UT Health Science Center at San Antonio, Greehey Children's Cancer Research Institute and Department of Cellular and Structural Biology
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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).
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 pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.
1. Remove Extraneous tissue from the outside of the eye
2. Remove the cornea and lens
3. Quarter the resulting eyecup, resulting in a 4-"petaled", flower-like structure
4. Cut each of the four "petals" in half, resulting in an eight-petal flower-like structure
5. Remove the neural retina
6. Mount you specimen(s) on a slide
7. Representative Results:
The result of this procedure should be a structure that looks like a flower and should be fairly symmetrical.
Figure 1. Whole mount RPE from a wildtype C67Bl/6J mouse. RPE from black or agouti animals should be dark brown in color and should have a smooth surface. It is normal to notice undulations in the topography of the petals. The pigmentation on any give specimen may be somewhat variable, due to variable density of pigmentation of both the RPE and the underlying choroid. White arrows point to hypopigmented "channels"- this is normal and is due to the underlying vasculature of the eye. Blue arrow points to physical damage to both the RPE and the underlying choroid.
Figure 2. Whole mount RPE from a dilute mouse. Specimens harvested from dilute animals can range in color from nearly transparent to café au lait and any one specimen is like to have variability within it, indicated in black circles here. In general, RPE harvested from younger animals are lighter and those harvested from older animals are darker. Black arrows indicate some of the underlying vasculature, which appears hypopigmented.
Figure 3. Phalloidin staining can be used to detect physical damage to the RPE. Phalloidin staining clearly outlines the cell membranes, showing the hexagonal shape of the epithelium. (A) Example from a black mouse. (B) Example from a dilute mouse.
Figure 4. A poorly dissected RPE from a dilute mouse. (A) Brackets indicate margins of cornea that are too wide, which can cause buckling and/ or folding. Within the black circle you can see excessive amounts of extraneous tissue that have not been removed from the back of the eye. They are particularly obvious because the sample is from a dilute animal. The petal on the bottom left is partially folded over. (B) This whole mount has the appearance of a pinwheel. Black arrows indicate some of the cuts that were made. Many of the cuts from the corneo-scleral divide toward the optic nerve head are not in line with the diameter. (C) Black arrows indicate how the cuts should have been made, in line with the diameter and perpendicular to the tangent.
The RPE is the site of the pun assay, 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 murine eye develops radially outward from the optic nerve7 and therefore, the relative distance of events from the optic nerve head correlates to the time in development in which they occurred.
The RPE is also of interest to those who study retinal development or retinal diseases. Although RPE cells can be cultured, there are significant limitations to doing so. Adult RPE cells are post-mitotic7 and predictably do not proliferate well in vitro. Furthermore, those cultures that do grow show many alterations, including but not limited to changes in pigmentation, morphology, and cell junctions8- some of the characteristics that are the focus of scientific investigation. Therefore, preparation of a whole mount of the RPE is a relevant technique for in vivo studies of development and diseases of the eye.
Dissection of this small tissue is very tricky at first. It is important to be relaxed and maintain the proper posture and positioning as shown in this video article. If a whole mount RPE looks like a pinwheel, this is because the long cuts were not made in line with the diameter, i.e. not perpendicular to a tangent. This is most likely to occur when the scissors are not held in front of and perpendicularly away from the body. A jagged appearance to the long cuts is likely due to a slight withdrawal of the scissor blades between cuts.
A rough or rugged appearance to the RPE surface is not normal and could be due to damage caused by rough handling of the specimen. Damage can be verified using phalloidin staining. White or clear spots are holes in the RPE and the underlying choroid, also caused by rough handling or puncture.
If the RPE appears rough and opaque, this is probably due to remnants of the neural retina. This can occur if the eyes are under- or over-fixed (less than 2 hours, more than 4 hours in 4% PFA). This may also occur in properly fixed eyes that have been in storage for an excessive period of time (>6 months). Dispase treatment can be used to remove such remnants, but can also cause the RPE to break up.
Detecting physical damage to the RPE is very difficult in dilute or albino specimens. We recommend using phalloidin staining on your early dissections so you can see any damage that may have occurred due to handling. To do this, block for 1 hour with 200μL 2% BSA in 1XPBS in a micro centrifuge tube. Next, add 2μL phalloidin and incubate in the dark at room temperature for 20 minutes. Then wash 3x with 1mL of 1XPBS for 30 minutes each at room temp in the dark. Mount slides as usual. *Note: the stain is very bright and we do not recommend using flouramount or similar flourescence-preserving media.
Despite all the cautioning, a whole mount RPE is not terribly delicate and can easily withstand the handling necessary to perform dissection, staining, immunohistochemistry, or immunofluorescence.
No conflicts of interest declared.
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.
|straight forceps||Roboz Surgical Instruments Co.||RS-4903||tip: .08 x .04 mm material: INOX|
|45° forceps||Roboz Surgical Instruments Co.||RS-5005||tip: .05 x.01 mm material: INOX|
|15° "up" forceps||Roboz Surgical Instruments Co.||RS-5045A||tip: .1 x.06 mm material: INOX|
|spring scissors||Roboz Surgical Instruments Co.||RS-5604||comb. tip width 0.3mm cutting edge length 3mm material: stainless steel|
|binocular dissecting microscope||Carl Zeiss, Inc.||Discovery V.8||use reflected light source|
|phalloidin||Invitrogen||A22283||Alexa Fluor 546|