1 Introduction

Multiple sclerosis (MS) is one of the most prevalent neurologic disorders leading to chronic disability among young adults [1, 2]. MS is considered an autoimmune disorder that causes a demyelinating inflammatory disease of the central nervous system where the destruction of oligodendrocytes and neurons results in heterogenic and accumulating clinical symptoms. To study pathologic features of MS, three main animal models are used: (1) disease induction by toxic agents like cuprizone, (2) viral models like Theiler’s murine encephalomyelitis virus (TMEV), and (3) different types of experimental autoimmune encephalomyelitis (EAE) [3]. These models mimic some of the features of MS, but do not recapitulate all of them. Yet, they are critical for the development of new therapeutic strategies.

EAE is among the most used models for our understanding of MS because of their histopathologic and immunologic similarities. EAE is a CD4+ T cell-mediated autoimmune disease characterized by perivascular CD4+ T cell and mononuclear cell infiltration and inflammation and subsequent primary demyelination of axons in the CNS, leading to progressive clinical signs such as tail and hind limb paralysis. Actively induced EAE in mice is elicited by immunization with CNS homogenates or peptides of myelin proteins like myelin oligodendrocyte glycoprotein (MOG) , which triggers an immune response against the CNS that induces inflammation and destruction of myelin or antigen-bearing structures that causes neurological abnormalities similar to those seen in patients with MS [3, 4].

We will now describe the protocol we have used to immunize mice from different strains using a MOG35–55 peptide which results in a monophasic response with symptoms that appear approximately 10–15 days after immunization [5,6,7].

2 Materials

  1. 1.

    Lyophilized MOG35–55: sequence:MEVGWYRSPFSRVVHLYRNGK-carboxyl. Purity: >98%. Peptide synthetized under request in the Peptide Synthesis Facility at the Universitat Pompeu Fabra (UPF).

  2. 2.

    Bovine serum albumin (BSA).

  3. 3.

    Complete Freund’s adjuvant (CFA).

  4. 4.

    Mycobacterium tuberculosis H37RA powder.

  5. 5.

    Bordetella pertussis toxin (BPT).

  6. 6.

    Animals: We have been using 2–4 months old male and female mice successfully at any time of the year. The genetic background in the different animals used has been: 129S1/SvImJ, C57BL/6 and C57BL/6 X SJL hybrid.

  7. 7.

    Needles: for the intraperitoneal (IP) injection, we use 0.5 × 16 mm 25 Gx5/8″, and for the subcutaneous (SC) injection 0.6 × 25 mm 23 Gx1”.

  8. 8.

    1 mL syringes.

  9. 9.

    Sonicator.

3 Method

3.1 Preparation of MOG35–55 Emulsion for EAE and BSA Emulsion for Sham-EAE Mice

Prepare immunizing emulsion for SC injection.

  1. 1.

    Calculate total amount of the solution that we will need for the mice to be studied in a single batch, knowing that 200 μL of 1:1 ratio of MOG35–55 and CFA solutions have to be injected to each mouse and that there is some loss of the viscous emulsion while preparing and injecting. We always prepare emulsion for ~5 animals more. For example, for 20 mice we prepare enough immunizing emulsion to inject 25 mice, i.e., 5 mL (2.5 mL of the MOG35–55 solution + 2.5 mL of the CFA solution):

  2. 2.

    Dilute lyophilized MOG35–55 peptide in sterile 10 mM PBS to a final concentration of 3 mg/mL.

  3. 3.

    Prepare CFA with a final concentration of Mycobacterium tuberculosis of 4 mg/mL. Since commercial CFA carries only 1 mg/mL, we must add 3 mg of Mycobacterium tuberculosis per mL of CFA that we want to prepare. Mix thoroughly with the pipette.

  4. 4.

    Mix thoroughly 1:1 MOG35–55 and CFA solutions in a glass vial and proceed to emulsify by sonication (0.75 cycles, amplitude 80%), keeping the solution in ice to avoid heating. It takes 4–6 min until the emulsion is well done. The solution should be white, stiff, and viscous with no separation of phases (seeNote 1 ).

    Alternatively, the emulsion can be prepared loading the MOG35–55 and CFA solutions into two separate Popper syringes, connected with a Luer connector and by continuous exchange between the two syringes. We initially prepared the emulsion with this method, but using the sonication method we have achieved very good, and faster results, and losing less emulsion.

  5. 5.

    For the Sham-EAE mice we proceed to prepare an emulsion in which the MOG35–55 is replaced by BSA.

3.2 Preparation of Bordetella Pertussis Toxin

Prepare BPT for IP injection. Each mouse is injected at day 0 (just after immunization) and 2 days later, with 500 ng of BPT per mouse in 200 μL saline or PBS (2.5 μg/mL).

  1. 1.

    Stock BPT 250 μg/mL is prepared in ddH2O. Prepare aliquots and store at −20 °C.

  2. 2.

    To prepare the necessary amount of BPT, freshly dilute 1:100 with saline or PBS to get the working solution (2.5 μg/mL).

3.3 Animal Immunization

  1. 1.

    On day 0, the EAE and Sham-EAE mice, under inhaled isoflurane anesthesia (induction 4%; maintenance 2%), are carefully injected subcutaneously into both hind flanks with 200 μL of emulsion (100 μL each). The injected emulsion should form a bubble under the skin. For this task, use a 1 mL syringe and 23G needle (seeNote 2 ).

  2. 2.

    Inject 200 μL of BPT by IP injection. For this task, use a 1 mL syringe and 25G needle.

  3. 3.

    A second dose of BPT is given on day 2 after immunization.

3.4 EAE Monitoring

  1. 1.

    Evaluate changes in body weight and clinical scoring. Record these parameters every other day at the beginning, and daily from 7th postimmunization day. Body weight decreases usually precedes EAE clinical signs and is a very objective and valuable measure (seeNote 3 ). The clinical score may be evaluated in a number of ways [4, 5, 8,9,10], for instance using a scale ranging from 0 to 5 following the criteria indicated in Table 1.

    Representative results of body weight changes and clinical score obtained in floxed IL-6 mice that are in C57Bl/6 background [11] are shown in Fig. 1. As expected, EAE clinical signs were obvious in MOG35–55 but not in BSA-immunized mice, beginning ~13 days after immunization in this particular experiment; onset of weight loss was noticed slightly before.

  2. 2.

    Euthanize mice by different methods depending on the type of analysis to be carried out, i.e., with (for detailed histopathological analysis) or without (for molecular analysis) tissue perfusion methods [5, 8]. For instance, one of the prototypical histopathological changes observed in the spinal cord is white matter demyelination, as shown in Fig. 2 (seeNote 4 ).

Table 1 Clinical score used for evaluating EAE
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Fig. 1
figure 1figure 1

Representative results of changes in body weight and clinical score obtained after induction of EAE in floxed IL-6 mice that are in C57Bl/6 background [11]. (a) Body weight gain. Mice immunized with MOG35–55 (flox/EAE) but not with BSA (flox/BSA) showed the prototypical body weight loss. (b) Clinical score. Mice immunized with MOG35–55 but not with BSA showed the expected signs of EAE, which started with tail loss of tonus and continued in an ascending fashion

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Fig. 2
figure 2figure 2

Luxol fast blue staining of spinal cord for myelin evaluation. Infiltrates (arrows) and demyelination is observed in EAE animals

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4 Notes

  1. 1.

    At this point we test to see if the emulsion has formed well by placing a single drop of emulsion into a layer of tap water in a Petri dish lid. If the drop disperses, then the emulsion has not formed properly. Keep going with the sonication until the drop does not disperse in fresh water. Once emulsion is formed it can be stored at 4 °C for a few hours or used immediately.

  2. 2.

    All experiments using mice must be approved by the respective institutional animal ethic committee. In this case, experiments were approved by the Autonomous University of Barcelona (UAB) Animal and Human Experimentation Ethics Committee. The criteria may change between institutions and countries, and also over time, so please refer to the respective guidelines.

  3. 3.

    All animals are kept under constant temperature and free access to food and water. When mice have clinical symptoms of EAE, hydrated food is placed on the cage floor to facilitate access to these mice.

  4. 4.

    In this experiment, the mice were killed 20 days postimmunization, i.e., at ~peak time. If the experiment lasted longer, the mice recover somewhat but normally will never fully recover (i.e., EAE is monophasic, without relapses), a well-known characteristic of the C57/Bl6 genetic background [3]. As stated above, if mice have very severe clinical symptoms of EAE, hydrated food must be placed on the cage floor to facilitate access to these mice. Also, the bladder should be checked to identify signs of pain and to help in cases of urine retention. Local guidelines must be followed regarding criteria for endpoints. For instance, body weight loss higher than 25–30% is a likely endpoint for humane killing of the animal.