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

All enucleated eyes were obtained from an abattoir and all experiments were performed in accordance with institutional biosafety laws.
<ol>
	<li>Secure enucleated eye down on a surface. Do this by placing tissue pins through the excess tissue around the eye and securing it down into a Styrofoam board.</li>
	<li>Dissect and detach perilimbal conjunctiva from eye.
	<ol>
		<li>Use fine forceps (0.3 forceps) and microscissors (Westcott scissors) to incise the conjunctiva at the limbus and bluntly dissect it off the sclera. Cut the conjunctiva along the limbus as the blunt dissection proceeds to allow further dissection.</li>
		<li>Remove conjunctiva all the way around the eye (360 degrees) to expose as much sclera as possible. 
		NOTE: It is helpful to leave a small amount of conjunctiva to facilitate fixating the eye with the surgical pins or a forceps during the rest of the procedure.</li>
	</ol>
	</li>
	<li>Create a full thickness scleral flap along one side of the eye (scalpel blade 69).
	<ol>
		<li>Make a ~5 mm scleral incision parallel to the limbus and 3 mm posterior to the limbus by gently cutting into the sclera with a scalpel (scalpel blade 11) until darkly pigmented choroid is visible. Be careful not to incise the choroid itself.</li>
		<li>Carefully dissect along the plane between the sclera and choroid, either with scissors (using blunt dissection) or with the scalpel, until it is possible to enlarge the potential space between these tissues by pushing the choroid gently inward.</li>
		<li>Make another scleral incision perpendicular to the first scleral incision with sharp microscissors, creating a T-shaped incision.</li>
		<li>Then continue to bluntly dissect in a circumferential fashion to remove the scleral tissue from the underlying choroid and gently push the choroid away from the sclera as mentioned above.</li>
		<li>Enlarge the scleral flap as needed.
		<ol>
			<li>Gently blunt dissect the choroid away from the sclera throughout the process.</li>
			<li>Enlarge the flap until the incision made perpendicular to the limbus reaches the optic nerve and the incision made parallel to the limbus is at least one-third of the eye circumference (45&#176;).</li>
			<li>Use the scleral flap as a source of leverage for manipulating the eye during blunt dissection. The counter-traction on the flap makes it easier for blunt dissection.</li>
		</ol>
		</li>
		<li>Repeat the previous step on the other scleral flap.</li>
	</ol>
	</li>
	<li>Cut away the scleral flaps to expose a large area of choroid.</li>
	<li>Continue the incision made in step 3 around the eye&#8217;s circumference (360 degrees).</li>
	<li>Remove the remaining choroid-retina tissue.
	<ol>
		<li>Within the exposed region, use a cotton-tip to gently brush off the choroid-retina tissue remaining. Alternatively, gently grab the tissue with forceps and peel it off the underlying vitreous body.</li>
		<li>If necessary make an incision into the choroid starting from the optic nerve. Then peel away the choroid gently to expose the retina and vitreous cortex.</li>
	</ol>
	</li>
	<li>Continue blunt dissecting the choroid away from the sclera and then peel away the choroid to obtain the whole, intact vitreous.</li>
	<li>Use the scleral rim attached to the cornea to position the whole vitreous in desired location. At this point, the vitreous is attached to the anterior sclera and lens.</li>
	<li>Blunt dissect the vitreous from the inside of the sclera, around the lens, removing all sclera.</li>
	<li>Use a blunt tool to scoop the lens away from the vitreous if need be. Use the sample for various vitreodynamic experiments.
	<ol>
		<li>Place sample in a glass beaker with known surface area exposed to air. Place oxygen sensitive probe at the edge of the sample. Use a micro-manipulator to move the probe to a known distance (r) into the sample.</li>
		<li>Use Fick&#8217;s laws of diffusion to obtain the theoretical diffusion equation. With the data collected in step 10.1, obtain the experimental diffusion coefficient/reaction term 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.

There are two critical steps that must be carefully performed during vitreous dissection. Step 3, creating a full thickness scleral flap, is crucial to the entire dissection. Care should be taken to not cut into the choroid when creating the full thickness scleral flap. The other critical step is dissecting away the sclera from the choroid. This step must be carefully done to prevent creating multiple holes in the choroid from which the vitreous can spill out. There is a way to modify the protocol and still dissect intact whole vitreous humor. Step 6 can be delayed until step 8. The choroid can be removed at the end after the vitreous tissue is positioned in the desired location.
The limitation of this procedure is that during dissection, there are only visual cues to suggest the success and accuracy of the intact vitreous dissection. Since the vitreous core and cortex are both transparent and undistinguishable to the naked eye, this can be challenging. By noticing the adhesion of the retina onto the vitreous it is possible to determine if successful dissection of the vitreous cortex has occurred. Otherwise, pharmacological substances, such as Kenalog, can be used to improve visualization15 of the vitreous but cannot readily allow one to distinguish between vitreous cortex and vitreous core. Another limitation of this technique is that it is most effective for the dissection of gel vitreous. Human vitreous gel is a non-regenerative tissue that undergoes liquefaction with age. This liquefaction prevents us from completely dissecting the whole, intact vitreous from the eye. Thus, our technique extends only to vitreous of animals, such as cows, pigs or rabbits, or eyes of young humans who do not have significant amounts of liquefaction. The vitreous humor in these animals tends to be completely in a gel state.
However, currently, there is no established method, that has been described in detail, for dissecting whole intact vitreous from cadaver eyes. Currently, the only vitreous dissection techniques that have been well detailed involve the dissection of unique but separate regions of the vitreous7 or the dissection of just the vitreous core6.
The applications of dissected intact vitreous humor are under appreciated and under-utilized. Surgical experience with the vitreous as well as clinical, histopathological and biochemical studies suggests that the chemical and structural properties of the vitreous can vary significantly16,17. Therefore, it is necessary to preserve the structure of the whole vitreous body to study the function of the vitreous in ocular physiology and anatomy. Intact vitreous can be explanted to a transparent globe for better visualization during experiments. They can then be used for a variety of mechanical/chemical tests to improve the measurements of physical/biomechanical properties of the vitreous humor. For example, we suggest that the dissected whole intact vitreous can be used in lieu of saline, viscous substitutes, or vitreous core in the cited rheology experiments6,18.

The goal of this method is to detail a technique to isolate a whole, intact vitreous body, with the vitreous core and cortex intact, from a cadaver eye, for the purposes of vitreodynamic analysis. As the field of vitreous physiology has grown, multi-disciplinary researchers, such as fluid mechanics researchers, are investigating the physical and biomechanical properties of the vitreous1. To this end, it is essential to detail a technique to isolate the whole, intact vitreous body to aid multi-disciplinary researchers.
Sebag et al.2 and others3 performed elegant whole vitreous dissections on human cadaver eyes and showed illustrations of the results. However, the technique used was not described in detail and non-experts would not be able to replicate the method independently. Other studies have harvested vitreous from cadaver eyes using simpler methods such as aspiration or partial dissection, both of which do not result in a whole, intact vitreous body. Gisladottis et al.4 and Xu et al.5 investigate permeability in vitreous humor harvested from cadaver eyes. However, since no method of vitreous extraction was described, it was assumed that they aspirated the vitreous humor with a syringe. Watts et al.6 went one step further by describing a method of isolating rabbit vitreous humor with a surgical technique. However, this method results in an isolation of just the vitreous core and not the vitreous cortex. Skeie et al.7 later organized the vitreous into 4 unique regions and elegantly described a method to dissect out each part for analysis. This technique however, does not result in an intact vitreous as a whole.
The current technique was developed to facilitate biophysical experiments that are currently only performed in cadaver eyes. Previous methods, as described above, are limited because 1) none completely isolate the whole vitreous body, 2) harvested vitreous core and cortex are homogenized, 3) vitreous anatomical structure is not maintained, or 4) dissection techniques are not adequately detailed for replication by researchers in other fields. In addition, due to the opacity of sclera and choroid, visualization of the vitreous body is limited in the intact eyeball. This limits the precision and feasibility of measurements that can be made inside the whole eye. In addition, the anatomical structures surrounding the vitreous can confound the study of biochemical and physical properties of the vitreous.
In recent years, the body of vitreous science has grown tremendously and there is reason to believe that the whole vitreous body has different properties than its individual parts. There is growing interest in investigating the physical, biomechanical, and chemical properties of the vitreous for vitreodynamics research, which has applications in clinical medicine such as drug delivery, intravitreal oxygenation8 and vitrectomy. Pharmacological vitreodynamics, which uses pharmacologic agents to manipulate the vitreous, can be used to improve vitrectomy outcomes9. Biomechanical properties are used to model vitreous fluid flow, which can be used to improve intravitreal drug delivery technologies10&#8211;12. Physical properties of various segments of the vitreous are crucial to understanding vitreo-retinal oxygen transport13. The proposed vitreous dissection technique can be used to study various properties of the intact vitreous humor. It enables bench-top experiments to be done on whole, intact vitreous bodies with better visualization.
In summary, current methods for study of the vitreous are either not adequately described, or result in an incomplete isolation the vitreous core and cortex. Therefore, there is a need to perform experiments in a transparent eye model while retaining the anatomy of the vitreous that exists in the cadaver eye.

<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.

Following the protocol will lead to a successful vitreous dissection with the core and cortex (Figure 3) intact. This is evident from the residual pieces of retina adhered to the vitreous cortex. Intact whole vitreous humor can be used in several ways for specific vitreodynamic experiments. In our case, the diffusion rate of oxygen in intact vitreous humor and it&#8217;s corresponding time constant was studied (Figure 2). Vitreous that was dissected (core and cortex) using our method was placed in a glass beaker with a known exposed surface area. This was compared to vitreous that was harvested using a previously published method that only isolates vitreous core6. All other factors were kept consistent across the experimental and control groups. The vitreous surface was exposed to air, which has a known oxygen tension (~160 mmHg). The oxygen tension within the vitreous was low (&#60;10 mmHg). Based on the rate of vitreous oxygen consumption determined by Shui et al.14 for vitreous in the gel state, we can use the equation for 1 dimensional oxygen transport (in steady or unsteady state forms). By measuring the oxygen tension a known distance within the vitreous surface, we can experimentally validate the diffusion coefficient. The oxygen tension was recorded with an oxygen probe such as Oxford, and the error rate was &#177;10%. A sample was collected every 30 sec. The presence of vitreous cortex affects the diffusion rate of oxygen into the mid- vitreous. In the presence of vitreous cortex, the diffusion of oxygen occurs over a longer time interval.
The physical properties of the vitreous core and cortex have an impact in health and disease. For example, localized, supplemental intraocular oxygenation has been proposed as a treatment for retinal ischemia8. Understanding the different diffusion coefficients for oxygen through vitreous cortex core will enable researchers to predict the actual rate of oxygen delivery to the retina.
<img alt="Figure 1" src="/files/ftp_upload/52759/52759fig1.jpg" /> 
Figure 1: Cross sectional view of the eye displaying relevant anatomical structures (modified from National Eye Institute, National Institutes of Health). The cornea is the transparent tissue that forms the anterior most section of the eye and provides a significant portion of the eye&#8217;s optical power. The limbus is the junction of the cornea, conjunctiva and sclera. The sclera extends from the limbus to the posterior most aspect of the eye where it meets the optic nerve. The conjunctiva is a thin epithelium that lines the very outside of the eye and forms the boundary between the external environment and the sclera. The internal components of the eye consist of the anterior chamber, lens, iris, ciliary body, posterior chamber, posterior cavity, retina and choroid. The vitreous core comprises the central region of the vitreous body, within the posterior cavity of the eye. The vitreous cortex is located around the vitreous body and is attached to the retina at several points including the pars plana, retinal vessels, optic disc and macula. The vitreous base is a three dimensional zone which extends from ~2 mm anterior to the ora serrata to 3 mm posterior to the ora serrata. The choroid is a vascular tissue layer that is located between the sclera and retina and provides blood supply to the outer retina. The retina is the neurosensory layer of the eye that subserves the light sending capacity of the eye. The sclera is the white opaque layer that provides bulk of the structural support to the eye wall.
<img alt="Figure 2" src="/files/ftp_upload/52759/52759fig2.jpg" /> 
Figure 2: Plot of oxygen tension of vitreous humor against time. Plot of oxygen tension in vitreous core (green line) compared to intact whole vitreous (blue line). An oxygen sensing probe was placed in the middle of the sample and the sample was exposed to air which has an oxygen tension of 160 mmHg.
<img alt="Figure 3" src="/files/ftp_upload/52759/52759fig3.jpg" /> 
Figure 3: Examples of isolated whole vitreous body. Top picture is an example of the whole intact vitreous body still attached to the scleral rim. Middle picture is an example of the whole intact vitreous body without sclera but with some remaining retinal tissue. Bottom is an example of whole, intact vitreous body.

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.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

11.4K 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.

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

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

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

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

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

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.

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

Cancer Research

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.

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 ...

Biology

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.

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 ...

Neuroscience

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.

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.

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

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

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.

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

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.

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

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the research laboratory. Here, we developed a protocol for acquiring high-quality liquid biopsies for molecular analyses from the aqueous humor and the vitreous from patients undergoing eye surgery. In this workflow, a Mobile Operating Room Lab Interface (MORLI) cart equipped with a computer, a barcode scanner, and lab instruments, including onboard cold storage, is used to obtain and archive human biological samples. A web-based data privacy-compliant database enables annotating each sample over its lifetime, and a cartesian coordinate system allows tracking each barcoded specimen in storage, enabling quick and accurate retrieval of samples for downstream analyses. Molecular characterization of human tissue samples not only serves as a diagnostic tool (e.g., to distinguish between infectious endophthalmitis and other non-infectious intraocular inflammation) but also represents an important component of translational research, allowing the identification of new drug targets, development of new diagnostic tools, and personalized therapeutics.

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

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