Effect of Risperidone and Cannabidiol on dental formation and development of periapical lesions in rats with behaviors similar or not to Autism Spectrum Disorder

• Grant number: 24/06726-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2025
• End date: August 31, 2027
• Field of knowledge: Health Sciences - Dentistry - Pediatric Dentistry
• Principal Investigator: Léa Assed Bezerra da Silva
• Grantee: Angélica Aparecida de Oliveira
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The aim of the present study is to evaluate the effects of Autism Spectrum Disorder (ASD) and the medications Risperidone, an atypical antipsychotic, and Cannabidiol, a phytocannabinoid, on tooth formation (in incisors) and the development of periapical lesions (in first molars) in offspring with or without behaviors resembling ASD, following the administration of valproic acid to pregnant rats to establish the model in the progeny. Initially, pregnant Sprague-Dawley female rats will receive a single dose of valproic acid at 400mg/kg subcutaneously on the 12th day of gestation to induce ASD in the offspring. At 40 days of age, the pups will undergo tests to assess neurobehavioral changes confirming the establishment of the ASD model. Subsequently, 144 pups will be divided into two groups (with or without ASD-like behaviors) and subdivided into 12 experimental groups (n=12). In the offspring with ASD-like behaviors, groups will be: G1 - Vehicle and healthy teeth without periapical lesion induction; G2 - Cannabidiol and healthy teeth without periapical lesion induction; G3 - Risperidone and healthy teeth without periapical lesion induction; G4 - Vehicle and induced periapical lesion; G5 - Cannabidiol and induced periapical lesion; and G6 - Risperidone and induced periapical lesion. Meanwhile, the offspring without ASD-like behaviors will be subdivided into: G7 - Vehicle and healthy teeth without periapical lesion induction; G8 - Cannabidiol and healthy teeth without periapical lesion induction; G9 - Risperidone and healthy teeth without periapical lesion induction; G10 - Vehicle and induced periapical lesion; G11 - Cannabidiol and induced periapical lesion; and G12 - Risperidone and induced periapical lesion. Samples of molars with or without induced periapical lesions and upper incisors will be collected for dental development analysis. The following analyses will be conducted: tests to evaluate neurobehavioral changes confirming the ASD model establishment (object recognition test, social interaction test, and open field test), and measurement and weighing of the animals. On the 40th day of life, the pups will receive Risperidone, Cannabidiol, or Vehicle via gavage for 14 days. After drug administration, periapical lesions will be induced in specific groups. After 21 days of lesion induction and euthanasia, assessments will include conventional microscopy using HE staining for descriptive and semi-quantitative analysis of the root canal and apical and periapical regions, morphometric analysis using fluorescent microscopy to evaluate the extent of periapical lesion areas, TRAP histochemistry for osteoclast counting and localization in the periapical lesion region, and evaluation of the gene expression of pro-inflammatory cytokines (IL1-², IL6, IL10, and TNF-±) and osteoclastogenesis markers (RANK, RANKL, and OPG) using qRT-PCR. Additionally, analyses of upper incisors will include macrophotographic analysis with digital caliper assistance, evaluation of microscopic characteristics of dental tissues using HE and Masson's Trichrome staining, immunohistochemistry for amelogenin, ameloblastin, tuftelin, KLK-4, MMP-20, and COX-2, in situ zymography for MMP-20, indirect immunofluorescence for RUNX2, and Knoop microhardness test. Lastly, analysis of neurogenesis and neuroplasticity will be conducted using specific markers. Results will be analyzed using GraphPad Prism 9 software. Data will undergo normality testing and, if normally distributed, will be subjected to analysis of variance (ANOVA), followed by Tukey's post-test. Additional tests may be applied as needed. The significance level will be set at 5%.