Mitochondrial dysfunction as a mediator of hippocampal apoptosis in a model of hepatic encephalopathy.

In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ (m)) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the presence of NH(4) (+) and calcium.

Enzymatic activities in brains of diabetic rats treated with vanadyl sulphate and sodium tungstate.

The hypothesis of the present study was that diabetes mellitus might affect brain metabolism. Streptozotocin (STZ)-induced diabetic rats, treated with vanadyl sulphate (V) and sodium tungstate (T) were employed to observe the aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) activities in brain homogenates. Significant increases in AST, ALT and CK activities were found in diabetic brain homogenates against controls, suggesting increments of transamination in brain and/or increases in cell membrane permeability to these enzymes. The increase in brain CK possibly expresses alterations in energy production. The decrease in CK activity caused by V and T treatment in diabetic rats suggests that both agents tend to normalize energy consumption. It is also possible that V and T-induced hypoglycemic effects cause metabolic alterations in brain.

Role of heme oxygenase/carbon monoxide pathway on the vascular response to noradrenaline in portal hypertensive rats.

1. Portal hypertension (PH), a major syndrome in cirrhosis, producing hyperdynamic splanchnic circulation and hyperaemia. In order to elucidate the contribution of heme oxygenase to the vascular hyporeactivity, we assessed the activity of heme oxygenase-1 (HO-1), measured the in vivo pressure response to noradrenaline (NA) and investigated the effects of blocking the carbon monoxide (CO) and nitric oxide (NO) pathways in a prehepatic model of PH in rats. 2. Portal hypertension was induced by partial portal vein ligation (PPVL). Noradrenaline was injected intravenously. Liver, spleen and mesentery homogenates were prepared for measurement of HO-1 activity and expression. Four groups of rats were used: (i) a sham group; (ii) a PPVL group; (iii) a sham group pretreated with Zn-protoporphyrin IX (ZnPPIX); and (iv) a PPVL group pretreated with ZnPPIX. Each group was studied before and after treatment with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). 3. For basal pressures and the pressure response to NA, inhibition of CO and NO pathways by ZnPPIX and L-NAME, respectively, produced an increase in mean arterial pressure (MAP) in sham-operated and in PH rats. Similarly, when both inhibitors were used together in either sham or PPVL rats, a greater increase in MAP was observed. 4. These results, together with the increased HO-1 activity and expression only in the PH group, have led us to suggest that the heme oxygenase/CO pathway is involved in the vascular response to NA in PH rats.

Decreased oxidative stress in prehepatic portal hypertensive rat livers following the induction of diabetes.

1. Oxidative stress (OS) is a biological entity indicated as being responsible for several pathologies, including diabetes. Diabetes can also be associated with human cirrhosis. Portal hypertension (PH), a major syndrome in cirrhosis, produces hyperdynamic splanchnic circulation and hyperaemia. The present study was designed to investigate the occurrence of OS in prehepatic PH rat livers following the induction of diabetes. 2. Five groups of rats were used: control, sham operated, chronic diabetes (induced with a single dose of streptozotocin at 60 mg/kg, i.p.), prehepatic PH and chronic diabetic plus prehepatic PH. The occurrence of OS was determined in liver homogenates by measuring hydroperoxide-initiated chemiluminescence and the activity of anti-oxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). 3. Prehepatic PH produced a significant increase in hydroperoxide-initiated chemiluminescence in the liver compared with control and sham-operated rats, whereas the liver in chronic diabetic rats showed no difference. However, chemiluminescence values decreased almost by 50% in the chronic diabetic plus prehepatic PH group. Concomitantly, the activities of the anti-oxidant enzymes in chronic diabetes, prehepatic PH and chronic diabetic plus prehepatic PH groups were decreased (P < 0.05 vs control and sham-operated groups). 4. Livers from the chronic diabetic group did not show any evidence of the occurrence of OS, whereas the prehepatic PH group showed the occurrence of OS. The association of PH and chronic diabetes resulted in a significant decrease in the occurrence of OS, which could be explained by an anti-oxidant response to an OS.

[Functional alterations in central nervous system of prehepatic portal hypertensive rats]. / Cambios funcionales en el sistema nervioso central de ratas hipertensas portales prehepaticas.

Prehepatic Portal Hypertension (PH) leads to morphologic changes in the rat Central Nervous System, including alterations of the blood brain barrier (BBB), and astrogliosis and angiogenesis in CA1 and CA4 hyppocampal fields. The present study investigates functional changes in portal hypertensive rats. Wistar Kyoto rats were used (240 g/bw) and allotted in two groups: GI (n = 8) portal hypertensive rats obtained through a regulated stenosis of the portal vein (Groszmann), and GII (n = 6), sham-operated rats. We have analyzed: BBB integrity with the Trypan Blue diffusion method (TB, Reynolds), protein concentration (PC) in Cerebrospinal Fluid (CSF) and plasma (Bradford method), electroencephalographic activity (EEG), cerebral edema expressed as brain water content (gravidimetric test), and behavior: Animex, righting reflex, pain reflex and Rotarod. TB was positive in GI in peripheral vascular areas in hippocampus, PC in CSF (ug/ml)(mean +/- SED) was GI: 40.6 +/- 6.8 and GII: 16.5 +/- 4.2 (p < 0.005), and the plasma levels were (mg/ml): GI: 108.8 +/- 7.6 and GII: 87.4 +/- 2 (NS). The EEG showed a higher power of the delta band in hypertensive rats (GI: 0.551 +/- 0.033 and GII: 0.342 +/- 0.031, p < 0.008), but water content was not different between GI and GII (water%/per/g/tissue) (GI: 79.21 +/- 0.2, GII: 78.95 +/- 0.18). These results, showing functional changes in the BBB and brain activity without behavioral alterations, suggest the development of a subclinic form of hepatic encephalopathy in our model of PH rats.

Cambios funcionales en el sistema nervioso central de ratas hipertensas portales prehepaticas. / [Functional alterations in central nervous system of prehepatic portal hypertensive rats]

Prehepatic Portal Hypertension (PH) leads to morphologic changes in the rat Central Nervous System, including alterations of the blood brain barrier (BBB), and astrogliosis and angiogenesis in CA1 and CA4 hyppocampal fields. The present study investigates functional changes in portal hypertensive rats. Wistar Kyoto rats were used (240 g/bw) and allotted in two groups: GI (n = 8) portal hypertensive rats obtained through a regulated stenosis of the portal vein (Groszmann), and GII (n = 6), sham-operated rats. We have analyzed: BBB integrity with the Trypan Blue diffusion method (TB, Reynolds), protein concentration (PC) in Cerebrospinal Fluid (CSF) and plasma (Bradford method), electroencephalographic activity (EEG), cerebral edema expressed as brain water content (gravidimetric test), and behavior: Animex, righting reflex, pain reflex and Rotarod. TB was positive in GI in peripheral vascular areas in hippocampus, PC in CSF (ug/ml)(mean +/- SED) was GI: 40.6 +/- 6.8 and GII: 16.5 +/- 4.2 (p < 0.005), and the plasma levels were (mg/ml): GI: 108.8 +/- 7.6 and GII: 87.4 +/- 2 (NS). The EEG showed a higher power of the delta band in hypertensive rats (GI: 0.551 +/- 0.033 and GII: 0.342 +/- 0.031, p < 0.008), but water content was not different between GI and GII (water

/per/g/tissue) (GI: 79.21 +/- 0.2, GII: 78.95 +/- 0.18). These results, showing functional changes in the BBB and brain activity without behavioral alterations, suggest the development of a subclinic form of hepatic encephalopathy in our model of PH rats.

[Does TNF-alpha contribute to liver disease physiopathology?]. / Contribuye el TNF-alpha a la fisiopatología de las hepatopatías?

The aim of the present paper is to establish the possible role of serum TNF in the pathophysiology of three experimental models of liver injury: paracetamol intoxication, cholestasis followed by paracetamol intoxication and cholestasis. We concluded that under our experimental conditions the serum TNF-alpha levels were not responsible for the inflammatory phenomena described in our previous paper as apoptosis.

Contribuye el TNF-alpha a la fisiopatología de las hepatopatías? / [Does TNF-alpha contribute to liver disease physiopathology?]

Theaim of the present paper is to establish the possible role of serum TNF in the pathophysiology of three experimental models of liver injury: paracetamol intoxication, cholestasis followed by paracetamol intoxication and cholestasis. We concluded that under our experimental conditions the serum TNF-alpha levels were not responsible for the inflammatory phenomena described in our previous paper as apoptosis.

Drug glucuronidation and hepatic lipid microsomal membrane profile in cholestatic rats followed paracetamol intoxication.

The uridin-diphosphoglucuronyl-transferase (UDP-GT) is a membrane-bound enzyme responsible for glucuronidation of endogenous and exogenous compounds. This work established the UDP-GT activity and its lipid membrane microenvironment in two experimental models: acute paracetamol intoxication, and cholestasis followed by acute paracetamol intoxication. Cholestasis was performed by bile duct ligation. After 7 days animals were injected with paracetamol (BDL-APAP group). Sham-operated rats were injected at day 7 with paracetamol (APAP group). Cholestatic and sham-operated rats injected with vehicle (BDL and control groups). UDP-GT activity was measured by a kinetic method for different substrates. Microsomal membrane phospholipid composition, cholesterol content and ultrastructure were determined. BDL-APAP group showed an increment in the UDP-GT activity except for chloramphenicol, morphine and paracetamol if compared to controls and to BDL group. The same increment was observed when BDL-APAP was compared to APAP except for chloramphenicol and lorazepam. Between BDL and APAP groups similar levels of activity were detected except for paracetamol. Microsomal phospholipid profile: phosphatidylcholine showed the lowest content in the BDL group, with a significant recovery in the BDL-APAP and APAP groups. Phosphatidylserine was markedly decreased in the APAP group compared to the rest and phosphatidylinositol was decreased in all the groups if compared to control values. An increment of phosphatidylethanolamine was seen in the APAP and BDL-APAP groups if compared to BDL and control values. A significant increment of microsomal cholesterol content was seen in BDL. Under these conditions, a different lipid microenvironment is produced, resulting in an increment of the enzyme activity for a variety of substrates.

[Benzodiazepines metabolism in various models of experimental liver diseases]. / Metabolismo de benzodiacepinas en distintos modelos de hepatopatías experimentales.

The aim of the present paper is to establish possible disturbances in benzodiazepines glucuronidation in two experimental models of liver injury: paracetamol acute intoxication and cholestasis followed by paracetamol acute intoxication. We concluded that, despite the alterations observed in liver microsomal lipid profile, glucuronidation remained similar to controls in paracetamol intoxicated rats. On the contrary, cholestatic animals followed by paracetamol intoxication showed an increment in the glucuronidation of the utilized substrated.

Norepinephrine uptake modifications in circumventricular organs, pons and myelencephalic areas and nuclei in prehepatic portal hypertensive rats.

Peripheral noradrenergic activity is enhanced in portal hypertension and correlates with the progression of the disease. However, little is known about the status of central norepinephrine (NE) in portal hypertension. The aim of the present work was to study the uptake of NE in several areas rich in NE in experimental prehepatic portal hypertension. The experiments were performed in vitro in several encephalic areas and nuclei, obtained according to the 'punch-out technique'. Results showed that in portal hypertensive rats NE uptake enhanced in all areas and nuclei studies (subfornical organ, organum vasculosum lamina terminalis, area postrema, locus coeruleus and nucleus tractus solitarius). The present results suggest that these encephalic areas and brainstem nuclei may be related to the development and/or maintenance of portal hypertension in this animal model.

An experimental model of liver damage and portal hypertension induced by a single dose of monocrotaline.

BACKGROUND/AIMS: Hepatic cirrhosis accompanied by portal hypertension is a common cause of death in human beings. The aim of the present study was to develop an experimental model of hepatic portal hypertension associated with liver damage. METHODOLOGY: To develop liver damage in rats, we used the toxic alkaloid monocrotaline. Two groups of male Wistar rats were used. Group 1 was injected with a single dose of monocrotaline (60 mg/kg of body weight) intraperitoneally. Group 2 was injected with an equal volume of saline solution. After 44 days, the animals underwent the following tests: splenoportography and measurement of portal pressure, hepatic serum biochemical tests, and light and electron microscopy. RESULTS: Group 1 showed a significant increase in splenic pressure, superior and inferior collateral circulation, and an increase in portal vein diameter. Serious alterations were detected in hepatic serum markers. Light microscopy showed different degrees of hepatocyte damage, varying from edema to focal necrosis. Ultrastructural changes were of membrane disruption, mitochondrial and nuclear alterations. CONCLUSIONS: The present experimental model could be useful in establishing the pathophysiological changes associated with portal hypertension due to liver damage.

Metabolismo de benzodiacepinas en distintos modelos de hepatopatías experimentales. / [Benzodiazepines metabolism in various models of experimental liver diseases]

The aim of the present paper is to establish possible disturbances in benzodiazepines glucuronidation in two experimental models of liver injury: paracetamol acute intoxication and cholestasis followed by paracetamol acute intoxication. We concluded that, despite the alterations observed in liver microsomal lipid profile, glucuronidation remained similar to controls in paracetamol intoxicated rats. On the contrary, cholestatic animals followed by paracetamol intoxication showed an increment in the glucuronidation of the utilized substrated.

Metabolismo de benzodiacepinas en distintos modelos de hepatopatías experimentales / Benzodiazepines metabolism in different models of experimental liver diseases

El objetivo de este trabajo es estabelecer posibles alteraciones en la glucuronización de benzodizcepinas en dos modelos experimentales de injuria hepática: La intoxicación aguda con paracetamol y la colestasis seguida de una intoxicación aguda con paracetamol. Por el contrario, los animales colestáticos seguidos de una intoxicación con paracetamol, mostraron un incremento en la glucuronización de los sustratos ensayados.

Metabolismo de benzodiacepinas en distintos modelos de hepatopatías experimentales / Benzodiazepines metabolism in different models of experimental liver diseases

El objetivo de este trabajo es estabelecer posibles alteraciones en la glucuronización de benzodizcepinas en dos modelos experimentales de injuria hepática: La intoxicación aguda con paracetamol y la colestasis seguida de una intoxicación aguda con paracetamol. Por el contrario, los animales colestáticos seguidos de una intoxicación con paracetamol, mostraron un incremento en la glucuronización de los sustratos ensayados. (AU)

Prostanoid production in endothelial and Kupffer liver cells from monocrotaline intoxicated rats.

UNLABELLED: A single dose of monocrotaline, a pyrrolizidine alkaloid, was injected into rats in order to produce 25 (Group I) and 45 (Group II) days later a progressive and so called delayed liver injury. The present study investigated the prostanoid production of Kupffer cells and endothelial cells separated from Monocrotaline and saline (Group III) injected rat livers. Kupffer cells: formation of 6 keto Prostaglandin F1 alpha, the major prostacycline metabolite, gradually decreased in Groups I vs II (P < 0.01) and in both Groups I and II vs Controls (P < 0.01). In addition Prostaglandin F2 alpha showed a significant increase in Groups I and II when compared to Group III, (P < 0.001), and Thromboxane B2 was present in both Groups of Monocrotaline treated animals, while it was not detectable in the control Group III. Endothelial cells: 6 keto Prostaglandin F1 alpha decreased in Groups 1 vs II. This differences was significant when compared, and compared to controls (Group III, P < 0.001). Prostaglandin E2 was detected only in Groups I and II. Prostaglandin F2 alpha and Thromboxane B2 could not be detected in any Group. Ultramicroscopy showed morphological cell damage in nonparenchymal cells in Monocrotaline intoxication in Group II, rats sacrificed 45 days after the injection, while it shows normal features in those treated animals sacrificed 25 days after the injection, as well as in control group. CONCLUSION: A single Monocrotaline injection produces, 25 and 45 days later, severe and progressive alterations in the prostanoid production in Kupffer and Endothelial cells, while ultramicroscopic alterations was only observed 45 days after the injection of Monocrotaline. A decreased production of vasodilators and the presence of vasoconstrictor prostanoids that can participate in the production of the circulatory derangements enhancing liver injury and portal hypertension were also observed.

Tyrosine hydroxilase activity in discrete brain regions from prehepatic portal hypertensive rats.

BACKGROUND/AIMS: Portal hypertension in patients and rat models are characterized by splanchnic and systemic hemodynamic alterations. Both the central and autonomic nervous systems are implicated in its pathophysiology. The aim of our research was to study the tyrosine hydroxylase activity and the rate limiting step in the biosynthesis of catecholamines in partial ligated portal hypertensive and in control rat brains. METHODOLOGY: The following seven discrete brain regions were investigated: Subfornical Organ, Organum Vasculosum Lamina Terminalis, Median Eminence, Periventricular Nucleus, Area Postrema, Locus Coeruleus and Nucleus Tractus Solitarius. RESULTS: The enzyme activity showed a significant increment in six nuclei and a decrease in Area Postrema Nucleus when portal hypertensive rats were compared to controls. CONCLUSIONS: These results suggest the participation of some discrete brain regions in the mechanism of hepatic portal hypertension under the present rat model.

Norepinephrine uptake is enhanced in discrete telencephalic and diencephalic areas and nuclei in prehepatic portal hypertensive rats.

Several evidences support the hypothesis that central catecholamines may play a significant role in the production and/or maintenance of different alterations that characterize portal hypertension. The aim of the present work was to study the possible modifications in norepinephrine (NE) metabolism in several telencephalic and diencephalic areas rich in NE in experimental prehepatic portal hypertension. NE uptake was studied as an index of NE metabolism. The experiments were carried out in vitro in encephalic areas and nuclei, obtained according to the punch-out technique. Results indicated that portal hypertensive rats showed an enhancement of NE uptake in olfactory bulb (OB), preoptic area (PA), and supraoptic, periventricular, paraventricular, and arcuate nuclei (SON, PeVN, PaVN, and AN, respectively) compared to sham-operated rats. However, no modifications on NE uptake was observed in the median eminence (ME). Present results suggest that the changes observed in central NE uptake may be related to the development and/or maintenance of the portal hypertensive state.