Duct-ligated Rats Research Articles

Preventive and therapeutic effects of angiotensin II type 1 receptor blocker on hepatic fibrosis induced by bile duct ligation in rats

The aim of this study was to examine the preventive and therapeutic effects of an angiotensin II type 1 receptor blocker, candesartan, on cholestasis-induced liver fibrosis. Candesartan was administered orally for 21 days immediately after bile duct ligation to evaluate its preventive effect, and for 21 days starting 3 weeks after bile duct ligation to evaluate its therapeutic effect. Fibrosis was assessed by measuring hepatic hydroxyproline (Hyp) content. The activated hepatic stellate cells (HSCs) were assessed by alpha-smooth muscle actin (alpha-SMA) immunostaining. The gene expression of collagen I, transforming growth factor-beta1 (TGF-beta1), and connective tissue growth factor (CTGF) in the liver was examined by real-time reverse transcriptase-polymerase chain reaction. As a preventive effect, candesartan reduced the hepatic Hyp content by 36%, alpha-SMA-positive cells by 65%, hepatic TGF-beta1 content by 35%, and the expression of collagen I by 72%, TGF-beta1 by 67%, and CTGF mRNA by 69%. As a therapeutic effect, candesartan reduced the hepatic Hyp content by 48%, TGF-beta1 content by 54%, and the expression of collagen I by 47%, TGF-beta1 by 43%, and CTGF mRNA by 53%. Significant decreases in lipid peroxidation markers, hepatic thiobarbituric acid-reactive substance, and 4-hydroxy-2-nonenal were observed in candesartan-treated rats. Candesartan attenuated liver fibrosis via suppression of collagen I and TGF-beta1 expression, HSC activation, and lipid peroxidation protein, showing its preventive and therapeutic effects on cholestasis-induced liver fibrosis.

Effects of melatonin or acetylsalicylic acid on gastric oxidative stress after bile duct ligation in rats

Antioxidant enzyme activities decrease after bile duct ligation. The aim of this study was to assess the effect of melatonin and acetylsalicylic acid on antioxidant enzyme activities in gastric oxidative stress induced by bile duct ligation. Sixty-four animals were divided into eight groups of eight rats each. Male Sprague-Dawley rats were subjected to either a sham operation or common bile duct ligation (BDL) before treatment with melatonin (MEL) or acetylsalicylic acid (ASA). Gastric superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, and malondialdehyde (MDA) and nitric oxide (NO) levels were determined by spectrophotometers and evaluated. Our results indicated that BDL caused a significant increase in lipid peroxidation, whereas coadministration of MEL with ASA significantly decreased MDA and NO levels in BDL rats. Moreover, coadministration of MEL and ASA increased antioxidant enzyme activities after the BDL, and these increases were statistically significant for CAT and GPx. On the other hand, the increase in SOD activity was not significant. Melatonin administration, either alone or together with acetylsalicylic acid, decreases lipid peroxidation and increases antioxidant enzyme activities in gastric tissues of rats after bile duct ligation. ASA administration, however, either alone or with a vehicle, increases lipid peroxidation and decreases antioxidant enzyme activities.

HEPATIC AND EXTRAHEPATIC SYNTHESIS AND DISPOSITION OF DINITROPHENYL-S-GLUTATHIONE IN BILE DUCT-LIGATED RATS

The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 micromol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of gamma-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (-39% and -33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases alpha, mu, and pi, as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.

The common bile duct ligation in rat: a relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing alpha-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.

Dose-related effects of dexamethasone on liver damage due to bile duct ligation in rats

To evaluate the effects of dexamethasone on liver damage in rats with bile duct ligation. A total of 40 male Sprague-Dawley rats, weighing 165-205 g, were used in this study. Group 1 (sham-control, n = 10) rats underwent laparotomy alone and the bile duct was just dissected from the surrounding tissue. Group 2 rats (untreated, n = 10) were subjected to bile duct ligation (BDL) and no drug was applied. Group 3 rats (low-dose dexa, n = 10) received a daily dose of dexamethasone by orogastric tube for 14 d after BDL. Group 4 rats (high-dose dexa, n = 10) received a daily dose of dexamethasone by orogastric tube for 14 d after BDL. At the end of the two-week period, biochemical and histological evaluations were processed. The mean serum bilirubin and liver enzyme levels significantly decreased, and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) values were significantly increased in low-dose dexa and high-dose dexa groups when compared to the untreated group. The histopathological score was significantly less in the low-dose and high-dose dexa groups compared to the untreated rats. In the low-dose dexa group, moderate liver damage was seen, while mild liver damage was observed in the high-dose dexa group. Corticosteroids reduced liver damage produced by bile duct obstruction. However, the histopathological score was not significantly lower in the high-dose corticosteroid group as compared to the low-dose group. Thus, low-dose corticosteroid provides a significant reduction of liver damage without increased side effects, while high dose is associated not with lower fibrosis but with increased side effects.

Tannic Acid Inhibits Cholangiocyte Proliferation after Bile Duct Ligation via a Cyclic Adenosine 5′,3′-Monophosphate-Dependent Pathway

Chronic cholestatic diseases are characterized by morphological changes involving cholangiocyte proliferation and functional alterations of secretory capacity. The plant polyphenol tannic acid inhibits the growth of malignant human cholangiocytes. However, the mechanisms by which tannic acid limits excessive cholangiocyte proliferation are unknown. In this study we assessed the effect of tannic acid on cholangiocyte proliferation after bile duct ligation in rats. Tannic acid feeding decreased cholangiocyte proliferation and ductal mass in vivo after bile duct ligation. These changes were associated with functional changes in bile secretion and with decreases of intracellular cyclic adenosine 5',3'-monophosphate. The anti-proliferative effect of tannic acid was associated with a reduction of ERK1,2 phosphorylation. Additionally, tannic acid feeding decreased protein kinase A phosphorylation and activity. Similar changes were observed in isolated cholangiocytes during in vitro incubation with tannic acid. Furthermore, forskolin abolished the anti-proliferative effect of tannic acid on cholangiocyte proliferation after bile duct ligation. In conclusion, the anti-proliferative effects of tannic acid in cholangiocytes involve modulation of ERK1,2 by a cyclic adenosine 5',3'-monophosphate-protein kinase A-dependent pathway. These data suggest that tannic acid may be useful in limiting excessive cholangiocyte proliferation and modulating secretion during cholestasis.

Cell Proliferation Activity of Proliferating Bile Duct after Bile Duct Ligation in Rats

The cell proliferation activity of proliferating bile ducts produced by bile duct ligation (BDL) in rats was examined histologically, immunohistochemically, and ultrastructurally. Proliferating bile ducts, which were similar to normal bile ducts, increased with time after BDL. The cell proliferation activity of proliferating bile ducts, measured using proliferating-cell nuclear antigen and 5-bromo-2'-deoxyuridine antibodies, tended to be high at 1 and 3 days after BDL and decreased progressively at 2 to 4 weeks after BDL. On the other hand, alpha-smooth muscle actin-positive myofibroblast-like cells increased continuously after BDL. These findings indicate that there is a negative correlation between the cell proliferation activity of proliferating bile ducts and that of myofibroblast-like cells.

Effects of Heme Oxygenase–1 Overexpression on Liver Injury after Bile Duct Ligation in Rats

Aims: Chronic cholestasis leads to liver injury and will finally progress to portal fibrosis, cirrhosis and end-stage liver disease necessitating liver transplantation. Heme oxygenases are the rate-limiting enzymes that catalyze the conversion of heme into biliverdin, CO and iron. Recently, there were several reports about protective effects of heme oxygenase–1 (HO–1) against oxidant-induced injury. AIM: Accordingly, this study investigated if cobalt protoporphyrin (CoPP), a HO–1 inducer, would reduce hepatic fibrosis caused by bile duct ligation (BDL). Methods: Either CoPP or saline were injected intraperitoneally into male SD-rats. Three days later, BDL or sham-operations were performed. Rats were sacrificed 3 weeks after BDL and livers were harvested for histology. Fibrosis was evaluated by sirius red staining and image analysis. Alpha-smooth muscular actin, which indicates activation of hepatic stellate cells, was detected by immunohistochemical staining, and cytokine and collagen-Iα (Col-Iα) mRNA was detected using RNase protection assays. Results: As expected, serum alanine transaminase increased about 10-fold one day after BDL. Surprisingly, enzyme release was not reduced in rats receiving CoPP. Liver fibrosis was evaluated 3 weeks after BDL and the sirius red-positive area was found to be increased to about 8.7%. However, in livers from bile duct ligated rats pretreated with CoPP sirius red-positive areas were increased to about 11.7%. Col-Iα mRNA was increased significantly about 20-fold by BDL, consistent with increases in collagen synthesis. Again, this effect was increased by HO–1 overexpression. Transforming growth factor-β and tumor necrosis factor-α were increased significantly after BDL but no difference was observed between the CoPP or saline pretreated rats. Conclusions: Taken together, it is concluded that hepatic fibrosis due to BDL is not reduced by the HO–1 inducer CoPP. In contrast, our data indicate that HO–1 overexpression increases liver injury in rats under conditions of experimental chronic cholestasis.

Protective Effect of Resveratrol Against Renal Oxidative Stress in Cholestasis

Background/aims. This experimental study was designed to evaluate histological changes of the kidney and renal tissue levels of malondialdehyde (MDA), reduced glutathione (GSH), and nitric oxide (NO) and the effect of resveratrol on these metabolites after bile duct ligation in rats. Methods. Secondary biliary cirrhosis was induced by bile duct ligation for 28 days. Swiss albino rats were divided into three groups. Group 1: Sham (n = 7), Group 2: Bile duct ligation (n = 7), Group 3: Bile duct ligation plus resveratrol (n = 7). Bile duct ligation (BDL) plus resveratrol group received 10 mgr/kg dose of resveratrol intraperitoneally daily throughout 28 days. Kidney tissues were harvested to determine the tissue levels of MDA, GSH, and NO activity. Liver and kidney tissues were removed for light microscopic evaluation. Results. Cholestasis was determined by biochemical and pathologic examination. In the resveratrol-treated rats, levels of MDA were significantly lower than those of the BDL group (p < 0.04). The levels of GSH in the resveratrol-treated rats were significantly higher than those in the BDL group (p < 0.01). The levels of NO in the resveratrol group were significantly lower than those in the BDL group (p < 0.01). Conclusion. The present study demonstrates that intraperitoneal administration of resveratrol in bile duct ligated rats maintains antioxidant defenses and reduces kidney oxidative damage. This effect of resveratrol may be useful in the preservation of renal oxidative stress in cholestasis.

Effect of ZVAD-fmk on hepatocyte apoptosis after bile duct ligation in rat

Retention and accumulation of toxic hydrophobic bile salts within hepatocyte may cause hepatocyte toxicity by inducing apoptosis. Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. Z-Val-Ala-Asp (OMe)-fluoromethyl ketone (ZVAD-fmk) is a cell-permeable irreversible inhibitor of caspase. The purpose of this study was to evaluate the possible effect of ZVAD-fmk on hepatocyte apoptosis after bile duct ligation in the rat. Male Sprague-Dawley rats, weighing 250-300 g, were randomized to five groups of five rats each. Group 1 underwent common bile duct ligation and simultaneous treatment with ZVAD-fmk (dissolved in dimethylsulfoxide (DMSO)). Group 2 underwent common bile duct ligation and simultaneous treatment with Z-Phe-Ala-fluoromethyl ketone ( ZFA-fmk, dissolved in DMSO). Group 3 underwent sham operation and simultaneous treatment with the same amount of DMSO. Group 4 underwent sham operation and simultaneous treatment with the same amount of normal saline. Group 5 underwent common bile duct ligation without other manipulation. After three days, liver tissue was harv-ested for histopathologic analysis and measurements of apoptosis. When compared with sham operation, common bile duct ligation significantly increased hepatocyte apoptosis (P = 0.008) and ductular proliferation (P = 0.007). ZVAD-fmk significantly diminished the increased hepatocyte apoptosis and ductular proliferation after common bile duct ligation (P = 0.008 and P = 0.007, respectively). ZFA did not show the same effects. Hepatocyte apoptosis and ductular proliferation significantly increased after common bile duct ligation. ZVAD-fmk effectively diminished the increased hepatocyte apoptosis and ductular proliferation after common bile duct ligation, whereas ZFA-fmk did not.

Protective effect of low dose of melatonin against cholestatic oxidative stress after common bile duct ligation in rats

To investigate the role of oxidative injury and the effect of exogenous melatonin administration on liver damage induced by bile duct ligation (BDL), and second, to evaluate the role of nitric oxide (NO), a free oxygen radical, in oxidative injury. Thirty-two Sprague-Dawley rats were assigned to four groups: sham operation (SO), BDL, BDL+melatonin, and BDL+vehicle. Cholestasis was achieved by double ligature of the common bile duct. Melatonin was injected intraperitoneally 500 microg/(kg.d) for 8 d. Hepatic oxidative stress markers were evaluated by changes in the amount of lipid peroxides, measured as malondialdehyde (MDA), and reduced GSH. Total nitrite (NOX) concentrations were determined in hepatic homogenates. Histopathological examination was performed using a histological scoring system. The histopathological changes including portal inflammation, necrosis, apoptosis, focal inflammation and fibrosis were severe in the BDL and BDL+vehicle groups. There were numerous large areas of coagulation necrosis. Histological Activity Index scores of these groups were significantly higher than that of the SO group. Treatment with melatonin reduced these alterations significantly. The degree of necro-inflammation and fibrosis showed significant difference between the BDL and BDL+melatonin groups. BDL was accompanied by a significant increase in MDA and NOX, and a significant decrease in GSH levels. Mean+/-SE values of MDA, GSH and NOX levels of SO group were 147.47+/-6.69, 0.88+/-0.33 micromol/g and 180.70+/-6.58 nm/g, respectively. The values of BDL group were 200.14+/-21.30, 0.65+/-0.02 micromol/g, and 400.46+/-48.89 nm/g, respectively, whereas the values of BDL+melatonin group were 115.93+/-6.8, 0.74+/-0.02 micromol/g, and 290.38+/-32.32 nm/g, respectively. Melatonin treatment was associated with a significant recovery of MDA, GSH and NOX levels. We have concluded that oxidative stress is associated with the pathogenesis of cholestatic liver damage and NO contributes to oxidative damage. Melatonin, even at low dose, is an efficient agent in reducing negative parameters of cholestasis.

Are Bile Acids Involved in the Renal Dysfunction of Obstructive Jaundice? An Experimental Study in Bile Duct Ligated Rats

Background. Surgery on patients with obstructive jaundice is associated with a significant risk of postoperative renal failure. Bile acids are implicated as nephrotoxins because they accumulate in the plasma and the kidney becomes their only excretory route in cholestasis. The experimental evidence favoring this proposal is inadequate and unconvincing. Therefore, we designed an animal experiment involving bile duct ligated (BDL) rats in which we could correlate variations in serum and urine bile acids with indices of nephrotoxicity and renal function. Hypothesis. Bile acids are putative nephrotoxins. Materials and Methods. Total serum and urine bile acid concentrations and profiles were determined using liquid chromatography/gas chromatography/mass spectrometry selected ion monitoring. Nephrotoxicity was assessed by renal histopathology and by determination of the urinary activities of the following enzymes: muramidase, glutamate dehydrogenase, alkaline phosphatase, N‐acetyl‐β‐D‐glucosaminidase, and lactate dehydrogenase. Renal function was assessed by measuring urine osmolality, daily osmolar excretion, sodium excretion (UNaV), potassium excretion (UKV), and total protein and albumin excretion. Results. Maximum plasma concentrations and renal clearance of bile acids occurred between the third or fourth postoperative day following BDL. This peak coincided with maximal disruption of proximal convoluted tubule architecture and postoperative changes in renal function—increased urine flow rate and decreases in urine osmolality and sodium excretion. Thereafter, 1) plasma levels of bile acids returned toward normal levels, 2) urinary bile acid clearance declined, 3) normal renal histology was restored, and 4) normal renal function was reestablished. Throughout this period, fluctuations in enzymuria were evident. However, these shifts did not coincide with plasma and urine bile acid concentrations and histological and functional changes. Discussion and Conclusions. Transient functional impairment of renal cation and water transport and nonspecific morphological changes in the proximal convoluted tubule occur 3 to 4 days following bile duct ligation in rats. These functional and morphological changes occurred when plasma total and urinary bile acids were at their peaks. Although it is tempting to equate association with causality, we cannot implicate bile acids as being responsible for the aberrations in renal function and structure following BDL. Accordingly, we have concluded that elevated plasma concentrations of bile acids are renal exacerbates acting in concert with other factors, be they prerenal or renal in origin to precipitate a cascade of events leading to postoperative renal failure in cholestasis.

Sho-saiko-to Prevents Liver Fibrosis Induced by Bile Duct Ligation in Rats

Hepatic fibrosis is an over-accumulation of extracellular matrix (ECM). It is a result of an imbalance between collagen synthesis and degradation. Matrix metalloproteinase (MMP) has degradative activity against collagen, but tissue inhibitors of metalloproteinase (TIMP) control the active forms of MMP by blocking the active site of MMP. In our study, we established the bile duct ligated model (BDL) in rats to evaluate anti-fibrotic potential of Chinese medicine sho-saiko-to (TJ-9). We assessed the drug's potential in inhibiting collagen accumulation, suppressing procollagen alpha1 types (I) and (III), and TIMP-1 mRNA expression. After administration of TJ-9, hyperbilirubinemia reduced approximately four-fold when compared with BDL-untreated group. TJ-9 also significantly reduced the collagen content and fibrogenic score, as well as downregulated elevated procollagen alpha1 types (I) and (III) and TIMP-1 mRNA level. Finally, we concluded that (1) TJ-9 significantly reduced cholestasis in rats with BDL, (2) TJ-9 markedly reduced the collagen content by 50%, and (3) TJ-9 exerted its antifibrogenic effect by downregulation of the mRNA expression of procollagen alpha1 types (I) and (III), and TIMP-1 in liver tissue.

Analysis of expression profiles of islet-associated transcription and growth factors during beta-cell neogenesis from duct cells in partially duct-ligated mice.

Beta-cell neogenesis from pancreatic duct cells has been reported to occur in duct-ligated rat. Nevertheless, detailed process of this phenomenon has not been clarified. To clarify the mechanism of beta-cell neogenesis, a partial pancreatic duct ligation mouse model was created. Proliferation of duct cells, beta-cell neogenesis, and expression of transcription factors and differentiation/growth factors were studied by immunohistochemistry, cDNA array, and RT-PCR methods. In the duct-ligated portion of the pancreas, newly formed islet-like cell clusters (ICCs) were observed arising from the ducts on day 7 and afterward. Transcription factors, such as pancreatic and duodenal homeobox gene-1 (PDX-1), paired box factor 6 (Pax6), islet1 and Nkx2.2-positive cells, and protein gene product 9.5 (PGP9.5) were also induced in duct lining cells. By cDNA microarray analysis, expression of insulin-like growth factor-1 (IGF-1) and transforming growth factor beta1 (TGF-beta1) were above control levels on day 5, and RT-PCR showed an increase from day 5 to day 28. IGF-1 and activin A-positive cells were detected in ducts. In addition, expression of betacellulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and TGF-alpha were also increased from day 3 or 5. These findings suggest that beta-cell or endocrine precursors are localized among duct lining cells. Induction of several islet cell-associated transcription factors and differentiation and/or growth factors may play important roles during beta-cell neogenesis in this model.

Immunolocalization of the Yb1 subunit of glutathione S-transferase in the adult rat epididymis following orchidectomy and efferent duct ligation.

In addition to the maturation of sperm, the epididymis also serves to protect sperm from harmful reactive oxygen species. To this end, various antioxidant enzymes are produced by the epididymis, such as glutathione S-transferases (GSTs), a family of dimeric proteins that catalyze the conjugation of glutathione to various electrophilic compounds, thus providing cellular detoxification. In the present study, the regulation of the Yb(1) subunit of GST was examined in Bouin-fixed epididymides of adult control, orchidectomized (O) rats with or without testosterone (T) supplementation and efferent duct-ligated (EDL) rats using light microscope immunocytochemistry with an anti-Yb(1)-GST antibody. The intensely reactive ciliated cells of the efferent ducts and principal cells of the epididymis showing a checkerboard staining pattern were unaltered in their expression of Yb(1)-GST after all experimental procedures, suggesting their regulation by factors other than of testicular origin. On the other hand, the intense reaction of narrow/apical cells and moderate reaction of basal cells of the proximal initial segment of control animals became negligible in O rats and was not restored with T supplementation. As staining was also absent after EDL, the data suggest that a luminal testicular factor(s), other than androgens, regulates expression of Yb(1)-GST in narrow/apical and basal cells of the proximal initial segment. Although basal cells of the caput and cauda epididymidis were unreactive after all experimental protocols, as also noted in controls, the intensely reactive basal cells of the corpus epididymidis of control animals became unreactive in O animals. However, Yb(1)-GST expression was restored to these cells with T supplementation, and as there was no effect on Yb(1)-GST expression after EDL, the data suggest that circulating testosterone or one of its metabolites regulates expression of Yb(1)-GST in basal cells of the corpus region. Taken together, these data indicate a differential regulation with respect to the expression of Yb(1)-GST in the various cell types and regions of the epididymis.

Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice.

The epithelium of the epididymal tubule has different biological functions in different regions of the tubule. Each region is further organized into lobules or intra-regional segments surrounded by connective tissue septa (CTS). Epididymal segmentation has received little direct attention, yet there is considerable evidence that expression of mRNA and protein often begins or ends precisely at the CTS border of a segment. How such 'on-off' regulation occurs coincident with the passing of the tubule from one segment to the next is unknown. This study examined the segmentation of epididymides in rats and mice. The average adult Sprague-Dawley rat and C57BL/6 mouse caput, corpus and cauda epididymides has seven, two and four, and three, one and two segments, respectively. The apoptosis response of the caput epididymal epithelium to deprivation of lumicrine factors 24 h after efferent duct ligation in rats and the epididymal expression of a marker protein, beta-galactosidase, in mice were segmented precisely. This validated both at a general response and at a specific protein level that many epididymal functions are regulated within segments. Blue dextran (molecular weight 20000) and erythrocine red (molecular weight 880) dyes infused into the interstitial space of specific segments by micropuncture were retained by the CTS of the segments. In similar micropuncture experiments, [(3)H]H(2)O (molecular weight 18) was able to diffuse into an adjacent segment relatively freely whereas [(14)C]polyethylene glycol (molecular weight 4000) could not. These studies indicate that the interstitium of intra-regional segments is organized into different physiological compartments and that these compartments play a role in regulating the epididymal epithelium.

Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice

The epithelium of the epididymal tubule has different biological functions in different regions of the tubule. Each region is further organized into lobules or intra-regional segments surrounded by connective tissue septa (CTS). Epididymal segmentation has received little direct attention, yet there is considerable evidence that expression of mRNA and protein often begins or ends precisely at the CTS border of a segment. How such 'on-off' regulation occurs coincident with the passing of the tubule from one segment to the next is unknown. This study examined the segmentation of epididymides in rats and mice. The average adult Sprague-Dawley rat and C57BL/6 mouse caput, corpus and cauda epididymides has seven, two and four, and three, one and two segments, respectively. The apoptosis response of the caput epididymal epithelium to deprivation of lumicrine factors 24 h after efferent duct ligation in rats and the epididymal expression of a marker protein, beta-galactosidase, in mice were segmented precisely. This validated both at a general response and at a specific protein level that many epididymal functions are regulated within segments. Blue dextran (molecular weight 20000) and erythrocine red (molecular weight 880) dyes infused into the interstitial space of specific segments by micropuncture were retained by the CTS of the segments. In similar micropuncture experiments, [(3)H]H(2)O (molecular weight 18) was able to diffuse into an adjacent segment relatively freely whereas [(14)C]polyethylene glycol (molecular weight 4000) could not. These studies indicate that the interstitium of intra-regional segments is organized into different physiological compartments and that these compartments play a role in regulating the epididymal epithelium.

Akt-NFkB pathway is activated in duct ligation-induced acute pancreatitis in rats

Bile-pancreatic duct ligation in rats excludes bile-pancreatic juice from gut and induces acute pancreatitis. CCK-A and c:holinergic receptor-mediated exocr/ne pancreatic hyperstimulation secondat 7 to bile-pancreatic juice exclusion ~ implicated in disease pathogenesis. Stimulation of these G protein coupled receptors can activate the Akt-NFkB pathway Nuclear Transcrip~ lion Factor kappa B (NFkB) is a downstream messenger of acinar cell signal transduction via the Akt pathway and is capable o/inr Nag adnar cell cytokina production. We hypothesize that the Akt-NFkB pathway is activated in figation-induced acute pancreatitis. METHODS: We studied rats w~th 1, 3, or 24 hra ot duct ligation or sham-operation (ducts dLssected but not ligated)(n= 5 rats/group). RESULTS: Compared to sham-controls, duct ligation induced acute pancreatitis as evidenced by hyperamylasemia and pancreatic morphologic changes. Electro-mobility shift assay showed a irma-dependent incraase in NFkB in pancreatic nuclear extracts after duct ligation (transcnption factor E-box was used as control; competing cold NFkB prone excluded non-specihc binding), immunobIot analysis of pancreatic homogehates showed mcreases in phospho-Akt and phospho-lkB that were also time-dependent and parallel with NFkB activation (actin immunobiots confirmed equal loadtirg of lanes) (lkB phosphorylation permits translocatinn of NFkB from the cytosol to the nucleus). CONCLUSIONS: 'Paere is a time-dependem and parallel increase in nuclear translocation of NFkB and acuvation of Akt and IkB after duct ligationdnduced acute pancreatitis in rats. These findings are consistent wuh the h)~otbesis that the Akt-NFkB pathway is activated in duct ligation induced acute pancreatitis. In this expemnentaI corollary of gallstone-reduced acute pancreatitts, bile-pancreatic juice exclusion-induced stimulation of acinar cell G protein coupled receptors could exacerbate acute pancreatitis by increased acinar cell production of pro-inflammatory cytokines via lbe Akt-NFkB pathway (Support: Carver Collaborative Pilot Grant; N1H #lK08DK062805)

Oxidative-stress-related changes in the livers of bile-duct-ligated rats.

The role of reactive oxygen species in liver fibrogenesis is not yet clarified. The aim of this study was to investigate oxidative-stress-related changes in cirrhotic rats. Cirrhosis was induced by bile duct ligation in Sprague-Dawley rats. Plasma malondialdehyde (MDA), hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG), hepatic mitochondrial respiratory functions and gene transcripts were measured at 2 and 4 weeks after surgery in bile-duct-ligated (BDL) and sham-operated-operated rats. The results showed progressive increases in the levels of plasma MDA, hepatic 8-OHdG and procollagen I and III mRNA expression, and progressive impairment of hepatic mitochondrial respiratory function in BDL rats at 2 and 4 weeks after ligation compared with sham-operated rats. Moreover, at 4 weeks after ligation, BDL rats exhibited reduced plasma glutathione and vitamin E levels, impaired hepatic mitochondrial electron transport enzyme activities and oxidative phosphorylation function. In addition, hepatic mRNA expression of transforming growth factor-beta1 was increased. Hepatomegaly, abnormal plasma alanine transaminase and aspartate transaminase levels, and portal hypertension were noted in BDL rats. Our results suggest that bile duct ligation in the rat induces mitochondrial dysfunction and biochemical and molecular changes related to oxidative stress in the liver.

Oxidative-Stress-Related Changes in the Livers of Bile-Duct-Ligated Rats

The role of reactive oxygen species in liver fibrogenesis is not yet clarified. The aim of this study was to investigate oxidative-stress-related changes in cirrhotic rats. Cirrhosis was induced by bile duct ligation in Sprague-Dawley rats. Plasma malondialdehyde (MDA), hepatic 8-hydroxy-2′-deoxyguanosine (8-OHdG), hepatic mitochondrial respiratory functions and gene transcripts were measured at 2 and 4 weeks after surgery in bile-duct-ligated (BDL) and sham-operated-operated rats. The results showed progressive increases in the levels of plasma MDA, hepatic 8-OHdG and procollagen I and III mRNA expression, and progressive impairment of hepatic mitochondrial respiratory function in BDL rats at 2 and 4 weeks after ligation compared with sham-operated rats. Moreover, at 4 weeks after ligation, BDL rats exhibited reduced plasma glutathione and vitamin E levels, impaired hepatic mitochondrial electron transport enzyme activities and oxidative phosphorylation function. In addition, hepatic mRNA expression of transforming growth factor-β1 was increased. Hepatomegaly, abnormal plasma alanine transaminase and aspartate transaminase levels, and portal hypertension were noted in BDL rats. Our results suggest that bile duct ligation in the rat induces mitochondrial dysfunction and biochemical and molecular changes related to oxidative stress in the liver.