Sandra Orozco-Suárez

Neuronal differentiation of chromaffin cells in vitro, induced by extremely low frequency magnetic fields or nerve growth factor: a histological and ultrastructural comparative study.

The application of nerve growth factor (NGF) to primary adrenal medulla chromaffin cell cultures induces phenotypic changes characterized mainly by the presence of neurites. A similar effect has been seen when these cells are stimulated by extremely low frequency magnetic fields (ELFMF). In this study, newborn rat chromaffin cells were cultured and subjected to NGF or ELFMF in order to compare their histological and ultrastructural characteristics. Cells cultured in the presence of NGF developed cytoplasmic projections and their distal ends showed growth cones as well as filopodia. With scanning and transmission electron microscopy, an increased submembranous electron density was observed in the nuclei of cells as well as irregular, wavy neuritic projections with a moderate number of varicosities, as well as the prevalence of intermediate filaments among the cytoskeleton components. Cells stimulated with ELFMF presented straighter neuritic extensions with a greater number of varicosities. With the transmission electron microscope, numerous neurotubules were observed, both in the cell soma and in their neuritic extensions. In both groups, growth cones were clearly identified by their ultrastructural characteristics. The differences seen in the cytoskeleton of cells stimulated with NGF or ELFMF suggest differential stimulation mechanisms possibly determining the biochemical, electrophysiological, and morphological characteristics in both types of cell cultures. J. Neurosci. Res. 53:569–582, 1998.

Antioxidant, antiinflammatory and antiapoptotic effects of dapsone in a model of brain ischemia/reperfusion in rats

Although dapsone (4,4′‐diaminodiphenylsulfone) has been described as a neuroprotective agent in occlusive focal ischemia in rats, its mechanism of action is still unknown. To explore this mechanism, oxidative, inflammatory and apoptotic processes were evaluated in the striatum of adult rats using a model of ischemia‐reperfusion (I/R), either with or without dapsone treatment. Male Wistar rats were submitted to transient middle cerebral artery occlusion for 2 hr, followed by reperfusion. Rats were dosed either with dapsone (12.5 mg/kg i.p.) or vehicle 30 min before or 30 min after the ischemia onset. Lipid peroxidation (LP) and nitrotyrosine contents were measured 22 hr after reperfusion, and myeloperoxidase activity was evaluated 46 hr after I/R. Different markers for apoptosis and necrosis were also evaluated both at 24 and 72 hr after I/R experimental procedure. LP increased by 37% in ischemic animals vs controls, and this effect was reversed by dapsone treatments. A similar effect was observed regarding nitrotyrosine striatal contents. Myeloperoxidase activity, a marker of inflammatory response, increased 3.7‐fold in ischemic animals vs. control rats, and dapsone treatment antagonized that effect. Although apoptosis was increased by the effect of ischemia at both evaluation times, dapsone antagonized that effect only at 72 hr after surgery. Dapsone antagonized all of the I/R end points measured, showing a remarkable ability to decrease markers of damage through antioxidant, antiinflammatory, and anti‐apoptotic effects.

Effects of high frequency electrical stimulation and R-verapamil on seizure susceptibility and glutamate and GABA release in a model of phenytoin-resistant seizures

The present study was focused to characterize the effects of intrahippocampal application of R-verapamil, a P-glycoprotein blocker, and High Frequency Electrical Stimulation (HFS) at 130 Hz, on seizure susceptibility and extracellular concentrations of glutamate and γ-aminobutyric acid (GABA) in hippocampus of kindled rats with drug-resistant seizures. Fully kindled rats classified in responsive and non-responsive to phenytoin were used for this purpose. In contrast with responsive animals, non-responsive rats showed lower afterdischarge threshold (ADT) values in pre-kindling conditions and required less number of kindling trials to achieve the kindled state. Once the animals attained the kindled state, both epileptic groups presented high glutamate and low GABA interictal release, effect more evident in non-responsive rats. In hippocampus of responsive animals, GABA levels demonstrated two increases at 120 and 240 min after the ictal event, a situation no detected for non-responsive rats. Kindled animals receiving hippocampal HFS showed augmented ADT, an effect associated with enhanced GABA release in responsive rats. Intrahippocampal perfusion of R-verapamil (5 mM) decreased the seizure susceptibility (high ADT values), enhanced the interictal GABA release and the postictal levels of glutamate and GABA in responsive and non-responsive rats. It is conclude that alterations of glutamate and GABA release in the epileptic hippocampus of non-responsive animals resemble those found in hippocampus of patients with refractory TLE. In addition, intrahippocampal application of HFS and R-verapamil modifies the amino acid release and reduces the seizure susceptibility of both, responsive and non-responsive rats.

Temporal lobe epilepsy causes selective changes in mu opioid and nociceptin receptor binding and functional coupling to G-proteins in human temporal neocortex.

There is no information concerning signal transduction mechanisms downstream of the opioid/nociceptin receptors in the human epileptic brain. The aim of this work was to evaluate the level of G-proteins activation mediated by DAMGO (a mu receptor selective peptide) and nociceptin, and the binding to mu and nociceptin (NOP) receptors and adenylyl cyclase (AC) in neocortex of patients with pharmacoresistant temporal lobe epilepsy. Patients with temporal lobe epilepsy associated with mesial sclerosis (MTLE) or secondary to tumor or vascular lesion showed enhanced [3H]DAMGO and [3H]forskolin binding, lower DAMGO-stimulated [35S]GTPgammaS binding and no significant changes in nociceptin-stimulated G-protein. [3H]Nociceptin binding was lower in patients with MTLE. Age of seizure onset correlated positively with [3H]DAMGO binding and DAMGO-stimulated [35S]GTPgammaS binding, whereas epilepsy duration correlated negatively with [3H]DAMGO and [3H]nociceptin binding, and positively with [3H]forskolin binding. In conclusion, our present data obtained from neocortex of epileptic patients provide strong evidence that a) temporal lobe epilepsy is associated with alterations in mu opioid and NOP receptor binding and signal transduction mechanisms downstream of these receptors, and b) clinical aspects may play an important role on these receptor changes.

Early metabolic reactivation versus antioxidant therapy after a traumatic spinal cord injury in adult rats.

Disability after traumatic spinal cord injury (TSCI) results from physical trauma and from “secondary mechanisms of injury” such as low metabolic energy levels, oxidative damage and lipid peroxidation. In order to prove if early metabolic reactivation is a better therapeutic option than antioxidant therapy in the acute phase of TSCI, spinal cord contusions were performed in adult rats using a well‐characterized weight drop technique at thoracic 9 level. After TSCI, pyrophosphate of thiamine or non‐degradable cocarboxylase (NDC) enzyme was used to maintain energy levels, antioxidants such as superoxide dismutase and catalase (ANT) were used to decrease oxidative damage and methylprednisolone (MP), which has both therapeutic properties, was used as a control. Rats were divided into one sham group and six with TSCI; one of them received no treatment, and the rest were treated with NDC, MP, NDC + MP, NDC + ANT or ANT. The ANT group decreased lactate and creatine phosphokinase levels and increased the amount of preserved tissue (morphometric analysis) as well as functional recovery (Basso, Beattie and Bresnahan or BBB motor scale). In contrast, NDC treatment increased lipid peroxidation, measured through thiobarbituric acid reactive substances (TBARS) levels, as well as spinal cord tissue destruction and functional deficit. Early metabolic reactivation after a TSCI may be deleterious, while natural early metabolic inhibition may not be a “secondary mechanism of injury” but a “secondary neuroprotective response”. While increased antioxidant defence after a TSCI may currently be an ideal therapeutic strategy, the usefulness of metabolic reactivation should be tested in the sub‐acute or chronic phases of TSCI and new strategies must continue to be tested for the early ones.

Anticonvulsive effect of vitamin C on pentylenetetrazol-induced seizures in immature rats ☆

Vitamin C helps to prevent brain oxidative stress and participate in the synthesis of progesterone. It also possesses a progesterone-like effect and acts synergistically with progesterone on the brain. Progesterone and its metabolites, but also vitamin C have been associated with anticonvulsant effects. We evaluated the progesterone concentration 30min and 24h after the last administration of vitamin C (500mg/kg, i.p. for five days). We also evaluated how vitamin C altered pentylenetetrazol (PTZ)-induced seizures by measuring the onset latency of seizures, percentage of incidence and mortality as well as amino acid levels after seizures. Vitamin C treatment alone increased basal progesterone concentrations to 531% after 30min compared to 253% after 24h. Furthermore, vitamin C significantly increased the latency to the first myoclonic, clonic and tonic seizure induced by PTZ (80mg/kg, i.p.) and decreased the percentage of incidence of clonic and tonic seizures as well as the mortality rate. Changes in tissue concentration of amino acids were primarily observed at 24h after vitamin C treatment. Our results suggest that vitamin C together with progesterone and/or its metabolites are involved in the protection against PTZ-induced seizures in immature rats.

Mu opioid receptor mRNA expression, binding, and functional coupling to G‐proteins in human epileptic hippocampus

Mu opioid receptors (MOR) are known to be involved in seizure activity. The main goal of the present study was to characterize the MOR mRNA expression, binding, as well as G protein activation mediated by these receptors in epileptic hippocampus of patients with pharmacoresistant mesial temporal lobe epilepsy (TLE). In contrast with autopsy samples, hippocampus obtained from patients with mesial TLE demonstrated enhanced MOR mRNA expression (116%). Saturation binding experiments revealed significantly higher (60%) Bmax values for the mesial TLE group, whereas the Kd values were not statistically different. Although mesial TLE group demonstrated high levels of basal binding for the G proteins (136%), DAMGO‐stimulated [35S]GTPγS binding did not demonstrate significant alterations. In conclusion, our present data provide strong evidence that the epileptic hippocampus of patients with pharmacoresistant mesial TLE presents significant alterations in MOR. Such changes may represent adaptive mechanisms to compensate for other as yet unknown alterations.

GABAergic Alterations in Neocortex of Patients with Pharmacoresistant Temporal Lobe Epilepsy Can Explain the Comorbidity of Anxiety and Depression: The Potential Impact of Clinical Factors

Temporal lobe epilepsy (TLE) is a chronic neurodegenerative disease with a high prevalence of psychiatric disorders. Temporal neocortex contributes to either seizure propagation or generation in TLE, a situation that has been associated with alterations of the γ-amino-butyric acid (GABA) system. On the other hand, an impaired neurotransmission mediated by GABA in temporal neocortex has also been involved with the pathophysiology of psychiatric disorders. In spite of these situations, the role of the necortical GABA system in the comorbidity of TLE and mood disorders has not been investigated. The present study was designed to identify alterations in the GABA system such as binding to GABAA and GABAB receptors and benzodiazepine site, the tissue content of GABA and the expression of the mRNA encoding the α1–6, β1–3, and γ GABAA subunits, in the temporal neocortex of surgically treated patients with TLE with and without anxiety, and/or depression. Neocortex of patients with TLE and comorbid anxiety and/or depression showed increased expression of the mRNA encoding the γ2-subunit, reduced GABAB-induced G-protein activation in spite of elevated GABAB binding, and lower tissue content of GABA when compared to autopsy controls. Some of these changes significantly correlated with seizure frequency and duration of epilepsy. The results obtained suggest a dysfunction of the GABAergic neurotransmission in temporal neocortex of patients with TLE and comorbid anxiety and/or depression that could be also influenced by clinical factors such as seizure frequency and duration of illness.

Role of Histaminergic System in Blood–Brain Barrier Dysfunction Associated with Neurological Disorders

Blood-brain barrier (BBB) disruption has been associated with several acute and chronic brain disorders such as Alzheimers disease, Parkinsons disease and epilepsy. This represents a critical situation because damaged integrity of the BBB is related to the influx of immune mediators, plasma proteins and other outside elements from blood to the central nervous system (CNS) that may trigger a cascade of events that leads to neuroinflammation. In this review, evidence that mast cells and the release of factors such as histamine play an important role in the neuroinflammatory process associated with brain disorders such as Alzheimers disease, Parkinsons disease and epilepsy is presented.

Opioid receptor binding in parahippocampus of patients with temporal lobe epilepsy: Its association with the antiepileptic effects of subacute electrical stimulation

Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with intractable mesial temporal lobe epilepsy (MTLE) with and without subacute high frequency electrical stimulation (HFS) in this brain area. Mu, delta and nociceptin receptor binding was determined by autoradiography in PHC of five patients (ESAE group) with MTLE history of 14.8 +/- 2.5 years and seizure frequency of 11 +/- 2.9 per month, two of them (40%) with mesial sclerosis. This group demonstrated antiepileptic effects following subacute HFS (130 Hz, 450 micros, 200-400 microA), applied continuously during 16-20 days in PHC. Values were compared with those obtained from patients with severe MTLE (history of 21.7 +/- 2.8 years and seizure frequency of 28.2 +/- 14 per month) in whom electrical stimulation did not induce antiepileptic effects (ESWAE group, n = 4), patients with MTLE in whom no electrical stimulation was applied (MTLE group, n = 4) and autopsy material acquired from subjects without epilepsy (n = 4 obtained from three subjects). Enhanced 3H-DAMGO (MTLE, 755%; ESAE, 375%; ESWAE, 693%), 3H-DPDPE (MTLE, 242%; ESAE, 80%; ESWAE, 346%) and 3H-nociceptin (MTLE, 424%; ESAE, 217%; ESWAE, 451%) binding was detected in the PHC of all epileptic groups. However, tissue obtained from ESAE group demonstrated lower opioid receptor binding (3H-DAMGO, 44.5%, p < 0.05; 3H-DPDPE, 47%, p < 0.05; 3H-nociceptin, 39.3%, p < 0.5) when compared with MTLE group. The present results indicate that a high effectiveness to the antiepileptic effects induced by HFS is associated with reduced opioid peptide binding.