Rat Bile Duct Research Articles

The biliary atresia susceptibility gene, EFEMP1, regulates extrahepatic bile duct elastic fiber formation and mechanics

IntroductionEGF-Containing Fibulin Extracellular Matrix Protein 1 (EFEMP1, also called fibulin-3) is an extracellular matrix protein linked in a genome-wide association study to biliary atresia, a fibrotic disease of the neonatal extrahepatic bile duct. Fibulin-3 is deposited in most tissues and Efemp1 null mice have decreased elastic fibers in visceral fascia; however, fibulin-3 does not have a role in the development of large elastic fibers and its overall function in the extrahepatic bile ducts remains unclear. MethodsWe used staining and histology to define the amount and organization of key extracellular matrix components in the extrahepatic bile ducts. We also repurposed pressure myography, a technique heretofore applied to the vasculature, to determine the contribution of elastin and fibulin-3 to extrahepatic bile duct mechanics. We examined extrahepatic bile duct structure and mechanics in three models: neonatal vs. adult rat ducts (n=6 each), elastase-treated adult rat ducts (n=6-7 each), and Efemp1+/- vs. wild type mouse ducts (n=6 each). ResultsWe demonstrated that fibulin-3 is expressed in the submucosa of both neonatal and adult mouse, rat and human extrahepatic bile ducts and that, in adult Efemp1+/- mouse ducts, elastin organization into fibers is decreased by approximately half. Pressure myography showed that Efemp1+/- ducts have altered mechanics compared to control ducts, with Efemp1+/- ducts displaying significant stretch compared to controls (p=0.0376); these changes in stretch are similar to those observed in elastase-treated compared to normal ducts (p<0.0001) and in neonatal ducts compared to adult ducts (p< 0.0001). ConclusionFibulin-3 has an important role in the formation of elastic fibers and the mechanical properties of the extrahepatic bile duct. This provides functional relevance for the biliary atresia susceptibility gene EFEMP1. Impact and ImplicationsThe gene EFEMP1 was found via a genome wide association study to be a susceptibility gene for the neonatal disease biliary atresia. EFEMP1 encodes the protein fibulin-3, which regulates elastic fiber organization in the extrahepatic bile duct (EHBD), the major site of disease in biliary atresia. We showed that neonatal EHBDs as well as mice heterozygous for Efemp1 have decreased numbers of elastic fibers, and that this alters EHBD mechanics. This work is important for understanding the mechanism of biliary atresia, in particular susceptibility to obstruction.

Among Other Tissues, Short-Term Garlic Oral Treatment Incrementally Improves Indicants of Only Pancreatic Islets of Langerhans Histology and Insulin mRNA Transcription and Synthesis in Diabetic Rats.

The source, mRNA transcription, and synthesis of insulin in the pancreas, in addition to the bile duct and liver, in streptozotocin (STZ)-induced diabetic rats (DR) in response to garlic oral treatment are not yet clear. This study investigated the accumulative effects of continued garlic oral treatment on changes in the pancreas, bile duct, and liver with regards to: 1-Insulin mRNA transcription, synthesis, and concentration in relation to changes in serum insulin (SI); 2-Insulinogenic cells insulin intensity and distribution, proliferation, and morphology. Fasting blood glucose (FBG) and insulin concentration in serum and pancreas (PI) and sources and mRNA transcription in the pancreas, bile duct, and liver in normal rats given normal saline (NR-NS) and DR given either NS (DR-NS) or garlic extract (DR-GE) before and after 1, 4, and 8 weeks of oral treatment were examined. Compared to NR-NS, DR-NS showed a significant increase in FBG and reductions in SI and PI and deterioration in islets histology, associated pancreatic insulin numerical intensities, and mRNA transcription. However, compared to DR-NS, the targeted biochemical, histological, and genetic variables of DR-GE were significantly and incrementally improved as garlic treatment continued. Insulin or its indicators were not detected either in the bile duct or the liver in DR-GE. 8 weeks of garlic oral treatment is enough to incrementally restore only pancreatic islets of Langerhans insulin intensity and insulinogenic cells proliferation, morphology, and distribution. These indices were associated with enhanced pancreatic insulin mRNA transcription and synthesis. Eight weeks of garlic treatment were not enough to stimulate insulinogenesis in either the bile duct or the liver.

Preliminary exploration of animal models of congenital choledochal cysts.

Various animal models have been used to explore the pathogenesis of choledochal cysts (CCs), but with little convincing results. Current surgical techniques can achieve satisfactory outcomes for treatment of CCs. Consequently, recent studies have focused more on clinical issues rather than basic research. Therefore, we need appropriate animal models to further basic research. To establish an appropriate animal model that may contribute to the investigation of the pathogenesis of CCs. Eighty-four specific pathogen-free female Sprague-Dawley rats were randomly allocated to a surgical group, sham surgical group, or control group. A rat model of CC was established by partial ligation of the bile duct. The reliability of the model was confirmed by measurements of serum biochemical indices, morphology of common bile ducts of the rats as well as molecular biology experiments in rat and human tissues. Dilation classified as mild (diameter, ≥ 1 mm to < 3 mm), moderate (≥ 3 mm to < 10 mm), and severe (≥ 10 mm) was observed in 17, 17, and 2 rats in the surgical group, respectively, while no dilation was observed in the control and sham surgical groups. Serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, and total bile acids were significantly elevated in the surgical group as compared to the control group 7 d after surgery, while direct bilirubin, total bilirubin, and gamma-glutamyltransferase were further increased 14 d after surgery. Most of the biochemical indices gradually decreased to normal ranges 28 d after surgery. The protein expression trend of signal transducer and activator of transcription 3 in rat model was consistent with the human CC tissues. The model of partial ligation of the bile duct of juvenile rats could morphologically simulate the cystic or fusiform CC, which may contribute to investigating the pathogenesis of CC.

Investigating the Effect of Serotonin II and III Receptor Inhibitors on the Chronotropic Changes of the Heart to Isoproterenol in Rat Models of Biliary Cirrhosis

Cirrhotic patients have hyperdynamic circulation and at-rest tachycardia, and agents that activate the sympathetic pathway, such as physical practice and pharmacologic stimulations, compared with the normal population, cannot cause enough increase in heartbeat, a condition known as cirrhotic cardiomyopathy. Concerning the presentation of 5-HT2 &amp; 5-HT3 receptors in rat hearts, we used Ketanserin as a 5-HT2 receptor inhibitor &amp; Tropisetron as a 5-HT3 receptor inhibitor to evaluate chronic therapeutic effects of 5-HT2 &amp; 5-HT3 antagonists on the cardiac chronotropic response of cirrhotic rats to adrenergics. Cirrhosis was induced by surgical ligation of the bile duct in Male Wistar rats, and another group remained sham. A week after bile duct ligation or sham surgery, the subjects were given an intraperitoneal injection of either saline or Tropisetron (2 mg/kg). In other BDL &amp; Sharm groups, the subjects were given an intraperitoneal injection of either saline or Ketanserin (6 mg/kg) every other 3 days in the last 3 weeks. Four weeks after bile duct ligation or sham surgery, the atria were isolated and chronotropic responsiveness to Isoproterenol was assessed using a standard organ bath. Our data showed that chronic treatment with Tropisetron (5-HT3 antagonist) in cirrhotic rats could decrease the cardiac chronotropic response. Chronic treatment with Tropisetron can cause a significant decrease in cardiac chronotropic response to Isoproterenol in healthy and cirrhotic rats, even lower than that in cirrhotic rats (without any special treatment). Chronic treatment with Ketanserin cannot change their impaired chronotropic response to Isoproterenol.&#x0D;

Development of pancreatic islet cells in the extrahepatic bile ducts of rats with experimentally-induced metabolic syndrome

Context There is data about the existence of some endocrine cells in the epithelial layer of the bile duct in humans and rats. Objective We evaluated Ghrelin-, Insulin-, Glucagon- and Somatostatin-positive cells in peribiliary glands, mast cells, and nerve fibres. Materials and methods Wistar rats were used for dietary manipulation with a 15% fructose solution for 12 weeks. Tissue samples were elaborated with immunohistochemistry for Insulin, Glucagon, Ghrelin, and Somatostatin. Glucose and lipid parameters were studied. Results In treated animals, Ghrelin+ and Insulin+ cells in perybiliary glands (PBGs) were significantly increased. In the male fructose group there was a significant increase of the homeostasis model assessment insulin resistance (HOMA-IR). Conclusions Stem/progenitor cells in extrahepatic bile tree (EHBT) could be a source of Insulin-producing cells in metabolic syndrome. Fructose treatment induces the increase of Ghrelin+ and Insulin+ cells in PBGs and the elevation of Insulin and Ghrelin plasma concentration.

Dynamics of pathomorphological changes in the liver of rats at different stages of experimental alcohol damage

Liver diseases represent one of the most common problems in gastroenterology. The liver, as the most important organ of metabolism, which plays a major role in anabolic and energy processes, takes part in the adaptive and compensatory reactions of the body under exogenous and endogenous adverse influences. Individual factors play a major role in the development of this disease, one of which is the duration of the effect of alcohol on the body. The duration of alcohol consumption affects the morpho-functional properties of the liver. When alcohol was given to the research animals, hypertrophic changes were seen in the linear dimensions of hepatocytes: in the 12th week of alcoholization, the nuclear area was 1.25 times larger, and the cytoplasmic area of hepatocytes was 1.16 times larger compared with the same indicators in the 6th week of alcoholization. Alcoholic hepatitis is characterized by protein-fatty degeneration, inflammatory lymphocytic infiltration, increase in the area of sinusoids and the size of hepatocytes, and, accordingly, the Vizotto coefficient (1.83 times in the 6th week and 2.10 times in the 12th week of the research). The alcohol consumption is accompanied by increase in the volume of the nucleus and cytoplasm of hepatocytes, decrease in the nuclear-cytoplasmic ratio with increase in pathohistological changes. With the lengthening of the terms of alcoholization, the number of binuclear hepatocytes decreased. Morphometric calculation of the number of fat cells per 100 hepatocytes showed that in the 12th week of the experiment, the number of fatty inclusions increased by 1.42 times, compared with 6 weeks of forced alcoholization. Fatty small- and large-droplet steatosis of hepatocytes was diffuse in nature. The duration of alcoholization also affects the liver vessels. The diameter of the central vein in the 6th week of the experiment was 1.52 times greater than in control rats, and 1.81 times in the 12th week of alcoholization. The bile ducts of rats of the experimental groups were also larger in diameter: by 37% after 6 weeks and 47% after 12 weeks of forced alcoholization. An increase in the area of the sinusoids and the diameter of the central vein indicates an impairment of the blood supply to the liver. The complexity of the structure of the liver and the diversity of its functions necessitate the use of a variety of diagnostic techniques and methodological approaches to assessing its activity in the normal condition, during a pathology. It will be promising to study the structure of the liver at the ultramicroscopic level of the effect of drugs on the treatment of alcoholic liver disease during different periods of alcoholization.

A mouse model of hepatic encephalopathy: bile duct ligation induces brain ammonia overload, glial cell activation and neuroinflammation

Hepatic encephalopathy (HE) is a common complication of chronic liver disease, characterized by an altered mental state and hyperammonemia. Insight into the brain pathophysiology of HE is limited due to a paucity of well-characterized HE models beyond the rat bile duct ligation (BDL) model. Here, we assess the presence of HE characteristics in the mouse BDL model. We show that BDL in C57Bl/6j mice induces motor dysfunction, progressive liver fibrosis, liver function failure and hyperammonemia, all hallmarks of HE. Swiss mice however fail to replicate the same phenotype, underscoring the importance of careful strain selection. Next, in-depth characterisation of metabolic disturbances in the cerebrospinal fluid of BDL mice shows glutamine accumulation and transient decreases in taurine and choline, indicative of brain ammonia overload. Moreover, mouse BDL induces glial cell dysfunction, namely microglial morphological changes with neuroinflammation and astrocyte reactivity with blood–brain barrier (BBB) disruption. Finally, we identify putative novel mechanisms involved in central HE pathophysiology, like bile acid accumulation and tryptophan–kynurenine pathway alterations. Our study provides the first comprehensive evaluation of a mouse model of HE in chronic liver disease. Additionally, this study further underscores the importance of neuroinflammation in the central effects of chronic liver disease.

Molecular mechanism of benign biliary stricture inhibition by rosiglitazone-activated peroxisome proliferator-activated receptor gamma.

The aim of this study was to investigate whether rosiglitazone-activated peroxisome proliferator-activated receptor gamma can inhibit the occurrence of benign biliary stricture and further elucidate the relevant molecular signaling mechanism. The primary cultured rat biliary fibroblasts following experiments were performed using within the fifth generation cells, which were separated from the bile ducts of Sprague-Dawley rats. The primary cultured rat biliary fibroblasts were co-cultured with 10 ng/mL transforming growth factor-beta 1 for stimulating collagen formation. Competent cells were transfected with siRNA that specifically target Smad3 or connective tissue growth factor to inhibit the expression of the corresponding proteins. The cells were incubated with 10 μmol/L rosiglitazone to activate peroxisome proliferator-activated receptor gamma. The cells were incubated with 10 μmol/L GW9662 in the pretreatment session to inactivate peroxisome proliferator-activated receptor gamma. ELISA was used to determine the levels of connective tissue growth factor and type I collagen in the cell supernatant. Western blotting was used to detect the levels of intracellular p-Smad3/t-Smad3. Rosiglitazone-activated peroxisome proliferator-activated receptor gamma inhibited the secretion of type I collagen induced by transforming growth factor-beta 1. Peroxisome proliferator-activated receptor gamma inhibitor GW9662 could significantly reverse the rosiglitazone-triggered inhibition of transforming growth factor-beta 1-induced type I collagen secretion by suppressing peroxisome proliferator-activated receptor gamma activation (p<0.01). Furthermore, we also found that the activation of peroxisome proliferator-activated receptor gamma was accompanied by the inhibition of transforming growth factor-beta 1-induced Smad3 phosphorylation (p<0.01), increased connective tissue growth factor expression (p<0.01), and production of type I collagen (p<0.01), all of which effects elicited by rosiglitazone could be reversed by peroxisome proliferator-activated receptor gamma inhibitor GW9662. Peroxisome proliferator-activated receptor gamma activated by rosiglitazone inhibits the transforming growth factor-beta1 -induced phosphorylation of Smad3 and the increased connective tissue growth factor expression as well as inhibits the secretion of type I collagen in biliary fibroblasts.

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

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. 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. 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. 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).

Bile Duct Regeneration with an Artificial Bile Duct Made of Gelatin Hydrogel Nonwoven Fabrics.

Although choledochojejunostomy is the standard technique for biliary reconstruction, there are various associated problems that need to be solved such as reflux cholangitis. Interposition with an artificial bile duct (ABD) to replace the resected bile duct maintains a physiological conduit for bile and may solve this problem. This study investigated the usefulness of an ABD made of gelatin hydrogel nonwoven fabric (GHNF). GHNF was prepared by the solution blow spinning method. The migration and activity of murine fibroblast L929 cells were examined in GHNF sheets. L929 cells migrated into GHNF sheets, where they proliferated and synthesized collagen, suggesting GHNF is a promising scaffold for bile duct regeneration. ABDs made of GHNF were implanted in place of resected bile duct segments in rats. The rats were killed at 2, 6, and 12 weeks postimplantation. The implantation site was histologically evaluated for bile duct regeneration. At postoperative 2 weeks, migrating cells were observed in the ABD pores. The implanted ABD was mostly degraded and replaced by collagen fibers at 6 weeks. Ki67-positive bile duct epithelial cells appeared within the implanted ABD. These were most abundant within the central part of the ABD after 6 weeks. The percentages of Ki67-positive cells were 31.7 ± 9.1% in the experimental group and 0.8 ± 0.6% in the sham operation group at 6 weeks (p < 0.05), indicating that mature biliary epithelial cells at the stump proliferated to regenerate the biliary epithelium. Biliary epithelial cells had almost completely covered the bile duct lumen at 12 weeks (epithelialization ratios: 10.4 ± 6.9% at 2 weeks, 93.1 ± 5.1% at 6 weeks, 99.2 ± 1.6% at 12 weeks). The regenerated epithelium was positive for the bile duct epithelium marker cytokeratin 19. Bile duct regeneration was accompanied by angiogenesis, as evidenced by the appearance of CD31-positive vascular structures. Capillaries were induced 2 weeks after implantation. The number of capillaries reached a maximum at 6 weeks and decreased to the same level as that of normal bile ducts at 12 weeks. These results showed that an ABD of GHNF contributed to successful bile duct regeneration in rats by facilitating the cell migration required for extracellular matrix synthesis, angiogenesis, and epithelialization. Impact Statement Development of an artificial bile duct (ABD) enables physiological biliary reconstruction and may solve clinical problems associated with choledochojejunostomy. In this study, we created ABDs with gelatin hydrogel nonwoven fabric and implanted them in place of resected bile duct in rats. We evaluated the process of bile duct regeneration as well as decomposition of the ABD and demonstrated successful regeneration of resected bile duct, highlighting the possibility of this novel biliary reconstruction method to replace choledochojejunostomy.

Newly Invented Micellized Vitamin K2 Recovered Prolonged Prothrombin Time under Obstructive Jaundice in Rats with Bile Duct Ligation.

In cholestatic liver diseases, coagulopathy is induced by malabsorption of vitamin K. Supplementation of vitamin K has previously been shown to prevent coagulopathy. In this study, we tested the efficacy of a newly invented micellized vitamin K2 (m-vitK2) in treating coagulopathy, using a rat bile duct ligation (BDL) model. Experiment 1: m-vitK2 (0.3 mg/kg) or m-vitK2 (0.3 mg/kg) mixed with taurocholic acid (TA) (10 mg/body) was orally administrated every day for 7 d from the fourth day after BDL (n=6 for each). Experiment 2: To evaluate absorption, m-vitK2 (0.3 mg/kg) with or without TA (10 mg/body) was orally administered on the fourth day after BDL and compared with the untreated control BDL (n=2 for each). These data were compared with sham-operated (n=6) and untreated control BDL rats (n=6). The m-vitK2 recovered prothrombin time (PT) in Experiment 1 (control 42.7±5.7 s vs. m-vitK2 24.0±9.3 s, p<0.05). Experiment 2 demonstrated that the mixture of m-vitK2 and TA enhanced absorption compared to m-vitK2 alone. Moreover, in Experiment 1, m-vitK2 mixed with TA completely recovered PT (control 42.7±5.7 s vs. m-vitK2+TA 14.9±1.2 s, p<0.01). Micelle sizes decreased with the m-vitK2 and TA treatment (m-vitK2 86.3±5.6 nm vs. m-vitK2+TA 71.9±4.7 nm, p<0.05). Orally administered, newly invented m-vitK2 recovered coagulopathy even under obstructive jaundice. TA decreased the mean micelle size and improved m-vitK2 absorption.

Refining Procedures within Regulatory Toxicology Studies: Improving Animal Welfare and Data.

Simple SummaryBefore any new medicine can be administered to humans, or new chemical marketed, some tests using animals such as fish, mice, rats, rabbits, dogs, or monkeys are performed in order to satisfy the legal requirements of international regulatory and government agencies. These assess the potential for harmful side effects in humans or species found in the environment and to explore how the compound is processed within the body. The housing conditions and procedures performed (such as dosing of compounds and removal of small blood samples) are designed to minimize any pain, suffering, distress, or lasting harm that may be experienced by the animals. These refinements improve animal welfare but can also improve the data quality. Examples of new processes, technologies, or equipment that have been introduced within some UK facilities are shared in this article and provide opportunities to benefit many more animals undergoing testing across the world in the future.During the development of potential new medicines or agrochemicals, an assessment of the safety profile to humans and environmental species is conducted using a range of different in silico and in vitro techniques in conjunction with metabolism and toxicity studies using animals. The required studies are outlined within international regulatory guidelines which acknowledge and support the application of the 3Rs to reduce the number of animals used or to refine the procedures performed when these studies are deemed to be necessary. The continued development of new technologies and adoption of best-practice approaches to laboratory animal housing and study procedures has generated a series of refinements that can be incorporated into animal studies throughout the package. These refinements benefit the welfare of fish, mice, rats, rabbits, dogs, minipigs, and non-human primates (NHPs) whilst maintaining or improving data quality within general toxicology, metabolism, and other studies and can also bring efficiencies to processes that benefit study costs and timings. Examples are shared which cover the following topics: social housing of dogs and NHPs, surgical refinements in the rat bile duct cannulation model for collection of data for metabolism studies, whether fasting is really required prior to clinical pathology sampling, and the use of microsampling for toxicokinetics.

Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates.

Hepatocyte nuclear factor-4α (HNF4α) presents in multiple isoforms generated using alternative promoter (P1 and P2) and splicing. Neither conservation of tissue distribution of HNF4α isoforms, nor presence of alternative promoter usage is known. In this study, to detect the expression of HNF4α in some species of animals, we have applied monoclonal antibodies against P1 (K9218) and P2 (H6939) promoter-driven and P1/P2 promoter-driven H1415 HNF4α for immunohistochemistry and western blot analysis. Antibody K9218 was observed in the hepatocytes, proximal tubules of the kidney, and epithelial cells in the mucosa of the small intestine and colon of rats, chicken, and tortoise, whereas antibody H6939 signal were detected in the stomach, pancreas, bile duct, and pancreatic duct of human and rats. The signal for antibody K9218 was recognized in tissues of a wide range of mammals, bird, reptile, amphibian, and fish as well. Antibody H1415 displayed a positive reaction in hepatocytes and intestinal epithelial cells in chicken and tortoise, whereas the bile duct, mucosal epithelial cells in the stomach, or pancreas in these animals were negative. Western blotting showed the binding of the antibody with HNF4α protein from each animal. The sequence of human HNF4α was 100% identical to murine and rat HNF4α, 88.9% to chicken, 77.8% to Xenopus HNF4α, and 81.5% to medaka. However, the specific part of human and invertebrate Drosophila HNF4 shares only 14.8% sequence identity. This antibody is useful for detecting HNF4α isoforms in a wide range of vertebrates, and suggests many insights into animal evolution.

The herbal medicines Inchinkoto and Saireito improved hepatic fibrosis via aquaporin 9 in the liver of a rat bile duct ligation model.

To determine if the administration of the Japanese herbal medicines Inchinkoto (ICKT) and Saireito (SRT) ameliorate hepatic fibrosis and derangement of hepatocyte aquaporins (AQPs) following bile duct ligation (BDL) in a rat model of obstructive cholestasis. Five groups of Wistar rats were used, and the groups included sham surgery (Sham group), BDL with no treatment (NT group), BDL plus ICKT (ICKT group), BDL plus SRT (SRT group), and BDL plus ICKT and SRT (SRT/ICKT group). Each herbal medicine was administered at 1g/kg/day on the first postoperative day. The serum levels and various clinical markers were measured with real-time polymerase chain reaction. Staining was used to evaluate the degree of fibrosis and the inflammatory responses. Serum aspartate aminotransferase and alanine aminotransferase in the ICKT and SRT/ICKT groups were significantly lower than those in the NT group. NF-κB mRNA expression was significantly decreased in the ICKT group and the SRT/ICKT group compared with the NT group. AQP9 mRNA expression was significantly increased in the ICKT group and the SRT/ICKT group compared with the NT group. The degree of Masson's trichrome staining in the SRT/ICKT group was significantly lower than that in the NT group. The degree of NF-κB staining in the SRT/ICKT group was significantly lower than that in the NT, ICKT, or SRT group. The postoperative administration of ICKT and SRT induced synergistic beneficial effects, resulting in the reduction of hepatic fibrosis via mechanisms involving the inhibition of NF-κB expression and the improvement of AQP9 downregulation.

Bsep expression\xa0in hilar cholangiocarcinoma of rat model

Develop a rat model of hilar cholangiocarcinoma for detecting bile salt export pump (Bsep) expression in hilar cholangiocarcinoma tissues, in order to provide a new therapeutic target for the gene therapy of hilar cholangiocarcinoma. Sixty male Wistar rats (body weight, 190 ± 8 g) were randomly divided into three groups (the experimental group, the control group and the sham operation group, n = 20 each) as follows: The three groups were fed a standard diet, the experimental group was injected by cholangiocarcinoma QBC939 cell suspension along the hilar bile duct into the bile duct bifurcation with microsyringe, the control group was injected by normal saline, the sham operation group did not inject anything. Every day assess the rats’ mental state, diet, and motion by using Basso–Beattie–Bresnahan and combined behavioral score. At 4 weeks, one rat of the experimental group was sacrificed after it was administered anesthesia, and we recorded changes in hilar bile duct size, texture, and form. This procedure was repeated at 6 weeks. After 6 weeks, hilar cholangiocarcinoma developed only in the experimental group, thereby establishing an experimental model for studying QBC939-induced hilar cholangiocarcinoma. Tumor formation was confirmed by pathological examination, and hilar bile duct tissues were harvested from both the groups. A real-time polymerase chain reaction assay and an immunohistochemical assay were used to analyze the expression of Bsep in hilar bile duct tissues of each group. From the second week, the rats in experimental group began to eat less, and their body mass decreased compared with control group and sham operation group. After 6 weeks, we detected hilar cholangiocarcinoma in the hilar bile duct tissues of 18 rats (90%) in the experimental group. In the experimental group with hilar cholangiocarcinoma, we found that the levels of total cholesterol, total bilirubin, and direct bilirubin were higher compared with those in the control group and sham operation group. Simultaneously, muddy stones emerged from the bile ducts of rats in the experimental group. The Bsep/Gapdh mRNA ratio in hilar cholangiocarcinoma, control group and sham operation group differed markedly. Light microscopy revealed a granular pattern of Bsep protein expression which reacted with the anti-Bsep antibody. Each section was randomly divided into six regions, with 80 cells were observed in every region. Sections with > 10% positive cells were designated positive, Sections with < 10% positive cells were designated negative. Each group included 4800 cells. In the experimental group, 1200 cells (25%) were positive, in the control group, 3648 cells (76%) were positive and in the sham operation group 3598 cells (75%) were positive, and this difference was statistically significant. Bsep expression significantly decreased in hilar cholangiocarcinoma of rats than those in control group and sham operation group, suggesting that drugs targeting Bsep are a new strategy for hilar cholangiocarcinoma.

Enhanced PRL-1 expression in placenta-derived mesenchymal stem cells accelerates hepatic function via mitochondrial dynamics in a cirrhotic rat model

BackgroundPlacenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model.MethodsPD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 using a nonviral sysyem (2 × 106 cells/animal) were intravenously administered into cirrhotic rats. The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed.ResultsPD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p < 0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy.ConclusionsOur findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

Morphological features of the wall of common bile duct under the conditions of experimental opioid exposure

The morphological condition of the bile ducts remains one of the most important problems of modern medical science. In order to obtain an analgesic effect in patients with acute cholangitis, opioids are often used. However, information on the effectiveness of opioids in the treatment of pathological conditions of the bile ducts is contradictory. The rapidly progressive destruction of the intrahepatic bile ducts associated with the use of narcotic agents has been described. Further study of the effect of opioids on the structural organization of the common bile duct is relevant. In order to establish the morphological state of the common bile duct under conditions of long-term opioid exposure, a study was performed on 24 sexually mature white male rats, aged 3.5-5.0 months and weighing 180-200 g, which were injected intramuscularly with Nalbuphine for 6 weeks. The study material is represented by histological specimens of the common bile duct of white rats. The “Aver Media” computer system was used to photograph microspecimens. The “ImageJ” computer program was used to measure the diameter of the lumen and the wall thickness of the common bile duct. After 2 weeks of Nalbuphine administration to white rats, plethora of wall microvessels and a significant increase in the longitudinal diameter of the lumen of the common bile duct were observed. After 4 weeks of the experiment, the common bile duct was dilated, the transverse and longitudinal diameters of its lumen almost doubled, pathological changes in its wall had all the signs of inflammation. In the later stages of the experiment (introduction of Nalbuphine for 6 weeks), the pathological changes increased and manifested by destructuring the wall of the common bile duct, disorganization of cholangiocytes, thinning of the cell layer due to detachment of cholangiocytes, polymorphism of their nuclei, destruction of intercellular junctions, stratification of its own plate, vacuolar dystrophy of the muscular membrane “varicose” expansion of venules, significant smooth muscle hyperplasia of arterioles, the presence of perivascular lymphocytic infiltrates in the duct wall.

Effect of phosphatidylserine on cirrhosis-induced hepatic encephalopathy: Response to acute endotoxemia in cirrhotic rats

Background/aimsIn cirrhosis, the levels of proinflammatory cytokines are high in the liver and blood. Endotoxin decreases level of consciousness in cirrhotic rats. Phosphatidylserine exists in the cell membrane structure and is essential for the survival of neurons. Phosphatidylserine receptor is found in phagocytic cells and also activates the signaling of membrane proteins in apoptotic process. Therefore this study was aimed to explore the hypothesis that hepatic encephalopathy is prevented by phosphatidylserine treatment and if so, whether this is associated with altered level of proinflammatory cytokines in the brain. MethodsCirrhosis was induced by surgical ligation of the bile duct in male Wister rats. The groups were treated with phosphatidylserine and saline for 4 weeks. Brain IL6, TNFα and the expression of phosphatidylserine receptor were assessed. Intraperitoneal injections of either saline or lipopolysaccharide (0.1 mg/kg) were administered to each group. Finally, animal behavior, blood ammonia and the expression of toll like receptor 4 were examined in the brain. ResultsCirrhosis in rats was associated with altered expression of toll-like receptor4 in brain cortex and phosphatidylserine treatment increases toll-like receptor4 receptor expression. Phosphatidylserine had anti-inflammatory effect in healthy rats but no effect in cirrhotic rats. Chronic phosphatidylserine treatment decreased blood ammonia in BDL cirrhotic rats treated with lipopolysaccharide. ConclusionThe brain of cirrhotic rat is more susceptible to acute endotoxemia and chronic phosphatidylserine treatment decreases blood ammonia and encephalopathy in cirrhotic rats by encountering endotoxin. Phosphatidylserine may boost immune system against endotoxin.

An improved method for the isolation and culture of rat pancreatic ductal epithelial cells.

BackgroundThis aim of this study was to explore a novel method that can be used to isolate and culture rat pancreatic ductal epithelial cells.MethodsRetrograde injection of indigo carmine solution into the bile duct of rats revealed the main pancreatic duct, which was isolated using the naked eye (without using a stereomicroscope). The main pancreatic duct was sequentially digested with three enzymes, and the digested cells were cultured in RPMI-1640 medium containing 10–15% fetal bovine serum. After 72 h, the primary pancreatic ductal epithelial cells started to adhere to the wall. The cells reached 70–80% confluence after approximately 7 days and were subsequently digested with 0.25% trypsin and subcultured. Cells of the second and fourth passages were harvested. Cytokeratin (CK)-7 and CK-19 protein expressions in the cells and pancreatic tissue were detected by Western blot analysis. q-PCR was employed to examine CK-7, CK-19, somatostatin, amylase, insulin, and glucagon mRNA expression in the cells and pancreatic tissue after the main pancreatic duct was removed.ResultsThe rats had one or two main pancreatic ducts meeting the bile ducts at a right or an acute angle. Rat pancreatic ductal epithelial cells isolated by this method grew well and showed a cobblestone-like appearance via microscopy. Western blot analysis showed that both the second and fourth passages of pancreatic ductal epithelial cells expressed CK-7 and CK-19 protein. The q-PCR results showed the expression of CK-7 and CK-19 genes in the second and fourth passages of pancreatic ductal epithelial cells, while the somatostatin, amylase, insulin, and glucagon genes were not expressed. The pancreatic tissue harvested after the removal of the main pancreatic duct did not express CK-7 or CK-19, while the somatostatin, amylase, insulin, and glucagon genes were expressed.ConclusionsThe preliminary results show that this method can be applied to successfully isolate and culture rat pancreatic ductal epithelial cells.

Protective effects of spironolactone against hepatic ischemia/reperfusion injury in rats.

In the present study, it was aimed to study the antioxidant effects of spironolactone (SPL) to determine its possible protective effects in hepatic ischemia reperfusion injury. Hepatic artery, portal vein, and bile duct of Wistar albino rats were clamped for 45 minutes under anesthesia to form an ischemia period. Then reperfusion was allowed and the rats were decapitated 60 minutes later. SPL (20 mg/kg, p.o.) or SF was orally administered for 30 minutes before ischemia. Rats in the control arm underwent sham surgery and were administered isotonic saline. Liver function was studied by measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-alpha (TNF-α), and interleukin 1beta (IL-1β) levels. Malondialdehyde (MDA), glutathione (GSH), luminol, and lucigenin levels, myeloperoxidase (MPO) and Na+-K+-ATPase enzyme activities were analyzed to study tissue injury under light microscope. While IR increased AST, ALT, TNF-α, and IL-1β levels and MDA, luminol, and lusigenin levels and MPO activities, it caused a decrease in GSH levels and Na+K+-ATPase activity. Spironolactone administration significantly improved these values. Protective effects of SPL against ischemia/reperfusion injury via various mechanisms suggest that this agent may become a novel treatment agent in clinical practice.