Experimental Autoimmune Encephalomyelitis in the Rat

Mark Mannie1, Robert H. Swanborg2, Jolie A. Stepaniak3

1 Department of Microbiology and Immunology, East Carolina University, Brody School of Medicine, Greenville, North Carolina, 2 Wayne State University, Detroit, Michigan, 3 Henry Ford Community College, Dearborn, Michigan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.2
DOI:  10.1002/0471142735.im1502s85
Online Posting Date:  April, 2009
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Abstract

There are several diverse rat models of experimental autoimmune encephalomyelitis (EAE) that can be used to investigate the pathogenesis and regulation of autoimmunity against CNS myelin. The disease course of these models ranges from an acute monophasic disease with limited demyelination to a chronic relapsing or chronic progressive course marked by severe demyelination. These models enable the study of encephalitogenic T cells and demyelinating antibody specific for major neuroantigens such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP), among other important CNS autoantigens. Overall, this unit provides an overview of common methods for induction of active and passive EAE, assessment and analysis of clinical disease, preparation and purification of myelin basic protein, and derivation of neuroantigen‐specific rat T cell lines. This unit also provides a brief discussion of the basic characteristics of these models. Curr. Protoc. Immunol. 85:15.2.1‐15.2.15. © 2009 by John Wiley & Sons, Inc.

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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Active EAE 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: Adoptive Transfer of EAE
  • Support Protocol 2: Establishment of MBP‐Specific T Cell Lines
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Active EAE with Myelin Basic Protein

  Materials
  • Lyophilized guinea pig MBP (see protocol 3)
  • Heat‐killed Mycobacterium butyricum (Difco)
  • Incomplete Freund's adjuvant (IFA, Difco)
  • LEW rats, preferably 8‐ to 10‐week‐old pathogen‐free females (Charles River Labs)
  • Probe sonicator
  • 12 × 75–mm test tube
  • 1‐ml Luer‐Lok glass syringe
  • 18‐G (optional) and 25‐G needles
  • Additional reagents and equipment for subcutaneous injection (unit 1.6)

Alternate Protocol 1: Induction of EAE with Spinal Cord Homogenate

  • 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

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 (unit 15.1), 4°C
  • 0.01 and 1 N HCl
  • Saturated ammonium sulfate [(NH 4) 2SO 4; unit 2.7], 4°C
  • 0.25 N NaOH
  • Waring Blendor (or equivalent) with a glass or stainless steel mixing chamber
  • 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 Buchner funnel, Fisher)
  • Whatman no. 90 and no. 4 filter paper (or equivalent)
  • Sorvall RC5B with SS‐34 rotor (or equivalent refrigerated high‐speed centrifuge)
  • Dialysis membrane, MWCO 6000 to 8000 (Spectrapor 1, Spectrum), boiled
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: Adoptive Transfer of EAE

  Materials
  • LEW (or DA) rats 10 days after immunization with MBP/CFA (see protocol 1, steps 1 to 5; donors)
  • HBSS ( appendix 2A), 4°C
  • Complete RPMI‐5 ( appendix 2A) supplemented with 25 mM HEPES (N‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid; RPMI‐5/HEPES)
  • Guinea pig MBP (see protocol 3) or concanavalin A
  • Unimmunized syngeneic rats (recipients)
  • 60‐mm petri dish
  • Sorvall T6000B centrifuge and H‐1000 B rotor
  • 50‐ml conical centrifuge tubes
  • 175‐cm2 tissue culture flasks (Falcon)
  • Additional reagents and equipment for preparing single‐cell suspensions (unit 3.1), testing cell viability by use of trypan blue exclusion ( appendix 3B), and intraperitoneal injection (unit 1.6)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Establishment of MBP‐Specific T Cell Lines

  Materials
  • Lewis (LEW) or Dark Agouti (DA) rats immunized 10 days previously with MBP/CFA (see protocol 1, steps 1 to 5)
  • HBSS ( appendix 2A)
  • Complete RPMI/NRS medium ( appendix 2A) supplemented with 25 mM HEPES (N‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid) and 1% (v/v) heat‐inactivated syngeneic normal rat serum (units 1.7& 2.4; RPMI/NRS)
  • Complete RPMI‐5/IL‐2, supplemented with 25 mM HEPES (RPMI‐5/HEPES), containing a source of recombinant rat IL‐2.
  • Unimmunized syngeneic rats (thymocyte donors)
  • Guinea pig MBP, final concentration of 20 µg/ml (see protocol 3)
  • Sorvall T6000B centrifuge and H‐1000B rotor
  • 25‐cm2 tissue culture flasks (Falcon)
  • Additional reagents and equipment for preparing single‐cell suspensions (unit 3.1), testing cell viability using trypan blue exclusion ( appendix 3B), harvest of lymphoid organs (unit 1.9), preparation of single‐cell suspension (unit 3.1), irradiating cells (unit 3.12), and adoptive transfer of EAE (see protocol 4)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

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Literature Cited

Literature Cited
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