This is the most important step in inducing EAE as the severity of the induced disease to a great extent depends on a thoroughly homogenised emulsion. The emulsion is typically a 1:1 ratio by volume of a solution containing the antigen of interest (in our laboratory this is typically recombinant MOG (rMOG) in female DA rats) and an emulsifier (usually incomplete Freund’s adjuvant (IFA) or complete Freund’s adjuvant (CFA)); each animal typically receives a 200 μl portion of the total emulsion (0.1 mg of antigen per animal). When calculating the amount of antigen needed, always allow for at least an extra volume of up to 200 μl as losing some of the emulsion while preparing for injection is inevitable.

To make the emulsion, two glass syringes (Hamilton, Bonaduz, Switzerland) are connected together using a metal connector. The plunger on one syringe is taken out before connecting to avoid vacuum build up. The connected syringes are then placed vertically in a glass container such as a beaker with the top syringe being the one with no plunger. The empty barrel is then filled by slowly adding the adjuvant and then the antigen using a pipette. The plunger is then put slowly back in. The two connected syringes are then held horizontally and the plunger is pushed firmly but slowly to facilitate mixing of the antigen with the adjuvant as the mixture passes through to the second syringe. The mixture is passed between the two syringes for at least 30–40 times or until the emulsion is thick and homogenous in texture. At this stage, the plunger of the syringe containing the emulsion is drawn back slightly to produce a gentle vacuum, and the empty syringe and connector are disconnected and removed to be replaced with a needle. To eliminate air bubbles remaining in the emulsion, which would result in under-dosing, a 23–25G needle is placed on the syringe and air bubbles are removed by holding the syringe vertically and tapping and expelling the air through the needle. If air bubbles have inadvertently been included, they can be difficult to dislodge if the emulsion is appropriately thick.

Under light anaesthesia (e.g., 2% isoflurane) the area of the back immediately adjacent to the base of the tail is shaved, and the skin scrubbed with iodine-based disinfectant.

Gently pull the skin from the shaved area to create a small “tent,” insert the tip of the sterile needle into the pulled tent making sure that the needle is in a subcutaneous space, and gently manipulate the needle laterally to create a small pocket for injection. Inject 200 μl of the emulsion, avoiding injecting intradermally. Allow the needle to remain in place for a few seconds and then gently remove it, closing the puncture wound between the fingertips while doing so. Clean the injection site using iodine and gently push the bolus of injected fluid rostrally to avoid the emulsion leaking out of the injection site. Transfer the animal to a recovery chamber.

In our experience, this EAE model works best in male DA rats, but it can also be employed in Lewis rats. The whole spinal cord of one adult male rat is sufficient for immunizing three adult male rats. Once the animal is sacrificed, the spinal cord is dissected free and the dura removed. Sterile saline should be applied as required to prevent the tissue from drying out. Each cord is weighed and the weight noted to ensure that sufficient cord tissue is obtained for the proposed usage, and the cord is then wrapped in a small piece of aluminium foil and left to freeze on dry ice. The frozen cords are stored at −80°C.

On the day of immunization, the required weight of spinal cord tissue is defrosted on ice. Using a sharp blade the cord is minced into smaller pieces to facilitate making the homogenate with adjuvant. In this model of EAE, we find it is best to use CFA to achieve effective disease. As explained in Sect. 2.2.1., the pieces of spinal cord will be added to CFA in the glass syringe and carefully emulsified as explained. The injection method is the same as described in Sect. 2.2.3.

In our experience, the first symptoms of EAE (rMOG and SCH rat models) occur between 9 and 11 days post immunization (DPI). Depending on the quality of the injected emulsion, a slightly earlier or later onset can occur, typically preceded by weight loss in the animals.

Eight or ten days post-immunization with rMOG (100 μg per ­animal in IFA), using techniques similar to those described above for induction of active EAE, animals are sacrificed by an overdose of carbon dioxide and the draining (inguinal) lymph nodes and spleens are harvested into sterile Hank’s buffered salt solution (HBSS). Spleens are used as a source of antigen-presenting cells (APCs) and do not have to be harvested from immunised animals. For efficiency, we harvest the tissue from 5 to 6 actively immunised animals. The lymph nodes and the spleens are dissociated ­separately using the plunger of a 2-ml syringe to pass the tissue through a 70-μm mesh cell strainer, and washed three times in sterile HBSS. The number of cells in the single cell suspension is then estimated using trypan blue staining and a haemocytometer.

Harvested lymphocytes are subjected to a two-phase in vitro protocol in order to produce a high number of activated, antigen-specific T cells. In the first phase, the lymph node cells are co-­cultured with APCs pre-incubated (pulsed) with the antigen (rMOG). During this process, any lymph node cells which were in vivo primed against rMOG will receive survival stimuli from APCs and change their morphology to that of activated (blast) cells. The blast cells are larger than non-activated lymphocytes and of lower density than non-activated lymphocytes, and thus they can be separated by density gradient centrifugation.

To produce blasts, washed and counted APCs are first ­irradiated with 30 Gy (Cobalt 60 source), centrifuged for 8 min at 680  ×  g at room temperature, and the pellet re-suspended in RPMI 1640 medium. The irradiated cells are incubated (pulsed) with antigen (20 μg/ml of rMOG) for 90 min at 37°C, in a 5% carbon dioxide atmosphere. After 90 min, the lymph node cells are added to antigen-pulsed APCs at a ratio of 1:6–1:10 (lymph node cells : APCs), to give a final concentration of cells in the co-culture of 3  ×  10⁶/ml of RPMI 1640 medium supplemented with 10% foetal calf serum (FCS). After 48–72 h, depending upon the specific response of the cells to culture conditions (see Note 4.2), cells from the culture are washed, re-suspended in 6 ml of HBSS, very carefully layered on 3 ml of Lymphoprep solution (Frenius Kabi Norge, Oslo, Norway) and then centrifuged for 30 min at 1,462  ×  g. At this stage, it is very important to not allow mixing of the two phases and to disable the centrifuge brake. After centrifugation, the blasts are carefully ­collected from the interface with a pipette, washed in HBSS and counted in a haemocytometer.

The second phase is the expansion of the blasts in a medium enriched with IL-2. The blasts are resuspended in RPMI 1640 medium supplemented with 20 U/ml of IL-2 and cultured for a further 48–72 h. The exact duration of culture with IL-2 depends on blast activation state and number (see Note 4.2). In general, blasts proliferate well at higher densities (70% covering of flask ­surface or more). When cells reach over 100% covering density in the culture flask, they are washed again and the whole cycle of ­re-stimulation with APCs is repeated. Several cycles of re-stimulation may be necessary to achieve the adequate numbers of cells needed for passive transfer. The vast majority of activated blasts produced in this way are CD4+ T cells.

For cell transfer, naive DA rats are anaesthetised (2% isoflurane in oxygen) and a small area of the tail is cleaned with iodine ­solution. A small incision is made on the lateral side of the tail base and a short length of the tail vein exposed and cleared of the ­surrounding connective tissue. The caudal end of the exposed vein is ligated using a 4.0 suture. A loop of suture is tied around the rostral end of the exposed vein, but not tightened. Using iris scissors a small cut is made in the vein, cutting approximately half of the vein circumference. This incision is used to insert a cannula (Fine Science Tools, Canada) attached to a 1-ml syringe containing 500–700 μl of a cell suspension in PBS. The cannula is then secured by tightening the knot at the rostral end of the exposed tail vein, and the cell suspension is slowly injected. After the cells are injected, the ­cannula is carefully removed, the rostral end of the exposed part of the vein ligated and the wound closed using a 4.0 suture. Sterile technique is applied throughout the procedure. Cells may also be injected via the intraperitoneal route.

We find that passive transfer of ten million encephalitogenic T cells generated using the described protocol induces very reproducible, monophasic EAE in DA rats. The disease onset is at day 4 or day 5 post-transfer. All animals reach the peak of the disease on day 6 post-transfer, characterised by a paralysed tail, inability to spread toes and ataxic gait. These signs of motor deficit last for 3 days. All animals completely recover by day 10 post-transfer. Pathological characteristics at the peak of disease are inflammatory infiltrates and demyelination, with only small numbers of degenerated axons present.

In C57BL6 mice, the donor group is immunized with MOG_35–55/CFA. After 11 days, spleens are isolated from the immunized mice and splenocytes are restimulated in vitro with MOG_35–55, IL-12 and anti-IFNγ antibodies for 3 days to generate encephalotigenic T cells. These are then injected to the recipient mice as before.

Each mouse is immunized with a total amount of 200 μg of MOG_35–55 in 200 μl of the emulsion, resulting in a final antigen concentration of 1 mg/ml. The emulsion contains a 1:1 ratio (by volume) of MOG_35–55 solution: CFA and is administered to each mouse by subcutaneous injection. In practice, the MOG_35–55 peptide is dissolved in sterile saline at a concentration that is twice the final concentration, to allow for the dilution. Due to loss of some of the emulsion in the dead space of the hub of the syringe, excess emulsion should initially be prepared. We calculate the total volume of emulsion needed by multiplying the number of mice to be immunized by 1.5, and multiply that number by 200 μl, then this number is divided by two which gives the ­volume of both the MOG_35–55 peptide and the CFA needed. The ­emulsion is prepared using two glass syringes following the procedure ­outlined in 2.2.1.

Pertussis toxin from Bordetella pertussis (Calbiochem, Nottingham, UK) is re-suspended in sterile saline to a concentration of 50 μg/ml stock solution. Dilutions are then made so that a final concentration of 30 (ng/ml) in sterile saline is achieved. Each mouse will receive 0.1 ml of pertussis toxin in saline i.p. on the day of immunization followed by a second dose 48 h later.

Mice are monitored daily to assess the magnitude of any neurological deficit and to detect weight gain or, more importantly, weight loss. In our laboratory, the clinical severity of EAE in mice is scored using a grading scale (0–7) that is outlined in Table 2. The symptoms of EAE tend to appear 9–14 days after immunization, but this timing can vary depending on the strain of mice used.