Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Summary

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Abstract

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous injection of an emulsion containing a peptide (IRBP_1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund's Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

Introduction

This protocol will demonstrate how to induce Experimental Autoimmune Uveitis (EAU) in the C57BL/6J mouse by a single subcutaneous injection of a retinal antigen in an emulsified adjuvant. Methods for monitoring and assessing disease progression will be detailed through fundoscopic imaging and histological examination, with measurement parameters outlined within. In addition, fluorescein angiography, a technique for examining retinal blood vessel structure and permeability will be discussed.

This EAU model recapitulates central features of non-infectious posterior uveitis in humans with regards to clinicopathologic characteristics and the ba....

Protocol

All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986, and institutional Animal Welfare and Ethical Review Body (AWERB) guidelines.

1. Housing C57BL/6J mice

1. House mice in a specific pathogen free environment, on a 12 hour light-dark cycle and food and water available ad libitum.
2. Perform all experiments on adult female C57BL/6J (females are preferentially chosen as there is an incidence of women to men 1.4 to 1 in uveitis patients). Randomize female C57BL/6J mice between 6-8 weeks old by weight and age. House the mice in individually ventilated cages (IVC) in ....

Results

In this protocol, we describe a step-by-step method for inducing a model of experimental autoimmune uveitis (EAU) by immunizing mice with a uveitogenic retinal peptide derived from IRBP. The assessment of disease employing widely used and readily accessible approaches are covered although these are not exclusive and may be added to, or partially replaced, by other imaging techniques. The first signs of EAU in C57BL/6J mice can be detected two weeks post-immunization and peak disease reached within three weeks as illustra.......

Discussion

Experimental animal models are necessary tools for studying disease pathogenesis and preclinical testing of novel therapeutic paradigms. In the current protocol, we have discussed a methodology for inducing, monitoring, and scoring EAU, an experimental model of intraocular inflammatory uveitis. This EAU model has more than 95% disease incidence when all procedures are performed according to the protocol outlined herein, and results in the development of chronic, monophasic EAU. To achieve this incidence level, we stress .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
antisedan	ZOETIS, USA		for waking up
Complete Freund’s Adjuvant; CFA	Sigma, UK	F5881	for immunisation
Domitor	Orion Pharma, Finland		for anesthesia
Flourescein	Sigma, UK	F2456	for Angiography
IRBP1-20	Chamberidge peptide, UK		peptide;antigen
Ketamine	Orion Pharma, Finland		for anesthesia
Micron III	Phoenix Research, USA		for fundoscopy
Mouse Serum	Sigma, UK	M5905	for immunisation
Mycobacterium terberculosis	Sigma, UK	344289	for immunisation
Pertussis Toxin	Sigma, UK	P2980	for immunisation
phenylephrine hydrochloride 2.5%	Bausch & Lomb UK	PHEN25	for dilation
Tropicamide 1%	SANDOZ		for dilation
Viscotears	WELDRICKS Pharmacy, UK	2082642	for eye lubrication