CatWalk Gait Test

The CatWalk XT quantitative gait analysis system (Noldus, Wageningen, The Netherlands) is a nonintrusive, accurate tool to determine gait ability in rodents. This method is described elsewhere ²¹, ²². In brief, it is based on rodent voluntary movement through an enclosed 1.3 m long glass walkway that is illuminated with green fluorescent lighting the walkway from the side and reflecting internally after the rodent comes in contact with the glass floor. High‐speed video camera was located under the walkway and was used to obtain footprint images. The rats were trained twice: 7 days prior to 6‐OHDA injection and on experimental day 7. Before each training session, the animals were placed on the walkway and allowed to habituate for 2 minutes. Testing was performed on experimental day 20 between 11:00 and 14:00, at least 1 hour after intranasal EVs administration. Testing was successful if the animal crossed the walkway without stopping and at least 3–4 each paw placements were recorded. Three complete runs across the walkway were recorded for each animal. CatWalk XT software was used to automatically analyze the video images of the runs. Data analysis was performed with a threshold value set at 40 arbitrary units (range 0–225) per pixel. The following parameters were analyzed according to ²¹, ²³: temporal (stand duration), spatial parameters attributed to individual paws (duty cycle, %), relative spatial relationships between paws (stride length) and interlimb coordination (step cycle). Duty cycle is calculated according to the formula: stand/(stand + swing) × 100% and it represents the stand as a percentage of step cycle.