Tissue Clearing for Three-dimensional Visualization of The Neurovascular Network in The Whole Mount Auricular Skin
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Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China

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This study was supported by grants from the CACMS Innovation Fund (CI2021A03407), The National Natural Science Foundation of China (82004492), and the Fundamental Research Funds for the Central Public Welfare Research Institutes of China (ZZ14-YQ-032).

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    Abstract:

    Objective To study the spatial correlation of the nerve fibers and blood vessels in the auricular skin by using the combination of histological staining technique and tissue clearing strategy.Methods The anterior and posterior auricular skins were carefully peeled away from the intervening cartilage and the auricular nerve fibers and blood vessels were directly immunofluorescent stained with protein gene product 9.5 (PGP 9.5) and phalloidin followed with tissue clearing treatment. After that, these auricular skins were mounted on the microscope slide in the whole mount pattern and examined under an epifluorescence microscope and a laser scanning confocal microscope.Results It was shown that the PGP 9.5+ nerve fibers aligning with phalloidin-labeled blood vessels ran from the base part of auricle to its peripheral region, forming the neurovascular network in the auricular skin. Beyond the conventional immunofluorescence staining, the additional tissue clearing treatment demonstrated the auricular nerve fibers and blood vessels better in morphological detail.Conclusion From the perspective of methodology, tissue clearing technique enhance the visualization of the immunofluorescence labeling within the auricular skin, which should be an effective approach for insight into the auricular neurovascular network in a three-dimensional view under the physiological and pathological conditions.

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ZHANG Kai-Wen, GUO Ya-Ting, SU Yu-Xin, WANG Yu-Qing, CUI Jing-Jing, ZHANG Jian-Liang, WANG Jia, BAI Wan-Zhu. Tissue Clearing for Three-dimensional Visualization of The Neurovascular Network in The Whole Mount Auricular Skin[J]. Progress in Biochemistry and Biophysics,2023,50(6):1473-1479

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History
  • Received:June 14,2022
  • Revised:March 31,2023
  • Accepted:July 25,2022
  • Online: June 20,2023
  • Published: June 20,2023