Name: Evisceration of Mouse Vitreous and Retina for Proteomic Analyses (Video) | JoVE Journal
Uploaded: 2011-04-03T15:00:00.000+00:00
Duration: 2 min 39 s
Description: Learn a dissection technique to isolate mouse vitreous, retina, and lens for proteomic analyses. This method uses centrifugation to separate tissues and enables reliable biochemical characterization.

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5 minutes

Funding was provided by Fight for Sight and Research to Prevent Blindness.

1. Anterior segment dissection. 
<ol>
	<li>Scleral tissue posterior to the limbus is grasped with a 0.22 forcep and the globe (eye ball) is stabilized.</li>
	<li>A microsurgical blade is used to make a linear incision in the cornea from limbus to limbus.</li>
	<li>A 0.12 Colibri forcep is then used to grasp the cornea at the incision.</li>
	<li>The anterior chamber fluid is then absorbed with a Weck-Cel surgical spear.</li>
</ol>
2. Lens evisceration. 
<ol>
	<li>A 0.12 Colibri forcep grasping the cornea is used to stabilize the globe.</li>
	<li>A fine curved needle holder (or curved dressing forceps) is inserted behind the lens toward the posterior aspect of the globe. The needle holder is partially closed, and pulled forward. Pressure is applied using the forceps on the external surface of the sclera, which pushes the lens forward through the corneal incision while leaving the eye wall intact. The vitreous appears as a translucent gel and is partially adherent to the lens.</li>
	<li>The lens-vitreous tissue is then placed into a filtered centrifugation tube containing 20 microliters of protease inhibitor cocktail (Roche) dissolved in PBS. </li> 
</ol>
3. Retina evisceration.
<ol>
	<li>Continue to stabilize the globe with 0.12 Colibri forceps grasping the corneal incision.</li>
	<li>A fine curved needle holder (or curved dressing forceps) is placed as far posterior to the globe as possible, near the optic nerve. The needle holder is partially closed, and pulled forward. Pressure is applied using the forceps on the external surface of the sclera, which pushes the retina forward through the corneal incision. The vitreous appears as a translucent gel adherent to the retina. The retina is visualized as a yellow, vascularized tissue. Any strips of pigmented tissue can be peeled away with forceps.</li>
	<li>The retina-vitreous tissue is then placed into the filtered centrifuge tube containing the lens-vitreous tissue. </li>
</ol>
4. Filtered centrifugation. 
<ol>
	<li>The filtered centrifuge tube is placed in a benchtop centrifuge. Spin at 14,000 x G for 12 minutes.</li>
	<li>Aspirate the eluant from the lower chamber, which is the vitreous. The retina remains in the upper chamber. These samples can be utilized for protein studies.</li>
</ol>
5. Representative Results
Vitreous samples were analyzed by SDS-PAGE (Figure 2). Samples were also used for an enzymatic activity assay (Figure 3).
<img src="/files/ftp_upload/2795/2795fig1.jpg" alt="Human vs mouse eye structure; diagram comparing lens and vitreous anatomy." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Mouse eye diagram. Cross-sectional schematic of the human and mouse eye shows the relative smaller vitreous volume in the mouse eye due to its larger lens.
<img src="/files/ftp_upload/2795/2795fig2.jpg" alt="SDS-PAGE protein electrophoresis result, vitreous vs retina lanes, molecular weight markers visible." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. One-dimensional SDS-PAGE of mouse vitreous and retina. Protein profiles of mouse vitreous, 13.6 mg/mL (lane 1), and retina, 11.35 mg/mL (lane 2). Gel electrophoresis was performed at 150 kV for 45 minutes, stained with Flamingo fluorescent gel stain and visualized using a VersaDoc Imaging system (BioRad, Hercules, CA).
<img src="/files/ftp_upload/2795/2795fig3.jpg" alt="Bar chart showing retinal activity; positive and negative control comparison; experimental results." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Superoxide dismutase (SOD) enzyme activity in the mouse and human vitreous. Measurement of total SOD activity (SOD activity colorimetric aasay kit, #AB65354, Abcam, Inc, Cambridge, MA) was performed on vitreous and retina collected by evisceration from DBA and albino mice. This was compared to SOD activity in human vitreous core and human retina collected from post-mortem human donor eyes to determine relative activity. Undiluted vitreous core samples were manually aspirated using a 23-gauge needle, 11 and retina was collected by flowering the eye and cutting the tissue away from the RPE/choroid complex. Although the assay does not distinguish between SOD isoforms, the vitreous SOD activity is likely to reflect SOD3, since it is the only extracellular isoform.12 The retina SOD activity is likely to reflect all three SOD isoforms: intracellular SOD1, mitochondrial SOD2, and extracellular SOD3. 12 For highly sensitive enzymatic assays, including this SOD assay, the possibility of cross-contamination of proteins is possible and requires further study. Error bars represent SEM.

<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>Gao, B. B., Chen, X., Timothy, N., Aiello, L. P. &amp, 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>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>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSmith%2C+RS+%22Systematic+evaluation+of+the+mouse+eye%3A+Anatomy%2C+pathology%2C+and+biomethods%22">Systematic evaluation of the mouse eye: Anatomy, pathology, and biomethods</a>. , CRC Press. 161-195 (2002).</li>
<li>Ihanamaki, T., Metsaranta, M., Rintala, M., Vuorio, E., Sandberg-Lall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+abnormalities+in+transgenic+mice+harboring+mutations+in+the+type+II+collagen+gene%5BTitle%5D)+AND+%22Eur.+J.+Ophthalmol%22%5BJournal%5D)">Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene.</a> Eur. J. Ophthalmol. 6, 427-435 (1996).</li>
<li>Kaarniranta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+for+Stickler%27s+syndrome%3A+ocular+phenotype+of+mice+carrying+a+targeted+heterozygous+inactivation+of+type+II+%28pro%29collagen+gene+%28Col2a1%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">A mouse model for Stickler's syndrome: ocular phenotype of mice carrying a targeted heterozygous inactivation of type II (pro)collagen gene (Col2a1.</a> Exp. Eye Res. 83, 297-303 (2006).</li>
<li>Marneros, A. G. &amp, Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-dependent+iris+abnormalities+in+collagen+XVIII%2Fendostatin+deficient+mice+with+similarities+to+human+pigment+dispersion+syndrome%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome.</a> Invest. Ophthalmol. Vis. Sci. 44, 2367-2372 (2003).</li>
<li>Martin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+persistent+hyperplastic+primary+vitreous+in+mice+lacking+the+arf+tumor+suppressor+gene%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene.</a> Invest. Ophthalmol. Vis. Sci. 45, 3387-3396 (2004).</li>
<li>Song, B. J., Tsang, S. H., Lin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy%5BTitle%5D)+AND+%22Gene.+Ther.+Mol.+Biol%22%5BJournal%5D)">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene. Ther. Mol. Biol. 11B, 229-262 (2007).</li>
<li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+of+Human+Vitreous+Body+Elements+for+Proteomic+Analysis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Dissection of Human Vitreous Body Elements for Proteomic Analysis.</a> J Vis Exp. , (2011).</li>
<li>Ciechanowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+synthesis+is+not+the+reason+for+decreased+activity+of+extracellular+superoxide+dismutase+in+patients+with+diabetes%5BTitle%5D)+AND+%22Arch+Med+Res%22%5BJournal%5D)">Impaired synthesis is not the reason for decreased activity of extracellular superoxide dismutase in patients with diabetes.</a> Arch Med Res. 36, 148-153 (2005).</li>
</ol>

Experiments on animals were performed in accordance with the Association for Research and Vision in Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research.

Transgenic mice are an important model for investigating retinal and vitreoretinal disease.6-10 The mouse vitreous body, however, comprises a significantly smaller proportion of the eye in comparison to the human vitreous due to the large lens in the mouse eye.5 This makes it difficult to isolate and purify the mouse vitreous. Understanding the protein changes associated with the vitreous during diseases such as proliferative diabetic retinopathy, age-related macular degeneration, uveitis, and retinal detachment will give insight into the mechanisms by which they progress. This visualized experimental protocol provides a means to obtain and purify the mouse vitreous body, while maximizing the protein yield for enzymatic and proteomic analyses.

<table><tbody><tr><td>15&amp;#x00B0;, BD Beaver Microsurgical Blade</td><td>BD Biosciences</td><td>374881</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Invitrogen</td><td>70011-044</td><td></td></tr><tr><td>0.22 Fine-Castroviejo Suturing Forceps</td><td>Storz Ophthalmics</td><td>E1805</td><td></td></tr><tr><td>0.12 Colibri forceps</td><td>Storz Ophthalmics</td><td>2/132</td><td></td></tr><tr><td>Microcon Centrifugal FilterUltracel YM-100 or YM-50</td><td>EMD Millipore</td><td>42412, 42415</td><td></td></tr><tr><td>SOD Activity Colorimetric Assay Kit</td><td>Abcam</td><td>Ab65354</td><td></td></tr><tr><td>Weck-Cel surgical spears</td><td>Medtronic Inc.</td><td>0008680</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche Group</td><td>11 836 170 001</td><td></td></tr><tr><td>Flamingo fluorescent gel stain</td><td>Bio-Rad</td><td>161-04910</td><td></td></tr></tbody></table>

evisceration of mouse vitreous and retina for proteomic analyses

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 ...

The human eye is filled with a liquid extracellular matrix called vitreous in the mouse eye. This space is mostly filled by the lens. To isolate the vitreous and retina, we first have to dissect the anterior segment, stabilize the eye with forceps, and use a super sharp blade to make a single incision across the cornea.A dull blade can tear the cornea and require a few strokes. A WEX cell sponge is used to absorb fluid in the anterior chamber. The arrows show where we'll squeeze the lens and vitreous out with forceps, partially close the forceps and pull forward.The lens looks like a clear sphere and trailing behind it will be the vitreous gel. In this example, the vitreous remains adherent to the back of the lens to isolate the retina forceps are placed further back near the optic nerve, squeeze gently and pull forward. The retina appears like a yellow gelatinous material.In some cases, the retina vitreous and lens will come out as a single tissue. The next step is to isolate and separate these three tissues using a filter centrifugation device. The filter piece goes in the top of an einor tube and we add some phosphate buffered saline.As each of the tissues are isolated, they're placed in the top upper well containing the filter. The lenses in the upper left corner and the retina vitreous is in the lower right place. The tubes in a micro centrifuge and spin at 14, 000 Gs for 12 minutes.Vitreous will spin to the bottom of the einor tube lens, and retina will stay in the upper. Well use forceps to separate the lens from the retina, then isolate the retina from the upper well. All these tissues can be placed in einor tubes and then processed for proteomics analyses.

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Learn a dissection technique to isolate mouse vitreous, retina, and lens for proteomic analyses. This method uses centrifugation to separate tissues and enables reliable biochemical characterization.

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

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