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The aim of this study was to assess the hemodynamic effects of acute and chronic administration of vapreotide, a somatostatin analog, in rats with intrahepatic portal hypertension induced by dimethylnitrosamine (DMNA) administration. Acute effects were evaluated at baseline and 30 min after placebo (N = 13) or vapreotide (8 μg/kg/hr, N = 13) infusions in DMNA rats. Chronic hemodynamic effects were evaluated using subcutaneous implants for five weeks in anesthetized DMNA rats (placebo: N = 13, vapreotide: N = 13) and in sham rats (placebo: N = 10, vapreotide: N = 10). Hemodynamic measurements included splenorenal shunt blood flow (SRS BF) by the transit time ultrasound (TTU) method and cardiac output by the combined dilution–TTU method. Acute administration of vapreotide significantly decreased SRS BF (−17.3 ± 19 vs −1.1 ± 14%, P < 0.05) and portal pressure (−8 ± 9 vs 0 ± 8%, p < 0.05) compared to placebo without systemic effects. Chronic administration of vapreotide significantly reduced the increase in SRS BF (2.4 ± 1.5 vs 1.2 ± 1.0 ml/min, P < 0.05) and cardiac index (50 ± 15 vs 33 ± 10 ml/min/100 g, P < 0.0001) while portal pressure and blood flow, and mean arterial pressure were not significantly changed compared to placebo. In conclusion, the acute administration of vapreotide decreased collateral circulation blood flow while chronic administration attenuated its development. Vapreotide seems to have a vasoconstrictive effect on collateral circulation.

In cirrhosis, delayed intestinal transit may be responsible for increased endoluminal bacterial overgrowth and increased bacterial translocation. Octreotide has been reported to reduce intestinal transit. Therefore, we evaluated whether octreotide administration influences bacterial translocation in a model of liver fibrosis secondary to dimethylnitrosamine (DMNA) administration. Twenty-nine conscious rats were randomly assigned to three groups (sham rats + placebo as controls, DMNA + placebo, DMNA + octreotide, 1.5 microg/kg thrice daily subcutaneously), and including portal pressure, intestinal transit (radioactive method), and bacterial translocation were measured. Three of four variables measuring intestinal transit suggested a significant delay in intestinal transit in DMNA rats compared to controls (eg, cumulated radioactivity 50%: controls: 5.3+/-1.5, DMNA + placebo: 3.2+/-1.2, DMNA + octreotide: 2.7+/-1.9, P < 0.01). This delay tended to be enhanced by octreotide but the effect was only significant with one of the intestinal transit variables. Bacterial translocation was significantly increased in DMNA rats compared to controls but octreotide did not increase translocation [eg, germ count (log) in lymph nodes: controls: 3.1+/-3.6, DMNA + placebo: 12.3+/-4.4, DMNA + octreotide: 10.6+/-6.0, P < 0.001]. There was no significant correlation of portal pressure, intestinal transit, and bacterial translocation in this study. In conclusion, our results show that, although octreotide worsens delayed intestinal transit, it has no influence on the level of bacterial translocation.

Recently, we developed a new method to measure collateral blood flow in rats: splenorenal shunt (SRS) blood flow (BF). The aims were to evaluate the reproducibility of SRSBF measurement in different models of portal hypertension, and to investigate the ability of SRSBF to disclose pharmacological changes.Hemodynamics were determined in anesthetized rats with secondary biliary, CCl4 or DMNA cirrhosis and portal vein ligation (PVL) under baseline and pharmacological (octreotide, vapreotide) conditions. The main measurements performed were: SRSBF by the transit time ultrasound (TTU) method and % portosystemic shunts (PSS) by the microsphere method.SRSBF was 6 to 10 times higher in portal hypertensive rats and was similar in the different models of cirrhosis but was higher in portal vein ligated rats than in cirrhotic rats (1.1+/-0.7 vs 0.6+/-0.7 ml x min(-1) x 100 g(-1), p=0.01). SRSBF was correlated with mesenteric %PSS (r=0.61, p0.01), splenic %PSS (r=0.54, p0.05), portal pressure (r= 0.32, p0.05) and the area of liver fibrosis (r=0.33, p0.05). Octreotide significantly decreased SRSBF (-23+/-20%, p0.01 vs placebo: -6+/-8%, NS). Vapreotide significantly decreased SRSBF but not mesenteric or splenic %PSS compared to placebo. The variations in SRSBF (-26+/-32%) and in splenic %PSS (0+/-15%) with vapreotide were significantly different (p0.05) and not correlated (r=-0.1, NS).Determination of SRSBF by TTU is an accurate way to measure collateral blood flow in different models of intra- and extra-hepatic portal hypertension in rats. Its sensitivity provides accurate measurement of pharmacological changes, unlike the traditional estimation of %PSS by the microsphere method.

The aim of this study was to assess the effect of early and chronic administration of a prostaglandin E1 analogue (misoprostol) in the prevention of liver fibrosis and portal hypertension.Liver fibrosis was induced by bile duct ligation. Controls had a sham operation. Bile-duct-ligated rats were divided into two groups: placebo (vehicle only) and misoprostol (10 microg/d by gavage) for 4 weeks after surgery. Liver fibrosis was assessed by the area of fibrosis (image analysis), liver hydroxyproline content and serum hyaluronate. Systemic and splanchnic hemodynamics were evaluated including spleno-renal shunt blood flow by the transit-time ultrasound technique.Mean arterial pressure was significantly lower in the misoprostol group (p0.01). There was an unexpected increase in fibrosis parameters in the misoprostol group compared to the placebo group, e.g. area of fibrosis: 9.5+/-4.0 vs 15.0+/-8.1% (p0.05). Spleno-renal shunt blood flow was significantly higher in the misoprostol group than in the placebo group (4.6+/-3.7 vs 2.2+/-2.0 ml/min, p0.05) while portal pressure was unchanged.The early and chronic administration of misoprostol enhances porto-collateral circulation blood flow and the development of liver fibrosis in bile-duct-ligated rats.

The aim of this study was to develop a technique that could serve as an index of portosystemic shunt (PSS) blood flow in portal hypertensive rats whose main shunt is the splenorenal shunt (SRS). The main hemodynamic measurements performed were: SRS blood flow by the transit-time ultrasound (TTU) method, percentage of PSS, and regional blood flows by the microsphere method. We determined the accuracy and reproducibility of SRS blood flow measurements under baseline and pharmacological (octreotide) conditions. SRS blood flow was compared with other hemodynamic characteristics. Two models of portal hypertension were used: secondary biliary and dimethylnitrosamine cirrhosis. The SRS blood flow was correlated with mesenteric (r = .76; P < .001) and splenic (r = .67; P < .01) PSS percentages. The intra- and interobserver agreements for SRS blood flow were excellent: ric = .99 and ric = .98, respectively. SRS blood flow was six times higher in portal hypertensive rats (0.6 ± 0.7 mL · min−1 · 100 g−1) than in sham rats (0.1 ± 0.1 mL · min−1 · 100 g−1 [P < .01]). Octreotide significantly decreased SRS blood flow but not mesenteric or splenic PSS percentages. SRS is the main PSS in rats. The measurement of SRS blood flow by TTU is accurate and reproducible. This method can be used to identify new mechanisms in hemodynamic studies that differ from those identified by the measurement of the percentage of PSS by the microsphere method, especially in pharmacological studies.

The aim of this study was to assess the effect of the early and chronic administration of interferon alpha in the prevention of hepatic fibrosis and portal hypertension.Rats with liver fibrosis due to bile duct ligation or CCl4 were divided into three groups: sham, placebo and interferon alpha2a 100,000 UI/day. Liver fibrosis was assessed by the area of fibrosis (image analysis), liver hydroxyproline and mRNA (fibronectin, procollagen alpha2(I)) contents, and serum hyaluronate. Systemic and splanchnic hemodynamics were also evaluated.Interferon alpha significantly decreased fibrosis in the CCl4 model only: area of fibrosis: 13.9+/-3.7 vs 10.5+/-3.3% (p0.05), hydroxyproline: 1.8+/-0.6 vs 1.2+/-0.2 mg/g wet liver (p0.001), respectively placebo vs interferon alpha. There was a significant correlation between the area of fibrosis and hydroxyproline liver content (r=0.77 in the biliary model and r=0.87 in the CCl4 model, p0.0001). Interferon decreased spleno-renal shunt blood flow (2.0+/-1.8 vs 0.9+/-0.7 ml/min; p0.05) but not portal pressure in the CCl4 model. No significant effects were observed in rats with biliary fibrosis.The early and chronic administration of interferon alpha prevents the development of liver fibrosis and porto-collateral circulation in the CCl4 model but not in the biliary model. However, the antifibrotic effects of interferon need to be confirmed in further studies.

The clearance of human cytomegalovirus (HCMV) was evaluated in infected patients under Ganciclovir (GCV) treatment, using a novel HCMV DNA quantitation assay (HCMV DNA hybrid capture system, Murex Diagnostics). Peripheral white blood cells (WBC) from whole blood specimens of seven AIDS patients, three kidney and two allogeneic bone marrow transplant (BMT) recipients suffering from HCMV disease, were assessed by this method. HCMV DNA 50 and 90% mean clearances were observed at 2.11 ± 1.97 and 6.22 ± 4.31 days, respectively, after initial GCV treatment. The viral DNA kinetics were correlated with positive and negative pp65 antigenaemia and viral blood culture. Two-fold higher clearances and initial DNA levels were observed in the AIDS group compared to the transplant group. Neither clinical nor virological relapses were observed under GCV treatment. HCMV DNA quantitation in WBC appears well adapted for a therapeutic follow up of patients with HCMV disease.

S-adenosylmethionine (SAM) is the principal biological methyl donor and precursor for polyamines. SAM is known to be hepatoprotective in many liver disease models in which TNF-alpha is implicated. The present study investigated whether and how SAM inhibited LPS-stimulated TNF-alpha expression in Kupffer cells (hepatic macrophages). SAM downregulated TNF-alpha expression in LPS-stimulated Kupffer cells at the transcriptional level as suggested by a transfection experiment with a TNF-alpha promoter-reporter gene. This inhibition was not mediated through decreased NF-kappaB binding to four putative kappaB binding elements located within the promoter. The inhibited promoter activity was neither prevented by overexpression of p65 and/or its coactivator p300 nor enhanced by overexpression of coactivator-associated arginine methyltransferase-1, an enzyme that methylates p300 and inhibits a p65-p300 interaction. SAM did not lead to DNA methylation at the most common CpG target sites in the TNF-alpha promoter. Moreover, 5'-methylthioadenosine (MTA), which is derived from SAM but does not serve as a methyl donor, recapitulated SAM's effect with more potency. These data demonstrate that SAM inhibits TNF-alpha expression at the level downstream of NF-kappaB binding and at the level of the promoter activity via mechanisms that do not appear to involve the limited availability of p65 or p300. Furthermore, our study is the first to demonstrate a potent inhibitory effect on NF-kappaB promoter activity and TNF-alpha expression by a SAM's metabolite, MTA.

The aim of this study was to assess the hemodynamic effects of acute and chronic administration of vapreotide, a somatostatin analog, in rats with intrahepatic portal hypertension induced by dimethylnitrosamine (DMNA) administration. Acute effects were evaluated at baseline and 30 min after placebo (N = 13) or vapreotide (8 /microg/kg/hr, N = 13) infusions in DMNA rats. Chronic hemodynamic effects were evaluated using subcutaneous implants for five weeks in anesthetized DMNA rats (placebo: N = 13, vapreotide: N = 13) and in sham rats (placebo: N = 10, vapreotide: N = 10). Hemodynamic measurements included splenorenal shunt blood flow (SRS BF) by the transit time ultrasound (TTU) method and cardiac output by the combined dilution-TTU method. Acute administration of vapreotide significantly decreased SRS BF (-17.3 +/- 19 vs - 1.1 +/- 14%, P0.05) and portal pressure (-8 +/- 9 vs 0 +/- 8%, p0.05) compared to placebo without systemic effects. Chronic administration of vapreotide significantly reduced the increase in SRS BF (2.4 +/- 1.5 vs 1.2 +/- 1.0 ml/min, P0.05) and cardiac index (50 +/- 15 vs 33 +/- 10 ml/min/100 g, P0.0001) while portal pressure and blood flow, and mean arterial pressure were not significantly changed compared to placebo. In conclusion, the acute administration of vapreotide decreased collateral circulation blood flow while chronic administration attenuated its development. Vapreotide seems to have a vasoconstrictive effect on collateral circulation.

Vasopressin has been used to treat arterial hypotension associated with hyperdynamic vasoplegic states, but detrimental effects on splanchnic circulation have been reported. We tested the effects of a low-dose vasopressin analogue, terlipressin (6 microg/kg), on systemic and splanchnic hemodynamics in fluid-challenged endotoxic rats (lipopolysaccharide, 30 mg/kg in 1 hr).Prospective, randomized, controlled experimental study with repeated measures.Investigational animal laboratory.A total of 77 rats were divided into five groups: group C, control (17 rats); group E, LPS (18 rats); group EF, LPS plus fluid challenge (18 rats); group EFT, LPS plus fluid challenge plus terlipressin (18 rats); and group ET, LPS plus terlipressin (seven rats).Rats were anesthetized, mechanically ventilated, and instrumented to measure heart rate, mean arterial pressure, and abdominal aortic and mesenteric vein indexed blood flows; ileal microcirculation was assessed by laser Doppler. After LPS infusion, rats experienced an endotoxic shock and were resuscitated after the allocation group. The fluid challenge was targeted to maintain mean arterial pressure of90 mm Hg and aortic blood flow at baseline values.Terlipressin significantly (p.05) increased mean arterial pressure without decreasing indexed aortic blood flow and heart rate in the fluid-challenged endotoxic rats (EFT) compared with EF rats and had detrimental effects in hypodynamic endotoxic rats (ET). Fluid challenge significantly (p.05) increased mesenteric vein blood flow in both the EF and EFT groups, and terlipressin had no detrimental effect on mesenteric blood flow. Terlipressin significantly (p.05) increased ileal microcirculation in fluid-challenged endotoxic rats (EF and EFT) but not in hypodynamic endotoxic rats (E and ET).Low-dose terlipressin in fluid-challenged endotoxic rats improved systemic and splanchnic hemodynamics and improved the ileal microcirculation.

Background/Aims : To assess the effects of the early and chronic administration of losartan -a specific angiotensin II receptor antagonist- in the prevention of hepatic fibrosis and portal hypertension. Methods/Results : (1) In CCl 4 rats, losartan at 5 and 10 mg/kg per day significantly decreased portal pressure (−11, −18%, respectively), splenorenal shunt blood flow (−60, −80%) and liver fibrosis (liver hydroxyproline and area of fibrosis) without significant changes in mortality and mean arterial pressure (MAP). (2) In bile duct ligated (BDL) rats, losartan at 5 mg/kg per day significantly decreased portal pressure (−14%), splenorenal shunt blood flow (−70%) and liver fibrosis. Losartan at 10 mg/kg per day significantly worsened liver and renal functions, mortality and liver fibrosis, without significant changes in portal pressure and splenorenal shunt blood flow. Losartan at 5 and 10 mg/kg per day significantly decreased MAP (−24, −30%). (3) In portal vein ligated (PVL) rats, losartan significantly decreased MAP (−12%) but did not change portal pressure or splenorenal shunt blood flow. Conclusions : In BDL and CCl 4 rats, losartan has beneficial effects on splanchnic hemodynamics and liver fibrosis. Losartan might decrease hepatic resistances in fibrotic liver. Losartan decreased MAP except in CCl 4 rats. Higher dosage of losartan had deleterious effects in BDL rats.

Interleukin (IL)-10 expression is induced in activated hepatic stellate cells (HSC) in vitro and in vivo. We analyzed expression of IL-10 receptor (IL-10R) and coreceptor cytokine receptor family (CRF2–4) in HSC. We aimed to clone and sequence partial cDNA for rat IL-10R and CRF2–4, determine their expression in activated rat HSC in vivo and in vitro, and examine the biological responsiveness of HSC to exogenous IL-10. PCR cloning and sequencing of partial rat IL-10R and CRF2–4 cDNAs revealed 86% homology with corresponding mouse sequences. In hepatic macrophages, Northern blot with cloned IL-10R cDNA detected an expected 3.5-kb transcript, and IL-10R and CRF2–4 mRNAs showed steady constitutive expression after in vitro lipopolysaccharide treatment or cholestatic liver injury. IL-10R mRNA expression, as confirmed by immunohistochemistry, was induced 20.1- and 8.6-fold in HSC from cholestatic livers and 7-day culture-activated HSC, respectively but CRF2–4 mRNA levels were unchanged. Under serum-free conditions, IL-10 had minimal effects on collagen production but reduced DNA synthesis, matrix metalloprotease-2 mRNA levels, and activity in HSC. With serum, IL-10 inhibited both collagen production and DNA synthesis but had no effect on procollagen-α1(I) mRNA levels. This shows concomitant induction of IL-10R but not CRF2–4 to that of IL-10 by activated HSC in vitro and in vivo and associated acquisition of the responsiveness to IL-10, entailing complex effects on HSC.

In cirrhosis there is a hyperdynamic circulation, which occurs mainly in the systemic and splanchnic regions. Using isolated-vessel models, previous studies have shown reduced aortic reactivity to vasoconstrictors in rats with cirrhosis. The aim of the present study was to evaluate and compare the vascular responsiveness to phenylephrine in arterial rings and the blood flows from different regions in rats with cirrhosis and controls. Reactivity was studied in isolated thoracic aortic, superior mesenteric arterial and carotid arterial rings from sham-operated and bile-duct-ligated rats by measuring the cumulative concentration-dependent tension induced by phenylephrine (10-9–10-4 M). Blood flows were measured by the radioactive microsphere method. In rats with cirrhosis, a significant hyporeactivity to phenylephrine was observed in both the aorta and the superior mesenteric artery compared with the corresponding arteries of normal rats. This hyporesponsiveness was corrected by Nω-nitro-l-arginine (0.1 mM). In contrast, carotid artery reactivity and the responses to Nω-nitro-l-arginine were similar in the cirrhotic and control groups. In each case, cardiac output and mesenteric arterial blood flow were significantly higher in cirrhotic than in normal rats. Cerebral blood flows were not significantly different between the two groups. In cirrhotic rats, arterial hyporeactivity may be a consequence of increased regional blood flow and increased production of nitric oxide.

Background/Aims: The action sites and kinetic effects of octreotide and terlipressin may be different. Therefore, we studied the hemodynamic effects of acute administration of these drugs alone or in combination in rats with portal hypertension due to portal vein ligation. Methods: In a first study performed in anesthetized rats, hemodynamics were measured before and after drug administration (placebo, octreotide: 8 μg·kg −1 ·h −1 for 30 min, terlipressin: 50 μg/kg bolus, terlipressin + octreotide at the same doses). The second study, performed in conscious rats, included the same groups and drug doses; hemodynamics were measured every 10 min for 1 h. The third study tested the effect of preinfusion of octreotide on responsiveneness to terlipressin. Results: Terlipressin produced more marked systemic effects than octreotide by decreasing heart rate and cardiac output and increasing mean arterial pressure. Terlipressin produced a greater decrease in portal pressure than octreotide: placebo: −3±5%, terlipressin: −42±8%, octreotide: −16±10%, combination: −44±8% (conscious rats at 20 min, p −4 ). The decrease in portal pressure was immediate and lasted at least 60 min with both drugs. Octreotide significantly decreased spleno-renal shunt blood flow (% variation): placebo: −6±8, terlipressin: −15.5±20, octreotide: −22.5±20, combination: −27±10 ( p −2 ). Octreotide preinfusion significantly increased the responsiveness of arterial pressure and heart rate to terlipressin. Conclusions: Terlipressin decreases portal pressure significantly more than octreotide, while only octreotide significantly decreases collateral blood flow. Simultaneous administration of these drugs does not have significant additive effects but has complementary effects. The preadministration of octreotide alters systemic reponse to terlipressin.

The aim of this study was to validate a new technique for the measurement of cardiac output (CO) based on ultrasound and dilution (COUD) in anesthetized rats. A transit time ultrasound (TTU) probe was placed around the rat carotid artery, and ultrasound velocity dilution curves were generated on intravenous injections of saline. CO by COUD were calculated from the dilution curves for normal and portal hypertensive rats in which CO was known to be increased. COUD was compared with the radiolabeled microsphere method and with direct aortic TTU flowmetry for baseline CO and drug-induced CO variations. CO in direct aortic TTU flowmetry was the ascending aorta blood flow measured directly by TTU probe (normal use of TTU flowmetry). The reproducibility of COUD within the same animal was also determined under baseline conditions. COUD detected the known CO increase in portal hypertensive rats compared with normal rats. CO values by COUD were correlated with those provided by microsphere technique or direct aortic TTU flowmetry (adjusted r = 0.76, P < 10−4 and r = 0.79, P < 0.05, respectively). Baseline CO values and terlipressin-induced CO variations were detected by COUD and the other techniques. Intra- and interobserver agreements for COUD were excellent (intraclass r = 0.99 and 0.98, respectively). COUD was reproducible at least 10 times in 20 min. COUD is an accurate and reproducible method providing low-cost, repetitive CO measurements without open-chest surgery. It can be used in rats as an alternative to the microsphere method and to direct aortic flowmetry.

AIM: To assess whether a liver specific nitric oxide (NO) donor (V-PYRRO/NO) would prevent the development of portal hypertension and liver fibrosis in rats with bile duct ligation (BDL). METHODS: Treatment (placebo or V-PYRRO/NO 0.53 μmol/kg per hour) was administered i.v. to rats 2 d before BDL (D-2) and maintained until the day of hemodynamic measurement (D26). Intra-hepatic NO level was estimated by measuring liver cGMP level. Effects of V-PYRRO/NO on liver fibrosis and lipid peroxidation were also assessed. RESULTS: Compared to placebo treatment, V-PYRRO/NO improved splanchnic hemodynamics in BDL rats: portal pressure was significantly reduced by 27% (P < 0.0001) and collateral circulation development was almost completely blocked (splenorenal shunt blood flow by 74%, P = 0.007). Moreover, V-PYRRO/NO significantly prevented liver fibrosis development in BDL rats (by 30% in hepatic hydroxyproline content and 31% in the area of fibrosis, P < 0.0001 respectively), this effect being probably due to a decrease in lipid peroxidation by 44% in the hepatic malondialdehyde level (P = 0.007). Interestingly, we observed a significant and expected increase in liver cGMP, without any systemic hemodynamic effects (mean arterial pressure, vascular systemic resistance and cardiac output) in both sham-operated and BDL rats treated with V-PYRRO/NO. This result is in accordance with studies on V-PYRRO/NO metabolism showing a specific release of NO in the liver. CONCLUSION: Continuous administrations of V-PYRRO/NO in BDL rats improved liver fibrosis and splanchnic hemodynamics without any noxious systemic hemo-dynamic effects.