A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.
Abstract
The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the punassay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.
Protocol
1. Remove Extraneous tissue from the outside of the eye Pour 1X PBS into the lid of a 35mm dish. The level should be just below the lip of the lid. Using straight forceps, transfer eye(s) from the storage tube to the 35mm dish. Eyes are submerged in PBS for two reasons: 1. The extraneous tissues will “float” away from the eye so you can see and remove them easily, and 2. During dissection, the eye will naturally maintain its spherical shape while in suspension, allowing you to work with this shape …
Discussion
The RPE is the site of the punassay, an in vivo assay of homology-directed repair. The pun assay has been used to study the effects of different DNA damages1,2 and DNA damage signaling and repair genes3,4,5 on the frequency of homology-directed repair. This assay is highly sensitive, detecting single-cell events on the RPE1 . It can also detect differences in the timing of homology-directed repair events during development6. The…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Institute of Environmental Health Sciences [K22ES012264 to A.J.R.B.] and an American Cancer Society InstitutionalResearch Grant [ACS-IRG-00-173-04]pilot projectaward [to A.J.R.B.]. We also thank members of the Bishop lab for critical reading of the manuscript and comments on the video and Adam Brown in particular, for the example of what not to do. We thank Dr. Donald McEwen of Greehey Children’s Cancer Research Institute for allowing us the use of his dissecting scope/ video camera set-up for filming of the dissection video. We thank Daron Brown at Corte Instruments for sharpening and repair of our microdissection tools.
Claybon, A., Bishop, A. J. R. Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium. J. Vis. Exp. (48), e2563, doi:10.3791/2563 (2011).