HE Wei, BAI Wanzhu, WANG Xiaoyu, SU Yangshuai, ZHANG Xiaoning, JING Xianghong, ZHU Bing

（Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700） 

Abstract: Objective: To investigate the characteristics of the active substances of the nerve fibersinnervating the auricular concha in the rats.  Methods:  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  via  transcardial  perfusion.  Then  the  tissues  of  the  auricular  concha  were collected.  Following  perfusion–fixation,  the  collected  tissues  were  post-fixed  in  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™,  120  mL,  Thermo  Scientific,  USA),  frozen,  and  cut  into  20μm  sections.  Then immunofluorescence  staining  techniques  were  performed  to  investigate  the  expression  of calcitonin-gene-related peptide  (CGRP),  Tyrosine  Hydroxylasen  (TH),  and  Vanilloid  Receptor Subtype 1 (VR1) ofthe sections. The nucleus was labelled with DAPI. Slides were recorded with confocal  imaging  system  (FV1200,  Olympus,  Japan)  and  analyzed  by  the  Olympus  Image Processing Software by an investigator. Approximately 4 randomized sections from each ratswere counted  for  the  sum  length  of  positive  nerve  fibers,  and  the  average  fluorescent  intensity. All immuno histochemistry for each staining combination was performed at the same time to ensure staining  consistency.  Results:  The  immunopositive  expression  of  VR1,  TH  and  CGRP  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  than  the  length  of  the  TH  or  VR1  positive  nerve  (P<0.01).  There  were  no  significant difference between the length of the TH and the VR1 positive nerve (P>0.05). As to the cutaneous and subcutaneous tissue dorsal to the auricular cartilage: the length of the VR1 or theTH positive nerve  was  longer  than  that  of  the  CGRP  positive  nerve  respectively  (P<0.01). There  were  no significant difference between the length of the VR1 and the CGRP positive nerve (P>0.05). The average  fluorescent  intensity  of VR1  the  CGRP  positive  nerve  was  more  than  that  of  the TH positive nerve (P<0.01). The average fluorescent intensity of VR1 positive nerve was more than that of the CGRP positive nerve (P<0.01). Conclusion: There were CGRP, VR1 and TH positive nerve fibers in the auricular concha of rats. The distribution  and the amount of the CGRP, VR1 and TH positive nerve fibers were different, and the expression of VR1 was the most.

Key words: Auricular acupuncture, Immunohistochemistry, CGRP, TH, VR1