Handling Techniques to Reduce Stress in Mice

My name is Dr.Thomas Prevot. I am a scientist in the Center for Addiction and Mental Health, or CAMH, and an assistant professor at the University of Toronto in the Department of Psychiatry. My group has a particular interest at reducing baseline anxiety and reactivity in mice in order to reduce data variability and improve animal welfare.

Mice have proven to be invaluable assets in basic science and preclinical research, however, very few labs around the world control for baseline anxiety and the reactivity of their laboratory animals, therefore, complicating replicability of data between labs. A major contributing factor to baseline anxiety is uncontrolled environmental variables, such as housing conditions, environmental enrichment, and exposure to stress. While standardization of housing conditions and enrichment has helped improve this issue, exposure to stress during interaction with experimenters is still largely unaddressed in mice.

Tail handling, the process of lifting the mouse by the tail, is still the mouse widely used mouse handling technique, despite it being particularly stressful. Though alternatives to tail handling have emerged including tunnel handling and cup handling. Adoption of these techniques has been slow.

Here we describe a novel technique, similar to cup handling, which can be implemented with as little as three days of habituation, or 3D handling. By gradually increasing the degree and complexity of interaction and progressing in response to the reactivity of the mouse to certain handling milestones, we can reduce stress and facilitate routine handling. We assess the efficacy of 3D handling in mice in behavioral tests, assessing anxiety-like behaviors, interaction with experimenters and peripheral stress hormone, corticosterone, levels, and provide a comparison with tunnel handling and tail handling techniques.

Gently open the cage and place the lid on the side. Introduce your gloved hand to the home cage before attempting to pick up the mouse. Allow the mouse habituate to the presence of the hand for about 30 seconds.

Then attempt to pick up the mouse in cupped hands. If the mouse can't easily be picked up, cup both hands together and use them to guide the mouse to the corner of the cage. Then use both hands to pick up the mouse.

Once the mouse is removed from the cage, keep your hands as flat and open as possible. Allow the mouse to freely explore. This provides a flat platform for the mouse to step onto and limits the risk of biting.

Hold the hand open and flat, with the palm up. Place your other hand adjacent to the mouse and allow the mouse to move freely from hand to hand, without any restraint. After one minute of handling with flat hands, relax the palm of your hand, and slightly cup the mouse in your head, prior to gently rolling the mouse between hands.

Continue for four minutes, alternating between gentle rolling between hands and free exploration of your open hands. During the last two minutes, perform a shelter test. Let the mouse move to edge of your hand and bring the two hands together.

Very slowly, cup your hands so the mouse fits inside a shelter formed by the hands. Make sure to leave an opening, so the mouse can escape if needed. Try to keep the mouse in the shelter for five to 10 seconds without any restraint.

Repeat the shelter test three to four times over the last two minutes. Do not rush the process. If the mouse appears stressed by being confined inside the hand, continue with rolling between hands and free exploration for 30 seconds, and then retry the shelter test.

Allow free exploration in hands for 30 seconds, then gently replace the mouse in its cage. Clean the bench top of any feces and urine with 70%ethanol or an appropriate cleaning solution. Clean the gloves of feces and urine as necessary, and when changing to a new cage, rinse gloves with 70%ethanol or change gloves.

By day two, it should already be feasible to remove the mouse from the cage in cupped hands. If the mouse seems reactive, perform the flat hand and free exploration of open heads for 30 seconds to a minute. Otherwise, proceed to the roll between hands and shelter test.

Perform the role between hands and shelter steps from day one for two to three minutes. Perform each step several times. If the mouse seems at ease in the shelter test, attempt to pet the mouse on the back and head.

If the mouse does not attempt to flee contact, proceed to the nose poke. Gently touch the mouse on the tip of the snout. If it does not bite or attempt to turn its head, this is a good sign of habituation.

For the petting and nose poke test, do not rush the process. If the mouse appears stressed by being confined inside the hands, or does not want to be touched, continue with the rolling between hands for a few minutes and then retry. Stop this session after about three minutes of handling, if the animal reacts well to the shelter, head petting and nose poke.

And if the animal appears to be willing to explore your hands without any signs of stress, such as attempts to escape, jumping from the hands or avoiding contact with the hands. If the mouse continues to exhibit signs of stress or is not reacting well to the shelter test or nose poke test, continue the session until reaching five minutes, as in day one. Replace the mouse in its cage and clean the bench top and gloves.

On the third day, proceed through the same steps that day two. Pick up the mouse in the palm of the hand. Transfer the mouse between hands.

Gently confine the mouse in cupped hands. If the mouse responds well to these steps, try petting the mouse and attempting the nose poke test. On the third day, the mouse should be relaxed enough to sit in the palm of their hand without moving off, and you should be able to proceed through these steps over approximately two to three minutes.

Replace the mouse in its cage, and clean the bench top and gloves. On the third day. if the animal will be restrained for experimental purposes, such as oral gavage or intra peritoneal injection, the mice can be subjected to the neck pinch test.

Grasp the mouse by the nape of the neck, between your thumb and forefinger and lift the mouse five centimeters above your hand for two to three seconds. This is normally an uncomfortable position for adult mice, and if the mice remain near immobile, they are well habituated to handling and will be easy to restrain for injections. Place the mouse back on your flat hand and allow it to freely explore for one minute.

Animals were tested for their willingness to voluntarily interact with the experimenter and the ease of handling in an experimental context. In study one, mice handled by either tube handling or 3D handling were less likely to flee when an experimenter attempts to pick up a mouse. Additionally, study one mice handled using the 3D technique spent more time in the same quadrant as the experimenter's hand, indicating an increased willingness to interact with the experimenter.

In study two, mice handled by either tube handling or 3D handling were less likely to flee when an experimenter attempts to pick up the mouse compared to tail handled mice. However, in study two, handling technique did not significantly influence the time spent in the same quadrant as the experimenter's hand. The effect of 3D and tunnel handling were compared to tail handling in two tests of anxiety-like behaviors, the novelty suppressed feeding test and the elevated plus maze.

In study one, in the novelty suppressed feeding test, there was a trend to reduced anxiety in tunnel handled and 3D handled mice. In the elevated plus maze, there was no overall effect of handling. When summarized as a Z-score, there was a significant reduction in overall anxiety in tunnel handled and 3D handled mice compared to tail handled mice.

In study two, there was no overall effect of handling on anxiety measures in the novelty suppressed feeding test or the elevated plus maze. When summarized as a Z-score, handling technique had no overall effect on anxiety. As a physiological measure of stress, we collected serum from tail handled, 3D handled and tunnel handled mice 15 minutes after a brief handling session and tested corticosterone levels.

There was no overall effect of handling in study one. In study two, 3D handling significantly reduced corticosterone levels in comparison to tail handling. Based on observations that handling techniques in mice are not widely recognized by the scientific community, we described the use of a novel handling technique.

In both studies presented, tunnel and 3D handling reduced attempts of mice to flee when being picked up, potentially facilitating routine handling. Additionally, in older male mice, this technique reduced anxiety-like behaviors and increased interaction with the experimenter. In young females.

this technique decreased corticosterone levels observed in response to handling. Implementing this technique has the potential to reduce data variability, improve animal welfare and facilitate routine handling.