Case Studies

Mouse model of amyotrophic lateral sclerosis (ALS)
Mouse cuprizone model of demyelination
Fungal infection in mice
Mouse models of rheumatoid arthritis
Here are some real life examples of the application of refinements to successfully avoid severe suffering:
Mouse model of amyotrophic lateral sclerosis (ALS)

Avoiding mortalityThe SOD1G93A mouse is a commonly used model of amyotrophic lateral sclerosis (ALS), a form of motor neuron disease which is rapidly progressive and fatal in humans. Current treatments only have a limited impact, so the SOD1G93A mouse is used to test potential therapies. Testing treatments that are intended to slow disease progression can require animals to experience clinical signs that can cause severe suffering. The life experiences of SOD1G93A mice were reviewed and refined, and a new Standard Operating Procedure set out for caring for the animals, including:

  • early screening of motor function and muscle characteristics, to select which drugs to take forward without mice suffering extreme motor deficits
  • righting reflex checked twice daily from 100 days (or earlier of motor problems are apparent)
  • non-particulate litter and nesting material, as animals may have difficulty grooming
  • long sipper tubes and mash provided in a dish on the cage floor, to cater for disabled animals
  • any dehydration treated with an intraperitoneal injection of sterile saline, followed by close monitoring
  • husbandry refinements, including carrying nesting material (not litter) over from soiled to clean cage to reduce aggression, changing gloves and cleaning work surfaces between handling male and female mice. Fun tunnels are provided but removed at 100 days to prevent disabled mice from becoming trapped

All investigators and animal technologists have a copy of the SOP, which has helped to avoid and reduce severe suffering.

Mouse cuprizone model of demyelination

Animals are used to study demyelinating diseases of the nervous system, in which the myelin sheath that insulates neurons (nerve cells) is damaged. These include multiple sclerosis, Charcot-Marie-Tooth disease and central nervous system neuropathies. In a commonly used model, mice are fed cuprizone, which causes neurons to degenerate throughout the nervous system including the brain. Potential drugs and therapies can then be evaluated for the disease under study.

Six weeks of 0.2% cuprizone in the diet causes a significant level of reversible demyelination in mice, which becomes irreversible if exposure to cuprizone is continuous for up to 12 weeks. Mice treated with cuprizone invariably exhibit
spontaneous seizures (fits) and mortality can be up to 50% in males. Treatment with cuprizone can therefore be severe.
A pharmaceutical company wanted to refine the use of cuprizone and, after evaluating the literature, they set up this protocol:

  • Female mice weighing at least 25g were used – according to the literature, female mice are less susceptible to cuprizone
  • 0.2% cuprizone was incorporated into chow pellets, which mice prefer to powdered diet and find easier to handle
  • Animals were housed in a ‘quiet’ area of the animal facilities with low traffic
  • The number of interventions was reduced by combining various activities within a single handling session, e.g. by weighing and checking the animals during cage clean
  • MRI techniques were used to assess demyelination more accurately and refine humane endpoints. Scanning sessions lasted 15 minutes, under isoflurane anaesthesia

As a result, the actual severity was ‘mild’ for all animals and the animal model was robust. MRI was also used to implement reduction, as longitudinal data was obtained from a single animal.

Fungal infection in mice

Fungal LD90 tests are conducted to characterise the infectivity of a fungus (e.g. Aspergillus) or to evaluate the effectiveness of antifungal treatments. For example, the antifungal treatment may be started 24 hours after infection and continued for seven days, with mortality then recorded over the following week.

In a study to evaluate the potential to use body temperature as a predictor of death in fungal (Candida) LD90 tests, male CD1 mice (4 to 5 weeks old and between 18 and 20 g) were immunosuppressed with cyclophosphamide and infected with either C. albicans, C. guillermondii or C. glabrata. Body temperature was measured using an implantable transponder and a high performance non-contact thermometer.

The non-contact thermometer gave reliable results and enabled an endpoint of 33.3 oC to be defined, which would have been able to predict mortality in 67 % of the mice.

Source: Warn et al. (2003) Laboratory Animals 37: 126-131

Mouse models of rheumatoid arthritis

A pharmaceutical company introduced the G6PI, CIA and CAIA mouse models of rheumatoid arthritis, which have the potential to cause severe suffering. This prompted a re-evaluation of the company’s welfare scoring sheets and husbandry refinement protocols, with the aim of reducing suffering. The scientists and animal technologists worked together to tailor and refine monitoring systems, husbandry and procedures.

Mice used in G6PI and CAIA studies were very carefully monitored by scientists and animal technologists, to identify indicators of adverse effects and collate data on weight loss and disease scores. The observations were specific to each model, although standardised terminology was created to describe indicators. As a result, the following refinements
were adopted:

  • the humane endpoint for weight loss was reduced from 25% to 20%, and another endpoint added of a 15% weight loss that persisted for 5 days
  • the tailored indicators (such as soft stools for CAIA) enabled study length to be reduced; e.g. the CIA studies were reduced from 30 days to 20
  • disease scores were revised to include a range of indicators, as opposed to paw volume only, capturing severity more effectively and enabling endpoints to be further refined
  • additional refuges are provided for DBA/1 male mice, eliminating aggression
  • non-tangling nesting material is provided
  • when mobility is restricted, longer sipper nozzles are fitted and food given in dishes on the cage floor
  • the Mouse Grimace Scale is used to help assess acute pain.
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Practical ways to reduce or avoid severe suffering include: