Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Our protocol using gait analysis is very useful in determining whether mouse motors of movement disorders mimic the symptom of patient with gait disturbance. So, gait analysis in the stress loading test can be performed easily, and without expensive equipment, to detect certain motor phenotypes in mice, with no spontaneous effects. Before initiating the behavioral test, record the weight of each mouse, and use a marking pen to label each tail.

When all of the animals have been weighed and marked, place the mice in the experimental room for at least 30 minutes, and set a clear, 25 by 25 by 40 centimeter hanging box, with a rotatable mesh lid, onto the lab bench. When the mice have acclimated, place one mouse into the center of the mesh lid, and carefully turn the lid upside-down. Then, measure the fall latency of the mouse from the mesh lid, before returning the mouse to it’s home cage.

When all of the mice have been tested, place a 9.9 by 42 centimeter piece of white paper onto the lab bench, and place a dark goal box at the distal end of the paper. Place other boxes, approximately the same length as the paper, on both sides of the runway to prevent escape. And add black and red ink to separate 35 milliliter Petri dishes.

To train the mice for the analysis, place a mouse at the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end of the paper to the goal box. When the mouse reaches the box, gently scruff the animal, tucking the back and tail between the ball of the thumb and the other fingers, to limit the movement of the hind limbs. And immerse the bottoms of the forelimbs in red ink, and the bottom of the hind limbs in black ink.

Then, immediately place the mouse back onto the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end to the goal box. After air-drying the foot printed paper, use a ruler to obtain three measurements of the stride length, by measuring the distances between the same parts of the paw prints. Of the front base-width, by drawing a line between consecutive, same-side, front footprints, and measuring the length of the vertical line from the pad of the front footprint, to the line drawn between the footprints.

And of the hind base-width, by drawing a line between consecutive right hind footprints, and measuring the length of the vertical line from the pad of the left hind footprint, to the line drawn between the other right footprints. For overlap, measure the distance between the pads of the front and hind footprints. To prepare a restraint tube, use a square drill to make 16 two millimeter holes in a 50 milliliter tube along the scale marks, and the backside of each scale mark.

Cut off the tip of the tube to make a five millimeter hole, for breathing, and make a four millimeter hole in the tube cap, for the tail. For the stress-loading test, scruff a mouse, and place the animal into the restraining tube head first, with the forelimbs gently confined. Pass the tail through the hole in the cap before capping the tube.

And place the mouse on the bench top for two hours at room temperature. At the end of the stress-loading period, remove the mouse from the tube, and return the animal to its home cage. As observed in these representative figures, heterozygous rapid-onset dystonia Parkinsonism model mice, demonstrate significantly shorter fore and hindlimb stride lengths, than do their wild-type litter mates at four weeks of age.

Stress heterozygous rapid-onset dystonia Parkinsonism model mice, also exhibit significantly shorter stride lengths of both limbs than those of stressed wild-type litter mates at eight weeks of age. Asymmetries of the stride lengths of both limbs are not significantly different, in any group of mice, at any age. The front base and overlaps of both limbs are also similar, in all groups, at all ages.

The hind base is significantly wider in these stressed heterozygous rapid-onset dystonia Parkinsonism model mice, than that observed in the stress wild-type animals at 10 weeks of age, indicating that stress causes motor deficits in heterozygous rapid-onset dystonia Parkinsonism model mice. No significant differences in hanging time, between wild-type and mutant, are observed in four to 10 week old animals. At 12 weeks of age, however, the hanging time of stress wild-type mice is significantly longer than that of the other animals, suggesting that the motor deficit in heterozygous rapid-onset dystonia Parkinsonism model mice, is distinguishable from wild-type mice by restraint test in older animals.

Place the mouse onto the paper immediately after the mouse hangs the bottoms of limbs in the ink to avoid the ink drying before the walking test. Additional tests for emotional characteristics, such as open field, elevated plus-maze, and whole swim test, can be performed to answer whether the gait abnormality result from only motor deficit. Our protocol can be used to explore the susceptibility to stress, by changing the number and the duration of the stress loading, and by comparing motor phenotypes between various loading conditions.

Our protocol can be used to explore the susceptibility to stress by changing the number and duration of the stress loadings, and by comparing motor phenotypes between various loading conditions.