Study of enteric neurons and enteric glial cells in Chronic Experimental Ulcerative Colitis in mice deficient for the P2X7 receptor (P2X7R-/-)

• Grant number: 23/03973-1
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): August 01, 2023
• Effective date (End): March 31, 2027
• Field of knowledge: Biological Sciences - Morphology - Anatomy
• Principal Investigator: Patricia Castelucci
• Grantee: Roberta Figueiroa de Souza
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Inflammatory bowel disease (IBD) is a term used to describe prolonged inflammation of the gastrointestinal tract, including Crohn's Disease (CD) and Ulcerative Colitis. Ulcerative colitis affects enteric neurons and causes continuous mucosal inflammation, extending from the rectum to the proximal colon. The P2X7 receptor (P2X7R) is activated by increased levels of extracellular ATP in intestinal inflammation and activates the NLRP3 inflammasome. The P2X7 receptor participates in the regulation of inflammatory response through the process of releasing pro-inflammatory cytokines, such as IL-1², IL-18 and TNF-alpha. The aim is to analyze enteric neurons and entric glial cells in chronic experimental ulcerative colitis in mice deficient for the P2X7 receptor gene (P2X7R -/-, P2X7RKO) and C57BL/6 Wild Type (WT). For this WT C57BL/6 and P2X7 receptor deficient mice (P2X7R-/-) will receive 2% (2g/100ml) of dextran sodium sulfate (DSS) dissolved in drinking water for 5 days followed by drinking water for the next 14 days. The mice will receive 3 cycles of DSS. Double and triple immunofluorescence staining techniques will be performed in order to analize a) the double labeling of P2X7R with neural nitric synthase neurons (nNOS), Calretinin (Carl), Calbindin (Calb), TNF1 receptor (TNFR1), TNF-alpha, the transcription factor NF-k², cleaved Caspase-3, NLRP3 and the glial cell marker GFAP; b) the number and the area of neurons/ganglion immunoreactive (ir) to nNOS, Carl, Calb; c) for the morphological analysis of the large intestine, the conventional histology method will be used; d) for the investigation of intestinal motility, the organ bath system will be used; e) to analyze apoptosis and/or necrosis, the TUNEL method will be used; f) for the study of signaling pathways, Western Blotting for P2X7 receptor, TNF1 receptor (TNFR1), NF-k² transcription factor, cleaved Caspase-3 and NLRP3 will be performed; g) for the study of cytokines TNF-alpha and IL-1² the Elisa method will be used. (AU)