Skin Biopsy

Morphologic Assessment of Skin Innervation

Normal and neuropathic calf biopsy
Confocal images of calf skin with normal and severe neuropathy. Nerves appear green, basement membrane (BM) appears red, and capillaries (CAP) appear magenta. Nerve fibers extend from trunks (NT) in the dermis and branch to form the subepidermal neural plexus (SNP) from which arise fine epidermal nerve endings (ENFs). In normal healthy humans, many ENFs arise from a full SNP to innervate the epidermis. In skin biopsies from subjects with severe neuropathy, the epidermis is often devoid of nerve fibers and the SNP consists of just a few nerve fibers, as seen in the lower image. Scale bars = 100 µm.


Primary antibodies (& lectin) used for Immunofluorescent staining
  • Protein Gene Product 9.5 (PGP 9.5) for all nerve fibers
  • Collagen type IV (Col IV) for basement membrane at the interface of the epidermis and dermis, as well as on vasculature and sweat glands
  • CGRP, Substance P, and VIP for labeling nerve fibers containing these neuromodulatory neuropeptides
  • Tryptase for labeling mast cells
  • Biotinylated Ulex Europaeus Agglutinin 1 (Ulex) is a lectin used for labeling superficial epidermis and endothelial cells
Species specific secondary antibodies are then added to detect these primary antibodies and biotinylated lectin.
We have characterized and quantified ENFs from five body locations in both normal subjects and subjects who have diabetes mellitus.
Normal skin cross sections at multiple locations
Skin cross sections of moderate neuropathy at various locations
These images demonstrate the difference in ENF density at the five body locations from a single healthy control subject (Normal) and a single diabetic subject (Diabetic) with neuropathy. The ENF density of the healthy subject contrasts with that of the subject with diabetic neuropathy - showing minimal  or no ENFs in the hand, calf and thigh (distal sites) as opposed the forearm and thigh (more proximal sites) where ENF density is similar to healthy subjects. This illustrates the length dependent nature of diabetic neuropathy where a distal to proximal gradient is seen. Scale bar = 100 µm for all images.

Epidermal Nerve Fiber (ENF) Density Analysis

  1. Immunofluorescently stained nerve fibers & basement membrane are imaged using a spinning disk confocal microscope and attached CCD camera.
  2. Nerve fiber counting to determine ENF density is performed by tracing epidermal nerve fibers in confocal images using MBF Biosciences NeuroLucida software according to Nerve Counting Rules (see below).
  3. The automated counts are then confirmed by visual counting by a qualified neurologist who also assesses tissue morphology.

Links and Documents:

Skin Biopsy Process Overview

Nerve Counting Rules

Pathology and Quantitation of Cutaneous Innervation (Chapter 34 of Peripheral Neuropathy Edited by:Peter J. Dyck, M.D. - included with permission)


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The analysis of skin biopsy tissue samples is an internationally1 accepted method to assess neuropathy that is mainly based on quantification of unmyelinated nerve fibers in the superficial layer of the skin, the epidermis.1,2  Different methods for analysis of epidermal nerve fibers (ENFs) obtained by skin biopsy were independently developed by Kennedy Laboratory (KLAB) at the University of Minnesota3 and the Griffin Laboratory at Johns Hopkins University.4 KLAB's interests include the changed structure and density of cutaneous nerves, especially ENFs, associated with diabetes, cancer chemotherapy and aging.5,6  We have developed a strong collaboration with Dermatology in the field of 'NeuroDerm', especially the innervation of the scalp associated with a variety of clinical syndromes. 

Our laboratory uses immunofluorescent staining methods to localize a variety of nerve and tissue antigens to aid in the assessment of cutaneous and gastrointestinal innervation in biopsied tissue. 

We acquire confocal images and trace single ENFs from their penetration of the dermal-epidermal basement membrane throughout the epidermis to their endings; commonly near the epidermal surface under stratum corneum. Our interest for diagnostic purposes is the change of ENF density and morphology.

Analysis of Nerves in a Skin Biopsy

We developed methods to quantify small fiber peripheral neuropathy for diagnosis, especially diabetic and chemotherapy-induced neuropathy, and to determine if treatment of neuropathy objectively benefits the patient. The main negative symptoms of neuropathy are decreased sense of touch, often called numbness, pain, warmth or cold. Positive symptoms are paresthesias, dysesthesias, burning and coolness. The nerve endings for these sensations are in the superficial layers of the skin where many can be sampled by skin biopsy. A 3mm (about 3/8 in.) skin biopsy is about as big as the head of a match. It doesn't require sutures. We cut the biopsies into 60 to 110 µm thick sections, and label the nerves and surrounding tissue with fluorescent antibodies so that we can see them either in a fluorescent microscope, or in a confocal microscope. The nerves are traced and quantified by number, length and branching with a computer from the confocal sections.

1   Lauria G, Hsieh ST, Johansson O, Kennedy WR, Leger JM, Mellgren SI, Nolano M, Merkies ISJ, Polydefkis M, Smith AG, Sommer C, Valls-Solé J. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federations of Neurological Societies and the Peripheral Nerve Society. European Journal of Neurology. 2010;1-15. PMID:20642627.
2   Kennedy, W.R., Wendelschafer-Crabb, G., Polydefkis, M., McArthur, J., (2005) Pathology and Quantitation of Cutaneous Nerves. In: Dyck PJ, Thomas PK. eds. Peripheral Neuropathy 4th ed. Philadelphia: Saunders, 869-896. ADD LINK
3   Kennedy, W.R. and Wendelshafer-Crabb, G.: "The innervation of human epidermis." J. Neurol. Sci. 115: 184-190, 1993.
4   McCarthy BG, Hsieh ST, Stocks A, Hauer P, Macko C, Cornblath DR, Griffin JW, McArthur JC. Cutaneous innervation in sensory neuropathies: evaluation by skin biopsy.Neurology. 1995 45:1848-1855. PMID: 7477980
5   Kennedy, W.R., Wendelschafer-Crabb, G., Johnson, T. Quantitation of epidermal nerves in diabetic neuropathy. Neurology 47:1042-1048, 1996
6   Kennedy, W.R., Wendelschafer-Crabb, G. Use of the skin biopsy method. EEG and Clinical Neurophysiology Elsevier Co. Suppl.;50:553-559, 1999