Figure 1.
siRNA-mediated knockdown of NgR.
qPCR of third ventricle samples show levels of NgR expression at 24, 48 and 72 hrs after single siRNA injection. Gene expression was normalized with Gapdh. Data are pooled from three independent experiments providing similar results (total number of mice for each data point = 6). Graph shows fold changes in NgR mRNA using qPCR analysis. Boxes represent the fifth to ninety-fifth percentiles around the median with whiskers for minimum and maximum values. Statistical analysis used two-way ANOVA and Bonferroni's post -test. Treatment had significant effects on the level of NgR gene expression. NgR gene expression was decreased significantly 24 h after siNgR injection compared to siControl, the asterisk indicates that the difference between the pairs denoted are significant at the confidence levels ***p<0.001.
Figure 2.
Extent of demyelination is decreased following NgR knock down.
Coronal sections of adult mouse brains (12 µm) treated with saline, LPC and LPC+siNgR were double stained with MOG (green) and NF200 (red) and nuclei are labeled with DAPI (blue). (A) Schematic picture of injection site and demyelination area. (B) Saline-treated chiasm at 7 days post injection (dpi), no detectable demyelination is seen 7 days after a single injection of saline (Control). (C–E) Demyelination at optic chiasm of LPC-treated animals at 3, 7 and 14 dpi, respectively. (F–H) Demyelination at optic chiasm of LPC+siNgR treated animals at 3, 7 and 14 dpi, respectively. (I) The extent of demyelination in different groups are quantitatively analyzed and presented as percent of total area. Control group represents demyelination level in animals treated with saline at dpi 7. Statistical analysis used two-way ANOVA and Bonferroni's post -test. Treatment and time had a significant effect on the remyelination process. In LPC treated-animals significant demyelination is seen at 3, 7 and 14 dpi compared to saline (***P<0.001). At 14 dpi, demyelination was partially reduced compared to 7 dpi. Between groups, there was a significant reduction of demyelination in LPC+siNgR 7 dpi compared to LPC 7 dpi (∧∧∧P<0.001) and in LPC+siNgR 14 dpi compared to LPC 14 dpi (∧∧∧P<0.001). Each data point shows data obtained from experiments carried out on three mice (n = 3), and represents Mean ± SEMs, Bars: 50 µm, Dashed line indicates lesion area.
Figure 3.
Macrophages and microglia response to LPC-induced demyelination.
Sections from lesion sites were stained for Iba-1 (green) and DAPI (blue) at 3 and 14 dpi in both LPC and LPC+siNgR treated groups. (A) The number of Iba-1+ cells in saline treated animals (Control) was low at 3 dpi. (B–C) Increased number of microglia and macrophages in the lesion site following LPC induced demyelination at 3 (B) and 14 dpi (C). (E–F) In siNgR treated mice in LPC induced lesion also the number of Iba-1+ cells was increased at 3 (E) and 14 dpi (F). (D) Iba1+ cells per area/mm2 are averaged from the counts of four sections of each chiasm (n = 3). Statistical analysis used two-way ANOVA with Bonferroni's post-test. The effects of treatment and time were significant. Further analysis using post-test showed significant difference between Control and LPC treated mice (p<0.001), and between Control and LPC+siNgR treated mice (p<0.001) at 3 and 14 dpi. The difference between the LPC and LPC+siNgR treated groups was non-significant (n.s.). Data are expressed as Mean ±SEMs, Bars: 50 µm.
Figure 4.
Functional recovery is induced by NgR inhibition in demyelinated optic chiasm.
(A) Visual evoked potential (VEP) sample recordings from electroencephalographic activity and its components. The trace represented is the average of 300 sweeps of 300 ms duration with 1 Hz frequency. P1 latency (red bar) was measured by Biochart software. (B) VEP sample from animals treated with saline inside the chiasm (control) recorded at 7 dpi. (C–E) Changes in the P1 wave latency at 3, 7 and 14 dpi in the LPC treated animals. (F–H) VEP sample recordings from LPC+siNgR treated animals at 3, 7 and 14 dpi. (I) Quantitative analysis of changes in P1 latency in different groups. Statistical analysis used two-way ANOVA with Bonferroni's post-test. Treatment and time had a significant effect in this study. In LPC treated animals P1- latency was increased at 3, 7 and 14 dpi compared to control (all, ***p<0.001) but was partially diminished at 14 dpi (E). In LPC+siNgR group, there was a significant increase in p-latency at 3 and 7 dpi compared to control (both, ***P<0.001). NgR inhibition induces functional recovery at 7 and 14 dpi compared to LPC 7 and LPC 14, respectively (both, P<0.001) and there was no significant change in p-latency between LPC+siNgR and Control at14 dpi. Data was pooled from three independent experiments on mice (n = 6), Bars: Mean ± SEMs.
Figure 5.
Increased BrdU+/Olig2+ cell numbers in demyelinated optic chiasm following NgR inhibition.
Double immunohistochemistry (IHC) of BrdU (red) and Olig2 (green)-labeled cells was done on coronal section of optic chiasm in different groups. (A) Time line of BrdU injection and sampling in different groups. Mice received seven injections of Brdu at intervals of 2 hrs, 24 hrs prior to demyelination, and were sacrificed 3, 7 or 14 days after LPC injection. (B–D) BrdU+/Olig2+ cells in optic chiasm of LPC-treated animals at 3 (B), 7 (C) and 14 (D) dpi. (E–G) Optic chiasm of LPC+siNgR treated animals at 3 (E), 7 (F) and 14 (G) dpi. (H) Low number of BrdU+/Olig2+ cells in optic chiasm of saline-treated animals at 7 dpi (Control). Arrows indicate double-labeled cells and Square shows the cells magnified in inset. Dashed line indicates optic chiasm border. (I) BrdU+/Olig2+ cells per area/mm2 are averaged from the counts of nine sections of each chiasm. Statistical analysis used two-way ANOVA with Bonferroni's post-test. The effects of treatment and time were significant (p<0.001). In LPC treated groups the number of BrdU+/Olig2+cells was increased over the time but changes were not significant compared to Control. In LPC+siNgR treated animals, the number of BrdU+/Olig2+cells at 7 and 14 dpi in the lesion site was increased compared to control (**p<0.01, ***p<0.001; respectively). Between groups significant changes exist at 7 dpi (*p<0.05) and 14 dpi (***p<0.001). Data are expressed as Mean±SEMs, n = 3, Bars: 100 µm.
Figure 6.
Increased numbers of BrdU+/GFAP+ cells within the lesion site following NgR inhibition.
(A–C) Immunofluorescent images of Optic chiasm in LPC-treated animals at 3, 7 and 14 dpi. The number of BrdU+/GFAP+ cells at LPC 3 dpi (A), LPC 7 dpi (B) and 14 dpi (C) was increased. (D–F) Optic chiasm images of LPC+siNgR treated animals at 3 (D), 7 (E) and 14 dpi (F). The number of BrdU+/GFAP+ cells at 7 and 14 dpi in the lesion site was increased. (G) The number of BrdU+/GFAP+ cells in optic chiasm of saline-treated animals (Control) at 7 dpi was low. Arrows indicate double-labeled cells and Square shows the cells magnified in inset. (H) BrdU+/GFAP+ cells per area/mm2 are quantified and averaged from the counts of nine sections of each chiasm. Statistical analysis of the differences between numbers of BrdU+/GFAP+ cells in the optic chiasm of all groups was done by two-way ANOVA followed by Bonferroni's post-test. Differences between groups were significant (p<0.001). Post-test showed that the number of BrdU+/GFAP+ cells in LPC treated animals at 7 dpi and 14 dpi was increased significantly compared to Control (both, p<0.05). In LPC+siNgR treated animals the number of BrdU+/GFAP+ cells at 7 and 14 dpi in the lesion site was increased significantly compared to Control (both, ***p<0.001). NgR inhibition enhance the number of BrdU+/GFAP+ cells in optic chiasm compered to LPC groups and this change was significant between siNgR 14 dpi and LPC 14 dpi (***p<0.001). Data are expressed as Mean ± SEMs, N = 3, Bars: 100 µm.
Table 1.
Total number of BrdU+ cells in third ventricle and optic chiasm in different groups.
Figure 7.
Third ventricle progenitor cell activation in response to LPC-induced lesions in optic chiasm is greater following NgR inhibition.
Double labeling of BrdU (red) with Olig2, GFAP or PSA-NCAM (green) was done in coronal sections of third ventricle area in different groups. (A–C) BrdU+/GFAP+ cells in the rims of third ventricle of LPC group at 3 (A), 7 (B) and 14 dpi (C). (D–F) BrdU+/GFAP+ cells in the rims of third ventricle of LPC+siNgR treated animals at 3 (D), 7 (E) and 14 dpi (F). (G–I) BrdU/Olig2 double staining in third ventricle of LPC+siNgR treated animals at 3 (G), 7 (H) and 14 (I) dpi (see fig. S1D–F for BrdU+/Olig2+ cells in LPC group). (J–L) BrdU/PSA-NCAM positive cells in the third ventricle of LPC+siNgR treated animals at 3 (J), 7 (K) and 14 dpi (L) (see fig. S1G-I for BrdU+/PSA-NCAM+ cells in LPC group). Square shows the cells magnified in inset. Arrows show double marker positive cells. (M–O) Histograms show quantification of double marker positive cells, BrdU+/GAFP+ (M), BrdU+/Olig2+ (N) and BrdU+/PSA-NCAM+ (O) in different groups. Double marker positive cells per area/mm2 are averaged from the counts of nine sections of each brain, n = 3. Statistical analysis used two-way ANOVA with Bonferroni's post-test. Differences between groups were significant (p<0.001). The number of BrdU+/GFAP+ cells in third ventricle of LPC 3, 7 and 14 dpi was increased but changes were not significant compared to Control 7dpi (mice received saline inside chiasm), but this type of cells in LPC+siNgR treated animals at 3, 7 and 14 dpi were considerably increased compared to Control (*p<0.05, ***p<0.001, **p<0.01, respectively) (M). The number of BrdU+/Olig2+ cells was considerably increased in siNgR-LPC group at 3, 7 and 14 dpi compared to Control (**p<0.01, ***p<0.001; respectively) (N). Additionally in these animals, at 7 dpi the number of BrdU+/Olig2+ was considerably increased compared to LPC 7 dpi (∧∧∧p<0.001) (N). The number of BrdU/PSA-NCAM positive cells in the third ventricle of LPC+siNgR treated animals was significantly increased at 3 and 7 dpi (both, p<0.01), while it was significantly increases in LPC treated animals at 7 and 14 dpi (p<0.05, p<0.01; respectively). Data are expressed as Mean ±SEM, Bars: 50 µm.