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Nitric Oxide Synthase in Healthy and Inflamed Human Dental Pulp
F. Di Nardo Di Maio,
Z. Lohinai1,
C. D’Arcangelo,
P. Esposito De Fazio,
L. Speranza2,
M.A. De Lutiis2,
A. Patruno2,
A. Grilli2 and
M. Felaco2,*
1 Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, 78/A Üllôi út, Budapest, Hungary, 1082; and
2 School of Dentistry, University of Chieti, 1 Via dei Vestini, Chieti, Italy, 66100;
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Figure 1. Hematoxylin-eosin staining of a healthy pulp tissue (A), hyperemic (B), and irreversible pulpitis (C) (oil immersion, magnification 100X). The diameters of the randomly selected arterioles were recorded only when they were perpendicular to the plane of section. The diameters of the arterioles showed a statistically significant increase in parallel with the development of an inflammatory pathological process. Data are presented as means ± SD of 5 pulps in each pulp condition, with 20 measurements in each group. The vascular diameter (A = 22 µm, B = 32 µm, C = 48 µm) differences were significant in all 3 groups (*p < 0.05 A vs. B, *p < 0.05 B vs. C, *p < 0.05 A vs. C).
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Figure 2. Immunohistochemical localization of eNOS and iNOS in healthy and inflamed human dental pulp. eNOS in: (A) normal pulp, (B) hyperemic pulp, and (C) irreversible pulpitis. iNOS in: (D) normal pulp, (E) hyperemic, and (F) irreversible pulpitis. Magnification, 25X. eNOS was visualized in the endothelial cells (arrows), odontoblasts (arrowheads), and in some fibroblasts (*) as well. In control pulp, the immunohistochemical localization revealed a lack of iNOS immunoreactivity. However, in hyperemic pulp and irreversible pulpitis, iNOS immunopositivity was characteristic mainly in the accumulated leukocytes and in the area adjacent to that of dense leukocytic infiltration (arrows). Densitometric analyses of eNOS and iNOS immunoreactivity in the 3 groups are presented as integrated optical density (IOD) and expressed as means ± SD of 5 pulps in each group, with 9 independent measurements in each pulp. In Table 1 (Appendix), significant differences are shown in all 3 groups of eNOS and iNOS IOD as well (*p < 0.05, A vs. B; *p < 0.05, B vs. C; *p < 0.05, A vs. C in both cases).
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Figure 3. Occurrence of mRNA of eNOS in 300 bp (upper part) and iNOS in 340 bp (lower part) by RT-PCR in human dental pulp in the healthy tissue (A), hyperemic (B), and irreversible pulpitis (C). The standard band is on the right lane (S 488 bp). The Fig. clearly shows the presence of eNOS 300 bp in all 3 groups, with the highest level in the hyperemic group. The presence of iNOS 340 bp in the healthy pulp is not detectable; however, a marked increase of iNOS 340 bp was found in hyperemic and irreversible pulpitis groups. 18S (488 bp) is the internal standard. Data are expressed as means ± SD of 5 pulps in each pulp condition. Significant differences of integrated optical density (IOD) of eNOS and also iNOS mRNA, indicated in Table 2 (Appendix), were found in all 3 groups (*p < 0.05, A vs. B; *p < 0.05, B vs. C; *p < 0.05, A vs. C in both cases).
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Figure 4. Western blot analysis of eNOS (upper part) and iNOS (lower part) proteins obtained from human healthy (A) and hyperemic (B) dental pulp, as well as from the pulp with irreversible pulpitis (C). The pulpal proteins were stained by antibodies against human eNOS antigen (133 kD) and iNOS antigen (130 kD). β-actin is used for control. All 3 groups contain the eNOS enzyme, with the highest amount in the hyperemic group. The iNOS protein was undetectable in the healthy group. A significant increase of iNOS enzyme was observed in the hyperemic and the irreversible pulpitis groups. The panels at right show the densitometric analysis of eNOS (upper part) and iNOS (lower part) protein bands. Data are expressed as means ± SD of 5 pulps in each group. The differences of integrated optical density (IOD) of eNOS and also iNOS bands, indicated in Table 3 (Appendix), were significant in all 3 groups (*p < 0.05, A vs. B; *p < 0.05, B vs. C; *p < 0.05, A vs. C in both cases).
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Journal of Dental Research, Vol. 83, No. 4,
312-316 (2004)
DOI: 10.1177/154405910408300408
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