Neuroimmune alterations in autism: a translational analysis focusing on the animal model of autism induced by prenatal exposure to valproic acid

Autism Spectrum Disorder (ASD) is a highly prevalent developmental disorder characterized by deficits in communication and social interaction and in stereotyped or repetitive behaviors. Besides the classical behavioral dyad, several comorbidities are frequently present in patients with ASD, such as anxiety, epilepsy, sleep disturbances and gastrointestinal tract dysfunctions. Although the etiology of ASD remains unclear, there is supporting evidence for the involvement of both genetic and environmental factors. Valproic acid (VPA) is an anticonvulsant and mood stabilizer that, when used during the gestational period, increases the risk of ASD in the offspring. The animal model of autism by prenatal exposure to VPA shows construct and face validity, since several changes seen in subjects with autism are also observed in the VPA animal model. Neuroimmune alterations are common both in autistic individuals and in animal models of autism. In addition, exposure to pathogens during the pregnancy is a known risk factor for ASD, and maternal immune activation can lead to autistic-like features in animals. Thus, immunological alterations in pregnancy could affect the developing embryo, since immune molecules can pass through the placental barrier. Here, we summarize important alterations in inflammatory markers, such cytokines and chemokines in patients with ASD and in the VPA animal model

Preventive effects of resveratrol against early-life impairments in the animal model of autism induced by valproic acid

Background: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by social interaction deficits and repetitive/stereotyped behaviors. Its prevalence is increasing, affecting one in 36 children in the United States. The valproic acid (VPA) induced animal model of ASD is a reliable method for investigating cellular, molecular, and behavioral aspects related to the disorder. Trans-Resveratrol (RSV), a polyphenol with anti-inflammatory and antioxidant effects studied in various diseases, has recently demonstrated the ability to prevent cellular, molecular, sensory, and social deficits in the VPA model. In this study, we examined the effects of prenatal exposure to VPA and the potential preventive effects of RSV on the offspring. Method: We monitored gestational weight from embryonic day 6.5 until 18.5 and assessed the onset of developmental milestones and morphometric parameters in litters. The generalized estimating equations (GEE) were used to analyze longitudinal data. Results: Exposure to VPA during rat pregnancy resulted in abnormal weight gain fold-changes on embryonic days 13.5 and 18.5, followed by fewer animals per litter. Additionally, we discovered a positive correlation between weight variation during E15.5-E18.5 and the number of rat pups in the VPA group. Conclusion: VPA exposure led to slight length deficiencies and delays in the onset of developmental milestones. Interestingly, the prenatal RSV treatment not only prevented most of these delays but also led to the early onset of certain milestones and improved morphometric characteristics in the offspring. In summary, our findings suggest that RSV may have potential as a therapeutic intervention to protect against the negative effects of prenatal VPA exposure, highlighting its importance in future studies of prenatal neurodevelopmental 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

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

Análise do efeito do dimetilsulfóxido (DMSO) pré-natal no núcleo medial póstero-dorsal da amígdala de ratos Wistar machos adultos

O composto dimetilsulfóxido (DMSO) é um veículo aprótico e anfipático usado em diversos estudos na pesquisa biomédica para solubilizar e carrear moléculas diferentes, podendo transportar compostos através de barreiras biológicas em administrações por via oral, intravenosa, intraperitoneal e tópica. Essa molécula também apresenta importantes propriedades analgésicas e anti-inflamatórias. No presente trabalho, avaliamos densidade, número e forma de espinhos dendríticos no subnúcleo póstero-dorsal da amígdala medial (MePD) de ratos Wistar adultos expostos ao DMSO durante o período gestacional. O DMSO, nesse caso, foi empregado como veículo do antioxidante e anti-inflamatório resveratrol (RSV), utilizado por nosso grupo como uma importante estratégia preventiva de características do tipo autista no modelo animal de autismo induzido por administração pré-natal de ácido valproico (VPA, solubilizado em salina 0,9%). O desenho experimental compreende 5 grupos de animais machos de 120 dias: (1) Näive (animais sem nenhuma intervenção); (2) Veículos DMSO (7,2 M, 1:2 em salina 0,9% v/v - 100 μL/kg/dia por via subcutânea, durante os dias Embrionários E6,5 ao E18,5) e salina 0,9% por via intraperitoneal, no dia E12,5; (3) RSV (3,6 mg/kg, uma injeção subcutânea diária do E6,5 ao E18,5) ou (4) VPA (600 mg/kg, uma injeção intraperitoneal no dia E12,5) e (5) RSV + VPA (administrados conforme indicado nos grupos 3 e 4). Para nossa surpresa, verificamos que os animais dos 4 grupos expostos aos tratamentos pré-natais apresentaram uma surpreendente e significativa diminuição na densidade e no número de espinhos dendríticos na MePD em relação ao grupo Näive. Esses dados indicam que o efeito observado está relacionado com a administração do veículo DMSO, visto que os grupos 2-5 apresentaram o mesmo perfil, independente ou não da administração de RSV ou VPA. A vulnerabilidade do MePD a estes fatores ambientais durante o período gestacional, pode estar associanda ao impacto de 13 injeções subcutâneas de DMSO em um período crítico da gestação, concomitante com um possível efeito sobre a atividade de receptores de estrogênio (ER), que sabidamente modulam densidade de espinhos dendríticos. Publicações anteriores do nosso grupo mostram que o RSV foi capaz de prevenir importantes alterações moleculares e comportamentais do tipo autista no modelo VPA. O presente trabalho indica que o mecanismo preventivo do RSV observado previamente não foi prejudicado pelas alterações encontradas na MePD. Assim, estudos futuros serão necessários para elucidar as vias biológicas envolvidas nessa drástica alteração em número e forma de espinhos dendríticos na MePD, como por exemplo o comportamento sexual, já bem muito bem relacionado com essa região. Os presentes dados também apontam para o uso cauteloso e a relevância de entender os efeitos bioativos de solventes comumente usados no campo da pesquisa.The compound dimethyl sulfoxide (DMSO) is an aprotic and amphipathic vehicle used in a variety of studies in biomedical research to solubilize and deliver different molecules. The delivery through biological barriers is one of the most important roles which DMSO is chosen as a vehicle by oral, intravenous, intraperitoneal and transdermal administration. However, subsequent studies demonstrated important analgesic and anti-inflammatory properties of DMSO exposure. In the present work, we evaluated the density, number and shape of dendritic spines in the medial posterodorsal subnucleus of medial amygdala (MePD) from adult Wistar rats exposed to DMSO during pregnancy. The DMSO was used as vehicle of Resveratrol (RSV), which presents antioxidant and anti-inflammatory roles, established by our research group as an important strategy to prevent autistic-like behaviors induced by the prenatal administration of valproic acid (VPA, solubilized in saline 0.9%). The experimental design comprises 5 groups: (1) Näive (no intervention); (2) Vehicles DMSO (7.2 M, 1:2 in saline 0.9% v/v - 100 μL/kg/day by subcutaneous administration from E6.5 to E18.5) and saline 0.9% by intraperitoneal administration in E12.5; (3) RSV (3.6 mg/kg by a diary subcutaneous administration from E6.5 to E18.5) and (5) RSV+VPA (administered as indicated by groups 3 and 4). Strikingly, we observed that animals from the 4 groups exposed to prenatal treatments presented an impressive and significative reduction in the density and number of dendritic spines in MePD compared to the Näive group. These data indicate that the observed effect could be possibly related to the DMSO vehicle administration, since the groups 2-4 presented the same profile, regardless the VPA or RSV administration. Besides, the vulnerability of MePD to environmental risk factors during pregnancy, associating the effect of 13 DMSO subcutaneous injections with a critical biological time, as well as the possible relationship with estrogen receptors (ER), which can modulate the density of dendritic spines. Recent studies from our research group demonstrated that RSV prevented important molecular and behavioral autistic-like feature in the VPA model. The present work showed that the preventive mechanism previously observed by RSV was not impaired by the alterations found in MePD. Therefore, future studies will be necessary to elucidate the biological pathways involved in these drastic alterations in number and shape of dendritic spines from MePD, as well as to clarify biological and behavioral aspects that could be influenced by changes in this subnucleus, for example, in sociosexual behavior. The data also highlights to a cautious use and to the relevance in understand the bioactive effects of solvents commonly used in research field

Análise do efeito do dimetilsulfóxido (DMSO) pré-natal no núcleo medial póstero-dorsal da amígdala de ratos Wistar machos adultos

O composto dimetilsulfóxido (DMSO) é um veículo aprótico e anfipático usado em diversos estudos na pesquisa biomédica para solubilizar e carrear moléculas diferentes, podendo transportar compostos através de barreiras biológicas em administrações por via oral, intravenosa, intraperitoneal e tópica. Essa molécula também apresenta importantes propriedades analgésicas e anti-inflamatórias. No presente trabalho, avaliamos densidade, número e forma de espinhos dendríticos no subnúcleo póstero-dorsal da amígdala medial (MePD) de ratos Wistar adultos expostos ao DMSO durante o período gestacional. O DMSO, nesse caso, foi empregado como veículo do antioxidante e anti-inflamatório resveratrol (RSV), utilizado por nosso grupo como uma importante estratégia preventiva de características do tipo autista no modelo animal de autismo induzido por administração pré-natal de ácido valproico (VPA, solubilizado em salina 0,9%). O desenho experimental compreende 5 grupos de animais machos de 120 dias: (1) Näive (animais sem nenhuma intervenção); (2) Veículos DMSO (7,2 M, 1:2 em salina 0,9% v/v - 100 μL/kg/dia por via subcutânea, durante os dias Embrionários E6,5 ao E18,5) e salina 0,9% por via intraperitoneal, no dia E12,5; (3) RSV (3,6 mg/kg, uma injeção subcutânea diária do E6,5 ao E18,5) ou (4) VPA (600 mg/kg, uma injeção intraperitoneal no dia E12,5) e (5) RSV + VPA (administrados conforme indicado nos grupos 3 e 4). Para nossa surpresa, verificamos que os animais dos 4 grupos expostos aos tratamentos pré-natais apresentaram uma surpreendente e significativa diminuição na densidade e no número de espinhos dendríticos na MePD em relação ao grupo Näive. Esses dados indicam que o efeito observado está relacionado com a administração do veículo DMSO, visto que os grupos 2-5 apresentaram o mesmo perfil, independente ou não da administração de RSV ou VPA. A vulnerabilidade do MePD a estes fatores ambientais durante o período gestacional, pode estar associanda ao impacto de 13 injeções subcutâneas de DMSO em um período crítico da gestação, concomitante com um possível efeito sobre a atividade de receptores de estrogênio (ER), que sabidamente modulam densidade de espinhos dendríticos. Publicações anteriores do nosso grupo mostram que o RSV foi capaz de prevenir importantes alterações moleculares e comportamentais do tipo autista no modelo VPA. O presente trabalho indica que o mecanismo preventivo do RSV observado previamente não foi prejudicado pelas alterações encontradas na MePD. Assim, estudos futuros serão necessários para elucidar as vias biológicas envolvidas nessa drástica alteração em número e forma de espinhos dendríticos na MePD, como por exemplo o comportamento sexual, já bem muito bem relacionado com essa região. Os presentes dados também apontam para o uso cauteloso e a relevância de entender os efeitos bioativos de solventes comumente usados no campo da pesquisa.The compound dimethyl sulfoxide (DMSO) is an aprotic and amphipathic vehicle used in a variety of studies in biomedical research to solubilize and deliver different molecules. The delivery through biological barriers is one of the most important roles which DMSO is chosen as a vehicle by oral, intravenous, intraperitoneal and transdermal administration. However, subsequent studies demonstrated important analgesic and anti-inflammatory properties of DMSO exposure. In the present work, we evaluated the density, number and shape of dendritic spines in the medial posterodorsal subnucleus of medial amygdala (MePD) from adult Wistar rats exposed to DMSO during pregnancy. The DMSO was used as vehicle of Resveratrol (RSV), which presents antioxidant and anti-inflammatory roles, established by our research group as an important strategy to prevent autistic-like behaviors induced by the prenatal administration of valproic acid (VPA, solubilized in saline 0.9%). The experimental design comprises 5 groups: (1) Näive (no intervention); (2) Vehicles DMSO (7.2 M, 1:2 in saline 0.9% v/v - 100 μL/kg/day by subcutaneous administration from E6.5 to E18.5) and saline 0.9% by intraperitoneal administration in E12.5; (3) RSV (3.6 mg/kg by a diary subcutaneous administration from E6.5 to E18.5) and (5) RSV+VPA (administered as indicated by groups 3 and 4). Strikingly, we observed that animals from the 4 groups exposed to prenatal treatments presented an impressive and significative reduction in the density and number of dendritic spines in MePD compared to the Näive group. These data indicate that the observed effect could be possibly related to the DMSO vehicle administration, since the groups 2-4 presented the same profile, regardless the VPA or RSV administration. Besides, the vulnerability of MePD to environmental risk factors during pregnancy, associating the effect of 13 DMSO subcutaneous injections with a critical biological time, as well as the possible relationship with estrogen receptors (ER), which can modulate the density of dendritic spines. Recent studies from our research group demonstrated that RSV prevented important molecular and behavioral autistic-like feature in the VPA model. The present work showed that the preventive mechanism previously observed by RSV was not impaired by the alterations found in MePD. Therefore, future studies will be necessary to elucidate the biological pathways involved in these drastic alterations in number and shape of dendritic spines from MePD, as well as to clarify biological and behavioral aspects that could be influenced by changes in this subnucleus, for example, in sociosexual behavior. The data also highlights to a cautious use and to the relevance in understand the bioactive effects of solvents commonly used in research field