We would like to thank Brittany Day and Jessica Onyak for their technical assistance and Dr. Liu for kindly letting us use his epifluorescent microscope.&#160;Acknowledgements of Support: NIH R15EY026255-01 and the Karl Kirchgessner Foundation.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Akron.
1. Using the Superior Rectus Muscle Landmark to Identify Retinal Orientation
NOTE: The superior rectus muscle is a landmark for the dorsal retina (Table 1). If the experiment does not require the marking of the dorsal retina, skip step 1 and continue to step 2.
<ol>
	<li>Follow your approved Institutional Animal Care and Use Committee protocol for mouse euthanasia.</li>
	<li>To identify the general orientation of the globe, make a burn mark on the dorsal cornea directly between the nasal and temporal canthi near the cornea-sclera border immediately after euthanasia (Figure 1A). Make the burn mark by heating up a cautery pen for ten seconds and then touching the tip of the pen to the dorsal cornea for less than a second. 
	NOTE: Holding the cautery pen to the cornea for too long will cause the globe to puncture. 
	NOTE: While some cautery pens do emit light, the cautery pen listed in the Table of Materials does not emit any light when heated, making it a safe option for dark-adapted experiments.</li>
	<li>For enucleation, use curved forceps to gently push the eye out of its socket and grip the globe from underneath. Do not cut the optic nerve to remove the globe; instead, slowly lift the globe from its socket while simultaneously moving it gently from left to right until the globe is released from the socket. 
	NOTE: This motion will allow the rectus muscles to remain attached to the globe when the globe is finally removed completely from the socket. The optic nerve will also often remain attached to the globe.</li>
	<li>Transfer the globe with the attached rectus muscles in a Petri dish containing dissection medium. Make sure to keep track of which eye is the left eye and which is the right eye. 
	NOTE: The dissector should use an appropriate dissection medium that aligns with their experimental protocol.</li>
	<li>Under the dissection scope, visually locate the dorsal corneal burn and identify the superior rectus muscle with which it is associated (Figure 1A).</li>
	<li>Using dissection scissors or a 20 G (0.9 mm x 25 mm) needle (see Table of Materials), puncture the cornea at the burn mark. Make a deep relieving cut into the globe toward the optic nerve to bisect the superior muscle. An isolated and reconstructed retina with this cut is shown in Figures 1B and 1C.</li>
	<li>Begin to isolate the retina by using two sets of forceps (see Table of Materials) to gently tear the hole made with the puncture in step 1.6 until part of the retina is exposed. 
	NOTE: It is important that this be done gently, as tearing too forcefully can cause the relieving cut to tear further.</li>
	<li>Use forceps to tease apart the retina from the sclera until the sclera has been completely removed. Remove the iris, lens, vitreous, and any remaining structures with forceps until the retina is completely isolated. 
	NOTE: The protocol may be paused here. If the tissue is going to be fixed for s-opsin immunohistochemistry, continue to step 3.5.</li>
</ol>
2. Using the Choroid Fissure Landmark to Identify Retinal Orientation
NOTE: The choroid fissure is present on the sclera on the back of the eye, and runs from the temporal pole to the nasal pole (Figures 2B and 2C; Table 1).
<ol>
	<li>Follow approved Institutional Animal Care and Use Committee protocol for mouse euthanasia.</li>
	<li>To identify the general orientation of the globe, make a burn mark on the dorsal cornea directly between the nasal and temporal canthi near the cornea-sclera border immediately after euthanasia (Figure 2A). Make the burn mark by heating up a cautery pen for ten seconds and then touching the tip of the pen to the dorsal cornea for less than a second. 
	NOTE: Holding the cautery pen to the cornea for too long will cause the globe to puncture.</li>
	<li>Enucleate the eye and transfer the globe in a Petri dish containing dissection medium. Make sure to keep track of which eye is the left eye and which is the right eye. 
	NOTE: The dissector should use an appropriate dissection medium that aligns with their experimental protocol.</li>
	<li>Visually locate and identify the choroid fissure on the back of the eye (Figure 2B, 2C). 
	NOTE: The choroid fissure is also visible inside the eyecup under infra-red light20.</li>
	<li>Orient the globe in the Petri dish so that the dorsal burn is located at the superior pole, as it would be if the eye were still in the mouse. 
	NOTE: The presence of the dorsal burn allows for the identification of the nasal and temporal side of the globe, as long as whether it is a right or left eye has been documented: If it is a right eye, the nasal choroid fissure will be to the right of the burn and the temporal choroid fissure will be to the left of the burn. If it is a left eye, the temporal choroid fissure will be to the right of the burn and the nasal choroid fissure will be to the left of the burn. 
	Using dissection scissors or a 20 G (0.9 x 25 mm) needle (see Table of Materials), make one puncture&#160;in the globe where the dorsal burn is located.</li>
	<li>Make a shallow relieving cut toward the optic nerve where the dorsal corneal burn is located. This cut will be perpendicular to the choroid fissure, allowing for identification of the dorsal retina after isolation (Figure 2D).</li>
	<li>Make the following two deep relieving cuts toward the optic nerve: one by lining the blades of the dissection scissors up with the temporal choroid fissure line on the back of the eye, and one by lining the blades of the dissection scissors up with the nasal choroid fissure line on the back of the eye. These cuts are shown on an isolated and reconstructed retina in Figure 2D and 2E. 
	NOTE: Alternatively, a deep cut can be made at the temporal choroid fissure and a shallow cut can be made at the nasal choroid fissure, making the dorsal corneal burn cut unnecessary. This allows for accurate orientation of the retina with fewer relieving cuts.</li>
	<li>Begin to isolate the retina by using two sets of forceps (see Table of Materials) to gently tear the hole made with the puncture in steps 2.7 and 2.8 until part of the retina is exposed. 
	NOTE: It is important that this be done gently, as tearing too forcefully can cause the relieving cut(s) to tear further.</li>
	<li>Use forceps to tease apart the retina from the sclera until the sclera has been completely removed. Remove the iris, lens, vitreous, and any remaining structures with forceps until the retina is completely isolated. 
	NOTE: The protocol may be paused here. If the tissue is going to be fixed for s-opsin immunohistochemistry, continue to step 3.5.</li>
</ol>
3. Labeling the S-opsin Gradient in the Mouse Retina
NOTE: The s-opsin photopigment expression is asymmetrically distributed to the ventral retina1, making it an excellent marker for the ventral half of the retina. This method is only useful for fixed and immunostained tissue (Table 1). The following steps can be applied to retinas that have been dissected using any of the aforementioned methods.
<ol>
	<li>Follow approved Institutional Animal Care and Use Committee protocol for mouse euthanasia.</li>
	<li>Immediately after euthanasia, enucleate the eye and place the globe in a Petri dish with dissection medium. Make sure to keep track of which eye is the left eye and which is the right eye in order to identify retinal orientation after the retina is dissected. 
	NOTE: The dissector should use an appropriate dissection medium that aligns with their experimental protocol.</li>
	<li>Begin to isolate the retina by using two sets of forceps (Table of Materials) to gently tear a hole in the cornea until part of the retina is exposed. 
	NOTE: It is important that this be done gently, as tearing too forcefully can cause the retina to tear.</li>
	<li>Use forceps to tease apart the retina from the sclera until the sclera has been completely removed. Remove the iris, lens, vitreous, and any remaining structures with forceps until the retina is completely isolated. 
	NOTE: The protocol may be paused here. If using the retina for an ex vivo experiment, conduct the experiment before performing the following steps.</li>
	<li>Using dissection scissors, make four relieving cuts in the retina so that it will lie flat. Mount the retina ganglion cell-side up on nitrocellulose membrane (Table of Materials) by gently pressing each corner of the retina onto the membrane with forceps. 
	NOTE: The location of the relieving cuts can be arbitrary when using the s-opsin gradient for retinal orientation.</li>
	<li>Using forceps, transfer the mounted retina to the first well in a 24-well plate (Table of Materials) filled with 1 mL of 4% paraformaldehyde (Table of Materials) for fixation. Place the 24-well plate on an orbital shaker at room temperature (Table of Materials) and fix the retina for exactly 40 min. 
	NOTE: All of the following wash and incubation steps should be completed with the 24-well plate on an orbital shaker.</li>
	<li>Wash the retina for 15 min at room temperature by transferring it to the second well filled with 1 mL of 0.1 M PBS. Repeat this step twice by sequentially transferring the retina to the 0.1 M PBS-filled third and fourth wells.</li>
	<li>Transfer the mounted retina to the fifth well containing 1 mL of blocking solution (1.7% Triton X-100 and 5.2% donkey normal serum in 0.1 M PBS; see Table of Materials) and incubate overnight at 4 &#176;C.</li>
	<li>Add the rabbit anti-s-opsin primary antibody (see Table of Materials) to the blocking solution at a concentration of 1:500 and incubate for three days at 4 &#176;C.</li>
	<li>Wash the excess primary antibody from the retina six times by sequentially placing it in six wells filled with 1 mL of 0.1 M PBS for 10 minutes each at room temperature.</li>
	<li>Place the retina in a well with fresh blocking solution (1.7% Triton X-100 and 5.2% donkey normal serum in 0.1 M PBS) and add donkey anti-rabbit Alexa-594 secondary antibody (see Table of Materials). Incubate the retina with the secondary antibody overnight at 4 &#176;C.</li>
	<li>Wash the excess secondary antibody from the retina six times by sequentially placing it in six wells filled with 1 mL of fresh 0.1 M PBS for 10 min each at room temperature.</li>
	<li>Using forceps, transfer the mounted retina to a Petri dish containing 0.1 M PBS. Release the retina from the nitrocellulose membrane by gently inserting the tips of forceps between the retina and the membrane until the retina is no longer attached.</li>
	<li>Mount the retina on a glass microscope slide by gently prodding it with forceps until the retina sticks to the glass and remove the slide from the Petri dish.</li>
	<li>Cover the retina on the slide with Aquamount and cover it with a #1.5 coverslip. Place the slide in a slide tray (see Table of Materials) and allow it to sit at room temperature for an hour.</li>
	<li>Return the slide to the refrigerator and store in a slide tray (see Table of Materials) at 4 &#176;C when not in use. After the slide has been coverslipped for 24 hours, use nail polish to seal the sides of the slide to prevent desiccation. 
	NOTE: The protocol can be paused here.</li>
</ol>
4. Using Reconstructed Retinas Immunostained with S-opsin to Identify Retinal Orientation
<ol>
	<li>Visualize the s-opsin gradient with either a confocal microscope or an epifluorescent microscope with a camera attachment (see Table of Materials) and image the retina so that the entire retina is visible in one image (Figures 1B, 2D, 3A, and 3D). This can be done by imaging the retina in sections at low magnification and then stitching the images together.</li>
	<li>Name the retinas so they are identifiable. For example, name the first retina to be reconstructed &#34;Retina1&#34;.</li>
	<li>Download and install ImageJ at https://imagej.nih.gov/ij/download.html.</li>
	<li>Create an individual folder for each retina that needs to be reconstructed, but leave the folders empty. For example, create a folder titled &#34;Retina1&#34;. All files needed to reconstruct this retina will be placed in this folder in the subsequent steps. 
	NOTE: The only files these folders should contain are the files that are to be analyzed by Retistruct. Any files other than the ones detailed below will make the retina unable to be opened by the Retistruct software.</li>
	<li>Open the image of the retina in ImageJ by selecting File &#8594; Open and then choosing &#34;Retina1&#34;.</li>
	<li>Without making any changes to the image, save it as &#34;image.png&#34; to the folder titled &#34;Retina 1&#34; by selecting File &#8594; Save As &#8594; PNG. 
	NOTE: The file must be named &#34;image.png&#34; in order for the Retistruct software to recognize the file as a retina for reconstruction.</li>
	<li>Use the Segmented Line Tool to outline the edges of the retina. By clicking on two adjacent spots on the border of the retina, the segmented line tool will essentially &#34;connect the dots&#34; between the two adjacent spots, creating an outline. Repeat until the entire retina has been outlined. Save the retina outline as &#34;outline.roi&#34; to the folder titled &#34;Retina1&#34; by selecting Analyze &#8594; Tools &#8594; ROI manager &#8594; Add[t] &#8594; More &#8594; Save. 
	NOTE: The edge of the retina can be identified where the s-opsin staining transitions to the background.</li>
	<li>Use the Segmented Line Tool as instructed in step 4.7 to outline the border of the optic disc. Save the optic disc outline as &#34;od.roi&#34; to the folder titled &#34;Retina1&#34; by selecting Analyze &#8594; Tools &#8594; ROI manager &#8594; Add[t] &#8594; More &#8594; Save. 
	NOTE: The optic disc is identified as the small hole in the middle of the retina, and will vary based on the quality of dissection. 
	NOTE: All of the files needed for Retistruct reconstruction (&#34;image.png&#34;, &#34;outline.roi&#34;, and &#34;od.roi&#34;) should now be saved the folder &#34;Retina1&#34;.</li>
	<li>To download, install, and open the Retistruct program, follow the instructions outlined in the Retistruct user guide found in the Supplementary Materials section of Sterratt, et al.24</li>
	<li>Once the Retistruct window has appeared, click the &#34;Open&#34; icon at the top left of the window and select the directory folder &#34;Retina1&#34;.</li>
	<li>An image window will pop up indicating that no scale bar exists. Click &#34;Close&#34; and an image of the retina will appear in the box. Visualize the outline of the retina by clicking the &#34;Properties&#34; button in the top right of the window and change outline color to a visible color (Figure 5A).</li>
	<li>IMPORTANT: Specify whether the retina is from a right eye or left eye in the panel on the left (Figure 5A).</li>
	<li>Click the &#34;Add Tear&#34; button on the left and specify where a tear or cut is in the retina by clicking on the three vertices of the tear (Figure 5A). This will create lines that connect the three vertices of the cut. Repeat for all cuts in the retina.</li>
	<li>Specify the dorsal retina by clicking on an arbitrary point of the retinal outline. An uppercase letter &#34;D&#34; will appear at that point on the outline (Figure 5B). 
	NOTE: The dorsal retina will be the darker half of the retina, opposite the s-opsin gradient. However, marking the dorsal retina in Retistruct is not a reliable method for identifying the dorsal half of the retina, so the marking of &#34;dorsal&#34; can be arbitrary in this step.</li>
	<li>Reconstruct the retina by clicking the &#34;Reconstruct Retina&#34; button at the top left of the screen (Figure 5B). A polar plot of the reconstructed retina will appear with the cuts visible in the same color as the outline (Figure 5C).</li>
	<li>Click the &#34;Save&#34; button on the right of the screen so that the reconstructed retina and all of the data associated with it is saved in the &#34;Retina1&#34; folder directory (Figure 5D).</li>
	<li>Save the reconstructed retina by clicking the &#34;PDF&#34; button in the right-hand panel (Figure 5D). A box will appear asking for size specifications. The default size is acceptable for the following steps. This action will save the reconstructed retina as &#34;image.polar.pdf&#34; in the &#34;Retina1&#34; folder directory.</li>
	<li>Open &#34;image.polar.pdf&#34; in a Paint program (or other image manipulation program) and use the &#34;Paint Bucket&#34; tool (or similar) to change the background of the reconstructed retina to black. Save the reconstructed retina as a .tif file, such as &#34;Retina1_reconstructed.tif&#34; in the &#34;Retina1&#34; folder directory. 
	NOTE: The protocol can be paused here.</li>
	<li>Download the MATLAB code for rotating the retina called &#34;Retina_Rotator.m&#34; (see Supplementary Materials). Place the code file in its own folder with no other files in the folder.</li>
	<li>Open MATLAB, version 2007b or later. Double click on the code file to open it in MATLAB. In the command window, type &#34;Retina_Rotator&#34; and then hit the enter key. A search window will appear. 
	NOTE: The code is specific for .tif files. If the file to be rotated is not in the correct format, the code will not rotate the retina correctly. See steps 4.17 and 4.18. for saving the reconstructed retina in the proper format.</li>
	<li>Open the file to be rotated. For example, choose &#34;Retina1_reconstructed.tif&#34;. The code will then analyze the reconstructed retina and will automatically save the rotated retina as &#34;Retina1_reconstructed_rotated.tif&#34; in the folder where the original file is located.</li>
	<li>After the code has finished analyzing the retina, a window will also appear showing the images of the retina before and after rotation for comparison (Figures 3B and 3C; Figures 3E and 3F). 
	NOTE: This code rotates the reconstructed retina so that the ventral (brightest) half is on the bottom and the dorsal (dimmest) half is on the top, thus accurately orienting the retina according to the s-opsin gradient1. If whether the retina is from a right eye or left eye has been documented, the location of the nasal and temporal poles can also be extrapolated from this method of orientation (Figure 3).</li>
</ol>

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<li>Lamb, T. D., Collin, S. P., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolution+of+the+vertebrate+eye%3A+Opsins%2C+photoreceptors%2C+retina+and+eye+cup%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Evolution of the vertebrate eye: Opsins, photoreceptors, retina and eye cup.</a> Nat Rev Neurosci. 8 (12), 960-976 (2007).</li>
<li>Sterratt, D. C., Lyngholm, D., Willshaw, D. J., Thompson, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standard+anatomical+and+visual+space+for+the+mouse+retina%3A+Computational+reconstruction+and+transformation+of+flattened+retinae+with+the+Retistruct+package%5BTitle%5D)+AND+%22PLoS+Comput+Biol%22%5BJournal%5D)">Standard anatomical and visual space for the mouse retina: Computational reconstruction and transformation of flattened retinae with the Retistruct package.</a> PLoS Comput Biol. 9 (2), 1002921(2013).</li>
</ol>

The authors have nothing to disclose.

There has been no comprehensive, standardized protocol for determining and labeling the orientation of the isolated mouse retina in anatomical space. The protocol detailed here attempts to fill this void by standardizing and detailing how to use deep anatomical landmarks as reference points to reliably identify retinal orientation. It has been shown that the deep anatomical landmarks in this protocol provide a more accurate and reliable method for orienting the mouse retina than superficial landmarks such as corneal burns22. Thus, studies that have relied on corneal burns for retinal orientation may have had greater errors in orientation than studies that have relied on landmarks such as the rectus muscles and choroid fissure. This discrepancy highlights the need for and significance of this standardized protocol with respect to interpreting results and making comparisons between studies that depend upon accurate retinal orientation. Overall, a standardized protocol will provide a common method for vision researchers to follow, thus eliminating the presence of a confounding variable in data acquisition that may occur with the use of non-standardized methods for identifying retinal orientation.
The methods presented here are easily repeatable and applicable to many types of experimental protocols. In fact, one of the greatest advantages of this protocol is its adaptability. Because the choroid fissure, s-opsin expression, and rectus muscle landmarks have all been found to reliably identify retinal orientation22 the landmark that best suits the experimental parameters can be chosen to optimize data acquisition (Table 1). Additionally, methods of dissection can be combined so as to further clarify the orientation of the retina. For example, choroid fissure cuts can be combined with s-opsin immunohistochemistry in order to orient all four poles of the retina: nasal and temporal hemispheres can be identified by the choroid fissure cuts, and s-opsin immunohistochemistry can identify ventral and dorsal hemispheres. Yet, the adaptability of this protocol may be constrained by the time-sensitive nature of physiology experiments. Because the time it takes to identify a landmark, make a corneal burn, and execute a relieving cut could result in significant tissue death in ex vivo experiments, some of these dissection methods may be less than optimal. Fortunately, once a dissector has become familiar with either the choroid fissure or superior rectus muscle dissection method, identifying the deep landmarks and making the relieving cuts quickly become a part of the dissection routine and do not significantly add to the length of dissection. Although we do acknowledge that the steps detailed here can add on time to extremely time-sensitive experiments, we suggest using the s-opsin gradient for post hoc retinal orientation when the viability of the tissue is no longer an issue (Figure 3). Staining the retina for s-opsin is an effective way to orient the retina, as it can identify all four poles: s-opsin staining divides the retina into dorsal and ventral poles and allows for identification of the nasal and temporal poles depending on whether the retina is from a right or left eye (Figure 3). Therefore, we believe this protocol delivers a reliable and repeatable set of methods for accurate retinal orientation that can fulfill any experimental parameters.
As with any modified retinal dissection, the validity of the dissection method is limited by the accuracy of the dissector and the quality of the tissue that has been isolated. If any tissue is lost during dissection or a retina is too mangled for accurate reconstruction, Retistruct and the MATLAB program will not be able to reliably reconstruct or orient the retina. It is therefore important to practice the dissection method before using it for data-collecting experiments. While the types of dissections explained here are not difficult, they must be practiced to ensure the repeatability of identifying retinal orientation with a particular landmark. Furthermore, it is essential that the dissector practice visually identifying the anatomical landmarks prior to beginning data collection to make certain that the correct landmark is being used. One way to check the accuracy of a particular dissector is to make either choroid fissure cuts or superior rectus muscle cuts and then compare the location of the cuts to the s-opsin gradient, since it is a fixed marker and thus is not dependent on the accuracy of dissection. Potential dissectors can also compare their reconstructed retinas to the examples of reconstructed retinas with accurate landmark cuts are shown in Figure 1 and Figure 2. Essentially, a potential dissector should perform the steps outlined in this protocol for a particular dissection type, whether it be the superior rectus muscle or choroid fissure method, and compare the results to the s-opsin gradient to establish the validity of a particular dissector. Because if the dissector is unsure about the location of the landmark, it may result in an inaccurate orientation of the retina that will, by default, affect data collection and interpretation.

An important and sometimes overlooked aspect of retinal neuroscience is the proper orientation and analysis of the isolated whole-mount retina, whether it be the orientation of a retina in an electrophysiology recording chamber or on a histological slide. This is particularly important for studies involving the mouse retina, which is currently the most widely used model for investigations of the mammalian visual system. Recent discoveries reveal that the mouse retina is not spatially uniform but has density and size gradients of functionally-distinct retinal cell types, such as melanopsin ganglion cells, transient OFF-alpha ganglion cells,&#160;and cone opsins1,2,3,4,5. Consequently, the method used to determine the orientation of the retina may affect the experimental results involving cell type or opsin distributions2,3,6, direction tuning of direction-selective ganglion cells7,8,9, and topographic patterns of retinal degeneration10,11,12,13,14. In fact, not reporting how retinal orientation is reported can cause discrepancies in the literature and confusion when attempting to compare data between studies. It is therefore vital that researchers report the method for identifying the orientation of the retina so that results of such studies can be accurately interpreted.
Retinal orientation is commonly identified by scoring the dorsal, ventral, nasal or temporal cornea prior to ocular enucleation1,3,12,15,16,17,18,19 or by cutting or staining deep anatomical eye landmarks such as the extraocular muscles6,7, the choroid fissure20,21, or the s-opsin gradient2,3. The rectus muscles can be used to identify the dorsal, ventral, nasal, and temporal retina by making a deep relieving cut that bisects the attachment of the either the superior rectus, inferior rectus, medial rectus, or lateral rectus muscle, respectively. However, for most experiments, using one rectus muscle is sufficient for orienting the retina22. The choroid fissure, which is a remnant of eye development, can be viewed as a faint horizontal line on the back of the eye. Each end of this line terminates at either the nasal or the temporal pole of the globe23. Finally, s-opsin expression is asymmetrically distributed to the ventral retina in mice, and s-opsin antibodies can be used to reveal the ventral retina in immunohistochemical experiments1.
Recent work by Stabio, et al.22 demonstrated that superficial ocular landmarks such as corneal burns are a less reliable method for orienting the retina in anatomical space, most likely due to human error and variability in making the corneal burn when using the temporal and medial canthi as reference points. In contrast, deep landmarks, such as the superior rectus muscle, choroid fissure, and the s-opsin gradient, have been shown to be more reliable and accurate landmarks for orienting the retina22. However, the identification of these anatomical landmarks requires unique dissection steps that are not described in detail in the literature. Thus, the goal of this protocol is to provide a comprehensive tutorial on how to use the superior rectus muscle, choroid fissure, and s-opsin gradient to accurately identify the spatial orientation of the mouse retina. In addition, we have included a comparison of the effectiveness of two s-opsin antibodies, as well as a protocol for s-opsin immunohistochemistry.
One additional challenge to studies relying on precise retinal orientation is the large relieving cuts required to flatten wholemount retinas on a recording chamber, dish, or slide. This can introduce challenges for the analysis of what is naturally a three-dimensional structure when it is imaged as a flat two-dimensional structure. A program called Retistruct24 can be used to return a flat wholemount retina to its three-dimensional structure before the data collected from it is analyzed. Thus, a section of this protocol is dedicated to highlighting the steps that are necessary for using the Retistruct software to reconstruct s-opsin immunostained mouse retinas. We have also included a section of protocol for using our custom MATLAB script, which was developed to accurately rotate and orient mouse retinas stained with s-opsin.

<table><tbody><tr><td>0.1 M Phosphate Buffered Saline</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P5244</td><td></td></tr><tr><td>Axioplan2 Epifluorescent Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Clear Nailpolish</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Corning LSE Low Speed Orbital Shaker</td><td>Sigma-Aldrich</td><td>CLS6780FP</td><td></td></tr><tr><td>Costar TC-Treated 24-well Plates</td><td>Sigma-Aldrich</td><td>CLS3524</td><td></td></tr><tr><td>Dissection Microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Donkey anti-Goat Alexa 594</td><td>Life Technologies&amp;nbsp;</td><td>A11058</td><td></td></tr><tr><td>Donkey anti-Rabbit Alexa 594</td><td>Life Technologies&amp;nbsp;</td><td>A21207</td><td></td></tr><tr><td>Donkey Normal Serum</td><td>Millipore</td><td>566460</td><td>Use at 5.2% (52 &amp;mu;L with 86 &amp;mu;L of 20% Triton X-100 and 863 &amp;mu;L of 0.1M PBS for 1 mL of blocking solution)</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Goat anti-s-opsin</td><td>Santa Cruz Biotechnologies&amp;nbsp;</td><td>sc-14363</td><td>Not commerically available as of 2017</td></tr><tr><td>Graefe Curved Forceps</td><td>Fine Science Tools</td><td>11052-10</td><td></td></tr><tr><td>ImageJ or FIJI</td><td>National Institute of Health</td><td>N/A</td><td>Freely available software</td></tr><tr><td>Low Temperature Cautery Ophthalmic Fine Tip Cauterizer</td><td>Bovie Medical Corporation</td><td>AA00</td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>N/A</td><td>At least version 2007b or later</td></tr><tr><td>Micro Cover Glasses&amp;nbsp;</td><td>VWR International</td><td>48393-241</td><td></td></tr><tr><td>Micro Slide Trays</td><td>VWR International</td><td>82020-913</td><td></td></tr><tr><td>Moira Ultra Fine Forceps</td><td>Fine Science Tools&amp;nbsp;</td><td>11370-40</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Use at 4% (25 &amp;mu;L and 875 &amp;mu;L of 0.1 M PBS for 1 mL of fixative)</td></tr><tr><td>PrecisionGlide Needle 20G (0.90 mm x 25 mm)&amp;nbsp;</td><td>BD PrecisionGlide</td><td>305175</td><td></td></tr><tr><td>Pyrex Glass Petri Dish</td><td>Sigma-Aldrich</td><td>CLS3160152</td><td></td></tr><tr><td>R</td><td>The R Project for Statistical Computing</td><td>N/A</td><td>Freely available software; version 3.4.3 or later</td></tr><tr><td>Rabbit anti-s-opsin</td><td>Millipore</td><td>ABN1660</td><td></td></tr><tr><td>Retiga R3 Microscope Camera</td><td>Qimaging</td><td>01-RET-R3-R-CLR-14-C</td><td></td></tr><tr><td>Retistruct</td><td>N/A</td><td>N/A</td><td>Freely available software&amp;nbsp; compatiable with Windows 7 or Windows 10</td></tr><tr><td>Shandon Aqua-Mount Slide Mounting Media</td><td>Fisher Scientific</td><td>14-390-5</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Use 1.7% (86 &amp;mu;L of 20% Triton-X with 52 &amp;mu;L of Donkey Normal Serum and 863 &amp;mu;L of 0.1 M PBS for 1 mL of blocking solution)</td></tr><tr><td>Vannas Spring Dissection Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>5MP USB Microscope Digital Camera</td><td>AmScope</td><td>MU500</td><td>To be used with the Olympus Dissection Microscope</td></tr></tbody></table>

where you cut matters: a dissection and analysis guide for the spatial orientation of the mouse retina from ocular landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

A single relieving cut that bisects the superior rectus muscle accurately and reliably identifies the dorsal retina (Figure 1). The choroid fissure accurately and reliably identifies the nasal and temporal retina with deep relieving cuts along the temporal and nasal choroid fissure (Figure 2). In this example, a relieving cut has also been made in the dorsal retina in order to identify the dorsal/ventral axis of the retina (Figure 2D, vertical arrow). The steps of these processes are shown for purpose of replication by future dissectors. A combination of s-opsin immunohistochemistry (Figure 3A and 3D), reconstruction with Retistruct software (3B, 3E) and accurate rotation with a custom MATLAB code (3C, 3F) allows for the identification of the ventral and dorsal halves of the retina, as well as the nasal and temporal poles if it is known whether the retina is from a right or left eye (Figure 3). We also compared two commonly used s-opsin primary antibodies for effectiveness in labeling s-opsin cones (Figure 4A-D): Both the goat anti-s-opsin primary antibody and the rabbit anti-s-opsin primary antibody effectively label s-opsin cones (Figure 4E) in the same mouse.
Relieving cuts were identified on s-opsin immunostained reconstructed retinas and their locations were compared to the orientation determined by the s-opsin gradient. Using our custom MATLAB code (see Supplemental Materials), retinas were accurately rotated so that the highest concentration of s-opsin staining is located ventrally, thus placing true dorsal at 90° (for superior rectus), true nasal at 0° (for nasal choroid fissure) and true temporal at 180° (for temporal choroid fissure). The value of each individual relieving cut angle was determined using the angle tool in ImageJ after retinas were rotated according to the s-opsin gradient. An average angle was calculated for each relieving cut type and the average value of each relieving cut type was then plotted on a polar plot (Figure 6). On average, superior rectus muscle cuts identified the dorsal pole at 96.3 ± 4.3° (n = 11) (Figure 6). The nasal choroid fissure identified the nasal pole at 6.7 ± 5.8° and the temporal choroid fissure identified the temporal pole at 172.0 ± 4.4° (n = 9; Figure 6).
<img alt="Microscope image; eye anatomy in retinal experiments; highlights dorsal burn, superior rectus." class="xfigimg" src="/files/ftp_upload/57861/57861fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Using the superior rectus muscle to accurately identify the dorsal retina of a right eye. (A) An example of a dorsal corneal burn near the corneal-scleral border made with a cautery tip pen (white arrow). The superior rectus muscle is also visible in this view (white arrow). (B) An example of a whole mounted retina with a relieving cut made in the dorsal retina by bisecting the superior rectus muscle. Arrow depicts the deep relieving cut made in the dorsal retina by bisecting the superior rectus muscle. The retina is stained with primary antibody goat anti-s-opsin (see Table of Materials) and secondary antibody donkey anti-goat Alexa 594 (see Table of Materials; excitation: 590 nm; emission: 620 nm) (cyan). Retina was imaged with an epifluorescent microscope with a Texas Red filter (595 nm). (C) A retina reconstructed in Retistruct and rotated with a custom MATLAB code (see Supplemental Materials) with the superior rectus muscle relieving cut visible (white arrow). D: dorsal, V: ventral, T: temporal, N: nasal. Scale bars = 1 mm. <a href="https://www.jove.com/files/ftp_upload/57861/57861fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Eye anatomy analysis; dorsal burn and choroid fissure; microscope images; retinal structure study." class="xfigimg" src="/files/ftp_upload/57861/57861fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Using the choroid fissure to accurately identify the nasal and temporal poles of the retina of a right eye. (A) An example of a dorsal corneal burn near the corneal-scleral border made with a cautery tip pen. (B) The choroid fissure visible on the back of the eye on the sclera (white arrow). The dorsal corneal burn is also visible in this view, located about 90° from the temporal choroid fissure. (C) The choroid fissure visible on the back of the eye on the sclera, traveling from the optic nerve to the corneal-scleral border. (D) A retina stained with goat anti-s-opsin (see Table of Materials) and secondary antibody donkey anti-goat Alexa 594 (see Table of Materials; excitation: 590 nm; emission: 620 nm) (cyan) with choroid fissure cuts (horizontal arrows) and the dorsal relieving cut (vertical arrow). Retina was imaged with an epifluorescent microscope with a Texas Red filter (595 nm). (E) A retina reconstructed in Retistruct and rotated with a custom MATLAB code (see Supplemental Materials) with the dorsal relieving cut and the nasal and temporal choroid fissure cuts visible (white arrows). D: dorsal, V: ventral, T: temporal, N: nasal. Scale bars = 1 mm. <a href="https://www.jove.com/files/ftp_upload/57861/57861fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Retinal mapping diagram; right and left retinas unrotated/rotated; structural analysis method." class="xfigimg" src="/files/ftp_upload/57861/57861fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Using the s-opsin gradient to identify all four poles of the retina. (A) An example of a retina dissected from a right eye that has been immunostained to label s-opsin and imaged with an epifluorescent microscope with a Texas Red filter (595 nm). The cuts in this retina are arbitrary since the topographical orientation is determined by the s-opsin gradient. (B) The results of reconstructing the retina in A with Retistruct. Notice that the s-opsin gradient is not correctly aligned because the retina has not been run through the custom MATLAB code (See Supplemental Materials). (C) The results of rotating the retina in A with the custom code. The retina has been rotated so that the highest concentration of s-opsin staining is located at the bottom and identified as the ventral retinal. Because the retina is from a right eye, the temporal pole is located 90° counterclockwise from the dorsal pole and the nasal pole is located 90° clockwise from the dorsal pole. (D) An example of a retina dissected from a left eye that has been immunostained to label s-opsin and imaged with a Texas Red filter (595 nm). The cuts in this retina are arbitrary since the topographical orientation is determined by the s-opsin gradient. (E) The results of digitally reconstructing the retina in D with Retistruct. Notice that the s-opsin gradient is not correctly aligned because the retina has not been rotated by the custom code. (F) The results of rotating the retina in D with the custom code. The retina has been rotated so that the highest concentration of s-opsin staining is located at the bottom and identified as the ventral retinal. Because the retina is from a left eye, the nasal pole is located 90° counterclockwise from the dorsal pole and the temporal pole is located 90° clockwise from the dorsal pole. D: dorsal, V: ventral, T: temporal, N: nasal. Scale bars = 1 mm. <a href="https://www.jove.com/files/ftp_upload/57861/57861fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Retinal cross-section with immunofluorescence, opsin distribution, graph immunopositive scores." class="xfigimg" src="/files/ftp_upload/57861/57861fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Comparison of two primary s-opsin antibodies in labeling s-opsin cones. (A) A retina stained with the goat anti-s-opsin primary antibody (see Table of Materials). (B) The other retina of the same mouse stained with rabbit anti-s-opsin primary antibody (see Table of Materials). (C) A representative region (0.1 x 0.1 mm2) from a retina stained with the goat anti-s-opsin primary antibody. Image taken on an epifluorescent microscope at 40X magnification. (D) A representative region (0.1 x 0.1 mm2) from a retina stained with rabbit anti-s-opsin (see Table of Materials), a primary antibody alternative. Image was taken on an epifluorescent microscope at 40X magnification. (E) Both antibodies label the same number of s-cone outer segments because there is no significant difference in the number of immunopositive s-cones that are stained by goat anti-s-opsin and rabbit anti-s-opsin at any of the tested retinal eccentricities (n = 2; ANOVA with post hoc Bonferroni test; p &gt;0.05). Scale bars = 1 mm (A-B); 25 µm (C-D). <a href="https://www.jove.com/files/ftp_upload/57861/57861fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy image analysis; tissue structure comparison; software interface with measurement data display." class="xfigimg" src="/files/ftp_upload/57861/57861fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: A visual guide for using the Retistruct software to reconstruct retinas immunostained with s-opsin. (A) A retina opened in Retistruct with the outline visible and a "Tear" added. Points of the "Tear" are indicated with superimposed white arrows. All cuts in this retina are arbitrary, as no particular landmark was used to mark retinal orientation during dissection. Important buttons are outlined in red. (B) A retina with all "Tears" added and the dorsal retina identified with "D" on the edge of the retina. Notice that the "Reconstruct Retina" button is now visible. Important buttons are outlined in red. (C) The process of reconstructing a retina. The polar plot of the reconstructed retina will appear on the right, showing the relieving cuts in cyan (blue arrows superimposed to clarify cut locations). (D) The final result of running a retina through Retistruct. The original wholemount retina remains on the left and the reconstructed retina appears on the right. The relieving cuts are visible in cyan (white arrows superimposed to clarify cut locations). <a href="https://www.jove.com/files/ftp_upload/57861/57861fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Directional diagram; shows angular averages of eye structures: Superior Rectus, Choroid Fissure." class="xfigimg" src="/files/ftp_upload/57861/57861fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: The superior rectus muscle and choroid fissure can be used to accurately orient the mouse retina. A polar plot of the angles obtained from either superior rectus muscle relieving cuts or choroid fissure cuts in retinas that have been reconstructed with Retistruct. Relieving cuts were identified on s-opsin immunostained reconstructed retinas and their locations were compared to the location of the s-opsin gradient. Using the custom MATLAB code to accurately rotate the retinas so that the highest concentration of s-opsin staining is located ventrally, true dorsal (90° for superior rectus), true nasal (0° for nasal choroid fissure) and true temporal (180° for temporal choroid fissure) were determined for each retina. The value of each individual relieving cut angle was determined in ImageJ and an average angle was calculated for each relieving cut type. Superior rectus muscle cuts identified the dorsal pole at 96.3 ± 4.3° (n = 11). The nasal choroid fissure identified the nasal pole at 6.7 ± 5.8° and the temporal choroid fissure identified the temporal pole at 172.0 ± 4.5° (n = 9). <a href="https://www.jove.com/files/ftp_upload/57861/57861fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Deep Landmark</td>
			<td>Corneal Burn Location</td>
			<td>Pole of Retina Identified</td>
			<td>Experimental Application</td>
		</tr>
		<tr>
			<td>Superior Rectus</td>
			<td>Dorsal</td>
			<td>Dorsal</td>
			<td>Live or Fixed</td>
		</tr>
		<tr>
			<td>Nasal Choroid Fissure </td>
			<td>Dorsal</td>
			<td>Nasal</td>
			<td>Live or Fixed</td>
		</tr>
		<tr>
			<td>Temporal Choroid Fissure </td>
			<td>Dorsal</td>
			<td>Temporal</td>
			<td>Live or Fixed</td>
		</tr>
		<tr>
			<td>S-opsin Gradient</td>
			<td>None</td>
			<td>Dorsal, Ventral, Nasal, Temporal</td>
			<td>Fixed</td>
		</tr>
	</tbody>
</table>
Table 1: Deep landmarks, the pole of the retina they identify, and whether they can be used for live or fixed tissue application.

Watch this Scientific Journal Video about Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks at JoVE.com

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

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Neuroscience

14.0K Views.  The University of Akron. Dit protocol biedt een uitgebreide dissectie en analyse gids voor het gebruik van diep oogbeschadigingen en/of bezienswaardigheden, s-opsin immunohistochemistry, Retistruct en aangepaste code nauwkeurig en betrouwbaar oriënteren het netvlies geïsoleerde muis in anatomische ruimte.

Video: Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

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.

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 ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Medicine

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

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, ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

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