Diese Arbeit wurde vom National Institutes of Health unterstützt Grants K08 NS061039-01A2, das Programm für Neurologie Research &amp; Discovery, and The A. Alfred Taubman Medical Research Institute an der University of Michigan. Diese Arbeit verwendet die Morphologie und Bildanalyse Kern der Michigan Diabetes Research and Training Center, National Institutes of Health Grants 5P90 DK-20572 aus dem National Institute of Diabetes and Digestive und Nierenkrankheiten finanziert. Die Autoren bedanken sich bei Robinson Singleton und Gordon Smith (University of Utah) für die großzügige Spende von menschlichen Hautproben, die anfängliche Entwicklung des nozizeptiven Biomarker Immunhistochemie Technik unterstützt danken.

Teil A: Immunhistochemie Herstellung von 96-Well-Platte und Prävention von Hintergrundfärbung Durchschlag Hautproben von menschlichen Probanden gesammelt und inkubiert für 12-24 Stunden in Fixierlösung (2% Paraformaldehyd mit 0,75 M L-Lysin-Lösung (pH 7,4) und 0,05 mM Natriumperiodat) bei 4 ° C wie zuvor 8 beschrieben. Die Proben werden dann in Phosphat kryogeschützt Kochsalzlösung (PBS) mit 20% Glycerin bei 4 ° C für bis zu 1 Woche, eingebettet in Eindeckmitteln optimale Schnittleistung Temperatur (OCT), dann im Schnitt in 50 um dicke Schnitte auf einem Kryostaten. Die unten beschriebenen Protokoll wird 8 Hautpartien, wird die maximale Anzahl von Hautpartien möglich, frei schwebenden Immunhistochemie in eine 96-Well-Platte zu unterziehen. TAG 1: 1. Prävention von nicht-spezifische Immunreaktivität in der Hornschicht <ol><li> Label-96-Well-Platte, wie in 1 gezeigt. </li&gt; <li> In 150 ul Bild-IT FX Signal (Bild-IT), wirksam zu blockieren Hintergrundfärbung 11,12, in jede Vertiefung in Reihe 1 der 96-Well-Platte. </li><li> In 150 ul 1X PBS in jedes Well in Reihe 2 und 3 der 96-Well-Platte. </li><li> Erwerben Sie zwei 50 um Abschnitte pro Patient: ein Abschnitt aus der Biopsie bei DL genommen, einen Abschnitt aus der Biopsie bei PT gemacht. Eine Impföse (LeLoop) wird verwendet, um Abschnitte von einem übertragen spülen, um die nächste zu morphologischen Schäden zu vermeiden. Sanft übertragen jede 50 um Abschnitt, mittels Impföse, zu einem individuellen und von Reihe 1, mit Bild-IT. Inkubieren Abschnitte in Bild-IT für 30 min bei RT auf einem flachen Rocker. </li><li> Spülen Abschnitte zweimal mit 1 × PBS (Zeilen 2 und 3) für 10 min bei RT. Zeigen Well-Platte auf einer flachen Rocker zwischen Spülungen. </li></ol> 2. Herstellung von 5% BSA Blocking Solution und 1% Spüllösung <ol><li> Während Biopsie Abschnitte in Bild-IT sind Inkubation, bereiten 5% Blocking Lösungtion (5% BSA, 0,3% TX-100, 0,1 M PBS-Vortex-Lösung bis BSA vollständig auflöst). </li><li> In 150 ul 5% BSA Blocking-Lösung in jedes Well der Reihe 4. </li><li> Mit einer Impföse übertragen Abschnitte in einzelnen Vertiefungen von 5% BSA-Lösung blockiert. Inkubieren Abschnitte in 5% BSA Blocking-Lösung für 1-2 h bei RT auf einem flachen Rocker. </li></ol> 3. Herstellung von 1% BSA Spüllösung und Verdünnung des primären Antikörper <ol><li> Während Teile in 5% BSA Blockierungslösung inkubiert werden, bereiten 1% Spüllösung (1% BSA, 0,3% TX-100, 0,1 M PBS-Lösung bis Vortex BSA vollständig auflöst). </li><li> Während Teile in 5% BSA-Lösung blockiert Inkubation, verdünnte Primärantikörper in 1% Spüllösung. <ol><li> Für acht Abschnitte (8 x 150 = 1.200 ul ul) insgesamt 1.200 ul benötigt wird. Die primären Antikörper werden in 1.500 ul (etwa 20% mehr Volumen) gemacht. </li><li> Verdünnungen der Primärantikörper: PGP, 1:500; Trk A,1:500 in 1% BSA Spüllösung. </li></ol></li></ol> 4. Inkubation von Sectioned Biopsien in primärer Antikörper <ol><li> In 150 ul verdünnten Primärantikörper in die dafür vorgesehenen Vertiefungen der 96-Well-Platte (Zeile 5). </li><li> Übertragen von Abschnitten 5% Blockierungslösung (Zeile 4) in die dafür vorgesehenen Vertiefungen primären Antikörper (Reihe 5). </li><li> Verschließen Sie die 96-Well-Platte mit Parafilm und Alufolie ein Austrocknen zu verhindern und Belichtung. </li><li> Inkubieren Sie die 96-Well-Platte O / N bei 4 ° C auf einem flachen Rocker. </li></ol> TAG 2: 5. Spülen Biopsien in 1% BSA Spüllösung <ol><li> In 150 ul 1% BSA Spüllösung in jede Vertiefung in Reihe 6, 7 und 8. </li><li> Spülen Abschnitte dreimal mit 1% BSA Spüllösung (Zeilen 6, 7 und 8) für 1 Stunde jedesmal bei RT gerührt. Titelbild Well-Platte mit Aluminiumfolie und auf flachen Rocker zwischen Spülungen. </li></ol> 6. Verdünnung der Sekundärantikörper &lt;ol&gt; <li> Während Teile in der letzten von 1% BSA Spüllösung spülen (Zeile 8) inkubiert, verdünnt Sekundärantikörper in 1% BSA Spüllösung: <ol><li> Für acht Abschnitte (8 x 150 = 1.200 ul ul) insgesamt 1.200 ul benötigt wird. Die sekundären Antikörper sind bis in 1.500 ul (etwa 20% mehr Volumen) gemacht. </li><li> Verdünnungen der sekundären Antikörper: für PGP (Alexa Fluor 488 Esel-anti-Kaninchen, 1:250), für Trk A (Alexa Fluor 647 Esel-anti-Ziege, 1:250) in 1% BSA Spüllösung. </li></ol></li></ol> 7. Inkubation von Sectioned Biopsien in sekundären Antikörper <ol><li> In 150 ul verdünnten Sekundärantikörper in ihr bezeichneten Vertiefungen der 96-Well-Platte (Zeile 9). </li><li> Übertragen Abschnitte von 1% BSA Blocking-Lösung (Reihe 8) in sekundären Antikörper gut (Zeile 9). </li><li> Verschließen Sie die 96-Well-Platte mit Parafilm und Alufolie ein Austrocknen zu verhindern und Belichtung. </li><li> Inkubieren Abschnitte in dem bezeichneten Sekundärantikörper O/ N bei 4 ° C auf einem flachen Rocker. </li></ol> 8. Spülen Biopsien in 1% BSA Spüllösung <ol><li> In 150 ul 1% BSA Spüllösung in jede Vertiefung in Reihe 10, 11 und 12. </li><li> Spülen Abschnitte dreimal mit 1% BSA Spüllösung (Reihen 10, 11 und 12) für 1 Stunde jedesmal bei RT gerührt. Titelbild Well-Platte mit Aluminiumfolie und auf einem flachen Rocker zwischen Spülungen. </li></ol> 9. Vorbereitung von Objektträgern und Montage Sectioned Biopsien <ol><li> Während Biopsie Abschnitte in der letzten von 1% BSA Spüllösung (Zeile 12) spülen sind Inkubieren, bereiten Objektträger. </li><li> Zeigen 50 ul 1% BSA Spüllösung auf einer Folie zu einer Zeit. </li><li> Entfernen Abschnitte von 1% BSA Spüllösung (Reihe 12), und legen Sie es in den 50 ul Tropfen 1% BSA Spüllösung auf dem designierten Objektträger. Nach der Optimierung der Position des Abschnitts, überschüssige 1% BSA Spüllösung mit einem wulstförmigen Glaspipette, wobei Vorkehrungenzu vermeiden, berühren Sie die Probe. </li></ol> HINWEIS: Vergewissern Sie Abschnitt nicht umgeklappt; die Probe sollte gegen die Oberfläche des Objektträgers flach. <ol start="4"><li> Platz 1 Tropfen verlängern Gold-antifade Montage Reagenz mit DAPI in der Nähe der Biopsie auf dem Objektträger. Werfen Sie einen 22x22 mm Mikroskop Deckglas und legen Sie sie vorsichtig über Biopsie und ein Tropfen verlängern Gold-antifade Reagenz mit DAPI Drop. Wiederholen Sie für jeden Abschnitt. </li><li> Lassen Objektträger trocken O / N bei RT im Dunkeln. </li></ol> HINWEIS: Entfernen Sie alle Luftblasen mit einer Pipettenspitze. Überschüssiges verlängern Gold-antifade Reagenz. Teil B: konfokale Bildgebung 10. Konfokalen Imaging <ol><li> Bild Fluoreszenz-Signale mit einer Olympus FluoView 500 konfokalen Laser-Scanning-Mikroskop mit einem 40x Öl-Immersions-(1.3 NA) Ziel-und Zoom zweimal mit FluoView Version 5.0 Software. </li&gt; <li> Verwenden Alexa Fluor 488 und Alexa Fluor 647, die 543-nm HeNe grünen Laser und 633-nm HeNe Red Laser bzw. anzuregen. Die Alexa Fluor 488 Signal wird durch einen grünen Nachschlagetabelle (LUT) und dem Alexa Fluor 647 von einem roten LUT dargestellt. </li><li> Stellen Sie die konfokale Öffnungen für jeden Detektor bei 400 um, um Signale zu verstärken. Sequential Scans sollten bei einer Auflösung von 1.024 x 1.024, um das Signal zu maximieren Trennung getroffen werden. </li><li> Erfassen dreidimensionaler z-Serie mit 1,2 um z-Schrittweiten (bezogen auf die optimale Scan-Einheit durch die Software berechnet FluoView) mit Kalman Mittelung (zwei Rahmen). </li></ol> Teil C: Dreidimensionale Visualisierung und Animation 11. Dreidimensionale (3D) Visualisierung und Animation <ol><li> Offene FluoView Dateien in Imaris x64-Software (Version 7.3, Bitplane AG) zur Visualisierung der 3D-Bild-Sets. </li><li> Passen Sie bei Bedarf angezeigt werden, um den Kontrast des Nervs spezifisches Signal zu verbessern. </li><li> Erstellen Sie einen surface die dermo-epidermalen Grenze zu visualisieren. Verwenden Sie das Tool in Contour Auslosung Modus, um die Grenze zu ziehen. </li><li> Zuweisen einer halbtransparenten Oberfläche zu der Grenze, um die zugrunde liegenden Fluoreszenzsignale anzusehen. </li><li> Standbilder der 3D-Fluoreszenz-Signale an Snapshot Bilder der 3D-Film zu erstellen. </li><li> Verwenden Sie die Animation, Funktion, um einen 3D-Film zu erstellen. Die Bilder können gedreht werden 360 Grad mehrmals, jedes Signal getrennt und fusionierte zeigen (Abbildungen 2, 3 und 4). Set Animation, Filme, bestehend aus 200 Bildern bei 15 Bildern pro Sekunde animiert erstellen. Speichern Sie jeden Film als AVI-Datei roh. </li><li> Komprimieren Sie die endgültige AVI-Film mit VirtualDub-Software (Version 1.9.4). </li></ol>

<ol>
	<li>Lauria, G., Holland, N., 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+innervation:+changes+with+aging,+topographic+location,+and+in+sensory+neuropathy.">Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy.</a> J. Neurol. Sci. 164 (2), 172-178 (1999).</li><li>Sullivan, K. A., Hayes, J. 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=Mouse+models+of+diabetic+neuropathy.">Mouse models of diabetic neuropathy.</a> Neurobiol. Dis. 28 (3), 276-285 (2007).</li><li>McArthur, J. C., Stocks, E. A., Hauer, P., Cornblath, D. R., Griffin, J. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor/p38+signaling+increases+intraepidermal+nerve+fiber+densities+in+painful+neuropathy+of+type+2+diabetes.">Nerve growth factor/p38 signaling increases intraepidermal nerve fiber densities in painful neuropathy of type 2 diabetes.</a> Neurobiol. Dis. 45 (1), 280-287 (2012).</li><li>Lauria, G., Lombardi, R., Camozzi, F., Devigili, G. <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+for+the+diagnosis+of+peripheral+neuropathy.">Skin biopsy for the diagnosis of peripheral neuropathy.</a> Histopathology. 54 (3), 273-285 (2009).</li><li>Vlckova-Moravcova, E., Bednarik, J., Dusek, L., Toyka, K. 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+among+pain,+sensory+loss,+and+small+nerve+fibers+in+diabetes.">The relationship among pain, sensory loss, and small nerve fibers in diabetes.</a> Diabetes Care. 29 (4), 883-887 (2006).</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=Axonal+swellings+predict+the+degeneration+of+epidermal+nerve+fibers+in+painful+neuropathies.">Axonal swellings predict the degeneration of epidermal nerve fibers in painful neuropathies.</a> Neurology. 61 (5), 631-636 (2003).</li><li>Herrmann, D. N., McDermott, M. 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>

Interessenkonflikte zu erklären.

Messung der IENFD wurde weithin verwendet, um den Grad von peripheren Neuropathien 13,14 bestimmen. Derzeit ist die am häufigsten verwendete Protokoll misst nur die Dichten von Nervenfasern, die die Basalmembran der Epidermis eindringen, es nicht zu berücksichtigen axonale Verzweigung und / oder morphologische Veränderungen der Nerven. Darüber hinaus hat IENFD aktuellen Analyse nicht gezeigt worden, um mit dem Vorhandensein von Schmerzen in PN 15 IENFD korrelieren. ...

Derzeit ist es üblich, für Ärzte, intraepidermalen Nervenfaser Dichten (IENFD) von der Haut Stanzbiopsien, die verwendet werden, um kleine Faser Neuropathie 3, 6-8 diagnostizieren kann quantifizieren. Biopsien aus dem distalen Schenkel (DL), 10 cm über den Malleolus lateralis und den proximalen Schenkel (PT), 20 cm unterhalb der Spina iliaca anterior 9. Alle IENF sind beschriftet mit Protein-Genprodukt 9.5 (PGP), ein pan-neuronalen Marker 10-12. Derzeit ist es üblich, kleine Faser mit Neuropathien diagnostizieren IENFD bestimmt durch Färbung mit PGP Hellfeldmikroskopie 6. Darüber hinaus haben mehrere Arbeitsgruppen Immunfluoreszenz Protokolle für PGP Immunhistochemie 7-9 verwendet. Kleine Faser Neuropathie ist häufig mit neuropathischen Schmerzen verbunden. Zur weiteren Verständnis der Rolle von IENF wichtig für Schmerzen Verarbeitung, entwickelten wir eine Technik, um Co-Label insgesamt IENF mit Fasern, die Schmerzen zu erzeugen. Nozizeptorentive IENF, speziell A &amp;dgr; und C-Fasern, durch die Co-Kennzeichnung IENF mit PGP und des nozizeptiven Marker, Tropomyosin-Rezeptor-Kinase A (Trk A) 5 untersucht werden. Trk A ist der Rezeptor mit hoher Affinität für NGF, die für die Entwicklung der Nozizeption ist. Die Trk A-positive nozizeptiven Nervenfasern sind peptiderge Fasern, express Substanz P (SP) und Calcitonin Gene Related Peptide (CGRP). Zuvor angewendet Lauria und Kollegen der doppelten Kennzeichnung Technik PN, Co-Kennzeichnung PGP-positive IENF mit einer nozizeptiven Markierung 10 zu studieren. In unserer früheren Studie haben wir gezeigt, dass Trk A-positive IENF, aber nicht Trk A-negative IENF, in einem Tiermodell der schmerzhaften diabetischen Neuropathie 5 wurden hochreguliert. Diese Co-Kennzeichnung Technik bietet die Möglichkeit, die Quantifizierung von nozizeptiven IENFD vergleichen, um IENFD und die Fähigkeit zur morphologischen Veränderungen mit PN zugeordnet studieren insgesamt. Die Fähigkeit, nozizeptiven IENF und Unternehmen visualisierenre Quantifizierung der gesamten IENFD zu nozizeptiven IENFD könnte Ziel Beweis für das Vorhandensein von Schmerz und möglicherweise Einblick in die Schwere der Schmerzen mit PN assoziiert ist. Diese Technik ist auch auf die Haut von Tiermodellen. Im Vergleich zu früheren Studien, beschreibt das aktuelle Protokoll Methoden zur 3D-Bildanalyse, die Schaffung der Möglichkeit, Fehler, die in 2D Bildanalyse auftreten könnten, zu vermeiden.

<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

Ein Schlag Biopsie der Haut wird üblicherweise zur intraepidermal Nervenfaser Dichten (IENFD) für die Diagnose der periphere Polyneuropathie 1,2 quantifizieren. Derzeit ist es üblich, 3 mm Hautproben von dem distalen Schenkel (DL) und dem proximalen Schenkel (PT) für die Bewertung der längenabhängigen Polyneuropathien 3 sammeln. Aufgrund der multidirektionalen Art IENFs, ist es schwierig zu prüfen überlappenden Nervenstrukturen durch die Analyse von zweidimensionalen (2D-) Bildgebung. Alternativ könnte dreidimensionale (3D)-Bildgebung eine bessere Lösung für dieses Dilemma. Im vorliegenden Bericht stellen wir Methoden für die Anwendung 3D-Bildgebung zu schmerzhaften Neuropathie (PN) zu studieren. Um IENFs zu identifizieren, werden Hautproben für Immunfluoreszenz-Analyse von Protein-Genprodukt 9.5 (PGP), ein pan-neuronalen Marker verarbeitet. Derzeit ist es üblich, kleine Faser Neuropathie mit IENFD abschrecken diagnostizierenabgebaut durch PGP Immunhistochemie mit Hellfeldmikroskopie 4. In der aktuellen Studie, angewendet wir doppelte Immunfluoreszenz Analyse insgesamt IENFD identifizieren, die mit PGP und nozizeptiven IENF, durch den Einsatz von Antikörpern, die Tropomyosin-Rezeptor-Kinase erkennen A (Trk A), die hohe Affinität Rezeptor für nerve growth factor 5. Die Vorteile der Co-Färbung IENF mit PGP und Trk A-Antikörper profitiert die Studie von PN deutlich Färbung PGP-positive, nozizeptiven Fasern. Diese fluoreszierenden Signale quantifiziert nozizeptiven IENFD und morphologische Veränderungen von IENF mit PN assoziiert zu bestimmen. Die Fluoreszenzbilder werden durch konfokale Mikroskopie erfasst und verarbeitet, für die 3D-Analyse. 3D-Bildgebung liefert Dreh-Fähigkeiten weiter zu analysieren morphologische Veränderungen mit PN verbunden. Zusammengenommen fluoreszierenden Co-Färbung, konfokale Bildgebung und 3D-Analyse eindeutig profitieren die Studie von PN.

Dabei haben wir das aktuelle Protokoll, um die Morphologie der IENF in PT und DL Hautbiopsien von Patienten mit PN studieren. Die Haut, aus drei Themen, wurde an der Universität von Utah gesammelt, um die Pathomorphologie mit PN zugeordnet demonstrieren. Die Themen umfassen: Fall 1: ein 51-jähriger Mann mit einer Geschichte von PN von Diabetes Typ 2 (Dauer: 14 Monate; Schmerz-Score: 51); Fall 2: ein 56-jähriger Mann mit einer Geschichte von PN von Diabetes Typ 2 (Dauer: 108 Monate; Schmerz-Score: 47) und Fall 3: ein ...

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

Um die Änderungen des nozizeptiven intraepidermal Nervenfasern (IENFs) in schmerzhafte Neuropathie (PN) zu untersuchen, haben wir Protokolle, die direkt untersuchen könnte dreidimensionalen morphologischen Veränderungen in nozizeptiven IENFs beobachtet. Dreidimensionale Analyse IENFs hat das Potenzial, um die morphologischen Veränderungen in IENF PN bewerten.

Dreidimensionale Bildgebung Nozizeptive Intraepidermale Nervenfasern im menschlichen Hautproben

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

13.0K Aufrufe.  University of Michigan. Um die Änderungen des nozizeptiven intraepidermal Nervenfasern (IENFs) in schmerzhafte Neuropathie (PN) zu untersuchen, haben wir Protokolle, die direkt untersuchen könnte dreidimensionalen morphologischen Veränderungen in nozizeptiven IENFs beobachtet. Dreidimensionale Analyse IENFs hat das Potenzial, um die morphologischen Veränderungen in IENF PN bewerten.

Serie: 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.

Menschliche neuroendokrinen Tumor-Zelllinien als dreidimensionales Modell für das Studium der menschlichen neuroendokrinen Tumor-Therapie

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 ...

Forschung

Medizin

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.

Eine dreidimensionale Zellkulturmodell zu primären humanen Knochenmark und seine Malignome Studieren

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.

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

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.

Dreidimensionale Alginat-Perle Kultur der Menschen Hypophysenadenom 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 ...

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.

Quantitative Visualisierung von Leukozyten-Infiltrat in einem Murin-Modell der Fulminant-Myokarditis durch Licht-Blatt-Mikroskopie

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.

Nozizeptiven objektiven in belüfteten Intensivpatienten: eine Machbarkeitsstudie mit Pupillenreaktion und der nozizeptiven Flexion Reflex

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.

Drei-dimensionale Druck einer komplexen Aorten Anomalie

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.

Verfeinerte CLARITY-basierte Gewebereinigung für die dreidimensionale Fibroblastenorganisation in gesunden und verletzten Mäuseherzen

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.

Hochauflösende dreidimensionale Abbildung der Fußpolstervaskulatur in einem murinen Hinterbein-Gangränmodell

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.

Geführte Endodontie: Dreidimensionale Planung und schablonengestützte Vorbereitung endodontischer Zugangskavitäten

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, ...