Hock immunization, a refined alternative for developing experimental autoimmune myasthenia gravis (EAMG) in mice

Experimental autoimmune myasthenia gravis (EAMG) is an active-immunization model that was first discovered by coincidence when rabbits injected with purified acetylcholine receptor for neuromuscular junction physiology studies, suddenly developed muscle weakness. In mice, this model typically involves bilateral subcutaneous injections of purified torpedo acetylcholine receptor (tAChR) in the hind footpads and the scapular regions, followed by one or two booster immunizations in the thighs and over the scapulas. However, footpad injections can cause severe discomfort and progressive debilitation. Hock immunization has shown to be a viable alternative in other models, offering comparable efficacy without impairing mobility caused by inflammation, thus reducing discomfort. To investigate the use of hock immunization in the mouse EAMG model, 7-week-old female C57BL6/6J (B6) mice were bilaterally injected over the scapulas and either in the hocks or footpads for the primary immunization. All animals received a booster immunization 4 weeks later, following standard guidelines. Inverted mesh measurements demonstrated that tAChR-immunized animals whether receiving hock or footpad injections, exhibited similar muscle weakness. This weakness correlated with reduced body weight, which reached statistical significance, only in the hock-immunized group. tAChR antibodies were detectable in both groups 4 weeks after primary immunization and continue raising until the end of the experiment. Additionally, tAChR immunized mice required significantly less curare to induce a decremental response of compound muscle action potentials. Notably, hock-immunized mice displayed consistent mechanical sensitivity over time, which was significantly lower compared to footpad-immunized mice. In conclusion, hock-immunization is a refined and effective alternative for developing the EAMG mouse model. It results in similar disease incidence and severity while minimizing immunization-related discomfort compared to the standard footpad method.