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

Todos os olhos enucleados foram obtidos a partir de um matadouro e todos os experimentos foram realizados de acordo com as leis de biossegurança institucionais. <ol><li> Fixe olho enucleado para baixo em uma superfície. Fazer isso colocando os pinos do tecido através do excesso de tecido em torno do olho e prendendo-o para baixo em uma placa de isopor. </li><li> Dissecar e retire conjuntiva perilimbal de olho. <ol><li> Use uma pinça fina (0,3 fórceps) e microtesoura (tesoura Westcott) para inciso conjuntiva no limbo e sem rodeios dissecá-lo fora da esclera. Cortar a conjuntiva ao longo do limbo como a dissecção romba prossegue para permitir posterior dissecação. </li><li> Remover conjuntiva a toda a volta do olho (360 graus) para expor tanto quanto possível esclera. NOTA: É útil para deixar uma pequena quantidade de conjuntiva para facilitar a fixação do olho com os pinos de uma pinça cirúrgica ou durante o resto do procedimento. </li></ol></li><li> Criar um retalho escleral de espessura total ao longo de umlado do olho (lâmina de bisturi 69). <ol><li> Adicione uma ~ 5 milímetros incisão escleral paralela ao limbo e 3 mm posterior ao limbo cortando suavemente para dentro da esclerótica com um bisturi (lâmina de bisturi 11) até coróide pigmentadas é visível. Tenha cuidado para não inciso a própria coróide. </li><li> Cuidadosamente dissecar ao longo do plano entre a esclera e coróide, ou com tesouras (utilizando uma dissecção romba) ou com o bisturi, até que seja possível aumentar o espaço de potencial entre estes tecidos, empurrando suavemente para dentro da coróide. </li><li> Fazer uma outra incisão escleral perpendicular à primeira incisão escleral com microtesouras afiadas, criando uma incisão em forma de T. </li><li> Em seguida, continuar a dissecar bruscamente de um modo circunferencial para remover o tecido a partir da coróide esclerótica subjacente e empurre o coróide longe da esclera como mencionado acima. </li><li> Ampliar a retalho escleral, conforme necessário. <ol><li> Suavemente sem corte dissecar a coróide longe da escleradurante todo o processo. </li><li> Aumentar a aba até que a incisão feita perpendicularmente ao limbo atinge o nervo óptico e a incisão feita paralela ao limbo é, pelo menos, um terço da circunferência do olho (45 °). </li><li> Use a retalho escleral como fonte de alavancagem para manipular o olho durante a dissecção romba. O contra-tração na aba torna mais fácil para dissecção romba. </li></ol></li><li> Repita o passo anterior no outro retalho escleral. </li></ol></li><li> Cortaram os retalhos esclerais para expor uma grande área de coróide. </li><li> Continuar a incisão feita no passo 3 em torno da circunferência do olho (360 graus). </li><li> Remover o tecido coróide-restante retina. <ol><li> Dentro da região exposta, use um algodão-ponta para escovar suavemente o tecido coróide-retina restante. Alternativamente, agarram delicadamente o tecido com uma pinça e retire-a do corpo vítreo subjacente. </li><li> Se for necessário fazer uma incisão na coróide starting do nervo óptico. Em seguida, retire a coróide suavemente para expor a retina eo córtex vítreo. </li></ol></li><li> Continuar sem corte dissecar a coróide longe da esclera e, em seguida, retire a coróide obter todo o vítreo intacta. </li><li> Utilizar o aro escleral fixada na córnea para posicionar todo o vítreo em local desejado. Neste ponto, o vítreo é ligada à esclerótica anterior e lente. </li><li> Blunt dissecar o vítreo a partir do interior da esclerótica, à volta da lente, a remoção de todos esclera. </li><li> Use uma ferramenta sem corte para colher a lente de distância do vítreo se for necessário. Use o exemplo para várias experiências vitreodynamic. <ol><li> Colocar a amostra num copo de vidro com uma área superficial conhecida exposta ao ar. Coloque sonda sensível ao oxigénio no bordo da amostra. Usar um micro-manipulador para mover a sonda para uma distância conhecida (R) na amostra. </li><li> Use Lei de Fick para obter a equação de difusão teórico. Com a data recolhidas no passo 10.1, obtenha o termo coeficiente de difusão experimental / reação etc. </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.

Existem dois passos críticos que devem ser cuidadosamente realizados durante a dissecção vítreo. Passo 3, a criação de uma aba escleral de espessura total, é crucial para toda a dissecção. Cuidados devem ser tomados para não cortar na coróide ao criar a espessura total retalho escleral. O outro passo crítico é dissecar afastado da esclera da coróide. Esta etapa deve ser feito com cuidado para evitar a criação de vários buracos na coróide de que o vítreo pode derramar. Existe uma maneira de modificar o...

O objectivo deste método é a de detalhe uma técnica para isolar todo um corpo vítreo, intacta, com o núcleo vítreo e córtex intacto, a partir de um olho de cadáver, para efeitos de análise vitreodynamic. Como o campo da fisiologia vítreo tem crescido, pesquisadores multidisciplinares, tais como mecânica dos fluidos pesquisadores, estão a investigar as propriedades físicas e biomecânicas do vítreo 1. Para este fim, é essencial detalhe uma técnica para isolar todo o, corpo vítreo intacta para ajudar os investigadores multidisciplinares. Sebag et al. 2 e 3 outros realizados inteiras elegantes dissecações de cadáveres vítreas em olhos humanos e mostrou ilustrações dos resultados. No entanto, a técnica utilizada não foi descrito em pormenor e não especialistas não seria capaz de se replicar independentemente do método. Outros estudos têm colhido vítreo de cadáveres olhos usando métodos mais simples, como aspiração ou dissecação parcial,ambas as quais não resultam em conjunto um corpo vítreo, intacta. Gisladottis et al. 4 e Xu et al. 5 investigar permeabilidade no humor vítreo colhida a partir de olhos de cadáveres. No entanto, uma vez que nenhum método de extracção vítreo foi descrito, assumiu-se que eles aspirado do humor vítreo com uma seringa. Watts et al. 6 foi um passo adiante, descrevendo um método de isolamento de coelho humor vítreo com uma técnica cirúrgica. No entanto, este método resulta num isolamento de apenas o núcleo vítreo e não no córtex vítreo. Skeie et al. 7 posteriormente organizou o vítreo em 4 regiões únicas e elegantemente descrito um método para dissecar cada parte para análise. Esta técnica no entanto, não resultar numa vítreo intacto como um todo. A técnica actual foi desenvolvido para facilitar experiências biofísicas que actualmente apenas são realizadas em cadáveres olhos. Os métodos anteriores, como descrito umbove, são limitados porque 1) nenhuma isolar completamente todo o corpo vítreo, núcleo e córtex vítreo 2) colhidas são homogeneizados, 3) estrutura anatômica vítreo não é mantido, ou 4) técnicas de dissecação não são adequadamente detalhados para a replicação por pesquisadores em outros campos . Além disso, devido à opacidade da esclera e coróide, visualização do corpo vítreo é limitado no globo ocular intacto. Isto limita a precisão das medições de viabilidade e que podem ser feitas dentro de todo o olho. Além disso, as estruturas anatómicas circundantes do vítreo pode confundir o estudo das propriedades bioquímicas e físicas do vítreo. Nos últimos anos, o corpo vítreo da ciência tem crescido muito e não há razão para acreditar que todo o corpo vítreo tem propriedades diferentes do que suas partes individuais. Há um crescente interesse em investigar as propriedades físicas, biomecânicos e químicas do vítreo para vitreodynamics research, que tem aplicações na medicina clínica, tais como a entrega de drogas, oxigenação intravítrea 8 e vitrectomia. Vitreodynamics farmacológicos, que utiliza agentes farmacológicos para manipular o vítreo, pode ser utilizada para melhorar os resultados de vitrectomia 9. As propriedades biomecânicas são utilizados para modelar o fluxo de fluido vítreo, que pode ser usado para melhorar as tecnologias de entrega de drogas intravítrea 10-12. Propriedades físicas de vários segmentos da vítreo são cruciais para a compreensão de transporte de oxigênio vítreo-retiniana 13. A técnica de dissecção do vítreo proposto pode ser utilizado para estudar diversas propriedades do humor vítreo intacta. Ele permite que os experimentos de bancada a ser feito em, corpos vítreos intactas inteiros com melhor visualização. Em resumo, os métodos actuais para o estudo do vítreo ou não são adequadamente descritos, ou resultar em um isolamento completo do núcleo vítreo e no córtex. Portanto, existe uma necessidade de executar experiments em um modelo de olho transparente, mantendo a anatomia do vítreo existente no olho cadáver.

<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.

Seguindo o protocolo irá levar a uma dissecção do vítreo bem sucedida com o núcleo e o córtex (Figura 3) intactos. Isto é evidente a partir dos pedaços residuais de retina aderiram ao córtex vítreo. Todo intacta humor vítreo pode ser usado de várias maneiras para experiências vitreodynamic específicos. No nosso caso, a taxa de difusão do oxigénio no humor vítreo intacto e é constante de tempo correspondente foi estudada (Figura 2). Vítreo que foi dissecado (núcleo e ...

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.

Whole humor vítreo Dissecção de Análise Vitreodynamic

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

11.4K visualizações.  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.

Vídeo: 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.

Dissecção da Human Elements Corpo Vítreo para Análise Proteômica

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

Medicina

Diagnóstico de linfoma vitreorretiniano usando DNA livre de células

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 em destaque: Avançando no diagnóstico de VRL usando extração de DNA livre de células do humor vítreo 

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.

Extração de DNA livre de células de espécimes de humor vítreo e aquoso para diagnóstico e monitoramento de linfoma vitreorretiniano

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

Pesquisa do câncer

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.

Evisceração de Vítreo Mouse e Retina de Análises Proteômica

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.

A Passo a Passo Protocolo de Cirurgia sub-retiniana em Coelhos

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.

<meta charset="utf8"></head><body>Dissecção microcirúrgica e limpeza de tecidos para imagens de retina inteira e vítreo intactas de alta resolução

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.

Dissecção microcirúrgica e limpeza de tecidos para imagens de retina inteira e vítreo intactas de alta resolução

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

Neurociência

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.

Cauterização em círculo completo do plexo vascular limbal no glaucoma induzido cirurgicamente em roedores

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.

Transplante de Epitélio de Pigmento De Retina em um modelo de primata não humano para doenças degenerativas da retina

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

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