Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Procedure for gait analysis
Procedure for grip strength measurement
* Grip strength measurements were conducted separately before or after gait analysis.
Step Procedure performed Equipment Data collected 1 Capture images of mice walking mouse treadmill system; digital video recorder - 2 Gait analysis is conducted by converting raw digital images into MPEG format that can be exported into Treadscan™ software computer stance, swing, and stride times; stride length and dynamic stance (track) width * Grip strength is measured grip strength meter peak tension
• Gait analysis system- hardware (Prototype)
- The system is configured to allow video capture from below the mice during treadmill locomotion. Hardware consists of a treadmill with a clear transparent belt and a digital video camera (Basler A301fc) mounted below. A rectangular, opaque plexiglas testing chamber (20 x 4 x 16.5 cm) is mounted over the tread and serves to keep the mouse within view of the camera. Illumination of the ventral side of the mouse is adjusted to obtain optimal contrast between the paws and body of the mouse. The digital camera operates at 100 frames per seconds (fps) with a resolution of 658 x 494 pixels and the camera output is fed directly (Firewire IEEE-1394) to a 2.4GHz, 1GB RAM computer.
- Digital video recording software is used to capture the raw video images and to convert them to MPEG format. The MPEG file is then exported to custom analysis software (Treadscan™ 1.0, Clever Sys, Inc., Reston, VA, USA)
• Gait analysis software system (CleverSys Inc., USA)
The Treadscan™ software determines when individual paws are in contact with the treadmill and then uses this determination to derive more than 20 different measures including standard time-domain gait parameters (stride, stance and swing time). Analysis is interactive and provides frame-by-frame feedback that allows the user to confirm that the mouse is walking with a consistent pattern and that the paws are tracked accurately. Measurements of improper strides (e.g. paw not being tracked, mouse moving forward or back on tread) can be flagged and excluded from the data output.
• Grip strength meter, Columbus Instruments
- 95% Ethanol
- Paper towels
Acclimation to test conditions
In general all mice are acclimated for at least 1 h in the testing room.
a. Standard video capture protocol. Camera settings, lighting and treadmill speed (20 cm/s for all studies) are preset and background images including distance calibration are captured, prior to introduction of the mouse.
b. Each mouse is placed into the testing chamber where it could be seen live on the computer screen and within 15 s the treadmill is turned on and operating at 20 cm/s.
c. The mouse walking is then recorded for a preset fixed number of frames (796 frames) after which the treadmill is stopped and the mouse removed from the testing chamber.
d. Each mouse is in the testing chamber for a maximum total of 45-60 s.
e. A step cycle or stride consists of two consecutive contacts of a given foot. At least 5 strides for each foot is included for a given data set. Only strides recorded when the mice are walking in a fixed position relative (i.e. at the preset speed) to the camera are included. Whenever possible consecutive strides are used. However, in the presence of intervening aberrant strides (e.g. jumps or partial steps), as do periods of "stop and go" walking, data from most mice included 2 or 3 sets of consecutive strides for a given trial.
f. Stored video recordings are subsequently analyzed to derive standard gait indices including stride, stance, and swing time.
g.The tread is cleaned between trials.
Investigator's note: The gait analysis device has been fully validated and the results have been published (Wooley et. al., 2005).
Procedure for measuring forelimb grip strength
a. Each mouse is gently lowered over the grip strength meter forelimb pull bar assembly (76 mm x 50 mm), such that only the front paws are able to grip the trapeze.
b. The speed and angle of pull manually applied to the mouse are standardized to reduce variability between measurements. Therefore, the body of the mouse is kept at a vertical angle, with a given standard weight applied (or taped to the tail) in a vertical direction to "break" the mouse's grip and to produce the peak force or tension.
c. Three consecutive forelimb grip strength trials are performed and recorded.
d. Any morphological defect or signs of inability to grip and successfully perform the test is noted.
e. The Trapeze is cleaned after each mouse tested.
Data collected by investigator
Grip strength (peak tension) in 6, 12, 18, and 24 mo old mice.
Gait analysis of the front and rear paws (speed, distance, and frequency of stride) in 6 and 18 mo old mice.
Wooley CM, Sher RB, Kale A, Frankel WN, Cox GA, Seburn KL. Gait analysis detects early changes in transgenic SOD1(G93A) mice. Muscle Nerve. 2005 Jul;32(1):43-50.