Active Immunization for Inducing Autoimmune Muscle-Specific Kinase (MuSK) Myasthenia Gravis in Mice

Summary

This article presents a comprehensive protocol for an experimental mouse model of autoimmune muscle-specific kinase (MuSK) myasthenia gravis, aimed at investigating the pathophysiology and potential therapeutic strategies for the disease.

Abstract

Myasthenia gravis (MG) is an autoimmune disorder affecting the neuromuscular junction. While most MG patients produce autoantibodies against the acetylcholine receptor (AChR), a subset of patients exhibits autoantibodies targeting the muscle-specific kinase (MuSK). MuSK MG is characterized by severe muscle weakness, treatment-resistant clinical manifestations, and myasthenic crises, often necessitating mechanical ventilation. Consequently, patients frequently require prolonged use of immunosuppressants, which are associated with long-term adverse effects. Given the severity and rarity of MuSK-MG, developing an experimental animal model is crucial for advancing new treatment modalities and a deeper understanding of the underlying pathophysiological mechanisms. Experimental autoimmune myasthenia gravis (EAMG) serves as an animal model for MuSK-MG, effectively but partly mimicking the clinical and immunological features of human MG. The induction of autoimmune animal models can be achieved through active or passive immunization. In the herein presented experimental protocol, active immunization was employed by subcutaneously administering the purified extracellular domain of human MuSK emulsified in complete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Immunization was performed at four sites, followed by a booster injection of MuSK on the 28th day. This adjuvant with M. tuberculosis enables activation of the immune system through TLR4 and enhanced phagocytosis of the administered antigen. This article comprehensively details the development and characterization of MuSK-EAMG from inception to conclusion.

Introduction

Myasthenia gravis (MG) is an autoimmune disease that develops through T-cell-dependent, antibody-mediated mechanisms. It is primarily caused by antibodies against nicotinic acetylcholine receptors (AChR) on the postsynaptic membrane of the neuromuscular junction (NMJ) in 80%-85% of patients, and by antibodies against muscle-specific kinase (MuSK) in approximately 5% of patients as well as other NMJ proteins. MG has become a prototype for autoimmune disorders and a model for understanding postsynaptic function¹. Animal models of autoimmune disorders can be divided into two main categories: (1) Spontaneous models, in which animals naturally devel....

Protocol

The protocol was approved by the Istanbul University Local Ethics Committee for Animal Experiments (HADYEK) with the decision number 2020/29. In our previous studies^7,8,9, the EAMG model was induced in 8-10-week-old BALB/c or B6 mice via active immunization with the MuSK protein. This article presents exemplary data obtained from different experiments using MuSK-immunized BALB/C mice (4-8 mice per group). The reagents and the equipment used are listed in the Table of Materials.

1. Production and preparation of human ....

Results

In evaluating and validating the MuSK Experimental Autoimmune Myasthenia Gravis (EAMG) model, several key parameters and experimental results are critical to demonstrate the successful induction and characterization of the disease model. Below are the representative results that confirm the establishment of the MuSK EAMG model.

Clinical scoring
Mice are monitored and scored for clinical symptoms of myasthenia gravis using a standardized clinical scoring system. This scorin.......

Discussion

Murine MuSK-EAMG mimics human MuSK-MG both in its clinical and immunopathogenic aspects in terms of muscle weakness, weight loss due to bulbar muscle weakness, circulating anti-MuSK antibodies, and reduced muscle AChR. As for milestones and anticipated timeline for this MuSK-EAMG model, clinical signs of muscle weakness should appear within the first week after the second immunization. Approximately 5% of mice may develop signs of disease after the first immunization. A >70% EAMG (at least grade 1) prevalence is expe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
125I-α-bungarotoxin	Invitrogen	BTX T1175
2-Methylbutane	Sigma-Aldrich	M32631
40 μm Cell Strainer	NEST	258369
96 well Maxisorp plate	Sigma-Aldrich	M9410
Adjuvant Incomplete	Becton Dickinson & Co.	263910
Anti-AChR alfa1	Alomone	ANC-001
Anti-mouse IgG HRP	Abcam	ab97023
Bullet Blender Storm	Next Advance		Tissue homogenizer
C3	Novus	NB200-540G
Carbonate-Bicarbonate Buffer	Merck	C3041
Cryostat	Leica	CM1860
Dynamometer Imada DST-50N	Muromachi	MK380Si
Fluorescence microscope	Leica	DM4M
Glass syringe with luer lock	ISOLAB	094.92.005
IgG	Abcam	ab96871
Microplate Photometer, Multiskan FC	Thermo Scientific	51119000
Mouse Surgical Kit	Kent Scientific Corparation	INSMOUSEKIT
Mycobacterium tuberculosis H37Ra	Becton Dickinson & Co.	231141
OCT Compound	Agar Scientific	AGR1180
Syringes, 1 mL	Becton Dickinson & Co.	309625
Syringes, 2 mL	B. Braun	7389
Universal animal restrainer	Merck	Z756911-1EA
WesternBright Sirius-Western blotting detection kit	Advansta	K-12043-D10	ECL