Standardization of the experimental autoimmune myasthenia gravis (EAMG) model by immunization of rats with Torpedo californica acetylcholine receptors — Recommendations for methods and experimental designs

Abstract

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR) is characterized by a chronic, fatigable weakness of voluntary muscles. The production of autoantibodies involves the dysregulation of T cells which provide the environment for the development of autoreactive B cells. The symptoms are caused by destruction of the postsynaptic membrane and degradation of the AChR by IgG autoantibodies, predominantly of the G1 and G3 subclasses. Active immunization of animals with AChR from mammalian muscles, AChR from Torpedo or Electrophorus electric organs, and recombinant or synthetic AChR fragments generates a chronic model of MG, termed experimental autoimmune myasthenia gravis (EAMG). This model covers cellular mechanisms involved in the immune response against the AChR, e.g. antigen presentation, T cell-help and regulation, B cell selection and differentiation into plasma cells. Our aim is to define standard operation procedures and recommendations for the rat EAMG model using purified AChR from the Torpedo californica electric organ, in order to facilitate more rapid translation of preclinical proof of concept or efficacy studies into clinical trials and, ultimately, clinical practice.

Differential RNA Expression Profile of Skeletal Muscle Induced by Experimental Autoimmune Myasthenia Gravis in Rats

“…Only, a few genes primarily associated with the immune system or inflammation were modified by EAMG, however these were focused on genes that would suppress inflammation. The observation is consistent with histology of muscle of human MG, and EAMG in the chronic stage, which does not have evidence of inflammation (Nakano and Engel, 1993; Losen et al, 2015; Tuzun et al, 2015). The expression of immunosuppressive genes provides the first insight into why EAMG, by extension human MG, lacks inflammatory infiltrates in muscle after the acute induction stage.…”

“…The rat and human immune systems possess unique characteristics, and the exogenous administration of autoantigen with adjuvant does not mimic the spontaneous development of the breakdown in tolerance of the human disease (Losen et al, 2015). However, the ultimate common pathway of antibody attack with complement activation is shared between EAMG and human MG.…”

“…EAMG induced in rodents by immunization with purified AChR mimics the human disease much better than administration of AChR antibodies (Losen et al, 2015). Within 6 weeks of a single immunization, rats generate AChR antibodies and then weakness, which improves with cholinesterase inhibition.…”

Utility of experimental animal models of myasthenia gravis for the elucidation of pathogenic mechanisms and development of new medications

“…19 They showed that rabbits immunized with AChR protein purified from the Electrophorus electricus electric organ developed MGlike symptoms, with flaccid paralysis and decremental electromyographic response. 21 Immunized rats show transient muscle weakness with high frequency after approximately 7 days. Subsequent to the publication of that pivotal study, mice and rats have been used for the induction of AChR-EAMG, and rats were found to be more susceptible than mice, with a high incidence of clinical signs of the disease.…”

“…In this model, the animals mount an active immune response against injected AChR, and MG is caused by cross-reactions of the antibodies with the animals' own muscle AChR. [21][22][23] Nevertheless, the mouse model has an advantage for the elucidation of MG pathogenesis due to the availability of diverse transgenic, knockout and mutant mouse strains. 20 In rats, EAMG can be induced by single immunization with AChR protein purified from Torpedo californica and emulsified in complete Freund's adjuvant.…”