Name: whole vitreous humor dissection for vitreodynamic analysis
Uploaded: 2015-05-24T14:00:00.000+00:00
Duration: 4 min 41 s
Description: Watch this Scientific Journal Video about Whole Vitreous Humor Dissection for Vitreodynamic Analysis at JoVE.com

The authors acknowledge the following funding sources, Whittier Foundation, Harrington Foundation, National Institutes of Health and Research to Prevent Blindness.

Alle Augen enukleiert wurden von einem Schlachthaus erhalten, und alle Experimente wurden in Übereinstimmung mit institutionellen Biosicherheit Gesetze durchgeführt. <ol><li> Sichere entkernte Auge nach unten auf eine Oberfläche. Tun Sie dies, indem Gewebestifte durch das überschüssige Gewebe um das Auge herum und befestigen Sie sie nach unten in eine Styroporplatte. </li><li> Sezieren und nehmen perilimbal Bindehaut aus dem Auge. <ol><li> Verwenden feinen Pinzette (0.3 Pinzette) und Mikroschere (Westcott Schere), die Bindehaut am Limbus einzuschneiden und stumpf sezieren von der Lederhaut. Schneiden Sie die Bindehaut entlang des Limbus, wie der stumpfe Präparation schreitet weiter Dissektion ermöglichen. </li><li> Entfernen Binde ganzen Weg rund um das Auge (360 Grad), um so viel wie möglich Sklera freizulegen. HINWEIS: Es ist hilfreich, eine kleine Menge von Bindehaut zu verlassen, um zu erleichtern Fixierung des Auges mit den chirurgischen Stiften oder einer Pinzette während der Rest des Verfahrens. </li></ol></li><li> Erstellen Sie eine volle Dicke Skleralappen entlang einerSeite des Auges (Skalpellklinge 69). <ol><li> Einen ~ 5 mm Skleraeinschnitt parallel zum Limbus und 3 mm hinter dem Limbus durch leichtes Einschneiden der Sklera mit dem Skalpell (Skalpell 11) bis dunkel pigmentierten Chorioidea sichtbar. Achten Sie darauf, die Aderhaut selbst einzuschneiden. </li><li> Sorgfältig seziert entlang der Ebene zwischen der Sklera und der Choroidea, entweder mit einer Schere (unter Verwendung stumpf) oder mit dem Skalpell, bis es möglich ist, das Potential Raum zwischen diesen Geweben zu vergrößern, indem der Choroidea sanft nach innen. </li><li> Eine weitere Skleraeinschnitt senkrecht zur ersten Skleraeinschnitt mit scharfen Mikroscheren, wodurch eine T-förmige Einschnitt. </li><li> Dann weiterhin unverblümt sezieren in Umfangs Mode, um die Skleragewebe von der darunter liegenden Aderhaut zu entfernen, und schieben Sie die Aderhaut weg von der Lederhaut, wie oben erwähnt. </li><li> Vergrößern Sie die Lederhautlappen nach Bedarf. <ol><li> Sanft leckersten Blunt der Aderhaut weg von der Lederhaut zu sezierenwährend des gesamten Prozesses. </li><li> Vergrößern der Klappe bis der Schnitt senkrecht zu dem Limbus vorgenommen erreicht den Sehnerv und der Einschnitt parallel zum Limbus ist zumindest ein Drittel des Auges Umfangs (45 °). </li><li> Verwenden Sie die Lederhautlappen als Quelle der Hebelwirkung für die Manipulation des Auges während stumpf. Die Gegen Traktion auf der Klappe erleichtert es stumpf. </li></ol></li><li> Wiederholen Sie diesen Schritt auf der anderen Lederhautlappens. </li></ol></li><li> Schneiden Sie die Lederhautlappen um einen großen Bereich der Aderhaut freizulegen. </li><li> Weiterhin den Einschnitt in Schritt 3, die um das Auge des Umfangs (360 Grad) aus. </li><li> Entfernen Sie die verbleibenden Aderhaut-Retina-Gewebe. <ol><li> Innerhalb der freigelegten Bereich, mit einem Watte-Spitze vorsichtig abbürsten die verbleibende Aderhaut-Retina-Gewebe. Alternativ sanft greifen das Gewebe mit einer Pinzette und abziehen der darunter liegenden Glaskörper. </li><li> Falls erforderlich einen Einschnitt in die Aderhaut starting vom Sehnerven. Dann abziehen der Aderhaut sanft, um die Netzhaut und Glaskörper freizulegen. </li></ol></li><li> Weiter stumpfes Sezieren der Aderhaut weg von der Lederhaut und dann abziehen der Aderhaut, die ganze, intakte Glaskörper zu erhalten. </li><li> Verwenden Sie die Sklerarand an der Hornhaut angebracht, um die gesamte Glaskörper in gewünschten Stelle zu positionieren. An diesem Punkt wird der Glaskörper an der vorderen Sklera und Objektiv. </li><li> Blunt den Glaskörper von der Innenseite der Sklera zu zergliedern, um die Linse, Entfernen aller Sklera. </li><li> Verwenden Sie ein stumpfes Werkzeug, um die Linse von der Glaskörper schöpfen, wenn es sein muss. Verwenden Sie die Probe für verschiedene vitreodynamic Experimente. <ol><li> Man gibt die Probe in einem Becherglas mit bekannter Oberfläche der Luft ausgesetzt. Platzieren Sauerstoff empfindliche Sonde an der Kante der Probe. Verwenden Sie einen Mikromanipulator, um die Sonde zu einem bekannten Abstand (r) in die Probe zu bewegen. </li><li> Verwenden Fickschen Gesetzen der Diffusion, die theoretische Diffusionsgleichung erhalten. Mit dem data in Schritt 10.1 gesammelt, erhalten den experimentellen Diffusionskoeffizient / Reaktionszeit usw. </li></ol></li></ol>

<ol>
	<li>Siggers, J. H., Ethier, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+Mechanics+of+the+Eye.">Fluid Mechanics of the Eye.</a> Annual Review of Fluid Mechanics. 44 (1), 347-372 (2012).</li><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+human+vitreous+structure.">Age-related changes in human vitreous structure.</a> Graefes Arch Clin Exp Ophthalmol. 225 (2), 89-93 (1987).</li><li>Grignolo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrous+components+of+the+vitreous+body.">Fibrous components of the vitreous body.</a> AMA Arch Ophthalmol. 47 (6), 760-774 (1952).</li><li>Gisladottir, S., Loftsson, T., Stefansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+vitreous+humour+and+saline+solution+follow+the+Stokes+Einstein+equation.">Diffusion characteristics of vitreous humour and saline solution follow the Stokes Einstein equation.</a> G Graefes Arch Clin Exp Ophthalmol. 247 (12), 1677-1684 (2009).</li><li>Xu, J., Heys, J. J., Barocas, V. H., Randolph, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+diffusion+in+vitreous+humor:+implications+for+drug+delivery.">Permeability and diffusion in vitreous humor: implications for drug delivery.</a> Pharm Res. 17 (6), 664-669 (2000).</li><li>Watts, F., Tan, L. E., Wilson, C. G., Girkin, J. M., Tassieri, M., Wright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+micro-rheology+of+the+vitreous+humor+using+an+optically+trapped+local+probe.">Investigating the micro-rheology of the vitreous humor using an optically trapped local probe.</a> Journal of Optics. 16 (1), 015301 (2014).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+human+vitreous+body+elements+for+proteomic+analysis.">Dissection of human vitreous body elements for proteomic analysis.</a> J Vis Exp. (47), e2455 (2011).</li><li>Abdallah, W., Ameri, H., 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=Vitreal+oxygenation+in+retinal+ischemia+reperfusion.">Vitreal oxygenation in retinal ischemia reperfusion.</a> Invest Ophthalmol Vis Sci. 52 (2), 1035-1042 (2011).</li><li>Goldenberg, D., Trese, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+vitreodynamics:+what+is+it?+Why+is+it+important.">Pharmacologic vitreodynamics: what is it? Why is it important.</a> Expert Review of Ophthalmology. 3 (3), 273-277 (2008).</li><li>Choonara, Y. E., Pillay, V., Danckwerts, M. P., Carmichael, T. R., du Toit, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+implantable+intravitreal+drug+delivery+technologies+for+the+treatment+of+posterior+segment+eye+diseases.">A review of implantable intravitreal drug delivery technologies for the treatment of posterior segment eye diseases.</a> J Pharm Sci. 99 (5), 2219-2239 (2010).</li><li>Balachandran, R. K., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+drug+delivery+to+the+posterior+eye:+effect+of+active+transport+and+loss+to+choroidal+blood+flow.">Computer modeling of drug delivery to the posterior eye: effect of active transport and loss to choroidal blood flow.</a> Pharm Res. 25 (11), 2685-2696 (2008).</li><li>Smith, C. a., Newson, T. 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=A+framework+for+modeling+ocular+drug+transport+and+flow+through+the+eye+using+micro-CT.">A framework for modeling ocular drug transport and flow through the eye using micro-CT.</a> Phys Med Biol. 57 (19), 6295-6307 (2012).</li><li>Quiram, P. A., Leverenz, V. R., Baker, R. M., Dang, L., Giblin, F. J., Trese, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplasmin-induced+posterior+vitreous+detachment+affects+vitreous+oxygen+levels.">Microplasmin-induced posterior vitreous detachment affects vitreous oxygen levels.</a> Retina. 27 (8), 1090-1096 (2007).</li><li>Shui, Y., Holekamp, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+state+of+the+vitreous+and+ascorbate-dependent+oxygen+consumption:+relationship+to+the+etiology+of+nuclear+cataracts.">The gel state of the vitreous and ascorbate-dependent oxygen consumption: relationship to the etiology of nuclear cataracts.</a> Arch Ophthalmol. 127 (4), 475-482 (2009).</li><li>Burk, S. E., Da Mata, A. P., Snyder, M. E., Schneider, S., Osher, R. H., Cionni, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+vitreous+using+kenalog+suspension.">Visualizing vitreous using kenalog suspension.</a> J Cataract Refract Surg. 29 (4), 645-651 (2003).</li><li>Spaide, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Posterior+Vitreous+with+Dynamic+Focusing+and+Windowed+Averaging+Swept+Source+Optical+Coherence+Tomography.">Visualization of the Posterior Vitreous with Dynamic Focusing and Windowed Averaging Swept Source Optical Coherence Tomography.</a> Am J Ophthalmol. S0002-9394 (14), 00537-00536 (2014).</li><li>Domalpally, A., Gangaputra, S., Danis, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diseases of the Vitreo-Macular Interface. 21, 21-27 (2014).</li><li>Stocchino, R., Repetto, A., Cafferata, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+vitreous+humour+motion+within+a+human+eye+model.">Experimental investigation of vitreous humour motion within a human eye model.</a> Phys Med Biol. 50 (19), 4729-4743 (2005).</li></ol>

The authors have nothing to disclose.

Es gibt zwei wichtige Schritte, die sorgfältig im Glaskörper Dissektion durchgeführt werden müssen. Schritt 3, der eine gesamte Dicke Lederhautlappen, ist von entscheidender Bedeutung für die gesamte Sektion. Achten Sie darauf, nicht in die Aderhaut schneiden bei der Erstellung der gesamten Dicke Lederhautlappen werden. Die andere wichtige Schritt ist Sezieren entfernt die Lederhaut von der Aderhaut. Dieser Schritt muss sorgfältig durchgeführt werden Erstellen mehrerer Löcher in der Aderhaut, aus der die Glaskö...

Das Ziel dieser Methode ist es, detaillierter eine Technik, um eine ganze, intakte Glaskörper zu isolieren, mit dem glasartigen Kern und Cortex intakt von einem kadaver Auges, für die Zwecke der vitreodynamic Analyse. Da der Bereich der Glaskörper Physiologie hat, multidisziplinären Forscher, wie der Strömungsmechanik Forscher geworden, untersuchen die physikalischen und biomechanischen Eigenschaften des Glaskörpers 1. Zu diesem Zweck ist es wesentlich, detailliert eine Technik, die ganze, intakte Glaskörper zu isolieren, um multidisziplinäre Forscher unterstützen. Sebag et al. 2 und 3 durchgeführt, andere elegant ganze Glaskörper Dissektionen auf menschliche kadaver Augen und zeigte Darstellungen der Ergebnisse. Jedoch wurde die Technik verwendet, nicht im Detail, und Nicht-Experten beschrieben nicht in der Lage ist, das Verfahren unabhängig replizieren. Andere Studien haben Glaskörper aus kadaver Augen mit einfacheren Methoden wie Aspiration oder Teilzerlegung geerntet,von denen beide nicht in eine ganze, intakte Glaskörper führen. Gisladottis et al. 4 und Xu et al. 5 zu untersuchen Lässigkeit in Glaskörper aus kadaver Augen geerntet. Da jedoch kein Verfahren aus glasartigem Extraktion wurde beschrieben, wurde angenommen, daß sie die Glaskörperflüssigkeit mit einer Spritze abgesaugt. Watts et al. 6 ging noch einen Schritt weiter, indem ein Verfahren zur Isolierung Kaninchen Glaskörper mit einem Operationstechnik beschreibt. Führt dieses Verfahren jedoch in einer Isolation von nur dem Glaskern und dem Glaskörper nicht. Skeie et al. 7 später organisierte den Glaskörper in 4 einzigartigen Regionen und ein Verfahren beschrieben, um jeden Teil zur Analyse sezieren elegant. Diese Technik jedoch nicht in einer intakten Glaskörper als Ganzes führen. Die aktuelle Technik wurde entwickelt, um die biophysikalischen Experimenten, die derzeit nur in kadaver Augen durchgeführt werden, zu erleichtern. Früheren Verfahren, beschrieben alsBove, sind begrenzt, da 1) keine die gesamte Glaskörper vollständig zu isolieren, 2) geerntet glasigen Kern und Cortex homogenisiert, 3) glasartigen anatomischen Struktur nicht beibehalten wird, oder 4) Dissektion Techniken sind nicht ausreichend für die Replikation von Forschern in anderen Bereichen detailliert . Darüber hinaus aufgrund der Opazität des Sklera und Aderhaut, die Visualisierung des Glaskörpers in der intakten Augapfels beschränkt. Dies begrenzt die Genauigkeit und Realisierbarkeit von Messungen, die innerhalb des ganzen Auges gemacht werden können. Zusätzlich können die anatomischen Strukturen umgebenden Glaskörper das Studium der biochemischen und physikalischen Eigenschaften der glasartigen verwechseln. In den letzten Jahren hat der Körper aus glasartigem Wissenschaft enorm gewachsen und es gibt Grund zu glauben, dass die gesamte Glaskörper hat andere Eigenschaften als ihre Einzelteile. Es gibt ein wachsendes Interesse bei der Untersuchung der physikalischen, biomechanische und chemischen Eigenschaften des Glaskörpers für vitreodynamics research, die Anwendungen in der klinischen Medizin, wie Arzneimittelabgabe, intravitreale Sauerstoffversorgung 8 und Vitrektomie hat. Pharmakologische vitreodynamics, das pharmakologische Mittel verwendet, um den Glaskörper zu manipulieren, können zur Vitrektomie Ergebnisse 9 zu verbessern. Biomechanischen Eigenschaften verwendet werden, um glasartige Fluidströmung, die verwendet werden können zur Verbesserung der intravitrealen Darreichungsformen 10-12 modellieren. Die physikalischen Eigenschaften der verschiedenen Segmente des Glaskörpers sind entscheidend für das Verständnis vitreoretinalen Sauerstofftransport 13. Die vorgeschlagene glasigen Dissektionstechnik verwendet, um verschiedene Eigenschaften des intakten Glaskörper zu untersuchen. Es ermöglicht Tischexperimente auf ganze, intakte Glaskörper mit besseren Visualisierung erfolgen. Zusammenfassend aktuellen Methoden zur Untersuchung des Glaskörpers sind entweder nicht hinreichend beschrieben, oder führen zu einer unvollständigen Trennung der Glaskern und Kortex. Daher besteht ein Bedarf, e auszuführenXperiments in einem transparenten Augenmodell unter Beibehaltung der Anatomie des Glaskörpers, die im kadaver Auge vorliegt.

<table><tbody><tr><td>0.3 forceps</td><td>Storz Opthalmics</td><td>E1793</td><td></td></tr><tr><td>Westcott Tenotomy Scissors Curved Right</td><td>Storz Opthalmics</td><td>E3320 R</td><td></td></tr><tr><td>Scalpel Handle No. 3</td><td>VWR</td><td>25607-947</td><td></td></tr><tr><td>Scalpel Blade, #11, for #3 Handle</td><td>VWR</td><td>470174-844</td><td></td></tr></tbody></table>

whole vitreous humor dissection for vitreodynamic analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

Anlehnung an das Protokoll zu einem erfolgreichen glasigen Dissektion mit dem Kern und Cortex (Abbildung 3) intakt zu führen. Dies ergibt sich aus den Reststücken Retina in den Glas cortex haftet. Intakte ganze Glaskörper kann auf verschiedene Weise für spezifische vitreodynamic Experimente eingesetzt werden. In unserem Fall ist die Diffusionsgeschwindigkeit von Sauerstoff in intakten Glaskörper und entsprechende Zeitkonstante wurde (Figur 2) untersucht. Glaskörper, die präparier...

Watch this Scientific Journal Video about Whole Vitreous Humor Dissection for Vitreodynamic Analysis at JoVE.com

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Ganze Glaskörper Dissection für Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

whole-vitreous-humor-dissection-for-vitreodynamic-analysis

Research

JoVE Journal

Medicine

11.3K Aufrufe.  University of Southern California. The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Serie: Whole Vitreous Humor Dissection for Vitreodynamic Analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Dissection of Human Glaskörper Elemente für Proteomanalyse

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Forschung

Medizin

Diagnose des vitreoretinalen Lymphoms mit zellfreier DNA

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Autor im Rampenlicht: Weiterentwicklung der VRL-Diagnose durch zellfreie DNA-Extraktion aus Glaskörper 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

Zellfreie DNA-Extraktion von Glaskörper- und Kammerwasserproben zur Diagnose und Überwachung des vitreoretinalen Lymphoms

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...

Krebsforschung

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

Ausweiden der Maus Glaskörper und Netzhaut für Proteomik-Analysen

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Biologie

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Eine Schritt für Schritt-Protokoll für Subretinale Chirurgie in Kaninchen

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Neurowissenschaften

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Vollkreiskauterisierung des limbalen Gefäßplexus bei chirurgisch induziertem Glaukom bei Nagetieren

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinale Pigmentepitheltransplantation in einem nicht-menschlichen Primatenmodell für degenerative Netzhauterkrankungen

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

OCT-basierte multimodale Bildgebung von menschlichen Spenderaugen für die AMD-Forschung

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Autor im Rampenlicht: Ex-vivo-OCT-basierte multimodale Bildgebung von menschlichen Spenderaugen für die Erforschung der altersbedingten Makuladegeneration  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

Ex Vivo OCT-basierte multimodale Bildgebung menschlicher Spenderaugen zur Erforschung der altersbedingten Makuladegeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

Ganze Glaskörper Dissection für Vitreodynamic Analysis

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Dissection of Human Glaskörper Elemente für Proteomanalyse

Zellfreie DNA-Extraktion von Glaskörper- und Kammerwasserproben zur Diagnose und Überwachung des vitreoretinalen Lymphoms

Zellfreie DNA-Extraktion von Glaskörper- und Kammerwasserproben zur Diagnose und Überwachung des vitreoretinalen Lymphoms

Zellfreie DNA-Extraktion von Glaskörper- und Kammerwasserproben zur Diagnose und Überwachung des vitreoretinalen Lymphoms

Ausweiden der Maus Glaskörper und Netzhaut für Proteomik-Analysen

Eine Schritt für Schritt-Protokoll für Subretinale Chirurgie in Kaninchen

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Vollkreiskauterisierung des limbalen Gefäßplexus bei chirurgisch induziertem Glaukom bei Nagetieren

Retinale Pigmentepitheltransplantation in einem nicht-menschlichen Primatenmodell für degenerative Netzhauterkrankungen

Ex Vivo OCT-basierte multimodale Bildgebung menschlicher Spenderaugen zur Erforschung der altersbedingten Makuladegeneration