We are developing new methods to diagnose minimal nerve injury and neuropathy, especially diabetic neuropathy, and to determine if treatment of neuropathy objectively benefits the patient. The main symptom of neuropathy is sensory loss, usually called numbness: decreased ability sense touch, pain, warmth or cold. The nerve endings for these sensations are in the superficial layers of the skin. We sample these nerves by skin biopsy. A skin biopsy is about as big as the head of a match. It doesn't require sutures. We label the nerves with fluorescent antibodies so that we can see them either in a fluorescent microscope, or in a confocal microscope. The nerves are counted, then measured with a computer.

This is a section of a biopsy captured on a confocal microscope. The nerves, shown in green, almost reach the surface of the epidermis. Blood vessels are shown in red.

The Biopsy:

Obtaining a biopsy is a simple procedure. The site is anesthetized with lidocaine and a 2 to 3mm punch biopsy instrument is used to extract the skin sample. The subject feels a slight pressure, but no pain. The sample is removed and preserved in a fixative. This wound does not require a suture. Antibiotic ointment and a band-aid are sufficient.

Immunofluorescent Staining:

Nerves, capillaries, and basement membrane are localized by immunohistochemistry in our laboratory. A specific antibody binds to the protein. A labeled antibody is applied to the first antibody to make the complex visible in a fluorescent microscope. By using different antibodies, labeled with distinct fluorescent dyes, up to three times the structures can be examined in the same section.

After the slides are stained, we view them with a fluorescent microscope. It has a low light intensity camera on top that can project its image either to a video recorder for storage, to a monitor for group viewing, or to a computer for image analysis.

Confocal Microscopy:

A lot of our work is based on confocal microscopy where we collect 16 to 60 images that are all in focus. Visualization of triple staining is possible. And all images are stored digitally. In the diagram, the laserbeam reflects down through the objective lens to the section. Photons from in-focus, fluorescent compounds reach the detector through a narrow aperture at the top. Out of focus photons strike the edge of the aperture. This results in sharply focused images of very thick sections. The focused laser beam is scanning the tissue to create an optical section. It is refocused deeper for each optical section. Usually, 16 to 60 optical sections 2um thick images are collected. This is called a z-series. The entire z-series can be projected to give a 3-dimensional image of the relationship between nerves and adjacent tissue structures.

Quantification of Nerves in Sweat Glands:

Confocal images are obtained of sweat glands triple-stained to visualize its sweat tubules, capillaries, and its dense nerve supply. Using specialized computer software on a Silicon Graphics workstation, the confocal z-series can be converted into a layer of polygons of known size. From this, a 3D reconstruction of the sweat gland can be constructed. From this reconstruction, we can derive surface areas and volumes of the capillaries and sweat tubules, and most importantly, of the nerves themselves. * Click on the image at the right to see an animation of the 3D model.

Quantification of Nerves in the Epidermis:

This is a 3D view of nerves rising through the epidermis to the surface of the skin.

We use the Neurolucida software package from MicroBrightField to focus through the confocal images and trace the nerves in three dimensions (as shown in the diagram below).

These nerve tracings are combined into a model from which number of nerve structures, three dimensional length of each structure and number of branch points in each structure are derived. After the nerves are traced, this model can be visualiezed from any perspective.

The Image Volumes software package is used to measure volume of epidermis. The epidermis of each confocal image is first traced and filled, and these tracings are combined to give total epidermal volume.

University of Minnesota Medical School
Department of Neurology
Last updated: January 14, 1998
Webmaster: eric@kennedysg.med.umn.edu