Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats

Free and conjugated noradrenaline concentrations were measured in portal-venous and arterial plasma from sham-operated rats or rats with portal hypertension. Two types of portal hypertension in rats were evaluated: in portal vein stenosed rats, the liver was normal, whereas cirrhosis developed in bile duct ligated rats. In cirrhotic rats, arterial free noradrenaline level was higher than in both sham-operated and portal-stenosed rats, this indicating that enhanced sympathetic nervous activity depends on the development of cirrhosis. In all groups of rats, portal venous plasma free noradrenaline was higher than arterial plasma level, indicating a production of noradrenaline by splanchnic organs. Arterial noradrenaline level may be mainly dependent on this splanchnic production in case of portal hypertension. Sulfoconjugated and glucuronoconjugated noradrenaline plasma levels were similar in the three groups of rats. This shows that alteration in conjugation is not likely to be a major factor in the abnormal circulating levels of free noradrenaline observed in cirrhotic rats.     

Portal hemodynamic changes after hepatocyte transplantation in acute hepatic failure

Hepatocyte transplantation has been proposed as an alternative for rescuing patients with acute hepatic failure. However, portal hemodynamic changes and issues of safety after hepatocyte transplantation in acute hepatic failure have not been systemically evaluated because of the lack of a suitable experimentation system. In this study, we created a novel spring-guidewire introducer needle to simplify the technique for long-term portal cannulation in F-344 rats. The portal cannula was capable of being used for blood sampling, infusion of hepatocytes, and measurement of portal hemodynamic changes. One week after portal cannulation, rats were injected withD-galactosamine (1.35 g/kg, i.p.) to induce hepatic failure. Hepatocytes (2×10⁷) were infused intraportally 24–26 h after induction of liver injury. Portal pressures were recorded for up to 60 min after hepatocyte transplantation. Intraportal infusion of 2×10⁷ hepatocytes caused an instantaneous onset of portal hypertension. The magnitude of the rise in portal pressure was similar in both normal rats and rats with acute hepatic failure (33.0±7.1 vs. 37.7±0.5 mm Hg; p=0.23). However, the resolution rate of portal hypertension was remarkably delayed in rats with acute hepatic failure, and the portal pressure was significantly higher than that in normal rats 60 min after hepatocyte transplantation (25.0±2.8 vs. 14.5±2.4 mm Hg; p=0.007). In conclusion, we have established a simple new technique for long-term portal cannulation of rats. Our studies provide critical insights into the delayed resolution of portal hemodynamics after hepatocyte transplantation in subjects with acute hepatic failure.     

Increased plasma volume in two models of portal hypertension in the rat: cirrhosis of the liver and partial portal vein ligation

Portal hypertension has been studied in the rat to see if it is associated to altered blood volume composition, as it has been shown in other species. Plasma volume was measured by isotope dilution using 99mTc labelled albumin in three groups of male Sprague-Dawley rats: normal rats (controls), partially ligated portal vein rats and rats with Cl4C induced cirrhosis. Plasma volume was significantly higher in rats with portal hypertension due to partially ligated portal vein and cirrhosis than in control animals. Similarly, the calculated blood volume was also significantly higher in the portal hypertensive animals than in control group. Portal hypertension in the rat, therefore, has been demonstrated to be associated to a marked hypervolemia and this finding should be taken into consideration in haemodynamic and pharmacokinetic studies in portal hypertensive rat models.     

Effect of portacaval surgical anastomosis on systemic and splanchnic hemodynamics in portal hypertensive, cirrhotic rats

The effect of surgical end-to-side portacaval anastomosis (PCSA) on systemic and splanchnic circulation has been studied in cirrhotic rats with portal hypertension (CCl4-phenobarbital method) and in control animals. Hemodynamics have been measured using the microsphere technique, with a reference sample for the systemic hemodynamic measurements, and intrasplenic injection for portal systemic shunting rate measurements. Compared with controls, sham-operated (SO) cirrhotic rats showed a hyperdynamic circulation with increased cardiac output (CO) and decreased mean arterial pressure and peripheral resistances. PCSA in control rats induced only a small change in systemic hemodynamics, with parallel decreases in arterial pressure and peripheral resistances, and a small, nonsignificant increase in CO. In cirrhotic rats, PCSA induced a decrease of CO to values similar to those of control rats, with an increase in total peripheral resistances. PCSA induced an increase in hepatic arterial blood flow in control and in cirrhotic rats, portal pressure becoming in this latter group not different from that of control rats. Blood flow to splanchnic organs was higher in SO cirrhotic than in SO control animals. Thus portal venous inflow was also increased in SO cirrhotic rats. PCSA induced an increase in portal venous inflow in control rats, which was only significant in cirrhotic rats when expressed as a percentage of CO. In SO control animals, a significant correlation was observed between total peripheral resistances and splanchnic arteriolar resistances and between CO and splanchnic blood flow. These correlations were not observed in cirrhotic rats. These results do not support the hypothesis that hyperdynamic circulation shown by cirrhotic rats is based on increases in splanchnic blood flow and (or) massive portal systemic shunting.     

Histochemistry and ultrastructure of gastric submucosal vasculature in portal hypertensive rats

We studied histochemical and ultrastructural characteristics of the gastric submucosal blood vessels in portal hypertensive (PHT) rats. PHT was induced by two-stage ligation of the portal vein. Control rats were sham operated (SO). On the fifth day after surgery portal vein blood pressure was measured and rats were killed under nembutal anesthaesia. Gastric specimens were obtained for histological, histochemical and ultrastructural assessment. PHT rats showed thickening and increased cellularity of submucosal vessels including increase in number and size of endothelial cells. All these mesenchymal cells were vimentin-positive. Thickening of vascular wall in submucosal vessels was also observed ultrastructurally together with prominent elaboration of luminal surface of endothelial cells. These changes were not observed in SO rats. All changes in PHT rats reflect vasculopathic involvement of gastric wall in portal hypertension.     

Hemodynamic effects of Salvia miltiorrhiza on cirrhotic rats

Salvia miltiorrhiza (Sm) administration has been shown to reduce hepatic fibrosis in rats. We investigated the hemodynamic effects of Sm on bile duct ligated (BDL) rats. Hemodynamic, histological, and vascular contractile studies were conducted in rats 4 weeks after bile duct ligation. An aqueous extract of Sm (0.2 g twice per day) or vehicle was administered for 4 weeks to BDL rats. Sm treatment in BDL rats significantly reduced histological grades of fibrosis and ameliorated the portal hypertensive state (including portal venous pressure, superior mesenteric artery blood flow, cardiac index, and total peripheral resistance) as compared with vehicle treatment. Moreover, Sm treatment enhanced the vascular sensitivity of mesenteric arteries to phenylephrine in BDL rats. Sm treatment had no effect on plasma biochemical profiles of either BDL or normal rats. Our results suggest that 4-week Sm treatment ameliorates the portal hypertensive state in BDL rats.     

Splanchnic and systemic hemodynamic alterations in chronic, progressive portal hypertensive rats

Systemic and splanchnic hemodynamics were studied by using the radioactive microsphere technique, in rats in which a chronic and progressive portal or intrahepatic hypertension was produced by the placement of a nonconstricting, well fitted ligature around the portal or suprahepatic vein when the rat weighted about 100 g. The hemodynamic measurements were performed 80-90 days after ligature placement. Suprahepatic ligated rats presented portal and intrahepatic hypertension, but nonportal-systemic shunts (PSS). The only hemodynamic disturbance observed was a decrease in renal blood flow. Portal ligated rats showed a wide range of PSS and were divided in two subgroups. The subgroups with high PSS rate (greater than 10%) showed increased cardiac output and plasma renin content, as well as decreased splanchnic blood flow, portal venous inflow, hepatic blood flow and renal blood flow. Low portal-systemic shunts subgroups showed decreased cardiac output while its distribution was similar to the control rats. There was no correlation between portal pressure and shunt rate. Low shunt groups, furthermore, showed increased levels of plasma renin concentration.     

Rapamycin Attenuates Splenomegaly in both Intrahepatic and Prehepatic Portal Hypertensive Rats by Blocking mTOR Signaling Pathway

Background

Spleen enlargement is often detected in patients with liver cirrhosis, but the precise pathogenetic mechanisms behind the phenomenon have not been clearly elucidated. We investigated the pathogenetic mechanisms of splenomegaly in both portal hypertensive patients and rats, and tried to identify the possible therapy for this disease.

Methods

Spleen samples were collected from portal hypertensive patients after splenectomy. Rat models of portal hypertension were induced by common bile duct ligation and partial portal vein ligation. Spleen samples from patients and rats were used to study the characteristics of splenomegaly by histological, immunohistochemical, and western blot analyses. Rapamycin or vehicle was administered to rats to determine the contribution of mTOR signaling pathway in the development of splenomegaly.

Results

We found that not only spleen congestion, but also increasing angiogenesis, fibrogenesis, inflammation and proliferation of splenic lymphoid tissue contributed to the development of splenomegaly in portal hypertensive patients and rats. Intriguingly, splenomegaly developed time-dependently in portal hypertensive rat that accompanied with progressive activation of mTOR signaling pathway. mTOR blockade by rapamycin profoundly ameliorated splenomegaly by limiting lymphocytes proliferation, angiogenesis, fibrogenesis and inflammation as well as decreasing portal pressure.

Conclusions

This study provides compelling evidence indicating that mTOR signaling activation pathway plays a key role in the pathogenesis of splenomegaly in both portal hypertensive patients and rats. Therapeutic intervention targeting mTOR could be a promising strategy for patients with portal hypertension and splenomegaly.     

Longitudinal prostaglandin E(2) generation in various organs during evolution of experimental portal hypertension

It has been suggested that the arteriolar vasodilatation and hyperdynamic circulation observed in rats with partial portal vein ligation (PPVL) is caused by increased splanchnic and systemic delivery of vasodilator substances. The aims of our study were to determine organ-specific generation of prostaglandin E(2) (PGE(2)) in rats with PPVL during the evolution of portal hypertension. Rats with PPVL and sham-operated (S) rats were studied in the first, third, fourth and 14th postoperative days. They were anesthetized and splenic pulp pressure and blood pressure were measured. Spleen, colon and lungs were removed and the splenic, pulmonary and mucosal colonic PGE(2) were determined. All PPVL rats developed sequential hemodynamic changes compatible with evolving portal hypertension. Splenic pulp pressure was higher in PPVL rats compared with S rats during all days of the study. Within the group of PPVL the splenic pulp pressure was higher in the first postoperative day and decreased in the ensuing days. No changes in splenic and colonic PGE(2) generation were noted during the study period. Pulmonary PGE(2) generation increased significantly in the first postoperative day in PPVL rats compared with S rats. However, similar increase was observed on the third postoperative day in S rats. PGE(2) probably has no role in splanchnic hemodynamic changes during evolution of portal hypertension. Pulmonary PGE(2) generation may increase as a response to increased portal pressure, or to abdominal surgery.     

Plasma catecholamines and urinary excretion of their main metabolites in three models of portal hypertension

We have measured, by a specific radioenzymoassay, the plasma concentration of dopamine (DA) and norepinephrine (NE) and by gas chromatography the urinary excretion of some catecholamine metabolites (HVA, homovanillic acid, DOPAC, dihydroxyphenyl acetic acid; VMA, vanilmandelic acid, and DOPEG, dihydroxyphenyl glycol) in three groups of rats with portal hypertension: cirrhotic rats (CR), rats with progressive portal hypertension (PPH) and rats with progressive hepatic congestion (PHC). The three groups of rats had portal hypertension. PPH and PHC had also intrahepatic hypertension. CR rats showed an increased urinary excretion of NE and DA metabolites with a normal plasma concentration of these catecholamines, suggesting an increased turnover of NE and DA in this experimental model. PPH animals had a high plasma DA concentration with a decreased urinary excretion of catecholamine metabolites. PHC showed high plasma DA and NE levels with normal or increased urinary excretion of its metabolites. These results suggest that an increased neural activity is present in the early stages of experimental cirrhosis in rats and this alteration does not seem directly related to the portal hypertension but perhaps to the intrahepatic hypertension or to the hepatocellular damage.     

Role of acute elevation of portal venous pressure by exogenous glucagon on gastric mucosal injury in rats with portal hypertension

Time-course studies revealed the increased susceptibility of the gastric mucosa to noxious injury in portal hypertension correlates with the level of elevated portal venous pressure and hyperglucagonemia. Whether acute elevation of portal venous pressure by exogenous glucagon aggravates such injury is not known. We tested the hypothesis that glucagon in a dose sufficient to acutely elevate portal venous pressure aggravates noxious injury of the gastric mucosa in rats with portal hypertension. Infusion of a portal hypotensive dose of somatostatin should reverse these changes. In anesthetized rats with portal vein ligation, glucagon, somatostatin or the combination was administered intravenously in a randomized, coded fashion. Acidified ethanol-induced gastric mucosal injury was determined. Portal venous pressure and gastric mucosal perfusion and oxygenation (reflectance spectrophotometry) were monitored to confirm the effects of the respective intravenous treatments. Exogenous glucagon exacerbated acidified ethanol-induced gastric mucosal injury. The exacerbation was attenuated by somatostatin. These changes paralleled the portal hypertensive and hypotensive effects of glucagon and somatostatin, respectively. Our data suggest that a unique mechanism is triggered with the onset of portal hypertension. In an antagonistic manner, glucagon and somatostatin modulate this novel mechanism that controls portal venous pressure and susceptibility of the gastric mucosa to noxious injury.     

Locomotor Activity and Temperature Rhythms in Cirrhotic and Portal Hypertensive Rats

This study was performed to examine the circadian variations in body temperature and locomotor activity in two rat models of liver damage and portal-systemic collateralization, it is, cirrhosis by common bile duct ligature and portal vein ligation. Locomotor activity and temperature were measured telemetrically, and the degree of portal-systemic shunting was evaluated by the radioactive microsphere technique. In cirrhotic rats a significant increase in portal pressure and portal-systemic shunting occurred, with extensive liver damage and ascitis. These changes were accompanied by a derangement of the activity rhythm (decrease in total activity, night/day ratio and Qp) and an increase in the amplitude of the temperature rhythm. In portal vein-ligated rats, portal vein pressure and portal-systemic shunting increased significantly, with no changes in any of the rhythm parameters analyzed (total, diurnal and nocturnal activity pulses, night/day activity ratio and Qp for activity; mesor, amplitude and free-running period for temperature). The results indicate that liver dysfunction, and not merely portal hypertension or portal-systemic shunting, is the main factor affecting daily rhythms in cirrhotic rats.     

Locomotor Activity and Temperature Rhythms in Cirrhotic and Portal Hypertensive Rats

This study was performed to examine the circadian variations in body temperature and locomotor activity in two rat models of liver damage and portal-systemic collateralization, it is, cirrhosis by common bile duct ligature and portal vein ligation. Locomotor activity and temperature were measured telemetrically, and the degree of portal-systemic shunting was evaluated by the radioactive microsphere technique. In cirrhotic rats a significant increase in portal pressure and portal-systemic shunting occurred, with extensive liver damage and ascitis. These changes were accompanied by a derangement of the activity rhythm (decrease in total activity, night/day ratio and Qp) and an increase in the amplitude of the temperature rhythm. In portal vein-ligated rats, portal vein pressure and portal-systemic shunting increased significantly, with no changes in any of the rhythm parameters analyzed (total, diurnal and nocturnal activity pulses, night/day activity ratio and Qp for activity; mesor, amplitude and free-running period for temperature). The results indicate that liver dysfunction, and not merely portal hypertension or portal-systemic shunting, is the main factor affecting daily rhythms in cirrhotic rats.     

Renal hyperemia in portal hypertension is not mediated by gastrointestinal peptides

The objectives of this study were to characterize the time course of development of the renal hyperemia induced by chronic portal vein stenosis (PVS) in the rat, and to assess the possibility that vasoactive blood-borne gastrointestinal peptides mediate the renal hyperemia in established portal hypertension. Blood flow to the kidneys was measured with radioactive microspheres over a ten day time course. On day 2, no difference in renal blood flow (RBF) was observed in PVS rats as compared with controls. However, by day 4, RBF significantly increased by 35% in PVS vs. control animals. On day 6, the renal hyperemia in PVS rats reached a maximal value that was 42% higher than controls. A steady state hyperemia (approximately 40%) was maintained thereafter. Radioimmunoassay of plasma from control and established portal hypertensive rats (10 days samples) revealed that vasoactive intestinal polypeptide, substance P, cholecystokinin, gastrin, neurotensin, pancreatic polypeptide, beta-endorphin and peptide histidine-isoleucine amide are not elevated in arterial plasma of portal hypertensive rats. These data suggest that the renal hyperemia induced by chronic portal vein stenosis is apparent within 4 days of the onset of a hypertensive state and attains a steady state by day 8. Furthermore, at least eight blood-borne gastrointestinal peptides are not directly involved in the renal hyperemia associated with chronic portal hypertension.     

Impaired generation of prostaglandins from isolated gastric surface epithelial cells in portal hypertensive rats

We studied generation of prostaglandins E2 and 6-keto F1a by surface epithelial cell isolated from the gastric mucosa of portal hypertensive and sham-operated rats. Oxygenated cell suspensions containing 80 +/- 3% of surface epithelial cells were incubated for 30 min at 37 degrees C and the concentration of prostaglandin E2 and 6-keto-prostaglandin F1a in medium was measured by radioimmunoassay. Viability of the cells was assessed with Fast green exclusion at baseline and after 30-min and 60-min incubation. Within 30 minutes the surface epithelial cells obtained from portal hypertensive rats generated 22.0 +/- 1.6 (mean +/- SE) pg prostaglandin E2 and 40.7 +/- 4.7 pg 6-keto prostaglandin F1a, per 10(6) cells. These were significantly less than prostaglandin generation by cells obtained from sham-operated rats. The viability of the surface epithelial cells from portal hypertensive rats was also significantly reduced compared with sham-operated rats after 60 minute incubation. Reduced ability of the surface epithelial cells to generate prostaglandins may be one mechanism for increased susceptibility of portal hypertensive gastric mucosa to injury by noxious agents.     

Proinflammatory liver and antiinflammatory intestinal mediators involved in portal hypertensive rats

Proinflammatory (TNF-alpha , IL-1beta, and NO) and antiinflammatory (IL-10, CO) levels were assayed in serum, liver, and small bowel in order to verify a hypothetic inflammatory etiopathogeny of portal hypertension that could be the cause of its evolutive heterogeneity. Male Wistar rats were divided into one control group (n=11) and one group with a triple stenosing ligation of the portal vein (n=23) after 28 days of evolution. In one subgroup of portal hypertensive rats, portal pressure, collateral venous circulation, mesenteric vasculopathy, and liver and spleen weights were determined. In the remaining rats with portal hypertension TNF-alpha, IL-1beta, and IL-10 were quantified in liver and ileum by enzyme-linked immunosorbent assay. NO synthase activity was studied in liver and ileum. CO and NO were measured in portal and systemic blood by spectrophotometry and Griess reaction, respectively. Portal hypertensive rats with mayor spleen weight show hepatomegaly and mayor development of collateral circulation. Ileum release of IL-10 (0.30 +/- 0.12 versus 0.14 +/- 0.02 pmol/mg protein; P< .01) is associated with a liver production of both proinflammatory mediators (TNF-alpha: 2 +/- 0.21 versus 1.32 +/- 0.60 pmol/mg protein; P< .05, IL-1beta: 19.17 +/- 2.87 versus 5.96 +/- 1.84 pmol/mg protein; P=.005, and NO: 132.10 +/- 34.72 versus 61.05 +/- 8.30 nmol/mL; P=.005) and an antiinflammatory mediator (CO: 6.49 +/- 2.99 versus 3.03 +/- 1.59 pmol/mL; P=.005). In short-term prehepatic portal hypertension a gut-liver inflammatory loop, which could be fundamental in the regulation both of the portal pressure and of its complications, could be proposed.     

Mild increases in portal pressure upregulate vascular endothelial growth factor and endothelial nitric oxide synthase in the intestinal microcirculatory bed, leading to a hyperdynamic state

Increased nitric oxide (NO) is the main factor leading to the hyperdynamic circulation associated with advanced portal hypertension (PHT), but the initial mechanisms and the magnitude of increase in portal pressure required to trigger NO production are not known. We addressed these issues by studying systemic and splanchnic hemodynamics and endothelial NO synthase (eNOS) and VEGF expression in rats with different degrees of portal hypertension. Portal vein ligation (PVL) performed over needles of three different calibers (16-, 18-, and 20-gauge) yielded different degrees of PHT and portosystemic shunting. Compared with sham rats, all three groups of PVL rats exhibited features of hyperdynamic circulation. Rats with minimal portal hypertension (PVL with a 16-gauge needle) showed an early increase in VEGF and eNOS expression selectively at the jejunum. Immunofluorescence showed that VEGF expression was located in highly vascularized areas of the mucosa. Inhibition of VEGF signaling markedly attenuated the increase in eNOS expression. In conclusion, mild increases in portal pressure are enough to upregulate eNOS at the intestinal microcirculation, and this occurs, at least in part, through VEGF upregulation.     

Prostaglandin D2 stimulates vasoactive intestinal polypeptide release into rat hypophysial portal blood

The effect of prostaglandin D2 (PGD2) on vasoactive intestinal polypeptide (VIP) release from the hypothalamus was examined by determining plasma VIP levels in rat hypophysial portal blood. Intraventricular injection of PGD2 (5 micrograms/rat) caused a 3-fold increase in the concentration of plasma VIP in hypophysial portal blood in anesthetized rats. A PGD2 metabolite, 13,14-dihydro-15-keto PGD2, did not affect VIP levels in portal blood. The flow rate of hypophysial portal blood was not changed after the injection of PGD2. The intraventricular injection of PGD2, but not PGD2 metabolite, resulted in an increase in peripheral plasma prolactin (PRL) levels in the rat. These findings suggest that PGD2 plays a stimulatory role in regulating VIP release from the hypothalamus into hypophysial portal blood and causes PRL secretion from the pituitary in rats.     

Role of NPY for vasoregulation in the splanchnic circulation during portal hypertension

Vascular dysfunction in the splanchnic circulation during portal hypertension is characterized by enhanced NO-mediated vasorelaxation and vascular hyporeactivity to norepinephrine that lead to arterial vasodilation. NPY most likely counteracts both of these key features. Firstly, NPY appears to inhibit Ach- and PNS-induced vasorelaxation in mesenteric arteries. This effect is more pronounced in portal hypertensive rats as compared to control, and most likely reflects the inhibition of increased e- and nNOS-derived NO-synthesis during portal hypertensive conditions. Secondly, NPY sensitizes the mesenteric vasculature to alpha(1)-adrenergic vasoconstriction. Most importantly, in portal hypertensive rats but not in sham rats NPY markedly augments vascular contractility and thereby corrects vascular hyporeactivity. Both actions of NPY increase vascular tone and may well act synergistically in the splanchnic circulation during portal hypertension. Moreover, the vasoconstrictive effects of NPY are most pronounced at particularly high levels of alpha(1)-adrenergic activity. Therefore, it appears that NPY becomes increasingly important for optimizing adrenergic vasoconstriction at particularly high adrenergic drive and also for playing a predominant role for vascular homeostasis. Cirrhotic patients present with elevated circulating plasma levels of NPY, which appears to be independent from the severity of liver dysfunction and to correlate with portal pressure. This finding indicates enhanced NPY release during portal hypertension that may represent a compensatory mechanism aimed at counterbalancing arterial vasodilation by restoring the efficacy of endogenous catecholamines and inhibiting vasodilative drive in the splanchnic circulation.     

双歧杆菌对门静脉血内毒素影响初步研究

本实验以大鼠为动物模型,探讨在肠道微生态失调的状态下,用大量双歧杆菌(Bifidobacteria)灌服,借以拮抗外源性需氧革兰氏阴性杆菌的生长繁殖,从而使肠源性内毒素自门静脉的吸收减少。对正常鼠、脱污染鼠、再污染鼠、治疗鼠和非治疗鼠,进行不同肠段的双歧杆菌、需氧革兰氏阴性杆菌的测定和门静脉血内毒素测定。结果表明:双歧杆菌对肠道需氧革兰氏阴性杆菌的生长繁殖,具有明显的生物拮抗作用,同时伴有相应的门静脉血内毒素水平的降低。