Tissue-clearing technique and cutaneous nerve biopsies: Quantification of the intraepidermal nerve-fiber density using active clarity technique-pressure related efficient and stable transfer of macromolecules into organs

Dai Hyun Kim, Se Jeong Lee, Eunsoo Lee, Ji Hyuck Hong, Soo Hong Seo, Hyo Hyun Ahn, Byung Jo Kim, Woong Sun, Im Joo Rhyu

Research output: Contribution to journalArticle


Background and Purpose Cutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies. Methods Five healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD). Results The mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm2, respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm2, respectively, and 3.06, 2.87, and 47.21 fibers/mm2, respectively, in the patients with PHN. Conclusions This study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

Original languageEnglish
Pages (from-to)537-544
Number of pages8
JournalJournal of Clinical Neurology (Korea)
Issue number4
Publication statusPublished - 2019 Oct



  • Intraepidermal nerve-fiber density
  • Peripheral neuropathy
  • Tissue-clearing and labeling technique
  • ‌Cutaneous nerve biopsy

ASJC Scopus subject areas

  • Neurology
  • Clinical Neurology

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