Whole structural reconstruction and quantification of epidermal innervation through the suction blister method and skin-clearing technique

Abstract

Three-dimensional (3-D) analysis of intraepidermal nerve fibers (IENFs) is conducted to advance assessment methods for peripheral neuropathies and pruritic skin disorders. The skin-clearing technique was proven to be a reliable method for 3-D imaging of IENFs. Nonetheless, it still requires further improvement in the imaging process. The aim of this study was to standardize the 3-D evaluation method of IENFs and to suggest promising 3-D biomarkers for clinical application. A total of nine healthy individuals were prospectively enrolled. The newly adopted suction blister method was combined with the tissue-clearing technique. The detailed structure of the IENFs was reconstructed and quantified using the neuron tracing software. The suction blister method showed improved linear integrity of IENFs compared with those obtained from the previously used salt-split skin test. The 3-D parameters most significantly related to natural aging were the convex hull two-dimensional perimeter and the total length (both p = 0.020). The meaningful correlations were followed by total volume (p = 0.025), ends (p = 0.026), convex hull 3-D surface, and complexity (both p = 0.030). Thus, the 3-D parameters could be utilized as possible biomarkers to identify ambiguous pathologies of peripheral neuropathies and pruritic skin disorders.

Figure 1

Summarized processes of advanced skin-clearing protocol. The epidermis was separated from the dermis using the (a) suction blister method instead of (b) a previously adopted SSST. The separated epidermis subsequently proceeded to the cutaneous ACT-PRESTO procedures, including (c) fixation, (d) electrophoretic tissue clearing (ETC), and (e) RI matching. SSST Salt-split skin test, ACT Active clarity technique, PRESTO Pressure-related efficient and stable transfer of macromolecules into organs, RI Reflective index, RIMS Reflective index matching solution.

Figure 2

(a, b) Conventional two-dimensional (2-D) image of the intraepidermal nerve fibers (IENFs) and counting rules (Reproduced from Eur J Neurol 2005;12:747–758). (a) The punch-biopsied healthy human skin was cryo-sectioned with 80 μm thickness and immunostained with PGP9.5 (red) and type IV collagen (green) to specify cutaneous nerve fibers and dermo-epidermal junction (DEJ), respectively with a Plan-Apochromat 20 × /0.80 M27 lens (z-stack step: 1 μm). Scale bar, 50 μm. (a, b) The nerve fibers that definitely crossed the DEJ were regarded as significant data (white and black arrowheads) in quantifying intraepidermal nerve fiber densities (IENFDs). (c) Suction blister-based three-dimensional (3-D) image provided true volume information of IENFs in the significantly expanded area of skin (dimensions of x: 1023.51 μm; dimensions of y: 828.96 μm; dimensions of z: 113.50 μm) compared with the previous method, which was based on tissue section. The 3-D images were obtained with a 40 × HC PL APO CS2 40 × /1.30 lens (z-stack step: 0.35 μm). All 3-D images were obtained using 1.28 × zoom with a scan speed of 600 or 700 Hz and merged with multi-frame search mode. Scale bar, 100 μm. (d, e) The merged result was subdivided into 64 serial volume images with a confined XY size (x: 83.22 μm; y: 104.13 μm) using LAS X for effective identification of the origin and the course of each nerve fiber. Scale bar, 20 μm. epi epidermis, der dermis, PGP9.5 protein gene product 9.5, COLIV type IV collagen.

Figure 3

3-D analysis of intraepidermal nerve fibers (IENFs) using software designed for neuronal reconstruction and quantitative analysis, N360. (a) The volume information of IENFs obtained from a 34-year-old participant was (b) semi-manually reconstructed using the tree tracing with directional kernels method. The reconstructed information was utilized to provide quantitative parameters, including not only conventional numbers of nerve fibers, but also true 3-D data such as nodes, ends, length, volume, complexity, tortuosity, and various angles. The 3-D evaluation also provided the specific field of branched structures controlling a given amount of physical space represented as (c,d) convex hull 2-D area and (e,f) convex hull 3-D volume. Each IENF, such as the (c,e) light blue-colored and (d,f) dark blue-colored reconstructed structure was separately analyzed to provide detailed individual morphological changes according to the natural aging process. Scale bar, 20 μm.

Figure 4

The graphical presentation of age-related changes in previously well-known quantified parameters, (a,c) number of nerve fibers (Qty), and (b,d) nodes. (a,b) Each color in 2-D color maps reflected the degree of quantified results regarding higher values as bright yellow to lower one to dark blue. (a,b) The quantified results are presented as 3-D bar graphs, while the height of the bar shows the degree of parameters in each volume.

Figure 5

The graphical presentation of age-related changes in the new 3-D parameters, (a,e) convex hull 2-D perimeter, (b,f) total length, (c,g) volume, and (d,h) ends that showed the most significant correlations with natural aging. (a–d) Each color in 2-D color maps reflects the degree of quantified results regarding higher values as bright yellow to lower one to dark blue.