Alteração de permeabilidade de barreira hematoencefálica e possíveis mecanismos subjacentes no contexto do transtorno do espectro autista : efeito preventivo do resveratrol

O Transtorno do Espectro Autista (TEA) pode apresentar uma infinidade de condições clínicas associadas, como maior volume encefálico nos primeiros anos de vida em uma parcela dos pacientes; dessa forma, nos propusemos a avaliar, em modelo animal de autismo, processos possivelmente associados à formação de macrocefalia, bem como revisar na literatura os possíveis mecanismos subjacentes a essa dinâmica. No Capítulo I, avaliamos o conteúdo encefálico de água, a permeabilidade da barreira hematoencefálica (BHE), a expressão das aquaporina (AQP) 1 e 4 e da proteína ácida fibrilar glial (GFAP, um marcador de astrócitos) no modelo animal de TEA por exposição ao ácido valproico (VPA), e o potencial efeito do resveratrol (RSV). O tratamento com RSV preveniu a maior permeabilidade da BHE e o edema encefálico presentes no grupo VPA. A exposição ao VPA diminuiu os níveis da AQP1 no plexo coroide, na área somatossensorial primária, na região da amígdala e no córtex pré-frontal medial, reduziu os níveis de AQP4 no córtex pré-frontal medial e aumentou AQP4 na área somatossensorial primária (com prevenção pelo RSV), além de aumentar o número de astrócitos e a imunomarcação de GFAP na área somatossensorial primária e no córtex pré-frontal medial, com melhora funcional promovida pelo RSV. No Capítulo II, fizemos uma extensa revisão da literatura e observamos a translacionalidade das alterações imunológicas entre pacientes com TEA e o modelo de TEA por exposição ao VPA. As alterações neuroimunológicas são uma marca registrada no TEA; nesse sentido, hipotetizamos que a indução do modelo de TEA pelo VPA possivelmente envolve mecanismos de ativação imunitária materna e que as alterações neuroimunológicas podem estar por trás das alterações observadas no Capítulo I, uma vez que são relacionadas com disfunções de BHE. Finalmente, no Capítulo III, revisamos a contribuição das alterações no metabolismo das purinas na fisiopatologia do TEA, pois, apesar da etiologia desse transtorno permanecer desconhecida, algumas rotas biológicas já foram associadas ao desencadeamento e/ou progressão do TEA, como a sinalização purinérgica. Em resumo, demonstramos a importante contribuição do modelo VPA também nos estudos de disfunções de barreiras neurais – ampliando os conhecimentos e a aplicabilidade desse modelo –, bem como reforçamos o caráter neuroprotetor preventivo do RSV nas disfunções da BHE devido ao seu papel tanto na manutenção da integridade da BHE quanto na prevenção de alterações subjacentes observadas. Finalmente, a partir de investigações translacionais, postulamos que as disfunções neuroimunológicas podem promover as alterações tanto em barreiras neurais quanto em vias biológicas associadas ao TEA, como a sinalização purinérgica, o que pode contribuir, não só na fisiopatologia, mas também na manutenção das características associadas a esse transtorno.Autism Spectrum Disorder (ASD) can present a plethora of associated clinical conditions, such as increased brain volume in the first years of life in a portion of patients; therefore, we proposed to evaluate, in an animal model of autism, processes possibly associated with the formation of macrocephaly, as well as to review, in the literature, the possible mechanisms underlying this dynamic. In Chapter I, we evaluated brain water content, blood-brain barrier (BBB) permeability, aquaporin (AQP) 1 and 4, and glial fibrillary acidic protein (GFAP, a marker of astrocytes) expression in the animal model of ASD by exposure to valproic acid (VPA), and the potential effect of resveratrol (RSV). Treatment with RSV prevented the increased permeability of the BBB and the brain edema present in the VPA group. Exposure to VPA decreased AQP1 levels in the choroid plexus, primary somatosensory area, amygdala region, and medial prefrontal cortex, reduced AQP4 levels in the medial prefrontal cortex, and increased AQP4 in the primary somatosensory area (with prevention by RSV), in addition to an increase in the number of astrocytes and GFAP immunostaining in the primary somatosensory area and in the medial prefrontal cortex, with functional improvement promoted by RSV. In Chapter II, we revised the literature and looked at the translationality of immunological changes between ASD patients and the VPA exposure model of ASD. Neuroimmunological changes are a hallmark of ASD; in this sense, we hypothesized that the induction of the ASD model by the VPA possibly involves mechanisms of maternal immune activation and that neuroimmunological alterations may be behind the alterations observed in Chapter I, since they are related to BBB dysfunctions. Finally, in Chapter III, we also reviewed the contribution of changes in purine metabolism to the pathophysiology of ASD, because, although the etiology of this disorder remains unknown, some biological pathways have already been associated with the triggering and/or progression of ASD, such as purinergic signaling. In summary, we demonstrated the important contribution of the VPA model also in the studies of neural barriers dysfunctions – expanding the knowledge and applicability of this model –, as well as reinforcing the preventive neuroprotective character of RSV in BBB dysfunctions due to its role both in maintaining the BBB integrity and prevention of underlying changes observed. Finally, from translational investigations, we postulated that neuroimmunological dysfunctions could promote changes both in neural barriers and in biological pathways associated with ASD, such as purinergic signaling, which can contribute not only to the pathophysiology but also to the maintenance of the characteristics associated with this disorder

Avaliação de ratas submetidas a diferentes níveis de suplementação com folato durante o período gestacional

Nos últimos anos, a suplementação com ácido fólico (AF) vem sendo amplamente incentivada devido à sua importante prevenção dos defeitos no fechamento do tubo neural (DFTN). Atualmente, a Organização Mundial da Saúde (OMS) recomenda que as gestantes recebam AF na dosagem de 400μg por dia, até o final do primeiro trimestre. Porém, com as políticas públicas de fortificação de alimentos com AF, a ingestão deste pode estar excedendo o limite recomendado e são escassos os estudos avaliando as consequências dessa “super” exposição a longo prazo nas grávidas que utilizaram o AF como suplementação. Assim, nosso objetivo foi avaliar os efeitos da suplementação com AF sob parâmetros motores e de memória após o período de prenhez das ratas Wistar. Foram utilizadas vinte e uma ratas Wistar, e assim que confirmada a prenhez, divididas em três grupos experimentais: Controle (ração padrão de biotério), AF2 (2mg AF/Kg de dieta) e AF20 (20mg AF/Kg dieta). Tanto a ração quanto os animais foram pesados todos os dias, sendo as avaliações comportamentais através dos testes de Campo Aberto, Reconhecimento de Objetos e Esquiva Inibitória, as quais foram iniciadas após o desmame (n=7/grupo). Nossos resultados não mostram diferença entre os grupos, apenas entre dias dentro do mesmo grupo, quando analisado o ganho de peso diário e a quantidade de ração consumida durante a gestação. Quando avaliada a atividade locomotora e a memória aversiva, os grupos não apresentam quaisquer diferenças entre eles. No entanto, quando avaliada a memória de longa duração no teste de reconhecimento de objetos ambos os grupos suplementados apresentam um pior desempenho, sugerindo um déficit na formação da memória. No seu conjunto, nossos resultados demonstram que tanto a dosagem recomendada (2mg/Kg) quanto a em excesso (20mg/Kg), em animais tratados durante os 21 dias de gestação estão associados a um déficit na memória de reconhecimento, mas não na memória aversiva e nem na atividade locomotora.In recent years, supplementation with folic acid (FA) has been widely encouraged due to important prevention of neural tube defects (NTD). Currently, the World Health Organization (WHO) recommends that pregnant women receive a daily dose of 400μg to FA until the end of the first trimester. However, with the public policy of fortification of foods with AF, the intake of this may be exceeding the recommended limit and there are few studies evaluating the consequences of this "super" long-term exposure in pregnant women who used the FA as supplementation. Thus, our aim was to evaluate effects of supplementation to FA in both motor and memory parameters after the pregnancy period in female Wistar rats. Twenty-one Wistar rats were used, and so confirmed the pregnancy, divided into three groups: control (standard diet of biotery), FA2 (2 mg FA/kg diet) and FA20 (20 mg FA/kg diet). Both feed and the animals were weighed every day, and the behavioral assessments through Open Field, Object Recognition and Inhibitory Avoidance test, which began after weaning (n = 7/group). Our results show no difference between the groups, only between days within the same group, when analyzing the average daily gain and the feed intake during pregnancy. When measured locomotor activity and aversive memory, the groups do not present any differences between them. However, when evaluating the long-term memory in the object recognition test both groups supplemented have a poorer performance, suggesting a deficit in formation of memory. Taken together, our results demonstrate that both recommended dosage (2mg/kg) and excess (20 mg/kg) in treated animals during the 21 days of pregnancy are associated with a deficit in the recognition memory but not in memory or aversive and locomotor activity

Data on social transmission of food preference in a model of autism induced by valproic acid and translational analysis of circulating microRNA

This article contains data of Social Transmission of Food Preference in an animal model of autism and the evaluation of a set of microRNA analyzed in autistic patients and animal model of autism. The analyses of the absolute consumption of two flavored food by male rats prenatally exposed to valproic acid (VPA) and treated with resveratrol (RSV), showed that VPA animals show a trend to eat less of the flavored food presented by a demonstrator rat. We also identified 13 microRNA with similar levels among rodents' experimental groups, as well as 11 microRNA with no alterations between autistic and control subjects. Further evaluation of mechanisms of VPA and RSV actions on behavioral and molecular alterations can shed light in important biomarkers and etiological triggers of autistic spectrum disorders

Resveratrol prevents cellular and behavioral sensory alterations in the animal model of autism induced by valproic acid

Autism spectrum disorder (ASD) is characterized by impairments in both social communication and interaction and repetitive or stereotyped behaviors. Although its etiology remains unknown, genetic and environmental risk factors have been associated with this disorder, including the exposure to valproic acid (VPA) during pregnancy. Resveratrol (RSV) is an anti-inflammatory and antioxidant molecule known to prevent social impairments in the VPA animal model of autism. This study aimed to analyze the effects of prenatal exposure to VPA, as well as possible preventive effects of RSV, on sensory behavior, the localization of GABAergic parvalbumin (PV+) neurons in sensory brain regions and the expression of proteins of excitatory and inhibitory synapses. Pregnant rats were treated daily with RSV (3.6 mg/kg) from E6.5 to E18.5 and injected with VPA (600 mg/kg) in the E12.5. Male pups were analyzed in nest seeking behavior and in whisker nuisance task. At P30, the tissues were removed and analyzed by immunofluorescence and western blotting. Our data showed for the first time an altered localization of PV+-neurons in primary sensory cortex and amygdala. We also showed a reduced level of gephyrin in the primary somatosensory area of VPA animals. The treatment with RSV prevented all the aforementioned alterations triggered by VPA. Our data shed light on the relevance of sensory component in ASD and highlights the interplay between RSV and VPA animal model as an important tool to investigate the pathophysiology of ASD

Resveratrol prevents cytoarchitectural and interneuronal alterations in the valproic acid rat model of autism

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by several alterations, including disorganized brain cytoarchitecture and excitatory/inhibitory (E/I) imbalance. We aimed to analyze aspects associated with the inhibitory components in ASD, using bioinformatics to develop notions about embryonic life and tissue analysis for postnatal life. We analyzed microarray and RNAseq datasets of embryos from different ASD models, demonstrating that regions involved in neuronal development are affected. We evaluated the effect of prenatal treatment with resveratrol (RSV) on the neuronal organization and quantity of parvalbumin-positive (PV+), somatostatin-positive (SOM+), and calbindin-positive (CB+) GABAergic interneurons, besides the levels of synaptic proteins and GABA receptors in the medial prefrontal cortex (mPFC) and hippocampus (HC) of the ASD model induced by valproic acid (VPA). VPA increased the total number of neurons in the mPFC, while it reduced the number of SOM+ neurons, as well as the proportion of SOM+, PV+, and CB+ neurons (subregion-specific manner), with preventive effects of RSV. In summary, metabolic alterations or gene expression impairments could be induced by VPA, leading to extensive damage in the late developmental stages. By contrast, due to its antioxidant, neuroprotective, and opposite action on histone properties, RSV may avoid damages induced by VPA

Behavioral alterations in autism model induced by valproic acid and translational analysis of circulating microRNA

Autism spectrum disorder (ASD) is characterized by difficulties in social interaction, communication and language, and restricted repertoire of activities and interests. The etiology of ASD remains unknown and no clinical markers for diagnosis were identified. Environmental factors, including prenatal exposure to valproic acid (VPA), may contribute to increased risk of developing ASD. MicroRNA (miRNA) are small noncoding RNA that regulate gene expression and are frequently linked to biological processes affected in neurodevelopmental disorders. In this work, we analyzed the effects of resveratrol (an antioxidant and anti-inflammatory molecule) on behavioral alterations of the VPA model of autism, as well as the levels of circulating miRNA. We also evaluated the same set of miRNA in autistic patients. Rats of the VPA model of autism showed reduced total reciprocal social interaction, prevented by prenatal treatment with resveratrol (RSV). The levels of miR134-5p and miR138-5p increased in autistic patients. Interestingly, miR134-5p is also upregulated in animals of the VPA model, which is prevented by RSV. In conclusion, our findings revealed important preventive actions of RSV in the VPA model, ranging from behavior to molecular alterations. Further evaluation of preventive mechanisms of RSV can shed light in important biomarkers and etiological triggers of ASD

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment had no impact on VPA-induced upregulation of P2X4 and P2Y2 in hippocampus, and P2X4 in medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic modulation in behavioral, molecular, and immunological aberrations described in VPA model, and suggest that purinergic system might be a potential target for pharmacotherapy in preclinical studies of ASD