The authors have no acknowledgements.

Diese Protokolle an die Richtlinien des menschlichen Forschungsethikkommission unserer Institution. 1. Konventionelle Hornhautkollagen CXL (C-CXL) 1. Herstellung der Patient <ol><li> 5 Tage vor der Operation, legte 1% Pilocarpin fällt zweimal am Tag in dem behandelten Auge. </li><li> Im Operationssaal, unter aseptischen Bedingungen, liegen, den Patienten auf seinem / ihrem Rücken. </li><li> Lokalanästhesie zu verabreichen, wie Oxybuprocain 0,4%. </li><li> Reinigen Sie die Augen und die Haut um die Augen mit Jod antiseptisch zweimal. </li><li> Verwenden Sie einen Lidspekulum, um das Auge offen zu halten. </li></ol> 2. Epithelial Removal <ol><li> Markieren Sie die zentralen 9,0 mm der Hornhaut mit einem Kreis Hornhautmarker. </li><li> Entfernen Sie die zentrale 7,0 bis 9,0 mm von Hornhautepithel durch mechanische Debridement mit einem stumpfen Spachtel. </li></ol> 3. Riboflavin Anwendungs <ol><li> Übernehmen 0,1% Riboflavin mit 20% Dextran auf th<html> e Hornhaut jedes min für 20 min. </li></ol> 4. UVA-Bestrahlung <ol><li> Bestrahlen der Hornhaut mit einer 370 nm Wellenlänge UVA-Licht bei einer Strahlungsintensität von 3 mW / cm 2 (5,4 J / cm 2 Oberflächendosis) und an einem 5 cm Arbeitsabstand für 30 min. </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/53119/53119fig1.jpg" /> Fig. 1: UV-A-Bestrahlung C-CXL Die Hornhaut wird mit einer 370 nm Wellenlänge UVA-Licht bei einer Strahlungsintensität von 3 mW / cm 2 (5,4 J / cm 2 Oberflächendosis) und an einem 5 cm Arbeitsabstand für 30 Minuten bestrahlt. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <ol start="2"><li> Während der Bestrahlung angewendet Tropfen Riboflavin auf die Hornhaut alle 5 min. </li><li> Während der Bestrahlung hinzufügen Lokalanästhesie (Oxybuprocain 0,4%), wenn nötig. </li></ol>&quot;&gt; 5. Ende der Chirurgie <ol><li> Setzen antibiotische Tropfen (Tobramycin 0,3%) und künstliche Tränen (Hyaluronat Tropfen 0,18%) in das operierte Auge. </li><li> Legen Sie ein weiches Verbandkontaktlinse am Ende der Operation, bis Reepithelisierung beendet ist. Reepithelisierung dauert in der Regel 3 Tage. </li><li> Verschreiben Schmerzmittel wie Paracetamol (500 mg) und Codein (30 mg), 6 Tabletten pro Tag. </li><li> Nach Hornhaut Reepithelisierung, initiieren topische Therapie mit Steroiden (topische Dexamethason 1 mg / ml) und fahren Sie für 3-4 Wochen. Außerdem verwenden künstliche Tränen 4 Mal pro Tag für 1 Monat. </li></ol> 2. Beschleunigtes Hornhautkollagen CXL (A-CXL) 1. Herstellung der Patient <ol><li> 5 Tage vor der Operation, legte 1% Pilocarpin fällt zweimal am Tag in dem behandelten Auge. </li><li> Im Operationssaal, unter aseptischen Bedingungen, liegen, den Patienten auf seinem / ihrem Rücken. </li><li> Lokalanästhesie zu verabreichen, wie Oxybuprocain 0,4%. </li><li> Reinigen Sie den eihr und die Haut um die Augen mit Jod antiseptisch zweimal. </li><li> Verwenden Sie einen Lidspekulum, um das Auge offen zu halten. </li></ol> 2. Epithelial Removal <ol><li> Markieren Sie die zentralen 9,0 mm der Hornhaut mit einem Kreis Hornhautmarker </li><li> Entfernen Sie die zentrale 7,0 bis 9,0 mm von Hornhautepithel durch mechanische Debridement mit einem stumpfen Spachtel. </li></ol> 3. Riboflavin Anwendungs <ol><li> Gelten 0,1% Riboflavin ohne Dextran auf der Hornhaut alle 2 min während 10 min. </li></ol> 4. UVA-Bestrahlung <ol><li> Bestrahlen der Hornhaut mit einer 370 nm Wellenlänge UVA-Licht bei einer Bestrahlungsstärke von 30 mW / cm 2 (5,4 J / cm 2 Oberflächendosis) und an einem 5 cm Arbeitsabstand für 3 min. </li><li> Während der Bestrahlung hinzuzufügen topischer Anästhesie (oxybuproca &amp; # 239; ne 0,4%), falls erforderlich. </li></ol> 5. Ende der Chirurgie <ol><li> Zeigen antibiotische Tropfen (Tobramycin 0,3%) und künstliche Tränen (Hyaluronat Tropfen 0<html> 0,18%) in das operierte Auge. </li><li> Legen Sie ein weiches Verbandkontaktlinse am Ende der Operation, bis Reepithelisierung beendet ist. Reepithelisierung dauert in der Regel 3 Tage. </li><li> Verschreiben Schmerzmittel wie Paracetamol (500 mg) und Codein (30 mg), 6 Tabletten pro Tag. </li><li> Nach Hornhaut Reepithelisierung, initiieren topische Therapie mit Steroiden (topische Dexamethason 1 mg / ml) und fahren Sie für 3-4 Wochen. Außerdem verwenden künstliche Tränen 4 Mal pro Tag für 1 Monat. </li></ol> 3. Die Iontophorese (I-CXL) 1. Herstellung der Patient <ol><li> 5 Tage vor der Operation, legte 1% Pilocarpin fällt zweimal am Tag in dem behandelten Auge. </li><li> Im Operationssaal, unter aseptischen Bedingungen, liegen, den Patienten auf seinem / ihrem Rücken. </li><li> Lokalanästhesie zu verabreichen, wie Oxybuprocain 0,4%. </li><li> Reinigen Sie die Augen und die Haut um die Augen mit Jod antiseptisch zweimal. </li><li> Verwenden Sie einen Lidspekulum, um das Auge offen zu halten. </li></ol><html> _step &quot;&gt; 2. Positionieren Sie die Iontophorese-Gerät. <ol><li> Übernehmen Sie die klebrige passive Elektrode auf der Stirn unter dem Operationsfeld. </li><li> Übernehmen Sie die aktive Elektrode, einen Saugring, zu dem offenen Auge. Zentrieren den Saugring an der Peripherie der Hornhaut vor dem Loslassen der Absaugung. </li></ol><img alt="Figur 2" src="/files/ftp_upload/53119/53119fig2.jpg" /> Abbildung 2. Iontophorese-Gerät. Die passive Elektrode auf der Stirn unter dem Operationsfeld und der aktiven Elektrode, einem Saugring angewendet wird, wird mit dem offenen Auge angewendet. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> 3. Riboflavin Anwendungs <ol><li> Füllen Sie den Saugring mit hypoosmolare 0,1% Riboflavin ohne Dextran. </li></ol><html> jpg &quot;/&gt; Abbildung 3. Riboflavin Anwendung in I-CXL. Der Saugring ist mit hypoosmolare 0,1% Riboflavin ohne Dextran gefüllt. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <ol start="2"><li> Beginnen, den elektrischen Strom von 0,2 mA und schrittweise auf 1,0 mA erhöht insgesamt Iontophorese von 5 min (Abbildung 4). </li></ol><img alt="Figur 4" src="/files/ftp_upload/53119/53119fig4.jpg" /> Abbildung 4. Iontophorese-Gerät für Riboflavin-Penetration. Der elektrische Strom wird zunächst 0,2 mA und nach und nach auf 1,0 mA erhöht. Insgesamt Iontophorese Zeit beträgt 5 Minuten. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig4large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> ep &quot;&gt; 4. UVA-Bestrahlung <ol><li> Bestrahlen der Hornhaut mit einer 370 nm Wellenlänge UVA-Licht bei einer Bestrahlungsstärke von 10 mW / cm 2 (5,4 J / cm 2 Oberflächendosis) und an einem 5 cm Arbeitsabstand für 9 min. </li><li> Während der Bestrahlung hinzuzufügen topischer Anästhesie (oxybuproca &amp; # 239; ne 0,4%), falls erforderlich. </li></ol> 5. Ende der Chirurgie <ol><li> Zeigen antibiotische Tropfen (Tobramycin 0,3%) und künstliche Tränen (Hyaluronat Tropfen 0,18%) in das operierte Auge. </li><li> Verschreiben Schmerzmittel wie Paracetamol (500 mg) und Codein (30 mg), 6 Tabletten pro Tag. </li><li> Nach der Operation einzuleiten topische Therapie mit Steroiden (topische Dexamethason 1 mg / ml) und fahren Sie für 3-4 Wochen. Außerdem verwenden künstliche Tränen 4 Mal pro Tag für 1 Monat. </li></ol>

<ol>
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S., 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=Pharmacokinetics+and+safety+of+transcorneal+iontophoresis+of+tobramycin+in+the+rabbit.">Pharmacokinetics and safety of transcorneal iontophoresis of tobramycin in the rabbit.</a> Invest Ophthalmol Vis Sci. 29 (9), 1397-1401 (1998).</li><li>Vinciguerra, 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=Transepithelial+iontophoresis+corneal+collagen+cross-linking+for+progressive+keratoconus:+initial+clinical+outcomes.">Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes.</a> J Refract Surg. 30 (11), 746-753 (2014).</li><li>Caporossi, A., 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=Riboflavin-UVA-induced+corneal+collagen+cross-linking+in+pediatric+patients.">Riboflavin-UVA-induced corneal collagen cross-linking in pediatric patients.</a> Cornea. 31 (3), 227-231 (2012).</li><li>Buzzonetti, L., Petrocelli, G., Valente, P., Larossi, G., Ardia, R., Petroni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iontophoretic+transepithelial+corneal+cross-linking+to+halt+keratoconus+in+pediatric+cases:+15-month+follow-up.">Iontophoretic transepithelial corneal cross-linking to halt keratoconus in pediatric cases: 15-month follow-up.</a> Cornea. 34 (5), 512-515 (2015).</li><li>Baiocchi, S., Mazzotta, C., Cerretani, D., Caporossi, T., Caporossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+crosslinking:+riboflavin+concentration+in+corneal+stroma+exposed+with+and+without+epithelium.">Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium.</a> J Cataract Refract Surg. 35 (5), 893-899 (2009).</li><li>Wollensak, G., Iomdina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+histological+changes+after+corneal+crosslinking+with+and+without+epithelial+debridement.">Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement.</a> J Cataract Refract Surg. 35 (3), 540-546 (2009).</li><li>Soeters, N., Wisse, R. P., Godefrooij, D. A., Imhof, S. M., Tahzib, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+versus+epithelium-off+corneal+cross-linking+for+the+treatment+of+progressive+keratoconus:+a+randomized+controlled+trial.">Transepithelial versus epithelium-off corneal cross-linking for the treatment of progressive keratoconus: a randomized controlled trial.</a> Am J Ophthalmol. 159 (5), 821-828 (2015).</li></ol>

The authors have nothing to disclose.

CXL mit UVA-Bestrahlung und Riboflavin ist die Standardtherapie für die Verhaftung der Progression des Keratokonus. Riboflavin ist ein Photosensibilisator, die chemische kovalente Bindungen (Querverbindungen) induziert, wenn sie mit UV-A-3 bestrahlt. In der Hornhaut, erzeugt dieses Phänomen Vernetzungen zwischen Kollagenfibrillen, die Hornhaut Steifigkeit zu erhöhen. Obwohl dieses Phänomen ist gut beschrieben, bis jetzt hat es keine direkten Beweise für intrakorneale Vernetzungen. Dennoch haben mehrere S...

Keratokonus ist eine bilaterale und progressive Hornhaut Ektasie der Regel in 1 in 2.000 in der allgemeinen Bevölkerung 1 was zu Modifikation der Form der Hornhaut ausgewiesen und damit verminderte Sicht 2. Keratokonus ist in der Regel in der frühen Pubertät Gegenwart und fortschreitet, bis die dritten bis vierten Lebensjahrzehnt, wenn die Krankheit in der Regel dazu neigt, sich zu stabilisieren, wenn auch das Fortschreiten kann während des gesamten Lebens des Patienten variabel sein. Durch Anhalten Keratokonus Progression soll Vernetzung bei aufzuschieben oder zu vermeiden Keratoplastik. Bisher die einzige effiziente und sichere Behandlung von progressiven Keratokonus in klinischen Studien nachgewiesen ist die konventionelle Hornhautkollagenvernetzung (C-CXL) Protokoll, das zur Erhöhung der Steifigkeit und damit zu stoppen Keratokonus Progression 3-8 zielt. Um den Betrieb Zeit und andere mögliche Risikofaktoren der C-CXL wie infektiöse Keratitis oder stromale Trübung 9 zu reduzieren, mehrere verbesserte Protokollebeschrieben worden. Zuerst wird in beschleunigter CXL (A-CXL), eine höhere Strahlungsintensität von UVA wird auf die Hornhaut über einen verringerten Zeit 10 geliefert. Zweitens, um die Notwendigkeit für epitheliale Debridement zu vermeiden, wurden transepithelialen Ansätze verwendet worden. Leider haben sie nur begrenzten Erfolg, verglichen mit dem herkömmlichen Protokoll 11. Die jüngste transepithelialen Verfahren zur Hornhaut Riboflavin Lieferung im Laufe CXL ist die Iontophorese (I-CXL), aber rigorose Evaluierung dieser Behandlung wurde noch nicht durchgeführt 12. Iontophorese ist eine nicht-invasive Technik, bei der ein kleiner elektrischer Strom angelegt wird, um das Eindringen eines ionisierten Medikaments durch ein Gewebe zu verbessern. In CXL durch Iontophorese wird das Riboflavin ionisiert, um die Hornhaut durch das Epithel zu durchdringen. In-vivo-Konfokalmikroskopie (IVCM) ist ein Verfahren zur Abbildung der Kornea, das die zelluläre Veränderungen abnormer Hornhäute in Krankheiten wie Keratokonus 13 hervorheben können. Tatsächlich IVCMhat Änderungen auf alle Schichten der Hornhaut bei Keratokonus mit einer bestimmten Verringerung in der Dichte der Untergrund Nervenplexus demonstriert und stromalen Keratozyten 13-15. Plus IVCM hat sich als sehr günstig für Mikrostrukturanalyse der Hornhaut nach der C-CXL 16. Die Hornhaut-Demarkationslinie als hyperreflektive Linie in vorderen Segment der optischen Kohärenztomographie (OCT AS) gesehen 1 Monat nach der C-CXL in einer Tiefe von 300 um 17,18 beschrieben. IVCM folgenden C-CXL liefert Informationen über Hornhaut strukturelle Veränderungen, einschließlich der Abwesenheit von cornealen Keratozyten zu einer Tiefe von 300 um. Die Tiefe dieser zellfreien Zone, wie auch die Tiefe der Demarkationslinie innerhalb der Hornhautstroma zeigte auf AS Oktober, scheint mit der effektiven Tiefe des CXL-Behandlung 19, und die Messung der Hornhaut Demarkationslinie Tiefe in AS 1. Oktober in Verbindung gebracht werden Monat nach CXL hat sich als effiziente klinische vorgeschlagenVerfahren zur Bewertung der Wirksamkeit CXL 18. In der vorliegenden Studie untersuchen wir die Wirksamkeit von drei verschiedenen Protokollen der Hornhautkollagenvernetzung (konventionell, beschleunigt und Iontophorese) mit Messung der Hornhaut Stromatumoren Demarkationslinie von AS Oktober und konfokale Mikroskopie. Wir weiterhin verwendet IVCM um quantitativ zu analysieren Hornhaut-Mikrostruktur Veränderungen nach drei Behandlungen.

<table>
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Riboflavin&#160;&#160;&#160;&#160;&#160;&#160;&#160; Product number</td>
			<td></td>
		</tr>
		<tr>
			<td>C-CXL</td>
			<td>Sooft SPA, Montegiorgio, Italy</td>
			<td>Ricrolin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 468465-6</td>
			<td></td>
		</tr>
		<tr>
			<td>A-CXL</td>
			<td>Avedro Inc, Waltham, Massachusetts</td>
			<td>VibeX&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 520-01863-006</td>
			<td></td>
		</tr>
		<tr>
			<td>I-CXL</td>
			<td>Sooft SPA, Montegiorgio, Italy</td>
			<td>Ricrolin+&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 975481-6</td>
			<td>Passive electrode: PROTENS ELITE 4848LE/ Active electrode: IONTOFOR CXL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>UVA Machine</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Vega</td>
			<td></td>
			<td></td>
			<td>UVA: 3 mW/cm2 30 min</td>
		</tr>
		<tr>
			<td>KXL System</td>
			<td></td>
			<td></td>
			<td>UVA: 30 mW/cm2 10 min</td>
		</tr>
		<tr>
			<td>X-Vega</td>
			<td></td>
			<td></td>
			<td>UVA: 10 mW/cm2 9 min</td>
		</tr>
	</tbody>
</table>

three different protocols of corneal collagen crosslinking in keratoconus: conventional, accelerated and iontophoresis

Keratokonus ist eine bilaterale und progressive Hornhaut Ektasie. Um ihr Fortschreiten verlangsamen, hat Hornhautkollagenvernetzung (CXL) kürzlich als effiziente Behandlungsmöglichkeit eingeführt. In der biologischen und chemischen Wissenschaften, bezieht sich Vernetzung, neue chemische Bindungen zwischen reaktiven Molekülen gebildet. Daher ist das Ziel der Hornhautkollagen CXL, um die Bildung von Vernetzungen zwischen den Kollagenfibrillen in dem Hornhautstroma synthetisch erhöhen. Trotz der Tatsache, dass der Wirkungsgrad des herkömmlichen CXL (C-CXL) Protokoll ist bereits in mehreren klinischen Studien konnte gezeigt werden, könnte es aus Verbesserungen der Dauer des Verfahrens und die Entfernung von Hornhautepithel nutzen. Daher, um eine zusammenhängende Evaluierung der beiden neuen und optimierten CXL Protokolle bieten, untersuchten wir Keratokonus Patienten, die eine der drei CXL-Behandlungen unterzogen hatten: Iontophorese (I-CXL), beschleunigte CXL (A-CXL) und herkömmlichen CXL ( C-CXL). A-CXL ist eine 6 mal schneller CXL Verfahren usingen ein zehn Mal höhere UVA-Strahlung, aber immer noch mit einem Epithel Entfernung. Iontophorese ist eine transepitheliale nichtinvasive Technik, bei der ein kleiner elektrischer Strom angelegt wird, um das Eindringen von Riboflavin in der gesamten Hornhaut zu verbessern. Mit Hilfe des vorderen Augenabschnitts der optischen Kohärenztomographie (AS Oktober) und in vivo konfokale Mikroskopie (IVCM) schließen wir, dass in Bezug auf die Tiefe der Behandlung Penetration bleibt herkömmlichen CXL-Protokoll zum Standard für die Behandlung von progressiven Keratokonus. Beschleunigte CXL scheint eine schnelle, effektive und sichere Alternative zu dünner Hornhaut zu behandeln. Die Anwendung der Iontophorese wird noch untersucht und sollte mit größerer Vorsicht betrachtet werden.

<html> Die Hornhaut Demarkationslinie war in AS Oktober sichtbar in 92% der Fälle bei einer mittleren Tiefe von 301,6 &amp; mgr; m (SD, 73.6) <img alt="Figur 5" src="/files/ftp_upload/53119/53119fig5.jpg" /> Abbildung 5. Trennungslinie nach der C-CXL. Hochauflösende Hornhaut vorderen Segment der optischen Kohärenztomographie-Scan (AS OCT) Visualisierung der Hornhaut-Stroma-Demarkationslinie bei einer mittleren Tiefe von 358 &amp; ...

Watch this Scientific Journal Video about Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis at JoVE.com

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

Drei Verschiedene Protokolle der Hornhautkollagenvernetzung in Keratokonus: Konventionelle, beschleunigte und Iontophorese

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

three-different-protocols-corneal-collagen-crosslinking-keratoconus

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JoVE Journal

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19.8K Aufrufe.  Quinze-Vingts National Ophthalmology Hospital. Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

Serie: Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Färbeprotokollen for Human Pankreasinseln

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Forschung

Medizin

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Eine Alkali-brennen Verletzungsmodell der Hornhaut Neovaskularisation bei der Maus

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Echokardiographische Ansätze und Protokolle für die umfassende phänotypischen Charakterisierung von Herzklappenerkrankungen bei Mäusen

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induktion von beschleunigter Atherosklerose bei Mäusen: Das "Wire-Injury"-Modell

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Epitheliale Korneaabnutzung mit okulärer Burr als Modell für die Hornhaut Wundheilung

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

Herz-Magnetresonanz zur Beurteilung von vermutetem Herzthrombus: Konventionelle und aufkommende Techniken

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Ein Porcine Corneal Endothelial Organ Culture Modell mit Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, hoarseness or asthma, which can be caused by laryngopharyngeal reflux (LPR). Due to their multifactorial origin, those symptoms can be a great diagnostic and therapeutic challenge. Esophageal pH-monitoring is commonly used to determine abnormal esophageal acid exposure and confirm the diagnosis of GERD. However, for better evaluation of acid exposure above the upper esophageal sphincter, a new laryngopharyngeal pH measurement system is now available and may lead to more reliable results in patients with predominantly extraesophageal symptoms. This article aims to present a standardized protocol for simultaneous pH measurement using esophageal and laryngopharyngeal pH probes in order to obtain acid exposure scores from both measurements.

Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously.

Simultane Laryngopharynx- und konventionelle pH-Überwachung der Speiseröhre

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, ...

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

3D-Bildgebung von PDL-Kollagenfasern während kieferorthopädischer Zahnbewegungen im Unterkiefermine modell

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Dreidimensionale Kollagenmatrix-Gerüstimplantation als Leberregenerationsstrategie

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...