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.

1. Remove Extraneous tissue from the outside of the eye
<ol>
	<li>Pour 1X PBS into the lid of a 35mm dish. The level should be just below the lip of the lid.</li>
	<li>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 rather than against it.</li>
	<li>Using both pairs of angled forceps (45&deg; and 15&deg;), gently remove as much muscle and connective tissue as possible, pulling the tissue against the grain (i.e. toward the cornea). Some connective tissue will remain. Effort should be made to squeeze the eyeball as little as possible.</li>
	<li>Trade your 15&deg; forceps for spring scissors. While holding the eye steady with 45&deg; forceps, use the spring scissors to cut away remaining muscle and connective tissue, as well as the optic nerve. *Tip: Use the outside edge of the scissors to push the tissue against the grain (toward the corneo-scleral divide) and then trim. Continue until all extraneous tissue has been removed from the eye.</li>
</ol>
2. Remove the cornea and lens
<ol>
	<li>With the cornea facing up, use the 45&deg; forceps to pinch up a small fold in the center of the cornea.</li>
	<li>Using the spring scissors, make a small incision at the base of the flap. Let go of the flap.</li>
	<li>Holding the eyes still with your forceps, insert the lower scissor blade into and perpendicular to the incision and make a cut toward the corneo-scleral divide. Try to keep your lower blade under the iris so both the cornea and iris are cut. Do not cut all the way to the corneo-scleral divide. Keep scissors inserted in the eye.</li>
	<li>Using your 45&deg; forceps, carefully rotate the eye so that your scissors are parallel to the corneo-scleral divide and make a small cut. Slowly rotate the eye 360&deg;, making small incisions all the way around. Again, try to keep the lower blade of your spring scissors under the iris so both the iris and cornea are cut. Lift the iris and cornea away from the rest of the eye and discard them.</li>
	<li>Tilt the eye on its side and press down gently on the posterior half of the eye, forcing the lens out. Discard the lens. The inside of the remaining eyecup is lined by the neural retina, which should look smooth and opaque.</li>
</ol>
3. Quarter the resulting eyecup, resulting in a 4-"petaled", flower-like structure
<ol>
	<li>The eye should be positioned such that the opening of the eyecup is perpendicular to your body. Use your 45&deg; forceps to steady and position the eyecup.</li>
	<li>Holding your spring scissors in front of you so they are perpendicular to your body, insert the lower blade of your spring scissors into the top edge of the open eye cup. Using your 45&deg; forceps, carefully align the eye cup so that a single, straight cut can be made from the corneo-scleral divide toward the optic nerve head. All cuts should be perpendicular to the corneo-scleral divide and unless specified, should go as close to the optic nerve head as possible without cutting into it.</li>
	<li>Rotate the eyecup 180&deg;, maintaining its perpendicularity with your body. Use technique in step 3.2 to make a second perpendicular cut.</li>
	<li>Rotate the eyecup and repeat the technique described in 3.2 to make two more cuts. All four cuts should be as close to 90&deg; apart as possible. Keep in mind that the eye is not perfectly spherical so there will be some variability.</li>
</ol>
4. Cut each of the four "petals" in half, resulting in an eight-petal flower-like structure
<ol>
	<li>Transfer the quartered eyecup from the 35mm dish to a clean slide. With the eyecup facing up, scoop under it with the 45&deg; forceps. Carefully lift it out of the liquid and place it on a clean slide.</li>
	<li>Using both pairs of angled forceps, gently open out the eye, with the neural retina facing up. DO NOT REMOVE THE NEURAL RETINA- it helps the eye maintain some of its spherical shape so you are not working against it. It also protects the RPE from being accidently damaged in the following procedures.</li>
	<li>Rotate the slide until one of the petals is perpendicular to your body.</li>
	<li>Use your 15&deg; forceps, gently grab the corner of the petal. The corner of the petal should be cornea, not neural retina or RPE. Lift up on the corner gently, allowing it to retain its curvature.</li>
	<li>Insert the bottom blade of your spring scissors between the petal and the slide. Line up your scissors such that a single, straight, perpendicular cut can be made from the corneo-scleral divide toward the optic nerve head. Cut as close to the optic nerve head as possible without cutting into it. Let go of the petal, allowing it to lie on the slide.</li>
	<li>Rotate the slide 90&deg;, so that the next petal is perpendicular to your body. Repeat step 4.5.</li>
	<li>Repeat steps 4.5 and 4.6 twice more. The resulting structure should look like an eight-petal flower.</li>
</ol>
5. Remove the neural retina
<ol>
	<li>Pipette 100-200 &mu;L of 1X PBS onto the specimen. This will prevent the neural retina from sticking as you peel it off of the RPE.</li>
	<li>Typically the neural retina is loosely attached to the RPE. However, it does take practice and skill to remove only the neural retina and not the RPE. Gently grasp a petal of the neural retina with one pair of angled forceps while stabilizing (holding down) the rest of the eye with the other pair of angled forceps. Pull the neural retina away from the rest of the eye. Continue this procedure until the entire neural retina has been removed. *Tip: stick the tip of your 15&deg; forceps into the optic nerve head to anchor the eye. Then use the 45&deg; forceps to sweep up underneath the neural retina and lift it off and away. It is usually best to lift the neural retina from the optic nerve head toward the corneo-scleral divide. Be sure to pull away any loose remnants of the iris. The iris is quite sticky and can otherwise get stuck to the RPE.</li>
	<li>Rinse the specimen with 1X PBS. Pipette 100-200 &mu;L of 1X PBS into the specimen and suck it back up. Discard the PBS. Repeat as necessary to remove dust and debris.</li>
</ol>
6. Mount you specimen(s) on a slide
<ol>
	<li>Pipette 80-90 &mu;L of 90% glycerol onto a new slide in a rectangular shape. Use the side of the pipette tip to make an even, rectangular smear of glycerol. (This is the procedure for mounting two RPE on one slide.)</li>
	<li>Insert the lower arm of 15&deg; forceps under the specimen. Gently lift the specimen off the dissecting slide.</li>
	<li>Slowly lower the specimen into the glycerol. Gently wiggle the forceps as you pull them out from under the specimen.</li>
	<li>Repeat steps 6.1-6.3 for the other specimen.</li>
	<li>Rotate the slide so that the long side is perpendicular to your body. Hold your cover glass at a 45&deg; angle to the slide so that it is touching the slide. Move it toward the glycerol smear until they touch. Maintaining contact between the cover glass and slide, slowly move the cover glass away from the smear, toward the edge of the slide. Line up the long sides of the slide and the cover glass so that they are parallel. Hold the corners of the cover glass between your thumb and forefinger.</li>
	<li>Pick up the 15&deg; forceps in your other hand. Cradle the upper (free) edge of the cover glass in the crook of the arm. Very slowly, lower the cover glass. When contact is made between the cover glass and the glycerol, stop and wait for the glycerol to migrate. Repeat this lowering and waiting until the RPE whole mounts are fully covered. This is to prevent the highly viscous glycerol from capturing air bubbles. DO NOT PUSH DOWN ON THE COVERGLASS - the glycerol will squeeze out from under it, making adhesion very poor.</li>
	<li>With clear nail polish, paint around the perimeter where the cover glass and slide meet.</li>
	<li>Place the slide on top of an empty rack to dry.</li>
</ol>
7. Representative Results:
The result of this procedure should be a structure that looks like a flower and should be fairly symmetrical.
<img src="/files/ftp_upload/2563/2563fig1.jpg" alt="Figure 1" /> 
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.
<img src="/files/ftp_upload/2563/2563fig2.jpg" alt="Figure 2" /> 
Figure 2. Whole mount RPE from a dilute mouse. Specimens harvested from dilute animals can range in color from nearly transparent to caf&eacute; 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.
<img src="/files/ftp_upload/2563/2563fig3.jpg" alt="Figure 3" /> 
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.
<img src="/files/ftp_upload/2563/2563fig4.jpg" alt="Figure 4" /> 
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.

<ol>
	<li>Bishop, A. J., Kosaras, B., Sidman, R. L., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzo(a)pyrene+andX-rays+induce+reversions+of+the+pink-eyed+unstable+mutation+in+the+retinal+pigmentepithelium+of+mice.">Benzo(a)pyrene andX-rays induce reversions of the pink-eyed unstable mutation in the retinal pigmentepithelium of mice.</a> Mutat. Res.. 457, 31-31 (2000).</li><li>Reliene, R. H. l. a. v. a. c. o. v. e., Mahadevan, A., Baird, B., M, W., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diesel+exhaust+particles+cause+increased+levels+of+DNA+deletions+after+transplacental+exposure+in+mice.">Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice.</a> Mutat. Res. 570, 245-2452 (2005).</li><li>Bishop, A. J., Barlow, C., Wynshaw-Boris, A. J., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm+deficiency+causes+increased+frequency+of+intrachromosomal+homologous+recombination+in+mice.">Atm deficiency causes increased frequency of intrachromosomal homologous recombination in mice.</a> Cancer Res. 60, 395-399 (2000).</li><li>Brown, A. D., Claybon, A. B., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+WRN+helicase+domain+is+not+required+for+spontaneous+homologous+recombination-mediated+DNA+deletion.">Mouse WRN helicase domain is not required for spontaneous homologous recombination-mediated DNA deletion.</a> J. Nucleic Acids. , (2010).</li><li>Claybon, A., Karia, B., Bruce, C., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP1+suppresses+homologous+recombination+events+in+mice+in+vivo.">PARP1 suppresses homologous recombination events in mice in vivo.</a> Nucleic Acids Res. , (2010).</li><li>Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53-,+and+Gadd45a-deficient+mice+show+an+increased+frequency+ofhomologous+recombination+at+different+stages+during+development.">p53-, and Gadd45a-deficient mice show an increased frequency ofhomologous recombination at different stages during development.</a> Cancer Res. 63, 5335-5343 (2003).</li><li>Bodenstein, L., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+development+of+the+mouse+retinalpigment+epithelium.+Part+I.+Cell+and+tissue+morphometrics+and+topography+of+mitoticactivity.">Growth and development of the mouse retinalpigment epithelium. Part I. Cell and tissue morphometrics and topography of mitoticactivity.</a> Dev Biol. 121, 192-204 (1987).</li><li>Burke, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+phenotype+and+the+RPE:+is+the+answer+blowing+in+the+Wnt?.">Epithelial phenotype and the RPE: is the answer blowing in the Wnt?.</a> Prog Retin. Eye Res. 27, 579-595 (2008).</li></ol>

No conflicts of interest declared.

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...

<table><tbody><tr><td>straight forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-4903</td><td>tip: .08 x .04 mm material: INOX</td></tr><tr><td>45&amp;#x00B0; forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-5005</td><td>tip: .05 x.01 mm material: INOX</td></tr><tr><td>15&amp;#x00B0; &quot;up&quot; forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-5045A</td><td>tip: .1 x.06 mm material: INOX</td></tr><tr><td>spring scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-5604</td><td>comb. tip width 0.3mm cutting edge length 3mm material: stainless steel</td></tr><tr><td>binocular dissecting microscope</td><td>Carl Zeiss, Inc.</td><td>Discovery V.8</td><td>use reflected light source</td></tr><tr><td>phalloidin</td><td>Invitrogen</td><td>A22283</td><td>Alexa Fluor 546</td></tr></tbody></table>

dissection of a mouse eye for a whole mount of the retinal pigment epithelium

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.

Watch this Scientific Journal Video about Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium at JoVE.com

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

dissection-mouse-eye-for-whole-mount-retinal-pigment

Research

JoVE Journal

Neuroscience

49.2K Views.  Greehey Children's Cancer Research Institute and Department of Cellular and Structural Biology. A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Video: Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Medicine

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

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 ...

Biology

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...