Involvement of Toll-like receptors 4 (TLR4) located in microglial cells in the behavioral consequences of stress exposure in mice

• Grant number: 23/01933-2
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2023
• End date: April 30, 2025
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Sabrina Francesca de Souza Lisboa
• Grantee: Laura Colete Cunha
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 17/19731-6 - Identification of epigenetic mechanisms induced by stress which modulate endocannabinoid signaling and neuroimmunological mechanisms as new therapeutic targets to treat the posttraumatic stress disorder (PTSD), AP.JP

Abstract

The Neuroimmune System has gained relevance in the etiology of psychiatric disorders associated with stress, such as post-traumatic stress disorder, depression and schizophrenia, and, therefore, is the target of studies for the development of new drugs. It is observed that exposure to homotypic stress induces increased expression of toll-like receptors 4 (TLR4) in microglia and their activation. The objective of this study will be to analyze the behavioral response and activation of microglia in situations of repeated homotypic stress, observing whether there is activation of the TLR4 pathway, and whether the genetic and/or pharmacological modulation of this pathway is capable of reversing behavioral damage. For this purpose, CSFR1Cre+/-/TLR4fl/fl transgenic animals and TLR4 KO animals (knockouts) will be used, in addition to animals treated with the TLR4 inhibitor, TAK242.All procedures were approved by the local ethics committee (Faculty of Pharmaceutical Sciences of Ribeirão Preto-FCFRP/USP; CEUA number 19.1.731 .60.9). Male C57Bl/6 (WT) and TLR4 KO mice aged 8-10 weeks were subjected to restraint stress for 10 days, 2 hours per day (9:00-11:00 am). One day after the end of the stress, the animals were submitted to the Open Field Test (ACT) followed by the Elevated Cross Maze (LCE). The novelty suppressed feeding test (TASPN) was performed the next day, after 24 hours of food deprivation. On the last day, the mice were submitted to the Splash Test followed by the Forced Swimming Test (TNF). After TNF, all mice were euthanized and brain tissues (hippocampus and prefrontal cortex) were collected for further analysis. The result of this preliminary study revealed that TLR4KO animals are protected against depressive-like behavior but not against anxious-like behavior after repeated 10-day restraint stress. More pharmacological studies are needed to determine the influence of the TLR4 receptor on behavior, therefore animals treated with TAK242, a TLR4 inhibitor, will also be used. The animals will be treated 1h before stress with TAK242 at a dose of 3mg/kg.Still, in order to elucidate the role of this receptor specifically in animal microglia CSFR1Cre+/-/TLR4fl/fl (conditional knockouts) will be used. These animals will be treated with tamoxifen for 5 consecutive days. After tamoxifen injection, activation of the Cre-recombinase enzyme will promote excision of the TLR4 gene from all CSFR1+ cells. Since the peripheral CSFR1+ cells are quickly replaced by myeloid cells, but as the microglial renewal rate is very slow, after 15 days of treatment only the microglial cells will have TLR4 deleted.