Biomedical subjects
Publications and source records attributed to Alberto Machado.
The selective loss of a particular subset of neurons is a common feature of neurodegenerative disorders. A failure in respiratory chain complex activities in mitochondria seems to be a causative factor. The aim of this review is to describe the most important toxins affecting the mitochondrial function, which could be involved in the incidence of some of these diseases: MPTP, rotenone and 3-nitropropionic (3-NPA).
Elongation Factor-2 (eEF-2) is the protein that catalyzes the translocation of the ribosome through mRNA. Not all oxidants affect eEF-2, which is extremely sensitive to oxidative stress caused mainly by lipid peroxidant compounds such as cumene hydroperoxide and t-butyl hydroperoxide. Lipid peroxides constitute a potential hazard to living organisms because of their direct reactivity with a variety of biomolecules and the ability to decompose into free radicals and reactive aldehydes. In this "in vitro" study, we show the effect of three of these aldehydes on the levels of hepatic eEF-2. The results suggest that the toxicity associated with prooxidant-mediated hepatic lipid peroxidation on protein synthesis can originate from the interaction of the aldehydic end products of lipid peroxidation with eEF-2.
The objective of the work was to study the relationship between the oxidative state of the mother and the newborn at the moment of birth. We measured oxidative stress markers (carbonyl groups, lipid peroxides and total antioxidant capacity (TAC)) and found a good correlation between the oxidative state of the normal mother and the neonate, since a high mother oxidative stress corresponds to an even higher oxidative stress of the newborn in umbilical cord blood. We also found that smoking mothers and their newborns had a higher concentration of the carbonyl group, lipid peroxides and less TAC. Newborns from these mothers weighed significantly less than others at birth. These data suggest a need for interest in monitoring the oxidative state of mothers during the pregnancy period, especially taking into account that the oxidative level could be involved in later risks of metabolic diseases for both mother and newborn.
To evaluate the potential role of endogenous zinc in the pathophysiology of epilepsy, we injected kainic acid into the medial septum, which evokes seizure activity and delayed hippocampal degeneration. Different approaches were used. In the hippocampus, we found a movement of zinc from the synaptic compartment to CA1 pyramidal neurons and astrocytes after kainate. The same was true in the amygdala. We found that in those areas showing intense zinc bleaching there was also a loss of reactive astrocytes, which supports the view that release of synaptic zinc induces astrocytic cell death. We have also tested whether the kainate-induced zinc movement from the synaptic compartment to neuronal or glial cells alters the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, trkB. There was a prominent loss of expression of trkB mRNA in areas that coincided precisely with those displaying astrocyte loss and zinc bleaching. In the amygdala, these events were accompanied by a high upregulation of BDNF mRNA. To demonstrate further a role of synaptic zinc in hippocampal pathology, we used two different approaches. We first injected different doses of zinc chloride in the CA1 area. At lower doses (0.1-10 nmol), zinc chloride selectively induced apoptosis in CA1 pyramidal neurons and dentate granular neurons. In a second approach, we found that hippocampal zinc chelation was effective in protecting CA1 pyramidal neurons against kainate-induced cell death.
We have tested the effect of deprenyl on the neurotoxicity induced by the injection of quinolinic acid within the striatum. Deprenyl was unable to prevent these quinolinic acid-induced damages, but enhanced the loss of several gamma-aminobutyric acid (GABA) positive subpopulations, the loss of the astroglial population and the activation of microglia produced by quinolinic acid. These effects are produced by deprenyl potentiation of dopamine actions since dopamine depletion produced by previous injection of the dopaminergic toxin 6-hydroxydopamine within the medial forebrain bundle overcomes deprenyl effects and the involvement of dopamine in the quinolinic acid-induced toxicity in striatum. In these conditions, quinolinic acid toxic action in striatum is significantly lower and similar in the animals treated with or without deprenyl. All these data justify why deprenyl worsen some pathological signals of disorders involving excitotoxicity. This also may be involved in other secondary effects described for deprenyl.
Oxidative stress seems to play an important role in the pathophysiology of essential hypertension. Due to its antioxidant features, we studied the protective action of folic acid in hypertensive patients, their food supplemented for 2 weeks with this vitamin. Several oxidative stress parameters were measured in the serum of these patients. The results show that folic acid supplementation improves levels of oxidative stress markers in individuals with hypertension, overall in those patients whose initial parameter values were highest.
We have studied the regulation of BDNF mRNA expression in the corticostriatal and nigrostriatal systems following neurotoxin ablation of striatal targets induced by quinolinic acid (QA) or 2S:2'R:3'R:-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV) injections. Striatal lesions were verified by quantifying the loss of glutamic acid decarboxylase (GAD) mRNA expression. Levels of BDNF mRNA were analyzed at 6, 12, and 24 h postlesion. Both lesions paradigms highly induced BDNF mRNA in the ipsilateral cerebral cortex at 6 and 12 h postlesion to drop to control levels at 24 h postlesion. These inductions were mostly restricted to cortical layers II/III and V and ipsilateral insular and piriform cortices, which provide the main cortical inputs to the striatum. Analysis of neuronal activation on these areas demonstrated high levels of cFos mRNA in response to the excitotoxic striatal lesions. Dual labeling of cFos and BDNF mRNAs demonstrated a positive correlation between cortical neuronal activation and expression of BDNF mRNA. Consequently, expression of BDNF in cortical areas projecting to striatum is dependent on both target integrity and neuronal activity. Regulation of BNDF mRNA in substantia nigra, the second major source of BDNF to striatal cells, highly differed from that seen in cerebral cortex. Analysis of cellular expression alone or in combination of BDNF, cFos, tyrosine hydroxylase and GAD mRNAs demonstrated that expression of BDNF in substantia nigra is dependent on target integrity and independent of neuronal activity. In addition, we have studied regulatory mechanisms of BDNF mRNA in the subthalamic nucleus.
Intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation, induces degeneration of dopaminergic neurons, along with an inflammatory process that features activation of microglial cells and loss of astrocytes. To test the involvement of dopamine (DA) in this degeneration induced by LPS, we treated albino Wistar rats with different concentrations of alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase (TH) activity. Results showed that alpha-MPT prevented LPS-induced loss of TH immunostaining and expression of mRNA for TH and DA transporter; it also prevented substantial activation of microglial cells. Loss of the astroglial population, a marker of damage in our model, was also prevented. This protective effect resulted from inhibition of TH and the consequent decrease in DA concentration, because treatment with L-DOPA/benserazide, which bypasses TH inhibition induced by alpha-MPT, reversed the protective effect produced by this drug. These results point out the important contribution of DA to the vulnerability and degeneration of dopaminergic neurons of the substantia nigra. Knowledge about the involvement of DA in this process may lead to the possibility of new protection strategies against this important degenerative process.
The oxidant status of an individual is assessed by determining a group of markers in noninvasive samples. One limitation when measuring these biomarkers is that they do not give information about tissue localization of oxidative stress. The present study was undertaken to establish whether the serum oxidative stress biomarkers are indicative of oxidative stress in tissues of an individual. To accomplish this, we determined a few generic markers of oxidation in serum and tissues of six groups of rats treated experimentally, to modulate their oxidative stress status. The correlation between serum and tissue levels was calculated for each marker. Also, for each tissue, the correlation between the values of these oxidative stress biomarkers was analysed. Our results show that only lipid peroxides in serum could be useful to predict the oxidative stress in tissues. No correlation was found between any of the oxidative stress markers in serum.
We have evaluated the potential neuroprotectant activity of minocycline in an animal model of Parkinson's disease induced by intranigral injection of lipopolysaccharide. Minocycline treatment was very effective in protecting number of nigral dopaminergic neurons and loss of reactive astrocytes at 7 days postlesion. Evaluation of microglia revealed that minocycline treatment highly prevented the lipopolysaccharide-induced activation of reactive microglia as visualized by OX-42 and OX-6 immunohistochemistry. Short-term RT-PCR analysis demonstrated that minocycline partially prevented the lipopolysaccharide-induced increases of mRNA levels for interleukin-1alpha and tumor necrosis factor-alpha. In addition, lipopolysaccharide highly induced protein nitration as seen by 3-nitrotyrosine immunoreactivity in the ventral mesencephalon. Minocycline treatment strongly diminished the extent of 3-nitrotyrosine immunoreactivity. We also found a direct correlation between location of IgG immunoreactivity-a marker of blood-brain barrier disruption-and neurodegenerative processes including death of nigral dopaminergic cells and reactive astrocytes. There was also a precise spatial correlation between disruption of blood-brain barrier and 3-nitrotyrosine immunoreactivity. We discuss potential involvement of lipopolysaccharide-induced formation of peroxynitrites and cytokines in the pathological events in substantia nigra in response to inflammation. If inflammation is proved to be involved in the ethiopathology of Parkinson's disease, our data support the use of minocycline in parkinsonian patients.
Aquaporin-4 (AQP4) is the most abundant aquaporin in the brain and it is widely accepted that this AQP is solely expressed by astrocytes and ependymal cells. AQP4 is particularly enriched in plasma membranes of ependymal cells and astrocyte membrane domains facing blood vessels and pia. AQP4 has gained much attraction due to its involvement in the physiopathology of brain edema, a major cause of death in humans. Consequently, it is of paramount importance to ascertain the phenotypic nature of AQP4 mRNA-expressing cells in the CNS before attempting future clinical studies aimed at minimizing the development of brain edema. We have used intranigral injections of lipopolysaccharide (LPS), a potent immunostimulant that causes disruption of the blood brain barrier, vasogenic edema, loss of reactive astrocytes and activation of microglial cells. These LPS-induced features are ideal for testing the possibility that reactive microglial cells express AQP4 in the adult brain. We have studied AQP4 at the mRNA and protein level. We provide strong evidence that reactive microglial cells highly express AQP4 mRNA and protein in response to LPS injections.
Aquaporins (AQPs) are water channels that mediate the efficient movement of water across the membrane. Among different AQPs, AQP4 is the predominant water channel in the brain and is thought to play a significant role in the physiopathology of brain edema. Brain edema is a major clinical problem since it largely accounts for the cause of death in patients suffering from traumatic brain injury or cerebrovascular accidents. In this review, we have tried to summarize comprehensive information about the pathological events conducting to brain edema. In addition, we also have summarized active investigation in the field of AQPs and cerebral edema to find plausible explanations and to deduce potential future directions for the treatment of this clinical condition.
We have evaluated the ability of the injured nigrostriatal dopaminergic system to produce highly reactive hydroxyl radicals ((*)OH) by the electrochemical detection of salicylate hydroxylation. Unilateral transection of the medial forebrain bundle transiently increased the formation of (*)OH in substantia nigra (SN) but not in striatum during the first 48 h postlesion, when most relevant changes in terms of oxidatively modified proteins take place. Short-term adaptive axotomy-induced changes in substantia nigra included downregulation of nigral tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA expression and more intense TH immunoreactivity. Maintained inhibition of monoamine oxidase activity with deprenyl totally prevented the axotomy-induced formation of (*)OH, thus demonstrating the dopaminergic nature of these radicals. In contrast, deprenyl treatment, which is associated with a diminution in free radical production, failed to delay the onset of dopaminergic degeneration. This observation highlights the importance of being extremely cautious when analyzing parameters of oxidative stress and extrapolating them as a primary cause of cell death in the context of neurodegeneration. Long-term adaptive changes included a dramatic downregulation of DAT mRNA expression along with a moderate decrease in TH mRNA levels in SN. We anticipate a key regulatory role of the DAT to maximally optimize dopaminergic transmission in the synaptic cleft under conditions of degeneration.
Different pre-treatment methods have been compared for two-dimensional mapping of individual rat cerebrospinal fluid samples based on acetone, trichloroacetic acid/acetone and methanol/acetone precipitation of proteins. Acetone precipitation following incubation with DTT gave the highest protein recovery (72%) and the largest number of protein spots (92 +/- 4) as well as minimizing the time taken.
Seven days after the injection of different concentrations of thrombin into the nigrostriatal pathway, a strong macrophage/microglial reaction was observed in the substantia nigra (SN), indicated by immunostaining, using OX-42 and OX-6 antibodies, and by the induction of iNOS, IL-1alpha, Il-1beta and TNF-alpha. Moreover, selective damage to dopaminergic neurones was produced after thrombin injection, evidenced by loss of tyrosine hydroxylase immunostaining and tyrosine hydroxylase mRNA-expressing cell bodies, and the unaltered transcription of glutamic acid decarboxylase mRNA in the SN and striatum. These thrombin effects could be produced by its ability to induce the activation of microglia described in in vitro studies, and are in agreement with the effects described for other proinflammatory compounds. Thrombin effects are produced by its biological activity since they almost disappeared when thrombin was heat-inactivated or injected along with its inhibitor alpha-NAPAP. Thrombin is a multi-functional serine protease rapidly produced from prothrombin at the sites of tissue injury, and also upon breakdown of the blood-brain barrier, which strongly suggests it could easily enter into the CNS. These results could have special importance in some degenerative processes of the nigrostriatal dopaminergic system.
We have analyzed the regulation of brain-derived neurotrophic factor (BDNF) mRNA expression in the nigrostriatal system following neurotoxin ablation of striatal targets by means of kainate (KA) or quinolinic acid (QA) injections. Loss of nigral target cells in the striatum was accompanied by significant induction of BDNF mRNA levels in the ipsilateral substantia nigra (SN) at 12 and 24 h post lesion. Dual tyrosine hydroxylase (TH) and BDNF mRNA in situ hybridization (ISH) confirmed the dopaminergic nature of the BDNF mRNA expressing cells. Analysis of neuronal activity in terms of cFos mRNA expression demonstrated intense induction of this marker in the ipsilateral SN pars reticulata (SNPR), but not in SN pars compacta. Dual glutamic acid decarboxylase (GAD) and cFos mRNA ISH confirmed this view. Colchicine injections into the medial forebrain bundle to specifically disrupt neuronal trafficking between SN and striatum induced BDNF mRNA levels in the ipsilateral SNPC, thus demonstrating that nigral expression of BDNF mRNA is dependent of striatal target tissue. In addition, we found significant elevations of BDNF in the subthalamic nucleus following striatal excitotoxic lesion, which may bring novel roles of BDNF in the basal ganglia complex.
We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.
1-Methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), an endogenous monoamine, which prevents the neurotoxic effect of 1-methyl-4-phenylpyridinium ion (MPP(+)) and other endogenous neurotoxins, has been described as being enzymatically formed in the brain by the 1-MeTIQ synthesizing enzyme (1-MeTIQse). In this paper, we report the brain's regional distribution of this enzyme in 3- and 24-month-old rats. The results show that the activity is spread throughout the brain, the highest activity being in the dopaminergic areas (striatum and substantia nigra) and in the cortex. During aging there was a 1-MeTIQse activity reduction ( approximately 50%) in the areas implicated in the ethyology of Parkinson disease (substantia nigra, striatum) and in the cerebral cortex.