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.

所有的眼睛摘除从屠宰场获得的所有实验均按照生物安全制度执行法律。 <ol><li>安全摘除下来的眼睛在表面上。通过将组织通过针眼部周围多余的组织和确保它分解成一个泡沫塑料板做这个。 </li><li>解剖和分离眼结膜perilimbal。 <ol><li>用细镊子（0.3钳）和microscissors（韦斯科特剪刀）切开结膜角膜缘，并直言不讳地剖析它关闭巩膜。沿着切开角膜缘结膜为钝性分离进入允许进一步解剖。 </li><li>删除结膜一路眼睛周围的（360度）暴露尽可能多巩膜越好。 注:这是有帮助的留下少量的结膜，方便程序的剩余时间内行了吧眼睛的手术针或镊子。 </li></ol></li><li>沿着创建一个全层巩膜瓣眼（手术刀刀片69）的一面。 <ol><li>使巩膜切口平行于角膜缘和3mm后至角膜缘一约5毫米乘轻轻切成巩膜用手术刀（手术刀刀片11），直到暗着色脉络膜是可见的。小心不要切割脉络膜本身。 </li><li>沿着巩膜和脉络膜之间的平面小心解剖，或者用剪刀（使用钝器解剖）或用手术刀，直到它可以通过推脉络膜轻轻向内放大这些组织之间的潜在空间。 </li><li>进行另一个巩膜切口垂直于第一巩膜切口尖锐microscissors，创建一个T形切口。 </li><li>然后继续在圆周方式直截了当地解剖到从底层脉络膜去除巩膜组织和轻轻地从巩膜推脉络膜远如上所述。 </li><li>根据需要扩大巩膜瓣。 <ol><li>轻轻钝解剖脉络膜远离巩膜在整个过程中。 </li><li>放大襟翼直到作出垂直于角膜缘的切口到达视神经和切口平行于角膜缘的至少三分之一的眼周（45°）的。 </li><li>使用巩膜瓣作为杠杆的过程中钝性分离操纵眼睛的来源。在瓣反牵引力更容易为钝性剥离。 </li></ol></li><li>在重复其他巩膜瓣上一步。 </li></ol></li><li>切掉巩膜瓣，露出大面积脉络。 </li><li>继续在步骤3的眼部周围的圆周（360度）所作的切口。 </li><li>除去剩余的脉络膜视网膜组织。 <ol><li>在暴露的区域，用棉尖轻轻刷过脉络膜视网膜组织剩余。另外，抢轻轻用钳子组织剥开下面的玻璃体。 </li><li>如果有必要做一个切口进入脉络膜ST从视神经arting。然后剥离脉络膜轻轻揭露视网膜和玻璃体皮质。 </li></ol></li><li>继续钝解剖脉络远离巩膜，然后剥离脉络膜获得整体，完整的玻璃。 </li><li>使用附连到角膜巩膜缘定位在所需的位置，整个玻璃体。此时，玻璃体附着到前巩膜和透镜。 </li><li>钝从巩膜的内部解剖玻璃体，围绕透镜，清除所有巩膜。 </li><li>用生硬的工具，舀镜头远离玻璃，如果需要的话。使用示例各种vitreodynamic实验。 <ol><li>放置样品与暴露于空气中的已知表面积的玻璃烧杯中。放置氧敏感探针在样品的边缘。使用一个微型机械手将探头移动到一个已知距离（r）到样品。 </li><li>使用菲克定律获得的理论扩散方程。随着DATA收集在步骤10.1，获得实验扩散系数/反应项等。 </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.

有迹象表明，必须在玻璃体切除认真执行两个关键步骤。步骤3中，创建完整厚度巩膜瓣，关键的是要在整个清扫。应注意建立的全部厚度巩膜瓣时不切入脉络膜。另一个关键的步骤是解剖远离脉络膜巩膜。这一步必须谨慎进行，以防止在创建从中可以玻璃洒出脉络膜多个孔。有一种方法来修改协议，仍然完好无损解剖全玻璃体。步骤6可以被延迟，直到步骤8的脉络膜可以在末端被除去后玻璃体组?...

该方法的目的是对细节的技术以分离一个整体，完好玻璃体，与玻璃体芯和皮层完好，从尸体眼睛，对于vitreodynamic分析的目的。作为玻璃体生理学领域已经成长，多学科的研究者，如流体力学研究人员，正在研究玻璃体1的物理和力学性能。为此，有必要对细节的技术以分离的整体，完好玻璃体，以帮助多学科研究。 塞巴格等 2等3进行优雅整个玻璃体解剖上人尸体眼睛和显示结果的说明。然而，所使用的技术中的细节和非专家没有描述将不能够独立复制的方法。其他研究也收获了使用简单的方法，如吸入或局部解剖尸体眼玻璃体，这两者不会导致一个整体，完好玻璃体。 Gisladottis 等 4和Xu 等 5探讨通透性玻璃体从尸眼收获。然而，由于玻璃体提取无法进行了说明，人们认为它们吸出玻璃体用注射器。瓦等 6更进一步通过描述分离兔眼玻璃体液与手术技术的方法。然而，这种方法会导致在刚玻璃体芯的隔离，而不是玻璃体皮质。 Skeie 等 7后举办的玻璃体到4个独特的区域和优雅描述的方法来剖析出各个部分进行分析。这种技术然而，这并不导致完好玻璃体作为一个整体。 目前的技术，以方便，目前仅在尸眼进行的生物物理实验。以前的方法，如所描述的波夫，是有限的，因为1）无完全隔离整个玻璃体，2）收获玻璃体芯和皮层匀浆，3）玻璃体解剖结构不能维持，或4）解剖技术没有充分研究人员在其他领域详述用于复制。此外，由于巩膜和脉络膜，玻璃体的可视化的不透明度在完整眼球被限制。这限制了可以在整个眼内进行的测量的精度和可行性。此外，周边玻璃体的解剖结构可混淆的玻璃体的生化和物理性能的研究。 在最近几年，玻璃体科学的身体就突飞猛进和有理由认为整个玻璃体具有不同的性质不是其各个部分。有一个在为vitreodynamics researc调查玻璃体的物理，力学，化学性能日益增长的兴趣H，其中有应用在临床医学如药物输送，玻璃体内氧8玻璃体切割术。药理vitreodynamics，它使用药理物质来操纵玻璃体，可用于改善玻璃体成果9。生物力学特性用于模拟玻璃体流体流，其可以被用来改善玻璃体内给药技术10-12。玻璃体的各部分的物理性能是至关重要的理解玻璃体视网膜氧迁移13。所提出的玻璃体切除术可用于研究完整玻璃体的各种属性。它使台式实验与更好的可视化进行整体​​上的，完整的玻璃体。 总之，目前的方法对于玻璃体的研究要么没有充分描述的，或导致不完全的隔离玻璃体芯和皮层。因此，有必要以执行电子在一个透明的眼睛模型xperiments同时保持存在于尸体眼睛玻璃体的解剖结构。

<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.

以下协议会导致一个成功的玻璃体切除与芯和皮层（ 图3）保持不变。这是从剩余片视网膜附着于玻璃体皮质明显。完好整个玻璃体可以以多种方式对特定vitreodynamic实验中使用。在我们的情况下，氧气在完整玻璃体的扩散速度和它的相应的时间常数进行了研究（ 图2）。玻璃体被解剖用我们的方法（芯和皮层）置于与已知的暴露表面积的玻璃烧杯中。这是相比于玻璃体被?...

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.

全玻璃体液清扫Vitreodynamic分析

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 次观看.  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.

视频: 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.

人类蛋白质组分析玻璃体元素剖析

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

科研

医学

使用游离 DNA 诊断玻璃体视网膜淋巴瘤

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.

作者聚焦：使用玻璃体液中的游离 DNA 提取推进 VRL 诊断 

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.

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

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

癌症研究

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.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

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 ...

生物学

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.

一种用于兔视网膜下手术分步协议

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 ...

神经科学

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.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

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.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

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

用于 AMD 研究的基于 OCT 的人类供体眼多模态成像

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.

作者聚焦:基于离体 OCT 的人类供体眼多模态成像，用于年龄相关性黄斑变性的研究  

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.

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

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

全玻璃体液清扫Vitreodynamic分析

摘要

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此视频中的章节

人类蛋白质组分析玻璃体元素剖析

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

一种用于兔视网膜下手术分步协议

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

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

全玻璃体液清扫Vitreodynamic分析

摘要

探索更多视频

此视频中的章节

人类蛋白质组分析玻璃体元素剖析

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

一种用于兔视网膜下手术分步协议

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

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性