Dorsal Column Fiber Regeneration Analysis

Dorsal hemisection was performed, as described (Liz et al. 2014 (link)) using a micro ophthalmic scalpel (Feather, Safety Razor Co. Ltd) in Thy1-cre⁺Kiaa0319^+/+ and Thy1-cre⁺Kiaa0319^F/F mice (n = 8 mice/group). Animals recovered for 5 weeks and 4 days prior to euthanasia, 2 μL of 1% cholera toxin-B (List Biologicals, Campbell, CA, USA) were injected in the left sciatic nerve. Serial spinal cord sagittal sections were collected for free floating immunohistochemistry with anti-cholera toxin-B (CT-B) (1:30 000; List Biologicals). Antigen detection was performed with biotinylated horse anti-goat (1:200; Vector) and streptavidin Alexa 568 (1:1000, Invitrogen). Image acquisition was done with a Laser Scanning Confocal Microscope (Leica SP5) and image analysis was performed with Fiji. Regeneration of dorsal column fibers was quantified by counting the total number of CT-B⁺/YFP⁺ axons within the glial scar. The length of the longest CT-B⁺/YFP⁺ axon found rostrally to the injury site was measured using as the origin a vertical line placed at the rostral end of the dorsal column tract.