Selective biliary occlusion in rodents: description of a new technique.

Background: Modern therapy concepts are of limited success in patients with cholestasis (e.g., biliary occluding malignancies). Therefore, we established a new animal model enabling simultaneous investigation of liver regeneration and hepato-biliary remodelling in biliary obstructed and biliary non-obstructed liver lobes. Methods: Biliary occlusion of different extent was induced in 50 male rats: Ligation and transection of the common bile duct (100% of liver, tBDT, n=25); or of the left bile duct (70% of liver, sBDT, n=25). At postoperative days 1, 3, 7, 14 and 28 we assessed the hepatic histomorphological alterations, proliferative repair, progress of liver fibrosis (HE, BrdU, EvG) and signs of liver regeneration (liver lobe weight gain). In addition, we determined systemic markers of hepatocellular injury (ASAT, ALAT), cholestasis (Bilirubin) and synthetic liver function (INR). The animals were monitored daily (body weight gain, stress score, survival). Results: All animals survived until the planned date of sacrifice. sBDT induced in the biliary occluded liver lobes similar histomorphological alterations, proliferative repair and progress of liver fibrosis like tBDT. In the biliary non-ligated liver lobes in sBDT animals we noticed a temporarily enhanced biliary proliferation and a persistent low grade liver fibrosis in the periportal area. Conclusions: Our model of sBDT represents a safe and valid method to induce selective cholestasis. The model enables further comparative investigation of liver regeneration in different extents of occlusive cholestasis (e.g., mimicking biliary occluding malignancies).

Species specific morphological alterations in liver tissue after biliary occlusion in rat and mouse: Similar but different.

BACKGROUND: The selection of the appropriate species is one of the key issues in experimental medicine. Bile duct ligation is the mostly used experimental model in rodents to explore special aspects of occlusive cholestasis. We aimed to clarify if rats or mice are suitable for the same or different aspects in cholestasis research. METHODS: We induced biliary occlusion by ligation and transection of the common bile duct (tBDT) in rats and mice (each n = 25). Recovery from surgical stress was assessed by daily scoring (stress score, body weight). At five different time points (days 1, 3, 7, 14, 28 after tBDT) we investigated hepatic morphometric and architectural alterations (Haematoxylin-Eosin staining, Elastica van Gieson staining) and the proliferative activities of parenchyma cells (Bromodeoxyuridine staining); as well as established systemic markers for liver synthesis, hepatocellular damage and renal dysfunction. RESULTS: We found substantial differences regarding survival (rats: 100%, 25/25 vs. mice 92%, 22/25, p = 0.07) and body weight gain (p<0.05 at postoperative days 14 and 28 (POD)). Rats showed a faster and progressive hepatobiliary remodelling than mice (p<0.05 at POD 7+14+28), resulting in: i) stronger relative loss of hepatocellular mass (rats by 31% vs. mice by 15% until POD 28; p<0.05 at POD 7+14+28); ii) rapidly progressing liver fibrosis (p<0.05 at POD 14); iii) a faster and stronger proliferative response of parenchyma cells (hepatocytes: p<0.05 at POD 1+14+18; cholangiocytes: p<0.05 at POD 1+3+7+28); and iv) only tiny bile infarcts compared to mice (p<0.05 at POD 1+3+7+14). Both species showed comparable elevated markers of hepatocellular damage and serum bilirubin. CONCLUSION: The key difference between rats and mice are the severity and dynamics of histological alterations, possibly accounting for their different susceptibilities for (septic) complications with low survival (mice).

Corrosion Cast and 3D Reconstruction of the Murine Biliary Tree After Biliary Obstruction: Quantitative Assessment and Comparison With 2D Histology.

Background: Obstructive cholestasis can lead to significant alterations of the biliary tree depending on the extent and duration of the biliary occlusion. Current experimental studies reported about advanced techniques for corrosion cast and 3D reconstruction (3D-reco) visualizing delicate microvascular structures in animals. We compared these two different techniques for visualization and quantitative assessment of the obstructed murine biliary tree with classical 2D histology. Methods: Male mice (n = 36) were allocated to 3 different experiments. In experiments 1 and 2, we injected two different media (Microfil© for 3D-reco, MV; Batson's No.17 for corrosion cast, CC) into the extrahepatic bile duct. In experiment 3 we sampled liver tissue for 2D histology (HE, BrdU). Time points of interest were days 1, 3, 5, 7, 14, and 28 after biliary occlusion. We used different types of software for quantification of the different samples: IMALYTICS Preclinical for 3D scans (MV); NDP.view2 for the digital photography of CC; HistoKat software for 2D histology. Results: We achieved samples in 75% of the animals suitable for evaluation (MV and CC, each with 9/12). Contrasting of terminal bile ducts (4th order of branches) was achieved with either technique. MV permitted a fast 3D-reco of the hierarchy of the biliary tree, including the 3rd and 4th order of branches in almost all samples (8/9 and 6/9). CC enabled focused evaluation of the hierarchy of the biliary tree, including the 4th to 5th order of branches in almost all samples (9/9 and 8/9). In addition, we detected dense meshes of the smallest bile ducts in almost all CC samples (8/9). MV and CC allowed a quantitative assessment of anatomical details of the 3rd and 4th order branches of almost every sample. The 2D histology identified different kinetics and areas of proliferation of hepatocytes and cholangiocytes. Complementary usage of 3D-reco, corrosion casting and 2D histology matched dense meshes of small bile ducts with areas of intensive proliferative activity of cholangiocytes as periportal proliferative areas of 4th and 5th order branches (∼terminal bile ducts and bile ductules) matched with its morphological information the matching assessment of areas with increased proliferative activity (BrdU) and a partial quantification of the characteristics of the 4th order branches of the biliary tree. Conclusion: The 3D-reco and corrosion casting of the murine biliary tree are feasible and provide a straightforward, robust, and reliable (and more economical) procedure for the visualization and quantitative assessment of architectural alterations, in comparative usage with the 2D histology.

The Interplay Between Biliary Occlusion and Liver Regeneration: Repeated Regeneration Stimuli Restore Biliary Drainage by Promoting Hepatobiliary Remodeling in a Rat Model.

Background and Aims: Patients with malignant biliary obstruction do not seem to benefit from "two-stage hepatectomy" due to an impairment of liver regeneration. We designed a novel model of "repeated regeneration stimuli" in rats mimicking a "two-stage hepatectomy" with selective or complete biliary occlusion mimicking Klatskin tumors III° or IV°. Using this new model, we wanted to investigate (1) the impact of preexistent cholestasis of different extent on the time course of liver regeneration and (2) the dynamics of hepatobiliary remodeling under regeneration conditions. Materials and Methods: Rats were subjected to a sequence of three operations: surgical induction of biliary occlusion, followed by "repeated regeneration stimuli" consisting of ligation of the left branch of the portal vein (supplying 70% of the liver volume, sPVL) as first stage and a 70%-hepatectomy (70%PHx) as second stage. Biliary occlusion (1st procedure) was induced by ligating and transection of either the common (100%, tBDT) or the left bile duct (70%, sBDT). A sham operation without ligating the bile duct was performed as control (0%, Sham). Two weeks later, on day 14 (POD14), the sPVL (2nd procedure) was performed. Another week later (POD 21), the 70%PHx (3rd procedure) took place and animals were observed for 1 week (POD 28). The first experiment (n = 45 rats) was dedicated to investigating liver regeneration (hypertrophy/atrophy), proliferative activity and hepatobiliary histomorphology (2D-histology: HE, BrdU) in the future liver remnant (FLR). The second experiment (n = 25 rats) was performed to study the dynamics of hepatobiliary remodeling in livers with different regenerative pressure (tBDT only POD21 vs. tBDT only POD 28 vs. tBDT + sPVL vs. tBDT + 70%PHx vs. tBDT + sPVL + 70%PHx) using µCT scans of explanted livers. Effect of biliary occlusion: Total biliary occlusion (tBDT) led to a 2.4-fold increase in whole liver volume due to severe biliary proliferation within 14 days. In contrast, partial biliary occlusion (sBDT) caused only a volume gain of the obstructed liver lobes due to biliary proliferates, resulting in a minor increase of total liver volume (1.7-fold) without an increase in bilirubin levels. Liver regeneration and atrophy: As expected, sPVL caused substantial volume gain (tBDT: 3-fold; sBDT: 2.8-fold; Sham 2.8-fold) of FLR and a substantial volume loss (tBDT: 0.9-fold; sBDT: 0.6-fold; Sham: 0.4-fold) of the portally deprived "future resected lobes" compared to the preoperative liver volume. The subsequent 70%PHx promoted a further volume gain of the FLR in all groups (tBDT: 4-fold; sBDT: 3-fold; Sham 3-fold compared to original volume) until POD 28. Hepatobiliary remodeling: After tBDT, we identified histologically three phases of hepatobiliary remodeling in the FLR. Following tBDT, biliary proliferates developed, replacing about 15% of the hepatocellular tissue. After sPVL we found incomplete restoration of the hepatocellular tissue with a visible reduction of the biliary proliferates. The 70%PHx led to an almost complete recovery of the hepatocellular tissue in the FLR with a nearly normal liver architecture. In contrast, after sBDT and Sham we observed a near normal liver morphology in the FLR at all time points. CT-scanning of the explanted livers and subsequent 3D reconstruction visualized the development of extrahepatic biliary collaterals. Collaterals were detected in 0/5 cases 1 week after sPVL (first regeneration stimulus), and in even more cases (3/5) 1 week after the 70%PHx (second regeneration stimulus). Histological workup identified the typical biliary cuboid epithelium as inner lining of the collaterals and peribiliary glands. Conclusion: Liver volume of the FLR increased in cholestatic rats mainly due to biliary proliferates. Application of repeated regeneration stimuli in the style of a "two-stage hepatectomy" promoted almost full restoration of hepatocellular tissue and architecture in the FLR by reestablishing biliary drainage via formation of biliary collaterals. Further exploration of the dynamics in hepatobiliary modeling using this model might help to better understand the underlying mechanism.

The significance of avoiding household food waste - A means-end-chain approach.

Many humans suffer from hunger, while edible food is discarded. This study aims at showing the importance of avoiding food waste in households and its causes by applying the means-end-chain analysis. Additional the means-end-chain approach should be examined in how far the method is suitable to get insights towards this topic. Consumer backgrounds in terms of feelings and attitudes regarding food waste should be shown, with the particular question why food waste personally is important. The data collection occurred utilizing the hard laddering method within a quantitative online survey. The results indicate that avoiding food waste is important for the greater part of consumers, as many claim to have a bad conscience, seeing it as morally wrong and reprehensible to waste food. A sample breakdown of gender, age and income points differences among these groups in regards to psychological consequences and value systems. Financial and environmental aspects have a lesser impact on attitudes and feelings regarding food waste in households.

Reversible biliary occlusion in a small animal model: first description of a new technique.

BACKGROUND: Experimental models with reversible biliary occlusion resulted in a high mortality of the animals, up to 20-60% according to the literature. Our aim was to assess a safe and valid technique for reversible biliary occlusion with a low mortality. METHODS: We randomized 30 rats into two groups: with bile duct occlusion (BDO, n=18) and with sham manipulation of the extrahepatic bile duct (control, n=12). We used a removable vascular clip for temporary occlusion of the extrahepatic bile duct. The clip was removed on postoperative day (POD) 2. On POD 2, 3, and 5, we measured the hepatocellular injury and metabolic function markers in serum. Activation of mononuclear cells (HIS36) and expression of regeneration markers [cytokeratin 19, hepatic growth factor (HGF)-α, and HGF-ß] were determined by immunohistochemistry. RESULTS: The survival rate was 96.67% (1/30); one animal died. The mortality in the BDO group was 6% (1/18) and that in the control group was 0% (0/12). BDO resulted in a sharp increase of hepatocellular injury and cholestatic parameters on POD 2 with a rapid decline till POD 3. Significantly strongest activation of Kupffer cells and expression of proliferation markers were found until POD 5 after BDO. CONCLUSION: The clip technique is a safe, cheap, and valid method for reversible biliary occlusion with an extremely low mortality.

Approaches of the German food industry for addressing the issue of food losses.

In the food industry the subject of food losses is of great importance due to economic balance and an efficient application of resources as well as the development of an efficient food chain system. This paper presents the explorative results of a quantitative survey of leading companies of the German food industry to evaluate the relevance and handling of this issue. The investigation reveals that the topic food losses have a high significance in the food industry which will probably increase in future. A sample breakdown by branches indicates that the issue has the highest relevance for companies in the confectionery industry. These companies as well as those in the meat and fish industry want to consider the subject prospectively more powerful in their companies. Across the food industry, there is no communication to consumers of the efforts concerning food losses. And companies in the confectionery industry and in the fruit and vegetable industry rather want to engage more powerful in this topic if consumers' interest increases. But in order to minimize food losses at all stages along the supply chain, communication and collaboration at all stages is essential, especially the communication to consumers. Thus, it has to be verified whether a suitable communication can lead to advantages in competition and become an important issue for companies to differentiate from competitors.

Cholestasis-induced adaptive remodeling of interlobular bile ducts.

UNLABELLED: 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.

Intrahepatic Vascular Anatomy in Rats and Mice--Variations and Surgical Implications.

INTRODUCTION: The intra-hepatic vascular anatomy in rodents, its variations and corresponding supplying and draining territories in respect to the lobar structure of the liver have not been described. We performed a detailed anatomical imaging study in rats and mice to allow for further refinement of experimental surgical approaches. METHODS: LEWIS-Rats and C57Bl/6N-Mice were subjected to ex-vivo imaging using µCT. The image data were used for semi-automated segmentation to extract the hepatic vascular tree as prerequisite for 3D visualization. The underlying vascular anatomy was reconstructed, analysed and used for determining hepatic vascular territories. RESULTS: The four major liver lobes have their own lobar portal supply and hepatic drainage territories. In contrast, the paracaval liver is supplied by various small branches from right and caudate portal veins and drains directly into the vena cava. Variations in hepatic vascular anatomy were observed in terms of branching pattern and distance of branches to each other. The portal vein anatomy is more variable than the hepatic vein anatomy. Surgically relevant variations were primarily observed in portal venous supply. CONCLUSIONS: For the first time the key variations of intrahepatic vascular anatomy in mice and rats and their surgical implications were described. We showed that lobar borders of the liver do not always match vascular territorial borders. These findings are of importance for the design of new surgical procedures and for understanding eventual complications following hepatic surgery.