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 JoVE Biology

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

1, 1, 1

1Department of Physiology and Biophysics, University of California, Irvine (UCI)

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    Summary

    This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

    Date Published: 7/04/2007, Issue 5; doi: 10.3791/224

    Cite this Article

    Beeton, C., Garcia, A., Chandy, K. G. Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis. J. Vis. Exp. (5), e224, doi:10.3791/224 (2007).

    Abstract

    Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).

    Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.

    The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.

    Protocol

    Induction and monitoring of chronic-relapsing EAE

    The emulsion is a 1:1 mixture of antigen in aqueous solution added to supplemented complete Freund's adjuvant. It is very important to add the antigen to the adjuvant and not the other way round!!!

    There is a lot of waste when making and injecting an emulsion. Always prepare 1.5 or 2 times more than what you need.

    Use autoclaved mortar, pestle, glass syringes, and bridges. All plastics should be sterile. The preparation can be done on a regular laboratory bench.

    1. Preparation of the supplemented adjuvant

    Weigh 30 mg of Mycobacterium tuberculosis (Difco catalog # 231141) and place into a mortar. Make into thin powder without pressing too hard to avoid breaking the bacteria. Add 10 ml of complete Freund's adjuvant H37Ra (Difco catalog # 231131) and mix. This supplemented adjuvant now contains 4 mg/ml Mycobacterium tuberculosis. Transfer to a tube.

    2. Preparation of the spinal cord homogenate

    Collect spinal cords from DA rats (age/gender indifferent) and store frozen at -80°C Weigh the frozen spinal cords to have enough to mix 1:1 (weight:volume) with the supplemented adjuvant. Mince the spinal cords as fine as possible with a razor blade, transfer to the mortar used in section 1, and make a paste with the pestle.

    3. Preparation of the emulsion

    1. Place supplemented complete Freund's adjuvant in a tube. Vortex at high speed. Add the spinal cord homogenate drop by drop while vortexing. When all of the spinal cord homogenate is added, vortex for 5 more minutes. The mixture should turn light pink.

    2. Put the emulsion in a 5 ml glass syringe and link it to another 5 ml glass syringe using a 18G bridge (Fisher catalog # 14-825-17L). Send the emulsion from a syringe to the other until it becomes hard (5-10 min). If more than 5 ml of emulsion is prepared, use several 5 ml syringes DO NOT use larger syringes.

    3. The emulsion can be stored in the syringes at 4°C for 3 weeks. I recommend preparing it at least 12 hours in advance to check that it does not break down. It should remain thick and not separate in 2 phases. The color will change overnight to a light yellow or beige.

    4. Immunization of the rats

    1. Mix the emulsion in the syringes for a few minutes and transfer to the syringe used for the injection. Inject 200 µl subcutaneously at the very base of the tail using 23G needles and 3 ml Luer-Lock syringes under short-term anesthesia.

    2. The recipients are 7-9 weeks old female DA rats. We get our rats from Harlan-Sprague Dawley.

    3. Rats should be observed twice daily and weighed daily. Clinical signs are expected 7-15 days after injection of the emulsion.

    Clinical scoring:

    0:         no disease

    0.5:      distal limp tail

    1:         limp tail

    2:         mild paraparesis, ataxia

    3:         moderate paraparesis, the rats trips from time to time

    3.5:      one hind limb is paralyzed, the other moves

    4:         complete hind limb paralysis

    5:         complete hind limb paralysis and incontinence

    6:         moribund, difficulty breathing, does not eat or drink. Euthanize immediately.

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    Discussion

    There are a few important considerations for this protocol.

    Genetics of DA rats will differ slightly depending on which breeder they are bought from. These differences may increase or decrease the susceptibility of rats to EAE induction. If your rats are more susceptible to EAE you will want to reduce the strength of the immunization by using non-supplemented complete Freund's adjuvant or even incomplete Freund's adjuvant. You may also consider reducing the amount of emulsion injected or diluting the spinal cord homogenate with saline before preparing the emulsion. If your rats do not develop clinical signs of EAE using the full-strength immunizing emulsion check the cleanliness of your housing facility. Rats infected with parasites (such as pinworm) will not develop EAE. You may consider housing your animals under cleaner conditions in autoclaved cages with filter-tops, and giving them acidified water ad libitum (the protocol for preparing acidified water is attached).

    The emulsion must be water-in-oil so that the oil in the adjuvant completely coat the antigen you add drop by drop. An emulsion prepared the other way round (oil-in-water) will become as thick but will not be encephalitogenic.

    Rats are friendly animals if they are accustomed to their handler, it is therefore a good idea to handle them gently on a daily basis before injection of the encephalitogenic emulsion. This will reduce stress and the risk of a bite.

    Animals with severe EAE (score of 3 or more) need special care. Rats should never be picked by the tail but rather by the body. This is even more important for reducing stress in sick animals. It is very important to ensure that all animals have access to food and water by putting food and gel packs in the bedding and providing long-sipper tubes on the water bottles. Rats with EAE will continue eating and drinking, if an animal stop either eating or drinking consult your institute's veterinarian or euthanize the animal. Sick rats should be separated from healthy animals to ensure they are not constantly trampled, but avoid leaving rats alone since they are social animals and need company.

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    Disclosures

    CB and KGC are co-founders and consultants for Airmid Inc.

    Materials

    Name Type Company Catalog Number Comments
    Complete Freund’s Adjuvant Reagent Fisher Scientific DF 311-360-5 Manufactured by Difco
    Mycobacterium tuberculosis H37Ra Reagent Fisher Scientific DF3114-33-8 Manufactured by Difco
    DA rat Animal Harlan Laboratories Females, 7-9 weeks old.
    Glass syringes - 5 ml Tool Fisher Scientific 14-823-10 B
    Metal bridge - 18G Tool Fisher Scientific 14-825-17L
    Luer Lok plastic syringes - 3 ml Tool BD Biosciences 309585
    Needles - 20G 1 1/2 Tool Fisher Scientific 14-826-5C

    Comments

    21 Comments

    Dear Dr. Beeton,
    I watched your video with great interest.
    I am a researcher at the CEA, Orsay, France, and we are interested in inducing EAE in DA rats. I would kindly like to ask you a few questions in this regard:
    1. Should the meninges be removed prior to the freezing of the spinal cord, to avoid aggregates/ clumps in the emulsion?
    ². Is it possible to reduce the spinal cord into fine powder by using a mortar and pestle in liquid nitrogen rather than using a razor and cutting it over dry ice?
    3. Can the emulsion be prepared by using a polytron or some other homogenisation system rather than the glass syringes and the connecting bridges (we seem to have a problem to obtain some of these tools).
    4. Is it possible to use rats older than 7-9 weeks or dŒs susceptibility decrease with increasing age?
    Thank you in advance.
    Reply

    Posted by: AnonymousFebruary 5, 2008, 11:20 AM

    We use whole spinal cords, without removing any parts.
    Since we published this video and following advice from Dr. Holmdahl (Lund, Sweden), we have started using fresh spinal cords from ~1² weeks old DA rats. The emulsions give more homogeneous diseases when working with a large group of animals (> 40 recipients). We have not tried using a mortar and pestle. I don't think it would work for fresh cords but might on frozen cords.
    I haven't tried any other methods for preparing the emulsion. As a graduate student in France I was able to buy glass syringes but we had to ask facilities to make metal bridges to connect the syringes by using two metal needles.
    Recipients should be young adults for best susceptibility to EAE. We have never tried using older DA rats but in other rat models of EAE (using Lewis rats) we have found that the older the recipients, the less susceptible to EAE they were. Note that not all DA rats are identical, there are small variations from vendor to vendor so you may want to try rats from several vendors in your area to find which are more susceptible to EAE.
    If you have any more questions you are welcome to contact me by email at beeton@bcm.edu.
    Reply

    Posted by: Christine B.February 5, 2008, 4:02 PM

    Dear Dr. Beeton, thank you for very interesting video. I would like to ask you , if you have any experience with the EAE induction in mice using this protocol. Thank You for the answer. Sincerely Jan Pazour
    Reply

    Posted by: AnonymousJune 30, 2008, 9:31 AM

    I have never used this protocol in mice. For mice I have only used the MOG 35-55 peptide to induce EAE [in C57BL/6 mice]. Using whole spinal cord should work in mice but the protocol would have to be adjusted. You would first have to ensure you are working with the right strain and then you would have to work out the doses and type of adjuvant. Most protocols in mice also call for an additional injection of pertussis toxin and very often mice need a booster injection to develop EAE. This can be a rather tricky protocol to adapt to mice. Sorry that I could not give you a more helpful answer.
    Reply

    Posted by: Christine B.June 30, 2008, 10:38 AM

    Dear all, could there be any difference in using M.butyricum instead of M. tuberculosis H37RA containing adjuvant in inducing EAE in wistar rats (mylein basic protein- MP bio-vendor). I plan to use the former.Please suggest.

    raghul
    toxicologist
    SGS -LSS india
    Reply

    Posted by: AnonymousAugust 25, 2008, 1:24 PM

    Dear Raghul, I have no experience inducing EAE in Wistar rats so am unsure of the results. When inducing acute EAE in Lewis rats with MBP we used CFA made with M. butyricum and supplemented it with 3 mg/ml M. tuberculosis and this worked well with 100% EAE incidence and a homogeneous disease. Best wishes, Christine
    Reply

    Posted by: Christine B.August 25, 2008, 5:46 PM

    what is the concentration of the HCl for drink wather?
    Reply

    Posted by: Andres Q.September 15, 2009, 2:40 PM

    To prepare acidified drinking water, fill a clean carboy with 10 liters of water. Add 5 ml of 1²N (concentrated) hydrochloric acid (HCl) to the water and stir. The pH of the solution should be in the range of ².4-3.0.
    For more safety, ensure that your stock of concentrated acid is not used for anything else but preparing acidified drinking water. This will reduce the risk of accidental contamination with toxic chemicals.
    Do not check the pH directly in your carboy but rather in a sample taken into a beaker.
    This acidified drinking water is not quite as acidic as common sodas many people drink every day. It will not damage the lining of the Œsophagus of the rats.
    Reply

    Posted by: AnonymousSeptember 15, 2009, 3:03 PM

    Dear Dr Beeton,
    I found and watched with great interest you movie about induction and clinical
    scoring of chronic-relapsing EAE in rats and i would like to ask you if
    you have some hystological or/and MRI findings to prove that the major
    pathophisiologycal changes, induced by the immunization, occur in the brain
    and not in the spinal cord? How much this animal model mimics what is
    happening in human brain in MS? I am interested more by demyelination
    which i can observe using DT-MRI.

    Thank you in advance
    Reply

    Posted by: AnonymousOctober 5, 2009, 8:32 AM

    We have not done any histology the brain of these animals as our focus was on the spinal cord.
    We used the brains to isolate mononuclear cells and found more with a CCR7- differentiated phenotype in EAE rats than controls. This suggest brain-infiltration and therefore a potential for demyelinating lesions, but I can't guaranty it.
    This model is definitely demyelinating in the spinal cord (see figure S6 in the online supplement of the paper Matheu, Beeton et al. Immunity ²008, ²9:60²).
    Christine
    Reply

    Posted by: AnonymousOctober 5, 2009, 1:54 PM

    Dear Dr. Beeton, thank you for very interesting video. Do you have the protocol to induce EAE in mice? Can you please provide injecting protocol for mice. Thank you.
    Reply

    Posted by: AnonymousJuly 12, 2010, 1:24 PM

    The protocol will depend on the mouse strain you are interested in. Some strains are more susceptible to EAE than others. A good place to start is the following protocol: http://www.penningerlab.com/uploads/media/EAE_induction_01.pdf
    Reply

    Posted by: AnonymousSeptember 30, 2011, 11:34 PM

    Dear Dr Beeton
    Thanks for the vieo, it is very helpful. I have just one question regarding the part you use as "SPINAL CORD". Is it seperated from spinal column or you just use spinal colum containing spinal cord?
    Reply

    Posted by: AnonymousNovember 12, 2011, 5:01 AM

    You want to use the spinal cord itself, removed from the bone. It is pretty easy to collect since you don't need to keep it in a single piece (as you would for histology for example). You can simply scrape it out.
    Hope this helps!
    Christine
    Reply

    Posted by: AnonymousNovember 14, 2011, 4:36 PM

    Dear Dr Beeton
    Thanks for the video. Please let me know how to make acidified water and HOW LONG THE ANIMAL SHOUL DRINK SUCH A WATER BEFORE INDUCTION OF EAE?
    Regards
    Reply

    Posted by: AnonymousNovember 27, 2011, 3:58 AM

    For preparation of the acidified water, please refer to my answer to a similar question from Andres Quintanar, posted 09/15/²009.
    We put our rats on acidified water as soon as we receive them, which is 4-7 days before we induce EAE.
    Reply

    Posted by: AnonymousNovember 27, 2011, 4:40 PM

    Dear Dr Beeton
    Thanks for the video. It is really helpful. Can we use Sprague-Dawley and Wistar Rats. Do we need in your model to use pertussis toxin.
    Reply

    Posted by: AnonymousDecember 5, 2011, 6:32 AM

    Hello Dear Christine Beeton
    Thanks for your useful video. I would be grateful if you response my question: In this video when you work with Mycobacterium tuberculosis H37RA for making it thin powder, you didn't wear mask, is it safe for you?

    Regards
    Reply

    Posted by: AnonymousJanuary 22, 2012, 5:34 AM

    Hello Dear Professor Beeton
    I have another question dear Dr Beeton: Should we perfuse the rat before spinal cord isolation (for EAE induction) or no the perfusion is just necessary when we want to isolate the cells from spinal cord or brain? I want to isolate the guina pig spinal cord for EAE induction and I don't know the perfusion is necessary or no.
    Best Regards
    Reply

    Posted by: AnonymousMarch 10, 2012, 7:46 AM

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