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

The overall goal of this procedure is to perform immunohistochemistry on human skin biopsy sections to study morphological changes of intra epidermal nerve fibers associated with painful neuropathy through three dimensional imaging. This is accomplished by first performing a three day floating section immunohistochemistry protocol on individual human skin biopsy sections. The second step is to image 50 micron thick skin sections using a confocal microscope.

Next, the flow view image files are run with MRS software to create a three dimensional movie file of the original immunofluorescent images. The final step is to use vdw software or similar to compress the movie file. Ultimately, three dimensional imaging results allow for the opportunity to study morphological changes associated with painful neuropathy.

At present intraop epidermal nerve fiber density is used as a quantitative method to diagnose neuropathy. Co labeling of these intradermal nerve fibers with the nociceptive biomarker has the potential to shed light on pain associated with this complication. The current two dimensional imaging protocol has multiple limitations, including the overlapping of nerve fibers.

In contrast, three dimensional imaging provides opportunity for further analysis of morphological changes associated with neuropathy such as axonal, swelling and blanching, as well as the measurements of axonal volume. It is important to note that measures must be taken in order to obtain quality images to generate meaningful three-dimensional animations That this method can provide insight into painful neuropathy. It can also be applied to other samples such as thick organ cultures or three dimensional cell cultures to examine and measure complex changes in three dimensional morphology.

After fixing and sectioning human skin biopsies as outlined in the written portion of this protocol label A 96 well plate to be used for staining as shown in this image. Then add 150 microliters of image ITFX signal into each well in row one of the labeled 96 well plate and pipette 150 microliters of one XPBS into all of the wells in row two and three. Use an inoculating loop to transfer a 50 millimeter section taken from the distal leg biopsy of a patient to one of the wells containing image.

It then transfer a 50 millimeter section taken from the proximal five of the same patient to the next well containing image. It repeat this procedure with the distal leg and proximal thigh sections from three further patients. Incubate the sections in, image it on a flat rocker for 30 minutes while biopsy sections are incubating in image it prepare a 5%blocking solution and vortex the solution until the BS A completely dissolves after the 30 minutes have elapsed.

Transfer the sections to the PBS in row two and rock for 10 minutes, followed by rinsing in the PBS in row three for 10 minutes. Again with rocking next pipette 150 microliters of 5%BS, A blocking solution into each well of row four and transfer the sections into these wells incubate sections in 5%BSA blocking solution for one to two hours at room temperature on a flat rocker. During this incubation, prepare 1%rinsing solution vortexing until the BSA completely dissolves and then dilute the primary antibodies to the required concentration in this solution.

Then add 150 microliters of diluted primary antibodies to the wells in row five of the 96 well plate. And after the incubation period has elapsed, transfer the sections from the blocking solution into the appropriate primary antibody wells. Next, seal the plate with param and aluminum foil to avoid drying and light exposure and incubate overnight at four degrees Celsius on a flat rocker the following day, add 150 microliters of 1%BSA rinsing solution into each well in row six, seven, and eight, and pass the sections through these washes for one hour with rocking it room temperature while ensuring that the plate is protected from light during each wash while the sections are incubating in the last rinse of 1%BSA rinsing solution dilute secondary antibodies in 1%BSA rinsing solution according to the instructions in the written portion of the protocols.

Once the incubation time has elapsed, add 150 microliters of diluted secondary antibodies into the designated wells of row nine of the 96. Well plate transfer the sections into these wells and cover the plate with paraform and aluminum foil and incubate overnight at four degrees Celsius on a flat rocker. The next day, add 150 microliters of 1%BSA rinsing solution into each well in row 10, 11, and 12, and rinse the sections in this solution for one hour each as before.

While biopsy sections are incubating in the last rinse of 1%BSA rinsing solution, prepare microscope slides by pipetting 50 microliters of 1%BSA rinsing solution on one slide at a time. Remove the section from the last rinse and place it in the 50 microliter drop of 1%BSA rinsing solution on the designated microscope slide. After optimizing the position of the section, remove excess 1%BSA rinsing solution with a bulb glass pipette taking precautions to avoid touching the specimen.

Place one drop of prolonged gold anti fade mounting reagent with a DPI near the biopsy on the microscope slide. Then take a 22 by 22 millimeter microscope glass cover slip and gently place it over the biopsy and drop of mounting reagent. Remove any air bubbles using a pipette tip and wipe away excess prolonged gold.

Antifa reagent. Repeat for each section and allow the microscope slides to dry overnight at room temperature in the dark the next day. Image fluorescent signals using a confocal microscope, an Olympus flow view 500 laser scanning confocal microscope with a 40 x oil immersion objective and zoom two times with flow view version 5.0 software is used here.

Use Alexa floor 4 88 and Alexa floor 6 47 to excite the 543 nanometer helium neon green laser and the 633 nanometer helium neon red laser respectively. Set the confocal apertures for each detector at 400 microns to enhance signals. Sequential scans should be taken at a resolution of 10 24 by 10 24.

To maximize signal separation, capture three dimensional Z series using 1.2 micron Z step intervals based on the optimal scan unit, calculated by the flow view software with kalman two frames averaging first open flow view files in MRSX 64 software or similar. To visualize the 3D image sets, adjust the display as needed to enhance contrast of the nerve specific signal. Create a surface to visualize the dermal epidermal boundary.

Use the contour tool in draw mode to draw the boundary. Assign a semi-transparent surface to the boundary in order to see the underlying fluorescent signals and capture still images of the 3D fluorescent signals. To create snapshot images of the 3D movie, use the animation feature to create a 3D movie set animation to create movies consisting of 200 frames, animated at 15 frames per second.

Save each movie as a raw A VI file compress the final A VI movie with virtual dub software or similar. This video shows 3D images of nociceptive intra epidermal nerve fibers. This first image shows PGP positive intra epidermal nerve fibers labeled green, representing total intra epidermal nerve fibers, and also track a positive intra epidermal nerve fibers labeled red, representing nociceptive intra epidermal nerve fibers.

And finally emerged image demonstrating PGP positive nociceptive intra epidermal nerve fibers in a skin sample from case one, the scale bar represents 20 microns. The following video shows 3D images of axonal swellings in intra epidermal nerve fibers of painful neuropathies. Axonal swellings indicated by arrowheads can be seen along the intra epidermal nerve fibers labeled by PGP with a green signal within the subdermal plexus.

In a skin sample from case two, the scale bar represents 10 microns. This 3D image shows axonal branching in intra epidermal nerve fibers of painful neuropathy. Axonal branching indicated by arrowheads can be seen along the intra epidermal nerve fibers labeled by PGP with a green signal in a skin sample from case three.

The scale bar represents 20 microns Once mastered, this technique can be completed over four days, three and a half hours each day for immunohistochemistry and 35 minutes per sample for confocal imaging and 3D animation. While attempting this procedure, it's important to remember to properly transfer and mount tissues, taking precautions to avoid ripping or tearing the specimen samples Following this procedure. Other methods like three dimensional tric analysis for assessment of cutaneous innovation can be performed in order to answer additional questions.

Like, does the branching of abdominal nerve fibers play an important role in the development of painful neuropathy?