The density and distribution of substance P-like immunoreactive (SP-LI) and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers in rat temporomandibular joint (TMJ) were investigated in whole-mount preparations and frozen sections by immunohistochemistry with the avidin-biotin-peroxidase complex method. Both types of immunoreactive nerves were observed primarily in the joint capsule, the peripheral articular disc, the synovial membrane, and the periosteum. The distribution of CGRP-LI nerves was similar to that of SP-LI nerves. The anterior portion of the joint capsule and disc was most densely innervated, followed by the posterior, lateral, and medial portions. In addition, CGRP-LI nerves were more numerous and more dense in immuno-intensity than SP-LI nerves. In the synovial membrane, many SP- and CGRP-LI nerves terminated in the subsynovial layer, but some branches extended into the superficial synovial lining layer close to the joint cavity. Immunolabeled nerves were prominently located in the disc attachment and peripheral portion of the disc, and occasional nerves were located in the dense collagenous disc band as an actual disc. However, no fibers were detected in the central disc band. Thus, most of the disc was not innervated by any nerves. The present study provides a morphological basis for the possible roles of neuropeptides in endocytosis by synoviocytes, regulation of blood flow in the synovial membrane, nociception mechanisms of the TMJ, and modulation of the inflammatory response in the TMJ.

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We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.