Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

Summary

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

Abstract

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy ^1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies ³. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.

In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy ⁴. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor ⁵. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

Introduction

At present, it is common practice for physicians to quantify intraepidermal nerve fiber densities, (IENFD) from skin punch biopsies, which can be used to diagnose small fiber neuropathies ^{3, 6-8}. Biopsies are taken from the distal leg (DL), 10 cm above the lateral malleolus, and the proximal thigh (PT), 20 cm below the anterior iliac spine ⁹. All IENF are labeled using protein gene product 9.5 (PGP), a pan neuronal marker ^10-12. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP staining with brightfield microscopy ⁶. Additionally, several research groups have used immunofl....

Protocol

Part A: Immunohistochemistry

Preparation of 96-well plate and prevention of background staining Punch skin biopsies are collected from human subjects and incubated for 12-24 hr in fixative solution (2% paraformaldehyde with 0.75 M L-Lysine solution (pH 7.4) and 0.05 mM sodium periodate) at 4 °C as previously described ⁸. Samples are then cryoprotected in phosphate buffered saline (PBS) with 20% glycerol at 4 °C for up to 1 week, embedded in mounting media optimal cutting temperature (OCT), then sectioned into 50 μm thick sections on a cryostat. The protocol described below is designed for 8 skin sections, the maximum numb....

Results

We applied the current protocol to study the morphology of IENF in PT and DL skin biopsies from patients with PN. The skin, from three subjects, was collected at the University of Utah to demonstrate the pathomorphology associated with PN. The subjects include: Case 1: a 51-year-old male with a history of PN of type 2 diabetes (duration: 14 months; pain score: 51); Case 2: a 56-year-old male with a history of PN of type 2 diabetes (duration: 108 months; pain score: 47); and Case 3: a 66-year-old male with a history of PN.......

Discussion

Measurement of IENFD has been widely used to determine the degree of peripheral neuropathies ^13,14. At present, the most commonly used protocol only measures the densities of nerve fibers that penetrate the basement membrane of the epidermis; it does not take into consideration axonal branching and/or morphological changes of the nerves. In addition, current IENFD analysis has not been shown to correlate IENFD with the presence of pain in PN ¹⁵.

We previously reported th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X PBS	Fisher Scientific	BP399-4	To make up 1X PBS
Image-IT FX Signal	Invitrogen	I36933	Image-IT
Protein Gene Product 9.5 (Polyclonal rabbit)	AbD Serotec	7863-0504	PGP
Tropomyosin Related-Kinase A (Polyclonal goat)	R&D Systems	AF1056	Trk A
Alexa Fluor 488 donkey α-rabbit	Invitrogen	A21206	AF488 donkey α-goat
Alexa Fluor 647 donkey α-goat	Invitrogen	A21447	AF647 donkey α-goat
Albumin, from Bovine Serum	Sigma-Aldrich	A7906-100	BSA
Triton X- 100	Sigma-Aldrich	T9284	TX-100
Non-calibrated Loop	LeLoop	MP 199025	inoculating Loop
96-well assay plate	Corning Incorporated	3603	Well plate
Prolong Gold antifade reagent with DAPI	Invitrogen	P36931	DAPI
Microscope Cover Glass 22x22 mm	Fisher Scientific	12-541-B	Coverslips
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15	Microscope Slides
Olympus Fluoview Laser Scanning Confocal Microscope	Olympus	FV500	Confocal Microscope
Optimum Cutting Temperature	Sakura	4583	OCT
Leica cryostat	Leica	CM1850	Cryostat