Prolonged Cold Preservation Research Articles

Establishment of a prolonged pancreas preservation model for islet isolation research in mice

Establishing a prolonged pancreas preservation model in a small animal is important for islet isolation research. Use of a rat pancreas model has been reported, but no published reports have used a mouse pancreas for prolonged cold preservation prior to islet isolation. For the model, a mouse is preferred over a rat because of its small size, well-known immune characterization and variety of gene-modulated models. In the present study, we established a prolonged pancreas preservation model in a mouse for islet isolation research. The collagenase solution was injected successfully after 24 and 48 h cold preservations in University of Wisconsin solution, and islets could be isolated from both groups of preserved pancreata. The islet yields from the control, 24 h preserved, and 48 h preserved pancreata were 183.9 ± 13.9, 128.5 ± 15.5, and 24.6 ± 12.9 per pancreas, respectively. The propidium iodide–positive area assay was significantly increased in both preserved groups, and insulin secretion levels in response to 20.0 mM glucose and stimulation indices were significantly decreased in the 48 h preserved group. Inflammation-related genes mRNA levels were significantly upregulated in the 24 h preserved group, as previously shown in the human model. Thus, this model might be useful for prehuman islet isolation screening research, reserving research using human pancreata for the most promising approaches.

Reduction of cold ischemia–reperfusion injury by graft-expressing clusterin in heart transplantation

Cold ischemia-reperfusion injury (IRI) is a major factor for early graft dysfunction and is associated with rejection episodes in heart transplantation. Clusterin (CLU) is a cytoprotective protein with chaperone activity. This study was designed to examine the impact of donor-expressing CLU on cold IRI. Donor hearts from wild-type C57BL/6J (H-2(b); B6 WT) vs CLU knockout C57BL/6J (H-2(b); B6 KO) mice were stored at 4°C for 8 hours, followed by heterotopic transplantation to B6 WT mice. The functional recovery of heart grafts was determined by scoring palpation, and tissue injury was determined by release of creatine kinase (CK) and lactate dehydrase (LDH) and also by histology. Heart cells constitutively expressed CLU, and mature CLU protein was localized mostly in the endothelium as well as on the cell surface of cardiac myocytes. As compared with CLU-deficient hearts, WT hearts were more resistant to cold injury during cold preservation, and had a better functional recovery after prolonged cold preservation and transplantation. The improved graft function of CLU-expressing grafts correlated significantly with reduced neutrophil infiltration and cardiac injury, including myocytic apoptosis and necrosis. Furthermore, in vitro examination showed that ectopic expression of CLU in cultured myocytes increased cell membrane stability after exposure to cold temperature and prevented cell death. CLU expression renders donor hearts resistance to cold IRI in transplantation, suggesting that upregulation of CLU expression in donor hearts may have potential for protecting heart grafts from cold IRI.

A cell penetrating heme oxygenase protein protects heart graft against ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is an unavoidable barrier that significantly affects outcome of solid organ transplantation. Here, we establish a protein transduction system to extend graft preservation time and to prevent I/R injury in heart transplantation. We generated a recombinant heme oxygenase-1 (HO-1) protein containing a modified protein transduction domain (PTD). PTD could cross cover cell membrane and carry target molecule to parenchymal cells of cold-preserved heart grafts. The newly generated PTD-HO-1 protein localized mainly in subcellular membrane organelle and nucleus after delivery that significantly prolonged cold preservation of heart grafts. This effect was associated with significantly less endothelial cell activation, less neutrophil and macrophage infiltration in PTD-HO-1-transduced heart grafts after reperfusion as compared with controls. In addition, transduction of PTD-HO-1 protein to heart graft significantly suppressed the I/R injury-associated myocardiocyte apoptosis. The infarct areas of heart graft after I/R injury were significantly reduced after PTD-HO-1 protein treatment. We show here for the first time that PTD can maintain its biological activities during cold preservation. Transduction of cell penetrating HO-1 protein significantly prolongs the cold preservation time and protects the graft from the I/R injury. This approach represents a novel method for the improvement of the overall outcome of organ transplantation.

Comparison of Outcomes with HTK and UW Solutions in Renal Transplantation

To the Editor: We appreciate the comments of Drs. Opelz and Döhler regarding our submission to the American Journal of Transplantation (1Comparison of Histidine-Tryptophan-Ketoglutarate and University of Wisconsin preservation in renal transplantation.Am J Transplant. 2008; 8: 567-573Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The authors correctly point out that our series, which encompassed 634 deceased donor and 950 living donor transplants, was not sufficiently powered to detect a difference in UW versus HTK performance in organs cold-preserved for more than 24 h. Our study did show, to a statistically significant degree, apparent equipoise in patient and graft survival between the whole of the UW and HTK groups. Separate multivariate analyses of outcomes validated the data set by showing contributions of donor and recipient factors consistent with previous reports from the literature (2De Boer J De Meester J Smits JMA et al.Eurotransplant randomized multicenter kidney graft preservation study comparing HTK with UW and Euro-Collins.Transplant Int. 1999; 12: 447-453Crossref PubMed Scopus (102) Google Scholar,3Agarwal A Murdock P Fridell JA Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution in prolonged cold preservation of kidney allografts.Transplantation. 2006; 81: 480-482Crossref PubMed Scopus (57) Google Scholar). Finally, our study assessed correlation between storage solution type and delayed graft function, and HTK use was actually seen by Cox multivariable analysis to be associated with decreased risk of delayed graft function. The results of the Collaborative Transplant Study, to which Drs. Opelz and Döhler allude, did show a detrimental effect of preservation with HTK over UW in prolonged ischemia times (4Opelz G Döhler B Multicenter analysis of kidney preservation.Transplantation. 2007; 83: 247-253Crossref PubMed Scopus (166) Google Scholar). This excellent work reports on 16 years of registry data. As in our own experience, the authors noted shifts in preservative fluid type and the proportion of organs stored for longer than 24 h over the course of the collection period. While this registry data can be used to calculate a hazard ratio to control for the era effect in our own study, the applicability of such a factor derived from an aggregate of heterogeneous data to a single center’s practices is not certain. Nonetheless, Drs. Opelz and Döhler correctly comment on an important weakness of our report, the potential confounding effects of secular trends on our results. We clearly discussed this in the original manuscript. In summary, final proof of the superiority of HTK over UW or vice versa as a renal preservative remains elusive. In the absence of any new randomized trial of sufficient power and granularity to assess all factors and outcomes of renal transplantation using the two solutions, large reports such as the Collaborative Transplant Study and smaller series such as our own provide complementary information on renal preservation. Our experience shows little, if any, difference between the fluids within the scope of our usual ischemia times, and as HTK has cost and ease of use benefits (5Englesbe MJ Heidt D Sung R Pietroski R Does using HTK solution for cold perfusion of cadaveric kidneys save money.Transplantation. 2006; 82: 580-581Crossref PubMed Scopus (7) Google Scholar), it remains our standard choice for cold preservation of renal allografts.

Carbon Monoxide Ameliorates Renal Cold Ischemia-Reperfusion Injury With an Upregulation of Vascular Endothelial Growth Factor by Activation of Hypoxia-Inducible Factor

We have previously shown that carbon monoxide (CO) inhalation at a low concentration provides protection against cold ischemia-reperfusion (I/R) injury after kidney transplantation. As vascular endothelial growth factor (VEGF) may promote the recovery process of impaired vascular endothelial cells during I/R injury, we examined whether protective effects of CO involved VEGF induction and its upstream hypoxia-inducible factor (HIF)-1 activation. Lewis rat kidney graft, preserved in University of Wisconsin at 4 degrees C for 24 hr, was orthotopically transplanted into syngeneic recipient. Recipients were continuously maintained in air or exposed to CO (250 ppm) for 1 hr before and 24 hr after transplant. Prolonged cold preservation resulted in progressive impairment of kidney graft function with early inflammatory responses. Carbon monoxide significantly protected kidney grafts from cold I/R injury, improved renal function and enhanced recipient survival. Real-time reverse transcriptase-polymerase chain reaction revealed upregulation of HIF-1alpha and VEGF in the CO-treated kidney grafts as early as 1 hr after reperfusion. Western blot showed CO significantly upregulated VEGF expression 1 to 3 hr after kidney transplantation. Considerably more VEGF-positive cells were observed mainly in tubular epithelial cells in CO-treated, but not air-exposed, kidney grafts at 3 hr after reperfusion. YC-1, HIF-1alpha inhibitor, completely abrogated the actions of CO on VEGF induction and reversed the protective effects afforded by CO. Nitric oxide production in the grafts was increased by CO, however, abolished by YC-1. These results demonstrate that the protective effect of CO against renal cold I/R injury may involve VEGF upregulation through its upstream signal, HIF-1 activation.

Evaluation of preservation solutions by ESR-spectroscopy: Superior effects of University of Wisconsin over Histidine–Tryptophan–Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.

Donor Dopamine Pretreatment Inhibits Tubulitis in Renal Allografts Subjected to Prolonged Cold Preservation

In the present study, we used the Brown-Norway (BN) to Lewis model as a model for acute rejection, to test the hypothesis that dopamine (DA) treatment of BN donors significantly reduces the inflammatory response after renal transplantation. BN and Lewis rats (isograft controls) were treated for 24 hr with DA (5 microg/kg/min) or NaCl (0.9%), respectively. After 24 hr of cold storage in University of Wisconsin (UW) solution, renal allografts were orthotopically transplanted into Lewis recipients. All recipients received immunosuppression until they were sacrificed. Allografts were harvested one, three, five, and 10 days after transplantation and analyzed by light microscopy, immunohistochemistry (CD3, major histocompatibility complex [MHC] class II, ED1, P-selectin and intercellular adhesion molecule [ICAM]-1) and by RNase protection assay for cytokine mRNA. Ten days after transplantation Banff tubulitis scores were significantly lower in DA-treated than in NaCl-treated allografts. No significant differences were found in Banff interstitial infiltration scores. The numbers of MHC class II+ and CD3+ cells were significantly decreased in DA-treated animals as assessed by immunohistochemistry. No differences were found in the number of ED1+, P-selectin+, and ICAM-1+ cells. The expression of Ltalpha, tumor necrosis factor, interleukin-1beta, and interleukin-2 mRNA was significantly reduced in DA-treated animals. Our data indicate that donor DA treatment significantly inhibits tubulitis in renal allografts subjected to prolonged cold preservation. A reduced number of infiltrating MHC class II+ and CD3+ cells together with decreased cytokine expression could diminish renal scarring, reduce allograft immunogenicity, and hence improve transplantation outcome.

Impact of cold preservation on leukocyte adhesion to the transplanted rat lung

Little is known about leukocyte migration in the early phase after ischemia-reperfusion injury. Recently, there has been much focus on in vivo visualization of cell kinetics. Previously, we established a method for the in vivo visualization of leukocyte circulation using a Green Fluorescent Protein transgenic rat. Herein, we developed a simplified rat heterotopic lung transplantation model that can be mastered by beginners within a few weeks. Using this system, we studied the impact of preservation on adhesion of circulating leukocytes to the transplanted lung. The graft of a fresh group was compared with that of a preserved group. Morphological and immunohistochemical analyses were also performed. We successfully visualized few adherent leukocytes in the fresh graft, whereas adherent cells were attached in the preserved graft within a few minutes. In conclusion, the prolonged cold preservation time promotes leukocyte adhesion, resulting in increased microvascular injury after lung transplantation.

SDF-1 Plays a Key Role in the Repairing and Remodeling Process on Rat Allo-Orthotopic Abdominal Aorta Grafts

Background Our previous study demonstrated that prolonged cold preservation promoted neointima formation and remodeling but delayed the subsequent arteriosclerosis of rat abdominal aorta grafts. The mechanisms of this phenomenon remain obscure. In this study, we investigated whether stromal cell derived factor-1 (SDF-1) could play a role in recruiting stem cells to repair and remodel the damaged intima of abdominal aorta grafts. Methods Male Spague-Dawley rats received abdominal aorta grafts from male Wistar rats. Hematoxylin and eosin staining was performed to assess the structure of graft aortas by measuring the neointimal thickness. Immunohistochemical staining detected SDF-1 expression. RT-PCR demonstrated the expression of CXCR4, the only known natural receptor of SDF-1 expression on stem cells. Results The neointimal thickness of the SDF-1 antibody-treated group was inconspicuous; a significant relationship existed between the expression of SDF-1 and the neointimal thickness of the grafts. Furthermore, no CXCR4 was detected in normal abdominal aortas, but it was observed in the grafted abdominal aorta. Conclusion Prolonged cold ischemia may delay the graft’s arteriosclerosis by selectively chemoattracting stem cells to the damaged intima through SDF-1, the presence of which may predict graft arteriosclerosis and the subsequent development of chronic graft dysfunction (CGD). The SDF-1 antibody slowed the endothelial chimerism by blocking this chemoattration. In addition to mycophenolate mofetil and FK 506, SDF-1 antibody might be a new potential effective strategy to decrease the frequency of CGD.

Selective Neutralization of the Chemokine TCA3 Reduces the Increased Injury of Partial Versus Whole Liver Transplants Induced by Cold Preservation

Given the shortage of liver donors and the development of techniques for partial liver transplantation, we compared chemokine expression and inflammatory cell infiltration of partial versus whole grafts in a mouse syngeneic liver transplant model. Orthotopic liver transplantation, using whole or partial murine liver grafts, was performed following cold preservation in ViaSpan solution for periods of one to eight hours. Partial grafts showed more severe cold ischemia/reperfusion injury and greater inflammatory cell infiltration than whole grafts, and was accompanied by the marked intrahepatic upregulation of multiple chemokines. Quantitative analysis showed that compared with expression in whole grafts harvested after the same period of cold ischemia, partial grafts had eightfold more T-cell activation gene (TCA)-3 (CCL1) chemokine messenger RNA (mRNA) expression (P<0.01) and sixfold more inducible protein (IP)-10 chemokine (CCL10) mRNA expression (P<0.01), as well as increased expression of the chemokine receptors CCR8 (receptor for TCA3) and CXCR3 (receptor for IP-10; P<0.01). Blockade of TCA3 by neutralizing monoclonal antibody significantly decreased intragraft IP-10 expression (P<0.05) but not tumor necrosis factor-alpha or interleukin-6 expression in partial grafts, and significantly decreased cold ischemia/reperfusion injury (P<0.05) and associated neutrophil and T-cell infiltration (P<0.01). These data demonstrate that the chemokine TCA3/CCL1 is important to the pathogenesis of ischemic injury of experimental partial liver grafts, and that its therapeutic targeting within such grafts can overcome the deleterious effects of prolonged cold preservation and restore liver function to the level achieved using whole liver grafts.

Hypothermia-Induced Loss of Endothelial Barrier Function Is Restored after Dopamine Pretreatment: Role of p42/p44 Activation

Donor dopamine usage is associated with improved immediate graft function after renal transplantation. Although prolonged cold preservation results in an increased vascular permeability, the present study was conducted to examine in vitro and in vivo if dopamine pretreatment influences endothelial barrier function under such conditions. To assess cold preservation injury in vitro and in vivo, cultured human umbilical vein endothelial cells (HUVEC) and Lewis donor rats were pretreated with dopamine or isotonic saline prior to cold storage. Injury was determined by lactate dehydrogenase (LDH) release, histology, and functional analysis. In vitro cold storage resulted in intercellular gap formation in both untreated and dopamine pretreated HUVEC. In the latter monolayer integrity was completely restored upon rewarming and paracellular transport of fluorescein isothiocyanate-dextran was significantly reduced. In dopamine treated HUVEC, intercellular gap formation was independent of cell death and was associated with redistribution of junctional proteins and condensation of cytoskeleton proteins. In untreated HUVEC proteolysis and cell death were clearly evident after hypothermia. Closing of intercellular gaps was dependent on p42/p44 activation. Regeneration of adenosine triphosphate was only observed in dopamine pretreated cells. Only in dopamine treated Lewis renal allografts subjected to cold storage, activation of p42/p44 occurred upon rewarming. These grafts had a better renal function and displayed less inflammatory cells five days after transplantation. Our study demonstrates beneficial effects of dopamine treatment on cold storage induced endothelial barrier disturbances. This may contribute to the positive effects of catecholamines on immediate graft function of renal allografts in men.

Effect of mycophenolate mofetil on rat kidney grafts with prolonged cold preservation

The impact of mycophenolate mofetil (MMF) on initial renal transplant function is not well characterized. We tested how MMF may modulate graft function and survival in a syngeneic rat kidney transplantation model after prolonged cold preservation. Donor kidneys were preserved in University of Wisconsin for either 24 or 39 h prior to transplantation into nephrectomized rats. Recipients received MMF (20 mg/kg/day) or vehicle. Mycophenolic acid (MPA) blood concentrations were measured by high-performance liquid chromatography. The inflammatory response, tubular epithelial proliferation, and histologic damage 3 days post-transplantation were assessed microscopically. In the 24 h cold storage (c.s.) group serum-creatinine was measured. In the 39 h c.s. group 1-week recipient survival was determined. After 24 h of c.s., recipient survival was 100%. The number of T-cell infiltrates was low and not influenced by MMF, whereas renal ED1+ cell infiltration was significantly suppressed by MMF. Tubular cell proliferation was enhanced by MMF. Serum-creatinine levels and renal histology were comparable between MMF and vehicle-treated animals. In the 39 h c.s. group, recipient survival was 20% in MMF-treated vs 90% in vehicle-treated animals (P=0.001). MMF effectively suppressed inflammatory cell infiltration and inhibited tubular cell proliferation. MMF-induced structural damage was most striking in the renal papilla. In rat kidney grafts with moderate preservation injury (24 h c.s.), MMF, given at an immunosuppressive dose, showed predominantly antiinflammatory effects without compromising graft function. In grafts with severe preservation injury (39 h c.s.), MMF caused irreversible structural damage and inhibited tubular cell regeneration resulting in renal failure.

Comparison of Histidine-Tryptophan Ketoglutarate Solution and University of Wisconsin Solution in Prolonged Cold Preservation of Kidney Allografts

Although University of Wisconsin (UW) solution is the standard preservation solution for organ transplantation, Histidine-Tryptophan Ketogluatarate (HTK) solution has been increasingly used. This study compared HTK or UW for cold static storage of kidney allografts. In all, 149 renal transplants were performed with cold ischemic times (CI) greater than 16 hr (UW 87, HTK 62) and a subset analysis was performed with CI over 24 hr (HTK 31, UW 38). Data from receiving renal transplant centers focused on delayed graft function (DGF), patient and allograft survival. In CI greater than 16 hr, graft and patient survival were comparable. HTK cohort had lower DGF. In CI greater than 24 hr, there was no difference in patient survival, a trend towards improved graft survival in HTK, and decreased rate of DGF in HTK. This data suggests that UW and HTK have at least similar efficacy in kidney preservation at longer ischemic times.

The influence of ischaemia on the biliary tract.

Damage of the biliary tract at the end of a period of hepatic preservation prior to liver transplantation has been observed in man. This damage may be important in the development of early biliary complications. A model of hepatic biliary ischaemia in the pig has been developed which permits comparison between the influences of warm and cold ischaemic damage on the biliary tract. The major damage to the biliary tract in the pig in this model appears to occur after prolonged cold preservation.

Bcl-xL Gene Transfer Inhibits Bax Translocation and Prolongs Cardiac Cold Preservation Time in Rats

Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4 degrees C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0+/-2.6% versus 47.7+/-7.0% in saline control and 48.6+/-6.1% in vector control, P<0.01) after 2-hour reperfusion at 37 degrees C with the Langendorff device and significantly decreased creatine kinase release (0.82+/-0.27 IU, versus 1.57+/-0.33 and 1.50+/-0.37 IU in saline and vector controls, respectively; P<0.05). In heart transplantation, overexpression of Bcl-xL inhibited Bax translocation from the cytosol to the mitochondria, resulting in decreased cytochrome c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.

Schwann-cell injection of cold-preserved nerve allografts

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

Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation

Cold ischemia-warm reperfusion injury of liver grafts has been investigated thoroughly, but its underlying mechanism remains poorly understood. Here we show that autophagy is involved not only during cold preservation but also during warm reperfusion following transplantation. Immunohistochemistry using an antibody against LC3, a microtubule associated protein 1 light chain 3 and a marker of autophagosomes, showed dot-like weak staining in hepatocytes of rat liver grafts during cold preservation. Since University of Wisconsin solution for graft preservation lacks amino acids, the induction of autophagy in hepatocytes was similar to that under starvation conditions. Intense immunopositive punctate structures were detected abundantly in the hepatocytes 30 min after the beginning of reperfusion. LC3-positive granules were often co-localized in ED2-positive Kupffer cells at 60 min of the reperfusion phase. The molecular form of LC3 was mainly LC3-II, a membrane-bound form, during reperfusion, especially at 30 min of the phase. Electron microscopic examination demonstrated numerous vacuolar structures in hepatocytes at 30 min of the reperfusion period, while some hepatocytes with such vacuolar structures were present in the sinusoidal lumen. At the late stage of the reperfusion period, Kupffer cells contained phagocytosed cells that possessed numerous autophagic vacuoles/autolysosomes and nuclei with condensed chromatin. Our results showing the presence of autophagic vacuoles/autolysosomes in hepatocytes of liver grafts after the start of reperfusion suggest that warm reperfusion acted as a stress stimulus to hepatocytes. Moreover, the stress response of hepatocytes may be involved in their degeneration process.

Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation

The current models of liver ischemia/reperfusion injury (IRI) in mice are largely limited to a warm ischemic component. To investigate the mechanism of hepatic "cold" IRI, we developed and validated a new mouse model of prolonged cold preservation followed by syngeneic orthotopic liver transplantation (OLT). Two hundred and forty-three OLTs with or without rearterialization and preservation in University of Wisconsin solution at 4 degrees C were performed in Balb/c mice. The 14-day survivals in the nonarterialized OLT groups were 92% (11/12), 82% (9/11), and 8% (1/12) after 1-hour, 6-hour and 24-hour preservation, respectively. In contrast, hepatic artery reconstruction after 1-hour, 6-hour, and 24-hour preservation improved the outcome as evidenced by 2-week survival of 100% (12/12), 100% (10/10), and 33% (4/12), respectively, and diminished hepatocellular damage (serum alanine aminotransferase /histology). Moreover, 24-hour (but not 1-h) cold preservation of rearterialized OLTs increased hepatic CD4+ T-cell infiltration and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin 2, interferon-gamma) production, as well as enhanced local apoptosis, and Toll-like receptor 4/caspase 3 expression. These cardinal features of hepatic IRI validate the model. In conclusion, we have developed and validated a new mouse model of IRI in which hepatic artery reconstruction was mandatory for long-term animal survival after prolonged (24-h) OLT preservation. With the availability of genetically manipulated mouse strains, this model should provide important insights into the mechanism of antigen-independent hepatic IRI and help design much needed refined therapeutic means to combat hepatic IRI in the clinics.

Prolonged cold preservation promotes the recipient's cell participating in neointima formation but delays the later graft arteriosclerosis in rat model

Chronic graft dysfunction is the greatest barrier to long-term graft survival, although the immediate outcome in organ transplantation has been greatly improved. Graft arteriosclerosis is a prominent feature of chronic graft dysfunction. Recipient progenitor cells have been shown to participate in neointimal development in graft arteriosclerosis. The present study investigated the role of recipient endothelial cells in the repair and remodeling after a cold preservation injury in an orthotopic cross-sex abdominal aortic allotransplantation model, namely female Wistar to male Sprague-Dawley rats. Grafts were preserved for 48 hours in 4°C University of Wisconsin (UW) solution for a prolonged cold ischemia (PCI) group or preserved for <1 hour in the control group; or for <1 hour in the presence of feeding with cyclosporine (CyA). A direct in situ polymerase chain reaction (ISPCR) for the SRY gene showed SRY-marked endothelial and smooth muscle-like cells in neointima at 2 weeks in the PCI group, at 4 weeks in the control group, and rarely at 3 months in the CyA group. Staining by H&E showed the aortic graft intima to be thicker in the PCI than in the control group at 4 weeks, but thinning thereafter. The SRY-positive cells correlated with intimal thickness in the PCI and the control group ( r = .801 and .825; P < .05 and <.05, respectively), but not in the CyA group ( r = .247, P > .5). Our data suggest that prolonged cold preservation promotes recipient cell participation in graft arteriosclerosis after endothelium injury. The early neointimal formation via recipient cells incorporated into arteriosclerotic neointima may delay later intimal thickening. In the aortic allotransplantation model, prolonged cold ischemia may be beneficial for long-term graft survival due to early endothelial replacement. We hypothesize that controlled injury to the graft may serve as a new strategy for treatment of intimal thickening.

Attenuation of ischemia-reperfusion injury in a canine model of autologous renal transplantation.

This study examined the potential therapeutic effects of a combination therapy consisting of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and N-acetyl cysteine (NAC) to attenuate ischemia-reperfusion (I/R) injury in a canine model of autologous renal transplantation. Male mongrel dogs (15-20 kg) underwent left nephrectomy followed by flushing and static preservation of the kidney in University of Wisconsin (UW) solution for 48 hr. The treatment group received AICAR (50 mg/kg) plus NAC (100 mg/kg) intravenously before the left nephrectomy. The compounds were added to the UW solution as well. All dogs underwent right nephrectomy 48 hr later followed by autotransplantation of the preserved left kidney. Treated dogs received a second dose of AICAR and NAC before implantation of the renal autograft. The treated dogs had excellent urine output posttransplant, with peak serum creatinine of 7.26 mg/dL on postoperative day (POD) 3 that normalized after 14 days. The control group were anuric and developed clinical symptoms of uremia on POD 1. Morphologic evaluation supported the protective effects of combination therapy. Immunohistochemical analysis revealed decrease of tumor necrosis factor-alpha, interferon-gamma, and inducible nitric oxide synthase; and TUNEL assay showed decreased apoptosis in the treated group. Combination therapy with AICAR and NAC attenuates renal I/R injury and improves the outcome of the transplanted kidney after prolonged cold preservation.