Metabotropic Glutamate Receptor Blockade Reduces Preservation Damage in Livers from Donors after Cardiac Death

We previously demonstrated that the blockade of mGluR5 by 2-methyl-6(phenylethynyl)pyridine (MPEP) reduces both cold and warm ischemia/reperfusion injury. Here we evaluated whether MPEP reduces the hepatic preservation injury in rat livers from cardiac-death-donors (DCDs). Livers from DCD rats were isolated after an in situ warm ischemia (30 min) and preserved for 22 h at 4 °C with UW solution. Next, 10 mg/Kg MPEP or vehicle were administered 30 min before the portal clamping and added to the UW solution (3 µM). LDH released during washout was quantified. Liver samples were collected for iNOS, eNOS, NO, TNF-α, ICAM-1, caspase-3 and caspase-9 protein expression and nuclear factor-erythroid-2-related factor-2 (Nrf2) gene analysis. Lower LDH levels were detected in control grafts versus DCD groups. An increase in eNOS and NO content occurred after MPEP treatment; iNOS and TNF-α content was unchanged. ICAM-1 expression was reduced in the MPEP-treated livers as well as the levels of caspase-3 and caspase-9. Nrf2, oxidative stress-sensitive gene, was recovered to control value by MPEP. These results suggest that MPEP can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage. MPEP protects against apoptosis and increased eNOS, whose overexpression has been previously demonstrated to be protective in hepatic ischemia/reperfusion damage.

Hypothermic preservation of rat hearts using antifreeze glycoprotein

Antifreeze proteins are an effective additive for low-temperature preservation of solid organs. Here, we compared static hypothermic preservation with and without antifreeze glycoprotein (AFGP), followed by nonfreezing cryopreservation of rat hearts. The heart was surgically extracted and immersed in one of the cardioplegia solutions after cardiac arrest. Control rat hearts (n=6) were immersed in University of Wisconsin (UW) solution whereas AFGP-treated hearts (AFGP group) (n=6) were immersed in UW solution containing 500 μg/ml AFGP. After static hypothermic preservation, a Langendorff apparatus was used to reperfuse the coronary arteries with oxygenated Krebs-Henseleit solution. After 30, 60, 90, and 120 min, the heart rate (HR), coronary flow (CF), cardiac contractile force (max dP/dt), and cardiac diastolic force (min dP/dt) were measured. Tissue water content (TWC) and tissue adenosine triphosphate (ATP) levels in the reperfused preserved hearts were also assessed. All the parameters were compared between the control and AFGP groups. Compared with the control group, the AFGP group had significantly (p<0.05) higher values of the following parameters: HR at 60, 90, and 120 min; CF at all four time points; max dP/dt at 90 min; min dP/dt at 90 and 120 min; and tissue ATP levels at 120 min. TWC did not differ significantly between the groups. The higher HR, CF, max dP/dt, min dP/dt, and tissue ATP levels in the AFGP compared with those in control hearts suggested that AFGP conferred superior hemodynamic and metabolic functions. Thus, AFGP might be a useful additive for the static/nonfreezing hypothermic preservation of hearts.

Application of Cryopreservation Technique in the Preservation of Rat Limbs

Objective: This study aims to observe the physiological and pathological changes of severed fingers (limbs) under different storage conditions through animal experiments, and to screen out the best preservation conditions. Medthods: Sixty healthy adult male Sprague-Dawley rats were selected and evenly divided into 4 preservation groups, including conventional low-temperature dry (CLTD), the university of wisconsin (UW) solution, cryopreservation and cryopreservation + UW solution preservation group. After harvesting the limbs, were preservated for 72 h and 7 days, respectively. Then the limbs were thawed and replanted in situ. Sciatic nerves were collected for hematoxylin and eosin (HE) staining, and observed the changes in tissue morphology. Results: Replantation was successful in 11 out of 15 rats (73%) in cryopreservation + UW group, and the walking function of the 9 (82%) rats in cryopreservation + UW group were significantly better than that of the cryopreservation preservation group. In addition, the HE staining results shown that the CLTD group nerve bundles were morphologically damaged, and there were more acellular structures and tissue fragments; the UW group nerve bundles were less injured and the perineurium was more complete and orderly; The nerve bundles in the cryopreservation group and cryopreservation + UW group are tightly arranged and the tissue morphology is regular; Compared with the cryopreservation + UW group, the complete of the cryopreservation group is not well. Conclusions: The cryopreservation technology combined with UW solution is a new and effective method for the severed limbs preserving.

Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage–Associated Kidney Transplantation

BackgroundKidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown.MethodsTo investigate PKCδ’s potential role in injury during cold storage–associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice.ResultsWe observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage–associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage–associated transplantation injury.ConclusionsThese results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage–associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

Comparison of Surgical and Cadaveric Intestine as a Source of Crypt Culture in Humans

Human small intestinal crypts are the source of intestinal stem cells (ISCs) that are capable of undergoing self-renewal and differentiation to an epithelial layer. The development of methods to expand the ISCs has provided opportunities to model human intestinal epithelial disorders. Human crypt samples are usually obtained from either endoscopic or discarded surgical samples, and are thereby exposed to warm ischemia, which may impair their in vitro growth as three-dimensional culture as spheroids or enteroids. In this study we compared duodenal samples obtained from discarded surgical samples to those isolated from whole-body preserved cadaveric donors to generate in vitro cultures. We also examined the effect of storage solution (phosphate-buffered saline or University of Wisconsin [UW] solution) as well as multiple storage times on crypt isolation and growth in culture. We found that intestinal crypts were successfully isolated from cadaveric tissue stored for up to 144 h post-procurement and also were able to generate enteroids and spheroids in certain media conditions. Surgical samples stored in UW after procurement were sufficiently viable up to 24 h and also allowed the generation of enteroids and spheroids. We conclude that surgical samples stored for up to 24 h post-procurement in UW solution allowed for delayed crypt isolation and viable in vitro cultures. Furthermore, in situ, hypothermic preservation in cadaveric duodenal samples permitted crypt/ISC isolation, and successful culture of spheroids and enteroids from tissues held for up to 6 days post-procurement.

University of Wisconsin solution for the xeno-free storage of adipose tissue-derived microvascular fragments

Aim: Adipose tissue-derived microvascular fragments (ad-MVF) are vascularization units for regenerative medicine. We investigated whether University of Wisconsin (UW) solution is suitable for their xeno-free storage. Materials & methods: Murine ad-MVF were cultivated for 24 h in 4°C or 20°C UW solution and 20°C endothelial cell growth medium (control). The ad-MVF were seeded onto collagen–glycosaminoglycan scaffolds, which were analyzed in dorsal skinfold chambers by intravital fluorescence microscopy and histology. Results: All implants exhibited microvascular networks on day 14 with the highest functional microvessel density in controls. Ad-MVF cultivation in UW solution at 4°C resulted in an improved scaffold vascularization compared with cultivation at 20°C. Conclusion: UW solution is suitable for the hypothermic storage of ad-MVF.

Phenothiazines Enhance the Hypothermic Preservation of Liver Grafts: A Pilot in Vitro Study

In vitro liver conservation is an issue of ongoing critical importance in graft transplantation. In this study, we investigated the possibility of augmenting the standard pre-transplant liver conservation protocol (University of Wisconsin (UW) cold solution) with the phenothiazines chlorpromazine and promethazine. Livers from male Sprague-Dawley rats were preserved either in UW solution alone, or in UW solution plus either 2.4, 3.6, or 4.8 mg chlorpromazine and promethazine (C+P, 1:1). The extent of liver injury following preservation was determined by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, the ratio of AST/ALT, morphological changes as assessed by hematoxylin-eosin staining, apoptotic cell death as determined by ELISA, and by expression of the apoptotic regulatory proteins BAX and Bcl-2. Levels of glucose (GLU) and lactate dehydrogenase (LDH) in the preservation liquid were determined at 3, 12, and 24 h after incubation to assess glucose metabolism. Oxidative stress was assessed by levels of superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA), and inflammatory cytokine expression was evaluated with Western blotting. C+P augmentation induced significant reductions in ALT and AST activities; the AST/ALT ratio; as well as in cellular swelling, vacuolar degeneration, apoptosis, and BAX expression. These changes were associated with lowered levels of GLU and LDH; decreased expression of SOD, MDA, ROS, TNF-α, and IL-1β; and increased expression of Bcl-2. We conclude that C+P augments hypothermic preservation of liver tissue by protecting hepatocytes from ischemia-induced oxidative stress and metabolic dysfunction. This result provides a basis for improvement of the current preservation strategy, and thus for the development of a more effective graft conservation method.