Sample IACUC Protocol
Evaluation of Compound Effects on EAE Development in C57BL/6 Mice
Introduction and use permission
This document is offered as a sample IACUC protocol. Your institutional IACUC committee may require changes or additions to this protocol – this document is only a starting point.
Permission is hereby granted to use, modify, and reproduce this protocol without limitation. Credit to Hooke Laboratories is requested as a courtesy, but is not required.
Suggestions for improvement or changes are welcome; please send them to us at .
Species justification
While MS models have been described in many animal species, mouse models are as effective as any other animal models in the elucidation of the mechanisms of MS and in prediction of the success of therapeutic compounds in human patients.
Mouse models have several advantages compared to models in other species:
- The small size of mice allows the use of small amounts of test compounds.
- The greatest number of genetically modified animals exist for mouse species.
- The use of mice has low costs compared to other experimental animals.
Protocol keywords
Autoimmunity, blood collection, complete Freund's adjuvant, MOG, EAE, experimental autoimmune encephalomyelitis, injections, mouse, mice, rodents, tissue collection, MS, multiple sclerosis
Technical terms and abbreviations
| CNS | Central nervous system |
| CFA | Complete Freund's adjuvant (mineral oil with killed M. tuberculosis) |
| EAE | Experimental autoimmune encephalomyelitis |
| IFA | Incomplete Freund's adjuvant |
| MS | Multiple sclerosis |
Background
Experimental autoimmune (allergic) encephalomyelitis (EAE) is considered to be the best model of multiple sclerosis (MS), a chronic, often disabling disease of the human central nervous system (CNS). Over 6000 papers have been published in scientific journals on this model.
EAE is characterized by immune responses against CNS tissue and can be induced in animals by immunizing them against proteins of CNS.
Study design
Mice will be immunized with MOG35-55 peptide or MOG1-125 emulsified in complete Freund's adjuvant (CFA) by injecting them subcutaneously at two sites on the back (0.1 mL of emulsion/site).
On the same day, and again the following day, mice will receive intraperitoneal injection of pertussis toxin in PBS, at up to 600 ng/mouse/dose (0.1 mL).
Some animals develop skin lesions at the site of injection of CFA. These lesions appear to be related to the amount of killed Mycobacterium tuberculosis in the adjuvant, rather than to the volume of adjuvant – lesions do not develop if animals are injected with incomplete Freund's adjuvant. The amount of killed Mycobacterium tuberculosis used is the lowest amount that will reliably induce EAE.
No skin lesions are observed after pertussis toxin injection.
EAE will develop in mice 7-14 days after immunization (Day 0).
Animals which develop EAE will become paralyzed. Paralysis is usually chronic, with the most severe paralysis lasting 2-4 days.
Daily observation and scoring of mice starts on Day 7 and continues until the end of the study.
Scoring is on the scale of 0 to 5, according to the table below. Mice may be given “in-between” scores (i.e. 0.5, 1.5, 2.5, 3.5) when the clinical picture lies between two defined scores.
In some cases atypical EAE symptoms develop. There is no universal consensus on how to score these cases. These can occur, for example, when inflammatory lesions develop in parts of the brain affecting balance.
When one or more mice in a cage initially receive a score of 0.5 or greater, the supportive care is initiated by placing a “HydroGel” tag on the cage card holder. This tag indicates to animal husbandry personnel that easily accessible food and water is to be provided by placement of pelleted or wet food and HydroGel (ClearH2O, Portland ME) on the floor of the cage. Often mice progress from level 0.5 to 1.5 or 2 overnight.
This supportive care is continued until the “HydroGel” tag is removed by the person responsible for scoring mice. Supportive care may be discontinued when all mice in a cage have returned to a score of 2 or less. Usually the tag will stay on the cages throughout the study.
EAE Scoring
Typically, EAE is scored on scale 0 to 5. Most researchers also give mice "in-between" scores (i.e. 0.5, 1.5, 2.5, 3.5) when the clinical picture lies between two defined scores.
The scoring method differs slightly depending on the stage of disease (onset/peak vs. recovery), for each individual mouse.
Reliable EAE scoring requires skill which comes after considerable experience. To avoid unconscious bias in scoring, we strongly recommend that mice should be scored blind, by a person unaware of which mice have received which treatment.
We recommend the following scoring guidelines for mice during onset and peak of EAE:
Mouse EAE scoring – onset and peak
| Score | Clinical observations |
|---|---|
| 0.0 | No obvious changes in motor function compared to non-immunized mice. When picked up by base of tail, the tail has tension and is erect. Hind legs are usually spread apart. When the mouse is walking, there is no gait or head tilting. |
| 0.5 | Tip of tail is limp. When picked up by base of tail, the tail has tension except for the tip. Muscle straining is felt in the tail, while the tail continues to move. |
| 1.0 | Limp tail. When picked up by base of tail, instead of being erect, the whole tail drapes over finger. Hind legs are usually spread apart. No signs of tail movement are observed. |
| 1.5 | Limp tail and hind leg inhibition. When picked up by base of tail, the whole tail drapes over finger. When the mouse is dropped on a wire rack, at least one hind leg falls through consistently. Walking is very slightly wobbly. |
| 2.0 | Limp tail and weakness of hind legs. When picked up by base of tail, the legs are not spread apart, but held closer together. When the mouse 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. - OR - Mouse appears to be at score 0.0, but there are obvious signs of head tilting when the walk is observed. The balance is poor. |
| 2.5 | Limp tail and dragging of hind legs. Both hind legs have some movement, but both are dragging at the feet (mouse trips on hind feet). - OR - No movement in one leg/completely dragging one leg, but movement in the other leg. - OR - EAE severity appears mild when picked up (as score 0.0-1.5), but there is a strong head tilt that causes the mouse to occasionally fall over. |
| 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. - OR - ALL of:
|
| 3.5 | Limp tail and complete paralysis of hind legs. In addition to: Mouse 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. - OR - Mouse is moving around the cage, but the hind quarters are flat like a pancake, giving the appearance of a hump in the front quarters of the mouse. |
| 4.0 | Limp tail, complete hind leg and partial front leg paralysis. Mouse is minimally moving around the cage but appears alert and feeding. Often euthanasia is recommended after the mouse scores 4.0 for 2 days. However, with daily s.c. fluids most C57BL/6 mice may recover to 3.5 or 3.0, while SJL mice may fully recover even if they reach score 4.0 at the peak of disease. When the mouse is euthanized because of severe paralysis, a score of 5.0 is entered for that mouse for the rest of the experiment. |
| 4.5 | Complete hind and partial front leg paralysis, no movement around the cage. Mouse is not alert. Mouse has minimal movement in the front legs. The mouse barely responds to contact. Euthanasia is recommended. When the mouse is euthanized because of severe paralysis, a score of 5.0 is entered for that mouse for the rest of the experiment. |
| 5.0 | Mouse is spontaneously rolling in the cage (euthanasia is recommended). - OR - Mouse is found dead due to paralysis. - OR - Mouse is euthanized due to severe paralysis. |
In the recovery stage of EAE, most mice will have a tail that is no longer limp but is not normal either; it feels rigid and is "hooked". The hind legs may start moving (pedaling), but the mouse cannot walk. Either change makes scoring difficult.
We recommend the following modifications to the above scoring criteria for these mice:
Mouse EAE scoring – modified
| Score | Clinical observations |
|---|---|
| 0.0 | When held by the base of tail, tail is somewhat “hooked” and rigid, but tail makes complete rotations around the body axis (“helicopter”). Mouse is healthy. No signs of wobbling. |
| 0.5 | Mouse appears normal but tail is “hooked” and rigid. Tail does not make complete rotations around the body axis (“helicopter”). Mouse is healthy. No signs of wobbling. |
| 3.0 | Mouse is found on its side (as described for score 3.5 above), but there is excessive hind leg movement. Mouse cannot walk. - OR - Mouse has a wobbly walk (as described for score 2.5 above), and is unable to take more than two steps without falling on its side. The mouse is unable to right itself. - OR - Mouse has poor movement in the hind legs (as described for score 2.5 above), and has partial front leg paralysis evidenced by head held lower than normal and mouse's inability to right itself when placed on its side. |
| All other scores | Subtract 0.5 from the score of all mice with either a rigid, “hooked” tail or pedaling of hind legs. |
Criteria used to euthanize mice
If an animal scores 5, it is immediately euthanized.
If an animal is severely paralyzed (score 4 or 4.5), it will be given s.c. fluid – 1 mL of 0.9% NaCl or Ringer's solution – and will be re-evaluated at the same time (± 1 hour) the following day. If that animal scores 4 or higher again, it will be euthanized immediately afterwards. This is done to ensure that no animal spends more than 24 hours with a score of 4 or higher.
In addition to EAE scoring, overall clinical appearance of the mice will be used as a criterion to euthanize mice. This criterion may override the EAE scoring criterion.
In cases where mice will be bled during the experiment, approximately 0.1 to 0.2 mL of blood will be collected from the retro-orbital plexus under complete anesthesia (isofluorane). The sample will be used to analyze cytokine levels or leukocyte levels in blood and/or concentration of a therapeutic compound in plasma or serum.
For major terminal surgery, deep surgical anesthesia will be achieved using i.p. administration of tribromoethanol. This will be done when CNS tissue needs to be collected for immunohistochemistry. Once mice are fully anesthetized, as tested by absence of any reflex withdrawal of the leg after firm pinching of the foot with forceps, perfusion will be performed, first with 5 to 10 mL of PBS, and then with 10 to 20 mL of 4% paraformaldehyde. The tissue will then be collected.
At the end of the study, animals will be euthanized using CO2 asphyxiation.
In some studies after the animals are euthanized various organs may be collected for in vitro analysis. In this case after CO2asphyxiation animals may be perfused with PBS to eliminate blood from blood vessels.
Treatment
Any time during the period of disease induction, development, or even before disease is induced, mice may receive experimental drugs. These drugs may be administered via:
- Oral gavage – up to twice/day, up to 10 mL/kg, using a 1 inch long 25G gavage needle
- Intraperitoneal injection – up to once/day, up to 10 mL/kg, using a 5/8 inch 27G needle
- Subcutaneous injection – up to once/day, up to 10 mL/kg, using a 5/8 inch 27G needle
- Intravenous injection - up to once/day, up to 10 mL/kg, using a 5/8 inch 27G needle
Hazardous agents:
Pertussis toxin, killed Mycobacteria tuberculosi – will be handled in biosafety cabinet using appropriate personal protective devices (lab coat, gloves, eye protection).
Version: 2014-10
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