Article Figures & Data
Figures
- Figure 1.
In vitro detection of rhBDNF and rhNT-3 transcripts (A) and BDNF-transduced (B) and NT-3-transduced (C) SC release. ELISA revealed 35-fold and 62.5-fold increases in BDNF and NT-3, respectively, released by BDNF and NT-3-SCs compared with native SCs (Student's t test; *p < 0.001 compared with native SCs).
- Figure 2.
In vitro quantification of the number of BrdU+- and GalC+-labeled cells in control and transduced SCs. To assay proliferation, cells were grown for 72 h in DMEM-FBS. To assay differentiation, cells were grown for 72 h in DMEM-N2 without FBS and NDFβ. The percentage of BrdU+ cells was strongly reduced in BDNF- and NT-3-expressing SCs compared with controls and GFP-expressing cell populations. The percentage of GalC+ cells was strongly increased in the BDNF and NT-3 and, to a lesser extent, in the GFP-expressing cells and controls (Student's t test; *p < 0.001 compared with control).
- Figure 3.
Effect of SC transplantation on motor performances. A, Rotarod assay scores. B, Grid walk assay scores. The groups transplanted with HIV-BDNF SCs or the HIV-NT-3 SCs showed a dramatic improvement of their motor score from 7 d p.i. From this stage, their score improved significantly over the demyelinated ungrafted and the unlesioned animals. ANOVA showed a group effect (p < 0.001), indicating overall differences between the various transplantation groups.
- Figure 4.
Effect of NT-3 and BDNF on CNS and PNS remyelination. A–L, Double immunolabeling of P0 and PLP in ungrafted lesions (A, E, I) and GFP-SC-grafted (B, F, J), BDNF-SC-grafted (C, G, K), and NT-3-SC-grafted (D, H, L) lesions 21 d p.i. E–L are enlargements of the boxed areas in A–D. The lesion area is delineated by the dashed lines. Staining for PLP and P0 did not overlap. M–P, Double immunolabeling for P0 and GFP in an ungrafted lesion (M) and in GFP-SC-grafted (N), BDNF-SC-grafted (O), and NT-3-SC-grafted (P) lesions showing colocalization of P0 and GFP in myelin-like segments. Quantification of remyelination by Schwann cells and oligodendrocytes at 15 (Q) and 21 (R) d p.i. BDNF and NT-3 improved CNS and PNS remyelination (n = 10). Scale bars: (in A) A–D, 50 μm; (in E, I) E–L, 25 μm; (in M) M–P, 10 μm. *p < 0.01 compared with control.
- Figure 5.
The effect of NT-3 on endogenous glial cell proliferation and differentiation. A, B, Double immunolabeling for NG2 (green) and BrdU (red) illustrating NG2+ cell proliferation 7 d p.i. in GFP-SC-grafted (A) and NT-3-SC-grafted (B) lesions. Insets illustrate NG2+/BrdU+ cells. C, Quantitative evaluation of glial cell proliferation showing that BDNF promoted proliferation of NG2+ and GFAP+ cells, whereas NT-3 favored proliferation of NG2+ cells (Student's t test; *p<0.001 compared with lesioned mice). D, Semiquantitative RT-PCR analysis of PLP/DM20 and PLP mRNA transcripts at 21 d p.i. DM20/PLP ratios were strongly reduced in BDNF- and NT-3-SC-grafted lesions, suggesting OPC differentiation (Student's t test; *p<0.001 compared with control mice). Scalebar: (in A) A, B, 50μm.
- Figure 6.
Effect of BDNF and NT-3 on astrogliosis 7 d p.i. Double immunodetection of GFAP (red) and laminin (green) in ungrafted (A) and in GFP-SC-grafted (B), BDNF-SC-grafted (C), and NT-3-SC-grafted (D) lesions revealed a decrease of GFAP and the absence of basal lamina-like structures in response to NT-3. Scale bar: (in A) A–D, 100 μm.
- Figure 7.
Effect of BDNF and NT-3 on axonal preservation. A–D, Luxol fast blue staining illustrates the general view of the lesions at 21 d p.i. E–H, Enlargement of each lesion showing double immunolabeling for 2F11 (red) and PLP (green): A, E, ungrafted lesion; B, F, GFP-SCs; C, G, BDNF-SCs; D, H, NT-3-SC-grafted lesions. The number of linear axonal profiles increased progressively from E to H. I–J, Quantification of linear axonal profiles in lesions 21 d p.i. (I) and GAP-43 structures in lesions 7 d p.i (J). The number of linear axonal profiles and the number of GAP-43+ structures in the demyelinated lesion grafted with BDNF and NT-3-SCs were higher than in ungrafted lesions (Student's ttest; *p<0.001 compared with lesioned mice). Scalebars:(in A) A–D, 100 μm; (in E) E–H, 50 μm.
Tables
- Table 1.
Effect of growth factor overexpression on SC survival, recruitment, and differentiation in vivo
Series Anti-human nuclei+ cells GFP+ cells PO+ structures GFP+/PO+ structures GFP-/PO+ structures Lesion 0 0 6 ± 2.7 0 6 ± 2.7 GFP 254 ± 32 188 ± 47 48 ± 11 26 ± 7 22 ± 5.8 BDNF 328 ± 49 264 ± 72 457 ± 82 31 ± 6 426 ± 71 NT-3 421 ± 27 368 ± 82 492 ± 91 127 ± 45 365 ± 82 -
Data represent the number of GFP+ cells and PO+ and GFP+/PO+ structures ± SEM per lesion in the four experimental series (n = 20 per series). Statistical differences were obtained between all groups for p < 0.001.
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