Figure 1.
Histological characterization of NP tissue from IVD of rats at different ages.
Frozen tissue sections from (A, D) 1 m, (B, E) 12 m and (C, F) 21 m-old rats were stained with (A, B, C) Hematoxylin and Eosin (H&E) for cell structure and (D, E, F) Safranin O (SO) for proteoglycan content. Representative images are shown and the inserts of small images shown higher magnifications of interested regions. Bar: 50 µm. m: month. Arrows in subfigures indicate vacuolated morphology. (G) The percentage of cells with vacuolated morphology indicates an age-dependent change in NP tissue. **p<0.01 as compared to 1 m NP (one-factor ANOVA, n = 8). (TIF)
Table 1.
Average fold-differences in relative mRNA levels of selected genes between both tissue regions (AF & NP of 1 m rat) and ages (1 m & 12 m of NP) analyzed by cDNA microarray (n = 4, p<0.01, fold-differences greater than or equal to 2 folds between groups by one-factor ANOVA).
Figure 2.
Realtime RT-PCR for relative mRNA level (2−ΔΔCt) of NP markers.
(A) Basp1; (B) Ncdn; (C) Nrp-1; (D) Brachyury T; (E) CD155; (F) CD221 in rat IVD tissues at different ages (1 m, 12 m and 21 m). All values of fold-difference were normalized to AF tissue (1 m) for comparison between different tissue regions and ages. m: month. * p<0.05, ** p<0.01, two-factor ANOVA. (TIF)
Figure 3.
Immunostaining illustrated region-dependent and age-related changes in the expression of NP markers.
(A) Basp1; (B) Ncdn; (C) Nrp-1; (D) CD221 in rat IVD tissues at different ages (1 m, 12 m and 21 m). Bar: 20 µm; m: month. (TIF)
Figure 4.
Immunostaining and flow cytometry detection for NP markers.
Immunostaining (A, C, E) and flow cytometry (B, D, F) illustrated region-dependent and age-related changes in the expression of NP markers (A, B) CD24; (C, D) CD90; (E, F) Brachyury T in rat IVD tissues at different ages (1 m, 12 m and 21 m). Bar: 20 µm; m: month. Representative histograms of flow cytometry at left illustrate the relative fluorescence intensity of NP markers on X-axis for freshly isolated cells of 1 m rats (cell surface: CD24 and CD90; nucleus: T). The numbers appeared in each histogram indicate the percentage of positive fluorescence labeled cells and mean fluorescence intensity (MFI) for each cell type. (First left black line: isotype control, red line: AF cells, blue line: NP cells). (TIF)
Table 2.
Realtime PCR probes and primers of NP-markers (from Applied Biosystems) and corresponding antibodies for protein analysis.