노화된 흰쥐 삼차신경절에서 Calcitonin Gene-Related Peptide 면역반응세포의 분포 및 형광강도에 관한 공초점레이저주사현미경적 연구

A Confocal Laser Scanning Microscopic Study on the Distribution and Fluorescent Intensity of Calcitonin Gene-Related Peptide Immunoreactive Cells in the Trigeminal Ganglion of Aged Rats

노화된 흰쥐 삼차신경절에서 calcitonin gene-related peptide (CGRP) 면역반응세포의 분포와 CGRP 면역반응세포의 형광강도를 알기 위해 대조군 (3개월)과 노화군 (36개월)으로 나누어 공초점레이저주사현미경으로 관찰하여 다음과 같은 결과를 얻었다. 1. 삼차신경절에서 CGRP 면역반응세포체의 수는 대조군이 28.6개이었고, 노화군이 19.4개 (P<0.001)이었다. 2. 광단층절편 (optical single sectioning)에서 삼차신경절에 발현하는 CGRP 면역반응세포체의 형광염색에 대한 강도는 대조군의 경우 적색 (높은 형광강도)이 제일 많았고, 백색 (중등도 형광강도), 회색 (낮은 형광강도)의 순이었으며, 노화군의 경우 백색(중등도 형광강도)이 제일 많았고, 회색 (낮은 형광강도), 적색 (높은 형광강도)의 순이었다. 3. 광연속절편 (optical serial sectioning, 1 μm 두께)에서 삼차신경절에 발현하는 CGRP 면역반응세포체의 형광염색에 대한 강도는 대조군 16개의 절편에서 형광강도가 높은 적색이 모두 관찰되었고, 노화군 16개의 절편중에서는 형광강도가 높은 적색이 일부분 또는 16개의 절편에서 작은 영역의 적색이 관찰되었다.

The purpose of this study was to investigate the distribution and fluorescent intensity of the calcitonin gene-related peptide (CGRP) -immunoreactive cell bodies in the trigeminal ganglion of aged rats by means of confocal laser scanning microscopy. Eight control (3 months) and 8 aged (36 months) Sprague-Dawley rats were used. Trigeminal ganglion was removed and immersed in the 4% paraformaldehyde in 0.01 M phosphate buffer (pH 7.4). Serial frozon 30 μm thick sections were cut on cryostat. The rabbit antihuman CGRP was used as a primary antibody (1 : 2,000 in 0.01M PBS at 4&#65533;C). The fluorescence isothiocyanate (FITC) -conjugated anti-rabbit IgG was used as a secondary antibody (1 : 1,000 in 0.01 M PBS). The slides were observed under a Carl Zeiss LSM410 confocal laser scanning microscopy. Samples were excited by a 488 nm argon laser, and the images were filtered by a longpass 515 nm filter. Threedimensional images were constructed from 16 serial images (each 1 μm thick) made by automatic optical sectioning. Unprocessed optical sections with an image size of 512×512 pixels and a pixel size of 0.2 μm were obtained and stored on a hard disk or optical disk. Color picture were printed on a video copy processor (Mitsubishi, Japan). The results were as follows; 1. The number of the CGRP-immunoreactive cell bodies in the trigeminal ganglion was 28.6 in the control rat and 19.4 in the aged rat. 2. In the optical single section, red (high fluorescent intensity) CGRP-immunoreactive cell bodies were the most abundant, followed by white (moderate fluorescent intensity), gray (low fluorescent intensity) in descending order in the control rat, whereas white CGRP-immunoreactive cell bodies were the most abundant, then gray, and red in desending order, in the aged rat. 3. In optical serial sections, red (high f luorescent intensity) CGRP-immunoreactive cell bodies were observed in all of the 16 sections of the control rat, whereas they were observed in a part of the 16 sections of the aged rat.