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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 11  »  Immunohistochemical localization of cyclooxygenase-2 in radicular cysts
Immunohistochemical localization of cyclooxygenase-2 in radicular cysts
Introduction - Materials and methods.



C.-H. Tsai, F.-M. Huang, L.-C.Yang, M.-Y.Chou & Y.-C. Chang
Department of Oral Pathology, Department of Dentistry, Chung Shan Medical University Hospital, and 3School of Dentistry, Chung Shan Medical University, Taichung, Taiwan.

Introduction.
Radicular cysts are common and comprise between52to 68% of all the cysts affecting the human jaw (Shear 1992, Bando et al. 1993). They are believed to result from an inflammatory process in the periapical tissues associated with necrotic and infected pulps (Shear 1985, Matthews et al. 1988). Chronic periapical lesions contain epithelial cells (Tencate 1972, Langeland et al. 1977,Yanagisawa 1980) believed to be derived from the cell rests of Malassez, which proliferate in some lesions and are presumed to serve as the source of epithelium that lines the lumen in certain lesions that develop into radicular cyst.
The inflammatory mediator prostaglandin E2 (PGE2), known to exert diverse physiological actions in different tissues, is considered to be involved in the pathogenesis of pulpal disease (Okiji et al. 1987, Okiji et al. 1989). Cyclooxygenase (COX), known as prostaglandin endoperoxide synthase, is a key enzyme in prostaglandin (PG) biosynthesis. Currently there are two known human isoforms of COX, designated as COX-1 and -2 (Hla et al.1999).Many of the inflammatory and inducible effects of COX are mediated through COX-2, whilst many of the constitutive or house keeping effects of this enzyme are mediated through COX-1 (Smith 1989). This is indicated by the tissue specific expression of each isoform; COX-1 is constitutively expressed in nearly all human tissues, whereas COX-2 is at low or undetectable levels in healthy tissues (Dubois et al.1998) and is upregulated in inflamed tissues (Morton & Dongari-Bagtzoglou 2001).
Recently, in vitro studies have shown that COX-2 may playa important role in the pathogenesis of pulpal/periapical lesions (Chang et al. 2003a,b, in press). To our knowledge, COX-2 expression in radicular cyst has not been previously reported. Detailed knowledge of COX-2 may be important for understanding the molecular mechanism for the pathogenesis of radicular cysts. Therefore, the present study was undertaken to investigate the in situ localization of COX-2 in radicular cysts.

Materials and methods.
Thirty formalin-fixed, paraffin-embedded specimens of radicular cysts were included in this study. The diagnosis of radicular cyst was based on clinical (swelling over periapical region), radiographic (radiolucency, well defined border over apex) and histological examination (cell wall with epithelium lining). All patients had received cyste nucleation at the Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan.
The tissue blocks were cut at 5 mm and subjected to the peroxidase-labelled streptavidin-biotin technique. The sections were immersed in 3%methanol-hydrogen peroxide solution for 10 min to block endogenous peroxidase activity and incubated with anti-COX-2 antibody (Transduction Laboratory, Lexington, KY, USA; dilution 1 : 50) or Alpha-1-antichymotrypsine (a-ACT) (DAKO, Carpinteria, USA; dilution1 : 50), a specific antibody for macrophages, for 18 h at 4 8C with 1% bovine serum albumin. After washing in 20 mmol L_1 Tris- HCl buffer (pH 7.4) containing 0.9% NaCl, the sections were incubated at room temperature with
  1. biotinylated multilink swine antigoat, mouse and rabbit immunoglobulins (DAKO) diluted 1 :150 in Tris-HCl for 30 min;
  2. washed with Tris-HCl twice for10 min;
  3. incubated for 30 min with horseradish peroxidaseconjugated streptavidin (DAKO) diluted 1 : 50 in Tris- HCl;
  4. washed with Tris-HCl twice for10 min;
  5. incubated for 3 min with 0.01% diaminobenzidine tetrahydro- chloride (Sigma, St. Louis, MO, USA) and 0.03% H2O2 in 20 mmol L_1 Tris-HCl buffer at pH 7.4; and
  6. rinsed in distilled H2O for 10 min and counterstained with AEC (3-amino-9-ethylcarbazole) (DAKO).
Negative controls included serial sections from which either the primary or secondary antibodies were excluded. Four biopsy specimens of inflamed gingiva were used as positive controls (Morton & Dongari- Bagtzoglou 2001).
One section from each radicular cyst specimen was stained with haematoxylin and eosin to access the presence of the inflammatory infiltrates. Each specimen was graded at 400x  magnification as: grade I (normal), less then10 inflammatory cells per field; grade II (moderately inflamed),10-50 inflammatory cells per field; and grade III (highly inflamed), >50 inflammatory cells per field. Grading of each specimen was based on the average inflammatory cell number in three consecutive microscopic fields starting from the epithelial-connective tissue border and proceeding gradually deeper into lamina propria.
Processed immunohistochemically for COX-2 expression, sections graded as ‘low’ were represented by less than 50% positive stained cells; sections graded ‘high’ exhibited positive stained cells over 50% on three sections per tissueat 400x magnification. Fisher’s exact test (two-tail) was applied for the statistical analysis of the results. A P-value of <0.05 was considered to be statistically significant.