Efeito do resveratrol sobre parâmetros astrocíticos e permeabilidade de barreira hematoencefálica no modelo animal de autismo induzido por exposição pré-natal ao ácido valproico

O Transtorno do Espectro Autista (TEA) é uma desordem do neurodesenvolvimento de elevada prevalência, afetando 1:59 crianças de até 8 anos de idade nos Estados Unidos. De acordo com a 5ª edição do Manual Diagnóstico e Estatístico de Transtornos Mentais (DSM-5), o diagnóstico do TEA é dado pela díade comportamental composta por prejuízos na comunicação e interação social, bem como por interesses restritos, comportamentos repetitivos e estereotipados. Além das alterações comportamentais, o TEA pode apresentar muitas comorbidades associadas e um importante achado clínico é a presença de maior volume encefálico nos primeiros anos de vida. Embora a etiologia do TEA permaneça desconhecida, sabe-se da importância de fatores genéticos e ambientais, como a exposição pré-natal ao ácido valproico (VPA). O resveratrol (RSV) é uma molécula anti-inflamatória, anti-oxidante e neuroprotetora conhecida por prevenir comportamentos sociais no modelo animal de autismo induzido por exposição pré-natal ao VPA. O objetivo desse trabalho foi avaliar os efeitos do VPA na permeabilidade da barreira hematoencefálica (BHE), nas células GFAP+ e na aquaporina 4 (AQP4) no córtex pré-frontal medial (CPFm) de ratos machos de 30 dias e o possível efeito protetivo do RSV. Ratas Wistar prenhes receberam doses diárias de 3.6 mg/kg de RSV ou DMSO (veículo) via subcutânea do dia embrionário 6.5 (E6.5) até E18.5 e no dia E12.5 foi administrada uma dose intraperitoneal de 600 mg/kg de VPA ou solução salina (0,9%). No dia pós-natal 30 (P30) os animais foram eutanasiados por sobredose anestésica de cetamina (300 mg/kg) e xilazina (40 mg/kg) e destinados a diferentes técnicas experimentais. Os animais avaliados para a permeabilidade da BHE ao corante azul de Evans foram injetados via intraperitoneal com uma solução de azul de Evans 2% diluído em salina (4 mg/kg), anestesiados e submetidos a perfusão transcardíaca com salina e paraformaldeído para posterior remoção e secção do encéfalo e análise de fluorescência. Para a imunofluorescência, os animais foram anestesiados, perfundidos e o encéfalo foi removido e seccionado. Animais do grupo VPA apresentaram maior permeabilidade da BHE ao corante azul de Evans e o RSV preveniu essa alteração. Os grupos que receberam VPA apresentaram aumento no número absoluto de células GFAP+ nas camadas profundas do pré-límbico (PrL), além de um efeito sinérgico do RSV e do VPA no CC (superficial), no PrL (superficial) e no infralímbico (IL – profundo). Na razão de células GFAP+ foi visto um aumento no número relativo nas camadas profundas e uma diminuição nas camadas superficiais do CC e do PrL no grupo VPA e o RSV foi capaz de prevenir essa alteração no PrL. Também foi observado uma diminuição no conteúdo de fluorescência de AQP4 em todas as regiões analisadas do CPFm no grupo VPA. Dessa forma, esse trabalho demonstrou alterações significativas na permeabilidade da BHE, no número e localização de células GFAP+ e no conteúdo de fluorescência de AQP4 no CPFm do modelo VPA, bem como o uso do RSV como uma ferramenta para a investigação de mecanismos envolvidos na fisiopatologia do TEA.Autism spectrum disorder (ASD) is a neurodevelopmental disorder of high prevalence, affecting 1:59 children up to 8 years of age in the United States. According to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), the ASD diagnosis is given by the behavioral dyad composed by impairments in communication and social interaction, as well as restricted interests, repetitive and stereotyped behaviors. In addition to behavior alterations, ASD may present many associated comorbidities and an important clinical finding in this context is the presence of increased encephalic volume in the first years of life. Although the etiology of ASD remains unclear, the importance of genetic and environmental factors such as prenatal exposure to valproic acid (VPA) are known. Resveratrol (RSV) is an anti-inflammatory, antioxidant and neuroprotective molecule known to prevent social behaviors in the animal model of autism induced by prenatal exposure to VPA. The aim of this study was to evaluate the effects of VPA in blood-brain barrier permeability (BBB), GFAP+ cells and aquaporin-4 (AQP4) in the medial prefrontal cortex (mPFC) of 30-day-old male rats and the possible protective effect of RSV. Pregnant Wistar rats received from embryonic day 6.5 (E6.5) up to E18.5 daily doses of 3.6 mg/kg RSV or DMSO (vehicle) via subcutaneous and on day E12.5 an intraperitoneal dose of 600 mg/kg VPA or saline solution (0.9%). On the postnatal day 30 (P30) the animals were euthanized by anesthetic overdose of ketamine (300 mg/kg) and xylazine (40 mg/kg) and destined to different experimental techniques. The animals evaluated for BBB permeability to the Evans blue dye were injected intraperitoneally with a solution of Evans blue 2% diluted in saline (4 mg/kg), anesthetized and submitted to transcardiac perfusion with saline and paraformaldehyde for posterior removal and section of the brain and fluorescence analysis. For immunofluorescence, the animals were anesthetized, perfused and the brain was removed and sectioned. VPA group animals presented greater BBB permeability to the Evans blue dye and RSV prevented this alteration. The groups that received VPA presented an increase in the absolute number of GFAP+ cells in the deep layers of the prelimbic (PrL), in addition to a synergistic effect of RSV and VPA in CC (upper), PrL (upper) and infralimbic (IL – deeper). In the GFAP+ cell ratio was seen an increase in the relative number in deep layers and a decrease in the upper layers of CC and PrL in the VPA group and RSV was able to prevent this chance in PrL. A decrease in the AQP4 fluorescence content was also observed in all mPFC regions analyzed in the VPA group. Thus, this work demonstrated significant changes in BBB permeability, number and location of GFAP+ cells and in the fluorescent content of AQP4 in the mPFC of the VPA model, as well as the use of RSV as a tool for the investigation of mechanisms involved in ASD pathophysiology

Expression profiles of meiotic genes in male vs. female gonads and gametes : insights into fertility issues

Gametes are specialized cells that, at fertilization, give rise to a totipotent zygote capable of generating an entire organism. Female and male germ cells undergo meiosis to produce mature gametes; however, sex-specific events of oogenesis and spermatogenesis contribute to specific roles of gametes in reproductive issues. We investigate the differential gene expression (DGE) of meiosis-related genes in human female and male gonads and gametes in normal and pathological conditions. The transcriptome data for the DGE analysis was obtained through the Gene Expression Omnibus repository, comprising human ovary and testicle samples of the prenatal period and adulthood, additionally to male (nonobstructive azoospermia (NOA) and teratozoospermia), and female (polycystic ovary syndrome (PCOS) and advanced maternal age) reproductive conditions. Gene ontology terms related to meiosis were associated with 678 genes, of which 17 genes in common were differentially expressed between the testicle and ovary during the prenatal period and adulthood. Except for SERPINA5 and SOX9, the 17 meiosis-related genes were downregulated in the testicle during the prenatal period and upregulated in adulthood compared to the ovary. No differences were observed in the oocytes of PCOS patients; however, meiosis-related genes were differentially expressed according to the patient’s age and maturity of the oocyte. In NOA and teratozoospermia, 145 meiosis-related genes were differentially expressed in comparison to the control, including OOEP; despite no recognized role in male reproduction, OOEP was co-expressed with genes related to male fertility. Taking together, these results shed light on potential genes that might be relevant to comprehend human fertility disorders

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