A Method for Histological, Enzyme Histochemical and Immunohistochemical Analysis of Periapical Diseases on Undecalcified Bone with Teeth

Abstract

Svrha je rada bila ispitati i primijeniti postupak histološke, histokemijske i imunohistokemijske raščlambe serijskih rezova nedekalcificiranih psećih zuba s okolnim koštanim tkivom čeljusti. Na premolarima pasa mješanaca induciran je pulpitis i apikalni parodontitis bakterijskom florom iz usne šupljine. Trepanirana pulpna komorica 50 je dana izložena djelovanju oralne mikroflore. Pošto je životinja žrtvovana, nedekalcificirana je kost s dekoroniranim eksperimentalnim zubima uložena u metilmetakrilat i rezana wolfram karbidnim nožem na debljinu 5 - 7 μm. Rezovi su za histološku raščlambu bojeni toluidinskim plavilom, te metodom bojenja kisele fosfataze i tartarat - rezistentne kisele fosfataze. Za detekciju CD 45+ limfocita uporabljeno je imunohistokemijsko bojenje. Debljina rezova 5 - 7 μm omogućila je na histološkim i histokemijskim pripravcima citološku raščlambu promjena u pulpi i periapeksu svjetlosnim mikroskopom in situ. Imunohistokemijsko bojenje nije bilo zadovoljavajuće. Postupak ulaganja nedekalcificirane kosti i zuba u metilmetakrilat i njihovo rezanje wolfram-karbidnim nožem zadovoljavajuća je tehnika za histološke i neke histokemijske raščlambe za potrebe istraživanja u endodonciji. Nužno je poboljšati metodu imunohistokemijskoga bojenja.The objective was to examine and apply a method for serial sectioning of undecalcified canine teeth with surrounding jawbone and to analyse it histologically, enzyme histochemically and immunohistochemically. Pulpitis and apical periodontitis were induced in a mongrel dog’s premolar teeth by exposing the pulp to the oral environmental influence for 50 days. After animal sacrifice undecalcified bone with the decoronated experimental teeth were embedded in methylmethacrylate and sectioned with a tungsten carbide knife at 5-7 μm. Sections were stained with toluidin blue (TB) for histological analysis and by a method of staining non-specific acid phosphatase (ACP), and tartrat-resistant acid phosphatase (TRAP). Immunohistochemical staining was performed for detection of CD 45+ lymphocytes. Because the specimens were 5-7 μm thick it was possible to perform a detailed cytological analysis of changes in the pulp and periapex in situ with a light microscope on histological and enzyme histochemical specimens. Immunohistochemical staining was not satisfactory. The method of embedding undecalcified bone and teeth in methylmethacrylate and sectioning with a tungstencarbide knife is satisfactory for histological and some enzyme histochemical analysis in endodontic research. However, immunohistochemical staining needs improvement