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 autoimmune reaction directed against the myelin sheath, causing demyelination of the peripheral nerves. In these diseases, activated T cells cross the blood nerve barrier and generate an immune reaction within the PNS. Activation of macrophages within the nerve then causes demyelination either directly via phagocytic attack or indirectly via secreted inflammatory mediators, resulting in clinical disabilities such as paralysis and sensory dysfunction⁵. While demyelinated axons retain the ability to be remyelinated following demyelination, remyelination is often delayed or incomplete, resulting in susceptibility of the naked axons to irreversible damage, which is the major cause of permanent clinical disability. Currently, the most effective treatments are immunomodulatory, but despite their efficacy, in many cases the recovery is often slow and ~25% patients will experience residual functional deficits that significantly reduce their quality of life^6,7.

EAN is a widely used animal model of demyelinating peripheral neuropathy that has provided valuable insights into pathogenesis and a means to assess novel therapeutic agents⁴. This model can be induced in different species such as rabbits, rats, mice, and guinea pigs, and is induced by immunization with neurogenic antigens. However, ultimately the successful EAN induction depends on an appropriate immune response for disease to occur. Given the species (and inter-species/strain) variations in immune function, multiple combinations of antigens and adjuvants have been developed to successfully induce EAN. In terms of murine genetic tools, the C57BL/6 is the most widely used; however, the traditional P2 protein peptide 57-81 (P2_57-81) that results in disease in the susceptible SJL mouse strain⁸ is unable to illicit pathogenesis leading to functional deficits in the C57BL/6 strain. Fortunately, sensitization paradigms using the P0_106-125 or P0_180-199 peptides, delivered in adjuvant combined with the injection of pertussis toxin can overcome this barrier, enabling sophisticated genetic tools to be utilized in the murine EAN model.

Here, a simple method for the induction of EAN in the C57BL/6 mice is presented. In addition, a comprehensive detailed approach by which to evaluate the functional and neuropathological deficits associated with the disease is provided. The P0_180-199 peptide⁹ was chosen in preference to the P0_57-81 alternative¹⁰. The P0_180-199 model has been described to produce less severe clinical signs compared with the P0_57-81 alternative¹⁰, and is therefore likely to withstand the introduction of potentially deleterious genetic perturbations, recover from surgical procedures (such as osmotic pump implantation), and is amenable to treadmill gait function testing⁴. However, the treadmill gait function tests and histological protocols described here could easily be applied when studying the disease in a P0_57-81 induced variant.

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 Table of Materials) solution of 1.6 µg/mL using the sterile 0.1 M mouse-isotonic phosphate buffered saline (MT-PBS).
   2. Anesthetization with isoflurane:
      1. Place the mouse (C57BL/6, male, 6-8 weeks) in the anesthetization chamber and adjust the oxygen flow to 1 L/min.
      2. Turn on the isoflurane vaporizer, adjust it to 2.5% for anesthetization, and monitor breathing and wait for 2 min, or until primary reflexes (corneal and hind limb) are no longer responsive 
   3. Remove the mouse from the anesthetization chamber and administer 250 µL of the above Pertussis Toxin solution via an intraperitoneal (i.p.) injection using a 0.5 mL syringe with a 30^1/2 G needle.
   4. Prepare the injectable inoculum for immunization:
      1. Prepare Solution A using 2 mg/mL solution of P0_180-199 peptide (with > 98% purity, sequence S-S-K-R-G-R- Q-T-P-V-L-Y-A-M-L-D-H-S-R-S) in 0.9% saline.
      2. Prepare Solution B using 20 mg/mL solution of Mycobacterium tuberculosis (see Table of Materials) in Freund's complete adjuvant (FCA, comprising: 15% mannide monoolate + 85% of paraffin oil and 0.5 mg/mL of desiccated killed and dried M Mycobacterium butyricum).
      3. To make the final inoculum for injecting, combine equal volume parts of Solutions A and B in a bead beater and mix them at a maximum speed for 1 min at room temperature.
         NOTE: Solutions A and B can be kept as stock and stored at 4 °C for a maximum of one month but the combined final inoculum must be freshly prepared on the day of the mouse injection.
2. On Day 0, administer 50 µL of the inoculum (preparation outlined in step 1.1.4) into a mouse via a subcutaneous injection using a 23 G needle.
   NOTE: The injection can be placed between the shoulder blades, or between the hind limb and tail over the caudal dorsum.
3. On Day 1, make the Pertussis Toxin solution of 1.2 µg/mL (in MT-PBS) and administer 250 µL into a mouse via i.p. injection using a 0.5 mL syringe with a 30^1/2 G needle.
4. On Day 3, repeat step 1.3 above.
   NOTE: Stock solutions A and B can be made up and stored at 4 ˚C for a maximum of one month. However, the final injectable inoculum, produced by combining stock solutions A and B, must be done on the day of the injection.
5. On Day 8, administer 50 µL of the inoculum (preparation outlined in step 1.1.4) into a mouse via a subcutaneous injection (see step 1.2 above), at the same injection site as in step 1.2 (for each animal)

2. Clinical Scoring

1. Perform clinical scoring out daily starting on Day 0.
2. Give each mouse a score of 0, 1, 2, 3, or 4 according to the published criteria⁴. See Table 1 for EAN clinical scoring.
   NOTE: For consistency, an effort should be made to score mice at the same time daily by the same researcher.

3. Motor Function Assessment

NOTE: The motor performance is assessed in parallel with clinical scoring for the same cohort of animals. The motor function assessment apparatus must be connected to a computer that has a gait function imaging system (see Table of Materials) installed. It is also recommended that all mice to be assessed should be habituated to the running task prior to EAN induction. To do this, practice runs (2 test runs per mouse) are performed three days prior to disease induction (Day -3).

1. Turn on the motor function assessment apparatus and switch on the light button.
2. Scruff the mouse firmly and ink its feet by lowering it onto a container filled with red ink while holding its tail.
   NOTE: This step is required for the C57BL/6 strain, but can be skipped if a mouse strain with a white coat color is being used.
3. Place the mouse into the walking compartment and set the treadmill speed at 15 cm/s.
4. Turn on the treadmill and click the "record" button to capture the running motion of the mouse using the gait function imaging system (see Table of Materials).
5. Use a timer and measure each running for 36 s. After 36 s, stop recording and stop the treadmill.
   NOTE: Mice that cannot successfully complete the running task for this 36 s interval are considered to have failed.
6. Save the video file in the designated folder.
7. Repeat the above steps for each mouse to be assessed. Test the mice with the same running task every 3 days.
8. Analyze the edited video files using software for gait function parameters (see Table of Materials).
   NOTE: The details of how to analyze different motor parameters vary amongst software so please refer to the manufacturer's instruction before analysis. To gain histological evidence of axonal and myelin damage via either immunohistochemistry or electron microscopy, animal tissues can be taken at the any stage post motor function assessment depending on the specific aims of research.

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