Drug addiction: new targets for an old problem

Classically, exposure to psychotropic stimulants has been primarily associated with damage to dopaminergic neuronal terminals and oxidative stress. However, it is now widely accepted that the interaction between neuronal and glial cells plays a critical role in the development of drug addiction. In this sense, exposure to psychostimulants has been repeatedly shown to produce inflammation in the addicted brain. The existing data evidences that the overall contribution of inflammation to the build up of addiction remains unclear. Contrary to the common held view, our data revealed that methamphetamine (METH), a potent psychostimulant frequently associated to neuroinflammation, cannot stimulate microglia in a cell-autonomous manner. In addition, recent findings evidenced microglia as a multifunctional cell, critical for shaping and refining neuronal connectivity through synaptic plasticity. We hypothesised that the long-term adverse neuropsychiatric consequences occurring within the brain’s reward circuitry under psychostimulat exposure may be due, at least in part, to the underlying neuroinflammatory process, and that limiting it would be relevant to control the addictive behaviour. We show that preventing exacerbated microglial activation and associated neurotoxicity is beneficial at several levels, and that this knowledge can be easily translated into clinical applications.info:eu-repo/semantics/publishedVersio

The insula, a key brain area for bladder pain control, is modulated by stress

Stress may cause or aggravate Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) symptoms. Thus, stress models are being refined to be used as systemic in counterpoint to bladder centric animal models We hypothesised that different stress insults modulate the insula activity diversely. Two models were used in female Wistar rats, Maternal Deprivation Model (MDM, stress applied to new-borns and studies done at 6M) and Water Avoidance Stress test (WAS, stress is applied to 6M rats for 1h every 10 days and studies carried afterwards). Mechanical pain threshold was evaluated in L6/S1 dermatomes which share the spinal cord segments of bladder sensory innervation. Bladder function was evaluated by cystometry. Then Insulas were removed. One was immersed in RNAlater, homogenized, RNA extracted, and expression of different biomarkers assessed by real-time PCR. The other insula was immunoreacted against microglia markers Iba1 and Cd68. The analysis of positive cells used LASAF and ImageJ software. Morphometric analysis and 3D reconstructions used IMARIS software.info:eu-repo/semantics/publishedVersio

Methamphetamine promotes a-tubulin deacetylation in endothelial cells: The protective role of acetyl-L-carnitine

Methamphetamine (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

Acetil-L-Carnitina como neuroprotetor na excitotoxicidade do glutamato

A excitotoxicidade consiste na ativação excessiva de recetores de glutamato, causada pela acumulação extracelular deste neurotransmissor. A ativação dos recetores de glutamato promove o aumento excessivo da concentração intracelular de cálcio e morte neuronal associada à ativação de protéases como calpaínas e caspases - acontecimentos típicos de patologias como isquémia cerebral, Alzheimer, Parkinson e Huntington. A acetil-L-carnitina (ALC) é um éster da L-carnitina com funções neuroprotetoras, mas o mecanismo envolvido permanece desconhecido.info:eu-repo/semantics/publishedVersio

Primeiro estudo sobre a produção pictórica de Vitorino Ribeiro : géis de limpeza de PVA-Bórax

O presente relatório diz respeito ao estágio curricular desenvolvido na Divisão Municipal de Museus (DMM) da Câmara Municipal do Porto e com foco no estudo sobre a produção pictórica do pintor Vitorino Ribeiro (1849-1928) e a exploração de metodologia de limpeza com hidrogéis PVA-Bórax. A primeira parte do estágio realizou-se nas Reservas Municipais de Ramalde e teve como objetivo o levantamento e inventariação da Coleção de pintura Joaquim Vitorino Ribeiro, assim como o estudo técnico, artístico e histórico de quatro esbocetos do artista, pertencentes a um conjunto de 24 obras todas da mesma época. Estas pinturas têm características em comum como, por exemplo, representarem temáticas condizentes com a fase de formação artística, carimbos do fabricante, carimbos indicadores dos formatos normalizados das telas, a utilização de grades fixas, preparações comerciais sobre as quais o artista aplicava imprimaduras acinzentadas nas carnações. A investigação reúne, então, o estudo sobre Vitorino Ribeiro e o seu percurso como artista e as influências técnicas artísticas e históricas dos seus Mestres e colegas contemporâneos. Trata-se do primeiro estudo feito ao acervo de pintura pertencente à Coleção Vitorino Ribeiro doada à Câmara Municipal do Porto, assim como a um pequeno conjunto de obras selecionadas pelas suas caraterísticas superficiais. Já a segunda parte do estágio destinou-se à exploração da metodologia de limpeza através de hidrogéis de PVA-Bórax sobre quatro esbocetos e à intervenção de conservação e restauro integral de um desses esbocetos que apresentava maiores problemas. O critério para a escolha das quatro obras baseou-se nas diferentes características superficiais: um esboceto com um verniz final não-original e em más condições; um esboceto sem qualquer revestimento final; um esboceto com pentimenti e com áreas da preparação expostas e por fim, um esboceto com manchas concêntricas brancas à base de calcite e sulfato de bário, segundo µ-FTIR. Estas manchas foram encontradas em mais obras pertencentes à coleção, pelo que o seu estudo se revestiu de particular importância, embora sejam necessários mais meios e estudos para aprofundar as causas e soluções para este fenómeno. Pretendeu-se dar um contributo para o conhecimento deste pintor tão pouco estudado, assim como da coleção de pintura existente na DMM, potenciando a sua divulgação e a sua re-descoberta.The present internship report at the Divisão Municipal de Museus (DMM) of the Câmara Municipal do Porto, focusing on the study of the pictorial production of the Portuguese painter Vitorino Ribeiro (1849-1928) and the exploration of a cleaning methodology using PVA-Borax hydrogels. The first part of the internship aimed at surveying and inventorying the Joaquim Vitorino Ribeiro painting collection, as well as conducting technical, artistic, and historical studies of four sketches by the artist, comprising a set of 24 works, all from the same period. These paintings share common characteristics, such as themes consistent with the artist's formative phase, manufacturer stamps, stamps indicating standardized canvas sizes, the use of fixed stretchers, and commercial preparations over which the artist applied grayish underpaintings to the carnations. The research thus gathers the study of Vitorino Ribeiro and his journey as an artist, as well as the technical and historical artistic influences of his master’s and contemporary colleagues. It represents the first study conducted on the painting collection belonging to the Vitorino Ribeiro collection donated to the Câmara Municipal do Porto, as well as a small selection of works chosen for their surface characteristics. The second part of the internship was dedicated to the exploration of the cleaning methodology using PVA-Borax hydrogels on four sketches and to the integral conservation and restoration intervention of one of these sketches that presented greater problems. The criterion for choosing the four works was based on the different surface characteristics: a sketch with a non-original final varnish and in poor condition; a sketch without any final coating; a sketch with pentimenti and with exposed areas of the preparation and finally, a sketch with white concentric stains based on calcite and barium sulphate, according to µ-FTIR. These stains were found in more works belonging to the collection, so their study was of particular importance, although more means and studies are needed to deepen the causes and solutions for this phenomenon. The aim was to contribute to the knowledge of this under-studied painter, as well as the painting collection at DMM, promoting its dissemination and re-discovery

NanoSPECT/MRI: a “new generation” high performance tool in pre-clinical imaging

Abstract Body: PreClinical Imaging it is an each-day evolving field, getting more and more important at distinct areas. The Biotechnology field is between those that taken the biggest benefit. With so much interest, it is not surprising that investment consented on Research and Development is also evolving at the same time that this specific branch of business into the Imaging Industry is getting more and more attention and growing in importance. Hybrid Imaging is also getting more and more interest in the last decade and actually there are different vendors proposing interesting technical solutions, making things easier for one that has the possibilities – and the knowledge about each will be the most adequate for the specific purpose on each situation! – to choose one (or more) of those solutions. This paper relates with the public presentation of images produced from the world first model of a new pre-clinical imaging system, an new equipment that mixes Nuclear Medicine and MRI Magnetic Resonance Imaging techniques, using as base the sub-millimeter Nano-SPECT (Single Photon Emission Computed Tomography), so an high-level performance system – the world reference, produced by MEDISO Company, in Hungary - from Nuclear Medicine and a new MRI component, optimized for Neurosciences, but able to perform adequately on other critical biological fields. As practical example from the possibilities being introduced, images from mice, which are being enrolled on a Neuroscience project, will be showed and will be discussed, but also will be compared with other images being produced on other, actually more current, technical solutions, in order to try to demonstrate the advantages and disadvantages of this specific new approach. Since this small – quick and unpretentious – comparison will be done, it is meant to make possible understanding the place and the real possibilities of this equipment, which it is expected to become available on the world market between the end of this year and the 1st Quarter from next year

Acetyl-L-Carnitine Prevents Methamphetamine-Induced Structural Damage on Endothelial Cells via ILK-Related MMP-9 Activity

Abstract 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.info:eu-repo/semantics/publishedVersio

The impact of psychostimulants on central and peripheral neuro-immune regulation: a scoping review of cytokine profiles and their implications for addiction

It is now well-accepted that psychostimulants act on glial cells causing neuroinflammation and adding to the neurotoxic effects of such substances. Neuroinflammation can be described as an inflammatory response, within the CNS, mediated through several cytokines, reactive oxygen species, chemokines and other inflammatory markers. These inflammatory players, in particular cytokines, play important roles. Several studies have demonstrated that psychostimulants impact on cytokine production and release, both centrally and at the peripheral level. Nevertheless, the available data is often contradictory. Because understanding how cytokines are modulated by psychoactive substances seems crucial to perspective successful therapeutic interventions, here, we conducted a scoping review of the available literature. We have focused on how different psychostimulants impact on the cytokine profile. Publications were grouped according to the substance addressed (methamphetamine, cocaine, methylphenidate, MDMA or other amphetamines), the type of exposure and period of evaluation (acute, short- or long-term exposure, withdrawal, and reinstatement). Studies were further divided in those addressing central cytokines, circulating (peripheral) levels, or both. Our analysis showed that the classical pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were those more investigated. The majority of studies have reported increased levels of these cytokines in the central nervous system after acute or repeated drug. However, studies investigating cytokine levels during withdrawal or reinstatement have shown higher variability in their findings. Although we have identified fewer studies addressing circulating cytokines in humans, the available data suggest that the results may be more robust in animal models than in patients with problematic drug use. As a major conclusion, an extensive use of arrays for relevant cytokines should be considered to better determine which cytokines, upon the classical ones, may be involved in the progression from episodic use to the development of addiction. A concerted effort is still necessary to address the link between peripheral and central immune players, including from a longitudinal perspective. Until there, the identification of new biomarkers and therapeutic targets to envision personalized immune-based therapeutics will continue to be unlikely.info:eu-repo/semantics/publishedVersio

Chronic ketamine administration impairs mitochondrial complex I in the rat liver

Aim Ketamine 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. Main methods 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. Key findings 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. Significance 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.info:eu-repo/semantics/publishedVersio

TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity

Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1β. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.We would like to thank Dr. João Relvas laboratory for the help with N9 microglia cell culture; Dr. Sofia Lamas for the guidance on the animal welfare and support with animal experiments (Animal facility, i3S); and to LC Sciences for the miRNA microarray data and analysis. The mass spectrometry technique was performed by Hugo Osório at the i3S Proteomics Scientific Platform with support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01–0145-FEDER-022125). This work was funded by project NORTE-01–0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.P.B. and J.B. are supported by FCT–Fundação para a Ciência e Tecnologia, through BiotechHealth PhD program fellowship (PD/BD/135490/2018) and Areas of Basic and Applied Biology PhD program fellowship (PD/BD/135450/2017), respectively.info:eu-repo/semantics/publishedVersio