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㎛ 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℃, and placed into biotinylated antirabbit Ig G as a
secondary anti body with dilution of 1:2000 in 0.01M PB and incubated in
ABC(avidinbiotin complex). The peroxidase reaction was visualized by incubating the
sections in 0.05% 3.3 diaminobenzidine tetrahydrochloride containing
0.02%jH2O2.
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㎛ 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 analized.
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 containg nerve fiber canbe important role in the
response to dental injury and pain regulation.
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