The overall goal of this protocol is to assess the well-being of mice after they underwent procedures including general anesthesia and to provide scientifically sound data to support the severity classification according to EU directive 2010/63. This protocol can help answer key questions on animal welfare in the field of laboratory animal science such as the degree of distress caused by procedures involving general anesthesia. The main advantage of this protocol is that well-being is evaluated of each individual mouse in an animal-based scientific manner and this protocol can easily be adapted and integrated into other planned studies.
We first had the idea for this protocol when we speculated that if repeated anesthesia cause more stress in laboratory mice than a single exposure, scientifically based data would be missing. In this step, prepare a glass box with a floor area of approximately 220 millimeters by 290 millimeters and a height of 390 millimeters. Cover the floor with approximately 0.5 centimeters of bedding material.
Then scatter a handful of used bedding material from the home cage on top of the new bedding material to reduce distress caused by the new environment. Provide with food and water. Gently transfer the mouse into the observation cage and allow the mouse to habituate to the new environment for at least 30 minutes.
For the mouse grimace scale, use a high-definition camera for photography. Continuously take about 30 to 40 photographs for each time point within one to two minutes. For testing burrowing behavior according to Deacon and Jirkof, prepare burrows by placing 140 grams food pellets in a standard opaque plastic water bottle.
Next, place the plastic water bottle filled with food pellets parallel to the back wall of the observation cage. After two hours, weigh the food pellets remaining in the burrow. Carry out the test twice two days prior to the procedure as baseline.
In this step, prepare a cage with new bedding material for the 24-hour observation period in which mice are housed individually. The 24-hour observation period begins after mouse grimace scale and burrowing behavior. The duration of single housing is kept to minimum.
In the mean time, home cage activity, food intake, nest building behavior, and the levels of fecal corticosterone metabolites are measured. Next, scatter a handful of used bedding material without feces from the home cage on top of the new material in order to reduce distress. Provide a standardized square cotton nestlet of a defined weight as environmental enrichment only.
After that, mount the infrared sensor on top of the cage for measuring home cage activity. Provide standard food pellets and water ad libitum and place the mouse in the cage. On the following morning, score the nest according to the five-point scale by Deacon approximately two hours after the light turns on.
In addition, weigh any untorn nestlet piece that accounts for at least 5%of the initial nestlet weight. Next, place a gridded cage top in the cage at an angle of 45 degrees to the longer side of the cage. Start the timer and monitor or video record the mice for 10 minutes from a distance of approximately 1.5 meters.
Note all times when the mouse climbs onto the cage top with all four paws on the cage top or leaves the cage top with one or more paws on the cage floor followed by analysis. Single experience of anesthesia as well as repeated anesthesia increased the mouse grimace scale difference scores compared to the control 30 minutes after exposure. At 150 minutes after the last anesthesia, mouse grimace scale difference scores of all mice returned to control levels.
For burrowing behavior, only repeated anesthesia significantly reduced the percentage of weight of food pellets the mice removed from the burrow versus the control. For nest building behavior, there were no significant differences in the nest scores between a single experience of anesthesia, repeated anesthesia, and the control. In the free exploratory paradigm, repeated anesthesia significantly reduced the number of explorations compared to control and the total duration of exploration versus a single anesthesia one day after the last anesthesia.
Eight days after the last anesthesia, there were no longer differences between the study groups. While attempting this protocol, it's important to remember to handle mice by tunnel and/or cupping methods. Picking up mice by the tail can induce stress and anxiety which in turn affects well-being and can also influence the results of the protocol.
This protocol can be easily adapted to and integrated into planned studies to assess the impact of a procedure on the well-being of mice. Depending on the procedure and study design, the most suitable test of this protocol should be chosen. After watching this video, you will have a good understanding of how to assess well-being of mice underwent the procedure of general anesthesia.
The protocol helps to classify the severity of the procedure in an animal-based scientific manner.
