The Journal of Neuroscience, September 20, 2006, ():
Neurotrophin-3 Gradients Established by Lentiviral Gene Delivery Promote Short-Distance Axonal Bridging beyond Cellular Grafts in the Injured Spinal Cord
J. Neurosci. Taylor et al.
26: 9713
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. Maps of lentiviral vectors for NT-3 and GFP expression. Lentiviral construct for the expression of NT-3 (Lenti-NT-3, top) also expressed GFP from an internal ribosome entry site (IRES), allowing vector-transduced cells to be identified by GFP labeling. Control vectors expressed GFP alone (Lenti-GFP, bottom). RSV: Rous Sarcoma Virus promoter/enhancer, PBS: primer binding site, gag: part of the gag coding sequence, RRE: rev response element, cPPT: HIV central polypurine tract, CAG Promoter: CMV enhancer/chicken β-actin promoter, WPRE: Woodchuck posttranscriptional response element.
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Supplementary Figure 2. Axons bridge, rather than circumvent, dorsal column lesion sites. (A) A region of extensive vector transduction rostral to a lesion site (indicated by GFP labeling) promotes extension of a large number of (B) CTB-labeled axons within the (C) GFAP-defined lesion site (outlined by arrows). (D) Many axons (arrowheads) cross from the graft (delineated by dashed lines) into a region of (E) GFAP labeling, suggesting that axons have bridged the lesion site. Scale bars: A-C, 500 µm; D,E, 100 µm.
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Supplementary Figure 3. Lesion completeness. (A) Caudal and (B) rostral regions of the nucleus gracilis in lesioned animals show an absence of ascending sensory axons in the brainstem, confirming completeness of lesions, while (C) caudal and (D) rostral regions of the nucleus in intact control animals show robust labeling of untransected axons in the nucleus. Scale bar: A-D, 200 µm.
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Supplementary Figure 4. Quantification of rostral axonal growth over 50 µm intervals in animals that received Lenti-NT-3 injections. The number of axons at each distance from the lesion site decreases with increasing distance from the rostral GFAP-defined lesion border. Values are mean ± SEM.
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Supplementary Figure 5. Characterization of MSC grafts. (A) Nissl staining shows integration of grafted cells and filling of the cavity (outlined by dashed lines). (B) Marrow stromal cells pre-labeled with BrdU before grafting and subsequently detected by BrdU immunocytochemistry (arrowheads) within the lesion site, 4 weeks post-grafting. (C) The majority of Iba1-labeled microglia/macrophages in the graft (red) did not co-localize with BrdU labeling (green). Occasionally, IBA1-labeled cells contained some BrdU-labeling, representing either MSC differentiation or phagocytosis of graft cells (box). (D) High magnification of boxed area in C. In contrast, double labeling for BrdU and GFAP or BrdU and NeuN did not reveal any detectable co-localization (data not shown). Scale bars: A, 500 µm; B, 25 µm; C, 20 µm; D, 5 µm.
