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

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.

Quantitative characteristics of intraepidermal nerve fibers by skin biopsy in healthy Chinese / 中华神经医学杂志

Objective To establish the quantitative standard of intraepidermal nerve fibers in healthy Chinese and provide reference basis for the early diagnosis of peripheral neuropathy through the large sample system of quantitative research by skin biopsy.Methods Adult skin samples of different anatomical locations,age and gender were obtained from 192 healthy volunteers who performed skin biopsies in our hospital from May 2009 to July 2013;they were divided into 6 groups according to different anatomic sites (distal upper arm,distal forearm,opisthenar,proximal thigh,distal crus and acrotarsium) and 4 groups according to the ages (patients under 20 years old,patients of 21-40 years old,patients of 41-60 years old and patients older than 61 years).Quantitative observation of the intraepidermal nerve fibers was performed by skin biopsy and immunohistochemical technique.Half males and half females were chosen.Results The comparison of intra epidermal nerve fiber density (IENFD) among thigh,leg,dorsum of foot and the comparison among upper arm,forearm,dorsum of hand showed statistical significance (P＜0.05).Along with the limbs part from proximal to distal,the IENFD in the upper and lower limbs both manifested a degressive tendency by degrees (normal reference value ranges of each anatomic site:upper arm＞320.00 fibers/mm2,forearm＞190.57 fibers/mm2,opisthenar＞184.37 fibers/mm2,thigh＞418.36 fibers/mm2,crus＞157.35 fibers/mm2,and acrotarsium＞ 140.00 fibers/mm2).The IENFD among patients under 20 years old,patients of 21-40 years old,patients of 41-60 years old and patients older than 61 years did not show statistically significant differences (P＞0.05).The IENFD between patients under 20 years old and patients older than 61 years showed statistical significance (P＜0.05).The IENFD in male and female adults did not show statistical significance (P＞0.05).Conclusions A significant difference of IENFD is observed among different anatomic sites.Ages has small effect on IENFD,and gender has no effect on IENFD.