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- Prenatal exposure to cocaine and enriched environmentPublication . Magalhães, Ana; Summavielle, Teresa; Melo, Pedro; Rosa, Rui; Tavares, Maria Amélia; Sousa, Liliana deExposure to cocaine throughout gestation may produce several deleterious outcomes in the offspring that include effects on neurotransmitter systems and structure of the central nervous system. Such changes are most likely correlated with behavioral alterations. Environmental enrichment (EE) in early stages is a factor that affects structural and behavioral development. This article examines the effects, upon social interactions, of EE during the first month of life in rats prenatally exposed to cocaine. Wistar dams were subcutaneously exposed to 60 mg/kg of cocaine divided in two daily doses from gestational day (GD)8 to GD22. Pair-fed controls were given saline vehicle in the same protocol. Offspring were distributed to the different environments in four experimental groups. Group 1: offspring from dams prenatally exposed to cocaine as previously described and reared in EE from postnatal day (PND)1 to PND28; Group 2: pups from cocaine-exposed dams and reared in a standard environment (SE); Group 3: pups from pair-fed saline-exposed dams and reared in EE; Group 4: offspring from saline-exposed dams and reared in SE. On PND21, 24, and 28, rats were examined in several social behavioral categories (play fighting, social investigation, comfort behaviors, and solicitation to play) for 10 min. Animals reared in SE do not display any differences due to treatment in the behavioral categories analyzed. Control offspring reared in EE presented decreased play fighting, decreased solicitation to play, and decreased social investigation compared to the control SE group, while cocaine-exposed animals reared in EE did not present these variations. These results suggest that EE rearing may unmask hidden effects of prenatal cocaine exposure.
- Chronic ketamine administration impairs mitochondrial complex I in the rat liverPublication . Venâncio, Carlos; Antunes, Luis; Félix, Luís; Rodrigues, Paula; Summavielle, Teresa; Peixoto, FranciscoKetamine can induce hepatotoxicity which has been suggested to be dependent on mitochondrial impairment. This study investigated the long-term effects of chronic low-dose ketamine on liver mitochondrial function, oxidative stress parameters, liver histology and glycogen content. Adult rats were administered with saline or ketamine (5 or 10 mg/kg) twice a day for a fourteen-day period in order to mimic chronic treatments. Effects between groups were compared ten days after the treatment had ended. Liver mitochondrial function was monitored in isolated mitochondrial extracts through evaluation of respiration parameters and activity of respiratory complexes, as well as oxidative stress, through lipid peroxidation, protein oxidation and superoxide dismutase activity. The hepatic histology and liver glycogen content were also evaluated. Ketamine groups showed a decreased evolution in body weight gains during the treatment period. Ketamine had no effect either on serum liver enzymes or on the oxidative stress parameters of liver mitochondria. Ketamine decreased the hepatic glycogen content, inhibited mitochondrial complex I and oxygen consumption when glutamate–malate substrate was used. These findings reflect a long-term mitochondrial bioenergetic deterioration induced by ketamine, which may explain the increased susceptibility of some patients to its prolonged or repeated use.
- 3D vs 2D Cell Cultures in the Evaluation of Radiobiological Effects of Exposition to Low Doses - Medical Imaging Levels - of Ionizing RadiationPublication . Costa, Pedro; Caires, Hugo; Lemos, Joana; Cunha, Lídia; Bravo, Joana; Bravo, Isabel; Silva, Regina; Summavielle, Teresa; Metello, Luís F.Pretending to develop advanced biological models to study biological effects of low doses of ionizing radiation and following the actual policies on Animal Sciences, based on 3 R’s Rule (to Reduce, Refine and Replace) – that limits as much as possible the application of animal models – scientific research using cellular models is constantly increasing. Nevertheless, the intrinsic limitations of actual cellular models quite often had been recognized on a significant number of papers pointing a significant number of non-concordances between results obtained using in vitro and in vivo studies. Actually, an increasing number of authors admit that three-dimensional cell culture (and spheroid cell culture in particular) could represent an interesting solution and a step further on use of cellular models. The work here to be presented reflects the first phase on the use of this methodology on the study, evaluation and quantification of cellular effects of low doses – starting on medical imaging level - of exposition to ionizing radiation.
- Maternal stress and vulnerability to depression: coping and maternal care strategies and its consequences on adolescent offspringPublication . Alves, Renata L.; Portugal, Camila C.; Lopes, Igor M.; Oliveira, Pedro; Alves, Cecília J.; Barbosa, Fernando; Summavielle, Teresa; Magalhães, AnaDepressive mothers often find mother-child interaction to be challenging. Maternal stress may further impair mother-child attachment, which may increase the risk of negative developmental consequences. We used rats with different vulnerability to depressive-like behavior (Wistar and Kyoto) to investigate the impact of stress (maternal separation-MS) on maternal behavior and adolescent offspring cognition. MS in Kyoto dams increased pup-contact, resulting in higher oxytocin levels and lower anxiety-like behavior after weaning, while worsening their adolescent offspring cognitive behavior. Whereas MS in Wistar dams elicited higher quality of pup-directed behavior, increasing brain-derived neurotrophic factor (BDNF) in the offspring, which seems to have prevented a negative impact on cognition. Hypothalamic oxytocin seems to affect the salience of the social environment cues (negatively for Kyoto) leading to different coping strategies. Our findings highlight the importance of contextual and individual factors in the understanding of the oxytocin role in modulating maternal behavior and stress regulatory processes.
- Multimodal classification of anxiety based on physiological signalsPublication . Vaz, Mariana; Summavielle, Teresa; Sebastião, Raquel; Ribeiro, Rita P.Multiple studies show an association between anxiety disorders and dysregulation in the Autonomic Nervous System (ANS). Thus, understanding how informative the physiological signals are would contribute to effectively detecting anxiety. This study targets the classification of anxiety as an imbalanced binary classification problem using physiological signals collected from a sample of healthy subjects under a neutral condition. For this purpose, the Electrocardiogram (ECG), Electrodermal Activity (EDA), and Electromyogram (EMG) signals from the WESAD publicly available dataset were used. The neutral condition was collected for around 20 min on 15 participants, and anxiety scores were assessed through the shortened 6-item STAI. To achieve the described goal, the subsequent steps were followed: signal pre-processing; feature extraction, analysis, and selection; and classification of anxiety. The findings of this study allowed us to classify anxiety with discriminatory class features based on physiological signals. Moreover, feature selection revealed that ECG features play a relevant role in anxiety classification. Supervised feature selection and data balancing techniques, especially Borderline SMOTE 2, increased the performance of most classifiers. In particular, the combination of feature selection and Borderline SMOTE 2 achieved the best ROC-AUC with the Random Forest classifier.
- Methamphetamine promotes a-tubulin deacetylation in endothelial cells: The protective role of acetyl-L-carnitinePublication . Fernandes, Sílvia; Salta, Sofia; Summavielle, TeresaMethamphetamine (METH) is a powerful psychostimulant drug used worldwide for its reinforcing properties. In addition to the classic long-lasting monoaminergic-disrupting effects extensively described in the literature, METH has been consistently reported to increase blood brain barrier (BBB) permeability, both in vivo and in vitro, as a result of tight junction and cytoskeleton disarrangement. Microtubules play a critical role in cell stability, which relies on post-translational modifications such as a-tubulin acetylation. As there is evidence that psychostimulants drugs modulate the expression of histone deacetylases (HDACs), we hypothesized that in endothelial cells METH-mediation of cytoplasmatic HDAC6 activity could affect tubulin acetylation and further contribute to BBB dysfunction. To validate our hypothesis, we exposed the bEnd.3 endothelial cells to increasing doses of METH and verified that itleads to an extensivea-tubulin deacetylation mediated by HDACs activation. Furthermore, since we recently reported that acetyl-L-carnitine (ALC), a natural occurring compound, prevents BBB structural loss in a context of METH exposure, we reasoned that ALC could also preserve the acetylation of microtubules under METH action. The present results confirm that ALC is able to prevent METH-induced deacetylation providing effective protection on microtubule acetylation. Although further investigation is still needed, HDACs regulation may become a new therapeutic target for ALC.
- Microglia dysfunction caused by the loss of rhoa disrupts neuronal physiology and leads to neurodegenerationPublication . Socodato, Renato; Portugal, Camila C.; Canedo, Teresa; Rodrigues, Artur; Almeida, Tiago O.; Henriques, Joana F.; Vaz, Sandra H.; Magalhães, João; Silva, Cátia M.; Baptista, Filipa I.; Alves, Renata L.; Coelho-Santos, Vanessa; Silva, Ana Paula; Paes-de-Carvalho, Roberto; Magalhães, Ana; Brakebusch, Cord; Sebastião, Ana M.; Summavielle, Teresa; Ambrósio, António F.; Relvas, João B.Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.
- Acetyl-L-Carnitine prevents methamphetamine-induced structural damage on endothelial cells via ILK-related MMP-9 activityPublication . Summavielle, Teresa; Fernandes, S.; Salta, S.; Bravo, J.; Silva, A.P.Methamphetamine (METH) is a potent psychostimulant highly used worldwide. Recent studies evidenced the involvement of METH in the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised function. The involvement of the matrix metalloproteinases (MMPs) in the degradation of the neurovascular matrix components and tight junctions (TJs) is one of the most recent findings in METH-induced toxicity. As BBB dysfunction is a pathological feature of many neurological conditions, unveiling new protective agents in this field is of major relevance. Acetyl- L-carnitine (ALC) has been described to protect the BBB function in different paradigms, but the mechanisms underlying its action remain mostly unknown. Here, the immortalized bEnd.3 cell line was used to evaluate the neuroprotective features of ALC in METH-induced damage. Cells were exposed to ranging concentrations of METH, and the protective effect of ALC 1 mM was assessed 24 h after treatment. F-actin rearrangement, TJ expression and distribution, and MMPs activity were evaluated. Integrin-linked kinase (ILK) knockdown cells were used to assess role of ALC in ILK mediated METH-triggered MMPs’ activity. Our results show that METH led to disruption of the actin filaments concomitant with claudin-5 translocation to the cytoplasm. These events were mediated by MMP-9 activation in association with ILK overexpression. Pretreatment with ALC prevented METH-induced activation of MMP-9, preserving claudin-5 location and the structural arrangement of the actin filaments. The present results support the potential of ALC in preserving BBB integrity, highlighting ILK as a new target for the ALC therapeutic use.
- Preclinical Imaging: an Essential Ally in Modern BiosciencesPublication . Cunha, Lídia; Horvath, Ildiko; Ferreira, Sara; Lemos, Joana; Costa, Pedro; Vieira, Domingos; Veres, Dániel S.; Szigeti, Krisztián; Summavielle, Teresa; Máthé, Domokos; Metello, Luís F.Translational research is changing the practice of modern medicine and the way in which health problems are approached and solved. The use of small-animal models in basic and preclinical sciences is a major keystone for these kinds of research and development strategies, representing a bridge between discoveries at the molecular level and clinical implementation in diagnostics and/or therapeutics. The development of high-resolution in vivo imaging technologies provides a unique opportunity for studying disease in real time, in a quantitative way, at the molecular level, along with the ability to repeatedly and non-invasively monitor disease progression or response to treatment. The greatest advantages of preclinical imaging techniques include the reduction of biological variability and the opportunity to acquire, in continuity, an impressive amount of unique information (without interfering with the biological process under study) in distinct forms, repeated or modulated as needed, along with the substantial reduction in the number of animals required for a particular study, fully complying with 3R (Replacement, Reduction and Refinement) policies. The most suitable modalities for small-animal in vivo imaging applications are based on nuclear medicine techniques (essentially, positron emission tomography [PET] and single photon emission computed tomography [SPECT]), optical imaging (OI), computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy imaging (MRSI), and ultrasound. Each modality has intrinsic advantages and limitations. More recently, aiming to overcome the inherent limitations of each imaging modality, multimodality devices designed to provide complementary information upon the pathophysiological process under study have gained popularity. The combination of high-resolution modalities, like micro-CT or micro-MRI, with highly sensitive techniques providing functional information, such as micro-PET or micro-SPECT, will continue to broaden the horizons of research in such key areas as infection, oncology, cardiology, and neurology, contributing not only to the understanding of the underlying mechanisms of disease, but also providing efficient and unique tools for evaluating new chemical entities and candidate drugs. The added value of small-animal imaging techniques has driven their increasing use by pharmaceutical companies, contract research organizations, and research institutions.
- Possible modulation of dopaminergic neurotransmission function by acetyl-L-carnitinePublication . Cunha, L.; Szigeti, K.; Mathe, D.; Summavielle, T.; Metello, L.Acetyl‐L‐Carnitine (ALC) has a putative neuroprotective effect being used in a variety of conditions. Nevertheless, the underlying molecular mechanisms, particularly regarding the induction of changes in neurotransmitter systems, are still not fully understood. We aim to contribute for the elucidation of the mechanisms by which ALC alters neurotransmitter release, using a cell line and an animal model of exposure to methamphetamine (METH). PC12 cells were incubated with several doses of ALC (0.01 to 1.0 mM) alone or in combination with METH 1.0 or 100 µM for 24h or 72h. When combined, ALC preceded METH administration in 30 minutes. Dopamine (DA) content was determined by high performance liquid chromatography. C57BL/6J mice were used for in vivo assays to assess DA striatal binding. Mice were divided into 4 groups, according to different treatments: group 1 (control), group 2 (ALC, 100 mg/kg), group 3 (METH, 10 mg/kg) and group 4 (ALC+METH). Images were acquired in a SPECT/CT scanner (NanoSPECT/CT, Mediso, Hungary) 70 minutes after 123I‐IBZM injection. Regions of interest were drawn in the striata and in the cerebellum to determine the striatal binding ratio. Increased intracellular levels of DA were observed in PC12 cells at 24h and 72h after the administration of ALC. Cells treated with METH 100 µM displayed decreased intracellular levels of DA. ALC prevented the METH‐induced decrease in DA concentration (p<0.0001). On the other hand, a single dose of 10 mg/kg of METH induced a decrease in striatal D2R binding ratios comparing to control group (between 20% and 30%). Interestingly, over time, ALC was able to reverse the decrease on the radiotracer binding induced by METH. The present study indicates a possible effect of ALC over METH‐induced DA release.