Tice Macedo

Mitochondrial dysfunction and caspase activation in rat cortical neurons treated with cocaine or amphetamine.

Drug abuse is associated with brain dysfunction and neurodegeneration, and various recreational drugs induce apoptotic cell death. This study examined the role of the mitochondrial apoptotic pathway in psychostimulant-induced neuronal dysfunction. Using primary neuronal cultures, we observed that amphetamine (IC50=1.40 mM) was more potent than cocaine (IC50=4.30 mM) in inducing cell toxicity. Apoptotic cell death was further evaluated using cocaine and amphetamine concentrations that moderately decreased cell reduction capacity but did not affect plasma membrane integrity. Compared to cocaine, amphetamine highly decreased the mitochondrial membrane potential, as determined using the fluorescent probe rhodamine-123, whereas both drugs decreased mitochondrial cytochrome c. In contrast to amphetamine, cocaine cytotoxicity was partly mediated through effects on the electron transport chain, since cocaine toxicity was ameliorated in mitochondrial DNA-depleted cells lacking mitochondrially encoded electron transport chain subunits. Cocaine and amphetamine induced activation of caspases-2, -3 and -9 but did not affect activity of caspases-6 or -8. In addition, amphetamine, but not cocaine, was associated with the appearance of evident nuclear apoptotic morphology. These events were not accompanied by differences in the release of the apoptosis-inducing factor (AIF) from mitochondria. Our results demonstrate that although both amphetamine and cocaine activate the mitochondrial apoptotic pathway in cortical neurons, amphetamine is more likely to promote apoptosis.

Methamphetamine‐Induced Early Increase of IL‐6 and TNF‐α mRNA Expression in the Mouse Brain

The mechanisms by which methamphetamine (METH) causes neurotoxicity are not well understood. Recent studies have suggested that METH‐induced neuropathology may result from a multicellular response in which glial cells play a prominent role, and so it is plausible to suggest that cytokines may participate in the toxic effects of METH. Therefore, in the present work we evaluated the effect of an acute administration of METH (30 mg/kg in a single intraperitoneal injection) on the interleukin (IL)‐1β, IL‐6, and tumor necrosis factor (TNF)‐α mRNA expression levels in the hippocampus, frontal cortex, and striatum of mice. We observed that METH did not induce changes in the IL‐1β mRNA expression levels in both hippocampus and striatum, with immeasurable levels in the frontal cortex. Regarding IL‐6, METH induced an increase in the expression levels of this cytokine in the hippocampus and striatum, 1 h and 30 min post injection, respectively. In the frontal cortex, the increase in IL‐6 mRNA levels was more significant and remained high even after 2 h. Moreover, the expression levels of TNF‐α were increased in both hippocampus and frontal cortex 30 min post METH administration, with immeasurable levels in the striatum. We conclude that the pro‐inflammatory cytokines IL‐6 and TNF‐α rapidly increase after METH administration, providing a new insight for understanding the effect of this drug of abuse in the brain.

Street heroin induces mitochondrial dysfunction and apoptosis in rat cortical neurons

Cortical function has been suggested to be highly compromised by repeated heroin self‐administration. We have previously shown that street heroin induces apoptosis in neuronal‐like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6‐monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly‐ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non‐selective caspase inhibitor z‐VAD‐fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT‐2 rho0 cells. Nonetheless, in street heroin‐treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl‐2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.

Neurotoxicity of heroin–cocaine combinations in rat cortical neurons

Cocaine and heroin are frequently co-abused by humans, in a combination known as speedball. Recently, chemical interactions between heroin (Her) or its metabolite morphine (Mor) and cocaine (Coc) were described, resulting in the formation of strong adducts. In this work, we evaluated whether combinations of Coc and Her affect the neurotoxicity of these drugs, using rat cortical neurons incubated with Coc, Her, Her followed by Coc (Her+Coc) and Her plus Coc (Her:Coc, 1:1). Neurons exposed to Her, Her+Coc and Her:Coc exhibited a decrease in cell viability, which was more pronounced in neurons exposed to Her and Her+Coc, in comparison with neurons exposed to the mixture (Her:Coc). Cells exposed to the mixture showed increased intracellular calcium and mitochondrial dysfunction, as determined by a decrease in intracellular ATP levels and in mitochondrial membrane potential, displaying both apoptotic and necrotic characteristics. Conversely, a major increase in cytochrome c release, caspase 3-dependent apoptosis, and decreased metabolic neuronal viability were observed upon sequential exposure to Her and Coc. The data show that drug combinations potentiate cortical neurotoxicity and that the mode of co-exposure changes cellular death pathways activated by the drugs, strongly suggesting that chemical interactions occurring in Her:Coc, such as adduct formation, shift cell death mechanisms towards necrosis. Since impairment of the prefrontal cortex is involved in the loss of impulse control observed in drug addicts, the data presented here may contribute to explain the increase in treatment failure observed in speedball abusers.

Neuroprotective properties ofValeriana officinalis extracts

Valeriana officinalis have been used in traditional medicine for its sedative, hypnotic, and anticonvulsant effects. There are several eports in the literature supporting a GABAergic mechanism of action for valerian. The rationale of the present work is based on the concept that by decreasing neuronal network excitability valerian consumption may contribute to neuroprotection. The aim of our investigation was to evaluate the neuroprotective effects ofV. officinalis against the toxicity induced by amyloid beta peptide 25–35 [Aβ(25–35)]. Cultured rat hip-pocampal neurons were exposed to Aβ(25–35)(25 μM) for 24–48 h,after which morphological and biochemical properties were evaluated. The neuronal injury evoked by Aβ, which includes a decrease in cell educing capacity and associated neuronal degeneration, was prevented by valerian extract. Analysis of intracellular free calcium ([Ca2+]i)indicated that the neuroprotective mechanisms may involve the inhibition of excess influx of Ca2+ following neuronal injury. Moreover, membrane peroxidation in rat hippocampal synaptosomes was evaluated, and our data indicate that valerian extract partially inhibited ascorbate/iron-induced peroxidation.In conclusion we show evidence that the signalling pathways involving [Ca2+]i and the redox state of the cells may play a central ole in the neuroprotective properties ofV. officinalis extract against Aβ toxicity. The novelty of the findings of the present work, indicating neuroprotective properties of valerian against Aβ toxicity may, at the long-term, contribute to introduction of a new elevant use of valerian alcoholic extract to prevent neuronal degeneration in aging or neurodegenerative disorders.

Brain Blood Flow SPET Imaging in Heroin Abusers

Abstract:  To assess whether chronic heroin abuse may generate vascular central nervous deficits, we studied the profile of vascular alterations in 17 heroin addicts (14 males mean age 31 years, range 23–39 years and 3 females mean age 33 years, range 30–35 years) before and, in one of them, 10 weeks after an ultra‐rapid heroin detoxification. Using the functional technique of single‐photon emission tomography (SPET) with 740 MBq of 99mTc‐hexametazine (HMPAO) and computational brain‐mapping techniques by means of a Talairach analysis, we determined the pattern of vascular brain alterations associated with chronic heroin abuse. Compared with controls, subjects who had used heroin chronically showed a decrease of global brain perfusion that was more significant in the frontal cortex—mainly in orbito‐frontal regions, as well as in the occipital and temporal lobes. All patients showed marked asymmetric perfusion of the basal ganglia and the majority of them showed also an asymmetric perfusion of cerebellum. In addition, there were small activated areas dispersed in the occipital lobe (3 of 17) and apex region (4 of 17). In conclusion, decreased perfusion in heroin addicts was found in regions involved in the control of attention, motor speed, memory and visual–spatial processing. The prefrontal cortex is involved in decision making and inhibitory control, processes disturbed in heroin addicts who have stopped heroin consumption. A reduction in regional perfusion may reflect ongoing subtle neurocognitive deficits, which are consistent with the maintenance of asymmetry of the basal nuclei.

May Exercise Prevent Addiction

Amphetamines exert their persistent addictive effects by activating brains reward pathways, perhaps through the release of dopamine in the nucleus accumbens (and/or in other places). On the other hand, there is a relationship between dopamine and all behavioural aspects that involve motor activity and it has been demonstrated that exercise leads to an increase in the synthesis and release of dopamine, stimulates neuroplasticity and promotes feelings of well-being. Moreover, exercise and drugs of abuse activate overlapping neural systems. Thus, our aim was to study the influence of chronic exercise in the mechanism of addiction using an amphetamine-induced conditioned-place-preference in rats. Adult male Sprague-Dawley rats were randomly separated in groups with and without chronic exercise. Chronic exercise consisted in a 8 week treadmill running program, with increasing intensity. The conditioned place preference test was performed in both groups using a procedure and apparatus previously established. A 2 mg.kg-1 amphetamine or saline solution was administered intraperitonially according to the schedule of the conditioned place preference. Before conditioning none of the animals showed preference for a specific compartment of the apparatus. The used amphetamine dose in the conditioning phase was able to produce a marked preference towards the drug-associated compartment in the group without exercise. In the animals with exercise a significant preference by the compartment associated with saline was observed. These results lead us to conclude that a previous practice of regular physical activity may help preventing amphetamine addiction in the conditions used in this test.

Methamphetamine Changes NMDA and AMPA Glutamate Receptor Subunit Levels in the Rat Striatum and Frontal Cortex

Methamphetamine (METH) is a powerful psychostimulant whose noxious effects depend largely on the pattern of abuse. METH‐induced glutamate release may overactivate N‐methyl‐d‐aspartate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptors (NMDAR and AMPAR, respectively) causing excitotoxicity. In the brain, these receptors are also known for their essential role in mediating memory consolidation. Therefore, we assessed glial fibrillary acidic protein (GFAP) expression as a marker for astrogliosis and neurodegeneration by using Fluoro‐Jade C (F‐J C) staining. Moreover, we investigated the effect of two METH regimens on NMDAR NR1 and NR2A and on AMPAR GluR2 subunit expression in the rat striatum and frontal cortex 24 h after drug treatment. Adult Sprague‐Dawley rats were injected subcutaneously (s.c.) on six consecutive days with saline (control and acute groups) or with an increasing dose of METH (10, 15, 15, 20, 20, 25 mg/kg/day; ED group). On the seventh day, both METH groups were given a “bolus” of 30 mg/kg METH, whereas controls received saline. We evaluated the expression levels of GFAP by both Western blot and immunohistochemical assays and concluded that there was no difference from control levels. In addition, neither drug regimen resulted in neurodegeneration within 24 h of last METH administration. In the frontal cortex of the acute group, NR1 expression level was decreased, and both NR2A and GluR2 were increased. Also, in the striatum of the acute group, the expression level of GluR2 was significantly increased, and both GluR2 and NR2A levels were augmented in the striatum of the ED group. Taken together, these results suggest a protective mechanism by decreasing permeability and/or functionality of AMPAR and NMDAR to counteract METH‐induced glutamate overflow in the brain. Moreover, these results may explain, in part, the mnemonic deficits and psychotic behavior associated with METH abuse.

Eslicarbazepine Acetate (BIA 2-093)

AbstractPurpose: To investigate the bioavailability and bioequivalence of three different formulations of eslicarbazepine acetate (BIA 2-093): 50 mg/mL oral suspension (test 1), 200mg tablets (test 2) and 800mg tablets (reference). Design, subjects and methods: Single-centre, open-label, randomised, three-way crossover study in 18 healthy subjects. The study consisted of three consecutive periods separated by a washout period of 7 days or more. Each subject received a single dose of eslicarbazepine acetate 800mg on three different occasions: 16mL of oral 50 mg/mL suspension, four 200mg tablets or one 800mg tablet. Results: Eslicarbazepine acetate was rapidly and extensively metabolised to BIA 2-005. Maximum BIA 2-005 plasma concentrations (Cmax) and area under the plasma concentration-time curve from time 0 to infinity (AUC∞) were, respectively (arithmetic mean ± SD), 18.0 ± 4.6 μg/mL and 325.7 ± 64.9 μg · h/mL for test 1, 16.0 ± 4.0 μg/mL and 304.2 ± 66.0 μg · h/mL for test 2, and 17.0 ± 4.1 μg/mL and 301.1 ± 60.0 μg · h/mL for the reference formulation. Point estimate (PE) and 90% confidence intervals (CIs) for AUC∞ test 1/reference geometric mean ratio were 1.09 and 1.01, 1.15; for Cmax ratio, PE and 90% CI were 1.07 and 0.97, 1.15. When test 2 and the reference formulations were compared, the PE and 90% CI were 0.99 and 0.94, 1.07 for the AUC∞ ratio, and 0.94 and 0.86, 1.02 for the Cmax ratio. Bioequivalence of test versus reference formulations is thus accepted for both AUC∞ and Cmax because the 90% CIs lie within the acceptance range of 0.80–1.25. Conclusion: The pharmacokinetic profiles of eslicarbazepine acetate oral 50 mg/mL suspension, 200mg tablet and 800mg tablet formulations were essentially similar, and the formulations can be considered bioequivalent.

Migraine, Serum Serotonin and Platelet 5–HT2 Receptors

In spite of recent theories about the aetiopathogenesis of migraine, serotonin continues to play a central role, explaining the efficacy of almost all migraine prophylactic drugs. In migraineurs with and without aura we measured (by HPLC-EC) the serum serotonin (5–HT) and 5–hydroxyindoleacetic acid (5–HIAA) levels between as well as during headache attacks. Between attacks of migraine with aura and at the beginning of attacks of both types of migraine the serum 5–HT and 5–HIAA concentration was significantly increased. These results were corroborated by 3H-spiperone binding to platelet membranes: in migraineurs with aura in the attack-free interval, there was a significant decrease in its Bmax, which suggests down-regulation of 5–HT2 receptors. In conclusion, we have verified that migraine with aura differs biochemically from migraine without aura.