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Effect of NOS Inhibitor on Cytokine and COX2 Expression in Rat Pulpitis

¹ Pulp Biology and Endodontics, Department of Restorative Sciences,
² Molecular Pathology, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical & Dental University, and
³ Center of Excellence (COE) Program for Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical & Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan;

View larger version (44K): [in a new window]   Figure 1. Histology of experimentally induced rat pulpitis and iNOS mRNA expression in experimentally induced rat pulpitis. LPS application to the pulp tissue of rat upper incisors for 6 hrs (A,B) caused severe inflammation, and the infiltration of many neutrophils into the pulp tissue can be seen (n = 3). Saline application (C,D) did not induce severe inflammation. Bars in the low-power fields (A,C) and those in the high-power fields (B,D) indicate 500 µm and 80 µm, respectively. iNOS mRNA expression (n = 3 in each group) was evaluated with RT-PCR, and the relative intensity of expression against ß-actin in each experimental period was statistically compared, by ANOVA and Tukey-Kramer tests (p < 0.05), with that in untreated pulp samples. The relative intensities in each period were: 0 ± 0, 0 hr; 0.4 ± 0.21, 3 hrs; 1.0 ± 0.24, 6 hrs; 0.6 ± 0.15, 9 hrs; 0.075 ± 0.03, 12 hrs; and 0 ± 0, 24 hrs (mean ± SD) (E). Competitive RT-PCR on LPS-treated pulp (6 hrs, n = 3) and LPS-treated liver (6 hrs, n = 3) shows that the expression of iNOS mRNA in the LPS-treated pulp is almost the same as that in the LPS-treated liver (F). The same results were observed in 3 separate experiments.

View larger version (42K): [in a new window]   Figure 2. iNOS+ cells in the inflamed pulp. (A) LPS application for 6 hrs (n = 3) caused infiltration of many iNOS+ cells with heterogeneous morphology into the inflamed coronal pulp. Many ED1+ macrophages and OX6+ antigen-presenting cells were also present. Saline application instead of LPS (n = 3) did not cause iNOS+ cell infiltration. ED1+ cells and OX6+ cells were observed in the saline-applied pulps, but their number was quite small compared with those in the LPS-applied pulps. Bars in the low- and high-power fields indicate 200 µm and 50 µm, respectively. The upper portions of these images are close to the exposed surface. (B) Double-staining with ED1 and anti-iNOS antibodies reveals that most of the iNOS+ cells were also ED1+ (closed arrowheads, ED1+ macrophages; open arrowheads, iNOS+ cells; open arrows, ED1 and iNOS double-positive cells). In contrast, the population of OX6+ cells (closed arrowheads) was completely different from the population of iNOS+ cells (open arrowheads).

View larger version (31K): [in a new window]   Figure 3. Cytokine and COX2 mRNA expression in inflamed pulp and the effects of L-NAME on their expression. (A) Cytokine and COX2 mRNA expression was evaluated with RT-PCR, and the density of each band was measured with Scion Image software. The peak expression of the pro-inflammatory cytokines (IL1, IL1ß, and TNF) and COX2 occurred at 6 hrs after the application of LPS (n = 3). The expression of IL10, which is one of the anti-inflammatory cytokines, was first observed at 6 hrs. The relative intensities of chemical mediators in each period were: (IL1a) 0.2 ± 0.11, 0 hr; 0.41 ± 0.17, 3 hrs; 0.83 ± 0.3, 6 hrs; 0.23 ± 0.15, 9 hrs; 0.17 ± 0.14, 12 hrs; 0.15 ± 0.1, 24 hrs; (IL1b) 0.2 ± 0.02, 0 hr; 0.81 ± 0.03, 3 hrs; 1.84 ± 0.38, 6 hrs; 1.22 ± 0.38, 9 hrs; 0.85 ± 0.13, 12 hrs; 0.88 ± 0.12, 24 hrs; (IL6) 0 ± 0, 0 hr; 1.21 ± 0.29, 3 hrs; 1.37 ± 0.43, 6 hrs; 1.02 ± 0.18, 9 hrs; 0.73 ± 0.17, 12 hrs; 0.27 ± 0.18, 24 hrs; (IL10) 0 ± 0, 0 hr; 0 ± 0, 3 hrs; 0.43 ± 0.11, 6 hrs; 0.27 ± 0.03, 9 hrs; 0.09 ± 0.01, 12 hrs; 0.08 ± 0.01, 24 hrs; (TNF) 0 ± 0, 0 hr; 0.2 ± 0.15, 3 hrs; 1.03 ± 0.41, 6 hrs; 0.6 ± 0.24, 9 hrs; 0.04 ± 0.01, 12 hrs; 0 ± 0, 24 hrs; and (COX2) 0.02 ± 0.01, 0 hr; 0.62 ± 0.07, 3 hrs; 0.75 ± 0.1, 6 hrs; 0.66 ± 0.09, 9 hrs; 0.01 ± 0.01, 12 hrs; and 0.01 ± 0.01, 24 hrs (mean ± SD). The relative intensities of each period were statistically compared, by ANOVA and Tukey-Kramer tests (p < 0.05), with those in untreated pulp samples. (B) Cytokine and COX2 mRNA expression in inflamed pulp (6 hrs after LPS application) treated with or without L-NAME (n = 3, respectively) was evaluated by RT-PCR, and the density of each band was measured with Scion Image software. Each pro-inflammatory cytokine/COX2 mRNA expression was inhibited by the application of L-NAME. In contrast, IL6 and IL10 mRNA expression was not influenced by the application of L-NAME. The relative intensities of chemical mediators at 6 hrs after LPS application in the absence and presence of L-NAME were: (IL1) 0.85 ± 0.13 and 0.26 ± 0.19; (IL1b) 1.7 ± 0.2 and 0.26 ± 0.17; (IL6) 1.2 ± 0.2 and 1.0 ± 0.29; (IL10) 0.42 ± 0.12 and 0.4 ± 0.14; (IL12) 0.2 ± 0.14 and 0 ± 0; (TNF) 0.81 ± 0.23 and 0.26 ± 0.13; and (COX2) 0.9 ± 0.09 and 0.49 ± 0.21 (mean ± SD). The relative intensities of each mediator in the presence of L-NAME were statistically compared, by ANOVA and Tukey-Kramer tests (p < 0.05), with those in the absence of L-NAME.