The Benefits of Fibrinolysis Combined with Venous Systemic Oxygen Persufflation (VSOP) in a Rat Model of Donation after Circulatory Death and Orthotopic Liver Transplantation.

Organ shortage has led to the increasing utilization of livers retrieved from donors after circulatory death (DCD). These pre-damaged organs are susceptible to further warm ischemia and exhibit minimal tolerance for cold storage. The aim was thus to examine the effects of fibrinolysis combined with Venous Systemic Oxygen Persufflation (VSOP) on the preservation of DCD livers in vivo. Livers of male Lewis rats were explanted after 45 min of warm ischemia, cold-stored for 18 h, and transplanted into a recipient animal. Livers were left untreated or underwent either VSOP or fibrinolysis via Streptokinase (SK) or received combined SK and VSOP. Combined treatment exhibited improved microvascular flow at 168 h (p = 0.0009) and elevated microperfusion velocity at 24 h post-transplantation (p = 0.0007). Combination treatment demonstrated increased portal venous flow (PVF) at 3 and 24 h post-transplantation (p = 0.0004, p < 0.0001), although SK and VSOP analogously achieved increases at 24 h (p = 0.0036, p = 0.0051). Enzyme release was decreased for combination treatment (p = 0.0002, p = 0.0223) and lactate dehydrogenase (LDH) measurements were lower at 24 h post-transplantation (p = 0.0287). Further supporting findings have been obtained in terms of serum cytokine levels and in the alterations of endothelial injury markers. The combination treatment of SK + VSOP might provide improved organ integrity and viability and may therefore warrant further investigation as a potential therapeutic approach in the clinical setting of DCD.

Effects of asparaginases and L-carnitine on Western-diet-induced hepatosteatosis in mice.

Background: Asparaginases are common chemotherapeutic agents used for the treatment of acute lymphoblastic leukemia as a single or combinational therapy. Accompanying hepatotoxicity makes its use in elderly patients with pre-conditions, as obesity or other hepatopathies, difficult. Various hepatoprotective compounds like, L-carnitine, are discussed to ameliorate the induced hepatotoxicity. Methods: Here we aimed to establish a mouse model to study the effect of asparaginases (L-asparaginase and Oncaspar) and L-carnitine on Western-diet-induced hepatosteatosis in mice. Dose-escalation studies were performed to analyze asparaginases induced hepatotoxicity in C57BL/6 mice with normal or fatty livers. Subsequently, the effect of L-carnitine to improve the induced toxicity was tested. Results: Our results showed mild-to-moderate hepatotoxic effects while the Western-diet induced a higher degree of vacuolization and hepatocyte damage in liver tissue. Testing of L-carnitine in the established models did not show any protective effect on the toxicity or impairment of the efficacy of asparaginases. Conclusion: The here established models were able to demonstrate the asparaginase-induced hepatotoxic effects which were enhanced by the Western-diet. However, to test potential ameliorating drugs, the models might need some improvements.

Apolipoprotein E4 Is Associated with Right Ventricular Dysfunction in Dilated Cardiomyopathy-An Animal and In-Human Comparative Study.

ApoE abnormality represents a well-known risk factor for cardiovascular diseases. Beyond its role in lipid metabolism, novel studies demonstrate a complex involvement of apoE in membrane homeostasis and signaling as well as in nuclear transcription. Due to the large spread of apoE isoforms in the human population, there is a need to understand the apoE's role in pathological processes. Our study aims to dissect the involvement of apoE in heart failure. We showed that apoE-deficient rats present multiple organ damages (kidney, liver, lung and spleen) besides the known predisposition for obesity and affected lipid metabolism (two-fold increase in tissular damages in liver and one-fold increase in kidney, lung and spleen). Heart tissue also showed significant morphological changes in apoE-/- rats, mostly after a high-fat diet. Interestingly, the right ventricle of apoE-/- rats fed a high-fat diet showed more damage and affected collagen content (~60% less total collagen content and double increase in collagen1/collagen3 ratio) compared with the left ventricle (no significant differences in total collagen content or collagen1/collagen3 ratio). In patients, we were able to find a correlation between the presence of ε4 allele and cardiomyopathy (χ2 = 10.244; p = 0.001), but also with right ventricle dysfunction with decreased TAPSE (15.3 ± 2.63 mm in ε4-allele-presenting patients vs. 19.8 ± 3.58 mm if the ε4 allele is absent, p < 0.0001*) and increased in systolic pulmonary artery pressure (50.44 ± 16.47 mmHg in ε4-allele-presenting patients vs. 40.68 ± 15.94 mmHg if the ε4 allele is absent, p = 0.0019). Our results confirm that the presence of the ε4 allele is a lipid-metabolism-independent risk factor for heart failure. Moreover, we show for the first time that the presence of the ε4 allele is associated with right ventricle dysfunction, implying different regulatory mechanisms of fibroblasts and the extracellular matrix in both ventricles. This is essential to be considered and thoroughly investigated before the design of therapeutical strategies for patients with heart failure.

Intra-iliac bone marrow injection as a novel alternative to intra-tibial inoculation in rat model.

BACKGROUND: Intra-bone marrow injection (IBMI) in rats is adopted in many studies for stem cell and hematopoietic cell transplantation. IBMI in the tibia or the femur results in severe distress to the animal. Therefore, this study aims to evaluate intra-iliac injections as an alternative approach for IBMI. METHODS: Twenty-seven Sprague Dawley rats were assigned into 3 groups, 9 rats each, for 4 weeks. The control group rats were not injected. Tibia group rats were injected intra-tibial and the iliac group rats were injected intra-iliac with saline. Behavioral, radiological, histopathological, and stress evaluation was performed. Total bilirubin, cortisol, and insulin-like growth factor-1 (IGF1) were measured. RESULTS: Behavioral measurements revealed deviation compared to control, in both injected groups, on the 1st and 2nd week. By the 3rd week, it was equivalent to control in the iliac group only. Bilirubin and cortisol levels were increased by intra-tibial injection compared to intra-iliac injection. The IGF-1 gene expression increased compared to control at 1st and 2nd weeks in intra-iliac injection and decreased by intra-tibial injection at 2nd week. The thickness of the iliac crest was not different from the control group, whereas there were significant differences between the control and tibia groups. Healing of the iliac crest was faster compared to the tibia. In the 3rd week, the tibia showed fibrosis at the site of injection whereas the iliac crest showed complete bone reconstruction. CONCLUSION: Intra-iliac injections exert less distress on animals, and by 3 weeks, they regained their normal activity in comparison to intra-tibial injections.

Effect of Stent Strut Interval on Neointima Formation After Venous Stenting in an Ovine Model.

OBJECTIVE: The impact of stent design on venous patency is not well studied. The purpose of this study was to investigate the effect of stent material burden on endothelial coverage of stented venous segments, which may contribute to vessel healing and patency. METHODS: Segmented self expanding bare nitinol stents (18 × 50 mm) comprising 5 mm long attached metallic rings separated by 2, 5, or 8 mm gaps were implanted in the inferior vena cava (IVC) of 10 sheep. These stents were designed and manufactured for the purposes of this study. At six, 12, and 24 weeks after implantation the animals were euthanised and the stented vessels harvested for histomorphometric analysis. Three sections from the metallic part as well as the gaps between the struts were reviewed for quantification of endothelialisation after six, 12, and 24 weeks. The intimal thickness over and between the stent struts was measured. The endothelialisation score (graded from 1 for complete luminal endothelialisation to 5 for absence of endothelial cells) was determined. RESULTS: All stents were successfully deployed and all 10 sheep survived until the time of harvesting. Macroscopic inspection after 24 weeks showed only partial endothelialisation over stents with 2 mm and 5 mm skipped segments, whereas the stents with 8 mm skipped segments were totally incorporated into the vein wall. After 24 weeks, the mean (SD) neointimal thicknesses over stent struts with 2 mm, 5 mm, and 8 mm skipped segments were 254.0 (51.6), 182.2 (98.1), and 194.6 (101.1) µm, respectively. Comparison of endothelialisation scores of stents over time showed statistically significantly better endothelialisation over stents with 8 mm gaps after 12 and 24 weeks. CONCLUSION: Stent designs providing structural support to veins with larger gaps between the scaffold material appear to lead to faster and more complete endothelialisation as well as a thinner intimal layer.

The gut bacterium Extibacter muris produces secondary bile acids and influences liver physiology in gnotobiotic mice.

Extibacter muris is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few in vivo models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM12 with or without E. muris. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM12 strains were capable of deconjugating primary bile acids in vitro. E. muris produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA in vitro. Ursodeoxycholic, chenodeoxycholic, and ß-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with E. muris, which showed higher relative abundances after HF diet feeding. The presence of E. muris had minor, diet-dependent effects on Oligo-MM12 communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in E. muris-colonized mice. E. muris colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in E. muris-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by E. muris.In summary, E. muris stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids in vivo.

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access.

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

Apolipoprotein E deficient rats generated via zinc-finger nucleases exhibit pronounced in-stent restenosis.

The long-term success of coronary stent implantation is limited by in-stent restenosis (ISR). In spite of a broad variety of animal models available, an ideal high-throughput model of ISR has been lacking. Apolipoprotein E (apoE) deficient rats enable the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. Whereas apoE deficient rats have been proposed as animal model of atherosclerosis, to date it is unknown whether they also develop pronounced ISR. We sought to assess ISR after abdominal aorta stent implantation in apoE deficient rats. A total of 42 rats (16 wildtype, 13 homozygous apoE-/- and 13 heterozygous apoE+/- rats) underwent abdominal aorta stent implantation. After 28 days blood samples were analyzed to characterize lipid profiles. ISR was assessed by histomorphometric means. Homozygous apoE-/- rats exhibited significantly higher total cholesterol and low-density cholesterol levels than wildtype apoE+/+ and heterozygous apoE+/- rats. ISR was significantly pronounced in homozygous apoE-/- rats as compared to wildtype apoE+/+ (p = <0.0001) and heterozygous apoE+/- rats (p = 0.0102) on western diet. Abdominal aorta stenting of apoE-/- rats is a reliable model to investigate ISR after stent implantation and thus can be used for the evaluation of novel stent concepts. Apolipoprotein E (apoE) deficient rats have been proposed as animal model of atherosclerosis. We investigated the development of restenosis 28 days after stent implantation into the abdominal aorta of wildtype apoE+/+, homozygous apoE-/- and heterozygous apoE+/- rats, respectively. Homozygous apoE-/- rats exhibited significantly higher LDL and significantly lower HDL cholesterol levels compared to wildtype apoE+/+ and heterozygous apoE+/- rats. Restenosis after stent implantation was significantly pronounced in western-diet-fed homozygous apoE-/- rats, accompanied by a significantly increased neointimal thickness. Thus, apoE knockout rats exhibit elevated restenosis and might provide a novel tool for testing of innovative stent concepts.

The Impact of a Nitric Oxide Synthase Inhibitor (L-NAME) on Ischemia⁻Reperfusion Injury of Cholestatic Livers by Pringle Maneuver and Liver Resection after Bile Duct Ligation in Rats.

The Pringle maneuver (PM) has been widely used to control blood loss during liver resection. However, hepatic inflow occlusion can also result in hepatic ischemia-reperfusion injury (IRI), especially in patients with a cholestatic, fibrotic, or cirrhotic liver. Here we investigate a nitric oxide synthase (NOS) inhibitor N-Nitroarginine methyl ester (L-NAME) on IRI after the PM and partial hepatectomy of cholestatic livers induced by bile duct ligation (BDL) in rats. Control group (non-BDL/no treatment), BDL + T group (BDL/L-NAME treatment) and BDL group (BDL/no treatment) were analyzed. Cholestasis was induced by BDL in the L-NAME and BDL group and a 50% partial hepatectomy with PM was performed. L-NAME was injected before PM in the BDL + T group. Hepatocellular damage, portal venous flow, microcirculation, endothelial lining, and eNOS, iNOS, interleukin (IL)-6, and transforming growth factor-ß (TGF-ß) were evaluated. Microcirculation of the liver in the BDL + T group tended to be higher. Liver damage and apoptotic index were significantly lower and Ki-67 labeling index was higher in the BDL + T group while iNOS and TGF-ß expression was decreased. This was corroborated by a better preserved endothelial lining. L-NAME attenuated IRI following PM and improved proliferation/regeneration of cholestatic livers. These positive effects were considered as the result of improved hepatic microcirculation, prevention of iNOS formation, and TGF-ß mRNA upregulation.

The impact of several hydraulic fracking chemicals on Nile tilapia and evaluation of the protective effects of Spirulina platensis.

Hydraulic fracturing (fracking) chemicals are used to maximize the extraction of hard-to-reach underground energy resources. Large amounts of fracking fluid could escape to the surrounding environments, including underground and surface water resources, during the chemical mixing stage of the hydraulic fracturing water cycle due to equipment failure or human error. However, the impact of pollution resulting from operational discharges is difficult to assess in aquatic ecosystems. In this study, pathological investigations, chromosomal aberrations, DNA damage, and biochemical and hematological parameters were used to evaluate the effects of such chemicals on Nile tilapia. Chromosomal aberrations are considered very sensitive genetic markers of exposure to genotoxic chemicals and are used as indicators of DNA damage. The appearance of different types of chromosomal aberrations (gaps and breaks) due to chemical exposure was significantly reduced by treatment with spirulina. Various deleterious findings in Nile tilapia, in the current study, could attributed to the presence of fracking chemicals in the aquatic environment. However, the presence of spirulina in the diet reduced the hazards of such chemicals. In addition, cytogenetic studies in the current work revealed the importance of spirulina in ameliorating the genotoxic effects of a mixture of some chemicals used in fracking.

Sheep models for evaluation of novel patch and prosthesis material in vascular surgery: tips and tricks to avoid possible pitfalls.

BACKGROUND: In vascular surgery, novel synthetic prosthesis materials for patch-angioplasties, interpositions, bypasses and shunts are continuously under development and optimization. The characteristics of an ideal vascular prosthesis would display long-term patency, biocompatibility, durability, low porosity, lack of stich hole bleeding, ease of handling, kink resistance, infection resistance and reasonable costs. The aim of this study was to establish and report a reliable sheep model including potential pitfalls where those parameters could be analyzed. Before surgery, sheep were acclimatized for 4-8 weeks, during which parasite infections were treated and blood and serum parameters monitored. Twenty-four sheep underwent surgery, and carotid patch-angioplasties (n = 12), graft interpositions (n = 6) or arteriovenous prosthetic shunts (n = 6) were implanted. Half of the animals in each group were sacrificed after 2 weeks and the other half after 8 weeks. The implants were analyzed for patency, endothelialization, thrombogenicity and biocompatibility by clinical observation, blood flow measurement and pathological and histopathological (H&E, EvG) as well as immunohistochemical (Ki67, CD31) evaluations. RESULTS: Health monitoring of the sheep revealed a parasitic burden with endoparasites in all animals. Some animals showed thereby infestations in the bile duct causing fibrotic cholangitis with calcifications in the liver. In addition, sarcosporidia were detected in histopathological specimen of the heart in all animals. Parasitic burden correlated with blood counts and serum bilirubin levels. Both were significantly reduced by albendazole treatment within the acclimatization time. Patches, interposition grafts, and straight shunts were successfully implanted bilaterally in all animals. The total average operation time was 136 ± 21 min. Most animals (23/24) showed good patency rates and general condition after implantation. Pathological and histopathological/immunohistochemical analyses were suitable to determine thrombogenicity, endothelialization, cellular/fibroblastic proliferation, biocompatibility, inflammatory cell infiltration, and thickness of neointima in the prosthesis material. CONCLUSIONS: We have developed a suitable experimental protocol with standardized and successful anesthesia- and surgical-procedures for patch-angioplasty, graft interposition, and arteriovenous prosthetic shunts. This sheep model allows testing of new prosthetic materials for biocompatibility, thrombogenicity, and endothelialization.

Novel synthetic adhesive as an effective alternative to Fibrin based adhesives.

AIM: To compare a novel, fully synthetic, polyurethane based glue (MAR-1) to fibrin sealant in a partial liver resection rat model. METHODS: After 50% resection of the lateral left liver lobe in male Wistar rats (n = 7/group/time point), MAR-1, Fibrin or NaCl was applied. After 14, 21 and 90 postoperative days, sealant degradation, intra-abdominal adhesions were scored, and histological examination of liver tissue was performed. RESULTS: (Mean ± SEM) (MAR-1 vs Fibrin vs NaCl). Bleeding mass was significantly higher in NaCl (3.36 ± 0.51 g) compared to MAR-1 (1.44 ± 0.40 g) and Fibrin (1.16 ± 0.32 g). At 14 and 90 d, bleeding time was significantly lower in MAR-1 (6.00 ± 0.9 s; 13.57 ± 3.22 s) and Fibrin (3.00 ± 0.44 s; 22.2 ± 9.75 s) compared to NaCl (158.16 ± 11.36 s; 127.5 ± 23.3 s). ALT levels were significantly higher in MAR-1 (27.66 ± 1 U/L) compared to Fibrin (24.16 ± 0.98 U/L) and NaCl (23.85 ± 0.80 U/L). Intrabdominal adhesions were significantly lower in MAR-1 (11.22% ± 5.5%) compared to NaCl (58.57% ± 11.83%). Degradation of the glue was observed and MAR-1 showed almost no traces of glue in the abdominal cavity as compared to the Fibrin (10% ± 5% 14 d; 7% ± 3% 21 d). Survival showed no significant differences between the groups. CONCLUSION: Compared to Fibrin, MAR-1 showed similar hemostatic properties, no adverse effects, and is biocompatible. Further studies on adhesion strength and biodegradability of synthetic sealants are warranted.

Protective Effects of Adenosine Receptor Agonist in a Cirrhotic Liver Resection Model.

OBJECTIVES: To investigate the role of CGS21680, a selective adenosine A2A receptor agonist, on a bile-duct-ligated cirrhotic liver resection model in rats. METHODS: Male Wistar rats were allotted into 3 groups (n = 7 per time-point): the control group, the bile duct ligation + CGS21680 group (BDL + CGS), and the bile duct ligation group (BDL). Biliary cirrhosis had been previously induced by ligature of the common bile duct in the BDL + CGS and BDL groups. After 2 weeks, the animals underwent partial hepatectomy (50%). The BDL + CGS group received a single dose of CGS21680 15 minutes prior to hepatectomy. Blood samples were collected and analyzed. RESULTS: Aspartate transaminase levels were found to be lower in the control vs BDL groups (1, 3, and 24 h) (P < 0.01) and the BDL + CGS (1 and 3 hours) (P < 0.01) and BDL + CGS vs BDL (24 hours) (P < 0.05) groups. Hepatic flow was measured and BDL showed significantly lower values at the 3, 24, and 168 h time-points compared to the control (P < 0.01) and BDL + CGS groups (P < 0.05 at 3 and 168 hours; P < 0.01 at 24 h). O2C velocity was reduced in the BDL compared to the control group (P < 0.001 at 3 hours; P < 0.01 at 24 and 168 hours) and the BDL + CGS group (P < 0.01 at 24 hours). Interleukin-6 levels were abrogated in the BDL + CGS (P < 0.05) and control (P < 0.01) groups versus BDL. Histone-bound low-molecular-weight DNA fragments in the BDL + CGS (P < 0.01) and control (P < 0.05) groups were low compared to the BDL group. CONCLUSIONS: Administration of CGS21680, an adenosine receptor agonist, after the resection of bile-duct-ligated cirrhotic livers led to improved liver function, regeneration, and microcirculation.

Cyclooxygenase Inhibitors as a New Therapeutic Strategy in Small Bowel Transplantation.

BACKGROUND: The long-term outcome of intestinal transplantations is still not favorable, which is partly due to the intestinal susceptibility to ischemia. There are several indications that the inflammatory response to ischemia-reperfusion injury is mediated by cyclooxygenases and that their inhibition may be associated with improved organ function. The aim of this study was to analyze if cyclooxygenase (COX) inhibitors could improve the early posttransplant outcome after orthotopic small bowel transplantation. METHODS: Small bowel transplantation was performed between rats to test the impact of nonselective (Piroxicam), preferential (Meloxicam), and selective COX-2 inhibitors (Parecoxib). The donor intestines were either perfused and stored with inhibitor or had inhibitor administered intravenously after transplantation. RESULTS: Using COX inhibitors, a sequential increase of posttransplantation intestinal integrity could be shown, with Parecoxib the least effective and Meloxicam the most effective treatment. These differences were in line with the downregulation of COX-2 activity by the inhibitors. Functionally, the same tendency could be seen in diminished expression of proinflammatory molecules, decreased leucocyte inflammation, and significantly improved graft microcirculation. In most cases, the intravenous administration was more effective. However, the COX inhibitors used were shown to cause relevant hepatotoxicity under nearly all conditions, but particularly under intravenous administration. Only Meloxicam in histidine-tryptophan-ketoglutarate was demonstrated to be a safe drug without hepatotoxic side effects. CONCLUSIONS: The activity of COX contributes to ischemia-reperfusion injury after intestinal transplantation. In this comparative study, the administration of the preferential COX-2 inhibitor Meloxicam via histidine-tryptophan-ketoglutarate showed the best graft-protective attributes and the lowest hepatotoxic side effects.

Optimal Timing for Venous Systemic Oxygen Persufflation Supplemented with Nitric Oxide Gas in Cold-Stored, Warm Ischemia-Damaged Experimental Liver Grafts.

BACKGROUND/AIM: Worldwide shortage of donor organs has increased the use of donation after cardiac death (DCD). The aim of this study was to analyze the best time point for venous systemic oxygen persufflation (VSOP) supplemented with nitric oxide (NO) gas during the 1st and 24th hour of cold storage (CS) in warm ischemia (WI)-damaged experimental liver grafts. MATERIALS AND METHODS: Liver grafts (n = 5) were retrieved after 30 min of WI induced by cardiac arrest and CS in histidine-tryptophan-ketoglutarate solution at 4°C. The 1st hour group was immediately persufflated with a VSOP plus NO (VSOP+NO) mixture for 1 h followed by 23 h of static CS (DCD+NO 1st hour). The 24th hour group entailed CS for 23 h followed by 1 h of VSOP+NO persufflation (DCD+NO 24th hour). CS livers without WI but with VSOP served as controls. CS livers with WI represented the fourth group (DCD). Viability of the liver grafts was assessed by normothermic isolated reperfusion for 45 min with oxygenated Krebs-Henseleit buffer. RESULTS: Data are presented as mean ± SEM (control vs. DCD vs. DCD+NO 1st hour vs. DCD+NO 24th hour). After 45 min of reperfusion, the DCD+NO 1st hour group showed significantly lower aspartate aminotransferase (13.4 ± 5.3, 63.2 ± 17.3, 25.6 ± 3.9, and 82.8 ± 27.3 U/l) and lactate dehydrogenase levels (289.4 ± 41.2, 2,139.4 ± 542.7, 577.2 ± 117.2, and 2,429 ± 221.6 U/l). Malondialdehyde levels were significantly abrogated (1.0 ± 0.3, 2.7 ± 1, 1.0 ± 0, and 3.9 ± 1.2 nmol/ml). Significantly higher levels of portal venous pressure were recorded in the DCD+NO 24th hour group (12.0 ± 1, 21.2 ± 3.1, 16.1 ± 1, and 23.2 ± 3.5 mm Hg). NO levels were recorded after 5 min of reperfusion (1.42 ± 0.17, 1.8 ± 0.2, 2.7 ± 0.2, and 2.6 ± 0.1 µmol/l). Bile production levels showed no statistical significance (23.2 ± 3.8, 27.3 ± 1.8, 43.5 ± 18, and 31 ± 2.5 µl/45 min). CONCLUSION: Our results present the beneficial effects of NO combined with VSOP during the 1st hour of CS of WI-damaged experimental liver grafts.

Oxygen Insufflation in University of Wisconsin Solution Ameliorates Reperfusion Injury in Small Bowel after Cold Storage and Reperfusion.

BACKGROUND Results in small bowel transplantation are inferior compared to other solid organ transplantations, among other reasons, due to a specific vulnerability to ischemia/reperfusion injury. New strategies are needed to improve organ storage. Here we compare static cold storage in University of Wisconsin solution to storage supplemented with molecular oxygen gas insufflation. MATERIAL AND METHODS Rat small bowel was retrieved and either stored unoxygenated (UW) or oxygenated (UW+O2) for 18 h at 4°C. Biochemical parameters, mucosal function, Toll-like receptor upregulation, and parameters of structural integrity were evaluated following isolated reperfusion in vitro for 30 min at 37°C. RESULTS Oxygenation showed: ATP concentration was 82 times higher; lactate dehydrogenase release was continuously lower over 30 min; malondialdehyde, a final product of lipid peroxidation (UW+O2 vs. UW; 2.7±0.92 nmol/mL vs. 17.22±10.1 nmol/mL; P<0.05) and nitric oxide concentration (0.87±0.27 µmol/L vs. 2.17±0.29 µmol/L; P<0.001) were significantly lower; whereas mucosal functional integrity (galactose uptake) was better preserved (0.47±0.18 mg/dL vs. 0.35±0.05 mg/dL). Amelioration of tissue damage could be demonstrated by reduced apoptosis (3.3±1.2 AU vs. 28.4±10 AU; P>0.05), and preserved subcellular integrity. Toll-like receptors were significantly less upregulated (TLR2 0.32±0.1 vs. 2.1±1.5-fold and TLR4 1.53±1.14 vs. 11.79±5.4-fold; P<0.05). CONCLUSIONS Oxygenated storage is superior to standard storage in University of Wisconsin solution in terms of energetics, tissue damage, and mucosal integrity.

Gut-liver axis improves with meloxicam treatment after cirrhotic liver resection.

AIM: To investigate the effect of meloxicam on the gut-liver axis after cirrhotic liver resection. METHODS: Forty-four male Wistar rats were assigned to three groups: (1) control group (CG); (2) bile duct ligation with meloxicam treatment (BDL + M); and (3) bile duct ligation without meloxicam treatment (BDL). Secondary biliary liver cirrhosis was induced via ligature of the bile duct in the BDL + M and BDL groups. After 2 wk, the animals underwent a 50% hepatectomy. In the BDL + M group 15 min prior to the hepatectomy, one single dose of meloxicam was administered. Parameters measured included: microcirculation of the liver and small bowel; portal venous flow (PVF); gastrointestinal (GI) transit; alanine aminotransferase (ALT); malondialdehyde; interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-ß1) and hypoxia-inducible factor 1 alpha (HIF-1α) levels; mRNA expression of cyclooxigenase-2 (COX-2), IL-6 and TGF-ß1; liver and small bowel histology; immunohistochemical evaluation of hepatocyte and enterocyte proliferation with Ki-67 and COX-2 liver expression. RESULTS: Proliferative activity of hepatocytes after liver resection, liver flow and PVF were significantly higher in CG vs BDL + M and CG vs BDL group (P < 0.05), whereas one single dose of meloxicam ameliorated liver flow and proliferative activity of hepatocytes in BDL + M vs BDL group. COX-2 liver expression at 24 h observation time (OT), IL-6 concentration and mRNA IL-6 expression in the liver especially at 3 h OT, were significantly higher in BDL group when compared with the BDL + M and CG groups (P < 0.01, P < 0.001, P < 0.01, respectively). Liver and small bowel histology, according to a semi quantitative scoring system, showed better integrity in BDL + M and CG as compared to BDL group. ALT release and HIF-1α levels at 1 h OT were significantly higher in BDL + M compared to CG and BDL group (P < 0.001 and P < 0.01, respectively). Moreover, ALT release levels at 3 and 24 h OT were significantly higher in BDL group compared to CG, P < 0.01. GI transit, enterocyte proliferative activity and number of goblet cells were in favor of meloxicam treatment vs BDL group (P < 0.05, P < 0.001, P < 0.01, respectively). Additionally, villus length were higher in BDL + M as compared to BDL group. CONCLUSION: One single dose of meloxicam administered after cirrhotic liver resection was able to cause better function and integrity of the remaining liver and small bowel.

Impact of oxygen free radicals in rat partial liver transplantation.

BACKGROUND: Due to the shortage of suitable organs, the demand for partial liver transplantation from living donors has increased worldwide. N-acetylcysteine (NAC) has shown protective effects as a free radical scavenger during hypothermic preservation and warm ischemia-reperfusion liver injury; however, no study has reported the effects in partial liver transplantation. The aim of this study was to analyze the impact of NAC on liver graft microcirculation and graft function after partial liver transplantation in rats. METHODS: Orthotopic partial liver transplantations were performed in 40 rats following cold storage in histidine-tryptophan-ketoglutarate solution for 3 h with 20 mM NAC (NAC group, n = 20) or without (control group, n = 20). We assessed portal circulation, graft microcirculation, and biochemical analyses of plasma at 1, 3, 24, and 168 h after portal reperfusion. RESULTS: (Control versus NAC, median and range): Portal venous pressure was significantly lower with NAC (P = 0.03). Microcirculation measured by laser Doppler was significantly improved with NAC throughout the time course (P = 0.003). Alanine aminotransferase levels were significantly lower in the NAC group (P < 0.05). Total antioxidative capacity was significantly higher in the NAC group at 1 h after reperfusion (Trolox equivalents: median, 3 µM; range, 2.9-6.7 versus median, 16.45 µM; range, 10.4-18.8). Lipid peroxidation was significantly abrogated in the NAC group (median, 177.6 nmol/mL; range, 75.9-398.1 versus median, 71.5 nmol/mL; range, 58.5-79 at 3 h). CONCLUSIONS: This study showed that NAC treatment during cold storage resulted in improved microcirculation and preservation quality of partial liver graft likely because of enhanced antioxidant capacity and reduced lipid peroxidation.

Hydroxyectoine ameliorates preservation injury in deceased after cardiac death donors in experimental liver grafts.

BACKGROUND: Due to the drastic shortage of organ donors, clinicians are increasingly considering the use of deceased after cardiac death donors (DCD). Compatible solutes like Ectoine and Hydroxyectoine are produced by extremophilic bacteria as a cell protectant to survive in harsh environments. We hypothesized that the addition of Hydroxyectoine to Histidine-Tryptophan-Ketoglutarate solution (HTK) could ameliorate cold ischemic preservation injury of DCD livers. MATERIAL AND METHODS: Rat livers were harvested from male Wistar rats weighing 250-300 g. Three experimental groups (n=5 per group) were studied: (1) CONTROLS: cold static storage in HTK for 24 h, (2) DCD: 30-min warm ischemia time and 24-h cold static storage in HTK, and (3) DCD+Hydroxyectoine: like DCD, but with 24-h cold static storage in HTK+Hydroxyectoine. Viability of the livers was assessed after 24 h of preservation by isolated perfusion for 45 min with oxygenated Krebs-Henseleit buffer solution. RESULTS: (mean ±SEM, Control vs. DCD vs. DCD+Hydroxyectoine) Parenchymal enzyme release was significantly lower in DCD+Hydroxyectoine compared to DCD (AST: 9±0.54; 56.8±2.05; 32.2±7.25 U/L, ALT: 9.5±0.5; 37.75±9.6; 17.5±4.17 U/L). Bile production at the end of 45 min reperfusion was significantly higher in DCD+Hydroxyectoine (5.16±1.32; 1.36±0.34; 10.75±2.24 µL/g liver weight/45 min). Malondialdehyde values were significantly lower in DCD+Hydroxyectoine (0.8±0.09, 1.14±0.18, 0.77±0.08 nmol/mL). Intercellular adhesion molecule-1 showed significantly lower values in DCD+Hydroxyectoine (219.07±51.79, 431.9±35.70, 205.2±37.71 pg/mL) and the portal venous pressure at 45 min was lower compared to DCD (20.41±0.12, 27.47±0.45, 22.08±0.78 mmHg). CONCLUSIONS: Our data provide evidence for the beneficial role of Hydroxyectoine-modified HTK solution for the preservation of DCD livers compared to HTK.