A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Summary

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)_180-199 peptide in combination with Freund's complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Abstract

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)_106-125 or P0_180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0_180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

Introduction

Peripheral neuropathies can be either genetic in origin or acquired, with acquired neuropathies having either metabolic, ischaemic, inflammatory, or toxic precipitants. These diseases are also usefully classified as either axonal or demyelinative in origin. The most common acquired demyelinating peripheral neuropathies are Acute Inflammatory Demyelinating Polyneuropathy (AIDP, also known as Guillain-Barré syndrome, GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)^1,2,3,4; both are pathogenetically characterized by an aut....

Protocol

All procedures described here were approved by the Florey Institute for Neuroscience and Mental Health (Melbourne Brain Centre) Animal Ethics Committee and follow the Australian Code of Practice for the Use of Animals for Scientific Purposes.

1. EAN Induction

NOTE: EAN can be successfully induced in male C57BL/6 mice aged between 6-8 weeks. The induction protocol takes 9 days in total. Day 0 refers to the day of the first immunization. For this protocol, injections were conducted under anesthesia (see step 1.1.2 below).

1. On Day -1:
   1. Prepare the Pertussis Toxin (see

Results

P0_180-199 peptide induced EAN in C57BL/6 mice leads to a monophasic disease with clinical score onset from 6 days post first immunization (dpi), and maximal score severity is observed from 25 dpi followed by some clinical score improvement from 40 dpi duration (Figure 1)^4,9. In terms of gait function, mice begin to fail at a simple running task as early as 6 dpi, and by 35 dpi mice have lit.......

Discussion

This report outlines a simple method to induce EAN using the P0_180-199 peptide in C57BL/6 mice, enabling the quantification of key neuropathological and functional deficits in mice induced with EAN. Distinct to the EAN induction protocol described here is the use of anesthesia while performing the immunization injections. The use of isoflurane anesthesia greatly enhances the ability to ensure that the total volume of inoculum is injected subcutaneously in the desired location with minimal error and stress to t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6, male, 6-8 weeks old	Australian Bioresources Cenre, WA, Australia
Pertussis toxin	List Biological Laboratories, Inc., CA, USA	#181
0.1 M mouse-isotonic phosphated buffered salined (MT-PBS)			Laboratories will have their own protocol.
Isoflurane	Pharmachem, QLD, Australia		Laboratories will have their own protocol for administration.
P0180–199 peptide	Wuxi Nordisk Biotech Co. Lt. SHG, CHN		P0180–199, sequence S–S–K–R–G–R– Q–T–P–V–L–Y–A–M–L–D–H–S–R–S
Heat killed Mycobacterium tuberculosis (strain H37RA)	Difco, MI, USA	#231141
Freund's complete adjuvant (FCA)	Difco, MI, USA	#263910
16% Paraformaldehyde (PFA)	Electron Microscopy Services	#15710	Dilute to 4% PFA day of tissue collection.
25% glutaraldheyde	ProSciTech Pty Ltd, QLD, Australia	#11-30-8	Dilute to 2.5% glutaraldehyde day of fixation.
Sodium azide	Chem-Supply Pty Ltd, SA, Australia	SL189	Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v).
Sucrose	Chem-Supply Pty Ltd, SA, Australia	SA030	Use at 30% (w/v).
Optimum cutting temperature (OCT) medium	Sakura Finetek, CA, USA	#4583
Normal donkey serum	Merck Millipore, MA, USA	#S30-100	Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory.
Triton-X 100	Sigma Aldrich, MI, USA	#90o2-31-1	Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory.
rabbit anti-amyloid precursor protein (APP)	Invitrogen (Life Technologies), CA, USA	S12700	Used at 1:400 or titrate in own lab.
rabbit anti-contactin-associated protein-1 (Caspr)	Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel		Used at 1:500 or titrate in own lab.
Appropriate Alexa Fluor conjugated secondary antibodies	Molecular Probes (Life Technologies), OR, USA	Various	Use at 1:200 or titrate in own lab. Choice of species the antibody was raised in and Alexa Fluor chosen is at the discretion of each laboratory.
Aqueous mounting solution	Dako (Agilent), CA, USA	#S3023	Each laboratory will have their own preference.
Name	Company	Catalog Number	Comments
Equipment
0.5 mL syringe with 301/2 g needles	BD	#326105
23 g needles	BD	#305143
Red ink pad			Any red ink pad or red food dye could be used to mark the animals' feet.
DigiGate apparatus (includes treadmill)	eMouse Specifics Inc. Framingham, MA
DigiGate Imaging System	eMouse Specifics Inc. Framingham, MA
Stopwatch			Any timer may be used.
DigiGait 8 Software	eMouse Specifics Inc. Framingham, MA
Dissecting microscope	Zeiss		Any appropriate dissecting microscope may be used.
Charged slides	Superfrost Plus, Lomb Scientific Pty Ltd	SF41296SP
Cyrostat	Leica		Any suitable cyrostat may be used.
Perfusion equipment and dissecting instruments			Labs will have their own perfusion protoctols.
Opaque humified chamber			Labs may produce their own using an opaque plastic container.
PAP pene	GeneTex (USA)		Wax pencil, or surface tension may also be used to create a well around the tissue section.
Confocal microscope	Zeis LSM780		Any confocal microscope with appropriate laser lines may be used.
FIJI/Image J	National Institues of Health		Available from www.fiji.sc