Juan Carlos Perazzo

Hippocampal mitochondrial dysfunction with decreased mtNOS activity in prehepatic portal hypertensive rats.

Portal hypertension is a major complication of human cirrhosis that frequently leads to central nervous system dysfunction. In our study, rats with prehepatic portal hypertension developed hippocampal mitochondrial dysfunction as indicated by decreased respiratory rates, respiratory control and mitochondrial nitric oxide synthase (mtNOS) activity in mitochondria isolated from the whole hippocampus. Succinate-dependent respiratory rates decreased by 29% in controlled state 4 and by 42% in active state 3, and respiratory control diminished by 20%. Portal hypertensive rats showed a decreased mtNOS activity of 46%. Hippocampal mitochondrial dysfunction was associated with ultrastructural damage in the mitochondria of hippocampal astrocytes and endothelial cells. Swollen mitochondria, loss of cristae and rupture of outer and inner membrane was observed in astrocytes and endothelial cells of the blood-brain barrier in parallel with the ammonia gradient. It is concluded that the moderate increase in plasma ammonia that followed portal hypertension was the potential primary cause of the observed alterations.

Hepatic encephalopathy: An approach to its multiple pathophysiological features

Hepatic encephalopathy (HE) is a neuropsychiatric complex syndrome, ranging from subtle behavioral abnormalities to deep coma and death. Hepatic encephalopathy emerges as the major complication of acute or chronic liver failure. Multiplicity of factors are involved in its pathophysiology, such as central and neuromuscular neurotransmission disorder, alterations in sleep patterns and cognition, changes in energy metabolism leading to cell injury, an oxidative/nitrosative state and a neuroinflammatory condition. Moreover, in acute HE, a condition of imminent threat of death is present due to a deleterious astrocyte swelling. In chronic HE, changes in calcium signaling, mitochondrial membrane potential and long term potential expression, N-methyl-D-aspartate-cGMP and peripheral benzodiazepine receptors alterations, and changes in the mRNA and protein expression and redistribution in the cerebral blood flow can be observed. The main molecule indicated as responsible for all these changes in HE is ammonia. There is no doubt that ammonia, a neurotoxic molecule, triggers or at least facilitates most of these changes. Ammonia plasma levels are increased two- to three-fold in patients with mild to moderate cirrhotic HE and up to ten-fold in patients with acute liver failure. Hepatic and inter-organ trafficking of ammonia and its metabolite, glutamine (GLN), lead to hyperammonemic conditions. Removal of hepatic ammonia is a differentiated work that includes the hepatocyte, through the urea cycle, converting ammonia into GLN via glutamine synthetase. Under pathological conditions, such as liver damage or liver blood by-pass, the ammonia plasma level starts to rise and the risk of HE developing is high. Knowledge of the pathophysiology of HE is rapidly expanding and identification of focally localized triggers has led the development of new possibilities for HE to be considered. This editorial will focus on issues where, to the best of our knowledge, more research is needed in order to clarify, at least partially, controversial topics.

Atrial natriuretic factor effects on norepinephrine uptake in discrete telencephalic and diencephalic nuclei of the rat

Atrial natriuretic factor (ANF) effects on norepinephrine (NE) uptake in olfactory bulb, preoptic, periventricular, supraoptic, paraventricular and arcuate nuclei and median eminence of the rat were studied. Experiments were carried out in vitro on nuclei punched out according to the Palkovitz and Brownstein technique. Results showed that 100 nM ANF enhanced NE uptake in all nuclei studied. These data suggest that ANF may be indirectly involved in the regulation of neuroendocrine processes, behavioral arousal, sexual behavior, water and electrolyte balance, arterial blood pressure, etc., through the modulation of central noradrenergic neurotransmission.

Atrial natriuretic factor enhances norepinephrine uptake in circumventricular organs, locus coeruleus and nucleus tractus solitarii of the rat.

The effects of atrial natriuretic factor (ANF) on norepinephrine (NE) uptake in circumventricular organs (organum vasculosum lamina terminalis, organum subfornicale and area postrema), locus coeruleus and nucleus tractus solitarii were studied in the rat. Experiments were carried out in vitro using nuclei obtained according to the punch-out technique. Results showed that 100 nM ANF enhanced NE uptake in all nuclei studied. These results suggest that ANF may be indirectly related to the control of cardiocirculatory functions, hydroelectrolyte balance, neuroendocrine secretions, nutrient and metabolic homeostasis, through the modulation of noradrenergic neurotransmission at the neuronal presynaptic level.

Oxidative stress and hippocampus in a low-grade hepatic encephalopathy model: protective effects of curcumin.

Aim:  The present study was performed on prehepatic portal hypertensive rats, a model of low‐grade hepatic encephalopathy, designed to evaluate whether oxidative stress was a possible pathway implicated in hippocampal damage and if so, the effect of an anti‐oxidant to prevent it.

Protection of ischaemic-reperfused rat heart by dimethylamiloride is associated with inhibition of mitochondrial permeability transition.

1 The aim of the present study was to assess whether protection afforded by the Na+/H+ exchanger blocker dimethylamiloride (DMA) is associated with inhibition of mitochondrial permeability transition (MPT). The effects of DMA were compared with those of cyclosporine (Cs) A, an inhibitor of MPT. 2 Rat hearts were Langendorff perfused with Krebs’–bicarbonate medium containing 10 mmol/L glucose and were subjected to 25 min no‐flow global ischaemia and 30 min reperfusion in the presence or absence of 10 µmol/L DMA or 0.2 µmol/L CsA. Cell viability was measured using tetrazolium stain. The MPT was determined by loading hearts with 2‐deoxy‐[3H]‐glucose (2DG), which enters mitochondria only during MPT. Total heart 2DG content as an estimation of the extent of tissue damage was also measured. To assess whether DMA has any direct effect on glycolysis, a cell‐free heart extract containing all the glycolytic enzymes was used. 3 Dimethylamiloride improved functional recovery (rate–pressure product) from 24 ± 7 to 68 ± 11% (P < 0.01) at reperfusion end, attenuated the increase in left ventricular end‐diastolic pressure (from 29 ± 7 to 6 ± 3% 10 min after reperfusion onset; P < 0.01), improved cell viability (from 21.2 ± 6.6 to 69.6 ± 7.1% at reperfusion end; P < 0.05) and lessened lactate accumulation at the end of ischaemia (119 ± 15 vs 163 ± 14 µmol/g dry weight; P < 0.05). Dimethylamiloride limited MPT : 2DG mitochondrial entrapment, being 33.1 ± 14.2 and 96.3 ± 14.0 at reperfusion end in the treated and control hearts, respectively (P < 0.05), and concomitantly raised total 2DG content (51.3 ± 4.4 vs 86.8 ± 1.7 × 103 d.p.m./g wet weight in control and treated groups, respectively; P < 0.05). Cyclosporine A improved functional recovery and attenuated the amplitude of ventricular diastolic pressure in ischaemic–reperfused hearts. It also reduced mitochondrial entrapment (67.3 ± 7.7%; P < 0.05 vs control) and increased total cell 2DG content (162.3 ± 1.3 × 103 d.p.m./g wet weight; P < 0.01 vs control) at the end of reperfusion. Dimethylamiloride did not affect glucose consumption and lactate production in the cell‐free heart extract. 4 In conclusion, DMA protects against the noxious effects of ischaemia–reperfusion and inhibits MPT, coinciding with present and previous findings concerning the effects of CsA. Dimethylamiloride also diminished lactate accumulation, although it did not exhibit any direct effect on glycolysis. These data suggest that blockade of Na+/H+ exchange by DMA attenuates the extent of MPT in ischaemic–reperfused rat heart.

Reyess Syndrome, Encephalopathy, Hyperammonemia and Acetyl Salicylic Acid Ingestion in a City Hospital of Buenos Aires, Argentina

Twelve cases of Reyes syndrome are presented with different degrees of encephalopathy, hyperammonemia and hypoglycemia; associated to acetyl salicylic acid (ASA) ingestion. The aim of the present retrospective study was to describe our experience in selected patients with Reyes syndrome associated to the ASA ingestion and to underline the influence of hyperammonemia on Reyes encephalopathy. All the cases presented moderate hyperbilirubinemia, elevated alanine aminotransferase, aspartate aminotransferase with an average of 302±205 UI/L and 285±149 UI/L respectively. Arterial blood ammonia averaged 172.4±71.3 μmol/L and glycaemia averaged 35.2±17.0 mg/dl. A high mortality was found in our series (41.7%). Considering that encephalopathy is the leading syndrome in these cases, the influence of ammonia on brain tissue was described. Glutamate is an excitotoxic neurotransmitter, capable to produce neuron and astrocyte damage and apoptosis. The presence of ASA could cause the onset of the mitochondrial permeability transition and the mitochondrial swelling in the astrocyte, leading to hyperammonemia. In Reyes syndrome, hyperammonemia and perhaps the increase of glutamate are the leading factors in the mechanism of brain damage and encephalopathy. Aspirin must be carefully administrated and controlled by professionals. Furthermore, parents must be informed about the risks in the use of this drug in children.

Changes in CNS cells in Hyperammonemic portal hypertensive rats

Rats with pre‐hepatic portal hypertension because of partial portal vein ligation develop minimal hepatic encephalopathy (MHE) with hyperammonemia, impaired blood–brain barrier, mild brain edema, and severe mitochondrial changes in the hippocampus. The aim of this study was to evaluate changes of different neural cells in the cerebral cortex and the hippocampus. Animals were divided into two groups, MHE and sham. Astrocytes were studied by immunostaining with glial fibrillary acidic protein and S100β protein; neurons were immunostained with neuronal nuclear marker, microtubule associated protein‐2, and NF‐200 and capillaries with Nestin. The hypoxia‐inducible factor 1α (HIF‐1α) and its downstream proteins, P‐glycoprotein (P‐gp) and erythropoietin receptor (Epo‐R), were also evaluated. Astrocytes were increased in area and number only in the hippocampus, while S100β increased in both brain areas in MHE animals. Microtubule associated protein‐2 and NF‐200 immunoreactivities (‐ir) were significantly reduced in both areas. Hippocampal Nestin‐ir was increased in MHE animals. These cellular changes were similar to those described in ischemic conditions, thus HIF‐1α, P‐gp, and Epo‐R were also evaluated. A high expression of HIF‐1α in cortical neurons was observed in the MHE group. It is likely that this hypoxia‐like state is triggered via ammonia occupying the binding domain of HIF‐1α and thereby preventing its degradation and inducing its stabilization, leading to the over‐expression of P‐gp and the Epo‐R.

Influence of fasting on the effects of diazoxide in the ischemic-reperfused rat heart

This investigation aimed to asses whether the mitochondrial ATP-sensitive potassium channel opener diazoxide could reproduce the protection conferred by ischemic preconditioning and to ascertain whether its effects are associated with changes in glycogen breakdown and glycolytic activity. Hearts of fed and 24-h fasted rats were perfused with 10 mM glucose containing medium and exposed to 25 min no-flow ischemia plus 30 min reperfusion. Diazoxide (10 μM) perfusion was begun 10 min before ischemia and continued throughout the experiment. Fasting accelerated reperfusion recovery of contraction, reduced the post-ischemic contracture and decreased lactate accumulation during ischemia but had no effects on glycogen levels and cellular viability. Diazoxide, did not affect glycogen catabolism but improved reperfusion recovery of contraction. Furthermore, diazoxide reduced ischemic lactate accumulation and contracture amplitude only in the fed group whereas it improved cell viability in the fed and fasted groups. These data indicate that: 1) reduced lactate production which may attenuate myocyte acidification might explain, at least in part, the beneficial effects of diazoxide on mechanical function, although data obtained with the fasted rat hearts indicate that other mechanisms must be involved as well; 2) the reduction of lactate production occurring in the fed group, does not seem to be related to glycogenolysis; and 3) since diazoxide improved cell viability in the fasted rat group where it did not reduce glycolytic activity, other mechanisms may be responsible for this cytoprotective effect.ResumenSe estudia en este trabajo si el diazóxido, que activa los canales mitocondriales de potasio sensibles al ATP, reproduce los efectos del precondicionamiento isquémico y si tales efectos se asocian con cambios en la glucogenolisis y la actividad glicolítica. Corazones perfundidos de ratas alimentadas y tras 24 h de ayuno se exponían a 25 min de isquemia con reperfusión de 30 min. La perfusión con diazóxido (10 μM) comenzaba 10 min antes de la isquemia y continuaba durante todo el experimento. El ayuno acelera la recuperación de la contracción al reperfundir, reduce la contractura post-isquémica y disminuye la acumulación isquémica de lactato, pero no tiene efectos sobre los niveles de glucógeno y la viabilidad celular. El diazóxido no afecta a la glucogenolisis, pero mejora la recuperación post-isquémica de la contracción. Además, el diazóxido reduce la acumulación isquémica de lactato y la amplitud de la contractura sólo en el grupo alimentado, pero mejora la viabilidad celular en ambos grupos. Los resultados indican que: 1) la reducción de la producción de lactato, que atenuaría la acidificación del miocito, explicaría, al menos en parte, los efectos beneficiosos del diazóxido sobre la función mecánica, aunque los resultados de las ratas en ayunas indican la implicación de otros mecanismos; 2) la reducción de la producción de lactato obtenida en el grupo alimentado no parece estar relacionada con la glucogenolisis; y 3) como el diazóxido mejora la viabilidad celular en los corazones de ratas en ayunas en los cuales no reduce la glicólisis, otros mecanismos serían responsables del efecto citoprotector

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

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 a, the major prostacyclin metabolite, gradually decreased in Groups I vs II (P50.01) and in both Groups I and II vs Controls (P50.01). In addition Prostaglandin F2 a showed a significant increase in Groups I and II when compared to Group III, (P50.001), and Thromboxane B2 was present in both Groups of Monocrotaline treated animals, while it was not detectable in the control Group III. Endothelial cells:6ketoProstaglandin F1 a decreased in Groups I vs II. This differences was significant when compared, and compared to controls (Group III, P50.001). Prostaglandin E2 was detected only in Groups I and II. Prostaglandin F2 a 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 sacrified 45 days after the injection, while it shows normal features in those treated animals sacrified 25 days after the injection, as well as in control group. Conclusion: Asingle 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.