<p style="text-align: center;">HE Wei, BAI Wanzhu, WANG Xiaoyu, SU Yangshuai, ZHANG Xiaoning, JING Xianghong, ZHU Bing</p><p style="text-align: center;">（Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700）&nbsp;</p><p style="text-align: justify;"><strong>Abstract</strong>: <strong>Objective</strong>: To investigate the characteristics of the active substances of the nerve fibersinnervating the auricular concha in the rats.&nbsp; Methods:&nbsp; Six Sprague Dawley rats, weighing 180-220 g, were introduced. The rats were fixed with 0.1 M phosphate-buffered saline (PBS) and 4% paraformaldehyde&nbsp; via&nbsp; transcardial&nbsp; perfusion.&nbsp; Then&nbsp; the&nbsp; tissues&nbsp; of&nbsp; the&nbsp; auricular&nbsp; concha&nbsp; were collected.&nbsp; Following&nbsp; perfusion–fixation,&nbsp; the&nbsp; collected&nbsp; tissues&nbsp; were&nbsp; post-fixed&nbsp; in&nbsp; 4% paraformaldehyde at 4 °C for 2 hours and cryoprotected in phosphate-buffered 20% sucrose at 4 °C for 24 hours. Following post-fixation, the tissue was embedded in artificial medium (Shandon Cryomatrix™,&nbsp; 120&nbsp; mL,&nbsp; Thermo&nbsp; Scientific,&nbsp; USA),&nbsp; frozen,&nbsp; and&nbsp; cut&nbsp; into&nbsp; 20μm&nbsp; sections.&nbsp; Then immunofluorescence&nbsp; staining&nbsp; techniques&nbsp; were&nbsp; performed&nbsp; to&nbsp; investigate&nbsp; the&nbsp; expression&nbsp; of calcitonin-gene-related peptide&nbsp; (CGRP),&nbsp; Tyrosine&nbsp; Hydroxylasen&nbsp; (TH),&nbsp; and&nbsp; Vanilloid&nbsp; Receptor Subtype 1 (VR1) ofthe sections. The nucleus was labelled with DAPI. Slides were recorded with confocal&nbsp; imaging&nbsp; system&nbsp; (FV1200,&nbsp; Olympus,&nbsp; Japan)&nbsp; and&nbsp; analyzed&nbsp; by&nbsp; the&nbsp; Olympus&nbsp; Image Processing Software by an investigator. Approximately 4 randomized sections from each ratswere counted&nbsp; for&nbsp; the&nbsp; sum&nbsp; length&nbsp; of&nbsp; positive&nbsp; nerve&nbsp; fibers,&nbsp; and&nbsp; the&nbsp; average&nbsp; fluorescent&nbsp; intensity. All immuno histochemistry for each staining combination was performed at the same time to ensure staining&nbsp; consistency.&nbsp;<strong> Results</strong>:&nbsp; The&nbsp; immunopositive&nbsp; expression&nbsp; of&nbsp; VR1,&nbsp; TH&nbsp; and&nbsp; CGRP&nbsp; was observed in the cutaneous and subcutaneous tissue of the concha of the rat. As to the cutaneous and subcutaneous tissue ventral to the auricular cartilage: the length of the VR1 positive nerve was longer&nbsp; than&nbsp; the&nbsp; length&nbsp; of&nbsp; the&nbsp; TH&nbsp; or&nbsp; VR1&nbsp; positive&nbsp; nerve&nbsp; (P&lt;0.01).&nbsp; There&nbsp; were&nbsp; no&nbsp; significant difference between the length of the TH and the VR1 positive nerve (P&gt;0.05). As to the cutaneous and subcutaneous tissue dorsal to the auricular cartilage: the length of the VR1 or theTH positive nerve&nbsp; was&nbsp; longer&nbsp; than&nbsp; that&nbsp; of&nbsp; the&nbsp; CGRP&nbsp; positive&nbsp; nerve&nbsp; respectively&nbsp; (P&lt;0.01). There&nbsp; were&nbsp; no significant difference between the length of the VR1 and the CGRP positive nerve (P&gt;0.05). The average&nbsp; fluorescent&nbsp; intensity&nbsp; of VR1&nbsp; the&nbsp; CGRP&nbsp; positive&nbsp; nerve&nbsp; was&nbsp; more&nbsp; than&nbsp; that&nbsp; of&nbsp; the TH positive nerve (P&lt;0.01). The average fluorescent intensity of VR1 positive nerve was more than that of the CGRP positive nerve (P&lt;0.01).<strong> Conclusion</strong>: There were CGRP, VR1 and TH positive nerve fibers in the auricular concha of rats. The distribution&nbsp; and the amount of the CGRP, VR1 and TH positive nerve fibers were different, and the expression of VR1 was the most.</p><p style="text-align: justify;"><strong>Key words</strong>: Auricular acupuncture, Immunohistochemistry, CGRP, TH, VR1</p><p><br/></p>