Tissue-Clearing Technique and Cutaneous Nerve Biopsies: Quantifcation of the Intraepidermal Nerve-Fiber Density Using Active Clarity Technique-Pressure Related Efcient and Stable Transfer of Macromolecules Into Organs
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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 fbers. 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-fber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. Te conventional methods of indirect immunofuorescence (IF) and bright-feld immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efcient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fber density (IENFD). Results Te mean IENFD values obtained by IF, bright-feld IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fbers/mm 2 , respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fbers/mm 2 , respectively, and 3.06, 2.87, and 47.21 fbers/mm 2 , respectively, in the patients with PHN. Conclusions Tis study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fbers but also yielded reliable quantitative IENFD data. Quantifcation of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.
Abstract
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 fbers. 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-fber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. Te conventional methods of indirect immunofuorescence (IF) and bright-feld immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efcient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fber density (IENFD). Results Te mean IENFD values obtained by IF, bright-feld IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fbers/mm 2 , respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fbers/mm 2 , respectively, and 3.06, 2.87, and 47.21 fbers/mm 2 , respectively, in the patients with PHN. Conclusions Tis study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fbers but also yielded reliable quantitative IENFD data. Quantifcation of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.
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