Name: three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies
Uploaded: 2013-04-29T13:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies at JoVE.com

Dette arbejde blev støttet af National Institutes of Health Tilskud K08 NS061039-01A2, Programmet for Neurology Research &amp; Discovery og The A. Alfred Taubman Medical Research Institute ved University of Michigan. Dette arbejde brugte Morfologi og Image Analysis Kernen i Michigan Diabetes Research og Training Center, finansieret af National Institutes of Health Grant 5P90 DK-20572 fra National Institute of Diabetes og Digestive og nyresygdomme. Forfatterne vil gerne takke Robinson Singleton og Gordon Smith (University of Utah) for deres generøse donation af humane hud prøver at støtte den indledende udvikling af nociceptive biomarkør immunhistokemi teknik.

Del A: Immunohistokemi Fremstilling af 96-brønds plade og forebyggelse af baggrundsfarvning Punch hudbiopsier indsamles fra forsøgspersoner og inkuberet i 12-24 timer i fikseringsopløsning (2% paraformaldehyd med 0,75 M L-lysin-opløsning (pH 7,4) og 0,05 mM natriumperiodat) ved 4 ° C som tidligere beskrevet 8.. Prøver bliver derefter kryobeskyttet i phosphatbufret saltvand (PBS) med 20% glycerol ved 4 ° C i op til 1 uge, indlejret i monteringsmedie optimal opskæring temperatu...

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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. 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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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Måling af IENFD har været meget anvendt til at bestemme graden af perifere neuropatier 13,14. På nuværende tidspunkt har kun den mest almindeligt anvendte protokol måler tætheder af nervefibre, der trænger basalmembranen af ​​overhuden, og det behøver ikke tage hensyn axonal forgrening og / eller morfologiske ændringer i nervesystemet. Desuden har den nuværende IENFD analyse ikke vist sig at korrelere IENFD med tilstedeværelsen af smerte i PN 15.. Vi har ...

På nuværende tidspunkt er det almindelig praksis for læger at kvantificere intraepidermal nerve fiber tætheder, (IENFD) fra hudens hudbiopsier, som kan anvendes til at diagnosticere små fiber neuropatier 3, 6-8. Biopsier taget fra den distale ben (DL), 10 cm over de laterale malleolus og den proximale lår (PT), 20 cm under den forreste iliacrygsøjle 9.. Alle IENF mærkes ved hjælp proteingen produkt 9,5 (PGP), et pan neuronal markør 10-12. På nuværende tidspunkt er det almindelig praksis at diagnosticere små fiber neuropatier hjælp IENFD bestemt ved PGP farvning med lysfelt mikroskopi 6.. Derudover har flere forskergrupper brugte immunfluorescerende protokoller for PGP immunhistokemi 7-9. Lille fiber neuropati er ofte forbundet med neuropatisk smerte. For yderligere at forstå den rolle IENF afgørende for smerte behandling, har vi udviklet en teknik til at co-label total IENF med fibre, der genererer smerte. Nociceptiv IENF specifikt Aδ og C-fibre, kan studeres gennem co-mærkning af IENF med PGP og nociceptive markør, tropomyosin-receptor-kinase A (Trk A) 5.. Trk A er den højaffinitetsreceptoren for nervevækstfaktor, der er afgørende for udviklingen af ​​nociception. TRK A-positive nociceptive nervefibre er peptiderge fibre, der udtrykker substans P (SP) og calcitoningenrelateret peptid (CGRP). Tidligere anvendte Lauria og kolleger dobbelt-mærkning teknik til at studere PN, co-mærkning PGP-positive IENF med en nociceptive markør 10.. I vores tidligere undersøgelse viste vi, at Trk A-positiv IENF, men ikke Trk A-negativ IENF blev opreguleret i en dyremodel af smertefuld diabetisk neuropati 5.. Dette samarbejde mærkning teknik giver mulighed for at sammenligne kvantificering af nociceptive IENFD til total IENFD og evnen til at studere morfologiske ændringer i forbindelse med PN. Evnen til at visualisere nociceptive IENF og virksomre kvantificering af den samlede IENFD til nociceptive IENFD kunne give objektive beviser for tilstedeværelsen af ​​smerte, og muligvis indsigt i sværhedsgraden af ​​smerter forbundet med PN. Denne teknik kan også anvendes til hud dyremodeller. I forhold til tidligere undersøgelser, beskriver den nuværende protokol metoder til 3D-billedanalyse, skaber mulighed for at undgå fejl, der kan opstå i 2D billedanalyse.

<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

En dorn biopsi af huden er almindeligt anvendt til at kvantificere intraepidermal nerve fiber tætheder (IENFD) til diagnose af perifer polyneuropati 1,2. På nuværende tidspunkt er det almindelig praksis at samle 3 mm hudbiopsier fra den distale ben (DL) og den proksimale låret (PT) til evalueringen af længde-afhængige polyneuropatier 3. Men på grund af den multidirektionelle karakter IENFs, er det udfordrende at undersøge overlappende nerve strukturer gennem en analyse af todimensionale (2D) afbildning. Alternativt kunne tredimensionale (3D) billeddannelse give en bedre løsning på dette dilemma. I den nuværende rapport præsenterer vi metoder til at anvende 3D-billedbehandling til at studere smertefuld neuropati (PN). For at identificere IENFs er hudprøver forarbejdet til immunofluorescent analyse af protein gen-produkt 9,5 (PGP), et pan neuronal markør. På nuværende tidspunkt er det almindelig praksis at diagnosticere små fiber neuropatier hjælp IENFD afskrækkeudvundet af PGP immunhistokemi hjælp brightfield mikroskopi 4.. I den aktuelle undersøgelse, anvendt vi dobbelt immunofluorescerende analyse for at identificere den samlede IENFD, bruger PGP, og nociceptive IENF, gennem anvendelse af antistoffer, der genkender tropomyosin-receptor-kinase A (Trk A) højaffinitetsreceptoren for nervevækstfaktor 5.. Fordelene ved co-farvning IENF med PGP og Trk A antistoffer gavner studiet af PN ved klart farvning PGP-positive, nociceptive fibre. Disse fluorescerende signaler kan kvantificeres for at bestemme nociceptive IENFD og morfologiske ændringer i IENF forbundet med PN. De fluorescerende billeder er erhvervet ved konfokal mikroskopi og forarbejdet til 3D analyse. 3D-billedbehandling giver roterende evner at analysere morfologiske ændringer i forbindelse med PN. Tilsammen fluorescerende co-farvning, konfokal billedbehandling og 3D-analyser klart gavne studiet af PN.

Vi anvendte den nuværende protokol for at studere morfologi IENF i PT-og DL hudbiopsier fra patienter med PN. Huden, fra tre emner, indsamlet blev på University of Utah for at demonstrere pathomorphology forbundet med PN. Emnerne omfatter: Case 1: en 51-årig mand med en historie af PN af type 2-diabetes (Varighed: 14 måneder, smerte score: 51) Case 2: en 56-årig mand med en historie af PN af type 2 diabetes (varighed: 108 måneder smerte score: 47), og sag 3: en 66-årig mand med en historie af PN af type 2 diabete...

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

For at undersøge ændringerne i nociceptive intraepidermal nervefibre (IENFs) i smertefulde neuropatier (PN), vi udviklede protokoller, der direkte kunne undersøge tredimensionelle morfologiske ændringer observeret i nociceptive IENFs. Tredimensional analyse af IENFs har potentiale til at vurdere de morfologiske ændringer IENF i PN.

Tre-dimensionel Imaging af Nociceptive Intraepidermal nervefibre i Human hudbiopsier

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

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