation Protects the Small Intestine in a Brain-dead Rat Model.

Background.Intestinal transplantation depends on donation after brain death (DBD). Luminal preservation (LP) has been beneficial against preservation injury in previous studies in animal models, but none include DBD. This study aims to investigate whether these benefits occur also with DBD.Methods.Wistar rats (male, N = 9) underwent brain death for 2 h. Thereafter, vascular perfusion was done with University of Wisconsin solution (UW). The small intestine was then explanted and randomized into 3 groups: control (empty segment), LP+PEG (with polyethylene glycol 3350 solution), or LP+UW (with UW), treated and tied shut. Ice-cold UW was used for cold storage. Samples were taken at procurement and after 4 (t = 4) and 8 h (t = 8) of preservation. Histopathological scorings were performed for intestinal preservation injury, subepithelial space, absence of epithelial lining, and hemeoxygenase-1 expression.Results.There was low-level mucosal injury (median intestinal preservation injury score 2) at procurement. At t = 4, bowels treated without LP had more damage than LP-treated samples (control score 4, LP+PEG 2 and LP+UW 2, P < 0.001 control versus LP+UW). At t = 8, no benefit of LP was observed (control 2, LP+PEG 3, LP+UW 2). Subepithelial space increased with time and the presence of LP; epithelial lining was better conserved in LP-treated samples. Hemeoxygenase-1 staining showed increased intensity with increased damage, irrespective of treatment.Conclusions.Luminal perfusion of the small intestine with UW or PEG protects the mucosa in brain-dead rats for up to 4 h. Fewer benefits of LP were found than previously described in non-DBD models. To mimic the clinical situation, DBD should be included in future animal studies on intestinal preservation.