Maria Amélia Tavares

Monoamine Oxidase-B Mediates Ecstasy-Induced Neurotoxic Effects to Adolescent Rat Brain Mitochondria

3,4-Methylenedioxymethamphetamine (MDMA)-induced neurotoxicity and the protective role of monoamine oxidase-B (MAO-B) inhibition were evaluated at the mitochondrial level in various regions of the adolescent rat brain. Four groups of adolescent male Wistar rats were used: (1) saline control, (2) exposed to MDMA (4 × 10 mg/kg, i.p.; two hourly), (3) treated with selegiline (2 mg/kg, i.p.) 30 min before the same dosing of MDMA, and (4) treated with selegiline (2 mg/kg, i.p.). Body temperatures were monitored throughout the whole experiment. Animals were killed 2 weeks later, and mitochondria were isolated from several brain regions. Our results showed that “binge” MDMA administration causes, along with sustained hyperthermia, long-term alterations in brain mitochondria as evidenced by increased levels of lipid peroxides and protein carbonyls. Additionally, analysis of mitochondrial DNA (mtDNA) revealed that NDI nicotinamide adenine dinucleotide phosphate dehydrogenase subunit I and NDII (nicotinamide adenine dinucleotide phosphate dehydrogenase subunit II) subunits of mitochondrial complex I and cytochrome c oxidase subunit I of complex IV suffered deletions in MDMA-exposed animals. Inhibition of MAO-B by selegiline did not reduce hyperthermia but reversed MDMA-induced effects in the oxidative stress markers, mtDNA, and related protein expression. These results indicate that monoamine oxidation by MAO-B with subsequent mitochondrial damage may be an important contributing factor for MDMA-induced neurotoxicity.

Long term alcohol consumption induces microtubular changes in the adult rat cerebellar cortex

The effects of prolonged alcohol consumption on the microtubules of Purkinje cell dendrites and granule cell axons were studied in adult rats fed alcohol for 1, 3, 6, 12 and 18 months and compared with respective age-matched controls. A significant consequential decrease in the number of dendritic microtubules in alcohol-fed rats was found when compared with the respective controls. Conversely, an increase in the number of these organelles was found in both ascending and parallel portion of the axons in the experimental animals. The possibility of a relationship between microtubular changes and previously reported cerebellar cortex alcohol-induced structural alterations is advanced.

A quantitative study of frontal cortex dendritic microtubules in patients with Alzheimer's disease

The dendritic microtubules (MT) of the frontal cortex layers II and III were studied in 9 patients with Alzheimers disease (AD) and the results compared with 9 case controls. Dendrites with abnormally oriented MTs and others depleted of these structures were seen. A significant reduction in the number of MTs per unit area was found in AD. It is suggested that microtubular changes in AD can interfere with neuroplasmic transport and thus, be implicated with the dendritic degeneration present in this disease.

Myelination changes in the rat optic nerve after prenatal exposure to methamphetamine

The use of psychostimulants during adolescence and early adult life has increased in recent years. It is known that these substances affect the sensory systems, and the optic nerve has been shown to be a target tissue. This work was conducted to evaluate the effects of prenatal exposure to methamphetamine (MA) on the developmental pattern of the rat optic nerve. Pregnant female rats were given 5 mg/kg body weight/day MA, s.c., in 0.9% saline from gestational days 8 to 22. The control group was injected with an isovolumetric dose of 0.9% saline. Animal model parameters, such as gestational body weight evolution, food intake and pups parameters were registered. The offspring were sacrificed at postnatal days (PND) 7, 14 and 21. Morphometric analyses were performed at light and electron microscopic levels on optic nerve cross sections; parameters measured included optic nerve diameter and area, axonal density, total number of axons and myelin thickness. Myelin basic protein (MBP) was measured by western blotting in optic nerve samples at PND14 and PND21. The animal model parameters, such as maternal and pup weight, showed no significant differences between MA and control groups. Optic nerve diameter was smaller at PND7 in the male MA group and in both male and female MA groups at PND21. The mean cross-sectional area was smaller at PND14 in the male MA group and in both male and female groups at PND21. The total number of myelinated axons did not vary between groups at any of the studied ages. The myelin thickness of the axons in MA-treated females was thinner when compared with the respective control group at PND21. No other differences were found concerning myelin thickness. There was a reduction of MBP protein expression in MA-injected females at PND14 and PND21. The combined results suggest that prenatal exposure to MA affects the myelination process.

Adaptative response of antioxidant enzymes in different areas of rat brain after repeated d‐amphetamine administration

d‐Amphetamine has been shown to be a potential brain neurotoxic agent, particularly to dopaminergic neurones. Reactive oxygen species indirectly generated by this drug have been indicated as an important factor in the appearance of neuronal damage but little is known about the adaptations of brain antioxidant systems to its chronic administration. In this study, the activities of several antioxidant enzymes in different areas of rat brain were measured after repeated administration of d‐amphetamine sulphate (sc, 20 mg/kg/day, for 14 days), namely glutathione‐S‐transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GRed), catalase, and superoxide dismutase (SOD). When compared to a pair‐fed control group, d‐amphetamine treatment enhanced the activity of GST in hypothalamus to 167%, GPx in striatum to 127%, in nucleus accumbens to 192%, and in medial prefrontal cortex to 139%, GRed in hypothalamus to 139%, as well as catalase in medial prefrontal cortex to 153%. However, the same comparison revealed a decrease in the activity of GRed in medial pre‐frontal cortex by 35%. Food restriction itself reduced GRed activity by 49% and enhanced catalase activity to 271% in nucleus accumbens. The modifications observed for the measured antioxidant enzymes reveal that oxidative stress probably plays a role in the deleterious effects of this drug in CNS and that, in general, the brain areas studied underwent adaptations which provided protection against the continuous administration of the drug.

Effects of Postnatal Cocaine Exposure and Environmental Enrichment on Rat Behavior in a Forced Swim Test

Abstract: This study examined the effects of environmental enrichment on rats exposed to cocaine during the first month of life, in several categories of behavior observed in a forced swim test. Wistar rats were divided in four groups. The first included pups that were subjected to injections of cocaine hydrochloride (15 mg/kg body weight/day, subcutaneously, in two daily doses, from postnatal days 1 to 27) and reared in an enriched environment (CocEE); the second, pups that were subjected to injections of cocaine (as previously described) and reared in a standard environment (CocSE); the third, pups that were subjected to saline injections and reared in an enriched environment (SalEE); the fourth, pups that were subjected to saline injections and reared in a standard environment (SalSE). On postnatal days 26 and 27, rats were tested in a swimming pool in two 5‐min sessions. The categories of behavior studied in this work were: fast swim, slow swim, struggling, diving, and immobility. Results showed that postnatal cocaine exposure decreased the time spent on fast swim during the two sessions and increased the immobility behavior during the second session in CocSE pups compared with SalSE pups. SalEE pups increased the time spent in fast swim, slow swim, and diving, and decreased the time spent in struggling and immobility during the two sessions compared with SalSE pups. CocEE animals spent more time in fast swim and struggling and less the time in immobility compared with CocSE pups. The present results suggest that postnatal cocaine exposure affects the ability of these animals to cope with stressful situations, and that environmental enrichment seems to enable the rats to adopt a more active strategy, one that allows them to better cope with this particular stress situation.

Correlation of axon size and myelin occupancy in rats prenatally exposed to methamphetamine

The abuse of methamphetamine (MA) and other psychostimulants is a social and medical problem. In particular, the use of these drugs by pregnant women results in an increased number of children exposed prenatally to psychostimulants. Our previous work has demonstrated that prenatal exposure to MA affects the normal development of the rat visual system due to alterations of biochemical mechanisms and oxidative stress. It was also demonstrated that prenatal exposure to MA affects the dopaminergic system of the rat retina and optic nerve (ON) myelination. The present work was conducted to evaluate the effects of prenatal exposure to MA on the development of the ON in terms of axon growth and the myelin sheath. Pregnant female rats were given 5 mg/kg/day MA, subcutaneously (s.c.), in 0.9% saline from gestational day (GD) 8 to 22. The pair-fed control group was injected s.c. with an isovolumetric dose of 0.9% saline. Qualitative analysis was performed using representative electron ultramicrographs. Quantitative analysis was performed at an electron microscopic level on ON cross sections; parameters measured included myelinated/unmyelinated ratio, outer axon mean area, inner axon mean area, myelin mean area, myelin occupancy and distribution of axons by size. The ON of prenatally MA-exposed rats presented a higher rate of deformed axons and slighter lamellar separation. At PND 21, the average outer axon area of MA-treated males was significantly reduced. The average inner axon area only showed a significant difference between MA and control males for axons with an area of less than 0.3 microm(2). The average myelin area of MA-treated males was significantly reduced, and in MA-treated females was only significantly reduced in axons with an area of less than 0.3 microm(2). The percentage of myelin occupancy was significantly affected in MA-treated males, and in MA-treated females in the group of axons with an area of more than 0.3 microm(2). At PND 14 no significant differences were found between MA and control groups. The spectrum of ON myelinated axon size of MA-treated animals was shifted to the left at PND 14 and PND 21 for both genders. These results are in agreement with previous animal studies of prenatal and perinatal exposure to drugs of abuse. Taken together, these data indicate that the ON is vulnerable to early exposure to MA which causes developmental changes and may interfere with the functioning of the visual system.

MDMA in Adolescent Male Rats

Abstract:  Long‐term behavioral consequences of the neurotoxicity produced by 3,4‐methylenedioxymethamphetamine (MDMA) in the adolescent rat are still mostly unknown. Here, adolescent male rats (postnatal day 45 PND [45]) were exposed to 10 mg/kg of MDMA, intraperitoneally, every 2 h for 6 h. Controls were given 0.9% saline in the same protocol. Ten days after exposure, the behavioral effects of MDMA were assessed in the elevated plus‐maze (n= 6 per group). After behavioral testing, animals were sacrificed and the amygdalae were dissected and processed for HPLC determination of dopamine (DA), serotonin (5‐HT), and metabolites. Results showed a significant decrease in the 5‐HT content (P < 0.05), but no significant alterations in DA or its metabolites. Behavioral observation in the elevated plus‐maze showed a decreased number of entries in the unprotected arms (P < 0.05), which were correlated to the number of entries and time spent in the central platform. Rearing was also decreased (P < 0.05). No differences were observed in head dips, grooming, or number of entries in the protected arms of the apparatus. Therefore, we conclude that, as in the adult rat, exposure to MDMA in the adolescent rat is associated to long‐term depletion of the 5‐HT content and increased anxiety‐like behavior.

Oxidative stress response in the adult rat retina and plasma after repeated administration of methamphetamine

Methamphetamine (MA) is a psychostimulant that target the sensory systems, with the neurosensory retina having been shown to be affected. In the brain, MA-related toxicity can be linked to oxidative stress; the same relationship has yet to be established for the retina. The aim of this study, therefore, was to evaluate the effects of repeated exposure to MA on oxidative stress parameters in the rat retina. Oxidative stress parameters in the blood plasma were also assessed. Male Wistar rats were given 5mg/kg MA every 2h for a period of 6h (i.e., 4 injections) daily between postnatal day (PND) 91 and 100. Evolution of body weight was registered. Rats were sacrificed at PND 110. Blood plasma was collected and immediately frozen for storage at -70 degrees C. The eyes were enucleated, and the retina and choroids rapidly dissected on ice under dim light also to be stored at -70 degrees C. Lipid peroxidation activity was measured by the thiobarbituric acid (TBA) test. Total antioxidant status, superoxide dismutase (SOD) activity, catalase (Cat) activity, and nitrogen oxides contents were also determined. Lipid peroxidation was significantly higher in the retina and blood plasma of the MA-treated rats. Total antioxidant levels were significantly lower in both retina and blood plasma of the MA-treated rats. The activity of SOD was significantly increased in the retina and blood plasma of MA-treated rats. Catalase activity did not differ between groups in either the retina or the blood plasma. Nitric oxide production was significantly higher in both the retina and blood plasma in the MA-treated animals. The overall findings show that the oxidative stress defence mechanisms in the retina are compromised by MA toxicity. The results are similar to those found in the brain, and, moreover, showed some correlation with the blood plasma.

Aqueous outflow system in familial amyloidotic polyneuropathy, Portuguese type

Familial amyloidotic polyneuropathy (FAP) is a hereditary disease which may present a wide range of ocular manifestations. Glaucoma is amongst the most serious complications of FAP. We report the results of ultrastructural study of the trabecular meshwork in a glaucomatous patient with the Portuguese form of FAP. This study showed that there was marked anatomical disruption of the uveoscleral, corneoscleral meshworks and juxtacanalicular tissue characterized by (a) accumulation of amyloid fibrils in the intertrabecular spaces; (b) degeneration of the endothelial cells; (c) homogeneous and/or multilayered plaques of basement membrane-like material loading the intertrabecular spaces or protruding to the lumen of the Schlemms canal; and (d) degeneration of unmyelinated nerve fibres. These morphological changes and an analysis of the literature suggest that mechanical and neuropathic events can be co-existing factors which enhance the resistance to aqueous humour outflow, leading to increased intraocular pressure and glaucoma in the Portuguese form of FAP.