Cerebral Peduncle

Growth and sprouting of BDA-positive CST fibers to stroke-denervated hemicord in response to stroke and intranasal anti-Nogo-A treatment were evaluated in three adjacent cross-sections at the caudal cervical enlargement C3 to C6. Midline-crossing fibers were manually counted in the dorsal and ventral commissure at the central canal (see Fig. 5B level M) and the branching of these fibers was evaluated at four defined regions within the gray matter (28 ), (see Fig. 5B levels D1 to D4). Six vertical lines (M, D1 to D4, and L) were superimposed on each spinal cord section using ROI manager in FIJI (ImageJ) as reference points for crossing axons. The first vertical line M was drawn through the central canal; L was drawn parallel to M and at the lateral rim of the gray matter. D1 to D4 were drawn parallel to M, at one-fifth, two-fifths, etc. of the distance between M and L (Fig. 5B). CST fibers in the gray matter have an irregular course, passing in and out of the plane of the section. To prevent multiple counting of single collaterals, only fibers that crossed M, D1, D2 D3, and D4 were counted on each section.
For the analysis of corticopontine projections to stroke-affected basilar pontine nuclei, three consecutive sections with clearly visible basilar pontine nuclei on the ipsilateral side to the BDA injection were chosen. Using ROI Manager in FIJI (ImageJ), four defined nuclei were selected on the ipsilateral to the BDA injection side and mirrored to the contralateral side of the section to ensure the proper location of the nuclei for the manual count of fibers (Fig. 6A). The central and medial nuclei were analyzed as one region of interest, the lateral was split in 2 – a lower, denser (lateral 1), and upper lighter (lateral 2) region (Fig. 6A). To correct for variations in BDA labeling, we normalized the data (pons and cervical spinal cord) to the number of BDA-labeled fibers counted in 10% of the area of the whole cerebral peduncle. The cerebral peduncle on the level of the midbrain had a cross-sectional area of about 300,000 µm². On the ipsilateral to the BDA injection side of the brain, in this area of the cerebral peduncle, labeled fibers in five regions of 6,000 µm² each were counted, which amount to 10% of the cerebral peduncle BDA-labeled corticofugal fibers. This value was later used as a normalization factor in the analysis of the midline crossing fibers.
Photothrombotic stroke, intrathecal administration of the antibodies, neuroanatomical tracing, and histology of labeled corticospinal tract were performed according to published protocols. More details regarding these procedures can be found in SI Appendix.