Name: a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments
Uploaded: 2017-11-09T13:00:00
Description: Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

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.

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 we...

<ol>
	<li>Newswanger, D. L., Warren, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 69 (10), 2405-2410 (2004).</li><li>Ruiz, E., Ramalle-Gomara, E., Quinones, C., Martinez-Ochoa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Guillain-Barre+syndrome+mortality+in+Spain+from+1999+to+2013.">Trends in Guillain-Barre syndrome mortality in Spain from 1999 to 2013.</a> Int J Neurosci. 126 (11), 985-988 (2016).</li><li>Walling, A. D., Dickson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 87 (3), 191-197 (2013).</li><li>Gonsalvez, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+and+Neuropathological+Testing+Paradigm+Reveals+New+Disability-Based+Parameters+and+Histological+Features+for+P0180-199-Induced+Experimental+Autoimmune+Neuritis+in+C57BL/6+Mice.">A Functional and Neuropathological Testing Paradigm Reveals New Disability-Based Parameters and Histological Features for P0180-199-Induced Experimental Autoimmune Neuritis in C57BL/6 Mice.</a> J Neuropathol Exp Neurol. 76 (2), 89-100 (2017).</li><li>Berg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome:+pathogenesis,+diagnosis,+treatment+and+prognosis.">Guillain-Barre syndrome: pathogenesis, diagnosis, treatment and prognosis.</a> Nat Rev Neurol. 10 (8), 469-482 (2014).</li><li>Koller, H., Schroeter, M., Kieseier, B. C., Hartung, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammatory+demyelinating+polyneuropathy--update+on+pathogenesis,+diagnostic+criteria+and+therapy.">Chronic inflammatory demyelinating polyneuropathy--update on pathogenesis, diagnostic criteria and therapy.</a> Curr Opin Neurol. 18 (3), 273-278 (2005).</li><li>Griffin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+the+motor-sensory+axonal+Guillain-Barre+syndrome.">Pathology of the motor-sensory axonal Guillain-Barre syndrome.</a> Ann Neurol. 39 (1), 17-28 (1996).</li><li>Taylor, W. A., Hughes, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+allergic+neuritis+induced+in+SJL+mice+by+bovine+P2.">Experimental allergic neuritis induced in SJL mice by bovine P2.</a> J Neuroimmunol. 8 (2-3), 153-157 (1985).</li><li>Zou, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P0+protein+peptide+180-199+together+with+pertussis+toxin+induces+experimental+autoimmune+neuritis+in+resistant+C57BL/6+mice.">P0 protein peptide 180-199 together with pertussis toxin induces experimental autoimmune neuritis in resistant C57BL/6 mice.</a> J Neurosci Res. 62 (5), 717-721 (2000).</li><li>Miletic, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P0(106-125)+is+a+neuritogenic+epitope+of+the+peripheral+myelin+protein+P0+and+induces+autoimmune+neuritis+in+C57BL/6+mice.">P0(106-125) is a neuritogenic epitope of the peripheral myelin protein P0 and induces autoimmune neuritis in C57BL/6 mice.</a> J Neuropathol Exp Neurol. 64 (1), 66-73 (2005).</li><li>Hafer-Macko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+motor+axonal+neuropathy:+an+antibody-mediated+attack+on+axolemma.">Acute motor axonal neuropathy: an antibody-mediated attack on axolemma.</a> Ann Neurol. 40 (4), 635-644 (1996).</li><li>Griffin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+nodal+changes+in+the+acute+motor+axonal+neuropathy+pattern+of+the+Guillain-Barre+syndrome.">Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain-Barre syndrome.</a> J Neurocytol. 25 (1), 33-51 (1996).</li></ol>

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...

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-Barr&#233; 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.

<table>
	<tbody>
		<tr>
			<td>C57BL/6, male, 6-8 weeks old</td>
			<td>Australian Bioresources Cenre, WA, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pertussis toxin</td>
			<td>List Biological Laboratories, Inc., CA, USA</td>
			<td>#181</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M mouse-isotonic phosphated buffered salined (MT-PBS)</td>
			<td></td>
			<td></td>
			<td>Laboratories will have their own protocol.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pharmachem, QLD, Australia</td>
			<td></td>
			<td>Laboratories will have their own protocol for administration.</td>
		</tr>
		<tr>
			<td>P0180&#8211;199 peptide</td>
			<td>Wuxi Nordisk Biotech Co. Lt. SHG, CHN</td>
			<td></td>
			<td>P0180&#8211;199, sequence S&#8211;S&#8211;K&#8211;R&#8211;G&#8211;R&#8211; Q&#8211;T&#8211;P&#8211;V&#8211;L&#8211;Y&#8211;A&#8211;M&#8211;L&#8211;D&#8211;H&#8211;S&#8211;R&#8211;S</td>
		</tr>
		<tr>
			<td>Heat killed Mycobacterium tuberculosis (strain H37RA)</td>
			<td>Difco, MI, USA</td>
			<td>#231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#39;s complete adjuvant (FCA)</td>
			<td>Difco, MI, USA</td>
			<td>#263910</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Services</td>
			<td>#15710</td>
			<td>Dilute to 4% PFA day of tissue collection.</td>
		</tr>
		<tr>
			<td>25% glutaraldheyde</td>
			<td>ProSciTech Pty Ltd, QLD, Australia</td>
			<td>#11-30-8</td>
			<td>Dilute to 2.5% glutaraldehyde day of fixation.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Chem-Supply Pty Ltd, SA, Australia</td>
			<td>SL189</td>
			<td>Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v).</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-Supply Pty Ltd, SA, Australia</td>
			<td>SA030</td>
			<td>Use at 30% (w/v).</td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (OCT) medium</td>
			<td>Sakura Finetek, CA, USA</td>
			<td>#4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Merck Millipore, MA, USA</td>
			<td>#S30-100</td>
			<td>Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory.</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma Aldrich, MI, USA</td>
			<td>#90o2-31-1</td>
			<td>Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory.</td>
		</tr>
		<tr>
			<td>rabbit anti-amyloid precursor protein (APP)</td>
			<td>Invitrogen (Life Technologies), CA, USA</td>
			<td>S12700</td>
			<td>Used at 1:400 or titrate in own lab.</td>
		</tr>
		<tr>
			<td>rabbit anti-contactin-associated protein-1 (Caspr)</td>
			<td>Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel</td>
			<td></td>
			<td>Used at 1:500 or titrate in own lab.</td>
		</tr>
		<tr>
			<td>Appropriate Alexa Fluor conjugated secondary antibodies</td>
			<td>Molecular Probes (Life Technologies), OR, USA</td>
			<td>Various</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Aqueous mounting solution</td>
			<td>Dako (Agilent), CA, USA</td>
			<td>#S3023</td>
			<td>Each laboratory will have their own preference.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL syringe with 301/2 g needles</td>
			<td>BD</td>
			<td>#326105</td>
			<td></td>
		</tr>
		<tr>
			<td>23 g needles</td>
			<td>BD</td>
			<td>#305143</td>
			<td></td>
		</tr>
		<tr>
			<td>Red ink pad</td>
			<td></td>
			<td></td>
			<td>Any red ink pad or red food dye could be used to mark the animals&#39; feet.</td>
		</tr>
		<tr>
			<td>DigiGate apparatus (includes treadmill)</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DigiGate Imaging System</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td></td>
			<td></td>
			<td>Any timer may be used.</td>
		</tr>
		<tr>
			<td>DigiGait 8 Software</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any appropriate dissecting microscope may be used.</td>
		</tr>
		<tr>
			<td>Charged slides</td>
			<td>Superfrost Plus, Lomb Scientific Pty Ltd</td>
			<td>SF41296SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyrostat</td>
			<td>Leica</td>
			<td></td>
			<td>Any suitable cyrostat may be used.</td>
		</tr>
		<tr>
			<td>Perfusion equipment and dissecting instruments</td>
			<td></td>
			<td></td>
			<td>Labs will have their own perfusion protoctols.</td>
		</tr>
		<tr>
			<td>Opaque humified chamber</td>
			<td></td>
			<td></td>
			<td>Labs may produce their own using an opaque plastic container.</td>
		</tr>
		<tr>
			<td>PAP pene</td>
			<td>GeneTex (USA)</td>
			<td></td>
			<td>Wax pencil, or surface tension may also be used to create a well around the tissue section.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeis LSM780</td>
			<td></td>
			<td>Any confocal microscope with appropriate laser lines may be used.</td>
		</tr>
		<tr>
			<td>FIJI/Image J</td>
			<td>National Institues of Health</td>
			<td></td>
			<td>Available from www.fiji.sc</td>
		</tr>
	</tbody>
</table>

a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments

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.

P0180-199 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...

Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

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

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&#39;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.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

The overall goal of this procedure is to successfully induce Experimental Autoimmune Neuritis or EAN in a mouse model for the functional and pathological analysis of the disease process. The EAN model can be used to address key questions relating to peripheral neuropathy. It's also a wonderful model to potentially investigate the efficacy of novel therapeutic agents.The main advantage of this approach is that it provides a simple and objective functional testing paradigm that can be applied when using the EAN model in the mouse. Demonstrating the procedure with me today is Sang Won Yoo, who is an undergraduate student in the neurotrophin and myelin biology lab at the University of Melbourne. Three days before the disease induction, turn on the motor function assessment apparatus and switch on the light button.Pick up the mouse and lower it onto a container of red food dye. Place the mouse into the walking compartment and set the treadmill speed to 15 centimeters per second. Turn on the treadmill and click record to use the gait function imaging system to capture the running motion of the mouse for 36 seconds.After 36 seconds, stop the recording and the treadmill and save the practice run video file of the practice run in the designated folder. One day before the first immunization, use a 0.5 milliliter syringe equipped with a 30-1/2 gauge needle to administer 1.6 micrograms per milliliter of Pertussis toxin in 250 microliters of mouse isotonic PBS IP to anesthetized six to eight-week-old male C57BL/6 mice. Then, score the mice for clinical signs of EAN on a scale from zero to four according to the table and perform a motor function assessment as just demonstrated.The next day, combine equal volumes of freshly prepared solutions of peptide in 0.9%saline and 20 milligrams per milliliter of Mycobacterium tuberculosis in complete Freund's adjuvant in a bead beader and mix the solutions at the maximum speed for one minute at room temperature. Load a syringe equipped with a 23 gauge needle with the inoculum and invert and shake the syringe before evacuating any air within the syringe shaft. Before delivering the inoculum, remove the hair just lateral to the midline between the scapulae and disinfect the exposed skin with 70%ethanol.Then, use a pair of Adson forceps to grasp the skin and deliver 50 microliters of the solution to the Pertussis toxin injected mouse via subcutaneous injection. The next morning, deliver 1.2 micrograms per milliliter of Pertussis toxin in 250 microliters of PBS IP as just demonstrated followed by another 1.2 micrograms per milliliter injection of Pertussis toxin IP 48 hours later. Three days later after the third Pertussis injection, administer another 50 microliters of freshly prepared inoculum at the same injection site as before and monitor the clinical score and motor function of the animals twice a week until the end of the experiment.P0 180-199 peptide-induced EAN in C57BL/6 mice leads to a monophasic disease with a clinical score onset from six days post first immunization and a maximal score severity at 25 days post first immunization, followed by some clinical score improvement at 40 days post first immunization. The mice begin to fail at a simple running task as early as six days post first immunization with little capacity to complete a simple treadmill running task by 35 days post immunization that does not improve in running ability at 55 days post first immunization. The examination of semi-thin sections of the sciatic nerves of peptide-induced EAN mice reveals areas of demyelination compared to sciatic nerves from age-matched healthy controls.High power transmission electron microscopy images allow the identification of specific neuropathological features such as demyelination, damage to the myelin lamella, and signs of axon stress such as mitochondrial swelling. These animals also display acute axonal damage as evidenced by traumatically elevated beta-amyloid precursor protein expression in the sciatic nerves that is not observed in age-matched healthy control tissues. A widening of the nodes and a disruption to the paranodes are also observed.Once mastered, the subcutaneous injections can be performed within about five to 10 minutes per mouse with minimum chance of any injection error. While attempting this procedure, it's really important that you observe the injection site to ensure that all of the inoculum remains within the injection site and that none of it leaks out. Video capture of treadmill gait is great because it enables other parameters to be computed such as swing phase or stride length and these measures may become important when testing the efficacy of novel therapeutics.After watching this video, you should have a really good understanding of how to induce EAN in C57BL/6 male mice. In addition, you should have a really good understanding of how to place subcutaneous injections safely and really accurately ensuring that there is no injecting error. Don't forget that working with needles and inoculum, there is the chance of injury especially when using sharps.So, to avoid any chance of injury, it's a really good idea to always use the forceps and never grasp skin or tissue by hand and also to use single-use syringes, never recapping in the process.

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