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.

Tutti gli occhi enucleati sono stati ottenuti da un macello e tutti gli esperimenti sono stati eseguiti in conformità con le leggi biosicurezza istituzionali. <ol><li> Fissare occhio enucleato in giù su una superficie. A tale scopo, mettendo i perni dei tessuti attraverso il tessuto in eccesso intorno all&#39;occhio e fissandolo in giù in una tavola di polistirolo. </li><li> Staccare e staccare congiuntiva perilimbare dall&#39;occhio. <ol><li> Utilizzare una pinza sottile (0,3) e pinze microscissors (forbici Westcott) per incidere la congiuntiva al limbus e senza mezzi termini sezionare fuori la sclera. Tagliare la congiuntiva lungo il limbus come la dissezione procede per consentire un&#39;ulteriore dissezione. </li><li> Rimuovere congiuntiva tutto intorno all&#39;occhio (360 gradi) per esporre il più sclera possibile. NOTA: È utile lasciare una piccola quantità di congiuntiva per facilitare fissarsi l&#39;occhio con i perni chirurgici o una pinza durante il resto della procedura. </li></ol></li><li> Creare un completo lembo sclerale spessore lungo unolato dell&#39;occhio (bisturi 69). <ol><li> Fare un ~ 5 millimetri incisione sclerale parallelamente al limbus e 3 mm posteriormente al limbus tagliando delicatamente nella sclera con un bisturi (lama da bisturi 11) fino coroide pigmentazione scura è visibile. Fare attenzione a non incidere coroide stesso. </li><li> Sezionare cautela lungo il piano tra la sclera e della coroide, sia con le forbici (utilizzando smussa) o con il bisturi, finché è possibile ampliare lo spazio potenziale tra questi tessuti spingendo coroide leggermente verso l&#39;interno. </li><li> Effettuare un&#39;altra incisione sclerale perpendicolare alla prima incisione sclerale con microscissors taglienti, creando una incisione a forma di T. </li><li> Poi continuare a sezionare bruscamente in modo circonferenziale per rimuovere il tessuto sclerale dalla coroide sottostante e spingere delicatamente la coroide dalla sclera come detto sopra. </li><li> Ingrandire il lembo sclerale, se necessario. <ol><li> Delicatamente Blunt sezionare la coroide lontano dalla scleradurante tutto il processo. </li><li> Allargare lo sportello finché l&#39;incisione fatta perpendicolarmente al limbus raggiunge il nervo ottico e incisione fatta parallelo al limbus è almeno un terzo della circonferenza dell&#39;occhio (45 °). </li><li> Utilizzare il lembo sclerale come fonte di leva per manipolare l&#39;occhio durante la dissezione smussa. Il contro-trazione sul lembo facilita scollamento. </li></ol></li><li> Ripetere il passaggio precedente sull&#39;altro lembo sclerale. </li></ol></li><li> Tagliare i lembi sclerali per esporre una vasta area della coroide. </li><li> Continuare l&#39;incisione fatta nel passaggio 3 circonferenza dell&#39;occhio (360 gradi). </li><li> Rimuovere il restante tessuto coroide-retina. <ol><li> All&#39;interno della regione esposta, usare un cotton-tip per spazzolare delicatamente il tessuto della coroide-retina rimanente. In alternativa, afferrare delicatamente il tessuto con una pinza e sbucciare fuori dal corpo vitreo sottostante. </li><li> Se necessario fare un&#39;incisione nella coroide stArting dal nervo ottico. Poi staccarsi coroide delicatamente per esporre la retina e la corteccia vitreo. </li></ol></li><li> Continuare smussato sezionare la coroide dalla sclera e poi staccarsi coroide per ottenere il tutto, vitrea intatto. </li><li> Utilizzare il cerchio sclerale attaccato alla cornea per posizionare l&#39;intero vetrosa in posizione desiderata. A questo punto, il vitreo è collegato alla sclera anteriore e lente. </li><li> Blunt sezionare il vitreo dall&#39;interno della sclera, intorno alla lente, eliminando tutti sclera. </li><li> Utilizzare uno strumento smussato per raccogliere la lente dal vitreo se necessario. Utilizzare il campione per vari esperimenti vitreodynamic. <ol><li> Mettere campione in un bicchiere di vetro con nota superficie esposta all&#39;aria. Inserire sonda sensibile all&#39;ossigeno al bordo del campione. Utilizzare un micromanipolatore per spostare la sonda ad una distanza nota (r) nel campione. </li><li> Utilizzare Leggi di Fick per ottenere l&#39;equazione di diffusione teorica. Con il data raccolta in fase di 10.1, ottenere il termine coefficiente di diffusione sperimentale / reazione etc. </li></ol></li></ol>

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	<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.

Ci sono due passaggi critici che devono essere accuratamente eseguite durante la dissezione vitreo. Fase 3, la creazione di un completo lembo sclerale spessore, è fondamentale per l&#39;intero dissezione. Si deve prestare attenzione a non tagliare in coroide quando si crea il pieno spessore lembo sclerale. L&#39;altro passaggio fondamentale è sezionando via la sclera dalla coroide. Questo passaggio deve essere fatto con attenzione per evitare la creazione di più fori nella coroide da cui vitreo può fuoriuscire. C&#3...

L&#39;obiettivo di questo metodo consiste nel dettaglio una tecnica per isolare un intero, corpo vitreo intatto, con il nucleo vitreo e corteccia intatta, da un occhio di cadavere, ai fini dell&#39;analisi vitreodynamic. Poiché il campo della fisiologia del vitreo è cresciuta, i ricercatori multidisciplinari, quali la meccanica dei fluidi ricercatori, stanno studiando le proprietà fisiche e biomeccaniche del corpo vitreo 1. A tal fine, è indispensabile dettaglio una tecnica per isolare tutto, corpo vitreo intatta per favorire ricercatori multidisciplinari. Sebag et al. 2 e altri 3 eseguiti eleganti intere dissezioni vetrose sugli occhi cadavere dell&#39;uomo e hanno mostrato le illustrazioni dei risultati. Tuttavia, la tecnica utilizzata non è stata descritta in dettaglio e non esperti, non sarebbe in grado di replicare il metodo indipendente. Altri studi hanno raccolto vitreo da occhi cadavere con metodi semplici, come l&#39;aspirazione o dissezione parziale,entrambi i quali non provocano un tutto, corpo vitreo intatto. Gisladottis et al. 4 e Xu et al. 5 indagare permeabilità nel vitreo raccolte da occhi cadavere. Tuttavia, poiché nessun metodo di estrazione vetrosa è stata descritta, si è ipotizzato che essi aspirati vitreo con una siringa. Watts et al. 6 andato un passo avanti descrivendo un metodo per isolare coniglio vitreo con una tecnica chirurgica. Tuttavia, questo metodo provoca un isolamento del solo nucleo vitreo e non la corteccia vitreo. Skeie et al. 7 poi organizzato il vitreo in 4 regioni uniche ed elegantemente descritto un metodo per sezionare fuori ogni parte per l&#39;analisi. Questa tecnica tuttavia, non comporta un vitreous intatto nel suo complesso. La tecnica attuale è stato sviluppato per facilitare esperimenti biofisici attualmente eseguite solo in occhi cadavere. I metodi precedenti, come descritto unbove, sono limitati perché 1) nessuno isolare completamente tutto il corpo vitreo, 2) raccolte nucleo vitreo e la corteccia sono omogeneizzati, 3) vitrea struttura anatomica non è mantenuta, o 4) tecniche di dissezione non sono adeguatamente dettagliate per la replica da parte dei ricercatori in altri campi . In aggiunta, a causa della opacità sclera e della coroide, visualizzazione del corpo vitreo è limitata nel bulbo oculare intatto. Questo limita la precisione e la fattibilità di misurazioni che possono essere fatte all&#39;interno occhio intero. Inoltre, le strutture anatomiche circostanti il ​​vitreo possono confondere lo studio delle proprietà biochimiche e fisiche del vitreo. Negli ultimi anni, il corpo della scienza vitreo è cresciuto enormemente e non vi è ragione di credere che tutto il corpo vitreo ha proprietà diverse rispetto le sue singole parti. Vi è un crescente interesse per lo studio delle proprietà fisiche, biomeccaniche e chimiche del vitreo per vitreodynamics research, che ha applicazioni in medicina clinica, come la somministrazione di farmaci, l&#39;ossigenazione intravitreale 8 e vitrectomia. Vitreodynamics farmacologici, che utilizza agenti farmacologici per manipolare il corpo vitreo, possono essere utilizzati per migliorare i risultati vitrectomia 9. Proprietà biomeccaniche vengono utilizzate per modellare il flusso di fluido vitreale, che può essere utilizzato per migliorare drug delivery tecnologie intravitreale 10-12. Le proprietà fisiche di diversi segmenti del vitreo sono cruciali per comprendere vitreo-retinica trasporto dell&#39;ossigeno 13. La tecnica dissezione vitreo proposto può essere usato per studiare varie proprietà del intatte vitreo. Permette da banco esperimenti da fare su tutto, corpi vetrosi intatti con una migliore visualizzazione. In sintesi, i metodi attuali per lo studio del vitreo sono o non adeguatamente descritte o causare un isolamento incompleta nucleo vitreo e corteccia. Pertanto, vi è la necessità di eseguire experiments in un modello di occhio trasparente, pur mantenendo l&#39;anatomia del vitreo che esiste negli occhi cadavere.

<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.

Seguendo il protocollo porterà ad una dissezione vetrosa di successo con il nucleo e la corteccia (figura 3) intatto. Questo è evidente dai pezzi residui di retina aderito alla corteccia vitreo. Intatto intera vitreo può essere utilizzato in diversi modi per specifici esperimenti vitreodynamic. Nel nostro caso, il tasso di diffusione di ossigeno intatta vitreo ed è costante di tempo corrispondente è stato studiato (Figura 2). Vetroso che è stato dissezionato (nucleo e la corteccia...

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.

Tutta vitreo Umore dissezione per l&#39;analisi Vitreodynamic

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

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Medicine

11.3K Views.  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.

Video: 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.

Dissezione di Human Elements corpo vitreo per l&#39;analisi proteomica

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

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Medicina

Diagnosi del linfoma vitreoretinico mediante DNA libero da cellule

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.

Autore in primo piano: Avanzamento della diagnosi di VRL mediante l'estrazione di DNA libero da cellule dall'umor vitreo 

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.

Estrazione di DNA senza cellule di campioni di vitreo e umor acqueo per la diagnosi e il monitoraggio del linfoma vitreoretinico

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

Ricerca sul cancro

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.

Eviscerazione delle Vitreous Mouse e Retina per analisi proteomica

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 ...

Biologia

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.

Un passo per passo protocollo per la chirurgia sottoretinico in Conigli

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 ...

Neuroscienze

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.

Cauterizzazione a cerchio completo del plesso vascolare limbare per il glaucoma indotto chirurgicamente nei roditori

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.

Trapianto di epitelio pigmentato retinico in un modello di primati non umani per malattie degenerative della retina

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

Imaging multimodale basato su OCT di occhi di donatori umani per la ricerca sull'AMD

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.

Autore in primo piano: Imaging multimodale ex vivo basato su OCT di occhi di donatori umani per la ricerca sulla degenerazione maculare legata all'età  

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 Imaging multimodale basato su OCT di occhi di donatori umani per la ricerca sulla degenerazione maculare legata all'età

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

Tutta vitreo Umore dissezione per l'analisi Vitreodynamic

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

Cauterizzazione a cerchio completo del plesso vascolare limbare per il glaucoma indotto chirurgicamente nei roditori

Trapianto di epitelio pigmentato retinico in un modello di primati non umani per malattie degenerative della retina

Ex Vivo Imaging multimodale basato su OCT di occhi di donatori umani per la ricerca sulla degenerazione maculare legata all'età