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.

Keywords: Acetylcholine receptor; Experimental autoimmune myasthenia gravis; Myasthenia gravis; Rat; Torpedo californica.

Fig. 1

Representative anti-tAChR (A) and anti-rat muscle AChR titers (B) after immunization with 40 μg tAChR in CFA (with 1 mg/mL Mycobacterium tuberculosis) on day 0 in 7-week old female Lewis rats. Anti-tAChR titers were detected approximately 2 weeks before anti-rat muscle AChR titers were measured. In the period between 35 and 56 days after immunization, anti-tAChR titers were two orders of magnitude higher compared to rat muscle AChR antibody titers. The variability of antibody titers seen here is typical of the EAMG model. The raw data used for the graph are available in Supplemental Table 2.

Fig. 2

Schematic representation of the relation between AChR loss and muscle weakness. Because of the safety factor of neuromuscular transmission (3 in this example), animals with an average AChR loss of up to 60% have no disease symptoms. Challenge of neuromuscular transmission with curare can reveal subclinical damage to the neuromuscular junction. The effect of therapeutical interventions can thereby be studied much more sensitively.