Characterisation of cold-induced mitochondrial fission in porcine aortic endothelial cells.

BACKGROUND: Previously, we observed that hypothermia, widely used for organ preservation, elicits mitochondrial fission in different cell types. However, temperature dependence, mechanisms and consequences of this cold-induced mitochondrial fission are unknown. Therefore, we here study cold-induced mitochondrial fission in endothelial cells, a cell type generally displaying a high sensitivity to cold-induced injury. METHODS: Porcine aortic endothelial cells were incubated at 4-25 °C in modified Krebs-Henseleit buffer (plus glucose to provide substrate and deferoxamine to prevent iron-dependent hypothermic injury). RESULTS: Cold-induced mitochondrial fission occurred as early as after 3 h at 4 °C and at temperatures below 21 °C, and was more marked after longer cold incubation periods. It was accompanied by the formation of unusual mitochondrial morphologies such as donuts, blobs, and lassos. Under all conditions, re-fusion was observed after rewarming. Cellular ATP content dropped to 33% after 48 h incubation at 4 °C, recovering after rewarming. Drp1 protein levels showed no significant change during cold incubation, but increased phosphorylation at both phosphorylation sites, activating S616 and inactivating S637. Drp1 receptor protein levels were unchanged. Instead of increased mitochondrial accumulation of Drp1 decreased mitochondrial localization was observed during hypothermia. Moreover, the well-known Drp1 inhibitor Mdivi-1 showed only partial protection against cold-induced mitochondrial fission. The inner membrane fusion-mediating protein Opa1 showed a late shift from the long to the fusion-incompetent short isoform during prolonged cold incubation. Oma1 cleavage was not observed. CONCLUSIONS: Cold-induced mitochondrial fission appears to occur over almost the whole temperature range relevant for organ preservation. Unusual morphologies appear to be related to fission/auto-fusion. Fission appears to be associated with lower mitochondrial function/ATP decline, mechanistically unusual, and after cold incubation in physiological solutions reversible at 37 °C.

HTK-N as a new preservation solution for human kidney preservation: Results of a pilot randomized controlled clinical phase II trial in living donor transplantation.

BACKGROUND: HTK-N was developed based on the traditional HTK preservation solution, resulting in stronger protection against reactive oxygen species as well as better tolerance to hypothermia and ischemia. Aim of the present study was to compare HTK-N to HTK in clinical kidney transplantation demonstrating safety and non-inferiority. METHODS: We performed a randomized controlled single blinded clinical phase II trial in patients undergoing living donor kidney transplantation. After retroperitoneoscopic nephrectomy kidneys were either perfused and stored with classical HTK solution or the new HTK-N solution. Primary endpoint was the glomerular filtration rate (eGFR according to CKD EPI) 3 months after transplantation. Secondary endpoints included graft and patient survival beside others. RESULTS: The study included 42 patients, of which 22 were randomized in the HTK-N group and 20 in the HTK group. The primary end point showed a mean eGFR of 55.4 ± 14.0 ml/min/1.73 m2 in the HTK group compared to a GFR of 57.2 ± 16.7 ml/min/m2 in the HTK-N group (P = .72). Regarding secondary endpoints, there were no apparent differences. Posttransplant graft and patient survival was 100%. CONCLUSION: This study is the first clinical application of HTK-N for kidney preservation and demonstrates non-inferiority compared to HTK in the setting of living donor kidney transplantation.

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution.

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Use of the new preservation solution Custodiol-MP for ex vivo reconditioning of kidney grafts.

Organ shortage and the increasing use of extended criteria donor grafts for transplantation drives efforts for more efficient organ preservation strategies from simple cold storage toward dynamic organ reconditioning. The choice of a suitable preservation solution is of high relevance in different organ preservation or reconditioning situations. Custodiol-MP is a new machine perfusion solution giving the opportunity to add colloids according to organ requirements. The present study aimed to compare new Custodiol-MP with clinically established Belzer MPS solution. Porcine kidneys were ischemically predamaged and cold stored for 20 hours. Ex vivo machine reconditioning was performed either with Custodiol-MP (n = 6) or with Belzer MPS solution (n = 6) for 90 minutes with controlled oxygenated rewarming up to 20°C. Kidney function was evaluated using an established ex vivo reperfusion model. In this experimental setting, differences between both types of perfusion solutions could not be observed. Machine perfusion with Custodiol-MP resulted in higher creatinine clearance (7.4 ± 8.6 mL/min vs. 2.8 ± 2.5 mL/min) and less TNC perfusate levels (0.22 ± 0.25 ng/mL vs. 0.09 ± 0.08 ng/mL), although differences did not reach significance. For short-term kidney perfusion Custodiol-MP is safe and applicable. Particularly, the unique feature of flexible colloid supplementation makes the solution attractive in specific experimental and clinical settings.

Methylene Blue Treatment of Grafts During Cold Ischemia Time Reduces the Risk of Hepatitis C Virus Transmission.

Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.

Resveratrol Does Not Protect from Ischemia-Induced Acute Kidney Injury in an in Vivo Rat Model.

BACKGROUND/AIMS: The natural polyphenol resveratrol (RSV) has been shown to ameliorate ischemia/reperfusion (I/R)-induced damage. Therefore, a rat model of I/R-induced AKI equipped with intensive monitoring was utilized to examine direct renal protection by RSV in vivo. METHODS: AKI was induced by bilateral renal clamping (45 min) followed by reperfusion (3 h). Solvent-free RSV was continuously infused intravenously (0.056 and 0.28 mg/kg) in a total volume of 7 ml/kg/h starting from 30 min before renal clamping. At a mean arterial blood pressure below 70 mmHg for more than 5 min, bolus injections of 0.5 ml 0.9% NaCl solution were administered repetitively (max. 5 ml/kg/h). RESULTS: No differences could be found between normoxic control groups with/without RSV. Bilateral renal clamping and subsequent reperfusion caused a progressive rise in creatinine, cystatin C, and CK, a decrease in cellular ATP content and diuresis. Infusion of RSV increased sirtuin 1 expression after ischemia/reperfusion and was associated with decreased blood pressure during ischemia and early reperfusion accompanied by an increased requirement of bolus injections as well as with increased expression of TNFα. CONCLUSION: RSV did not exert protective effects on I/R-induced AKI in the present short-term in vivo rat model. The lack of protection is potentially connected to aggravation of blood pressure instability.

Kidney transplantation after oxygenated machine perfusion preservation with Custodiol-N solution.

Custodiol-N, a new preservation solution, has been shown particularly suitable for hypothermic machine perfusion preservation (HMP) in isolated porcine kidneys. These preliminary results should be confirmed in an actual transplant model in vivo. Kidney function after 21 h of HMP was studied in an autotransplant model using Landrace pigs (25-30 kg; n = 6 per group). Perfusion was performed with oxygenated perfusate, using either Custodiol-N solution including 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS-1) as gold standard. Viability of the grafts was followed for 1 week after bilateral nephrectomy in the recipient pigs. HMP with CND resulted in less acute tubular injury, evaluated by levels of fatty acid-binding protein and better clearance function during the first 24 h after Tx than with KPS-1 (P < 0.05, resp.). Serum creatinine tended to be lower in the CND group during the whole observation period. Histological tissue scores one week after Tx were similar in both groups. Expression of endothelin-1 as well as of Toll-like receptor 4 15 min after reperfusion was lower in the CND group (P < 0.05), suggesting less endothelial stress response. The data provide first in vivo evidence for the suitability of Custodiol-N as an effective perfusate for renal machine perfusion.

Subnormothermic machine perfusion for preservation of porcine kidneys in a donation after circulatory death model.

Machine perfusion for preservation led to compelling success for the outcome of renal transplantation. Further refinements of methods to decrease preservation injury remain an issue of high interest. This study investigates functional and morphological aspects of kidneys preserved by subnormothermic (20 °C) machine perfusion (SNTM) compared with oxygenated hypothermic machine perfusion (HMPox) and cold storage (CS) in a donation after circulatory death (DCD) model. After 30 min of warm ischaemia, porcine kidneys were randomly assigned to preservation for 7 h by CS, HMPox or SNTM. Afterwards, kidneys were reperfused for 2 h with autologous blood in vitro for assessment of function and integrity. Application of SNTM for preservation led to significantly higher blood flow and urine output compared with both other groups. SNTM led to a twofold increased creatinine clearance compared with HMPox and 10-fold increased creatinine clearance compared with CS. Structural integrity was best preserved by SNTM. In conclusion, this is the first study on SNTM for kidneys from DCD donors. SNTM seems to be a promising preservation method with the potential to improve functional parameters of kidneys during reperfusion.

Treatment of Staphylococcus aureus infection with sphingosine in ex vivo perfused and ventilated lungs.

BACKGROUND: Ex vivo lung perfusion (EVLP) has expanded the donor pool for lung transplantation. Pulmonary Staphylococcus aureus infection, especially that caused by multidrug-resistant strains, is a severe threat to posttransplantation outcomes. Sphingosine is a lipid compound that exhibits broad-spectrum antibacterial activity. Therefore, we aimed to evaluate the effects of S aureus infection on EVLP and whether sphingosine administration during EVLP prevents infection with S aureus. METHODS: Eighteen pigs were randomly assigned to 3 groups: uninfected, infected with S aureus with NaCl treatment, or infected with sphingosine treatment. Bacterial numbers were determined before and after treatment. Sphingosine concentrations in the lung tissues were determined using biochemical assays. Lung histology, lung physiological parameters, perfusate content, lung weight, and cell death were measured to analyze the effects of infection and sphingosine administration on EVLP. RESULTS: Sphingosine administration significantly reduced the bacterial load. The concentration of sphingosine in the bronchial epithelium was elevated after sphingosine administration. S aureus infection increased pulmonary artery pressure and pulmonary vascular resistance. Lung edema, histology scores, lactate and lactate dehydrogenase levels in the perfusate, ΔPO2 in the perfusate, static lung compliance, and lung peak airway pressure did not differ among the groups. CONCLUSIONS: Infection of S aureus did not affect the lung function during EVLP but induced higher pulmonary artery pressure and pulmonary vascular resistance. Administration of sphingosine effectively eliminated S aureus without side effects in isolated, perfused, and ventilated pig lungs.

Steatosis does not impair liver regeneration after partial hepatectomy.

Hepatic steatosis is a key feature of non-alcoholic fatty liver disease (NAFLD). While storage of lipid droplet-bound triglycerides in simple steatosis is physiologically inert, non-alcoholic steatohepatitis (NASH) is associated with hepatocyte damage and apoptosis. Mitochondrial oxidation of free fatty acids (FFA), derived from lipid droplets and hepatocellular uptake, is a rapid and effective way of energy supply for proliferating cells and FFA esterification provides substrates for lipid synthesis and cell proliferation. Thus, we investigated whether simple steatosis induced by western diet (WD) improves liver regeneration after partial hepatectomy (PHx). WD feeding for 6 weeks caused simple steatosis with hepatic lipid droplet and triglyceride accumulation accompanied by induction of fatty acid transport proteins (FATP), death receptors (DR), pro- and anti-apoptotic genes, hepatocyte growth factor (Hgf) as well as increased serum leptin levels in a mouse model. After PHx, liver cell proliferation was higher in WD-fed mice and associated with FATP and Hgf induction. In addition, Erk1/2 (extracellular-related MAP kinase 1/2) dephosphorylation observed in standard diet (SD) mice was reduced in WD animals. PHx in steatotic livers did not affect hepatocyte apoptosis, despite DR upregulation. WD-induced steatosis enhances liver cell proliferation, which is accompanied by increased Hgf and leptin signaling as well as Erk1/2 phosphorylation. Induction of mild steatosis may therefore be beneficial for surgical outcome of hepatectomies.

Use of the new preservation solution Custodiol-N supplemented with dextran for hypothermic machine perfusion of the kidney.

Custodiol-N is a new preservation solution specifically designed to prevent free radical-induced tissue alterations and to protect vascular integrity of the graft. Thus, Custodiol-N appears particularly suitable as base solution for oxygenated machine preservation and its putative benefit for renal preservation by hypothermic machine perfusion (HMP) was investigated using a porcine in vitro model. Kidneys were retrieved from German Landrace pigs and preserved for 20 h by pulsatile oxygenated HMP on a Lifeport kidney transporter (syst. pressure 30 mmHg, 30cycles/min). Each graft was randomly assigned to the use of one of the following preservation solutions: Custodiol-N solution supplemented with 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS-1). Renal viability was evaluated upon reperfusion in vitro with diluted autologous blood from the donor for 120 min at 37°C. After 2h of postischemic reperfusion CND-preserved kidneys exhibited significantly higher renal blood flow and urine production. Oxygen consumption was also higher in the CND group than in KPS-1 kidneys. Clearance of creatinine increased during reperfusion of CND kidneys but declined in KPS-1 grafts ending in significantly higher values in CND kidneys. No differences between the groups were seen for enzyme release or fractional excretion of sodium. In conclusion the data presented provide first experimental evidence for adequate organ protective potential of CND in HMP as compared to the gold standard KPS-KPS-11.

Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro.

Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 °C in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC50) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 µM. In contrast, the exposure to malaoxon in the presence of 1,000 µM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.

Composition of ex vivo perfusion solutions and kinetics define differential cytokine/chemokine secretion in a porcine cardiac arrest model of lung preservation.

Background: Ex vivo lung perfusion (EVLP) uses continuous normothermic perfusion to reduce ischemic damage and to improve post-transplant outcomes, specifically for marginal donor lungs after the donation after circulatory death. Despite major efforts, the optimal perfusion protocol and the composition of the perfusate in clinical lung transplantation have not been identified. Our study aims to compare the concentration levels of cytokine/chemokine in different perfusion solutions during EVLP, after 1 and 9 h of cold static preservation (CSP) in a porcine cardiac arrest model, and to correlate inflammatory parameters to oxygenation capacities. Methods: Following cardiac arrest, the lungs were harvested and were categorized into two groups: immediate (I-EVLP) and delayed EVLP (D-EVLP), after 1 and 9 h of CSP, respectively. The D-EVLP lungs were perfused with either Steen or modified Custodiol-N solution containing only dextran (CD) or dextran and albumin (CDA). The cytokine/chemokine levels were analyzed at baseline (0 h) and after 1 and 4 h of EVLP using Luminex-based multiplex assays. Results: Within 4 h of EVLP, the concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-1α, and IL-1ß increased significantly (P < 0.05) in all experimental groups. The CD solution contained lower concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-2, IL-12, IL-10, IL-4, IL-1RA, and IL-18 (P < 0.05) compared with those of the Steen solution. The concentration levels of all experimental groups have correlated negatively with the oxygenation capacity values (P < 0.05). Protein concentration levels did not reach statistical significance for I-EVLP vs. D-EVLP and CD vs. CDA solutions. Conclusion: In a porcine cardiac arrest model, a longer period of CSP prior to EVLP did not result in an enhanced protein secretion into perfusates. The CD solution reduced the cytokine/chemokine secretion most probably by iron chelators and/or by the protecting effects of dextran. Supplementing with albumin did not further reduce the cytokine/chemokine secretion into perfusates. These findings may help in optimizing the preservation procedure of the lungs, thereby increasing the donor pool of organs.

Aggravation of cold-induced injury in Vero-B4 cells by RPMI 1640 medium - identification of the responsible medium components.

BACKGROUND: In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. RESULTS: Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. CONCLUSION: These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types.

Reduction of chronic graft injury with a new HTK-based preservation solution in a murine heart transplantation model.

OBJECTIVE: Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution. METHODS: Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0-4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-ß expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-ß expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis. RESULTS: The re-beating time was significantly shorter in hearts stored in the new solution (10.3±2.6 min vs. HTK 14.2±4.1 min; p<0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3±0.4 vs. HTK 1.6±0.5; p<0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8±0.2 vs. HTK 2.2±0.7), less interstitial fibrosis (4.8±1.9% vs. HTK 8.5±3.8%, p<0.05), less vasculopathy (14.7±6.9% vs. 22.0±23.2%; p=0.06) and lower TGF-ß1-expression (6.6±1.4% vs. HTK 12.0±4.6%). CONCLUSION: The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-L-histidine, glycine, alanine, arginine and sucrose.

HTK-N, a modified HTK solution, decreases preservation injury in a model of microsteatotic rat liver transplantation.

BACKGROUND: Ischemia/reperfusion injury is an obstacle especially in steatotic livers, including those with steatosis induced by acute toxic stress. Recently, a modified histidine-tryptophan-ketoglutarate (HTK) solution, HTK-N, has been developed. This solution contains N-acetylhistidine, amino acids, and iron chelators. This study was designed to test the effects of HTK-N on preservation injury to rat livers after acute toxic injury. METHODS: Microvesicular steatosis was induced by a single dose of ethanol (8 g/kg BW). Livers were harvested and stored at 4 °C for 8 h with HTK or HTK-N before transplantation. Tissue and blood samples were taken at 1, 8, and 24 h after reperfusion to compare serum liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), standard histology, and immunohistochemistry for myeloperoxidase (MPO), caspase-3, and inducible nitric oxide synthase. Survival was compared after 1 week. For statistics, Analysis of Variance and t test were used. RESULTS: HTK-N improved survival from 12.5% in HTK to 87.5% (p < 0.05). Furthermore, liver enzymes were decreased to 2-75% of HTK values (p < 0.05). Necrosis and leukocyte infiltration and MPO, caspase-3, and iNOS expression after transplantation were decreased (p < 0.05). CONCLUSIONS: This study demonstrates that HTK-N protects liver grafts with microvesicular steatosis caused by acute toxic injury from cold ischemic injury better than standard HTK most likely via inhibition of hypoxic injury and oxidative stress and amelioration of the inflammatory reaction occurring upon reperfusion.

Prolonged cold storage using a new histidine-tryptophan-ketoglutarate-based preservation solution in isogeneic cardiac mouse grafts.

AIMS: The aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based cold preservation solution in comparison with traditional HTK solution in a mouse cardiac transplant model and to assess the impact of chloride ions and of iron chelators. METHODS AND RESULTS: After 24 h cold ischaemia, traditional HTK-preserved hearts survived up to 13 days (4.4 ± 1.7 days; n = 8)). Hearts stored in the new solution without iron chelators (N46) showed significantly prolonged survival up to 2 months (N46: 11.9 ± 8.7 days; P < 0.01; n = 7) and with iron chelators (DesfLK) up to 3 months (N46 DesfLK: 12.7 ± 13.7 days; n = 6). Re-beating time was significantly shorter with the new solution (HTK: 14.5 ± 5.9 min, N46: 9.2 ± 2.7 min, N46 DesfLK: 7.1 ± 3.7 min; P < 0.01). The new solution showed significantly decreased release of creatine kinase (HTK: 25998 ± 8471 U/L, N46: 13829 ± 7679 U/L, N46 DesfLK: 3093 ± 597 U/L; P < 0.01 and n = 7 each) and lactate dehydrogenase (HTK: 5391 ± 1062 U/L, N46: 3428 ± 1890 U/L, P < 0.05; N46 DesfLK: 682 ± 344 U/L, P < 0.01) and decreased histological evidence of injury. A chloride-poor variant of the new solution showed inferior graft survival. CONCLUSION: The new solution markedly attenuates myocardial injury and yields better graft survival than traditional HTK solution. The presence of chloride ions is crucial for heart preservation. Some protective effects are obviously caused by iron chelators.

Recovery from cold-induced mitochondrial fission in endothelial cells requires reconditioning temperatures of ≥ 25◦C.

Mitochondrial integrity and function constitute a prerequisite for cellular function and repair processes. We have previously shown that mitochondria of different cell types exhibit pronounced fragmentation under hypothermic conditions. This fission, accompanied by a decline of cellular ATP content, showed reversibility at 37◦C. However, it is unclear whether other temperatures as currently discussed for reconditioning of organs allow this reconstitution of mitochondria. Therefore, we here study in a model of cultured porcine aortic endothelial cells how different rewarming temperatures affect mitochondrial re-fusion and function. After 48 h cold incubation of endothelial cells in Krebs-Henseleit buffer with glucose (5 mM) and deferoxamine (1 mM) at 4◦C pronounced mitochondrial fission was observed. Following 2 h rewarming in cell culture medium, marked fission was still present after rewarming at 10◦ or 15◦C. At 21◦C some re-fusion was visible, which became more marked at 25◦C. Networks of tubular mitochondria similar to control cells only re-appeared at 37◦C. ATP content decreased at 4◦C from 3.6 ± 0.4 to 1.6 ± 0.4 nmol/106 cells and decreased even further when rewarming cells to 10◦ and 15◦C. Values after rewarming at 21◦C were similar to the values before rewarming while ATP gradually increased at higher rewarming temperatures. Metabolic activity dropped to 5 ± 11% of control values during 4◦C incubation and recovered with increasing temperatures to 36 ± 10% at 25◦C and 78 ± 17% at 37◦C. Integrity of monolayers, largely disturbed at 4◦C (large gaps between endothelial cells; cell injury ≤ 1%), showed partial recovery from 15◦C upwards, complete recovery at 37◦C. Endothelial repair processes (scratch assay) at 25◦C were clearly inferior to those at 37◦C. These data suggest that reconditioning temperatures below 21◦C are not optimal with regard to reconstitution of mitochondrial integrity and function. For this goal, temperatures of at least 25◦C appear required, with 30◦C being superior and 37◦C yielding the best results.

Custodiol-MP for ex vivo lung perfusion - A comparison in a porcine model of donation after circulatory determination of death.

INTRODUCTION: Ex vivo lung perfusion (EVLP) is an established technique to evaluate and eventually recondition lungs prior to transplantation. Custodiol-MP (C-MP) solution is a new solution, designed for clinical machine perfusion, that has been used for kidneys. The aim of this study was to compare the effects of EVLP with Custodiol-MP on lung functional outcomes to the gold standard of EVLP with Steen Solution™. MATERIAL AND METHODS: In a porcine EVLP model of DCDD (Donation after Circulatory Determination of Death), lungs were perfused with Steen Solution™ (SS, n = 7) or Custodiol-MP solution supplemented with 55 g/l albumin (C-MP, n = 8). Lungs were stored cold for 4 h in low potassium dextran solution and subsequently perfused ex vivo for 4 h. During EVLP pulmonary gas exchange, activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AP) as well as levels of lactate in the perfusate were recorded hourly. RESULTS: Oxygenation capacity differed significantly between groups (averaged over 4 h: SS 274 ± 178 mmHg; C-MP 284 ± 151 mmHg p = 0.025). Lactate dehydrogenase activities and lactate concentrations were significantly lower in Custodiol-MP perfused lungs.In a porcine model of DCDD with 4 h of EVLP the use of modified Custodiol-MP as perfusion solution was feasible. The use of C-MP showed at least comparable lung functional outcomes to the use of Steen SolutionTM. Furthermore C-MP perfusion resulted in significantly lower lactate dehydrogenase activity and lactate levels in the perfusate and higher oxygenation capacity.

Antimicrobial effects of inhaled sphingosine against Pseudomonas aeruginosa in isolated ventilated and perfused pig lungs.

BACKGROUND: Ex-vivo lung perfusion (EVLP) is a save way to verify performance of donor lungs prior to implantation. A major problem of lung transplantation is a donor-to-recipient-transmission of bacterial cultures. Thus, a broadspectrum anti-infective treatment with sphingosine in EVLP might be a novel way to prevent such infections. Sphingosine inhalation might provide a reliable anti-infective treatment option in EVLP. Here, antimicrobial potency of inhalative sphingosine in an infection EVLP model was tested. METHODS: A 3-hour EVLP run using pig lungs was performed. Bacterial infection was initiated 1-hour before sphingosine inhalation. Biopsies were obtained 60 and 120 min after infection with Pseudomonas aeruginosa. Aliquots of broncho-alveolar lavage (BAL) before and after inhalation of sphingosine were plated and counted, tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Immunostainings were performed. RESULTS: Sphingosine inhalation in the setting of EVLP rapidly resulted in a 6-fold decrease of P. aeruginosa CFU in the lung (p = 0.016). We did not observe any negative side effects of sphingosine. CONCLUSION: Inhalation of sphingosine induced a significant decrease of Pseudomonas aeruginosa at the epithelial layer of tracheal and bronchial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated pig lungs.