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 JoVE Biology

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

1,2, 3, 1,2

1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons

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    Summary

    The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

    Date Published: 4/03/2011, Issue 50; doi: 10.3791/2795

    Cite this Article

    Skeie, J. M., Tsang, S. H., Mahajan, V. B. Evisceration of Mouse Vitreous and Retina for Proteomic Analyses. J. Vis. Exp. (50), e2795, doi:10.3791/2795 (2011).

    Abstract

    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.

    Protocol

    1. Anterior segment dissection.

    1. Scleral tissue posterior to the limbus is grasped with a 0.22 forcep and the globe (eye ball) is stabilized.
    2. A microsurgical blade is used to make a linear incision in the cornea from limbus to limbus.
    3. A 0.12 Colibri forcep is then used to grasp the cornea at the incision.
    4. The anterior chamber fluid is then absorbed with a Weck-Cel surgical spear.

    2. Lens evisceration.

    1. A 0.12 Colibri forcep grasping the cornea is used to stabilize the globe.
    2. 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.
    3. The lens-vitreous tissue is then placed into a filtered centrifugation tube containing 20 microliters of protease inhibitor cocktail (Roche) dissolved in PBS.

    3. Retina evisceration.

    1. Continue to stabilize the globe with 0.12 Colibri forceps grasping the corneal incision.
    2. 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.
    3. The retina-vitreous tissue is then placed into the filtered centrifuge tube containing the lens-vitreous tissue.

    4. Filtered centrifugation.

    1. The filtered centrifuge tube is placed in a benchtop centrifuge. Spin at 14,000 x G for 12 minutes.
    2. 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.

    5. Representative Results

    Vitreous samples were analyzed by SDS-PAGE (Figure 2). Samples were also used for an enzymatic activity assay (Figure 3).

    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.

    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).

    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.

    Discussion

    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.

    Disclosures

    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.

    Acknowledgements

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

    Materials

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

    References

    1. Bishop, P. N. Structural macromolecules and supramolecular organisation of the vitreous gel. Prog. Retin. Eye Res. 19, 323-344 (2000).
    2. Gao, B. B., Chen, X., Timothy, N., Aiello, L. P. &, Feener, E. P. Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. J. Proteome Res. 7, 2516-2525 (2008).
    3. Izuta, H. Extracellular SOD and VEGF are increased in vitreous bodies from proliferative diabetic retinopathy patients. Mol. Vis. 15, 2663-2672 (2009).
    4. Sebag, J. Molecular biology of pharmacologic vitreolysis. Trans. Am. Ophthalmol. Soc. 103, 473-494 (2005).
    5. Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. Systematic evaluation of the mouse eye: Anatomy, pathology, and biomethods. 161-195 CRC Press (2002).
    6. Ihanamaki, T., Metsaranta, M., Rintala, M., Vuorio, E., Sandberg-Lall, M. Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene. Eur. J. Ophthalmol. 6, 427-435 (1996).
    7. Kaarniranta, K. A mouse model for Stickler's syndrome: ocular phenotype of mice carrying a targeted heterozygous inactivation of type II (pro)collagen gene (Col2a1. Exp. Eye Res. 83, 297-303 (2006).
    8. Marneros, A. G. &, Olsen, B. R. Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome. Invest. Ophthalmol. Vis. Sci. 44, 2367-2372 (2003).
    9. Martin, A. C. Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene. Invest. Ophthalmol. Vis. Sci. 45, 3387-3396 (2004).
    10. Song, B. J., Tsang, S. H., Lin, C. S. Genetic models of retinal degeneration and targets for gene therapy. Gene. Ther. Mol. Biol. 11B, 229-262 (2007).
    11. Skeie, J. M., Mahajan, V. B. Dissection of Human Vitreous Body Elements for Proteomic Analysis. J Vis Exp. (2011).
    12. Ciechanowski, K. Impaired synthesis is not the reason for decreased activity of extracellular superoxide dismutase in patients with diabetes. Arch Med Res. 36, 148-153 (2005).

    Comments

    11 Comments

    very nice! very useful!
    What is the average protein concentration of vitreous from one eye?
    Reply

    Posted by: AnonymousApril 11, 2011, 1:54 AM

    The average concentration per eye is 1.5 mg/mL.
    Reply

    Posted by: Vinit M.April 20, 2011, 6:05 PM

    could I use this method of isolation lens and retina to culture for 1 week in media? Will there be any adverse effects I.e. bacterial contamination?
    Reply

    Posted by: AnonymousJuly 8, 2011, 9:29 AM

    You can use this method for the dissection. Bacterial contamination is always an issue with primary cultures. Use sterile instruments under a hood and cleanse the surfaces of the eye as much as possible with a solution of betadine or alcohol before cutting the cornea. First culture medium should contain antibiotics and fungizone to prevent contamination. I usually discontinue the use of fungizone after first passage but continue the use of the antibiotics with primary cultures.
    Reply

    Posted by: Vinit M.July 8, 2011, 9:36 AM

    To sterilize the ocular surface before dissection, you can apply a few drops of betadine (ophthalmic) to the cornea and then rinse with sterile PBS x6. Let us know how it works :-)
    Reply

    Posted by: Vinit M.July 12, 2011, 11:42 AM

    Can you first isolate the lens, and then go back and isolate the retina or do you have to do them both at one time?
    I have tried using a #11 belly blade disposable scalpel and I find it hard to tell if I have successfully made a good cut across the cornea because the eye is black. I do use an enlarging lens with a light to work under but I still find it difficult to tell if I have cut the cornea. Sometimes, if the cut totally across the cornea, i squeeze they eye and end upd squishing the lens- it never comes out. Will the opthalmic blace make th e difference in being successful
    Reply

    Posted by: Barbara S.December 1, 2011, 1:14 PM

    Can you first isolate the lens, and then go back and isolate the retina or do you have to do them both at one time?
    I have tried using a #11 belly blade disposable scalpel and I find it hard to tell if I have successfully made a good cut across the cornea because the eye is black. I do use an enlarging lens with a light to work under but I still find it difficult to tell if I have cut the cornea. Sometimes, if the cut totally across the cornea, i squeeze they eye and end upd squishing the lens- it never comes out. Will the opthalmic blace make th e difference in being successful
    Can you first isolate the lens and then go back and isolate the retina or do you have to do both at one time?
    Also, I have tried cutting the cornea using a disposable #11 belly blade but most of the time I am unsuccessful- hard to make a good cut. Will using the blade you recommend make a big difference?

    Reply

    Posted by: Barbara S.December 1, 2011, 1:19 PM

    1.There are two maxims for microsurgery: adequate illumination and adequate magnification. To complete the procedure, you really need to see the tissue well under a good binocular dissecting microscope to allow for stereo. A bright and sometimes eccentric (rather than coaxial) light from a goose neck source can sometimes highlight the tissues better.

    ². I would probably recommend a flat #11 blade. It will be easier to apply the very sharp tip to the tissue compared to a curved blade.

    3. When making the incision you should see a small amount of anterior chamber fluid efflux from the wound. This indicates that you have penetrated the full thickness of the cornea.

    4. Make the incision from one end of the limbus to the other. This should give an adequate wound size to allow the full lens to pass. To avoid crushing the lens, make sure that your forceps are far back enough to get behind the lens, and don²17;t close them completely before pulling anteriorly. The lens and retina can be isolated separately, but sometimes they stick together.
    Reply

    Posted by: Vinit M.December 4, 2011, 12:10 PM

    Hello
    Could we harvest some hyalocytes by using this method( vitroues evisceration) and culture those? I need to find a way of generating hyalocyte cell line from mouse eye.
    Thanks
    Reply

    Posted by: AnonymousMarch 15, 2012, 9:58 AM

    I think this is possible but challenging. Our filter was selected to separate any cells (from the retina) from the vitreous. In your case, you don't want to filter the vitreous and remove cells. I would consider eviscerating the lens with the vitreous, which often sticks to the lens. Then skip the filtration step and plate the lens and vitreous. The vitreous should solubilize in culture medium after a short time, and then you can remove the lens from the dish. Hopefully any hyalocytes would grow on the dish. Good luck and let us know how it works!
    Reply

    Posted by: Vinit M.March 22, 2012, 10:07 PM

    Hi,
    Vitreous evisceration from mouse is a tough task and your video article has made it look very simple.

    You are using a cut-off centrifugation column to separate vitreous from lens and retina. If you are using 100kD column then one would only able to get protein in vitreous that are below 100kD. But if my protein of interest is above 100kD, what is the best way of separating vitreous from lens ?

    Any advise ?

    Thanks
    Reply

    Posted by: Imran M.February 6, 2013, 3:10 PM

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