Experimental Autoimmune Myasthenia Gravis in the Mouse

Bo Wu1, Elzbieta Goluszko1, Ruksana Huda1, Erdem Tüzün1, Premkumar Christadoss1

1 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.8
DOI:  10.1002/0471142735.im1508s100
Online Posting Date:  February, 2013
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Abstract

Myasthenia gravis (MG) is a T cell–dependent antibody‐mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody‐mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques. Curr. Protoc. Immunol. 100:15.8.1‐15.8.26. © 2013 by John Wiley & Sons, Inc.

Keywords: myasthenia gravis; experimental autoimmune myasthenia gravis; acetylcholine receptor; neuromuscular junction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Inducing and Evaluating EAMG
  • Support Protocol 1: Extraction and Affinity Purification of AChR
  • Support Protocol 2: Preparation of Neurotoxin‐3‐Agarose Columns
  • Support Protocol 3: Evaluating EAMG Using Electromyography
  • Support Protocol 4: Measurement of AChR‐Antibodies by Radioimmunoassay (IgG)
  • Support Protocol 5: Evaluating EAMG by Measuring Muscle AChR Content via Radioimmunoassay
  • Support Protocol 6: Preparation of Mouse Muscle AChR
  • Evaluating IgG Content of Muscle Samples
  • Support Protocol 7: Detection of Neuromuscular Junction IgG and Complement Deposit
  • Support Protocol 8: Detection of C3 Concentration in Muscle Extract by ELISA
  • Support Protocol 9: Measurement of AChR‐Antibody Isotypes by ELISA (IgM, IgG, IgG1, IgG2b)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Inducing and Evaluating EAMG

  Materials
  • 200 µg purified Torpedo californica AChR (see protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete Freund's adjuvant (CFA; BD Difco)
  • 50 mg/ml sodium pentobarbital (Nembutal; Abbott Labs)
  • Ten 8 to 10‐week‐old C57BL6/J mice (male or female), marked for identification
  • 70% (v/v) ethanol
  • 300 µl/ml neostigmine bromide in PBS
  • 120 µg/ml atropine sulfate in PBS
  • Test tubes, sterile
  • 2‐ml glass syringes (VWR)
  • Microemulsifying needle connector (Fisher Scientific)
  • 1‐ml plastic tuberculin syringe with slip tip (Becton Dickinson Labware)
  • Heating pad
  • 25‐G needles (Becton Dickinson Labware)
  • 5 × 5–cm gauze sponges
  • Grid attached to a dynamometer (Chatillon Digital Force Gauge, DFIS 2, Columbus Instruments)
  • Additional reagents and equipment for intraperitoneal injection of mice (unit 1.6), collecting blood (unit 1.7), evaluating EAMG by electromyography ( protocol 4), by quantifying anti‐AChR antibodies by radioimmunoassay ( protocol 5), and/or by measuring muscle AChR content (see protocol 6), and evaluation of muscle IgG and complement content (see protocol 8)

Support Protocol 1: Extraction and Affinity Purification of AChR

  Materials
  • 250 g electroplax tissue from Torpedo californica (E‐PLAX‐F, Pacific Bio‐Marine Labs), frozen
  • Homogenization buffer (see recipe), 4°C
  • Triton X‐100
  • Neurotoxin‐3 affinity column ( protocol 3)
  • NaCl/Triton buffer, pH 8 (see recipe)
  • 0.2% cholate buffer (see recipe)
  • 1 M carbamylcholine buffer (see recipe)
  • Bradford protein assay kit (BioRad)
  • Bio‐Gel HT hydroxylapatite (BioRad)
  • 10 mM Tris buffer, pH 7.5 (see recipe)
  • 1.5 × 20‐cm Econo columns for low‐pressure chromatography (BioRad)
  • HT column wash buffer (see recipe)
  • Glycerol
  • Acetate buffer (see recipe)
  • 7.5% acrylamide SDS‐PAGE gel (unit 8.4)
  • 8‐ to 10‐week‐old C57BL6/J mice (male or female), marked for identification
  • Waring Blendor
  • 38.5‐ml polyallomer ultracentrifuge tubes with open top (Beckman)
  • High‐speed centrifuge amd JA‐20 rotor (Beckman) or equivalent
  • Ultracentrifuge and 60 Ti rotor (Beckman) or equivalent
  • 96‐well microtiter plate
  • Microtiter plate reader spectrophotometer
  • Cryoprotective vials
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and Coomassie blue staining of gels (unit 8.9)
NOTE: Perform steps 1 to 16 in a 4°C cold room or chromatography refrigerator (Revco).

Support Protocol 2: Preparation of Neurotoxin‐3‐Agarose Columns

  Materials
  • 30 to 50 mg neurotoxin‐3 from Naja naja kaouthia (Sigma)
  • Coupling buffer: 0.1 M sodium carbonate buffer, pH 8.3 (see recipe), containing 0.5 M NaCl
  • CNBr‐activated cross‐linked agarose (Sigma)
  • 1 mM HCl
  • 0.2 M glycine, pH 8
  • 0.1 M acetate buffer, pH 4.0 (see recipe), containing 0.5 M NaCl
  • NaCl/Triton buffer (see recipe)
  • 0.2% cholate buffer (see recipe)
  • Sintered‐glass filter with G3 porosity (VWR)
  • End‐over‐end rotator or equivalent
  • 1.5 × 20‐cm Econo columns for low‐pressure chromatography (BioRad)
CAUTION: Neurotoxin‐3 is extremely toxic (LD 50 <500 µg/kg). This compound blocks postsynaptic transmission in skeletal muscles without depressing acetylcholine release from the motor nerve endings. It binds to nicotinic AChR on the postsynaptic membrane of the neuromuscular junction, thus preventing binding of acetylcholine. In experimental animals, the toxin causes death by respiratory paralysis, with violent spasms during the final stages of asphyxia. The binding is irreversible in vivo, and therefore precautions should be taken when working with it. Always wear examination gloves and mask and take other precautions appropriate for handling neurotoxin.

Support Protocol 3: Evaluating EAMG Using Electromyography

  Materials
  • Sodium pentobarbital
  • Mouse to be evaluated for EAMG ( protocol 1)
  • Electrode gel
  • Styrofoam board
  • Band‐Aids
  • Electromyography machine (e.g., Cadwell 7400; Cadwell Labs, http://www.cadwell.com)
  • Electrodes (ground, stimulating, reference, and negative; Cadwell Labs)
  • Additional reagents and equipment for intraperitoneal injection of mice (unit 1.4)

Support Protocol 4: Measurement of AChR‐Antibodies by Radioimmunoassay (IgG)

  Materials
  • Mouse muscle AChR prepared from normal C57BL6/J mice (see protocol 7)
  • 0.5% (v/v) Triton X‐100 buffer (see recipe), 4°C
  • 100 µCi [125I]α‐bungarotoxin in 0.2 ml (240 Ci/mmol; Perkin‐Elmer)
  • Serum from experimental mice (e.g., protocol 1)
  • Normal mouse serum
  • Polyclonal rabbit anti–mouse Ig
  • 12 × 75–mm borosilicate glass tubes
  • Sorvall centrifuge and H‐1000B rotor (or equivalent)
  • γ counter (Beckman)

Support Protocol 5: Evaluating EAMG by Measuring Muscle AChR Content via Radioimmunoassay

  Materials
  • Muscle extract from EAMG and control mice (see protocol 7)
  • 0.5% Triton X‐100 buffer (see recipe)
  • 0.05 M benzoquinonium dibromide (Tocris Bioscience)
  • 100 µCi [125I]α‐bungarotoxin (240 Ci/mmol; Perkin Elmer)
  • Mouse anti‐AChR serum collected from immunized mice (see protocol 1)
  • Polyclonal rabbit anti–mouse Ig
  • 1.5‐ml microcentrifuge tubes
  • 4°C incubator
  • Sorvall centrifuge and H‐1000B rotor (or equivalent)
  • γ counter (Beckman)

Support Protocol 6: Preparation of Mouse Muscle AChR

  Materials
  • Normal uninjected (control) or EAMG C57BL6/J (experimental) mouse (see protocol 1)
  • Sodium pentobarbital
  • Homogenization buffer (see recipe), 4°C
  • Triton X‐100
  • Glycerol
  • Dissecting instruments
  • Waring Blendor with miniflask (or equivalent)
  • 15‐ and 50‐ml high‐speed‐compatible centrifuge tubes (Beckman‐Coulter, cat. no. 357007)
  • Beckman centrifuge and JA‐20 rotor (or equivalent)
  • Spatula or glass rod
  • Reciprocal shaker
  • 38.5‐ml ultracentrifuge tubes
  • Ultracentrifuge
  • 5‐ml cryovials
  • Additional reagents and equipment for euthanizing mice (unit 1.8)
NOTE: Perform procedure a 4°C cold room or chromatography refrigerator (Revco).

Support Protocol 7: Detection of Neuromuscular Junction IgG and Complement Deposit

  Materials
  • Normal uninjected (control) and EAMG C57BL6/J (experimental) mice (see protocol 1)
  • Liquid nitrogen
  • Tissue‐Tek O.C.T. (Optimal Cutting Temperature) Compound (Ted Pella)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Blocking solution: 5% (v/v) normal goat serum in PBS (see appendix 2A for PBS)
  • Tetramethylrhodamine‐conjugated α‐BTx solution (e.g., Molecular Probes; recommended dilution, 1:500)
  • Fluorescein isothiocyanate (FITC)‐conjugated anti‐mouse IgG (e.g., Southern Biotech; recommended dilution, 1:200)
  • FITC‐conjugated anti‐mouse C3 (e.g., ICN/Cappel; recommended dilution, 1:200)
  • FITC‐conjugated anti‐human MAC (e.g., anti‐human C9, Cedarlane; recommended dilution, 1:200; cross‐reacts with mouse MAC since human and mouse MAC components are highly homologous)
  • Acetone, chilled
  • Fluorescent mounting medium (optional)
  • DAPI (optional)
  • Insulated container for liquid nitrogen
  • Screw‐top microcentrifuge tubes
  • Long forceps
  • Cryomold
  • Cryostat (see unit 21.4)
  • Small brushes
  • Coated, precleaned microscope slides (will attract tissue sections electrostatically)
  • Humidity chamber (e.g., Tupperware box containing wet paper towels)
  • Glass Coplin staining jars with plastic screw caps (for 5 slides)
  • Hydrophobic marker
  • Gauze sponges
  • Coverslips
  • Fluorescence microscope
  • Additional reagents and equipment for euthanizing mice (unit 1.8) and immunohistochemistry (unit 21.4)

Support Protocol 8: Detection of C3 Concentration in Muscle Extract by ELISA

  Materials
  • Muscle extract from EAMG and control mice (see protocol 7)
  • Carbonate/bicarbonate buffer, pH 9.6 (see recipe)
  • Washing buffer for ELISA (see recipe)
  • HRP‐conjugated goat anti–mouse C3 (e.g., ICN Biomedicals/Cappel)
  • Dilution buffer for ELISA (see recipe)
  • Ready‐to‐use 3,3′,5,5‐tetramethylbenzidine (TMB) single‐solution chromogen/substrate (e.g., Invitrogen)
  • Stop solution: 1 N hydrochloric acid
  • 96‐well microtiter plate (e.g., Dynatech Immulon 2; Dynatech Labs)
  • Multichannel pipettor
  • Microtiter plate reader

Support Protocol 9: Measurement of AChR‐Antibody Isotypes by ELISA (IgM, IgG, IgG1, IgG2b)

  Materials
  • Mouse muscle AChR prepared from normal C57BL6/J mice (see protocol 7)
  • Carbonate/bicarbonate buffer, pH 9.6 (see recipe)
  • Blocking solution: 2% (w/v) bovine serum albumin in PBS (see appendix 2A for PBS)
  • Washing buffer for ELISA (see recipe)
  • Dilution buffer for ELISA (see recipe)
  • Mouse serum samples
  • Horseradish peroxidase (HRP)–conjugated goat anti‐mouse IgM, IgG, IgG1, IgG2b (e.g., Caltag Laboratories)
  • Ready‐to‐use 3,3′,5,5‐tetramethylbenzidine (TMB) single‐solution chromogen/substrate (e.g., Invitrogen)
  • Stop solution: 1 N hydrochloric acid
  • 96‐well microtiter plate (e.g., Dynatech Immulon 2; Dynatech Labs)
  • Multichannel pipettor
  • Microtiter plate reader spectrophotometer
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Figures

Videos

Literature Cited

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   Deng, C., Goluszko, E., Tüzün, E., Yang, H., and Christadoss, P. 2002. Resistance to experimental autoimmune myasthenia gravis in IL‐6‐deficient mice is associated with reduced germinal center formation and C3 production. J. Immunol. 169:1077‐1083.
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   Shenoy, M., Goluszko, E., and Christadoss, P. 1994. The pathogenic role of acetylcholine receptor alpha chain epitope within α146‐162 in the development of experimental autoimmune myasthenia gravis in C57BL6 mice. Clin. Immunol. Immunopathol. 73:338‐343.
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Key References
   Christadoss, 1989. See above.
  Detailed review of genetic control of EAMG.
   Christadoss et al., 2000. See above.
  Animal models of myasthenia gravis.
   Kaul et al., 1994. See above.
  First genetic evidence that MHC class II molecules are involved in EAMG and autoimmunity in general.
   Lindstrom et al., 1981. See above.
  Complete methodology for purification of AChR and measurement of antibodies to AChR.
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