Limb Trajectory Marker Analysis
Objective: To analyze limb trajectories during treadmill walking at low, intermediate, and high speeds using reflective markers placed on anatomical landmarks
Protocol Steps
Animal acclimation to treadmill
Mice were allowed to acclimate quietly on the treadmill lane
Note: Initial habituation phase before speed training
View evidence from paper
“First, mice were allowed to acclimate quietly on the lane for 20–30 min”
Initial speed training
Mice were introduced to walk at low speed to learn to avoid electrified grid at rear of lane
Note: Electrified grid set at minimal intensity (0.1 mA) to motivate locomotion
View evidence from paper
“They were then introduced to walk at 10–15 cm/s for 5 min. At that stage, the mice kept walking on the treadmill belt to avoid the electrified grid”
Progressive speed training
Mice were walked at increasing speeds until reaching 100 cm/s
Note: Three of nine mice failed to learn and were excluded from study
View evidence from paper
“Mice were walked at increasing speed. Once they reached 100 cm/s, they were tested 3 times at each speed”
Inter-limb coordination testing across speed range
Mice tested at 12 different treadmill speeds to study inter-limb coordination
Note: Tested 3 times at each speed to obtain at least 10 contiguous strides
View evidence from paper
“mice were tested at treadmill belt speeds of 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 cm/s”
Marker placement under anesthesia
Limbs shaved and reflective markers glued to anatomical landmarks on hindlimbs and forelimbs
Note: Markers placed on: hindlimb (iliac crest, hip, knee, ankle, MTP joints, toe); forelimb (scapula, shoulder, elbow, metacarpophalangeal joints, toe)
View evidence from paper
“Under isoflurane anesthesia (2–3%), limbs were shaved and reflective markers (2 mm diameter) were glued on the iliac crest, hip, knee, ankle, and metatarsophalangeal (MTP) joints and toe for the hindlimb, and on the scapula, shoulder, elbow, and metacarpophalangeal joints and toe for the forelimb”
Limb trajectory testing at three speeds
Mice walked at low, intermediate, and high speeds with reflective markers while being filmed
Note: Three speeds tested: 15 cm/s (low), 45 cm/s (intermediate), 90 cm/s (high)
View evidence from paper
“mice walked at low (15 cm/s), intermediate (45 cm/s), and high speeds (90 cm/s) with reflective markers”
High-frequency video recording
Mice filmed from left and right sides during treadmill locomotion at all speeds
Note: Recording at 200 frames per second
View evidence from paper
“All mice were filmed on the left and right sides by high-frequency (200 frames/s) cameras (Genie HM640, Dalsa Teledyne) during treadmill locomotion”
Video digitization and offline analysis
Films digitized using StreamPix software and analyzed offline
Note: Analysis performed after recording session
View evidence from paper
“Films were digitized with StreamPix 6.0 (Norpix) and analyzed offline”