TB Retrograde Labeling

To identify the neurons whose axons regenerated into the PNG, a separate cohort of animals received C2Hx lesion, virus injection (n = 4 for AAV5-GFP and n = 4 for AAV5-caRheb), and PN grafting into the C2Hx site, as described above. Four weeks after placing the proximal end of a PN graft into the C2Hx lesion site, the distal end of PN graft was exposed, trimmed by at least 1 mm, and exposed to GELFOAM saturated with 2% TB (Sigma-Aldrich). Animals were euthanized 1 week later by an overdose of Euthasol, then perfused transcardially with ice-cold 0.9% saline followed by ice-cold 4% paraformaldehyde (PFA). Spinal cord and brain tissues were dissected out, postfixed in 4% PFA overnight at 4°C, and cryoprotected in 30% sucrose.

The entire brainstem and spinal cord rostral to the C2Hx lesion was sectioned in a coronal plane at a thickness of 50 μm. Every other section was mounted serially onto glass slides, dried, and coverslipped with Fluoromount (Biomedical Specialties). To characterize the transduction efficiency and which neurons regenerated axons into the PNG, each mounted section was examined by fluorescence microscopy to detect the number and location of supraspinal and propriospinal neurons that were labeled with GFP and TB, respectively. The number of GFP⁺ and TB⁺ neurons in a specific region in each section was manually counted while blinded to treatment and subsequently summed for each animal and compared for significance using Student’s t test (p < 0.05 was the criterion for significance).