This work was supported by National Institutes of Health Grants K08 NS061039-01A2, the Program for Neurology Research &amp; 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 immunohistochemistry technique.

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 &deg;C as previously described 8. Samples are then cryoprotected in phosphate buffered saline (PBS) with 20% glycerol at 4 &deg;C for up to 1 week, embedded in mounting media optimal cutting temperature (OCT), then sectioned into 50 &mu;m thick sections on a cryostat. The protocol described below is designed for 8 skin sections, the maximum number of skin sections possible to undergo free-floating immunohistochemistry in one 96-well plate.
DAY 1:
1. Prevention of Non-specific Immunoreactivity in the Stratum Corneum
<ol>
 <li>Label 96-well plate as shown in Figure 1.</li>
 <li>Add 150 &mu;l of Image-IT FX Signal (Image-IT), effective for blocking background staining 11,12, into each well in Row 1 of the 96-well plate.</li>
 <li>Add 150 &mu;l of 1X PBS into each well in Row 2 and 3 of the 96-well plate.</li>
 <li>Acquire two 50 &mu;m sections per patient: one section from the biopsy taken at DL, one section from the biopsy taken at PT. An inoculating loop (LeLoop) is used to transfer sections from one rinse to the next to avoid morphological damage. Gently transfer each 50 &mu;m section, using an inoculating loop, into an individual well of Row 1, containing Image-IT. Incubate sections in Image-IT for 30 min at RT on a flat rocker.</li>
 <li>Rinse sections twice with 1X PBS (Rows 2 and 3) for 10 min at RT. Place well plate on a flat rocker between rinses.</li>
</ol>
2. Preparation of 5% BSA Blocking Solution and 1% Rinsing Solution
<ol>
 <li>While biopsy sections are incubating in Image-IT, prepare 5% blocking solution (5% BSA, 0.3% TX-100, 0.1 M PBS- Vortex solution until BSA completely dissolves).</li>
 <li>Add 150 &mu;l of 5% BSA blocking solution into each well of Row 4.</li>
 <li>Using an inoculating loop, transfer sections into individual wells of 5% BSA blocking solution. Incubate sections in 5% BSA blocking solution for 1-2 hr at RT on a flat rocker.</li>
</ol>
3. Preparation of 1% BSA Rinsing Solution and Dilution of Primary Antibodies
<ol>
 <li>While sections are incubating in 5% BSA blocking solution, prepare 1% rinsing solution (1% BSA, 0.3% TX-100, 0.1 M PBS- Vortex solution until BSA completely dissolves).</li>
 <li>While sections are incubating in 5% BSA blocking solution, dilute primary antibodies in 1% rinsing solution.
 <ol>
 <li>For eight sections (8 x 150 &mu;l = 1,200 &mu;l) a total of 1,200 &mu;l is needed. The primary antibodies are made up in 1,500 &mu;l (about 20% extra volume).</li>
 <li>Dilutions of the primary antibodies: PGP, 1:500; Trk A, 1:500 in 1% BSA rinsing solution.</li>
 </ol>
 </li>
</ol>
4. Incubation of Sectioned Biopsies in Primary Antibody
<ol>
 <li>Add 150 &mu;l of diluted primary antibodies into the designated wells of the 96-well plate (Row 5).</li>
 <li>Transfer sections from 5% blocking solution (Row 4) into the designated primary antibody wells (Row 5).</li>
 <li>Seal the 96-well plate with parafilm and aluminum foil to avoid drying and light exposure.</li>
 <li>Incubate the 96-well plate O/N at 4 &deg;C on a flat rocker.</li>
</ol>
DAY 2:
5. Rinse Biopsies in 1% BSA Rinsing Solution
<ol>
 <li>Add 150 &mu;l of 1% BSA rinsing solution into each well in Row 6, 7, and 8.</li>
 <li>Rinse sections three times with 1% BSA rinsing solution (Rows 6, 7, and 8) for 1 hr each time at RT. Cover well plate with aluminum foil and place on flat rocker between rinses.</li>
</ol>
6. Dilution of Secondary Antibodies
<ol>
 <li>While sections are incubated in the last rinse of 1% BSA rinsing solution (Row 8), dilute secondary antibodies in 1% BSA rinsing solution:
 <ol>
 <li>For eight sections (8 x 150 &mu;l = 1,200 &mu;l) a total of 1,200 &mu;l is needed. The secondary antibodies are made up in 1,500 &mu;l (about 20% extra volume).</li>
 <li>Dilutions of the secondary antibodies: for PGP (Alexa Fluor 488 donkey anti-rabbit, 1:250), for Trk A (Alexa Fluor 647 donkey anti-goat, 1:250) in 1% BSA rinsing solution.</li>
 </ol>
 </li>
</ol>
7. Incubation of Sectioned Biopsies in Secondary Antibody
<ol>
 <li>Add 150 &mu;l of diluted secondary antibodies into their designated wells of the 96-well plate (Row 9).</li>
 <li>Transfer sections from 1% BSA blocking solution (Row 8) into secondary antibody well (Row 9).</li>
 <li>Seal the 96-well plate with parafilm and aluminum foil to avoid drying and light exposure.</li>
 <li>Incubate sections in the designated secondary antibodies O/N at 4 &deg;C on a flat rocker.</li>
</ol>
8. Rinse Biopsies in 1% BSA Rinsing Solution
<ol>
 <li>Add 150 &mu;l of 1% BSA rinsing solution into each well in Row 10, 11, and 12.</li>
 <li>Rinse sections three times with 1% BSA rinsing solution (Rows 10, 11, and 12) for 1 hr each time at RT. Cover well plate with aluminum foil and place on a flat rocker between rinses.</li>
</ol>
9. Preparation of Microscope Slides and Mounting Sectioned Biopsies
<ol>
 <li>While biopsy sections are incubating in the last rinse of 1% BSA rinsing solution (Row 12), prepare microscope slides.</li>
 <li>Place 50 &mu;l of 1% BSA rinsing solution on one slide at a time.</li>
 <li>Remove sections from 1% BSA rinsing solution (Row 12), and place it in the 50 &mu;l 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 bulbed glass pipette, taking precautions to avoid touching the specimen.</li>
</ol>
NOTE: Make sure section is not folded over; the specimen should be flat against the surface of the microscope slide.
<ol start="4">
 <li>Place 1 drop of Prolong Gold antifade mounting reagent with DAPI near the biopsy on the microscope slide. Take a 22x22 mm microscope glass coverslip and gently place it over biopsy and a drop of Prolong Gold antifade reagent with DAPI drop. Repeat for each section.</li>
 <li>Let microscope slides dry O/N at RT in dark.</li>
</ol>
NOTE: Remove any air bubbles using a pipette tip. Wipe away excess Prolong Gold antifade reagent.
Part B: Confocal Imaging
10. Confocal Imaging
<ol>
 <li>Image fluorescence signals using an Olympus FluoView 500 laser scanning confocal microscope with a 40X oil-immersion (1.3 NA) objective and zoom two times with FluoView version 5.0 software.</li>
 <li>Use Alexa Fluor 488 and Alexa Fluor 647 to excite the 543-nm HeNe green laser and the 633-nm HeNe Red laser, respectively. The Alexa Fluor 488 signal is represented by a green look up table (LUT) and the Alexa Fluor 647 by a red LUT.</li>
 <li>Set the confocal apertures for each detector at 400 &mu;m to enhance signals. Sequential scans should be taken at a resolution of 1,024 x 1,024 to maximize signal separation.</li>
 <li>Capture three-dimensional z-series using 1.2 &mu;m z-step intervals (based on the optimal scan unit calculated by the FluoView software) with Kalman averaging (two frames).</li>
</ol>
Part C: Three-dimensional Visualization and Animation
11. Three-dimensional (3D) Visualization and Animation
<ol>
 <li>Open Fluoview files in Imaris x64 software (version 7.3, Bitplane AG) to visualize the 3D image sets.</li>
 <li>Adjust display as needed to enhance contrast of the nerve specific signal.</li>
 <li>Create a surface to visualize the dermal-epidermal boundary. Use the Contour tool in draw mode to draw the boundary.</li>
 <li>Assign a semi-transparent surface to the boundary in order to see the underlying fluorescent signals.</li>
 <li>Capture still images of the 3D fluorescent signals to create Snapshot images of the 3D movie.</li>
 <li>Use the Animation feature to create a 3D movie. The images can be rotated 360 degrees several times to show each signal separately and merged (Figures 2, 3, and 4). Set animation to create movies consisting of 200 frames animated at 15 frames per second. Save each movie as a raw AVI file.</li>
 <li>Compress the final AVI movie with VirtualDub software (version 1.9.4).</li>
</ol>

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V., Sommer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+validity+of+epidermal+nerve+fiber+densities+in+painful+sensory+neuropathies.">Diagnostic validity of epidermal nerve fiber densities in painful sensory neuropathies.</a> Muscle Nerve. 37 (1), 50-60 (2008).</li><li>Casanova-Molla, J., Morales, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+fluorescence+quantitation+provides+a+new+approach+to+assess+cutaneous+innervation.">Axonal fluorescence quantitation provides a new approach to assess cutaneous innervation.</a> J. Neurosci. 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No conflicts of interest to declare.

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...

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 immunofluorescent protocols for PGP immunohistochemistry 7-9. Small fiber neuropathy is commonly associated with neuropathic pain. In order to further understand the role of IENF essential for pain processing, we developed a technique to co-label total IENF with fibers that generate pain. Nociceptive IENF, specifically A&delta; and C fibers, can be studied through the co-labeling of IENF with PGP and the nociceptive marker, tropomyosin-receptor-kinase A (Trk A) 5. Trk A is the high affinity receptor for nerve growth factor that is essential for the development of nociception. The Trk A-positive nociceptive nerve fibers are peptidergic fibers that express substance P (SP) and calcitonin gene related peptide (CGRP). Previously, Lauria and colleagues applied the double-labeling technique to study PN, co-labeling PGP-positive IENF with a nociceptive marker 10. In our previous study, we demonstrated that Trk A-positive IENF, but not Trk A-negative IENF, were upregulated in an animal model of painful diabetic neuropathy 5. This co-labeling technique provides the ability to compare quantification of nociceptive IENFD to total IENFD and the ability to study morphological changes associated with PN. The capability to visualize nociceptive IENF and compare quantification of total IENFD to nociceptive IENFD could provide objective evidence for the presence of pain, and possibly insight into the severity of pain associated with PN. This technique is also applicable to skin of animal models. In comparison to previous studies, the current protocol describes methods for 3D image analysis, creating the opportunity to avoid errors that could occur in 2D image analysis.

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

three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies

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.

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...

Watch this Scientific Journal Video about Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies at JoVE.com

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

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. ...

three-dimensional-imaging-nociceptive-intraepidermal-nerve-fibers

Research

JoVE Journal

Medicine

13.1K Views.  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.

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

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Neuroscience

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Biology

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 &#181;m from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 &#181;m tissue sections under confocal microscopy.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

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

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Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research