Proteome variation of the rat liver after static cold storage assayed in an ex vivo model.

Cold storage is a common procedure for liver preservation in a transplant setting. However, during cold ischemia, the liver suffers molecular alterations that can affect its performance. Also, deleterious mechanisms set forth in the storage phase are exacerbated during reperfusion. This study aimed to identify liver proteins associated with injury during cold storage and/or normothermic reperfusion using the isolated perfused rat liver model. Livers from male rats were subjected to either (1) cold storage for 24 h, (2) ex vivo normothermic reperfusion for 90 min or (3) cold storage for 24 h followed by ex vivo normothermic reperfusion for 90 min. Then, the livers were homogenized and proteins were extracted. Protein expression between each experimental group and the control (freshly resected livers) was compared by two-dimensional (2D) gel electrophoresis. Protein identification was carried out by matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF/TOF) using MASCOT as the search engine. 23 proteins were detected with significantly altered levels of expression among the different treatments, including molecular chaperones, antioxidant enzymes, and proteins involved in energy metabolism. Some of them have been postulated as biomarkers for liver damage while others had been identified in other organs subjected to ischemia and reperfusion injury. The whole data set will be a useful resource for studying deleterious molecular mechanisms that result in diminished liver function during storage and subsequent reperfusion.

TAT-mediated transduction of bacterial redox proteins generates a cytoprotective effect on neuronal cells.

Cell penetrating peptides, also known as protein transduction domains, have the capacity to ubiquitously cross cellular membranes carrying many different cargos with negligible cytotoxicity. As a result, they have emerged as a powerful tool for macromolecular delivery-based therapies. In this study, catalytically active bacterial Ferredoxin-NADP+ reductase (LepFNR) and Heme oxygenase (LepHO) fused to the HIV TAT-derived protein transduction peptide (TAT) were efficiently transduced to neuroblastoma SHSY-5Y cells. Proteins entered the cells through an endocytic pathway showing a time/concentration dependent mechanism that was clearly modulated by the nature of the cargo protein. Since ferredoxin-NADP+ reductases and heme oxygenases have been implicated in mechanisms of oxidative stress defense, neuroblastoma cells simultaneously transduced with TAT-LepFNR and TAT-LepHO were challenged by H2O2 incubations to judge the cytoprotective power of these bacterial enzymes. Accumulation of reactive oxygen species was significantly reduced in these transduced neuronal cells. Moreover, measurements of metabolic viability, membrane integrity, and cell survival indicated that these cells showed a better tolerance to oxidative stress. Our results open the possibility for the application of transducible active redox proteins to overcome the damage elicited by oxidative stress in cells and tissues.

Hypothermic machine perfusion versus cold storage in the rescuing of livers from non-heart-beating donor rats.

The aim of this work was to compare the efficiency of cold storage (CS) and hypothermic machine perfusion (HMP) methods of preserving grafts excised from non-heart-beating donors that had suffered 45 minutes of warm ischemia. We developed a new solution for HMP to use in liver transplantation, based on BES, gluconate, and polyethylene glycol (BGP-HMP solution). After 24 h of HMP or CS, livers were reperfused at 37°C with Krebs-Henseleit solution with added dextran. For both procedures, portal pressure and flow were measured and the intrahepatic resistance (IR) was calculated. The pH oscillations and enzyme activities (LDH, AST, and ALT) were evaluated for the perfusion buffer during normothermic reperfusion. O2 consumption of the liver, glycogen production, and bile flow were also measured during the normothermic reperfusion period. Portal flow and IR showed statistical differences (P < 0.05) between the two groups (n = 5). HMP with BGP-HMP solution resulted in higher values of portal flow and lower IR than CS with HTK solution. Enzyme release after 90 min of reperfusion did not show statistical differences between groups. With regard to bile flow and O2 consumption, livers preserved by both processes were able to produce bile, but livers preserved with HMP were able to take up more O2 than livers preserved by CS.

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model.

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

Organ Preservation: Current Concepts and New Strategies for the Next Decade.

SUMMARY: Organ transplantation has developed over the past 50 years to reach the sophisticated and integrated clinical service of today through several advances in science. One of the most important of these has been the ability to apply organ preservation protocols to deliver donor organs of high quality, via a network of organ exchange to match the most suitable recipient patient to the best available organ, capable of rapid resumption of life-sustaining function in the recipient patient. This has only been possible by amassing a good understanding of the potential effects of hypoxic injury on donated organs, and how to prevent these by applying organ preservation. This review sets out the history of organ preservation, how applications of hypothermia have become central to the process, and what the current status is for the range of solid organs commonly transplanted. The science of organ preservation is constantly being updated with new knowledge and ideas, and the review also discusses what innovations are coming close to clinical reality to meet the growing demands for high quality organs in transplantation over the next few years.