Name: Dissection of Human Vitreous Body Elements for Proteomic Analysis (Video) | JoVE Journal
Uploaded: 2011-01-23T17:45:00.000+00:00
Duration: 5 min 5 s
Description: Learn precise dissection techniques for human vitreous body substructures including the anterior hyaloid, vitreous base, and cortex. This method enables researchers to isolate samples for proteomic analysis in ophthalmology studies.

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5 minutes

Funding was provided by Fight for Sight. Tissues were obtained from the Iowa Lions Eye Bank.

1. Anterior Segment Dissection.
<ol>
<li>The cornea is removed by first making an incision at the limbus into the anterior chamber using a 15° blade (Figure 1). Then one blade of curved cornea-scleral scissors is inserted into the anterior chamber. Circumferential cuts are made in the cornea, just anterior to the limbus.</li>
<li>0.12 Colibri forceps and Westcott scissors are used to cut the iris circumferentially anterior to the ciliary body.</li>
<li>The lens nucleus is removed with 0.12 Colibri forceps or a 15° blade (supersharp) and the cortex and capsule with forceps. </li>
</ol>
2. Vitreous Core Aspiration.
<ol>
<li>A 23-gauge needle on a 5-cc syringe is inserted into the mid-vitreous (Figure 1).</li>
<li>Approximately 1 mL or more of vitreous core is gently aspirated.</li>
<li>The sample is then placed in a microfuge tube and then into liquid nitrogen.</li>
</ol>
3. Anterior Hyaloid Dissection.
<ol>
<li>The anterior hyaloid is seen as a semi-transparent ring by adjusting the incident light from a goose neck microscope light. Previous aspiration of the vitreous core separates the anterior hyaloid from other structures for easier identification.</li>
<li>The sheet of elastic tissue is carefully pulled away from the ciliary body with Colibri or dressing forceps and cut with Vannas scissors. This maneuver is repeated.</li>
<li>The sample is then placed in a microfuge tube and then into liquid nitrogen.</li>
</ol>
4. Vitreous Base Dissection.
<ol>
<li>Using 0.12 forceps to stabilize the eye cup, Westcott scissors are used to "flower" the eye by making four equally spaced stress-relieving cuts from the ciliary body towards the optic nerve head.</li>
<li>The pars plicata of the ciliary body (Figure 2) is removed from each of the quadrants using Westcott scissors.</li>
<li>The vitreous base is then grasped on either side of the ora serrata with forceps over the pars plana and the retina (Figure 2).</li>
<li>Continuous traction on the vitreous base is applied with the forceps. Sequential cutting with Westcott scissors extracts a semi-transparent tissue with a "string of pearls" appearance as it is excised.</li>
<li>The sample is then placed in a microfuge tube and then into liquid nitrogen.</li>
</ol>
5. Vitreous Cortex Removal.
<ol>
<li>The pars plana is excised from each of the quadrants with Westcott scissors by cutting the leaflet approximately 3-mm posterior to the ora serrata (Figure 2).</li>
<li>Between the tissue leaflets, the vitreous cortex is visualized as a semi transparent film.</li>
<li>The elastic film is either grasped with 0.12 forceps or held by adherence to a Weck-Cel surgical sponge. The vitreous cortex is then pulled away from the retina and excised with Westcott scissors (Figure 1).</li>
<li>The sample is placed in a microfuge tube and then into liquid nitrogen. All samples are stored at -80° C until utilized for experimentation.</li>
</ol>
6. Representative Results
Tissue samples can be processed by a variety of methods for specific experiments. In our case, samples were submitted for protein analysis by SDS-PAGE (Figure 3).
 <img src="/files/ftp_upload/2455/2455fig1.jpg" alt="Eye anatomy diagram: cornea, lens, and vitreous body; educational biology keywords." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Cross-sectional view of the human eye depicting different substructures of the vitreous body. The most anterior vitreous is a thin collagenous layer called the anterior hyaloid. The vitreous core comprises the entire central region of the vitreous body. This portion of the vitreous is more aqueous in contrast to the vitreous base, which is viscous enough to be grasped by forceps and is firmly attached to the underlying ciliary body and retina. Encompassing the vitreous core is a very thin collagenous shell called the vitreous cortex.
 <img src="/files/ftp_upload/2455/2455fig2.jpg" alt="Ciliary body anatomy diagram showing retina, ora serrata, and vitreous base structure." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Vitreous base anatomy. The vitreous base is a semi-transparent substructure of the vitreous body located along the ora serrata (white arrows), which is the dividing line separating the ciliary body and retina. The anterior border of the vitreous base extends over the pars plana (white line) of the ciliary body. The posterior border of the vitreous base extends 2-3-mm posterior to the ora serrata (white dash). To excise the vitreous base, forceps are used to grasp the tissue and pull it away from the underlying ciliary body and retina. Once elevated, Westcott scissors are used to cut along the base.
 <img src="/files/ftp_upload/2455/2455fig3.jpg" alt="Gel electrophoresis results of vitreous body proteins; molecular weight markers on left." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. One-dimensional SDS-PAGE of Vitreous Body Elements. Total protein concentrations for the anterior hyaloid, vitreous base, vitreous core, and vitreous cortex were 11.24, 20.1, 16.61, 14.24 mg/mL, respectively. Gel electrophoresis was performed at 200 kV for 45 minutes, stained with Flamingo (Bio-Rad), and visualized using a VersaDoc Imaging system (Bio-Rad). The profiles for the different tissues show several similar bands, indicating either conserved or cross-contaminating proteins, as well as unique bands (asterisk), indicating differentially localized proteins.

<ol><li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog Retin Eye Res. 19, 323-344 (2000).</li>
<li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+biology+of+pharmacologic+vitreolysis%5BTitle%5D)+AND+%22Trans+Am+Ophthalmol+Soc%22%5BJournal%5D)">Molecular biology of pharmacologic vitreolysis.</a> Trans Am Ophthalmol Soc. 103, 473-494 (2005).</li>
<li>Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+analysis+of+inflammatory+immune+mediators+in+vitreoretinal+diseases%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases.</a> PLoS One. 4, e8158-e8158 (2009).</li>
<li>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P., Feener, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+vitreous+proteome+in+diabetes+without+diabetic+retinopathy+and+diabetes+with+proliferative+diabetic+retinopathy%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.</a> J Proteome Res. 7, 2516-2525 (2008).</li>
<li>Izuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+SOD+and+VEGF+are+increased+in+vitreous+bodies+from+proliferative+diabetic+retinopathy+patients%5BTitle%5D)+AND+%22Mol+Vis%22%5BJournal%5D)">Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients.</a> Mol Vis. 15, 2663-2672 (2009).</li>
<li>Azimzadeh, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formalin-fixed+paraffin-embedded+%28FFPE%29+proteome+analysis+using+gel-free+and+gel-based+proteomics%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.</a> J Proteome Res. 9, 4710-4720 (2010).</li>
</ol>

No conflicts of interest declared.

The vitreous body is a semi-transparent gel whose molecular composition is poorly understood, especially at the level of its substructures: the vitreous base, core, cortex, and anterior hyaloid. The vitreous core contains collagens II, V, IX, and XI, along with chondroitin sulphate proteoglycans, heparan sulphate proteoglycans, and hyaluronan.1,2 Protein biomarkers in the vitreous core have been associated with diseases such as diabetic retinopathy.3-5 How these proteins are differentially expressed in each of the substructures, and in many cases the specific protein identities, are not known. These details may give insight to the origin of proteins associated with specific vitreoretinal diseases and help target future therapies. Although the optimum post mortem interval for tissue dissection has not been determined, protein degradation may affect downstream experiments. For example, immunohistochemistry is affected in 12-hour postmortem eyes and some specific enzyme activities may be reduced within a few hours (unpublished observation). All tissues in this study were collected between 2 and 8 hours of death without significant changes in protein expression or suitability for proteomics analsyis. The liquid nitrogen freezing method of preservation is chosen over fixation in order to prevent small changes in protein structure caused by fixative cross linking, which has been demonstrated in other tissues by LC-MS/MS.6 Proteomic studies will depend on the ability to accurately dissect the different compartments of the vitreous, as demonstrated in this video experiment. We have validated the dissection technique using 1-dimensional SDS-PAGE. As our results suggest, there are differentially expressed proteins in the various vitreous body substructures. Identifying these proteins will provide a more detailed understanding of vitreous compartmentalization.

<table><tbody><tr><td>0.12 forceps</td><td>Storz Ophthalmics</td><td>E1502</td><td></td></tr><tr><td>5-cc syringe</td><td>BD Biosciences</td><td>309603</td><td></td></tr><tr><td>Straight Dressing Forceps With Serrations</td><td>Storz Ophthalmics</td><td>E1400</td><td></td></tr><tr><td>23 gauge needle</td><td>BD Biosciences</td><td>305145</td><td></td></tr><tr><td>Colibri forceps</td><td>Storz Ophthalmics</td><td>2/132</td><td></td></tr><tr><td>Castroviejo angled corneal scissors</td><td>Storz Ophthalmics</td><td>E3223</td><td></td></tr><tr><td>Vannas Curved Capsulotomy Scissors</td><td>Storz Ophthalmics</td><td>E3387</td><td></td></tr><tr><td>Weck-Cel surgical spears</td><td>Medtronic Inc.</td><td>0008680</td><td></td></tr><tr><td>Westcott Curved Tenotomy Scissors</td><td>Storz Ophthalmics</td><td>E3320</td><td></td></tr></tbody></table>

dissection of human vitreous body elements for proteomic 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 ...

The vitreous is an optically clear extracellular matrix that fills the inside of the eye. It can be divided into several substructures, including the anterior hyaloid behind the lens, the vitreous base at the anterior retina, the vitreous cortex forming an elastic shell and the vitreous core in the center. To dissect the vitreous, we first need to remove the anterior segment.A super sharp blade is used to incise the cornea at the limbus. Then cornea scleral scissors are used to cut the cornea just anterior to the limbus. The iris is dissected with curved scissors by pulling it from its face.Gentle pressure on the globe can expul the nucleus. To remove the vitreous core, use a 23 gauge needle on a five cc syringe and aspirate the mostly liquid vitreous. The anterior hyaloid becomes visible as a thin transparent membrane structure that can be grasped with forceps.It's an elastic structure just behind the lens. The inner circle is wit's ligament. Van scissors are used to cut the hyaloid while forceps apply traction.Lat tissue can be transferred to afu tube and the maneuver is repeated until all the tissue has been dissected. To access the remaining vitreous substructures, it is necessary to open the eye using curved westcott scissors. Each of the flaps are averted with forceps in a Petri dish.This image shows the anatomy of the vitreous base, a thin, transparent tissue that straddles the aura serrata. The aura serrata the dividing line between the ciliary body anteriorly and the retina posteriorly. Unlike other parts of the vitreous, the vitreous base is firmly adherent to the underlying ciliary body and retina pulling with forceps will actually elevate the tissue.To isolate the vitreous base, we first remove the pars plata using forceps and wescott scissors. The pars plata is this small area with ridges. Then to isolate the vitreous base, cut two to three millimeters posterior to the aura with westcott scissors while stabilizing the eye with forceps, hold and put traction on the vitreous base using forceps and cut sequentially with westcott scissors.There's a string of pearls appearance as the vitreous base is excised and it can be observed how this extracellular matrix might compartmentalize proteins. Different techniques can be used to dissect the shell of the vitreous. Contact with a WEX cell sponge will elevate the cortex.An edge of cortex can be found between the leaflets as shown here. Again, use the forceps to create traction and find VanNess scissors to sequentially cut the vitreous cortex. The cortex can also be elevated off the disc and here larger westcot scissors are used to to dissect the cortex as it is carefully peeled off the surface of the retina.Samples can then be placed in a micro and stored for later proteomic analysis.

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Research

JoVE Journal

Medicine

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Learn precise dissection techniques for human vitreous body substructures including the anterior hyaloid, vitreous base, and cortex. This method enables researchers to isolate samples for proteomic analysis in ophthalmology studies.

Dissection of Human Vitreous Body Elements for Proteomic Analysis

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