Experimental Autoimmune Uveoretinitis in the Rat and Mouse

Rachel R. Caspi1

1 National Eye Institute, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.6
DOI:  10.1002/0471142735.im1506s53
Online Posting Date:  May, 2003
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Abstract

Experimental autoimmune uveoretinitis (EAU) in rats and mice is a prototypic T cell‐mediated autoimmune disease that targets the neural retina and related tissues. The model is used to represent human sight‐threatening inflammatory eye diseases that are believed to have an autoimmune etiology, and to study basic mechanisms of tolerance and autoimmunity to organ‐specific antigens from immunologically privileged sites. In this unit, EAU is induced in rats and mice by immunization with uveitogenic peptide antigens emulsified in complete Freund's adjuvant (CFA). Clinical onset is observed by both external and microscopic examination. A protocol is provided for preparation of tissue sections of affected eyes for microscopic analysis. EAU can also be induced in the rat (as described) by adoptive transfer of lymphocytes from uveitic rats into unimmunized recipients, which obviates the use of CFA. To induce EAU in mice, Bordetella pertussis toxin (PTX) is included to overcome immunological resistance mechanisms.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of EAU in the Rat by Active Immunization
  • Alternate Protocol 1: Induction of EAU in the Rat by Adoptive Transfer
  • Basic Protocol 2: Induction of EAU in the Mouse by Active Immunization
  • Alternate Protocol 2: Induction of EAU in the Mouse by Adoptive Transfer
  • Support Protocol 1: Preparation of EAU Antigen/Adjuvant Emulsion by Mixing
  • Support Protocol 2: Preparation of EAU Antigen/Adjuvant Emulsion by Sonication
  • Support Protocol 3: Fundoscopic Examination in the Mouse
  • Support Protocol 4: Collecting Eyes for Histopathology
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of EAU in the Rat by Active Immunization

  Materials
  • Bovine IRBP peptide R16 emulsified in CFA (see protocol 5 or protocol 62)
  • Lewis rats of either sex, 6 to 8 weeks old
  • 1‐ml glass syringes with Luer‐Lok tips
  • 19‐G blunt‐tipped and 23‐G needles, sterile
  • Flashlight
  • Additional reagents and equipment for preparing adjuvant/antigen emulsion (see Support Protocols protocol 51 and protocol 62), animal restraint (unit 1.3), injecting animals (unit 1.6), euthanizing with CO 2 (unit 1.8), and collecting eyes for histopathology (see protocol 8)

Alternate Protocol 1: Induction of EAU in the Rat by Adoptive Transfer

  • Donor Lewis rats injected with bovine interphotoreceptor retinoid‐binding protein (IRBP) peptide R16 (see protocol 1, steps to )
  • RPMI 1640 medium with and without 10% (v/v) FBS (HyClone), ice cold
  • recipeComplete RPMI‐10 medium (see recipe)
  • Bovine IRBP peptide R16
  • Recipient Lewis rats, 6 to 8 weeks old
  • Disposable plastic syringes
  • Stainless steel mesh
  • 50‐ml centrifuge tubes
  • Sorvall RC‐3B centrifuge and H2000 swinging bucket rotor (or equivalent)
  • 12‐well tissue culture plates
  • Additional reagents and equipment for removing lymph nodes (unit 1.9), preparing a single‐cell suspension (unit 3.1), testing cell viability by trypan blue exclusion ( appendix 3B), counting cells ( appendix 3A), injecting animals (unit 1.6), euthanizing with CO 2 (unit 1.8), and collecting eyes for histopathology (see protocol 8)
NOTE: All reagents and equipment coming into contact with live 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.

Basic Protocol 2: Induction of EAU in the Mouse by Active Immunization

  Materials
  • B10.RIII mice, 6 to 8 weeks old, either sex
  • Human IRBP peptide 161 to 180 emulsified in CFA (see protocol 5 or protocol 62)
  • 1‐ml glass syringe with Luer‐Lok tip
  • 19‐G blunt‐tipped and 25‐G needles, sterile
  • Additional reagents and equipment for emulsifying antigen (see protocol 5 or protocol 62), restraining animals (unit 1.3), injecting animals (unit 1.6), inspecting the fundus (see protocol 7), euthanizing using CO 2 (unit 1.8), and collecting eyes for histopathology (see protocol 8)

Alternate Protocol 2: Induction of EAU in the Mouse by Adoptive Transfer

  • Donor B10.RIII mice injected with human IRBP peptide 161‐180 (see protocol 3, steps to )
  • recipeComplete RPMI‐10 medium (see recipe)
  • Recipient B10.RIII mice
  • Disposable plastic syringes
  • Petri dishes
  • Stainless steel mesh
  • 50‐ml centrifuge tubes
  • 75‐cm2 tissue‐culture flasks
  • Additional reagents and equipment for removing lymph nodes (unit 1.9), preparing a single‐cell suspension (unit 3.1), testing cell viability by trypan blue exclusion ( appendix 3B), counting cells ( appendix 3A), injecting animals (unit 1.6), euthanizing with CO 2 (unit 1.8), and collecting eyes for histopathology (see protocol 8)
NOTE: All reagents and equipment coming into contact with live 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 1: Preparation of EAU Antigen/Adjuvant Emulsion by Mixing

  Materials
  • 600 µg/ml bovine interphotoreceptor retinoid‐binding protein (IRBP) peptide R16 (for rat; sequence ADGSSWEGVGVVPDV) or human IRBP peptide 161 to 180 (for mouse; sequence SGIPYIISYLHPGNTILHVD)
  • recipeComplete Freund's adjuvant (CFA) prepared with 2.5 mg/ml Mycobacterium tuberculosis strain H37Ra (see recipe)
  • 10 to 50‐ml conical‐bottom polypropylene test tubes, sterile
  • Glass syringe with Luer‐Lok tip
  • 16‐G blunt‐tipped needle
  • Sorvall RC‐3B centrifuge and H2000 swinging bucket rotor (or equivalent)

Support Protocol 2: Preparation of EAU Antigen/Adjuvant Emulsion by Sonication

  Materials
  • 600 µg/ml bovine interphotoreceptor retinoid‐binding protein (IRBP) peptide R16 (for rat; sequence ADGSSWEGVGVVPDV) or human IRBP peptide 161 to 180 (for mouse; sequence SGIPYIISYLHPGNTILHVD)
  • recipeComplete Freund's adjuvant (CFA) prepared with 2.5 mg/ml Mycobacterium tuberculosis strain H37Ra (see recipe)
  • 20 to 50‐ml vials or test tubes, sterile
  • Sonicator equipped with a microtip probe (e.g., Misonix)
CAUTION: Use ear protection when performing the sonication.

Support Protocol 3: Fundoscopic Examination in the Mouse

  Materials
  • Ophthalmic dilating solutions: 1% Tropicamide and Neo‐Synephrine
  • Sterile physiological solution (e.g., saline, recipePBS, or “artificial tears”)
  • Binocular dissecting microscope with coaxial illumination
  • Microscope coverslips
  • Additional reagents and equipment for injection or inhalant anesthesia (unit 1.4)
NOTE: Dilating drops cause a temporary opacification of the lens within 15 to 20 min after application, so it is important to complete fundoscopic examination within that time.

Support Protocol 4: Collecting Eyes for Histopathology

  Materials
  • Rat or mouse with EAU
  • 4% (v/v) glutaraldehyde/ recipePBS ( appendix 2)
  • 10% (v/v) formaldehyde/ recipePBS
  • Small blunt‐tipped dissecting scissors and forceps
  • Curved forceps (for mice)
  • Glass or plastic vials
  • Additional reagents and equipment for CO 2 euthanasia (unit 1.8)
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Figures

Videos

Literature Cited

Literature Cited
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