EAE Induction by Active Immunization in Lewis Rats

Recommended protocol for use with:

Kit selection

These kits are recommended for study of experimental autoimmune encephalomyelitis (EAE), including testing efficacy of potential therapeutics. They will induce EAE in Lewis rats by active immunization with antigen in an emulsion with complete Freund's adjuvant (CFA).

Both kits are recommended for use with female Lewis rats at age 10 to 14 weeks, and contain gpMBP69-88 as the antigen; kit EK-3111 is supplied also with lyophilized pertussis toxin (PTX).

Each kit supplies enough material for 10 rats.

EK-3110 (without pertussis toxin)

EK-3110 is recommended when EAE relapses are not desired.

Typical EAE onset is 10 to 12 days after immunization, with peak of disease usually 3 days after onset for each rat. The peak lasts 1 to 3 days. All rats will fully recover by Day 20 after immunization, without relapses.

Rats are typically observed for 3 weeks.

EK-3111 (with pertussis toxin)

This kit is supplied with two vials of PTX.

With kit EK-3111 around 30% to 60% of rats will show an increase in EAE severity (relapse) after initial full recovery. This usually occurs 20-22 days after immunization.

EK-3111 will induce a more severe disease with onset 1 to 2 days earlier than with EK-3110. Between 30 and 60% of rats immunized with EK-3111 will develop relapse.

Immunization is followed by administration of PTX in PBS, first on the day of immunization (Day 0, using the first vial of PTX) and then again 2 days later (Day 2, using the second vial of PTX).

Typical EAE onset is 8 to 11 days after immunization, with peak of disease usually 3 days after onset for each rat. The peak lasts 1 to 3 days, followed by full recovery. Relapses will start 20 to 22 days after immunization and last 2 to 6 days, then rats will fully recover. Rats will not develop further relapses for at least 60 days.

Rats are typically observed for 4 weeks.

For information on choice of model and antigen, please see Hooke's Learning Center (http://hookelabs.com/learning).

Rat selection and handling

For the most uniform EAE development, use female Lewis rats at age 10 to 14 weeks. All rats should be the same age. Male rats can be used and will develop slightly milder EAE.

We have observed significant differences in EAE development in rats obtained from different breeders; this can change over time. As of this writing (see version date at the end of this protocol), in the United States we recommend Lewis rats from Charles River Laboratories.

Successful induction of EAE requires low-stress rat handling and husbandry procedures, good injection technique, use of appropriate rats, and good quality, stable, antigen emulsion. Stress decreases EAE susceptibility; minimizing rat stress is very important for successful EAE induction.

For consistent EAE, minimize rat stress as follows:

Administration of antigen emulsion (EK-3110 only)

Emulsion should be administered subcutaneously, at two sites, 0.1 mL/site (0.2 mL/rat total).

Good subcutaneous injection technique is important for successful EAE induction, both for proper emulsion administration and also to minimize animal stress. Because EAE susceptibility is influenced by stress, the same person should dose all groups in an experiment (because different people may inflict different amounts of stress).

Lower back injection
  1. Have assistant gently restrain rat against cage.

  2. Inject rat subcutaneously on one side of lower back with 0.1 mL of emulsion (Figure 1).

    Keep the needle inserted into the subcutaneous space for 10 to 15 seconds to avoid leakage of the emulsion. Alternatively, a light pull on the syringe plunger will prevent leakage.

  3. Inject rat subcutaneously on the other side of lower back with 0.1 mL of emulsion.

    Again, keep the needle inserted into the subcutaneous space for 10 to 15 seconds or lightly pull on the syringe to avoid leakage.

    Repeat (1-3) for all rats.

Administration of antigen emulsion and PTX (EK-3111 only)

Materials needed (per kit)

QtyDescription
1 Hooke Kit™ gpMBP69-88/CFA Emulsion PTX (EK-3111)
1 50 mL sterile polypropylene tube
10 Lewis rats, females, 10 to 14 weeks old
(Taconic Biosciences model LEWIS-F)
Sterile phosphate buffered saline (PBS)
(standard formulation, pH 7.4, calcium-free, magnesium-free)

Method overview

Use female Lewis rats at 10 to 14 weeks of age. Minimize rat stress during all procedures (see "Rat selection and handling" above).

  1. Acclimate rats for at least 7 days prior to immunization
  2. Day 0 – Prepare PTX solution (first vial of PTX)
  3. Day 0 – Inject antigen emulsion, s.c.
  4. Day 0 – Wait approximately 2 hours (not critical; 1 to 6 hours is acceptable)
  5. Day 0 – First injection of PTX solution, i.p.
  6. Day 2 – Prepare PTX solution (again, second vial of PTX)
  7. Day 2 – Second injection of PTX solution, i.p.

The following sections provide detailed procedures.

Preparation of pertussis toxin solution (EK-3111 only)

Note: This protocol version is for use only with Hooke PTX lot numbers 1003, 1004, or 1005. (Future lots may require a different pertussis toxin dose; see hookelabs.com/protocols for an updated protocol.)

PTX should be prepared fresh on Day 0 (using the first vial of PTX), and again fresh on Day 2 (using the second vial of PTX). Prepare PTX under sterile conditions using a biosafety cabinet.

The lyophilized PTX must be dissolved in PBS. A higher PTX dose induces more severe EAE; the amount of PBS may be adjusted to achieve more or less severe EAE.

Important: To ensure the same PTX concentration is administered to all rats, the pooled PTX solution for all rats in the experiment should be prepared on each dosing day in a single 50 mL tube as follows

  1. Put PBS into 50 mL tube: Use 3 mL PBS for each vial of PTX. Place the total PBS volume for all PTX vials to be used that day into a single 50 mL sterile polypropylene tube. (For example, if 3 vials of PTX will be used, put 9 mL PBS into the tube).

    Note: Adjust PBS volume as needed for local lab conditions and stress of dosing. (See "Titration for optimal dosage of pertussis toxin" below. The recommended dilution is for Hooke PTX lots 1003, 1004, or 1005 only; future lots may require different PBS dilution.)

  2. Dissolve PTX in each vial: Using the 3 mL syringe and 18G needle (provided), put 1 to 2 mL of PBS from the 50 mL tube into each PTX vial. Mix each vial well but gently until PTX is fully dissolved (do not vortex). Let sit for 2 minutes, mix well again.

  3. Pool solution in tube: Using the same 3 mL syringe, move solution from each vial back into the 50 mL tube.

  4. Rinse vials again: Put 1 to 2 mL of PTX solution (from 50 mL tube) into each PTX vial, then move the solution back into the 50 mL tube (this is done to ensure that high concentration of PTX is not left in the vial).

Administration of antigen emulsion (EK-3111 only)

Administer antigen according to EK-3110 procedure given above.

Wait approximately 2 hours before proceeding with first PTX administration.

(Exact timing is not critical; 1 to 6 hours delay is acceptable, but the delay should be the same for all groups.)

Administration of PTX

PTX will be administered intraperitoneally at 0.2 mL/dose. This will be repeated 48 hours later.

  1. Prepare PTX solution (see "Preparation of pertussis toxin solution" above).

  2. Draw 1 mL PTX solution into 1 mL syringe (provided).

  3. Mount a fresh 27G needle (provided).

  4. Inject each rat i.p. with 0.2 mL/rat (first PTX administration).

Repeat (2-4) for all rats.

  1. 44-48 hours later, repeat the PTX preparation and injection (steps 1-4 above) for all rats.

  2. Check rats for signs of EAE daily starting on day 7 after the immunization. (See Appendix A - EAE scoring guide.)

  3. As soon as the first signs of paralysis occur, provide rats with food pellets and wet food on the floor of the cage, and easily accessible water. HydroGel (ClearH2O, Portland ME) may be used as a source of water during the most severe paralysis.

All rats will develop obvious bumps of emulsion at the injection sites 2 to 4 days after injection.

The emulsion will remain at the site of injection for the duration of the experiment (typically 20 to 30 days).

Titration for optimal dosage of pertussis toxin

We strongly recommend that new customers establish the optimal PTX dose by inducing EAE in 7-10 rats in their own lab.

The optimal PTX dose needed to obtain EAE relapse is typically 400 to 500 ng/rat for each of the two PTX administrations. Larger doses of PTX will induce EAE which may be too severe. Lower doses of PTX may not induce relapses.

Finally, PTX potency varies from lot to lot; this affects the required dose. Hooke tests each lot of PTX in rats; the dilutions recommended in this protocol are the result of our testing.

To determine the best PTX dose for the lab conditions, adjust PTX concentration to 2 µg/mL; this gives 400 ng PTX in each 0.2 mL dose (6000/3*0.2).

Immunize 10 rats. Score rats daily starting 8 days after immunization, continuing until 28 days after immunization. (See Appendix A - EAE scoring guide.)

If EAE is too severe, reduce PTX concentration to ~1.4-1.7 µg/mL (280 to 340 ng PTX/dose).

If no relapses develop, a higher dose of PTX is required; additional PTX vials are available as Hooke cat. no. LT-0105.

References

[1] McFarlin DE, Blank SE, Kibler RF, J. Immunol. 1 13:712 (1974)
[2] Kardys E and Hashim GA, J Immunol 127:862 (1981)
[3] Mannie MD, Paterson PY, U'Prichard DC, Flouret G., Proc Natl Acad Sci USA 82:5515 (1985)
[4] Hashim GA, Day ED, Fredane L, Intintola P, Carvalho E, J Neurosci Res. 16(3):467 (1986)



Appendix A - Rat EAE Scoring Guide

Typically, EAE is scored on scale 0 to 5. Most researchers also give rats "in-between" scores (i.e. 0.5, 1.5, 2.5, 3.5) when the clinical picture lies between two defined scores.

In most cases rats are scored daily, starting 7 days after immunization (Day 7) and continuing until Day 20 (EK-3110).

We recommend the following scoring guidelines for rats:

Rat EAE scoring

Score Clinical observations
0.0

No obvious changes in motor function compared to non-immunized rats.

When picked up, the tail has tension and some movement when finger runs along the tail.

0.5

Tip of tail is limp.

When picked up, the tail has tension except for the tip. When walking, the tip of tail is dragged on the cage bottom.

1.0

Limp tail.

When picked up, the whole tail is limp. The walk is not affected.

1.5

Limp tail and hind leg inhibition.

When picked up, the whole tail is limp. Walking is very slightly wobbly.

2.0

Limp tail and weakness of hind legs.

When picked up, legs are held close together. When the rat is observed walking, it has a clearly apparent wobbly walk. One foot may have toes dragging, but the other leg has no apparent inhibitions of movement.

2.5

Limp tail and dragging of hind legs.

Both hind legs have some movement, but both are dragging at the feet (rat trips on hind feet).

  - OR -

No movement in one leg/completely dragging one leg, but movement in the other leg.

3.0

Limp tail and complete paralysis of hind legs (most common).

  - OR -

Limp tail and almost complete paralysis of hind legs. One or both hind legs are able to paddle, but neither hind leg is able to move forward of the hind hip.

  - OR -

Limp tail with paralysis of one front and one hind leg.

3.5

Limp tail and complete paralysis of hind legs.

Rat is moving around the cage, but when placed on its side, is unable to right itself. Hind legs are together on one side of body.

4.0

Limp tail, complete hind leg and partial front leg paralysis.

Rat is minimally moving around the cage but appears alert and feeding.

Often euthanasia is recommended after the rat scores 4.0 for 2 days. However, with daily s.c. fluids some rats can recover. When the rat is euthanized because of severe paralysis, a score of 5.0 is entered for that rat for the rest of the experiment.

4.5

Complete hind and partial front leg paralysis, no movement around the cage. Rat is not alert.

Rat has minimal movement in the front legs. The rat barely responds to contact.

Euthanasia is recommended. When the rat is euthanized because of severe paralysis, a score of 5.0 is entered for that rat for the rest of the experiment.

5.0

Rat is found dead due to paralysis.

  - OR -

Rat is euthanized due to severe paralysis.

Version: 2017-01