Lewis Rat Model of Experimental Autoimmune Encephalomyelitis

Kalliopi Pitarokoili1, Bjoern Ambrosius1, Ralf Gold1

1 Department of Neurology, St. Josef Hospital, Ruhr‐University Bochum, Bochum
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.61
DOI:  10.1002/cpns.36
Online Posting Date:  October, 2017
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Abstract

In this unit, we describe in detail the most common methods used to break immunological tolerance for central myelin antigens and induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats as an animal model of multiple sclerosis. The resulting disease course ranges from an acute monophasic disease to a chronic relapsing or chronic progressive course, which strongly resembles the human disease. These models enable the study of cellular and humoral autoimmunity against major antigenic epitopes of the myelin basic protein, myelin oligodendrocyte glycoprotein, or proteolipid protein. We provide an overview of common immunization protocols for induction of active and passive EAE, assessment and analysis of clinical score, preparation and purification of myelin basic protein, and derivation of neuroantigen‐specific rat T cell lines. Finally, we describe the major clinical characteristics of these models. © 2017 by John Wiley & Sons, Inc.

Keywords: animal models; autoimmune disease; central nervous system; demyelination; experimental autoimmune encephalomyelitis EAE; Lewis rat; multiple sclerosis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Active EAE in Lewis Rats with Myelin Basic Protein
  • Alternate Protocol 1: Induction of EAE with Spinal Cord Homogenate
  • Support Protocol 1: Preparation of Guinea Pig MBP
  • Basic Protocol 2: Adaptive Transfer of EAE with One Antigen‐Specific Restimulation
  • Support Protocol 2: Establishment of MBP‐Specific T Cell Lines with Further Restimulation Cycles
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Active EAE in Lewis Rats with Myelin Basic Protein

  Materials
  • Lyophilized guinea pig MBP (see protocol 3)
  • Freund's adjuvant, complete (CFA; available from Sigma, Gibco, and Difco); store at 4°C (CFA contains heat‐killed Mycobacterium tuberculosis which stimulates the innate immune response; avoid inhalation and contact with skin and eyes)
  • Incomplete Freund's adjuvant (IFA, Difco)
  • Lewis rats, preferably 8‐ to 10‐week‐old pathogen‐free females (Charles River Laboratories, The Jackson Laboratory, Harlan)
  • UV spectrophotometer (capable of measurement at 280 nm)
  • Vortex with an attachment capable of holding multiple microcentrifuge tubes (optional, depending on the method used for emulsifying adjuvant; available from IKA, cat. no. 3340000; microcentrifuge tube attachment, cat. no.: 3344300)
  • 12 × 75–mm test tube
  • 1‐ml Luer‐Lok glass syringe with 18‐G needle (optional)
  • Probe sonicator (optional; e.g., Hielscher UP200St)
  • Vaporizer for isoflurane
  • Flow meter for anesthesia
  • 1‐ml luer‐lock glass syringe with 25‐G needle (Becton Dickinson, cat. no. 305122)
  • Disinfection solution for the site of the subcutaneous injection
  • Additional reagents and equipment for injection of mice (Donovan & Brown, )

Alternate Protocol 1: Induction of EAE with Spinal Cord Homogenate

  Additional Materials (also see protocol 1)
  • Frozen guinea pig or rat spinal cord (e.g., Rockland or Harlan Bioproducts for Science)
  • 0.9% (w/v) NaCl
  • Modified CFA (see protocol 1, step 2)
  • Tissue homogenizer
  • Animal balance

Support Protocol 1: Preparation of Guinea Pig MBP

  Materials
  • Frozen guinea pig brains or spinal cords (e.g., Rockland Immunochemicals for Research or Harlan Bioproducts for Science)
  • 2:1 (v/v) chloroform/methanol, 4°C
  • 1 N HCl
  • Saturated ammonium sulfate [(NH 4) 2SO 4], 4°C
  • 0.25 N NaOH
  • Waring Blender (or equivalent) with a glass or stainless steel mixing chamber
  • 5‐ to 6‐liter flask
  • Propeller mixer (e.g., Dyna‐Mix, Fisher)
  • Vacuum filtration system [e.g., Gast vacuum pump with high‐pressure tubing and a trap, connected to a vacuum flask with a Büchner funnel (ThermoFisher Scientific]
  • Whatman no. 90 and no. 4 filter paper (or equivalent)
  • Magnetic stirrer (IKA, cat. no. 5019700) and stirring bar
  • Sorvall RC5B centrifuge with SS‐34 rotor (or equivalent refrigerated high‐speed centrifuge)
  • Dialysis membrane, MWCO 6000 to 8000 (Spectrapor 1, Spectrum), boiled
  • Lyophilizer (optional)
CAUTION: Chloroform/methanol, which is employed to remove lipids from the CNS tissue, is toxic, so take care to minimize breathing the vapors and wear rubber gloves to protect the skin.

Basic Protocol 2: Adaptive Transfer of EAE with One Antigen‐Specific Restimulation

  Materials
  • Donors: Lewis rats 10 days after immunization with MBP/CFA (see protocol 1, steps 1 to 5)
  • Hanks balanced salt solution (HBSS; Life Technologies, cat. no. 14170‐112), 4°C
  • RPMI‐1640 medium supplemented with 25 mM HEPES, serum‐free (e.g., Life Technologies)
  • Complete RPMI‐1640/NRS (see recipe)
  • Guinea pig MBP (see protocol 3) or concanavalin A (ConA, 2.5 μg/ml)
  • Unimmunized syngeneic rats (recipients)
  • Halothane
  • 60‐mm or 100‐mm Petri dish
  • 100‐µm nylon mesh
  • Plunger from 5‐ml syringe
  • 50‐ml conical centrifuge tubes
  • Sorvall RC5B centrifuge with SS‐34 rotor (or equivalent refrigerated high‐speed centrifuge)
  • Additional reagents and equipment for inhalation anesthesia of rats ( protocol 1), harvesting of spleen and lymph nodes (Reeves & Reeves, ), testing cell viability by trypan blue exclusion ( appendix 3B; Phelan, ), and injection of rodents (Donovan & Brown, )

Support Protocol 2: Establishment of MBP‐Specific T Cell Lines with Further Restimulation Cycles

  Materials
  • Lewis rats immunized 10 days previously with MBP/CFA (see protocol 1, steps 1 to 4)
  • Hanks balanced sault solution (HBSS; Life Technologies, cat. no. 14170‐112), 4°C
  • Complete RPMI/NRS medium (see recipe)
  • Guinea pig MBP ( protocol 3)
  • Complete RPMI‐5 (see recipe)
  • Rat IL‐2 (multiple vendors, including eBioscience, BD Biosciences, and R&D Systems)
  • Unimmunized syngeneic rats (thymocyte/spleen donors for restimulation)
  • Sorvall RC5B centrifuge with SS‐34 rotor (or equivalent refrigerated high‐speed centrifuge)
  • 50‐ml conical centrifuge tubes
  • 60‐mm Petri dishes
  • 60Co or 137 Cs γ irradiator (i.e., Gammacell 1000, Nordion)
  • 6‐well plates (Sigma‐Aldrich, cat. no. CL3738)
  • Additional reagents and equipment for harvesting of lymphoid organs (Reeves & Reeves, ), testing cell viability by trypan blue exclusion ( appendix 3B; Phelan, ), and adoptive transfer of EAE ( protocol 4)
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Figures

Videos

Literature Cited

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