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 P0180-199 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 P0180-199 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 life6,7.
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 (P257-81) 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 P0106-125 or P0180-199 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 P0180–199 peptide9 was chosen in preference to the P057-81 alternative10. The P0180–199 model has been described to produce less severe clinical signs compared with the P057-81 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 P057-81 induced variant.
Protocol
Representative Results
Discussion
This report outlines a simple method to induce EAN using the P0180-199 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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
DGG is a NHMRC Peter Doherty and Multiple Sclerosis Research Australia (MSRA) Early Career Fellow. JLF is supported by an MSRA Postdoctoral Fellowship. This work was supported by the Australian National Health and Medical Research Council (NHMRC) project grant #APP1058647 to JX.
Materials
| C57BL/6, male, 6-8 weeks old | Australian Bioresources Cenre, WA, Australia | ||
| Pertussis toxin | List Biological Laboratories, Inc., CA, USA | #181 | |
| 0.1M 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 |
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