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Medicine

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

Published: April 29th, 2013

DOI:

10.3791/50331

1Department of Neurology, University of Michigan , 2Department of Internal Medicine, University of Michigan

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.

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.

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....

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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 temper.......

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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.......

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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.......

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This work was supported by National Institutes of Health Grants K08 NS061039-01A2, the Program for Neurology Research & Discovery, and The A. Alfred Taubman Medical Research Institute at the University of Michigan. This work used the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center, funded by National Institutes of Health Grant 5P90 DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases. The authors would like to thank Robinson Singleton and Gordon Smith (University of Utah) for their generous donation of human skin samples to support the initial development of the nociceptive biomarker i....

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
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

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