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

Kim, Myung Kook

Abstract

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 micrometer thick sections were cut on cryostat. The rabbit antihuman CGRP was used as a primary antibody (1 : 2,000 in 0.01 M PBS at 4 degrees 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. Three-dimensional images were constructed from 16 serial images (each 1 micrometer thick) made by automatic optical sectioning. Unprocessed optical sections with an image size of 512X512 pixels and a pixel size of 0.2 micrometer 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 fluorescent 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.