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In vivo liver decellularization has become a promising strategy to study in vivo liver engineering. However, long-term survival after in vivo liver decellularization has not yet been achieved due t...

ZusammenfassungUm den Erfordernissen der medizinischen Versorgung aktuell und in Zukunft Rechnung zu tragen, wird die medizinische Aus- und Weiterbildung zunehmend kompetenzbasiert ausgerichtet. Grundlage bilden handlungsorientiert formulierte Lernziele, die in kompetenzorientierten Lernzielkatalogen zusammengeführt werden. Der Nationale Kompetenzbasierte Lernzielkatalog Medizin (NKLM) kann als fächerübergreifende Basis für ein Kerncurriculum herangezogen werden. Bereits 2013 sind für die chirurgischen Fachdisziplinen mit dem speziellen Teil des Nationalen Kompetenzbasierten Lernzielkataloges Chirurgie (NKLC) Handlungskompetenzen definiert worden, die die Studierenden nach Abschluss des Praktischen Jahres in Bezug auf konkrete Krankheitsbilder erreicht haben sollen. Mit dem nun ergänzten allgemeinen Teil des NKLC wurden disziplinübergreifend Kompetenzen definiert und von allen chirurgischen Fachdisziplinen konsentiert, die für sämtliche chirurgische Fachdisziplinen gleichermaßen Gültigkeit haben und von Vertreterinnen und Vertretern aller Fachdisziplinen in die Lehre einbezogen werden sollten. Der nun vollständige NKLC liegt den Fakultäten, Lehrenden und Lernenden zur Erprobung vor (verfügbar unter: https://www.dgch.de/index.php?id=190&L=528). Leitgedanke für den gesamten Entwicklungsprozess waren das Erreichen der grundsätzlichen Weiterbildungsbefähigung und die Patientensicherheit beim Berufseinstieg.

Zusammenfassung Hintergrund Die Verwendung videobasierter Analysekonzepte gewinnt in der medizinischen Ausbildung zunehmend an Bedeutung. In erster Linie dienen diese Tools dazu, Informationen über die individuelle Performance eines Kandidaten zu bekommen und Feedback zu geben. Die vorliegende Studie untersucht, ob die Videoanalyse praktischer Fertigkeiten auch für die Weiterentwicklung des chirurgischen Unterrichts genutzt werden kann. Material und Methoden Zunächst wurde die Durchführung einer chirurgischen Nahtübung (Dauer: 3 min) bei Studierenden des 10. Semesters (n = 38) videobasiert dokumentiert. Im Anschluss folgte die Analyse des Videomaterials anhand von 10 spezifischen Kriterien. Die Analyse diente dann als Grundlage für die Entwicklung fehlerpräventiver Übungen. Nachfolgend wurden die Effekte der fehlerpräventiven Übungen auf die Ergebnisqualität der Nahtübung im Rahmen eines Pilotversuchs in einem 2-Gruppen-Vergleich untersucht. Ergebnisse Die Videosequenzen wurden von 2 Experten unabhängig voneinander begutachtet. Es konnten typische Fehler bei der Handhabung des chirurgischen Instrumentariums, der Handhabung des Nahtmaterials sowie im Bewegungsablauf beobachtet werden. Die daraufhin entwickelten zusätzlichen Übungseinheiten fokussierten sich auf die identifizierten Fehlerbereiche (z. B. Handhabung des Nadelhalters und des Nahtmaterials). Die Ergebnisse des 2-Gruppen-Vergleichs (vor und nach Integration der fehlerpräventiven Übungen in das Kurskonzept) zeigten, dass das Absolvieren der zusätzlichen Übungseinheiten einen mittelstarken Effekt auf die Ergebnisqualität einer Nahtübung hatte (Cohens d = 0,73). Schlussfolgerung Die Videoanalyse praktischer Fertigkeiten scheint eine geeignete Basis für die Weiterentwicklung des chirurgischen Unterrichts darzustellen. Typische Fehler können in Bezug auf Art und Häufigkeit identifiziert werden und es lassen sich fehlerpräventive Übungen entwickeln, die einen positiven Effekt auf die Ergebnisqualität einer praktischen Aufgabe haben.

ZusammenfassungDer „Masterplan Medizinstudium 2020“ der Bundesregierung darf in der Chirurgie keinesfalls als „beiläufiges Werk unter Vielen“ unterschätzt werden. Daher nimmt die chirurgische Arbeitsgemeinschaft Lehre (CAL) der Deutschen Gesellschaft für Chirurgie (DGCH) in ihrem Positionspapier zu den geplanten Maßnahmen im „Masterplan Medizinstudium 2020“ Stellung und diskutiert die Herausforderungen, Konsequenzen und Aufgaben, vor die der „Masterplan Medizinstudium 2020“ die Fachvertreter der chirurgischen Fachgesellschaften und die in der Lehre engagierten Chirurgen stellt.

Background: Expectations towards surgeons in modern surgical practice are extremely high with minimal complication rates and maximal patient safety as paramount objectives. Both of these aims are highly dependent on individual technical skills that require sustained, focused, and efficient training outside the clinical environment. At the same time, there is an increasing moral and ethical pressure to reduce the use of animals in research and training, which has fundamentally changed the practice of microsurgical training and research. Various animal models were introduced and widely used during the mid-20th century, the pioneering era of experimental microsurgery. Since then, high numbers of ex vivo training concepts and quality control measures have been proposed, all aiming to reduce the number of animals without compromising quality and outcome of training. Summary: Numerous microsurgical training courses are available worldwide, but there is no general agreement concerning the standardization of microsurgical training. The major aim of this literature review and recommendation is to give an overview of various aspects of microsurgical training. We introduce here the findings of a previous survey-based analysis of microsurgical courses within our network. Basic principles behind microsurgical training (3Rs, good laboratory practice, 3Cs), considerations around various microsurgical training models, as well as several skill assessment tools are discussed. Recommendations are formulated following intense discussions within the European Society for Surgical Research (ESSR) and the International Society for Experimental Microsurgery (ISEM), based on scientific literature as well as on several decades of experience in the field of experimental (micro)surgery and preclinical research, represented by the contributing authors. Key Messages: Although ex vivo models are crucial for the replacement and reduction of live animal use, living animals are still indispensable at every level of training which aims at more than just a basic introduction to microsurgical techniques. Modern, competency-based microsurgical training is multi-level, implementing different objective assessment tools as outcome measures. A clear consensus on fundamental principles of microsurgical training and more active international collaboration for the sake of standardization are urgently needed.

// Anding Liu 1,2 , Enshuang Guo 3 , Jiankun Yang 1 , Renlong Li 3 , Yan Yang 1 , Shenpei Liu 1 , Jifa Hu 1 , Xiaojing Jiang 3 , Olaf Dirsch 2 , Uta Dahmen 2 , Jian Sun 4 and Mingwen Ouyang 5 1 Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Friedrich-Schiller-University Jena, Jena, Germany 3 Department of Infectious Diseases, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China 4 Department of Biliopancreatic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 5 Department of Anesthesiology, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, China Correspondence to: Mingwen Ouyang, email: // Keywords : ischemic preconditioning; steatosis; liver ischemia/reperfusion injury; autophagy; heme oxygenase-1, Pathology Section Received : April 25, 2016 Accepted : November 02, 2016 Published : November 10, 2016 Abstract Steatotic livers are more susceptible to ischemia/reperfusion (I/R) injury, which is ameliorated by ischemic preconditioning (IPC). Autophagy possesses protective action on liver I/R injury and declines in steatotic livers. The aim of this study was to test the hypothesis that the increased susceptibility of steatotic livers to I/R injury was associated with defective hepatic autophagy, which could be restored by IPC via heme oxygenase-1 (HO-1) signaling. Obesity and hepatic steatosis was induced using a high fat diet. Obesity impaired hepatic autophagy activity and decreased hepatic HO-1 expression. Induction of HO-1 restored autophagy activity and inhibited calpain 2 activity. Additionally, suppression of calpain 2 activity also restored autophagy activity. Mitochondrial dysfunction and hepatocellular injury were significantly increased in steatotic livers compared to lean livers in response to I/R injury. This increase in sensitivity to I/R injury was associated with defective hepatic autophagy activity in steatotic livers. IPC increased autophagy and reduced mitochondrial dysfunction and hepatocellular damage in steatotic livers following I/R injury. Furthermore, IPC increased HO-1 expression. Inhibition of HO-1 decreased the IPC-induced autophagy, increased calpain 2 activity and diminished the protective effect of IPC against I/R injury. Inhibition of calpain 2 restored autophagic defect and attenuated mitochondrial dysfunction in steatotic livers after I/R. Collectively, IPC might ameliorate steatotic liver damage and restore mitochondrial function via HO-1-mediated autophagy.

Aging is often associated with a decreased autophagic activity that contributes to the high sensitivity of aged livers to ischemia reperfusion injury (IRI). Blood from young animals can positively affect aged animals. This study was designed to evaluate the effect of young plasma in a model of liver IRI in aged rats. Aged rats were treated with pooled plasma collected from young rats before ischemia. Administration of young plasma restored aging-induced suppression in hepatic autophagic activity and reduced liver IRI. Inhibition of the young-plasma-restored autophagic activity abrogated the beneficial effect of young plasma against liver IRI. Similarly, young serum restored autophagic activity and reduced cellular injury after hypoxia/reoxygenation (H/R) in primary old rat hepatocytes. Mechanistic studies showed thatadministration of young plasma increased AMPK phosphorylation and led to unc-51-like autophagy activating kinase (ULK)1 activation. Furthermore, AMPK-inhibition abrogated the young serum-induced ULK1 activation and autophagic activity and diminished the protective action of young serum against H/R injury in primary old rat hepatocytes, whereas AMPK-activation potentiated the effects of young serum. Young plasma could restore age-impaired autophagy, at least in part, via AMPK/ULK1 signaling. Restoration of age-impaired autophagic activity may be a critical contributing mechanism to young-plasma-afforded protection against liver IRI in aged rats.-Liu, A., Yang, J., Hu, Q., Dirsch, O., Dahmen, U., Zhang, C., Gewirtz, D. A., Fang, H., Sun, J. Young plasma attenuates age-dependent liver ischemia reperfusion injury.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

Growth differentiation factor 11 (GDF11) has been implicated in a variety of aging conditions and the regulation of organ regeneration after injury; however, the role of GDF11 in liver ischemia reperfusion injury (IRI) is unknown. The aim of the current study was to investigate the possible role of GDF11 in liver IRI. We investigated the effects of GDF11 in liver IRI in both young (3 mo) and old (22 mo) mice in vivo, and in primary young and old mouse hepatocytes in vitro. Both serum and hepatic GDF11 protein expression levels increased with age and after IRI. Treatment with recombinant GDF11 significantly increased IRI-induced elevations of serum aminotransferase levels, worsened the histologic status of livers, and impaired liver regeneration. In contrast, inhibition of GDF11 activity with neutralizing Abs significantly decreased liver injury and improved liver regeneration after IRI. In vitro, treatment with recombinant GDF11 significantly delayed cell proliferation in cultured hepatocytes that were subjected to hypoxia/reoxygenation insult. Moreover, suppression of cell-cycle progression may be a key mechanism by which GDF11 inhibited hepatocyte regeneration. Collectively, rather than acting as a rejuvenating agent, GDF11 worsens hepatocellular injury and impairs liver regeneration after IRI.-Liu, A., Dong, W., Peng, J., Dirsch, O., Dahmen, U., Fang, H., Zhang, C., Sun, J. Growth differentiation factor 11 worsens hepatocellular injury and liver regeneration after liver ischemia reperfusion injury.

Multimodal treatment concepts including liver transplantation for hepatocellular carcinoma (HCC), extended resection methods and neoadjuvant chemotherapy for colorectal liver metastasis significantly improve patients’ outcome. However, surgery-induced hepatic ischemia-reperfusion injury (IRI) and chemotherapy-associated hepatotoxicity result in hepatocellular damage and compromised liver function. Activation of common key pathways in ischemic liver and hepatotoxic injury results in oxidative stress, inflammatory responses and apoptosis causing organ damage. Controlling liver damage before and during surgery is essential for the postoperative outcome. Nigella sativa has a long tradition as a natural remedy. In the essential oil, Thymoquinone (TQ) was identified as the main component and responsible for most of the therapeutic effects. Therefore, this systematic review aimed to summarize the hepatoprotective effects of TQ and its potential suitability to improve surgical outcome by reducing surgical ischemic injury and hepatotoxicity of neoadjuvant chemotherapy. The key findings can be summarized as TQ having strong antioxidant, anti-inflammatory, antifibrotic, anti-/proapoptotic and anticarcinogenic effects. Almost no side effects were reported irrespective of a large dose range, suggesting a wide therapeutic window. These results give rise to the expectation that TQ could evolve to a novel powerful drug to reduce hepatic injury.

Cholestasis is a common complication in liver diseases that triggers a proliferative response of the biliary tree. Bile duct ligation (BDL) is a frequently used model of cholestasis in rodents. To determine which changes occur in the three‐dimensional (3D) architecture of the interlobular bile duct during cholestasis, we used 3D confocal imaging, surface reconstructions, and automated image quantification covering a period up to 28 days after BDL. We show a highly reproducible sequence of interlobular duct remodeling, where cholangiocyte proliferation initially causes corrugation of the luminal duct surface, leading to an approximately five‐fold increase in surface area. This is analogous to the function of villi in the intestine or sulci in the brain, where an expansion of area is achieved within a restricted volume. The increase in surface area is further enhanced by duct branching, branch elongation, and loop formation through self‐joining, whereby an initially relatively sparse mesh surrounding the portal vein becomes five‐fold denser through elongation, corrugation, and ramification. The number of connections between the bile duct and the lobular bile canalicular network by the canals of Hering decreases proportionally to the increase in bile duct length, suggesting that no novel connections are established. The diameter of the interlobular bile duct remains constant after BDL, a response that is qualitatively distinct from that of large bile ducts, which tend to enlarge their diameters. Therefore, volume enhancement is only due to net elongation of the ducts. Because curvature and tortuosity of the bile duct are unaltered, this enlargement of the biliary tree is caused by branching and not by convolution. Conclusion: BDL causes adaptive remodeling that aims at optimizing the intraluminal surface area by way of corrugation and branching. (Hepatology 2016;63:951–964)

Background/Aim: Liver size regulation is based on the balance between hepatic regeneration and atrophy. To achieve a better understanding of intrahepatic size regulation, we explored the size regulation of a portally deprived liver lobe on a liver subjected to concurrent portal vein ligation (PVL) and partial hepatectomy (PHx). Materials and Methods: Using a surgical rat model consisting of right PVL (rPVL) plus 70% PHx, we evaluated the size regulation of liver lobes 1, 2, 3, and 7 days after the operation in terms of liver weight and hepatocyte proliferation. Portal hyperperfusion was confirmed by measuring portal flow. The portal vascular tree was visualized by injection of a contrast agent followed by CT imaging of explanted livers. Control groups consisted of 70% PHx, rPVL, and sham operation. Results: The size of the ligated right lobe increased to 1.4-fold on postoperative day 7 when subjected to rPVL + 70% PHx. The right lobe increased to 3-fold when subjected to 70% PHx alone and decreased to 0.3-fold when subjected to rPVL only. The small but significant increase in liver weight after the combined procedure was accompanied by a low proliferative response. In contrast, hepatocyte proliferation was undetectable in the right lobe undergoing atrophy after PVL only. The caudate lobe in the rPVL + 70% PHx group increased to 4.6-fold, which is significantly more than in the other groups. This increase in liver weight was paralleled by persisting portal hyperperfusion and a prolonged proliferative phase of 3 days. Conclusions: A discontinued portal blood supply does not always result in atrophy of the ligated lobe. The concurrent regenerative stimulus induced by 70% PHx seemed to counteract the local atrophy after a simultaneously performed rPVL, leading to a low but prolonged regenerative response of the portally deprived liver lobe. This observation supports the conclusion that portal flow is not necessary for liver regeneration. The persisting portal hyperperfusion may be crucial for the specific kinetics of prolonged liver regeneration after rPVL + 70% PHx in the portally supplied caudate lobe. Both observations deserve more attention regarding the underlying mechanism in further studies.

Differences in LPS responsiveness influence the outcome of patients with sepsis. The intensity of the response is highly variable in patients and strain dependent in rodents. However, the role of the liver for initiating the LPS response remains ill defined. We hypothesize that hepatic LPS uptake is a key event for initiating the LPS response. In the present study, the severity of the LPS-induced inflammatory response and the hepatic LPS uptake was compared in two rat strains (Lewis (LEW) rats and Brown Norway (BN) rats). Using a transplantation model, we demonstrated the decisive role of the liver. The expression of hepatic TNF-α, IL-6, and IL-1β mRNA levels in BN rats was significantly lower than that in LEW rats. LEW rats were sensitized to LPS via G-CSF pretreatment. Sensitization caused by G-CSF pretreatment induced severe liver injury and mortality in LEW rats, but not in BN rats (survival rate: 0% (LEW) versus 100% (BN), p<0.01). LEW rats presented with higher liver enzymes, more alterations in histology, and higher expression of caspase 3 and higher cytokines levels. One of the reasons could be the increased hepatic LPS uptake, which was only observed in LEW but not in BN livers. Using the transplantation model revealed the decisive role of the LPS responsiveness of the liver. Injection of LPS to the high-responding LEW recipient before transplantation of a low-responder BN liver resulted in a 50% survival rate. In contrast, injecting the same dose of LPS into the high-responding LEW recipient after transplanting the low-responding BN liver resulted in a 100% survival rate. The severity of inflammatory response in different strains might be related to the differences in hepatic LPS uptake. This observation suggests that the liver plays a genetically defined decisive role in modulating the inflammatory severity.

Background: Automated image analysis enables quantitative measurement of steatosis in histological images. However, spatial heterogeneity of steatosis can make quantitative steatosis scores unreliable. To improve the reliability, we have developed novel scores that are ""focused"" on steatotic tissue areas. Methods: Focused scores use concepts of tile-based hotspot analysis in order to compute statistics about steatotic tissue areas in an objective way. We evaluated focused scores on three data sets of images of rodent liver sections exhibiting different amounts of dietary-induced steatosis. The same evaluation was conducted with the standard steatosis score computed by most image analysis methods. Results: The standard score reliably discriminated large differences in steatosis (intraclass correlation coefficient ICC = 0.86), but failed to discriminate small (ICC = 0.54) and very small (ICC = 0.14) differences. With an appropriate tile size, mean-based focused scores reliably discriminated large (ICC = 0.92), small (ICC = 0.86) and very small (ICC = 0.83) differences. Focused scores based on high percentiles showed promise in further improving the discrimination of very small differences (ICC = 0.93). Conclusions: Focused scores enable reliable discrimination of small differences in steatosis in histological images. They are conceptually simple and straightforward to use in research studies.

The liver has the unique capability of regeneration from various injuries. Different animal models and in vitro methods are used for studying the processes and mechanisms of liver regeneration. Animal models were established either by administration of hepatotoxic chemicals or by surgical approach. The administration of hepatotoxic chemicals results in the death of liver cells and in subsequent hepatic regeneration and tissue repair. Surgery includes partial hepatectomy and portal vein occlusion or diversion: hepatectomy leads to compensatory regeneration of the remnant liver lobe, whereas portal vein occlusion leads to atrophy of the ipsilateral lobe and to compensatory regeneration of the contralateral lobe. Adaptation of modern radiological imaging technologies to the small size of rodents made the visualization of rodent intrahepatic vascular anatomy possible. Advanced knowledge of the detailed intrahepatic 3D anatomy enabled the establishment of refined surgical techniques. The same technology allows the visualization of hepatic vascular regeneration. The development of modern histological image analysis tools improved the quantitative assessment of hepatic regeneration. Novel image analysis tools enable us to quantify reliably and reproducibly the proliferative rate of hepatocytes using whole-slide scans, thus reducing the sampling error. In this review, the refined rodent models and the newly developed imaging technology to study liver regeneration are summarized. This summary helps to integrate the current knowledge of liver regeneration and promises an enormous increase in hepatological knowledge in the near future. © 2014 S. Karger AG, Basel

Liver dysfunction is a serious complication in the early phase following major liver resection or liver transplantation and might be aggravated by the translocation of bacteria and lipopolysaccharide (LPS). As a preventive strategy, granulocyte colony-stimulating factor (G-CSF) is prophylactically applied in patients who are subjected to major surgery. However, we previously demonstrated that G-CSF can induce LPS sensitization. In this study, we aimed to evaluate the effects of G-CSF pretreatment on hepatic microcirculatory disturbances and postoperative liver dysfunction after 70 % partial hepatectomy (PH) in rats. PH alone was well tolerated by all animals (100 % survival rate, slight liver damage and inflammation). LPS application after 70 % PH caused moderate inflammation, microcirculatory disturbances and hepatic damage and led to a 24-h survival rate of 30 % after the operations. In the G-CSF-LPS-PH group, all of the rats died within 4 h with severe inflammatory responses and liver damage (i.e., pronounced erythrocyte congestion and neutrophil infiltration). Portal hypertension and microcirculatory disorders (i.e., inhomogeneous perfusion, sinusoidal dilatation and reductions on functional capillary density) were more pronounced in the G-CSF-LPS-PH group. In conclusion, increased circulating LPS levels were associated with an imbalanced inflammatory response and microcirculatory dysfunction that preceded liver damage and subsequent dysfunction following surgery. G-CSF-pretreatment aggravated microcirculatory disturbances and liver damage, which might have been related to G-CSF-induced LPS sensitization.

Background Steatotic livers are particularly vulnerable to ischemia/reperfusion injury (IRI). One of the reasons is an underlying impairment of autophagy. Autophagy is regulated by glycogen synthase kinase 3b (GSK3b) and extracellular signal-regulated kinases (ERK1/2) pathways. Both of them are target proteins of a cell-protective drug, lithium chloride. Lithium chloride treatment reduces IRI in many organs including liver. Therefore, we aimed to investigate the effect of lithium chloride treatment on autophagy induction in steatotic rat livers. We also wanted to evaluate the related cell-protective effects on the enhanced hepatic IRI. Materials and methods After inducing hepatic steatosis, rats were injected with lithium chloride or normal saline for 3 d before being subjected to 70% selective warm ischemia for 60 min. After reperfusion, rats were observed for 30 min, 6, 24, and 48 h. Results Lithium chloride appeared to protect hepatocytes from IRI via its ability to induce autophagy by modulation of both GSK3b and ERK1/2 pathways. Hepatic damage was significantly decreased in the treatment group as indicated by a reduced inflammatory response, less apoptosis, less necrosis, and lower liver enzyme levels. Conclusions Simultaneous modulation of GSK3b and ERK1/2 pathways might be an interesting strategy to reduce IRI in steatotic livers with an impairment of autophagy.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (µCT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees. The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after µCT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Presence of hematopoietic stem-cell-derived hepatocytes after clinical liver transplantation was demonstrated repeatedly. The relevance of this controversial mechanism of regeneration was discussed. Regarding frequency, the demonstrated results were divergent. In the present study, we propose to investigate the influence of growth and regeneration on the frequency of hematopoietic stem-cell-derived hepatocytes in transplanted organs.Paraffin-embedded liver specimens, obtained as clinically indicated, from female grafts transplanted into male recipients were investigated. The presence of Y-chromosome in hepatocytes, detected by fluorescence in situ hybridization (FISH), was the indicator for recipient origin. Slides were evaluated by assessing the relative number of Y-chromosome containing hepatocytes within 50 images representing an average of 775 hepatocytes.In only 9 out of 81 specimens, single Y-chromosome positive hepatocytes were detected, resulting in a maximal frequency of 0.64%. Six positive specimens were obtained from full-size liver grafts and one from a partial liver graft. In the pediatric group, two positive samples were found. By staining additional sections from the nine positive specimens, no additional positive hepatocytes were detected suggesting an even lower frequency within the whole sample. We did not find any accumulation of Y-chromosome positive cells in any of the individually analyzed patient groups.Transdifferentiation of hematopoietic stem cells is an extremely rare event in liver growth and regeneration after transplantation. Due to the low number of positive events, the biological relevance seems questionable.

Variations among inbred rats in terms of anatomy and routine laboratory values can potentially blur surgical experimental results. Therefore, a retrospective analysis aiming at investigating hepatic and perihepatic anatomical variations, liver weight, body weight, liver weight/body weight ratio (LBWR), variations in routine laboratory values, and the influence of shipment and repeated sampling was performed. In our study, liver weight of rats seemed to be strain-specific. LBWR was weakly and negatively correlated with body weight in rats. A statistically significant difference in routine blood tests was found among normal rats grouped by different body weight or shipment. Weekly repeated sampling from the same rats revealed a statistically significant difference in a blood test. In conclusion, the fact that variation among rats or their environment can blur the results of a surgical experimental study should be kept in mind.

Recent in vitro studies of cold-induced cell injury have revealed the detrimental effects of extracellular chloride on cold-stored isolated rat hepatocytes; however, its influence on endothelial cells is beneficial. To determine which of these effects is predominant in vivo, we tested both a chloride-poor variant of a new histidine-tryptophan-ketoglutarate (HTK)–based preservation solution and a chloride-containing variant in a rat liver transplantation model. The study, which was carried out in a blinded fashion with 7 or 8 rats per group, was divided into 2 parts: (1) a comparison of survival in 3 series under different conditions [different microsurgeons, rat strains, cold ischemia times (3, 12, and 24 hours), and warm ischemia times] and (2) an assessment of the microcirculation (30-90 minutes after reperfusion), laboratory data, bile production, and histology. In each of the survival experiments, a (strong) tendency toward prolonged survival was observed with the new chloride-containing solution (50% versus 12.5%, 75% versus 37.5%, and 100% versus 71.4% [chloride-containing vs. chloride-poor], overall P < 0.05). Additionally, the sinusoidal perfusion rates (83.9% ± 4.0% versus 69.2% ± 10.8%, P < 0.01) and the red blood cell velocities in sinusoids (147.7 ± 26.7 versus 115.5 ± 26.0 μm/second, P < 0.05) and in postsinusoidal venules (332.4 ± 87.3 versus 205.5 ± 53.5 μm/second, P < 0.01) were clearly higher with chloride. Moreover, the serum activities of liver enzymes were slightly reduced (not significantly), and bile production was significantly increased. These results suggest an overall beneficial effect of chloride in HTK-based liver preservation solutions. Liver Transpl 17:650-660, 2011. © 2011 AASLD.

High mobility group box 1 (HMGB1) acts as an early mediator in inflammation and organ injury. Ischemia reperfusion (I/R) injury induces HMGB1 translocation and expression in ischemic areas. However, it is unknown whether selective warm liver I/R injury also induces the expression of HMGB1 in non-ischemic lobes. The present study aimed to test the hypothesis that selective liver I/R injury also causes HMGB1 translocation and up-regulates its expression in non-ischemic liver areas. In the present study, selective I/R injury was induced by clamping the median and left lateral liver lobes for 90 min followed by 0.5, 6 and 24 h reperfusion. We used male inbred Lewis rats; six animals for each point in time and six animals for the normal control group. Selective hepatic I/R injury induced morphological changes not only in ischemic lobes but also in non-ischemic lobes. HMGB1 translocation and expression was increased in a time-dependent manner in the ischemic lobes, and increased in with delayed onset in the non-ischemic lobes. Serum HMGB1 levels were increased after reperfusion. Furthermore, liver I/R injury up-regulated the expression of HMGB1 receptors (Toll-like receptor 4 and receptor for advanced glycation end products and pro-inflammatory cytokines (Tumor necrosis factor-alpha and interleukin-6) in both ischemic lobes, however, the up-regulation of these cytokines was more prominent in the ischemic lobes. In conclusion, selective warm I/R induces a substantial "sympathetic/bystander" effect on the non-ischemic lobes in terms of HMGB1 translocation and local cytokine production.

SUMMARY Quantification of liver regeneration is frequently based on determining the 5-bromo-2-deoxyuridine labeling index (BrdU-LI). The quantitative result is influenced by preanalytical, analytical, and postanalytical variables such as the region of interest (ROI). We aimed to present our newly developed and validated automatic computer-based image analysis system (AnalySIS-Macro), and to standardize the selection and sample size of ROIs. Images from BrdU-labeled and immunohistochemically stained liver sections were analyzed conventionally and with the newly developed AnalySIS-Macro and used for validation of the system. Automatic quantification correlated well with the manual counting result (r50.9976). Validation of our AnalySIS-Macro revealed its high sensitivity (.90%) and specificity. The BrdU-LI ranged from 11% to 57% within the same liver (32.96 6 11.94%), reflecting the highly variable spatial distribution of hepatocyte proliferation. At least 2000 hepatocytes (10 images at 2003 magnification) per lobe were required as sample size for achieving a representative BrdU-LI. Furthermore, the number of pericentral areas should be equal to that of periportal areas. The combination of our AnalySIS-Macro with rules for the selection and size of ROIs represents an accurate, sensitive, specific, and efficient diagnostic tool for the determination of the BrdU-LI and the spatial distribution of proliferating hepatocytes. (J Histochem Cytochem 57:1075–1085, 2009)

Reduced-size liver transplantation is associated with liver regeneration. This study was designed to analyze the influence of graft size on liver rejection and liver regeneration.Reduced-size liver transplantations were performed in the rejecting ACI to Lewis and the graft acceptance BN to Lewis strain combination. The BN to Lewis control group was treated with the immunosuppressive drug FK506.An accelerated liver rejection in the ACI to Lewis strain combination was found in small-for-size partial liver grafts. Graft weight to recipient liver weight ratio (GW/RLW) showed a positive correlation with survival time. In the BN to Lewis strain combination, lethal rejection was seen in small-for-size partial liver grafts. A critical immunologic GW/RLW of 33% was calculated. In rats dying from lethal rejection, GW/RLW and survival time showed a positive correlation. However, GW/RLW showed a negative correlation with hepatocellular proliferation. In regenerating livers, MHC II upregulation was also observed in the control group. All control animals survived small-for-size liver transplantation.The relative graft size seems to be a decisive factor influencing the kinetic of liver rejection and the induction of liver rejection. Relative critical immunologic liver mass was determined to be 33%.

Many physiological processes and pathological conditions in livers are spatially heterogeneous, forming patterns at the lobular length scale or varying across the organ. Steatosis, a common liver disease characterized by lipids accumulating in hepatocytes, exhibits heterogeneity at both these spatial scales. The main goal of the present study was to provide a method for zonated quantification of the steatosis patterns found in an entire mouse liver. As an example application, the results were employed in a pharmacokinetics simulation.For the analysis, an automatic detection of the lipid vacuoles was used in multiple slides of histological serial sections covering an entire mouse liver. Lobuli were determined semi-automatically and zones were defined within the lobuli. Subsequently, the lipid content of each zone was computed. The steatosis patterns were found to be predominantly periportal, with a notable organ-scale heterogeneity.The analysis provides a quantitative description of the extent of steatosis in unprecedented detail. The resulting steatosis patterns were successfully used as a perturbation to the liver as part of an exemplary whole-body pharmacokinetics simulation for the antitussive drug dextromethorphan. The zonated quantification is also applicable to other pathological conditions that can be detected in histological images. Besides being a descriptive research tool, this quantification could perspectively complement diagnosis based on visual assessment of histological images. Graphical abstractDisplay Omitted HighlightsA new spatial quantification shows heterogeneity of liver biomarkers on two scales.Steatosis was assessed in histological serial sections covering an entire mouse liver.The steatosis is distributed periportally with a notable variation across the organ.Steatosis distributions influence results of a whole-body pharmacokinetics simulation.This quantification can complement visual evaluation of histological images.

Background We adapted the anatomically oriented parenchyma-preserving resection technique for associating liver partition with portal vein ligation (PVL) for staged hepatectomy (ALPPS) in rats and examined the role of revascularization in intrahepatic size regulation. Methods We performed the procedures based on anatomic study. The ALPPS procedure consisted of a 70% PVL (occluding the left median, left lateral, and right lobes), parenchymal transection (median lobe) and partial (10%) hepatectomy (PHx; caudate lobe). The transection effect was evaluated by measuring the extent of hepatic atrophy or regeneration of individual liver lobes in the ALPPS and control groups (70% PVL and 10% PHx without transection). The survival rates after stage II resection and collateral formation within the portal vein system was examined. Results Anatomic study revealed a close spatial relationship between the demarcation line and the middle median hepatic vein. This enabled placing the transection plane without injuring the hepatic vein. Transection was achieved via stepwise clamping, followed by 2–3 parenchyma-preserving piercing sutures on both sides of the clamp. Ligated liver lobes atrophy was significantly enhanced after ALPPS compared with the control group. In contrast, both a significantly greater relative weight of the regenerated lobe and proliferation index on the first postoperative day were observed. All animals tolerated stage II-resection without complications. Portoportal collaterals were only observed in the control group. Conclusion We developed an anatomically precise technique for parenchymal transection. The lack of a dense vascular network between the portalized and deportalized lobes may play an important role in accelerating regeneration and atrophy augmentation.

It is well known that personal decision making in respect to organ donation is highly dependent on the balance of knowledge, trust, and fear. We wanted to explore the attitude of German medical students towards organ donation and investigate the relationship between knowledge, trust, and fear in this special subgroup. We conducted an online survey utilizing (1) the snowball effect of using Facebook groups and advertisement as well as (2) mailing lists of medical faculties in Germany for distribution. We surveyed 1370 medical students. 75.8 % (N = 988) of the participants stated to carry an organ donor card and allowed their organs to be donated. 1.8 % (N = 23) refused donation. 22.5 % (N = 293) did not carry an organ donor card. Analysis of the “decided” versus the “undecided” group revealed substantial differences regarding transplantation knowledge (mean knowledge score of 4.23 vs. 3.81; P

Living liver donation between 2 adults is sometimes hampered by the fact that either graft or remnant liver are considered to be too small to support the life of the donor and/or recipient. A graft-to-body-weight ratio larger than 0.8 is considered to be safe.1 However, there are a number of reports2–4 describing successful partial liver transplantations and extended liver resections with a smaller liver mass. To further extend liver resections and also living liver donation to donor-recipient pairs with a liver mass below the current safety margin, an animal model is needed to investigate the maximal reduction of liver mass compatible with survival. Dependent on the combination of lobes to be resected, the approximate reduction of liver mass is estimated. Reduction of the liver mass by 90% through extended hepatectomy in rats was introduced by Weinbren et al5,6 and was regarded to be a lethal model. Resection of individual lobes was performed by mass ligation, which carries the risk of either constricting the vena cava, when placed at the base of the lobes or the risk of bleeding when placed at some distance from the vena cava. The transition from crude mass ligation to a more delicate vessel-oriented approach reducing both risks was introduced by Kubota et al7 who were the first to achieve a 100% 1-week survival rate after 90% liver resection. Determination of the absolute minimal liver mass supporting the animal's life requires the development of a surgical approach allowing a further reduction of the liver mass beyond the 90% resection model while still keeping the animal alive. A few reports are available describing the 95% resection model, achieved by performing a 90% resection plus the additional removal of the upper caudate lobe, leaving only the lower caudate lobe behind.8–12 The maximal reduction of liver mass, which is surgically achievable, consists of the removal of all liver lobes, leaving only the stumps and the paracaval tissue as remnant liver. This represents a 97% resection. No report regarding such a model exists up to now. To determine the absolute minimal liver mass, a precise vessel-oriented, parenchyma-preserving surgical technique must be used. Detailed anatomic knowledge of the hepatic vascular system is required to develop such a precise vessel-oriented resection technique for extreme experimental liver surgery. Based on the three-dimensional modeling of the hepatic vascular anatomy, the vessel-oriented approach was further optimized.

The aim of this study was to assess the impact of donor gender on small-for-size (SFS) liver transplantation in male recipients using a rat model. Adult female or male Lewis rats were used as donors and male Lewis rats as recipients. Size-matched (SM) and SFS liver grafts from either male or female donors were transplanted into male recipients. Animals receiving SFS grafts were sacrificed at postoperative week 1, week 4, and week 12, respectively (n = 6-8 per group), those receiving SM grafts after 3 months. The cumulative survival rate (SVR) in the female-to-male (F-M) SFS group was significantly lower (62%; 13 of 21) compared with the male-to-male (M-M) group (90%; 18 of 20) (P0.05). Spontaneous death occurred in the F-M SFS combination either in the early postoperative period (3 weeks) in animals with confluent hepatic necrosis or in the late postoperative period (8 weeks) in animals with biliary obstruction. In contrast, no death was observed in the early posttransplantation period after M-M liver transplantation. The relative graft size in the SM F-M group was significantly higher (graft-to-recipient weight ratio [GRWR] 2.40% +/- 0.8%) than in the SFS M-M group (GRWR 1.35% +/- 0.2%; P0.001). Regardless of graft size, the outcome was worse in terms of SVR as well as regarding the incidence and severity of biliary complications in F-M compared with M-M liver transplantation. In conclusion, male recipients of female livers had a less favorable outcome irrespective of graft size. Confluent hepatic necrosis as well as biliary obstruction were perceived as consequence of a severe perfusion problem in F-M liver transplantation, which was possibly related to an enhancement of ischemia-reperfusion (I/R) injury by the lack of estrogen in male recipients of female grafts.

Background: The German transplantation system is in a crisis due to a lack of donor organs. Information campaigns are one of the main approaches to increase organ donation rates. Since 2012, German health insurance funds are obliged by law to inform their members about organ donation. We raised the hypothesis: The willingness to sign a donor card rises due to the subsequent increase of specific knowledge by receiving the information material of the health insurance funds. Objective: The objective of the study was to assess the influence of information campaigns on the specific knowledge and the willingness to donate organs. Methods: We conducted an online survey based on recruitment via Facebook groups, advertisements using the snowball effect, and on mailing lists of medical faculties in Germany. Besides the demographic data, the willingness to hold an organ donor card was investigated. Specific knowledge regarding transplantation was explored using five factual questions resulting in a specific knowledge score. Results: We recruited a total of 2484 participants, of which 32.7% (300/917) had received information material. Mean age was 29.9 (SD 11.0, median 26.0). There were 65.81% (1594/2422) of the participants that were female. The mean knowledge score was 3.28 of a possible 5.00 (SD 1.1, median 3.0). Holding a donor card was associated with specific knowledge ( P

PLoS ONE 10(11), e0141798 (2015). doi:10.1371/journal.pone.0141798, Published by PLoS, Lawrence, Kan.

Summary Recent studies showing the therapeutic effect of young blood on aging-associated deterioration of organs point to young blood as the solution for clinical problems related to old age. Given that defective autophagy has been implicated in aging and aging-associated organ injuries, this study was designed to determine the effect of young blood on aging-induced alterations in hepatic function and underlying mechanisms, with a focus on autophagy. Aged rats (22 months) were treated with pooled plasma (1 ml, intravenously) collected from young (3 months) or aged rats three times per week for 4 weeks, and 3-methyladenine or wortmannin was used to inhibit young blood-induced autophagy. Aging was associated with elevated levels of alanine transaminase and aspartate aminotransferase, lipofuscin accumulation, steatosis, fibrosis, and defective liver regeneration after partial hepatectomy, which were significantly attenuated by young plasma injections. Young plasma could also restore aging-impaired autophagy activity. Inhibition of the young plasma-restored autophagic activity abrogated the beneficial effect of young plasma against hepatic injury with aging. In vitro, young serum could protect old hepatocytes from senescence, and the antisenescence effect of young serum was abrogated by 3-methyladenine, wortmannin, or small interfering RNA to autophagy-related protein 7. Collectively, our data indicate that young plasma could ameliorate age-dependent alterations in hepatic function partially via the restoration of autophagy.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory rate and heart rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure. This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41 ± 2.94 cmH2O which was significantly higher (P

Computer-assisted automatic quantification (CAQ) was developed as an alternative method for the diagnosis of hepatic steatosis in order to compensate for observer-dependent bias. Here, we aim to demonstrate that CAQ can provide an accurate and precise result in analysis of fatty content, but that it is inappropriate to validate CAQ by comparison with conventional pathologist estimation (PE). Male rats were fed with a methionine-choline-deficient plus high-fat diet for three days, one week, or two weeks to induce mild, moderate, or severe steatosis. Samples were collected from all liver lobes. Severity of hepatic steatosis was assessed by an experienced pathologist who estimated the percentage of hepatocytes containing lipid droplets. Fatty content was quantified by PE, CAQ, and biochemical analysis (BA). CAQ, PE, and BA can correctly reflect severe fatty change. However, in the case of mild and moderate steatosis, PE could not reflect the true fatty content ( r between PE and BA was

BACKGROUND Extended partial hepatectomy (PH) in patients is leading to portal hyperperfusion but reduced hepatic arterial perfusion (HAP), and is invariably causing focal hepatic venous outflow obstruction (FHVOO). We observed in a rat model that PH in combination with right median hepatic vein ligation (RMHV-L) caused confluent parenchymal necrosis interspersed with viable portal tracts in the obstructed territory and large sinusoidal vascular canals in the border zone. Lack of HAP impaired the spontaneous course of recovery in terms of enlarged parenchymal necrosis, delayed regeneration, and the absence of draining vascular canals. We aimed to investigate whether pharmacological intervention modulates the imbalance between portal venous and hepatic arterial inflow, aggravates the liver damage, and delays the recovery process after FHVOO in liver-resected rats. METHODS Male Lewis rats were subjected to 70% PH and RMHV-L. Molsidomine or NG-nitro-L-arginine methyl ester (L-NAME) or saline were applied daily. Hepatic damage, microcirculation, regeneration, and vascular remodeling were evaluated at postoperative days 1, 2, and 7. Animals subjected to RMHV-L only were used as "no HAP" control. RESULTS Significant increase of portal venous inflow with a concomitant decrease in HAP was observed in all groups after PH. Molsidomine treatment did neither affect hepatic hemodynamics nor the spontaneous recovery. In contrast, L-NAME treatment further decreased HAP which impaired hepatic microcirculation, aggravated parenchymal damage, decelerated recovery, and impaired the formation of sinusoidal canals. CONCLUSIONS Reduction of HAP through inhibition of nitric oxide production worsened the recovery from FHVOO. Drugs increasing HAP need to be evaluated to reverse the hyperperfusion-induced impairment of the spontaneous course after FHVOO.

Liver dysfunction has been known to occur frequently in cases of sepsis. Excessive inflammation and apoptosis are pathological features of acute liver failure. Recent studies suggest that activation of glycogen synthase kinase- (GSK-) 3βis involved in inflammation and apoptosis. We aimed to investigate the protective effects of GSK-3βinhibition on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and SB216763 was used to inhibit GSK-3βin C57BL/6 mice. GSK-3βwas activated following CLP. Administration of SB216763 decreased mortality, ameliorated liver injury, and reduced hepatic apoptosis. The inhibition of GSK-3βalso reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release. Moreover, GSK-3βinhibition suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) but enhanced the transcriptional activity of cAMP response element binding protein (CREB) in the liver. In in vitro studies, GSK-3βinhibition reduced inflammatory cytokine production via modulation of NF-κB and CREB signaling pathways in lipopolysaccharide-stimulated macrophages. In conclusion, these findings suggest that GSK-3βblockade protects against CLP-induced liver via inhibition of inflammation by modulating NF-κB and CREB activity and suppression of hepatic apoptosis.

We aimed to evaluate whether using sodium hyaluronate carboxymethylcellulose membrane (Seprafilm™) can facilitate assessment of hepatic microcirculation via orthogonal polarization spectroscopy (OPS) by preventing intra-abdominal adhesions and whether Seprafilm™ as a foreign material can evoke local or systemic inflammatory reactions. After the right median hepatic vein was ligated, rats received either placement of Seprafilm™ or untreated with observation of 1 or 4 weeks (n = 6/group). Hepatic microcirculation was visualized. Systemic and local inflammatory reactions were evaluated by blood count, histology and immunohistochemical staining for CD68. Seprafilm™ significantly (P

The inflammatory response after liver ischemia/reperfusion (I/R) contributes to increased risk of liver failure after liver surgery. Strategies aimed to preventing inflammation could be beneficial in reducing liver I/R injury. Recent studies have demonstrated that peptide B"15-42 is able to decrease the injury of I/R in heart and kidney by inhibition of leukocyte migration and preserving endothelial barrier function. Prompted by these results, we hypothesized that B"15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury. Therefore, in this study, we aimed to evaluate the effects of B"15-42 in a model of liver I/R injury in rats. Rats were treated with B"15-42 at initiation of reperfusion and 2 h thereafter. Rats were killed at 0.5, 6, 24, and 48 h after reperfusion. Hepatic mRNA levels of fibrinogen-! (Fg!), Fg" ,F g+ were significantly increased after I/R. Treatment with Fg-derived B"15-42 ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels and fewer I/R-associated histopathologic changes. B"15-42 treatment decreased leukocyte infiltration and expression of hepatic inflammatory cytokines. Moreover, B"15-42 significantly reduced high-mobility group box 1 release and altered mitogen-activated protein kinase activation. In conclusion, B"15-42 treatment protected against liver warm I/R injury. The mechanism of protective action of B"15-42 seemed to involve its ability to reduce hepatic inflammatory response through preventing high-mobility group box 1 release and altering mitogen-activated protein kinase activation. KEYWORDS—Liver ischemia/reperfusion, fibrinogen, B"15-42, inflammatory response, HMGB1, MAPK

Aim To analyze time intervals of inflammation and regeneration in a cholestatic rat liver model. Methods In 36 Lewis rats, divided into six groups of 6 animals (postoperative observation periods: 1, 2, 3, 4, 6, 8 wk), the main bile duct was ligated with two ligatures and observed for the periods mentioned above. For laboratory evaluation, cholestasis parameters (bilirubin, γ-GT), liver cell parameters (ASAT, ALAT) and liver synthesis parameters (quick, albumin) were determined. For histological analysis, HE, EvG, ASDCL and HMGB-1 stainings were performed. Furthermore, we used the mRNA of IL-33, GADD45a and p-21 for analyzing cellular stress and regeneration in cholestatic rats. Results In chemical laboratory and histological evaluation, a distinction between acute and chronic cholestatic liver injury with identification of inflammation and regeneration could be demonstrated by an increase in cholestasis (bilirubin: 1-wk group, 156.83 ± 34.12 μmol/L, P = 0.004) and liver cell parameters (ASAT: 2-wk group, 2.1 ± 2.19 μmol/L.s, P = 0.03; ALAT: 2-wk group, 1.03 ± 0.38 μmol/L.s, P = 0.03) after bile duct ligation (BDL). Histological evaluation showed an increase of bile ducts per portal field (3-wk group, 48 ± 6.13, P = 0.004) during the first four weeks after bile duct ligation. In addition to inflammation, which is an expression of acute cholestasis, there was an increase of necrotic areas in the histological sections (2-wk group, 1.38% ± 2.28% per slide, P = 0.002). Furthermore, the inflammation could be verified by ASDCL (4-wk group, 22 ± 5.93 positive cells per portal field, P = 0.041) and HMGB-1 [2-wk group, 13 ± 8.18 positive cells per field of view (FoV), P = 0.065] staining. Therefore, in summary of the laboratory evaluation and histological studies, acute cholestasis could be found during the first four weeks after bile duct ligation. Subsequently, the described parameters declined so that chronic cholestasis could be assumed. For quantification of secondary biliary cirrhosis, eosin staining was performed, which did not reveal any signs of liver remodeling, thus precluding the development of a chronic cholestasis model. Additionally, to establish the chronic cholestasis model, we evaluated liver regeneration capacity through measurements of IL-33, p-21 and GADD45a mRNA. Conclusion We created a chronic cholestasis model. The point of inflammatory and regenerative balance was reached after four weeks. This finding should be used for experimental approaches dealing with chronic cholestatic liver damage.

We have investigated the vascular endothelial phenotypes found at various times posttransplant in murine B10-->C3H liver grafts. In this model, liver allografts are spontaneously accepted, and survive indefinitely unless the recipient is first allosensitized with a skin allograft, in which case the liver allografts are rejected within five days. In our previous studies, allograft inflammation was associated with the development of vascular endothelial reactivity with the mAbs MECA-32 and M/K-2 (anti-VCAM-1). We observed that vascular endothelia in both liver isografts and allografts develop reactivity with MECA-32 mAb within two days of transplantation, indicating endothelial activation in both situations. In contrast, only the endothelia in liver allografts develop VCAM-1 expression, as detected with M/K-2 mAb. VCAM-1 was expressed in both rejecting and accepting liver allografts, demonstrating that endothelial VCAM-1 expression is indicative of ongoing graft inflammation but not necessarily graft rejection. Liver parenchymal cells did not appear to develop reactivity with either antibody under any of the conditions tested. In contrast, bile duct epithelia developed M/K-2 reactivity (VCAM-1 expression), but not MECA-32 reactivity in liver allografts, but not isografts. These data demonstrate alloantigen-dependent and alloantigen-independent patterns of endothelial behavior in murine liver grafts that are quite similar to those found in murine cardiac grafts. Further, they demonstrate that the expression of VCAM-1 by graft endothelia is not diagnostic for acute rejection of liver allografts.

Background Liver regeneration consists of cellular proliferation leading to parenchymal and vascular growth. This study complements previous studies on cellular proliferation and weight recovery by (1) quantitatively describing parenchymal and vascular regeneration, and (2) determining their relationship. Both together are needed to (3) characterize the underlying growth pattern. Methods Specimens were created by injecting a polymerizing contrast agent in either portal or hepatic vein in normal or regenerating livers after 70% partial hepatectomy. 3D image data were obtained through micro-CT scanning. Parenchymal growth was assessed by determining weight and volume of the regenerating liver. Vascular growth was described by manually determined circumscribed parameters (maximal vessel length and radius of right inferior portal/hepatic vein), automatically determined cumulative parameters (total edge length and total vascular volume), and parameters describing vascular density (total edge length/volume, vascular volume fraction). The growth pattern was explored by comparing the relative increase of these parameters to the increase expected in case of isotropic expansion. Results Liver volume recovery paralleled weight recovery and reached 90% of the original liver volume within 7 days. Comparing radius-related vascular parameters immediately after surgical resection and after virtual resection in-silico revealed a slight increase, possibly reflecting the effect of resection-induced portal hyperperfusion. Comparing length-related parameters between post-operative day 7 and after virtual resection showed similar vascular growth in both vascular systems investigated. In contrast, radius-related parameters increased slightly more in the portal vein. Despite the seemingly homogeneous 3D growth, the observed vascular parameters were not compatible with the hypothesis of isotropic expansion of liver parenchyma and vascular structures. Conclusion We present an approach for the quantitative analysis of the vascular systems of regenerating mouse livers. We applied this technique for assessing the hepatic growth pattern. Prospectively, this approach can be used to investigate hepatic vascular regeneration under different conditions.

Liver is the main organ for lipopolysaccharide (LPS) clearance. Sensitization to LPS is associated with the upregulation of LPS-binding protein (LBP) in animal models. Therefore, we hypothesized that LBP could induce LPS sensitization through enhancing hepatic uptake of LPS. In this study, we examined the role of LBP in pathogenesis of LPS induced systemic inflammatory response syndrome (SIRS). LBP expression was upregulated after granulocyte colony stimulating (G-CSF) pretreatment. The effect of LBP was further confirmed by blockade of LBP using LBP blocking peptide – LBPK95A. After G-CSF pretreatment, upregulation of LBP was observed in bone marrow cells and liver. The G-CSF induced LBP upregulation caused LPS hypersensitization in rats as indicated by higher mortality and severer liver damage. Of note, LBP blockade increased the survival rate and attenuated the liver injury. The LBP induced LPS hypersensitization was associated with increased hepatic uptake of LPS and augmented hepatic expression of LPS receptors, such as toll-like receptor (TLR)-4. Furthermore, LBP mediated early neutrophil infiltration, which led to increased monocyte recruitment in liver after LPS administration. In conclusion, G-CSF induced LBP expression could serve as a new model for investigation of LPS sensitization. We demonstrated the crucial role of LBP upregulation in pathogenesis of LPS induced SIRS. peerReviewed

Lipopolysaccharide (LPS) binding protein (LBP) is up-regulated in inflammation and infection. Its use as a biomarker of severe infection and sepsis is currently discussed controversially. Here a novel LBP-ELISA was established to assess serum LBP-levels after various degree of in a rat partial hepatectomy (PH) model. The LBP enzyme-linked immunosorbent assay (ELISA) was designed based on the binding between LPS and LBP. LPS was employed as capture molecule. An anti-LBP antibody was used as detection antibody. Serum LBP levels were measured in serum obtained 24h after 30% PH, 70% PH and 90% PH in rats using newly established LBP ELISA method. Expression of hepatic LBP mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The detection range of the ELISA was from 0.1 μg/ml to 60 μg/ml. The correlation between ELISA and western blotting was strong (r=0.885, p0.0001). Hepatic LBP mRNA expression was upregulated after PH. Of note, elevations of serum LBP protein levels were positively correlated to the remnant liver mass (R=0.821, p0.0001), serum albumin levels (R=0.532, p0.05) and total protein levels(R=0.813, p0.0001). In conclusion, we established an economic and rapid ELISA assay for rat serum LBP quantification. Our results speak against using LBP as diagnostic marker of the acute phase response after liver resection as its elevation is related to the size and thereby the synthetic capacity of the small remnant liver.

Background As repeatedly operating rat liver transplantation (LTx) until animals survive is inefficient in respect to time and use of living animals, we developed a new training concept. Methods and Concepts Training was divided into four phases: pretraining-phase, basic-microsurgical-training phase, advanced-microsurgical-training phases, and expert-microsurgical-training phase. Two “productivity-phases” were introduced right after the basic- and advanced-microsurgical-training phases, respectively, to allow the trainee to accumulate experience and to be scientifically productive before proceeding to a more complex procedure. PDCA cycles and quality criteria were employed to control the learning-process and the surgical quality. Predefined quality criteria included survival rate, intraoperative, postoperative, and histologic parameters. Results Three trainees participated in the LTx training and achieved their first survival record within 4–10 operations. All of them completely mastered the LTx in fewer procedures (31, 60 and 26 procedures) as reported elsewhere, and the more complex arterialized or partial LTx were mastered by trainee A and B in additional 9 and 13 procedures, respectively. Fast progress was possible due to a high number of training in the 2 Productivity-phases. Conclusion The stepwise and PDCA-based training program increased the efficiency of LTx training, whereas the constant application and development of predefined quality criteria guaranteed the quality of microsurgery.