Resveratrol Prevents Molecular Aspects in Sensory Areas of Rats Prenatally Exposed to Valproic Acid

Background: Autism Spectrum Disorder (ASD) characterized by impairments in communication and social interaction in addition to repetitive or stereotyped behaviors. Another extremely important aspect for ASD are the sensory alterations present in more than 90% of the individuals with ASD like hyper-responsiveness to non-harmful stimuli (visual, tactile and auditory) and hypo-responsiveness to harmful stimuli, which implies a great loss in life quality. Although etiology is unknown, both genetic and environmental risk factors have been associated with the development of ASD, including the use of valproic acid (VPA) during pregnancy. Based on these observations, an animal model of autism by prenatal exposure to VPA was developed and validated by the reproducibility of behavioral, molecular and morphological changes observed in ASD individuals. It also includes alterations in redox, immune and inflammatory systems. We previously showed that a prenatal subchronic treatment of pregnant rats with the antioxidant and anti-inflammatory compound resveratrol (RSV), was able to preven important sensorial deficits induced by VPA in male offspring rats.

Objectives: The aim of the study was to perform a quantitative and organizational analysis of the effects of RSV and VPA on parvalbumin-positive GABAergic interneurons in sensory processing related regions – Primary Somatosensory Area and Amygdala Region.

Methods: Mating was undertaken overnight and confirmed in the following morning by the presence of spermatozoa in vaginal smears. Females received a single intraperitoneal injection of 600 mg/kg VPA or physiological saline on Embryonic day 12.5 (E12.5). For the RSV treatment females received daily subcutaneous injections of 3.6 mg/kg of RSV solution or the correspondent volume of DMSO from E6.5-E18.5. Experimental groups: Control, RSV, VPA and RSV+VPA. At 30 days-old, male rats were perfused with PF 4%, followed by brain dissection. The tissue was sliced in cryostat at -20ºC. Specific regions of interest were defined using Paxinos atlas. Immunofluorescence labelling: DAPI, Anti-NeuN and Anti-Parvalbumin (PV). The images were obtained by confocal microscopy and processed in ImageJ software with Cell Counter plug-in. Statistical analysis realized in GraphPad Prism 5 software, using one-way ANOVA test followed by Bonferroni’s poshoc and considering p<0.05 as significant.

Results: In Primary Somatosensory Area, VPA group exhibitedlaminar disorganization, associated to changes in PV⁺-neurons positioning, increasing in layer IV-V and decreasing in layer II-III. The prenatal administration of RSV not only promoted cortical reorganization at laminar and columnar level, but also prevented all alterations in layer II-III and IV-V induced by VPA. Moreover, considering total layers, no changes were observed in the amounts and density of PV⁺-neurons, suggesting a possible alteration in the migration of these interneurons between the cortical layers. In Amygdala Region, only the density of PV+-neurons were significant reduced in VPA group and RSV was able to prevent this alteration.

Conclusions: These data demonstrates for the first time not only alterations at a cellular level with expressive consequences in the excitatory/inhibitory balance, but also the neuroprotective effect of RSV. Further studies will focus on the mechanisms involved in the RSV-preventive effects.