A STUDY ON THE DISTRIBUTION OF CALCITONIN GENE-RELATED PEPTIDE CONTAINING NERVE FIBERS IN RAT PULP FOLLOWING DENTINAL INJURY

상아질 손상 후 흰쥐 대구치 치수의 calcitonin gene-related peptide(CGRP) 함유 신경섬유 분포에 관한 연구

The purpose of this study was to investigate the distribution of calcitonin gene-related peptide containing nerve fibers in rat pulp after dentinl injury by means of immunohistochemistry and confocal laser scanning microscope. The Spague-Dawley rats weighing about 250-300gm were used. The animals were devided into normal control and experimental groups. Experimental animals were sacrified 1, 2, 4, 7, 10, 21days after dentinal injury (dentin cutting, and then acid etching with 35% phosphoric acid) on the maxillary molar teeth. The maxillary teeth and alveolar bone were removed and immersed in the 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4), then were decalcified with 15% formic acid for 10 days. Serial frozen $50{\mu}m$ thick sections were cut on a cryostat. The rabbit CGRP antibody was used as a primary antibody with a dilution of 1:2000 in 0.01M PB. The sections were incubated for 48 hours at $4^{\circ}C$, and placed into biotinylated antirabbit Ig G as a secondary anti body with dilution of 1:200 in 0.01M PB and incubated in ABC(avidin-biotin complex). The peroxidase reaction was visualized by incubating the sections in 0.05% 3,3 diaminobenzidine tetrahydrochloride containing 0.02% $H_2O_2$. For the confocal laser scanning microscopic examination, Primary antibody reaction was same as immunoperoxidase stainning, but fluorescein isothiocyanate(FITC)-conjugate antirabbit IgG as a secondary antibody was used. The confocal laser scanning microscope was used for the examination. A series of images of optical sections was collected with a 20x objective at $3{\mu}m$ intervals throughout the depth of specimen. FITC fluerescence was registrated through a 488nm and 568nm excitation filter, and images were saved on optical disk. The stereoscopic images and three dimentionnal images were reconstructed by computer software, and then were analyzed. The results were as follows : 1. In normal control group, CGRP containing nerve fibers were coursed through the root with very little branching, and then formed a dense network of terminals in coronal pulp. 2. A slight increase in CGRP containing nerve fibers at 1 and 2day postinjury was noted subjacent to the injury site. In the 4day group, there were an extensive increase in the number of reactive fibers, followed by a partial return toward normal levels at 7~10 day postinjury, and return by 21days. 3. The sprouting of the CGRP containing nerve fibers was evident within 2day after dentinal injury, and by 4days there was a maximal increased, but was decreased at 7days and returned to normal 10~21 day postinjury. 4. In confocal laser scanning microscopic exammination, the distinct distribution pattern and sprouting reaction of CGRP containing nerve fibers were observed in stereoscopic images and three dimentional images. These results suggest that CGRP containing nerve fiber can be important role in the response to dental injury and pain regulation.