Axonal Regeneration in the Sciatic Nerve of a Mouse Following a Crush Injury

Begin with the sciatic nerve isolated from a mouse.

The neurons within the nerve were transfected to express fluorescent cytoplasmic proteins.

The nerve was crushed to induce an injury, which was marked with a suture.

The injured axon segments at the site degenerate, and recruited macrophages clear the debris.

Schwann cells proliferate to form guiding tracks and secrete neurotrophic factors that promote axon regrowth. The axons extend along these tracks, regenerating the nerve.

Fix the nerve to preserve cellular structures, then remove any attached non-neuronal tissues.

Wash with a buffer to eliminate residual fixatives.

Position the nerve on a microscope slide, apply a mounting medium, and place a coverslip.

Flatten the nerve to position the axons within the same focal plane for imaging.

Using an epifluorescence microscope, trace the axons from the crush site to their distal ends to

measure the regeneration length.

Separate the DRGs together with the nerve roots and sciatic nerve, carefully with micro scissors and micro forceps under the dissection microscope.

Then, transfer the sciatic nerve directly in 4% PFA overnight at four degrees celsius. To image and measure the fluorescence labeled sensory axons under the dissection microscope, strip off the attached tissue and membrane on the fixed sciatic nerve with micro scissors and micro forceps, carefully. Then, wash the nerve with PBS three times.

Next, place the sciatic nerve on a slide and keep it straight. Add 80 microliters of anti-fade solution around the nerve, then lay a cover slip on it. Flatten the whole mounted tissue with pressure.

Afterward, place the flattened tissue under the inverted epifluorescent microscope, equipped with an accessory for mosaic acquisition and image processing. When measuring the length of regenerated axons, trace all identifiable fluorescently labeled axons in the sciatic nerve from the crush site to the distal axon ends.