Experimental Autoimmune Encephalomyelitis (EAE) is used to study Multiple Sclerosis (MS) a chronic neuroinflammatory demyelinating disorder of the CNS. EAE induces a T cell and mononuclear cell inflammation, which leads to a progressive hind-limb paralysis (Robinson et al., 2014). Due to the severity of EAE, the welfare of animals should be taken into account through the planning of humane endpoints (HEP), providing appropriate perioperative care and minimizing stress factors. Several refinement (3R’s) (W.M.S. Russell and R.L. Burch 1959) methods were tested in order to minimize the number of animals euthanized due to HEP.
Materials and Methods:
Ten weeks old female C57BL/6J mice were housed in individually ventilated cages (IVC, Allentown) in groups of 3-6 individuals. Muscle weakness and symptoms of impaired coordination became evident approximately one week after active induction of EAE (Hooke Laboratories). Clinical scoring (scale 0-5) was done daily to follow the progress of the disease (Hooke Laboratories).
Refinements tested in this model:
Daily body weight and body temperature measurements
Anti-skid mattress on the bottom of the cages
Supportive feed (commercial and wet feed) and water bowls on the bottom of cages throughout the study (Figure 1.)
1-3 hrs on heating pad with supportive feed
Nest condition scoring, refined nesting material
Refined handling techniques (cup method) (Figure 2.)
Comparison of mice from two different origins
Body weight loss of > 20 %,
Clinical score ≥ 4.0
Absence of righting reflex at score 3.0
Animal not eating or drinking for 2 days.
Figure 1. Mice with anti-skid mattress and paper shavings (Scanbur/Datesand) as nesting material on the bottom of the cage to make it easier to move after hind limb paralysis. Nest building behavior was also tested as an indicator of animal welfare.
Figure 2. Animals were handled using cup method, which reduces stress experienced by mice (Gouveia and Hurst 2017).
Placing the mice on the heating pad with wet feed (Figure 3.) for 1-3 hours when body weight reduction was observed, reduced the number of animals being euthanized due to HEP (Table 1).
Figure 3. Partially paralyzed mice, which started eating when placed on warming pad. This “wet feed” was made by mixing commercial rodent pellets (TEKLAD) with water.
Table 1. Body weights were measured before and after heating pad treatment. This table shows minimum, maximum and average value of the weight changes. The average weight was 15.93 ± 0.90.
Weight change (g)
The frequency for ‘increased’ body weight was significantly higher than ‘not increased’ (table 2).
Table 2. Frequencies and percentages of weight changes from animals kept on heating pad due to body weight loss. 1 = weight increased, 2 = weight didn’t increase. The results showed significant difference (p<0.05, Chi-squared test, Microsoft Excel).
In this study 21/84 EAE-induced mice were euthanized due to HEP, compared to the same study model without refinement, where 54/72 reached HEP. Mice from different origins showed no statistical difference in the progress of EAE symptoms.
The combination of heating pad and wet feed was observed to be a successful method of improving animal welfare, when the physical condition (e.g. body temperature, weight, clinical score) deteriorated. The warmer environment apparently increases animals’ welfare to the point that will begin feeding. This led to a considerable number of animals that did not reach HEP and were not euthanized.
Daily rectal temperature measurements were also considered to be a good way to track the welfare in addition to the heating pad treatment. However, this would have also increased stress levels, so the idea was discarded.
Employment of the anti-skid mattress seemed to make moving easier by providing a good grip. On the other hand, some mice chewed the rubber mattress, so alternative material should be tested.
Nest condition is, in general, considered good indicator of mouse welfare (Gaskill et al., 2013), but in this study, animals were group housed and animals within the same cage had different clinical scores and therefore the nest scores were not completely reliable.
Paper shavings turned out to be a better nesting material than the alternative aspen nest material due to the fact that the nails of paralyzed limbs did not get caught in the paper shavings in the way observed in the aspen material.
Robinson A, Harp C, Noronha A and Miller S 2014: The Experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment. Handbook of Clinical Neurology 122: 173-189.
Russell WMS and Burch RL 1959: The Principles of Humane Experimental Technique: The Removal of Inhumanity: The Three R's. In The Principles of Humane Experimental Technique, Methuen, London. ISBN 0900767782.
EAE Induction by Active Immunization in C57BL/6 Mice. Version 2018-03-2: https://hookelabs.com/protocols/eaeAI_C57BL6.html
Gouveia K and Hurst J 2017: Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling. Sci Rep 2019: 7:44999. doi: 10.1038/srep44999.
Gaskill BN, Karas AZ, Garner JP and Pritchett-Corning KR 2013: Nest building as an indicator of health and welfare in laboratory mice. J of Vis Exp Dec 24; (82): 51012. doi: 10.3791/51012.