The Role of Autophagy for the Regeneration of the Aging Liver

Age is one of the key risk factors to develop malignant diseases leading to a high incidence of hepatic tumors in the elderly population. The only curative treatment for hepatic tumors is surgical removal, which initiates liver regeneration. However, liver regeneration is impaired with aging, leading to an increased surgical risk for the elderly patient. Due to the increased risk, those patients are potentially excluded from curative surgery. Aging impairs autophagy via lipofuscin accumulation and inhibition of autophagosome formation. Autophagy is a recycling mechanism for eukaryotic cells to maintain homeostasis. Its principal function is to degrade endogenous bio-macromolecules for recycling cellular substances. A number of recent studies have shown that the reduced regenerative capacity of the aged remnant liver can be restored by promoting autophagy. Autophagy can be activated via multiple mTOR-dependent and mTOR-independent pathways. However, inducing autophagy through the mTOR-dependent pathway alone severely impairs liver regeneration. In contrast, recent observations suggest that inducing autophagy via mTOR-independent pathways might be promising in promoting liver regeneration. Conclusion: Activation of autophagy via an mTOR-independent autophagy inducer is a potential therapy for promoting liver regeneration, especially in the elderly patients at risk

Peripheral Progenitor Cell Graft in the Rat: A Technique of Graft Processing

The aim of this study was to establish a procedure for blood progenitor cell graft processing in rats.  As a first step the mobilization protocol was optimized. The second step was dedicated to define the optimal  source for subsequent graft manufacturing: either peripheral blood or spleen. The third step was  designed to establish a protocol for purification of stem cells. The best mobilization results in terms of white blood cell count, granulocyte colony forming units (CFUG)  and CD90 positive progenitor cells were obtained after pre-treatment of the donors for 5 days with  recombinant human granulocyte colony stimulating factor (100 μg/kg) in combination with murine stem  cell factor (33 μg/kg). Splenectomy prior to mobilization increased the yield of stem cells from peripheral blood. The numbers of  CD90-positive progenitor cells recovered from the spleen of one rat after stem cell mobilization were sufficient  to generate one stem cell graft. Grafts containing 1 x 106 progenitor cells – and thus sufficient for transplantation - were obtained after Tcell  depletion and positive selection of CD90 positive cells. The grafts were characterized and showed a  purity exceeding 70%, a T-cell depletion of 3.6 log10 and a 3-fold increase in CFU-G compared to the yield  post mobilization.

Release of Danger Signals during Ischemic Storage of the Liver: A Potential Marker of Organ Damage?

Liver grafts suffer from unavoidable injury due to ischemia and manipulation before implantation. Danger signals such as high-mobility group box -1(HMGB1) and macrophage migration inhibitory factor (MIF) play a pivotal role in the immune response. We characterized the kinetics of their release into the effluent during cold/warm ischemia and additional manipulation-induced mechanical damage. Furthermore, we evaluated the relationship between HMGB1/MIF release and ischemic/mechanical damage. Liver enzymes and protein in the effluent increased with increasing ischemia time. HMGB1/MIF- release correlated with the extent of hepatocellular injury. With increasing ischemia time and damage, HMGB1 was translocated from the nucleus to the cytoplasma as indicated by weak nuclear and strong cytoplasmic staining. Enhancement of liver injury by mechanical damage was indicated by an earlier HMGB1 translocation into the cytoplasm and earlier release of danger signals into the effluent. Our results suggest that determination of HMGB1 and MIF reflects the extent of ischemic injury. Furthermore, HMGB1and MIF are more sensitive than liver enzymes to detect the additional mechanical damage inflicted on the organ graft during surgical manipulation

Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury

Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracellular HMGB1, the role of cytoplasmic HMGB1 in hepatic IRI remains elusive. We hypothesized that cytoplasmic HMGB1 interacts with binding proteins involved in the hepatocellular response to IRI. In this study, binding proteins of cytoplasmic HMGB1 during hepatic IRI were identified. Liver tissues from rats with warm ischemia reperfusion (WI/R) injury and from normal rats were subjected to cytoplasmic protein extraction. Co-immunoprecipitation using these protein extracts was performed to enrich HMGB1-protein complexes. To separate and identify the immunoprecipitated proteins in eluates, 2-dimensional electrophoresis and subsequent mass spectrometry detection were performed. Two of the identified proteins were verified using Western blotting: betaine–homocysteine S-methyltransferase 1 (BHMT) and cystathionine γ-lyase (CTH). Therefore, our results revealed the binding of HMGB1 to BHMT and CTH in cytoplasm during hepatic WI/R. This finding may help to better understand the cellular response to IRI in the liver and to identify novel molecular targets for reducing ischemic injury

Computational Modeling in Liver Surgery

The need for extended liver resection is increasing due to the growing incidence of liver tumors in aging societies. Individualized surgical planning is the key for identifying the optimal resection strategy and to minimize the risk of postoperative liver failure and tumor recurrence. Current computational tools provide virtual planning of liver resection by taking into account the spatial relationship between the tumor and the hepatic vascular trees, as well as the size of the future liver remnant. However, size and function of the liver are not necessarily equivalent. Hence, determining the future liver volume might misestimate the future liver function, especially in cases of hepatic comorbidities such as hepatic steatosis. A systems medicine approach could be applied, including biological, medical, and surgical aspects, by integrating all available anatomical and functional information of the individual patient. Such an approach holds promise for better prediction of postoperative liver function and hence improved risk assessment. This review provides an overview of mathematical models related to the liver and its function and explores their potential relevance for computational liver surgery. We first summarize key facts of hepatic anatomy, physiology, and pathology relevant for hepatic surgery, followed by a description of the computational tools currently used in liver surgical planning. Then we present selected state-of-the-art computational liver models potentially useful to support liver surgery. Finally, we discuss the main challenges that will need to be addressed when developing advanced computational planning tools in the context of liver surgery.Peer Reviewe

Focused scores enable reliable discrimination of small differences in steatosis

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

Обоснование технологии гидравлического разрыва пласта на примере Приобского нефтяного месторождения (ХМАО)

В данной работе представленны обоснования технологии гидравлического разрыва пласта в низкопродуктивных пластах Приобского месторождения. В результате было предложено проведение многостадийного ГРП с увеличением стадий до 8 и на основе углеводородного геля.This paper presents the substantiation of the technology of hydraulic fracturing in low-productive formations of the Priobskoye field. As a result, it was proposed to conduct a multi-stage hydraulic fracturing with an increase in stages up to 8 and based on a hydrocarbon gel

Cross-species variability in lobular geometry and cytochrome P450 hepatic zonation: insights into CYP1A2, CYP2D6, CYP2E1 and CYP3A4

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies