Abnormal empathy-like pro-social behaviour in the valproic acid model of autism spectrum disorder

Impairments in social behaviour are a defining feature of autism spectrum disorder (ASD). Individuals with ASD also usually present some difficulty to recognise or understand another person's feelings. Therefore, it is possible that altered empathy processing could hinder typical social interaction in ASD. Recently, robust paradigms confirmed that rodents show primordial forms of empathy-like behaviour. Therefore, in this work, we used one of these new protocols to test pro-social behaviour in the rat model of autism induced by Valproic Acid (VPA). We also evaluated possible beneficial effects of Resveratrol, since it can prevent social deficits in the VPA model. Rats were tested on their ability to open a restrainer to release a trapped conspecific. Exposure to VPA precludes the timely manifestation of this empathy-like behaviour, but does not affect its continuation after its first expression. We also found a significant correlation between average speed during the first day of test and becoming an Opener. Similarly, rats able to open the restrainer on the first day had an increased likelihood of repeating this behaviour in the later days of the testing programme. We did not find any protective effects of Resveratrol. Further investigation of empathy-like behaviour in the VPA model and in other models of autism could help to clarify the behavioural and neural processes underpinning the basic aspects of empathy alterations in autistic individuals. [Abstract copyright: Copyright © 2019. Published by Elsevier B.V.

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

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

Obtenção e caracterização de formulações de liberação controlada de atrazina e avaliação dos perfis de liberação

A aplicação de atrazina (ATZ) associada a sistemas poliméricos pode representar uma alternativa para diminuir o impacto ambiental decorrente de sua aplicação e aumentar sua eficiência agronômica. Visando avaliar a viabilidade da utilização futura destes sistemas associados em solos, foram preparadas formulações em dois diferentes tipos de matriz polimérica. As formulações em matriz inorgânica foram sintetizadas pelo método sol-gel. As formulações em matriz orgânica foram obtidas por solubilização com posterior secagem por aspersão. Foram avaliadas as características físico-químicas e os perfis de liberação de ATZ a partir das formulações. As formulações em matriz inorgânica apresentaram eficiências de encapsulação de ATZ entre 93 a 109 % e rendimento molar entre 97 e 107 %. As formulações em matriz orgânica apresentaram eficiências de encapsulação e rendimentos inferiores a 25 %, devido a perdas de herbicida durante o processo. A análise cromatográfica da ATZ extraída das formulações confirmou a presença de ATZ. O teor de umidade das formulações diminuiu com o aumento do teor de ATZ nas mesmas, o que pode estar relacionado com a baixa afinidade entre este herbicida e a água. O herbicida não apresentou ligação química com as matrizes testadas, porém a inserção do herbicida em matriz de sílica causou uma alteração na densidade eletrônica na molécula de ATZ. O número de cristais de ATZ livre e o diâmetro médio das partículas aumentam com a proporção água:TEOS e diminuiram com a proporção etanol:TEOS nas formulações em matriz inorgânica. A temperatura e a entalpia de fusão de ATZ aumentaram com a quantidade de ATZ nas formulações devido à ocorrência de maior número de cristais de ATZ. A cinética de liberação de ATZ nas formulações seguiu o modelo de Korsmeyer- Peppas e indicou a ocorrência simultânea de difusão e de erosão no processo. Entre as formulações preparadas, aquelas com maior teor de ATZ parecem ser as mais adequadas para emprego no campo devido a sua taxa de liberação mais lenta.The application of atrazine (ATZ) associated to polymeric systems may represent an alternative to reduce the environmental impact caused by the widespread application of this herbicide and simultaneously to increase the agronomic efficiency. Aiming the use of these particulated systems in soils, formulations were prepared by two different types of polymeric matrix. The formulations in inorganic matrix were prepared by the sol gel method, while formulations in organic matrix were obtained by spray drying. The physicochemical characteristics were evaluated and the release profiles of ATZ were measured. The inorganic matrix formulations presented encapsulation efficiency between 93 and 109 % and molar yield between 97 and 107 %. The organic matrix formulations presented encapsulation efficiency and yield less than 25 %, due to the loss of herbicide during the process. The presence of ATZ in the formulations was confirmed by chromatographic analysis. The water content of the formulations decreased with the increase of ATZ content, and that can be attributed to the low affinity between the herbicide and water. No chemical interaction between the herbicide and the matrix was observed. Nevertheless, the insertion of the herbicide in silica matrix caused an alteration in the electronic density of ATZ. The number of ATZ free crystals and the mean diameter of the particles increased with the increase of the water:TEOS proportion and decreased with the ethanol:TEOS proportion in the inorganic matrix formulations. The temperature and the melting enthalpy of ATZ increased with the ATZ content in the formulations due to the greater amount of isolated ATZ crystals. The release kinetics of ATZ from the formulations followed the Korsmeyer- Peppas model, and indicated that both diffusion and erosion acted on the release process. Among the formulations, those with the greater content of ATZ seemed to be the most adequate due to their slower release rate

Obtenção e caracterização de formulações de liberação controlada de atrazina e avaliação dos perfis de liberação

A aplicação de atrazina (ATZ) associada a sistemas poliméricos pode representar uma alternativa para diminuir o impacto ambiental decorrente de sua aplicação e aumentar sua eficiência agronômica. Visando avaliar a viabilidade da utilização futura destes sistemas associados em solos, foram preparadas formulações em dois diferentes tipos de matriz polimérica. As formulações em matriz inorgânica foram sintetizadas pelo método sol-gel. As formulações em matriz orgânica foram obtidas por solubilização com posterior secagem por aspersão. Foram avaliadas as características físico-químicas e os perfis de liberação de ATZ a partir das formulações. As formulações em matriz inorgânica apresentaram eficiências de encapsulação de ATZ entre 93 a 109 % e rendimento molar entre 97 e 107 %. As formulações em matriz orgânica apresentaram eficiências de encapsulação e rendimentos inferiores a 25 %, devido a perdas de herbicida durante o processo. A análise cromatográfica da ATZ extraída das formulações confirmou a presença de ATZ. O teor de umidade das formulações diminuiu com o aumento do teor de ATZ nas mesmas, o que pode estar relacionado com a baixa afinidade entre este herbicida e a água. O herbicida não apresentou ligação química com as matrizes testadas, porém a inserção do herbicida em matriz de sílica causou uma alteração na densidade eletrônica na molécula de ATZ. O número de cristais de ATZ livre e o diâmetro médio das partículas aumentam com a proporção água:TEOS e diminuiram com a proporção etanol:TEOS nas formulações em matriz inorgânica. A temperatura e a entalpia de fusão de ATZ aumentaram com a quantidade de ATZ nas formulações devido à ocorrência de maior número de cristais de ATZ. A cinética de liberação de ATZ nas formulações seguiu o modelo de Korsmeyer- Peppas e indicou a ocorrência simultânea de difusão e de erosão no processo. Entre as formulações preparadas, aquelas com maior teor de ATZ parecem ser as mais adequadas para emprego no campo devido a sua taxa de liberação mais lenta.The application of atrazine (ATZ) associated to polymeric systems may represent an alternative to reduce the environmental impact caused by the widespread application of this herbicide and simultaneously to increase the agronomic efficiency. Aiming the use of these particulated systems in soils, formulations were prepared by two different types of polymeric matrix. The formulations in inorganic matrix were prepared by the sol gel method, while formulations in organic matrix were obtained by spray drying. The physicochemical characteristics were evaluated and the release profiles of ATZ were measured. The inorganic matrix formulations presented encapsulation efficiency between 93 and 109 % and molar yield between 97 and 107 %. The organic matrix formulations presented encapsulation efficiency and yield less than 25 %, due to the loss of herbicide during the process. The presence of ATZ in the formulations was confirmed by chromatographic analysis. The water content of the formulations decreased with the increase of ATZ content, and that can be attributed to the low affinity between the herbicide and water. No chemical interaction between the herbicide and the matrix was observed. Nevertheless, the insertion of the herbicide in silica matrix caused an alteration in the electronic density of ATZ. The number of ATZ free crystals and the mean diameter of the particles increased with the increase of the water:TEOS proportion and decreased with the ethanol:TEOS proportion in the inorganic matrix formulations. The temperature and the melting enthalpy of ATZ increased with the ATZ content in the formulations due to the greater amount of isolated ATZ crystals. The release kinetics of ATZ from the formulations followed the Korsmeyer- Peppas model, and indicated that both diffusion and erosion acted on the release process. Among the formulations, those with the greater content of ATZ seemed to be the most adequate due to their slower release rate

Análise in silico de microRNA com perfis de expressão alterados no modelo animal de autismo induzido por exposição pré-natal ao ácido valpróico

Os Transtornos do Espectro do Autismo (TEA) reúnem um conjunto de alterações no desenvolvimento, caracterizado por dificuldades nos níveis de interação social, linguagem, comunicação, processo imaginativo e no repertório restrito de interesses e atividades. O autismo ainda possui etiologia desconhecida e até o momento nenhum tratamento ou marcador clínico para diagnóstico foi identificado. Apesar de alta herdabilidade, há uma alta heterogeneidade na sua arquitetura genética. Fatores ambientais podem contribuir com o aumento do risco do desenvolvimento do autismo, incluindo a exposição pré-natal a teratógenos como o ácido valpróico (VPA). MicroRNA são pequenos RNA não codificadores com aproximadamente 19-25 nucleotídeos que atuam como reguladores da expressão gênica, podendo agir no controle de diversos processos biológicos. O objetivo deste estudo foi avaliar os perfis de expressão de alguns miRNA no sangue total do modelo animal de autismo induzido por exposição pré-natal ao ácido valpróico (VPA). Além disso, as funções potenciais dessas moléculas foram avaliadas através da correlação com seus genes alvos, os quais poderiam, por sua vez, estar associados com os fundamentos da patofisiologia do autismo. Mudanças nos níveis de miRNA induzidas pelo VPA podem contribuir para algumas características relacionadas ao autismo. Os alvos preditos e validados dos miRNA que se mostraram alterados incluem alguns genes que estão envolvidos na síntese de proteínas, na resposta imune, inflamatória e à hipóxia, no desenvolvimento dos linfócitos e do sistema nervoso entérico e na transmissão sináptica. O presente estudo sugere que a avaliação da expressão de miRNA pode ser utilizada para a identificação potencial de rotas alteradas no autismo e podem constituir marcadores para a detecção e diagnóstico, além de auxiliar nas investigações sobre mecanismos de ação molecular algumas em vias de sinalização supostamente alterados no autismo.The Autism Spectrum Disorders (ASD) assemble a group of developmental disorders characterized by difficulties in levels of social interaction, language, communication, imaginative process and restricted repertoire of activities and interests. The etiology of autism has still unknown and no treatment or clinical markers for diagnosis were identified. Despite its high heritability, there is a high heterogeneity in its genetic architecture. Environmental factors may contribute to the increased risk of developing autism, including prenatal exposure to teratogens such as valproic acid (VPA). MicroRNA are small noncoding RNA with approximately 19-25 nucleotides that act as regulators of gene expression and may act in the control of many biological processes. The aim of this study was to evaluate the expression profile of miRNA levels in whole blood samples from animal model of autism by prenatal exposure to VPA. In addition, some potential roles of these miRNA were analyzed through correlation with their target genes, which could, in turn, be associated with the basis of the pathophysiology of autism. Changes in the miRNA levels induced by VPA may contribute to some features related to autism. The predicted and validated targets of the altered miRNA included some genes that are involved in protein synthesis, immune, inflammatory and hypoxia responses, lymphocytes and enteric nervous system development and synaptic transmission. The present study suggests that the assessment of the expression of miRNA may be used to perform the identification of potential pathways altered in autism and may constitute markers for the detection and diagnostics, in addition to studies related therapies and mechanistic investigations on signaling pathways putatively altered in autism

Modelo animal de autismo induzido por exposição pré-natal ao ácido valproico : estudos comportamentais, moleculares e estratégias terapêuticas

O Transtorno do Espectro Autista (TEA), segundo o DSM-5, se enquadra nos Transtornos do Desenvolvimento e é caracterizado por uma díade comportamental: 1) prejuízos na comunicação e interação social e 2) comportamentos repetitivos ou estereotipados. Apesar da etiologia do TEA ser desconhecida, tanto fatores genéticos quanto ambientais já foram associados à desordem, incluindo a utilização de ácido valproico (VPA) durante a gestação. Observando essa relação importante, desenvolveu-se um modelo animal de autismo induzido pela exposição pré-natal ao VPA, o qual já foi amplamente validado em aspectos comportamentais e moleculares. No primeiro capítulo desta tese, utilizamos o modelo animal de autismo obtido através de uma única injeção intraperitoneal de VPA (600mg/kg) no dia E12,5 nas ratas Wistar prenhes e testamos um tratamento pré-natal com resveratrol (RSV), através de injeções subcutâneas (3,6 mg/Kg) administradas nas ratas prenhes nos dias E6,5-E18,5. O tratamento pré-natal com RSV demonstrou ser capaz de prevenir alterações na sociabilidade recíproca, porém não teve impacto nos prejuízos na memória olfativa e comportamentos repetitivos induzidos pelo VPA. No que se refere aos dados de microRNA (miRNA), RSV foi capaz de prevenir a alteração na expressão de miR134-5p, o qual também se observou alterado em pacientes com TEA, juntamente com o miR138-5p, ambos com alvos associados à modulação do citoesqueleto na estrutura de espinhos dendríticos. Em conjunto, esses resultados demonstram que o RSV altera vias importantes no modelo, possivelmente através das suas características antioxidantes e anti-inflamatórias, as quais contrapõem os aspectos inflamatórios do VPA. Este trabalho permitiu o aprimoramento e melhor conhecimento da técnica de RT-qPCR para análise de miRNA, sendo tema do segundo capítulo da presente tese, reunindo diferentes estratégias que auxiliaram a superar potenciais problemas e interferentes no uso dessa técnica. Finalmente, no terceiro capítulo, utilizamos o modelo VPA para avaliar o efeito do tratamento pós-natal com suramina, através de uma única injeção subcutânea (20 mg/kg) administrada nos filhotes machos na idade P30. Este tratamento foi capaz de reverter alterações de sociabilidade, novidade social e comportamento do tipo ansioso, enquanto os prejuízos na sociabilidade recíproca, comportamento exploratório, estereotipia e processamento sensorial não foram revertidos por esse tratamento. Nos dados moleculares, a suramina não foi capaz de reverter o aumento de expressão nos receptores purinérgicos P2X4 (hipocampo e córtex pré-frontal medial) e P2Y2 (hipocampo), porém reverteu o aumento de IL-6 promovido pelo VPA. Assim, possivelmente a modulação comportamental associada à suramina parece não estar associada a interações específicas nos receptores purinérgicos, mas sim com uma modificação neuroimune através da interleucina pró-inflamatória IL-6, demonstrando a importância do sistema imunológico na fisiopatologia do TEA. De forma geral, a tese contribuiu para elucidar mecanismos envolvidos no desenvolvimento das características do tipo autista, demonstrando o papel relevante das alterações neuroimunes e da modulação de alvos por miRNA, as quais, em conjunto, podem contribuir para o desenvolvimento de métodos diagnósticos e adequação de estratégias farmacológicas voltados ao TEA. Palavras-chave: Comportamento animal, microRNA, neuroimune, PCR, resveratrol, sistema purinérgico, suramina, transtorno do espectro autista.According to DSM-5, Autism Spectrum Disorder (ASD) is a developmental disorder characterized by a behavioral dyad: 1) deficits in communication and social interaction, and 2) repetitive and stereotyped behaviors. Although the etiology of ASD is still unknown, both genetic and environmental factors have been associated with the disorder, including the use of valproic acid (VPA) during gestation. Observing this important relationship, an animal model of autism induced by prenatal exposure to VPA was developed, which has already been widely validated in behavioral and molecular aspects. In the first chapter of this thesis, we used the animal model obtained through a single intraperitoneal injection of VPA (600 mg/kg) at E12.5 in pregnant Wistar rats and tested a prenatal treatment with resveratrol (RSV) by subcutaneous injections (3.6 mg/kg) administered in the pregnant rats at E6.5 to E18.5. The prenatal treatment with RSV was able to prevent changes in the reciprocal sociability, but had no impact on the deficits in olfactory memory and repetitive behavior induced by VPA. Regarding the microRNA (miRNA) data, RSV treatment was able to prevent the alteration in the expression of miR134-5p, which also was altered in ASD patients along with miR138-5p, both with targets associated with cytoskeletal modulation in the structure of dendritic spines. Taken together, these results demonstrate that RSV alters important pathways in the model, possibly through its antioxidant and anti-inflammatory properties, which counteract the inflammatory aspects of VPA. This work allowed the improvement and better knowledge of the RT-qPCR technique for miRNA analysis, which was the theme of the second chapter of this thesis, combining different strategies to overcome potential problems and interferences in this methodology. Finally, in the third chapter we used the same animal model to evaluate the effect of postnatal treatment with suramin after a single subcutaneous injection (20 mg/kg) administered to male pups at P30. This treatment was able to revert VPA-induced deficits in sociability, social novelty, and anxiety-like behavior, whilst present no effect on impairments in reciprocal sociability, exploratory behavior, repetitive behavior and sensory processing. In the molecular data, suramin was not able to reverse the increase of expression in the purinergic receptors P2X4 (hippocampus and medial prefrontal cortex) and P2Y2 (hippocampus), but reversed the VPA-induced increase of proinflammatory interleukin IL-6. Thus, behavioral modulation associated with suramin appears to be related not with specific interactions in purinergic receptors, but with a neuroimmune modification through the IL-6, indicating the importance of the immune system in the ASD pathophysiology. In general, the thesis contributed to elucidate the mechanisms involved in the development of autistic-like features, demonstrating the relevant role of neuroimmune alterations and the modulation of targets by miRNA, which, together, may contribute to the development of diagnostic methods and improvement of pharmacological strategies related to ASD