Figure 1.
Histological and imaging evidence of progressive cervical spinal compression in twy/twy mice.
Microphotographs of hematoxylin and eosin (H&E)-stained sagittal (A) and transaxial (B–D) sections, and computed tomography (CT) scans (E, F) of the cervical spine of 12- (B), 18- (C) and 24-week-old twy/twy mice (A, D, E, F). Calcified lesions originating from the atlantoaxial membrane increased in size progressively with age, compressing the lateral and dorsal aspects of the spinal cord between C2 and C3 segments (*) calcified lesions. A spinal canal transverse area on CT at thoracic (Th) 1 level and the site of compression of a 24-week-old twy/twy mouse is surrounded by white dotted line in (E, F). The relative spinal canal and spinal cord transverse area was shown compared with that of Th1 vertebral level assessed by CT and H&E staining (G). The spinal canal and spinal cord transverse area decreased with advancing age. Scale bars = 500 µm (A); 200 µm (B–D). **p<0.01 (n = 3 for each time point).
Figure 2.
Increased prevalence of activated microglia/macrophages after spinal cord compression correlates to neuronal changes in twy/twy mice.
Immunofluorescence staining for the expression of CD11b (red) and NeuN (green) in 12- (B–C), 18- (A, E–G) and 24-week-old (H–J) twy/twy mice. In sagittal sections of the spinal cords of 18-week-old twy/twy mice, CD11b-positive cells were distributed mainly in the sites of maximal compression site, compared with those sites that were rostral or caudal to the cord compression (A). The CD11b-positive area increased with the worsening of spinal cord compression, both in the gray and white matter, especially in the anterior horn (C, F, I) and anterior/lateral column (D, G, J). The NeuN-positive area of mainly the gray matter was especially lower in 24-week-old twy/twy mice (K). Scale bars = 500 µm (A, B, E, H); 50 µm (C, D, F, G, I, J). *p<0.05, **p<0.01 (n = 5 for each time point). AH: anterior horn, PH: posterior horn, AC: anterior column, LC: lateral column, PC: posterior column. A–J microphotographs were taken using confocal laser scanning microscope.
Figure 3.
The prevalence of phenotypically activated microglia/macrophages in association with increased severity of spinal cord compression in twy/twy mice.
Immunofluorescence staining for the expression of iNOS and CD16/32 (green) for classically activated microglia/macrophages (M1 phenotype) and arginase-1 and CD206 (green) for alternatively activated microglia/macrophages (M2 phenotype) co-localized with CD11b (red) in the anterior column of 12- (A), 18- (B) and 24-week-old (C) twy/twy mice. The numbers of CD11b-, CD11b/iNOS- and CD11b/CD16/32-positive cells (arrow heads) increased with the worsening of spinal cord compression. The CD11b/arginase-1- and CD11b/CD206-expressing cells (arrow heads) were the predominant population. The differences between iNOS, CD16/32 and arginase-1, CD206 were statistically significant in 18- and 24-week-old twy/twy mice. In control ICR mice, the expression of these factors was same as in 12-week-old twy/twy mice (D). The M1/M2 antigen expression ratio was higher in 24-week twy/twy mice compared with younger mice and control mice (E). Scale bars = 50 µm (A–C). Data are mean±SD. *p<0.05 (n = 5 for each time point). A–C microphotographs were taken using confocal laser scanning microscope.
Figure 4.
The resting microglia population decreased in association with increased severity of spinal cord compression in twy/twy mice.
Semi-quantitative flow cytometric analysis of resting microglia and activated microglia/macrophages in the CD11b positive cells (10.4±0.7% of the spinal cord cells) according to the degree of spinal cord compression. Representative data for 12- (A), 18- (B) and 24-week-old (C) twy/twy mice. CD11bhigh cells in the spinal cord were sub-fractioned into a CD45low/GR-1negative population, identifying them as resting microglia; or CD45high/GR-1negative population, which identified them as activated microglia/macrophages. The numbers of resting microglia (CD11bhigh/CD45low/GR-1negative cells) were significantly lower in 24-week-old mice (D), while the numbers of activated microglia/macrophages (CD11bhigh/CD45 high/GR-1negative cells) were higher in 18- and 24-week-old twy/twy mice (E). The number of resting microglia and activated microglia/macrophages in control ICR mice was same as in 12-week-old twy/twy mice (D, E). Data are mean±SD. **p<0.01 (n = 3 for each time point).
Figure 5.
Expression of the M1 phenotype in activated microglia/macrophages correlated to increased severity of spinal cord compression in twy/twy mice.
Semi-quantitative flow cytometric analysis of iNOS, CD16/32, arginase-1, and CD206 in activated microglia/macrophages. Representative data for 12- (A), 18- (B), and 24- (C) week-old twy/twy mice. The number of iNOS positive and CD16/32 positive activated microglia/macrophages increased with the worsening of spinal cord compression. Arginase-1 positive and CD206 positive cell populations were the predominant in cells present 18- and 24-week-old twy/twy mice (D). Data are mean±SD. **p<0.01 (n = 3 for each time point).
Figure 6.
Expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and macrophage antigen (Mac)-2 correlated to the phenotype of activated microglia/macrophages and increased spinal cord compression in twy/twy mice.
Immunofluorescence staining for the expression of BDNF, NGF, and Mac-2 (red) colocalized with iNOS and arginase-1 (green) in the anterior column of 12- (A), 18- (B) and 24-week-old (C) twy/twy mice (n = 5 for each time point). The expression levels of neurotrophic factors and Mac-2-positive cells increased with the worsening of spinal cord compression. Neurotrophic factor and Mac-2 colocalized with arginase-1- and CD206-positive cells (arrow heads), but not with iNOS- and CD16/32-positive cells. Scale bars = 50 µm. A–C microphotographs were taken using confocal laser scanning microscope.
Figure 7.
The prevalence of CD4-positive cells in the spinal cord of twy/twy mice.
Immunostaining of infiltrating helper T cells increased with the worsening of spinal cord compression, especially in 18-week-old twy/twy mice (A–E). Panel (C) and (E) are high-power photographs of the anterior horn (boxed area). (F) CD4-positive area in control ICR, 12-, 18-, and 24-week-old twy/twy mice. Scale bar = 500 µm (A, B, D); 200 µm (C, E). Data are mean±SD. **p<0.01 (n = 3 for each time point).
Figure 8.
Cytokine expression and phagocytic activity in the twy/twy mouse.
Immunoblot analysis of T helper 1 (Th1), T helper 2 (Th2) cytokines, neurotrophic factors and phagocytic activity in control ICR, 12, 18, and 24-week-old twy/twy mice. (A) The expression of IFN-γ was not detected in all age groups, but the expression levels of TNF-α and IL-6 increased significantly with the severity of spinal cord compression. (B) The expression levels of Th2 cytokines were highest in 18-week-old and remained significantly elevated in 24-week-old twy/twy mice. (C) The expression levels of neurotrophic factors and Mac-2 (D) increased significantly with the severity of spinal cord compression. Each graph shows the relative band intensity normalized to that of β-actin. Data are mean±SD. *p<0.05 (n = 3 for each time point).