This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

Das vorgestellte Verfahren nach einem von der Institutional Review Board des Academic Medical Center Amsterdam, Registriernummer NL41985.018 genehmigten Forschungsprotokoll statt. Eine schriftliche Einverständniserklärung ist von allen Beteiligten erforderlich. 1. System <ol><li> Für dieses Experiment wurde mit einem Fourier-Domain-OCT-System, die bei einer 1,280-1,350 nm Wellenlängenband 13. Fourier-Bereich Niederkohärenz-Interferometrie ermöglicht eine kontinuierliche Abtastung, die im Vergleich zu der ersten Generation der Zeitdomäne OCT-Systeme die Datenerfassungsgeschwindigkeit erhöht. Hinweis: Das OCT-System mit einem faseroptischen Sonde schraubenförmig bei ~ 90 ° angeschlossen, Scannen. Sie hat einen Außendurchmesser von 2.7F (0,9 mm) und eine einführbare Länge von 135 cm. Die Sonde verbindet sich mit dem Oktober-Konsole über einen Antriebsmotor und Lichtsteuerung (Montage Dock) mit einem Pullback Bereich von 54 mm. Die erworbenen Oktober Datensätze bestehen aus 541 Schnittbilder (B-Bilder) with eine axiale Auflösung von 15 um (1, 2). </li><li> Um genaue und reproduzierbare Dämpfungsmessungen zu gewährleisten, kalibriert durch Messen μ OCT zur steigenden Konzentrationen bezogen auf Gewichtsprozent einer Fettemulsion, (zum Beispiel Intralipid), wie zuvor von Kodach et al. 14, 15 beschrieben. Kurz gesagt: <ol><li> Verdünnen Sie eine Standard-Charge von 20% Fettemulsion mit VE-H 2 O, um Konzentrationen von 0,125, 0,250, 0,5, 1,0, 2,0, 4,0, 10, 15 und 20 (Lager) Prozent. <ol><li> Setzen Sie den OCT-Sonde in 200 ml Fettemulsion Gemischs und erwerben ein OCT-Messung. </li><li> Querverweis extrahiert μ Oktober Werte mit bekannten Werten in der Literatur. </li></ol></li></ol></li></ol> 2. Time Out und Patientenlagerung <ol><li> Vor dem Start des Verfahrens, führen Sie eine &quot;Auszeit&quot; Check-Name, Geburtsdatum, Verfahren, procedural Seite Antikoagulans Verwendung und Allergien. </li><li> In Abhängigkeit von der Tumorlokalisation, legen Sie die Patienten entweder in Bauch- oder Seitenlage. Geben Sie dem Patienten mit angemessener Unterstützung und überprüfen, ob er / sie geht davon aus bequem in dieser Position über einen 20-40 min Periode sein. </li><li> Mit Ultraschall (US) 16, den Tumor zu lokalisieren und markieren Sie die Nadeleinstichpunkt auf der Haut mit dauerhafter Tinte. HINWEIS: Bei der Verwendung von Computertomographie (CT), mit einem flexiblen Nadelführung Schablone, um die bevorzugte Position der Zugangsnadel zu lokalisieren. </li></ol> 3. Desinfektion und sterilen Abdeckung <ol><li> Setzen Sie auf einen chirurgischen Kappe und Mundschutz. </li><li> Reinigen Sie die Haut um die Einstichstelle mit einem Chlorhexidin / Alkohol-Lösung, kümmert sich nicht um die zuvor platzierten Nadeleinstich Markierung zu entfernen (Schritt 2.3). Desinfektions einen weiten Bereich wird die Notwendigkeit für eine zusätzliche Reinigung bei unerwarteten Zugangsnadel Neupositionierung zu verhindern. </li><li> Mit reGard der sterilen Inhalt, öffnen Sie die perkutane Punktion Set enthält: eine 10-ml-Spritze, eine stumpfe Punktionsnadel, eine 21 G Injektionsnadel, ein Skalpell, eine 15 G koaxiale Führungsnadel, eine 18 G Trokarnadel und eine 16 G Kernbiopsiepistole. </li><li> Hände gründlich waschen, Anwendung Händedesinfektion danach. Setzen Sie auf einen OP-Kittel und sterile Handschuhe. </li><li> Bedecken Sie den Patienten in sterilen Tüchern. </li><li> Tragen Sie eine sterile Abdeckung um die Ultraschallsonde und befestigen Sie die Nadelführung an Ort und Stelle. </li></ol> 4. Oktober Vorbereitung <ol><li> Starten Sie das OAT-Konsole und geben Sie die Patientendaten in den Bereichen Patienten-ID bezeichnet, Nachname, Vorname und DOB (Geburtsdatum) mit Hilfe der Konsolenschnittstelle. </li><li> Hinsichtlich des sterilen Inhalts, entpacken Sie die Oktober-Paket enthält ein OCT-Sonde, eine sterile Dock Befestigungsabdeckung und eine 5 ml Luer-Lock-Spritze. </li><li> Übernehmen Sie die sterile Abdeckung am Oktober Konsolenmontage Dock. Führung des nicht-sterilen Montage Dock erfordert dieHilfe eines Assistenten. </li><li> Füllen Sie den 5-ml-Spritze mit 0,9% NaCl und befestigen Sie es am Spülanschluss. Spülen Sie die Sonde Oktober bis Wasser im distalen Teil der Sondenhülle erscheint. </li><li> Laden Sie die Oktober-Sonde in die Montage Dock. Nach dem Laden der Sonde drehen und emittieren rotes Licht bestätigt reibungslose Funktionieren. Lassen Sie die Sonde in der Schutzhülle beim Spülen und Laden, um das Risiko von Schäden zu minimieren. </li><li> Entfernen Sie die Oktober-Sonde aus dem Cover. Legen Sie die Sonde auf eine harte Oberfläche und mit einem Skalpell, um die Spitze zu verkürzen. Fixieren den distalen Teil der Sonde während des Schneidens, um Druck auf die optische Faser und das Prisma zu minimieren. Schneiden 5 mm distal von Prisma, mit dem ausgesendeten (rot) Licht zur Orientierung. </li></ol> 5. Puncture <ol><li> Betäuben die Haut und tiefen Schichten mit 2% Lidocain (20 mg / ml). Warten Sie einige Minuten unter Berücksichtigung der Lidocain wirksam werden. Bitten Sie den Patienten, wenn es irgendeinen Schmerz. </li><li> Mit der Nadelführung, legen Sie die15 G koaxiale Führungsnadel Überprüfung der Position durch Bildgebung. Wenn Platzierung zufriedenstellend ist, entfernen Sie den Verschluss (scharfe Nadelkern). </li><li> Legen Sie die 18 G Trokarnadel durch die Punktionsnadel, Durchstechen der Tumor. Auch überprüft die Position der Nadel mit Bildgebung. Wenn die Platzierung ist zufriedenstellend entfernen Sie die Verschluss. </li><li> Führen Sie die Sonde bis Oktober die Trokarnadel, bis das Gefühl Widerstand. </li><li> Während die Festsetzung der OCT-Sonde einfahren Trokarnadel, Freilegung der OCT-Sonde, um das Tumorgewebe. Halten Sie die Spitze des Trokarnadel innerhalb des Tumors minimiert Abknicken des OCT-Sonde während Atemzyklen. Dies senkt das Risiko einer Schädigung Sonde. </li><li> Oktober Scan: <ol><li> Führen Sie einen OCT-Scan, mit der Konsole bei 541 B-Scans pro Datensatz gesetzt. Das hier verwendete OCT-System wird eine automatische Rückzieh über eine Länge von 5,4 cm benötigt keine speziellen Parametereinstellungen durchzuführen. </li><li> Überprüfen Sie die Scan-Qualität, Artefakte und das Aussehen der festen Gewebes (1A). Artefakte erscheinen am häufigsten als kreisförmige Bänder, sich von der normalen Oktober Muster (Abbildung 1B). </li><li> Bringen Sie die Sonde, wenn Artefakte besteht, nachdem erneut zu scannen. </li></ol></li><li> Wiederholen Sie die Schritte 5.6 bis mindestens 3. Oktober Datensätze übernommen. </li><li> Entfernen Sie die Sonde und Oktober Trokarnadel, so dass die Punktionsnadel in-place. </li><li> Der Arm des Kernbiopsiepistole und legen Sie sie durch die Führungsnadel, die Überprüfung der Lage auf Bildgebung. </li><li> Wenn die Positionierung ist zufriedenstellend, Feuer der Biopsiepistole. </li><li> Zeigen Biopsiematerial in Behälter nach Pathologie Protokoll. Hier Ort Biopsien auf eine Petrischale mit einem Papiereinleger, ausreichend mit 0,9% NaCl gesättigt. </li><li> Überprüfen Sie die Stanzbiopsie Qualität und wiederholen Sie Schritt 5.9 und 5.10 bis genügend Material erhalten. </li></ol>

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Urol. 60, 501-515 (2011).</li><li>Ljungberg, 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=EAU+guidelines+on+renal+cell+carcinoma:+the+2010+update.">EAU guidelines on renal cell carcinoma: the 2010 update.</a> Eur. Urol. 58, 398-406 (2010).</li><li>Donat, S. 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=Follow-up+for+Clinically+Localized+Renal+Neoplasms.">Follow-up for Clinically Localized Renal Neoplasms.</a> AUA Guideline, J. Urol. 190, 407-416 (2013).</li><li>Faber, D. J., van der Meer, F. J., Aalders, M. C. G., van Leeuwen, T. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+tumorigenesis+in+urinary+bladder+with+optical+coherence+tomography:+optical+characterization+of+morphological+changes.">Detection of tumorigenesis in urinary bladder with optical coherence tomography: optical characterization of morphological changes.</a> Optics Express. 10, 1431-1443 (2002).</li><li>Barwari, K., 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=Differentiation+between+normal+renal+tissue+and+renal+tumours+using+functional+optical+coherence+tomography:+a+phase+I+in+vivo+human+study.">Differentiation between normal renal tissue and renal tumours using functional optical coherence tomography: a phase I in vivo human study.</a> BJU. 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T., 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=Volumetric+in+vivo+visualization+of+upper+urinary+tract+tumors+using+optical+coherence+tomography:+a+pilot+study.">Volumetric in vivo visualization of upper urinary tract tumors using optical coherence tomography: a pilot study.</a> J. Urol. 190, 2236-2242 (2013).</li><li>Wessels, R., 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=Optical+coherence+tomography+in+vulvar+intraepithelial+neoplasia.">Optical coherence tomography in vulvar intraepithelial neoplasia.</a> Journal of Biomedical Optics. 17, (2012).</li><li>Yun, S. H., Tearney, G. J., de Boer, J. F., Iftimia, N., Bouma, B. 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The authors of this article have nothing to disclose.

In dieser Veröffentlichung berichten wir über die Möglichkeit der perkutanen, Nadel Sitz Oktober der Niere. Dies ist ein wichtiger erster Schritt bei der Entwicklung der OCT in einer klinisch anwendbare Technik für Tumordifferenzierung als &quot;Optical Biopsy&quot; bezeichnet. Unsere ersten 25 Patienten wurden perkutane Oktober gezeigt, dass eine einfache und sichere Verfahren. Optisches Biopsie hat zwei Vorteile gegenüber herkömmlichen Stanzbiopsien. Zuerst wird die Echtzeit-Erfassung und Analyse von OCT-Daten l...

In den vergangenen Jahrzehnten haben einen stetigen Anstieg der Inzidenz von Nierentumoren 1,2 gezeigt. Bisher wurden Nierenmassenbehandlungsentscheidungen in erster Linie anhand der MRI und CT-Abbildungseigenschaften, Alter und Begleiterkrankungen vorliegen. Allerdings sind diese Diagnosemethoden und klinische Parameter fehlt die Raffinesse, um wirklich erfassen das maligne Potential eines Nierenmasse. Eine Stanzbiopsie oder Feinnadelpunktion mit genügend Gewebe für pathologische Auswertung (Diagnose) liefert objektive Tumordifferenzierung sowohl Sensitivität und Spezifität im Bereich von 95-100% 3. Daher Biopsie Akzeptanz bei der Bewertung von verdächtigen Nierentumoren 4,5. Allerdings Biopsien, ohne ausreichend Gewebe, um eine Diagnose oder bei normalen Nierenparenchym (non-Diagnose) treten mit einer Rate von 10-20% insgesamt, und sogar bis zu 30% in kleinen Nierentumoren (&lt;4 cm, SRM) aufzubauen, verzögert der Diagnoseprozess aufgrund der häufigen Notwendigkeit zusätzlicherBiopsie 3,5. Die optische Kohärenztomographie (OCT) ist ein neuartiges bildgebendes Verfahren, das das Potenzial, die oben genannten Hürden bei Nierenmassen Differenzierung überwunden hat. Basierend auf der Rückstreuung von Licht im nahen Infrarot liefert OCT-Bilder mit einer 15 um die axiale Auflösung bei einer wirksamen Gewebepenetration von 2-3 mm (Figur 1, 2). Der Verlust der Signalintensität pro Millimeter Gewebepenetration, einer Resultierenden von gewebespezifischen Lichtstreuung wird als Dämpfungskoeffizienten ausgedrückt (μ Okt: mm -1). Wie durch Faber et al 6 beschrieben. Histologischen Eigenschaften korreliert werden kann OCT Werte bietet eine quantitative Parameter zur Gewebedifferenzierung (Abbildung 3) μ. Während der Krebsentstehung, bösartige Zellen zeigen eine erhöhte Anzahl, größer und unregelmäßig geformte Kerne mit<html> einen höheren Brechungsindex und aktiver Mitochondrien. Durch diese Überexpression von Zellbestandteilen, ist eine Änderung in μ OAT erwarten beim Vergleich malignen Tumoren zu gutartigen Tumoren oder unberührt Gewebe 7. Kürzlich haben wir die Fähigkeit der oberflächlichen Oktober, zwischen gutartigen und bösartigen Nierentumoren 8,9 unterscheiden. Bei 16 Patienten wurden intraoperative Oktober Messungen von Tumorgewebe unter Verwendung eines extern platziert Oktober Sonde erhalten. Der Steuerarm OCT Messungen unbeeinflußt Gewebe in der gleichen Patienten umfaßt. Normalgewebe zeigten eine signifikant niedrigere mittlere Schwächungskoeffizient im Vergleich zu bösartigem Gewebe, bestätigt das Potenzial von OCT für Tumor-Differenzierung. Diese quantitative Analyse wurde in ähnlicher Weise in die Besoldungsgruppe andere Arten von bösartigem Gewebe, wie Urothelkarzinom 10,11 und Vulva epitheliale Neoplasie Differenzierung 12 angewendet. ent &quot;&gt; Wir wollen Oktober in eine optische Biopsie entwickelt, Echtzeit-Bildgebung mit on-the-spot Tumordifferenzierung kombiniert. Das Ziel der vorliegenden Studie ist es, eine perkutane, Nadel basierend beschreiben, Oktober Ansatz bei Patienten mit einer Diagnose feste steigernden Nierenmasse. Diese Methode Beschreibung ist unseres Wissens das erste, um die Möglichkeit von Nadel basierend Oktober von Nierentumoren zu bewerten.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

Unter den ersten 25 Tumoren (23 Patienten), wurden insgesamt 24 erfolgreiche Oktober Verfahren durchgeführt. In einem Fall führte eine Sonde eine Fehlfunktion der Unfähigkeit, einen OCT-Scan zu erwerben. Zwei Nebenwirkungen (AE) aufgetreten ist, die im Detail in der Diskussion beschrieben werden. Allgemeine Eigenschaften der Patienten sind in Tabelle 1 zu finden. Die OCT-Konsole vorinstalliert Software, die Echtzeit-OCT-Bilder für die sofortige qualitative Analyse der er...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

In Vivo, perkutane, Nadel Based, Optical Coherence Tomography von Nierentumoren

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

8.8K Aufrufe.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Serie: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Doppler Optical Coherence Tomography der retinalen Zirkulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Forschung

Medizin

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

Near Infrared Optical Projection Tomography für Assessments von β-Zellen Massenverteilung im Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generation und 3-Dimensional Quantifizierung der arterielle Läsionen in Mäuse mit optischen Projektionstomographie

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Optical Imaging von Hirntumoren und Arthritis mit fluoreszierenden SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Vorbereitung und Respirometrische Bewertung der Mitochondrien isoliert aus dem Skelettmuskelgewebe durch perkutane Nadelbiopsie Erhalten

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Transkutane Beurteilung der Nierenfunktion in Conscious Nagetiere

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depletion von Maus-Zellen aus menschlichem Tumorxenografte deutlich verbessert Downstream-Analyse von Zielzellen

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Höchste Auflösung Maus optische Kohärenztomographie, intraokulare Injektion in die retinale Gentherapie Forschung unterstützen

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Darstellung der Cx3cr1GLP/Gfp Reporter Mäuse mit Spectral-Domain optische Kohärenztomographie und Scanning Laser Ophthalmoskopie

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Beurteilung der Nierenfunktion in Mausmodellen der glomerulären Erkrankung

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...