Article Figures & Data
Figures
- Figure 1.
CNTF is upregulated after demyelination in the CC and participates in SVZ-derived cell recruitment to the lesion site. A, Western blot and semiquantitative analysis of CNTF expression in perilesional areas 2 and 7 d after LPC-induced demyelination. CNTF expression was normalized to α-tubulin and expressed as the percentage of the contralateral (unlesioned) side 2 dpi (protein samples from five lesioned adult mice for each time point). B, Illustration of the coinjection of LPC and CNTF neutralizing antibody in the CC and representation of the experimental design for histological analysis. C, Four days postinjection, the demyelinated area is revealed by the loss of MBP immunostaining and the densification of DAPI (delineated by a white dashed line). D, E BrdU labeling showing SVZ-derived cell recruitment toward the demyelinated area. F, Quantification of BrdU+ cells within the lesion site showing a significant decrease in cell recruitment in mice treated with CNTF-neutralizing antibody compared with IgG-treated mice. Data from nine IgG-treated mice, 10 anti-CNTF-injected mice in two independent experiments. All pictures are from brain sagittal sections. Scale bars, 200 μm.
- Figure 2.
Ectopic CNTF expression controls SVZ-derived cell migration. A, Representation of the experimental design. B, Illustration of the graft of mRFP HEK cells secreting CNTF in the CC. SVZ-derived cells recruited in the CC were quantified within a 250-μm-wide by 1000-μm-long (500 μm on both sides of the graft) area (white dashed line) around the CNTF-secreting cells (red dashed line). Control mice were grafted with mRFP HEK cells secreting no CNTF. C, D, BrdU labeling showing SVZ-derived cells recruited toward the graft of control cells (C) and CNTF-secreting cells (D). E, Quantification of BrdU+ cells within the CC (quantification area) showing an increase in SVZ-derived cell recruitment toward the ectopic source of CNTF compared with control source. F, G, DCX labeling showing the architecture of the RMS. H, Quantification showing longer RMS interruptions (gaps) in CNTF-secreting HEK-grafted mice. I, J, DCX labeling showing neuronal progenitors exiting the RMS to migrate to the CC. K, Quantification of the number of DCX+ cells present within a 100-μm-wide stripe above the RMS. More cells are migrating out of the RMS toward the ectopic source of CNTF compared with control source. L, M, BrdU and p-STAT3 labeling showing JAK–STAT3 pathway activation in BrdU+ cells in the CC. White arrows indicate BrdU+ p-STAT3+ cells. N, Quantification showing an increase of the percentage of p-STAT3+ cells relative to the BrdU+ population, in the presence of a source of CNTF. Note that the percentages of p-Akt+ and p-ERK1/2+ cells among BrdU+ cells are not modulated by the presence of CNTF ectopic expression. All pictures are from brain sagittal sections. V, Ventricle. Scale bars: B–G, L, M, 200 μm; I, J, 100 μm. Data are from five control grafted mice and five CNTF-secreting HEK-grafted mice in one experiment.
- Figure 3.
Effect of CNTF on SVZ cell migration in vitro. A, Western blot and semiquantitative analysis of STAT3 phosphorylation after the stimulation of in vitro amplified SVZ cells with recombinant CNTF (data from three independent experiments). p-STAT3 expression was normalized to nonphosphorylated STAT3 and expressed as a percentage of control (no stimulation). B, Quantification of SVZ cell migration through chemotaxis chambers (Boyden chambers) (data are from six independent experiments). After cell plating, different growth factors were used in the lower chambers to stimulate SVZ cell migration. SDF1 was used as a positive control. CNTF has the strongest effect at 100 ng/ml. Note that SVZ cell migration is not improved in absence of a gradient (CNTF in both compartments). C–F, SVZ neuronal progenitor migratory behavior was determined using the Matrigel assay. C, Illustration showing SVZ explants cultured in Matrigel in the vicinity of a bead soaked with CNTF or PBS for control. The length of chains formed by cells migrating out of the explant was measured in three independent measures proximally and distally to the bead. D, E, Neuronal progenitor migration pattern in the absence (D) or presence (E) of CNTF. F, Quantification of chain length proximal and distal to the source of CNTF (data are from 70 control explants and 30 CNTF explants from six independent experiments). The significant lengthening of proximal chains argues in favor of a chemoattractant effect of CNTF on SVZ neuronal progenitors. Scale bars, 100 μm.
- Figure 4.
Effect of CNTF on the migratory behavior of purified OPCs in vitro. A–C, Increased p-STAT3 (red) expression on purified OPCs (PDGFRα+, green) after CNTF exposure was visualized by immunofluorescence and directly quantified on four independent experiments. D, Quantification of purified OPC migration through Boyden chambers (data are from eight independent experiments). PDGF was used as a positive control. CNTF has the strongest effect at 10 ng/ml. Note that OPC migration is not improved in the absence of a gradient. E–I, The migratory behavior of OPCs was further determined using video time-lapse analysis on modified aggregation assays. E, F, OPC migration pattern in the absence (E) or presence (F) of CNTF. White stars indicate the source's position relative to the explant. Note that OPCs migrated preferentially in the direction of the CNTF source, whereas they were equally distributed in the control condition. G, Illustration showing OPC aggregates cultured in Matrigel in the vicinity of a bead soaked with CNTF or PBS for control. H, Quantification of the efficient distance traveled by OPCs proximal and distal to the source of CNTF. The longer cell migration distance proximal to the source of CNTF, compared with proximal control and distal CNTF, argues further in favor of a chemoattractant effect of CNTF on OPCs. I, Quantification of OPC migration speed proximal and distal to the source of CNTF. OPCs migrate faster proximal to the source of CNTF compared with proximal control and distal CNTF OPCs. Data are from three independent experiments. Scale bars: A, B, 50 μm; E, F, 100 μm.
Tables
- Table 1.
Cell proliferation and survival 4 d after CNTF neutralizing antibody and LPC injection in corpus callosum
Site Cells IgG Anti-CNTF p value A: at the lesion BrdU+ cells/mm3 166,158 ± 18,691 92,639 ± 16,921 0.022 % PH3+/BrdU+ cells 1.40 ± 0.49 1.41 ± 0.38% 0.96 % Casp3+/BrdU+ cells 0.36 ± 0.08 0.54 ± 0.24% 0.87 PH3+ cells/mm3 37,692 ± 5,842 31,988 ± 4,811 0.35 Casp3+ cells/mm3 3353 ± 346 3,818 ± 734 0.89 B: around the lesion Olig2+ BrdU− cells/mm3 20,814.9 ± 1,171.4 9,890.1 ± 1,568.5 0.033 % Ki67+/Olig2+ BrdU− cells 11.9 ± 0.7% 2.5 ± 2.2% 0.027 % Ki67+/Olig2+ BrdU+ cells 3.13 ± 3.1% 2.22 ± 2.2% 1 C: SVZ niche PH3+ cells/mm3 38,814.7 ± 3,087.8 39,073.7 ± 5,161.1 0.84 For site A, proliferation and survival indexes were determined using PH3 and Casp3 markers, respectively, among BrdU+ at the lesion site (see quantification area in Fig. 1C). Overall proliferation and survival indexes were determined by counting the number of PH3+ cells/mm3 and Casp3+ cells/mm3 at the lesion site. For site B, proliferation of OPCs was determined using Ki67 marker among BrdU+, Olig2+, and BrdU− Olig2+ cells. Olig2+ cells were quantified within a 50-μm-wide corridor surrounding the lesion site. For site C, cell proliferation was estimated within the SVZ by counting the number of PH3+ cells/mm3. Data are from nine IgG-treated mice and 10 anti-CNTF-injected mice in two independent experiments.
- Table 2.
Cell proliferation and survival after CNTF-secreting HEK cells grafting in corpus callosum
Site Cells Ctrl HEK CNTF-secreting HEK p value A: around the graft BrdU+ cells/mm3 3,462 ± 662 6,127 ± 573 0.028 % PH3+/BrdU+ cells 3.89 ± 1.38% 3.07 ± 1.49% 0.77 % Casp3+/BrdU+ cells 2.67 ± 2.67% 0.67 ± 0.67% 1 PH3+ cells/mm3 2,675 ± 372 4,108 ± 949 0.2 Casp3+ cells/mm3 483 ± 126 558 ± 158 0.91 B: around the graft % Ki67+/Olig2+ BrdU− cells 2.9 ± 1.1% 8.4 ± 2.1% 0.028 % Ki67+/Olig2+ BrdU+ cells 32.6 ± 7.8% 33.9 ± 1.8% 0.51 C: SVZ niche PH3+ cells/mm3 64,583.3 ± 10,958.1 72,916.7 ± 20,361.2 0.88 For site A, proliferation and survival indexes were determined using PH3 and Casp3 markers, respectively, among BrdU+ cells around the graft site (see quantification area in Fig. 2B). Overall proliferation and survival indexes were determined by counting the number of PH3+ cells/mm3 and Casp3+ cells/mm3 around the graft site. For site B, proliferation of OPCs was determined using Ki67 marker among BrdU+ Olig2+ and BrdU− Olig2+ cells. For site C, cell proliferation was estimated within the SVZ by counting the number of PH3+ cells/mm3. Data from five control grafted mice and five CNTF-secreting HEK grafted mice in one experiment.
- Table 3.
SVZ cell populations recruited after CNTF neutralizing antibody and LPC co-injection or CNTF ectopic expression
Site Cells IgG anti-CNTF p value Control HEK CNTF-secreting HEK p value A: in the CC % Mash1+/BrdU+ cells 9.4 ± 6.2 3.1 ± 1.0 0.69 % DCX+/BrdU+ cells 73.5 ± 3.3 85.4 ± 4.0 0.015 % GFAP+/BrdU+ cells 6.3 ± 0.6 2.9 ± 0.8 0.028 % Olig2+/BrdU+ cells 10.7 ± 0.8 8.6 ± 1.4 0.54 B: in the CC % Mash1+/BrdU+ cells No cells No cells % DCX+/BrdU+ cells 68.4 ± 4.5 57.7 ± 3.1 0.11 % GFAP+/BrdU+ cells 15.5 ± 2.5 17.7 ± 4.3 0.88 % Olig2+/BrdU+ cells 16.1 ± 1.4 24.6 ± 2.9 0.14 At site A, 4 d after lesion, BrdU+-recruited cells were phenotyped in the CC using Mash1 (SVZ-derived transitory amplifying cells), DCX (neuroblasts), GFAP (astrocytes), and Olig2 (oligodendrocytes) markers. At site B, following CNTF-secreting HEK cell grafting, BrdU+-recruited cells were phenotyped in the CC (see quantification area in Fig. 2B) using Mash1, DCX, GFAP, and Olig2 markers. Site A, Data are from five IgG-treated mice and five anti-CNTF-injected mice in one experiment. Site B, Data are from five control grafted mice and five CNTF-secreting HEK grafted mice in one experiment.
