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>

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Arch. Neurol. 55 (12), 1513-1520 (1998).</li><li>Griffin, J. W., McArthur, J. C., Polydefkis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cutaneous+innervation+by+skin+biopsies.">Assessment of cutaneous innervation by skin biopsies.</a> Curr. Opin. Neurol. 14 (5), 655-659 (2001).</li><li>Cheng, H. T., Dauch, J. R., Hayes, J. M., Yanik, B. M., Feldman, E. 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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. Methods. 200 (2), 190-198 (2011).</li><li>Wang, L., Hilliges, M., Jernberg, T., Wiegleb-Edstrom, D., Johansson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+gene+product+9.5-immunoreactive+nerve+fibres+and+cells+in+human+skin.">Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin.</a> Cell Tissue Res. 261 (1), 25-33 (1990).</li><li>Lauria, G., Morbin, 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=Expression+of+capsaicin+receptor+immunoreactivity+in+human+peripheral+nervous+system+and+in+painful+neuropathies.">Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies.</a> J. Peripher. Nerv. Syst. 11 (3), 262-271 (2006).</li><li>Penna, G., Fibbi, B., 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=Human+benign+prostatic+hyperplasia+stromal+cells+as+inducers+and+targets+of+chronic+immuno-mediated+inflammation.">Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immuno-mediated inflammation.</a> J. Immunol. 182 (7), 4056-4064 (2009).</li><li>Lentz, S. I., Edwards, J. L., 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=Mitochondrial+DNA+(mtDNA)+Biogenesis:+Visualization+and+Duel+Incorporation+of+BrdU+and+EdU+Into+Newly+Synthesized+mtDNA+In+Vitro.">Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro.</a> J. Histochem. Cytochem. 58 (2), 207-218 (2010).</li><li>Polydefkis, M., Hauer, P., Griffin, J. W., McArthur, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy+as+a+tool+to+assess+distal+small+fiber+innervation+in+diabetic+neuropathy.">Skin biopsy as a tool to assess distal small fiber innervation in diabetic neuropathy.</a> Diabetes Technol. Ther. 3 (1), 23-28 (2001).</li><li>Lauria, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibre+neuropathies.">Small fibre neuropathies.</a> Curr. Opin. Neurol. 18 (5), 591-597 (2005).</li><li>Sorensen, L., Molyneaux, L., Yue, D. 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P., 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=Epidermal+nerve+fiber+density,+axonal+swellings+and+QST+as+predictors+of+HIV+distal+sensory+neuropathy.">Epidermal nerve fiber density, axonal swellings and QST as predictors of HIV distal sensory neuropathy.</a> Muscle Nerve. 29 (3), 420-427 (2004).</li><li>Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27:+Neural+plasticity+after+nerve+injury+and+regeneration.">Chapter 27: Neural plasticity after nerve injury and regeneration.</a> Int. Rev. Neurobiol. 87, 483-505 (2009).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<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;D Systems</td>
			<td>AF1056</td>
			<td>Trk A</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey &alpha;-rabbit</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>AF488 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey &alpha;-goat</td>
			<td>Invitrogen</td>
			<td>A21447</td>
			<td>AF647 donkey &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

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Medicine

13.0K 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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, pulpitis or apical periodontitis may develop in the long term. Root canal treatment of teeth with severe PCO and pulpal or periapical pathosis is challenging for general practitioners and even for well-equipped endodontic specialists. To ensure detection of the calcified root canal and avoid excessive loss of tooth structure or root perforation, static navigation using templates ("Guided Endodontics") was introduced a few years ago. The general workflow includes three-dimensional imaging using cone-beam computed tomography (CBCT), a digital surface scan, and superimposition of both in a planning software. This is followed by virtual planning of the access cavity and the design of a template that will guide the drill to the desired target point. To do this, a true-to-scale virtual image of the drill must be placed in a way that the tip of the drill reaches the orifice of the calcified root canal. Once the template has been fabricated using computer-aided design and computer-aided manufacturing (CAD/CAM) or a 3D printer, guided preparation of the access cavity can be performed clinically. For research purposes, a postoperative CBCT image can be used to quantify the accuracy of the access cavity performed. This work aims to present the technique of static guided endodontics from imaging to clinical implementation.

Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth.

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...