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Oxidative Stress-induced DNA Damage in the Synovial Cells of the Temporomandibular Joint in the Rat

¹ Departments of Oral Anatomy and Cell Biology,
² Removable Prosthodontics, and
³ Fixed Prosthodontics, Faculty of Oral Science, Kyushu University Graduate School of Dental Science, Fukuoka 812-8582, Japan;

View larger version (68K): [in a new window]   Figure 1. Schematic diagram, immunolight micrographs, and quantitative scoring of synovitis in the synovitis-induced and non-induced TMJs. (a) Schematic diagram of the sagittal section of the central portion of the rat TMJ. The joint compartment is divided into upper and lower compartments (UC, LC) by the articular disc (Disc), and the synovial membrane lines parts of these compartments. The boxes indicate the areas described in the RESULTS section of this paper. (b-e) Light micrographs of serial 10-µm-thick sagittal sections of the posterior portion that faces the upper joint compartment of the synovitis-induced (b,c) and non-induced (d,e) rat TMJs, which were immunostained with either the anti-nitrotyrosine (NT) (b,d) or anti-iNOS (c,e) antibody. (b,d) Nitrotyrosine immunoreactivity is expressed at a higher level in the grade 2 (G2; synovial lining is 7 layers thick) synovitis of the synovitis-induced TMJ (synov-TMJ) (b) than in the grade 0 (G0; synovial lining is 1–3 layers thick) synovitis of the non-forced TMJ (non-TMJ) (d). F: synovial fold. (c,e) Increased iNOS immunoreactivity is seen in the G2 synovitis of the synovitis-induced TMJ (c), as compared with the grade 0 synovitis of the non-induced TMJ (e). Hematoxylin staining. Original magnification: x400 (b-e). Bar = 25 µm (b-e). (f) Scoring the thickness of the synovial cell layers of the synovitis-induced and non-induced rat TMJs. The thickness of the synovial cell layer (synovitis) is graded as follows: grade 0, 1–3 layers; grade 2, 4–6 layers; grade 2, 7 layers. The numbers of joints that were graded are indicated. LC: lower joint compartment. UC: upper joint compartment.

View larger version (130K): [in a new window]   Figure 2. Electron micrographs of the synovial membranes that face the joint compartment in synovitis-induced rat TMJs. The sections were prepared by the pre-embedding technique, with the same method and anti-nitrotyrosine antibody as in Figs. 1b and 1d. (a-d) Electron micrographs of synovial lining cells that face the joint compartment in synovitis-induced rat TMJs. (a,b) Ultrastructural localization of nitrotyrosine-immunoreactive products in the synovial type A cells (A cell). (b) High magnification of the synovial type-A cells. Nitrotyrosine-immunoreactive products are distributed widely in the cytoplasm of type A cells, with microvilli (small arrows) that protrude toward the joint compartment (JC). Nitrotyrosine-immunopositive products are present in the cytoplasm (white arrow) near the mitochondrion (M), vesicle (V), and vacuole (Va). The nitrotyrosine-positive vesicle (arrowhead) is fused with vacuoles. Nitrotyrosine immunoreactivity is also found on the plasma membrane of the cell (white arrowhead). The nitrotyrosine-positive reaction is also detected in the intercellular space of the synovial lining cells (arrow). (c,d) Ultrastructural localization of nitrotyrosine-immunoreactive products in the synovial type B cells (B cell). (d) Higher magnification of the region indicated by the arrow in Fig. 2c. Positive immunostaining for nitrotyrosine is found in the type B cells. Immunoreactive products (arrowheads) are present in the cytosol, vesicles, and rough endoplasmic reticulum (r-ER) within the cytoplasm. In the nuclei (N) of type B cells, many nitrotyrosine-positive products are deposited on the euchromatin of the nucleoplasm (large arrowheads). (e) Ultrastructural localization of nitrotyrosine-immunoreactive products in a fibroblast of the subsynovial layer. Nitrotyrosine immunoreactivity is evident in the cytoplasm (arrowhead) and nucleus (arrows). Not counterstained. Original magnification: (a,c,e) x5000; (b,d) x12,500. Bar = 1 µm (a,c,e) or 0.4 µm (b,d).

View larger version (100K): [in a new window]   Figure 3. Light micrographs of 10-µm-thick serial sagittal sections of the posterior portion that faces the upper compartment of synovitis-induced rat TMJs (synov-TMJ), with ISNT (a,b) or TUNEL (c,d) staining for DNA strand breaks. The sections were pre-treated without proteinase K (PK-) (a,c), or with PK (PK+) (b,d). (a) In the section without PK treatment, there is no reactivity for ISNT in the G1 (grade 1, synovial lining 4–6 layers thick) synovitis of the synovitis-induced TMJ. F: synovial fold. V: blood vessel. Inset: higher magnification of the area indicated by arrowheads in Fig. 3a. The nuclei (N) of the multilayered synovial lining cells are negative for ISNT. (b) In the sections that were treated with PK, ISNT-positivity is found in the synovial lining cells of synovitis-induced TMJs with G1 grade synovitis, and in the fibroblasts of the subsynovial layer. Inset: higher magnification of the area indicated by arrowheads in Fig. 3b. Strong reactivity for ISNT is observed in the nuclei of the multilayered synovial lining cells. (c,d) In the sections without PK (c) or with PK (d) treatment, the synovial lining cells of synovitis-induced TMJs with G1 synovitis are negative for TUNEL. Insets: higher magnification of the areas indicated by arrowheads in Figs. 3c and 3d. There is no TUNEL reactivity in the nuclei of the multilayered synovial lining cells. Eosin staining. Original magnification: x800 and x1600 (inset). Bar = 50 µm.

View larger version (148K): [in a new window]   Figure 4. Light micrographs of 10-µm-thick sagittal serial sections of the posterior portion that faces the upper compartment of non-forced rat TMJs (non-TMJ), with the ISNT (a,b) and TUNEL (c,d) methods for staining DNA strand breaks. The sections were pre-treated without proteinase K (PK-) (a,c), or with PK (PK+) (b,d). (a,b) In the section that was pre-treated without PK (a) or with PK (b), there is no reactivity for ISNT in the synovial lining cells of the synovitis-induced TMJs with G0 synovitis. F: synovial fold. UC upper joint compartment. (c,d) In the TUNEL-stained sections that were treated without PK (c) or with PK (d), the synovial lining cells of the synovitis-induced TMJs with G0 synovitis are negative for TUNEL. Eosin staining. Original magnification: x400. Bar = 25 µm.