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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.

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.

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 (PVC) 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 (NS) behavior and in whisker nuisance task (WNT). At P30, the tissues were removed and analyzed by immunofluorescence and western blotting. Our data showed for the first time an altered localization of PVC-neurons in primary sensory cortex and amygdala. We also showed a reduced level of gephyrin in the primary somatosensory area (PSSA) 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.

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.

O Transtorno do Espectro Autista (TEA) é uma desordem do neurodesenvolvimento caracterizada, segundo o DSM-V, por uma díade comportamental: déficits de comunicação e interação social além de comportamentos repetitivos ou estereotipados. Uma observação de extrema importância na clínica é a de maior volume encefálico nos primeiros anos de vida do paciente com TEA, sendo esse aumento revertido ao longo da vida, tendo normalizado já no início da adolescência. Porém, durante o desenvolvimento é frequente uma maior permeabilidade de barreiras neurais. Apesar da etiologia do autismo ser desconhecida, tanto fatores genéticos quanto ambientais já foram associados à desordem, incluindo a utilização de ácido valproico (VPA) durante a gestação. Por isso, nosso grupo tem utilizado um modelo animal de autismo induzido pela exposição pré-natal ao VPA. Por meio desse modelo, demonstramos que o tratamento pré-natal com resveratrol (RSV) foi capaz de prevenir comportamentos do tipo autista na prole do grupo VPA. O objetivo do presente trabalho foi avaliar a permeabilidade de barreiras encefálicas e a expressão e localização de aquaporina (AQP) 1, 4 e 9 em diferentes regiões do Sistema Nervoso Central (SNC) em modelo animal de autismo induzido por exposição pré-natal ao VPA. A obtenção dos animais passou por um processo inicial de acasalamentos, onde a confirmação da fecundação foi através da identificação de espermatozoides no líquido vaginal na manhã posterior ao pareamento. O modelo animal foi obtido através de uma única injeção intraperitoneal de VPA (600mg/kg) no E12,5 nas fêmeas prenhes. O tratamento com RSV (3,6 mg/Kg) ocorreu dos dias E6,5 ao E18,5 via subcutânea. Para a obtenção do material biológico dos filhotes machos com 30 dias, diferentes procedimentos foram realizados, de acordo com cada técnica, mediante preceitos éticos de experimentação animal para obtenção do tecido a fresco e para fixação com paraformaldeído 4%. No ensaio de permeabilidade de barreira, os animais do grupo VPA apresentaram maior entrada do corante azul de Evans nas regiões do plexo coroide e na área somatossensorial. Na análise da expressão proteica por western blotting, o grupo VPA apresentou diminuição de AQP1 e aumento de AQP4 na área somatossensorial e diminuição da AQP9 na região da amígdala, sendo só o aumento de AQP4 prevenido pelo tratamento com RSV. Com a técnica de imunofluorescência, observou-se diminuição somente na marcação da AQP1 no plexo coroide, juntamente com alteração morfológica dessa estrutura em ambos os grupos que receberam VPA. Esses resultados de maior permeabilidade e de alteração em AQP corroboram com os dados clínicos de edema e de maior volume encefálico descritos na literatura. Os mecanismos envolvendo esse perfil de expressão alterado no contexto do TEA ainda permanecem desconhecidos. Podemos concluir com o presente trabalho que os animais do grupo VPA possuem maior marcação do corante azul de Evans e alteração na expressão proteica de AQP em diferentes regiões do SNC, indicando possíveis alterações na permeabilidade encefálica no modelo animal de autismo. Autistic Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by a behavioral dyad: communication and social interaction deficits, as repetitive or stereotyped behaviors. A clinical observation of extreme importance is the greater encephalic volume early in life of ASD patients, being this reverted throughout and normalized in the beginning of the adolescence. However, during development a greater permeability of neural barriers is frequent. Although the etiology of autism is unknown, both genetic and environmental factors have been associated with the disorder, including use of valproic acid (VPA) during gestation. Therefore, our group has used an animal model of autism induced by prenatal exposure to VPA. By this model, we demonstrated that prenatal treatment with resveratrol (RSV) was able to prevent autistic behaviors in offspring of VPA animals. The aim of the present study was to evaluate the permeability of brain barriers and the expression and location of aquaporin (AQP) 1, 4 and 9 in different regions of the Central Nervous System (CNS) in animal model of autism induced by prenatal exposure to VPA. Animals were mated overnight and the fertilization was confirmed by the identification of spermatozoa in the vaginal fluid in the next morning after the pairing. The animal model of autism was induced by a single intraperitoneal injection of VPA (600mg/kg) at E12.5 in pregnant females. Treatment with RSV (3.6 mg/kg) occurred from days E6.5 to E18.5 subcutaneously. In order to obtain the biological samples of 30-day-old male offspring, different procedures were performed according to each technique, using ethical precepts of animal experimentation to obtain fresh tissue and for fixation with 4% paraformaldehyde. In the barrier permeability assay, animals of the VPA group showed increased Evans blue dye labeling in choroid plexus and somatosensory area. In the Western blotting protein expression analyses, the VPA group showed decreased AQP1 and increased AQP4 in somatosensory area and decreased AQP9 in amygdala region, but the RSV treatment was able to prevent only the increase in AQP4 levels. According immunofluorescence assay, only the AQP1 labeling was observed in choroid plexus, together with morphological alteration of this structure in VPA-exposed animals. These results of increased permeability and alteration in aquaporins levels corroborate the clinical data of edema and of larger encephalic volume described in the literature. However, mechanisms involving this altered expression profile in the context of ASD remain unknown. We conclude with the present work that the animals of the VPA group presented an increased Evans blue dye labeling and alteration in protein expression of aquaporins in different regions of the CNS, indicating possible alterations in brain permeability in animal model of autism.