Optimization of determinant factors associated with the efficiency of experimental autoimmune uveitis induction in C57BL/6 mice.

Background: Experimental autoimmune uveitis (EAU) is a widely used animal model for uveitis research. The C57BL/6 mouse strain is the most commonly used mouse strain in the research of genetic modification, but C57BL/6 mice are not sufficiently susceptible to EAU induction, partly due to experimental factors. This work aims to optimize relevant factors to improve the efficiency of EAU induction in C57BL/6 mice. Methods: To induce EAU, mice were immunized via intraperitoneal injection with pertussis (PTX) and subcutaneous injection with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified with complete Freund's adjuvant (CFA). The severity of inflammation was assessed using several approaches. The relevant experimental factors were evaluated, including methods of emulsification and doses of peptide and PTX. Results: Uveitis occurred at 8-12 days after immunization and reached its peak at 18-20 days, while T helper type 17 (Th17) cells peaked earlier at 14-18 days after immunization. Based on clinical and histological scores, 500 µg of IRBP peptide was the optimal dose required to induce EAU. The PTX dose demonstrated no influence on EAU incidence, but potentially affected the severity of uveitis. A single injection of 1,000 ng of PTX induced the most severe EAU and the highest proportion of Th17 cells. Compared to extruded emulsion, sonicated emulsion produced a higher incidence, higher histological score, and a 2-day-earlier onset of EAU. Electron microscopy showed a significantly different microstructure between the 2 emulsions. Conclusions: This work optimized the protocols of EAU induction and obtained a high and stable induction rate with severe inflammation in the C57BL/6 mouse strain. Our results facilitate future experimental research involving uveitis.