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.

Establishing mouse model of type 2 diabetes peripheral neuropathy and measuring its intra-epidermal nerve fiber density / 临床麻醉学杂志

Objective To establish mouse model of type 2 diabetes peripheral neuropathy and measure its intra-epidermal nerve fiber density (IENFD).Methods Male C57BL6 mouse were ran-domly divided into four groups:group HS (n =6):high-fat diet+single streptozotocin intraperitoneal injection (120 mg/kg);group H (n =6):high fat diet+buffer injection;group S (n =6):standard chow diet+single streptozotocin intraperitoneal injection (120 mg/kg);group C (n = 6 ):standard chow diet+buffer injection.The 24th week was the end point of the experiment,and random glucose, homeostasis model assessment of insulin resistance (HOMA-IR),mechanical threshold,and IENFD were measured.Results Group HS had significantly higher random glucose and HOMA-IR than other groups (P <0.01),had significantly lower mechanical threshold than other groups (P <0.05), had significantly lower IENFD than groups S and C at 24th week (P <0.05 );group H had signifi-cantly higher random glucose and HOMA-IR than group C at 24th week (P <0.01),had no signifi-cant difference in mechanical threshold compared with group S and group C,and had no significant difference in IENFD compared with group HS.Conclusion A mouse model of type 2 diabetes periph-eral neuropathy was successfully established,and the IENFD was found to be decreased significantly.

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.

Changes in corneal nerve fiber length and density before and after eye opening in mouse and its significance / 中华实验眼科杂志

Background Recently,there were many studies on corneal innervations during mammalian development.However,there were fewer studies on discussing corneal innervations before and after mouse eye openings.Objective The present study was to investigate the change in the regulation of corneal nerve fiber length and density before and after mouse eye openings to offer a basis for clinical research in human.Methods Thirty SPF C57BL/6 mice were divided into postnatal 1 day(P1 d),P7 d,P13 d(1 day before eye opening),P14 d(eye halfopened),P17 d(1 day after eye opening)and P23 d(7 day after eye opening)groups,with 5 mice and 10 eyes for each group.Entire corneal stretches were prepared and immunostaining with an anti-neuron-specific β-Ⅲ tubulin antibody was performed to label the corneal nerve fibers.Confocal microscopic pictures from the corneal dorsal-nasal region (DN),dorsal-temporal(DT),ventral-nasal region(VN)and ventral-temporal(VT)were taken using Delta Vision Core.From these pictures,the mouse corneal area,total length and density of nerve fibers in the 4 regions were calculated.The use of the animals complied with Statement of ARVO.Results Corneal areas of P1 d,P7d,P13 d,P14 d,P17 d and P23 d mice were(0.404±0.007),(1.362±0.154),(1.573±0.080),(1.603±0.046),(1.847±0.052),(2.445±0.798)mm2,respectively ; the total lengths of nerve fibers were(3.718±1.044),(19.065±3.350),(23.687±0.907),(27.309±2.477),(31.989±3.976),(41.214±1.573)mm,respectively ; the densities of nerve fibers were(9.592±1.138),(14.506±1.908),(15.088±1.241),(16.772±1.897),(16.821±2.102),(17.660±1.216)mm/mm2,respectively,all showing significant increases with age(F =22.906,P =0.000 ; F =0.424,P =0.000 ; F =2.375,P=0.000).A positive correlation of the increasing corneal areas and increasing lengths of nerve fibers was found(r=0.983,P＜0.01).Nerve fiber densities in the four corneal regions significantly increased with age(DN region:F =0.159,P =0.000 ; DT region:F =2.1 72,P =0.001 ; VN region:F =1.998,P =0.000 ; VT region:F=2.352,P=0.000).From P13 d to P14 d,the corneal nerve fiber densities in the DN region decreased by 6.0％ without significant difference(t =0.589,P =0.572); and the corneal nerve fiber densities in the DT region,VN region and VT region decreased by 4.6％,5.5％ and 0.1％,respectively,without significant difference from P14 d to P17 d(t=0.549,P=0.596;t=0.701,P=0.501 ;t=-0.100,P=0.919).Conclusions The development of nerve fibers in the whole cornea or the four corneal regions is influenced by eye opening in mouse to various extents.From P13 d to P14 d,the corneal nerve fiber densities in the DN region decreased by 6.0％ without significant difference.From P14 d to P17 d,the corneal nerve fiber densities in the DT region,VN region and VT region decrease by 4.6％,5.5％ and 0.1％,respectively,without significant difference.Afterwards,the growth of nerve fibers increased in pace and the growth rate is recovered.