This work was supported by grants from the Illinois Society to Prevent Blindness, National Institute of Health (EY031350 and EY001792) and an unrestricted grant from the Research to Prevent Blindness Foundation.

All animal studies were approved by the Office of Animal Care and Institutional Biosafety at the University of Illinois at Chicago. Seven-week-old male C57/BL6/J mice were housed in group cages in a pathogen-free environment on a 12 h-light/dark cycle and provided free food and water access. Mice were euthanized by CO2 asphyxiation, and the eyes were enucleated and processed immediately18. The animals were obtained from a commercial source (see Table of Materials). The essential tools needed for the study are depicted in Figure 1.
1. Delivery of the death-inducing insult
<ol>
	<li>Perform oxidative stress with TBH (ex vivo).
	<ol>
		<li>Euthanize the mice following institutionally approved protocols and enucleate their eyes18 (Figure 2A).</li>
		<li>Put the eyeballs directly into individual wells of a 24-well plate containing DMEM + 1% BSA, with or without 5 mM Tert-butyl hydroperoxide (TBH) (see Table of Materials); incubate for 1 h at 37 &#176;C. A positive control sample is treated with DNase (50 U/100 &#181;L) for 10 min to fragment DNA. 
		NOTE: The dose of TBH (the agent that induces oxidative stress) was chosen to induce a readily detectable and sub-maximal level of cell death within the isolated retinal vessels18.</li>
		<li>Fix the eyes in 10% buffered formalin overnight (16 h minimum).</li>
	</ol>
	</li>
	<li>Use cytokine cocktail to induce inflammation (in vivo).
	<ol>
		<li>Anesthetize the mice with an intraperitoneal injection of 100 mg/kg ketamine and 5 mg/kg xylazine.</li>
		<li>Use a customized 33 G needle (see Table of Materials) to inject 1 &#181;L/eye of the cytokine cocktail into the vitreous; the injection site is 2-3 mm from the limbus (Figure 2B). 
		NOTE: The cytokine cocktail contains 1:1:1 ratio of 1 &#181;g/mL TNF-&#945;, 1 &#181;g/mL IL-1&#946; and 1500 U/&#181;L IFN-&#947;18 (see Table of Materials).</li>
		<li>24 h after the injection, euthanize the mice (following institutionally approved protocols), enucleate their eyes and &#64257;x with 10% buffered formalin overnight. 
		&#8203;NOTE: The experiment can be paused after fixation for 1 week maximum and then restarted later.</li>
	</ol>
	</li>
</ol>
2. Retina isolation
<ol>
	<li>Cut open the eyeball.
	<ol>
		<li>Use straight forceps to grab the optic nerve gently (Figure 2C). Hold a micro-knife with the other hand to make an incision of 2-3 mm posterior to the limbus.</li>
		<li>Switch from the micro-knife to micro-scissors to cut parallel to the limbus while rotating the eyeball along with the optic nerve until the eyeball has been cut into two halves.</li>
		<li>Discard the anterior half of the eye, including the lens (Figure 2C).</li>
	</ol>
	</li>
	<li>Remove the sclera.
	<ol>
		<li>Use straight forceps to gently lift the sclera 1-3 mm off of the retina.</li>
		<li>Use micro-scissors to make two radial cuts in the sclera part of the way to the optic nerve. Avoid cutting the underlying retina.</li>
		<li>Use a pair of curved forceps to grab the scleral flap and tear it off the retina. The RPE layer will come off with the sclera.</li>
	</ol>
	</li>
	<li>Wash the retina.
	<ol>
		<li>Use a micro-spatula to transfer the isolated retina to a well within a 24-well dish that has been filled with double-distilled water.</li>
		<li>Gently shake the dish at mid-moderate speed at room temperature. Change the water every 30 min to 1 h, at least 4-5 times, then leave overnight.</li>
	</ol>
	</li>
</ol>
3. Retinal vasculature isolation
<ol>
	<li>Digestion: Replace the double-distilled water with 800 &#181;L of YL trypsin solution (3% trypsin in 0.1 M Tris buffer (pH 7.8)). Incubate at 37 &#176;C with gentle to no shaking for 4 h 15 min19 (Figure 2D). 
	NOTE: Avoid rapid shaking as this can damage the vasculature.</li>
	<li>Transfer: Dip the broad end of a glass transfer pipet with YL trypsin solution to transfer the retina to a 35 mm Petri dish containing lint-free, double-distilled water.</li>
	<li>Remove the outer nuclear layer (photoreceptors) (Figure 2E).
	<ol>
		<li>Flip the retina semi-sphere downward facing. Use the straight single-hair brush to gently press the retina to the bottom of the dish.</li>
		<li>Use the loop brush to gently brush away the photoreceptors of the retina. The brush strokes are applied in a direction from the optic nerve towards the periphery of the retina. The photoreceptors can detach in large sheets because they are not anchored to the rest of the retina by blood vessels.</li>
		<li>Using a 200 &#181;L pipette to collect and discard the sheets of neural tissue.</li>
	</ol>
	</li>
	<li>Remove the vitreous.
	<ol>
		<li>Flip the retina semi-sphere so that it faces upward. Use a pair of curved forceps (A) to grasp the vitreous as much as possible under a dissecting microscope. 
		NOTE: The vitreous look like a sheet of transparent tissue attached to the optic nerve or retina.</li>
		<li>Use another curved forceps (B) to grasp the end of the vitreous where it connects the optic nerve. Remove the vitreous by pulling forceps A away from forceps B.</li>
		<li>Discard the vitreous. Examine the remnant of the vitreous and repeat this step to remove all the vitreous as the residual will impede the next steps.</li>
	</ol>
	</li>
	<li>Remove the remaining neural and glial tissue (Figure 2F).
	<ol>
		<li>Flip the retina semi-sphere so that it is downward facing again. Once again, use the straight brush to gently press the retina to the bottom of the dish (do not use the tip of the hair).</li>
		<li>Use the loop brush to gently brush over the vasculature from the optic nerve head towards the periphery to remove the remaining neural tissue20.</li>
		<li>Spin the retina slowly with the straight brush and use the loop brush to remove all the small chunks of neural tissue on the retinal vasculature, till the vascular network is well cleaned (Figure 2G,H).</li>
	</ol>
	</li>
</ol>
4. Mounting the isolated retinal vasculature on a microscope slide
<ol>
	<li>Place a microscope slide.
	<ol>
		<li>Place a clean mounting cassette (see Table of Materials) under a dissecting microscope. Fill the cassette with double-distilled water.</li>
		<li>Use a black background under the dissecting microscope to help generate contrast to see the illuminated transparent vasculature.</li>
		<li>Use forceps to place a clean, labeled microscope slide into the bottom of the mounting cassette.</li>
	</ol>
	</li>
	<li>Transfer the retinal vasculature.
	<ol>
		<li>Dip the broad end of a glass transfer pipet with YL trypsin solution.</li>
		<li>Transfer the cleaned retinal vasculature and gently dislodge the vasculature into the double-distilled water within the mounting cassette and above the microscope slide.</li>
	</ol>
	</li>
	<li>Mount the retinal vasculature. 
	NOTE: While the vasculature is floating above the microscope slide, the isolated vasculature will acquire its normal bowl shape.
	<ol>
		<li>Use the loop brush to flip the retina semi-sphere facing upward and gently push the vasculature down onto the glass slide, and then use the hair to tack down the opened vasculature onto the center of the slide. The vasculature should stick to the slide as it touches.</li>
		<li>Flat mount the vasculature by brushing the bowl-shaped retinal vessel from the optic nerve to the periphery.</li>
		<li>Repeat brushing in all directions till the vasculature sticks to the slide completely.</li>
	</ol>
	</li>
	<li>Air dry the retinal vasculature.
	<ol>
		<li>Once the entire retinal vasculature attaches to the slide, take the slide out of the water either by gently lifting one edge or by slowly draining water at the corner of the cassette to minimize currents, then pulling it out (Figure 2I). 
		NOTE: The vasculature will become readily visible once it has once air-dried (Figure 2J).</li>
		<li>Mark the circumference of the retinal vasculature on the back of the slide with a marker pen.</li>
		<li>Proceed to stain the sample without pause.</li>
	</ol>
	</li>
</ol>
5. Death detection with TUNEL staining
NOTE: For details on this procedure, refer to Zheng et al.21. Representative images of ischemia +/- ox stress-induced apoptotic bodies in isolated retinal vessels are depicted in Figure 3.
<ol>
	<li>Rehydrate the isolated vasculature with PBS. Rinse it 3 times in PBS and then incubate with 1% Triton X-100 in PBS for 2 min on ice to permeabilize the isolated vasculature.</li>
	<li>Rinse the slides twice with PBS to remove the residual Triton X-100. Dry the area around the sample.</li>
	<li>Add 50 &#181;L of TUNEL reaction mixture (see Table of Materials) onto the sample. Incubate the slide in a humidified atmosphere for 60 min at 37 &#176;C in the dark.</li>
	<li>Rinse the slide 3 times with PBS to remove the TUNEL reaction mixture. Dry the area around the sample.</li>
	<li>Add a drop of DAPI mounting media to stain the nuclei with DAPI and mount the sample with a cover glass. Store at 4 &#176;C in the dark. 
	NOTE: The protocol could be paused for up to 1 week before capturing images.</li>
	<li>Photograph the resulting vasculature with a confocal &#64258;uorescence microscope (see Table of Materials). Capture six to eight randomly selected fields in the far periphery surrounding the optic nerve (Figure 4). 
	NOTE: Representative images of cytokine-induced apoptotic bodies in isolated retinal vessels are illustrated in Figure 5.</li>
	<li>Analyze the results.
	<ol>
		<li>For ex vivo, TBH-treated samples, perform the following steps.
		<ol>
			<li>Count the number of apoptotic bodies (TUNEL/DAPI double-positive species) in each field using Image J (see Table of Materials). Tally the mean of the apoptotic bodies in all of the fields of a single sample.</li>
			<li>Calculate the fold change in the number of apoptotic bodies between a randomly selected pair of non-DM and DM samples.</li>
			<li>Determine if there is a statistically significant difference using the two-tailed student t-test18. 
			NOTE: Stain the control and experimental samples (non-DM and DM) on the same occasion because the extent of TUNEL staining can vary, even when done in the same way. Since up to 10 retinas can be cleaned and mounted per day by an experienced user, plan to process an equal number of control and experimental retinas before starting this protocol.</li>
		</ol>
		</li>
		<li>For in vivo, cytokines cocktail treated samples, perform the following the steps.
		<ol>
			<li>Manually count the number of apoptotic bodies (TUNEL/DAPI double-positive species) in the entire retinal vasculature.</li>
			<li>Calculate the fold change in the number of apoptotic bodies between a randomly selected pair of non-DM and DM samples.</li>
			<li>Determine if there is a statistically significant difference using the two-tailed student t-test.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Novel Retina Protection Assay for Diabetic Retinopathy Pathogenesis Study

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The authors have no con&#64258;icts of interest to report.

In this study, an assay was established to detect resistance/vulnerability of the retinal vasculature to death induced by DM/DR-related insults such as ischemia/oxidative stress and cytokines. This manuscript provides a detailed description of this assay, which is a modification of several published protocols19,20,21.
The protocol encompasses several crucial stages. First, it is imperative to meticulously dissect the retina, ensuring the preservation of the vascular network and preventing substantial tears. This can be accomplished by making an incision 2-3 mm posterior to the limbus since the retina tightly adheres to the ora serrata, and separating it is challenging. Second, coat all instruments that come into contact with the vessels (e.g., single-hair brushes, transferring pipet, and forceps) with trypsin by dipping these tools into the YL trypsin solution throughout the procedure. This prevents the vasculature from adhering to the instruments that are used in this protocol. Third, because the microvasculature is nearly invisible under normal light, heightened vigilance is required during debris aspiration and transfer to the microscope slide in order to prevent accidental loss.
An appropriate degree of enzymatic digestion of the various layers of the retina is crucial; insufficient digestion prevents the separation of neuronal tissue from the vascular network, while excessive digestion dissolves the vascular plexus. Various digestion times ranging from 1 h19 to overnight 23 have been reported. Based on observations, a digestion time of 4 h and 15 min yields the most favorable results compared to durations of 2 h, 3 h, and 4 h. Prolonging the digestion beyond this point is unlikely to enhance the process and might instead compromise the integrity of the vasculature.
In cases where the digested retina adheres to the single-hair brush, dip the hair in YL trypsin solution multiple times. This reduces the brush&#39;s sticky areas. If the hairbrush still adheres to the vascular tissue, then inspect it for residual fragments of vitreous, and remove them with forceps.
The optimal moment for complete vitreous removal is after eliminating the photoreceptors, but prior to the removal of the remaining neural and glial tissue. The retina maintains rigidity until photoreceptors are removed. Extracting the vitreous at this stage could potentially tear the retina/vasculature. The delicate layers of residual neural and glial tissue play a pivotal role in preserving the curved form of the vascular structure. They prevent the retina from tearing at its center when the vitreous is detached from the optic nerve.
If the isolated vasculature does not adhere to the microscope slide at all, then it indicates that the section of the slide where adhesion is expected to occur is dirty. Try moving the vessels across the slide&#39;s surface to locate a sticky spot, switching to a different slide or meticulously cleaning the slide and trying again. If the vessels stick to the slide before they unfurl into their bowl-like shape, lift the vasculature from the slide in order to allow it to float freely in the water once again. Do this step with forceps that have been repeatedly dipped in the YL trypsin solution.
Several alternative techniques for isolating the vasculature have been reported, which would not be suitable for the protection assay described herein. For instance, osmotic lysis has been employed to isolate the vasculature from unfixed retina samples, facilitating biochemical investigations of the tissue24,25. However, this procedure may not preserve the anatomy of the vasculature as well as the approach employed in this article. Similarly, while the tissue print method to isolate large segments of microvasculature enables analysis of the vasculature&#39;s electrotonic architecture26, the entire vascular bed is typically not recovered.
This assay was developed because existing approaches to monitor capillary degeneration do not address the issue of protection. Capillary degeneration, which occurs after prolonged DM, indicates if DR has developed. In addition to diagnosing DR, this outcome is useful to assess if an agent/therapy prevents DR. However, existing capillary degeneration assays do not speak to the underlying mechanism of action of the agent. Such an agent may prevent pathological events that drive DR such as increased oxidative stress or cytokines. Alternatively, the agent may enforce protection by enhancing resilience to oxidative stress and cytokines and/or promote the repair of damage. This new protection assay can be used to determine if the beneficial effect of a given therapy involves enforcing the endogenous system that protects from DM-related death.
A drawback of this protection assay is that it does not distinguish the two cell types within the retinal vessels: endothelial cells (ECs) and pericytes (PCs). While the appearance of their nuclei in PASH-stained sections is cell-type specific (Figure 6), not every nucleus displays diagnostic features. Approximately 30% of the nuclei cannot be unambiguously defined as EC or PC at least in part because the two-dimensional images obtained from PASH-stained samples incompletely resolve the three-dimensional structure of the vascular plexus. This obstacle could be surmounted by additional analysis such as immunofluorescent staining with cell type-specific markers. Such images, which distinguish the two cell types could be co-stained with TUNEL to determine the resistance/vulnerability of each of the vascular cell types.
This vascular-focused assay does not provide any information regarding the neural retina. Additional assays could be developed to provide such information. For instance, instead of isolating the retinal vasculature, a single cell suspension of the entire retina could be generated and then analyzed (by fluorescent activated cell sorting) for resistance/vulnerability. The inclusion of cell type-specific markers (both neural and vascular) along with indicators of cell death would provide a more complete picture of the retinal cell types that have the capacity for protection from DM/DR-mediated death.
In conclusion, the protection assay described herein provides a powerful approach to investigating the mechanism responsible for the delay between the onset of DM and the manifestation of DR in mice.

Diabetic retinopathy (DR) is one of the microvascular complications of diabetes mellitus (DM), and the leading cause of blindness in working-age individuals in developed countries1. Major risk factors for diabetic retinopathy are the duration and degree of hyperglycemia2,3,4. While DM causes dysfunction of both the vascular and neural components of the retina5, diagnosis of DR is based on morphological features of the retinal vasculature6.
Hyperglycemia-induced oxidative stress is one of the drivers of DR pathogenesis7. Increased oxidative stress causes widespread damage, which compromises the functionality of the mitochondria and thereby further increases the level of reactive oxygen species. These events are accompanied by leakage of retinal vessels, an increase in the level of inflammatory cytokines, and death of both neural and vascular cell types within the retina. Loss of vascular cells, and hence the functionality of the extensive capillary network of the retina, results in hypoxia, a potent stimulus for a variety of responses5. Such responses include increased expression of vascular endothelial growth factor (VEGF) that drives both permeability and angiogenesis, cardinal features of the advanced, sight-threatening stages of DR - diabetic macular edema, and proliferative diabetic retinopathy6.
Certain features of DR suggest that an organism (both patients and experimental animals) has an intrinsic ability to resist this indication. Patients experience several decades of DM before they develop sight-threatening DR8,9,10,11,12,13. While rodent models of DM do not develop the advanced, sight-threatening stages of DR14, the initial/mild form of DR that manifests does so only after a period of weeks or months of DM15,16. Furthermore, in both patients and experimental animals, DR is progressive, and retinal dysfunction/damage increases as the duration of DM is prolonged. Finally, some patients with DM never develop DR. In certain instances, this is because such individuals do not experience diabetes long enough for DR to develop. In other instances, it is because they display extraordinary resistance to DR; as is the case with participants of the Medalist study, who do not develop DR after 50 or more years of DM17. Despite such compelling support for the existence of protection from DR and its enormous translational relevance, the mechanism underlying protection has not been aggressively investigated.
The protection assay described herein was developed to facilitate investigation of why DR is delayed from the onset of DM in diabetic mice. The key steps of this assay, applied to DM and non-DM mice, include (1) delivering a sub-maximal death-inducing insult to the eye (ex vivo or in vivo), (2) isolating the retinal vasculature, (3) staining the vasculature with TUNEL and DAPI, (4) photographing the resulting images and quantifying the percentage of TUNEL/DAPI double-positive species.

<table><tbody><tr><td>10% neutral buffered formalin</td><td>Fischer Scientific</td><td>SF100-4</td><td>Fixation</td></tr><tr><td>24-well plates</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>33 G needle</td><td>Hamilton</td><td>customized</td><td></td></tr><tr><td>Ammonium hydroxide&amp;nbsp;</td><td>Sigma</td><td>221228-1L-PCA</td><td></td></tr><tr><td>C57/BL6/J mice</td><td>The Jackson Laboratory</td><td>Jax #000664</td><td></td></tr><tr><td>Cytokine cocktail</td><td></td><td></td><td>consisted of a 1:1:1 ratio of 1 &amp;micro;g/mL TNF-&amp;alpha;, 1 &amp;micro;g/mL IL-1 &amp;beta; and and 1500 U/&amp;micro;LIFN- &amp;gamma;</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Any microscope that allows good visualization of the retina is adequate.</td></tr><tr><td>Dumont #3 forceps</td><td>Fine Science Tools (FST)</td><td>11231-30</td><td>Straight tips</td></tr><tr><td>Dumont #5 forceps&amp;nbsp;</td><td>Fine Science Tools (FST)</td><td>11253-25</td><td>Micro-blunted tips</td></tr><tr><td>Easy-Grip Tissue Culture Dishes</td><td>Falcon</td><td>353001</td><td>35 x 10 mm</td></tr><tr><td>Glass transfer pipet</td><td>Fischer Scientific</td><td>1367820A</td><td>snap off the thin end of a Pasteur pipet and fit the &amp;ldquo;broken&amp;rdquo; end with a rubber bulb.</td></tr><tr><td>Harris modified hematoxylin&amp;nbsp;</td><td>Sigma</td><td>HHS32</td><td></td></tr><tr><td>Image J</td><td>NIH, Bethesda</td><td></td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>In Situ Cell Death Detection Kit, Fluorescein</td><td>Milipore</td><td>11684795910</td><td>TUNEL reaction mixture</td></tr><tr><td>Micro cover glasses</td><td>VWR</td><td>48366-227</td><td>22 mm x 22 mm</td></tr><tr><td>Microknife&amp;nbsp;</td><td>Sharpoint</td><td>72-1551</td><td></td></tr><tr><td>Micro-spatula</td><td>Fine Science Tools</td><td>10091-12</td><td></td></tr><tr><td>Mounting cassette&amp;nbsp;</td><td></td><td></td><td>Any transparent cassette that is slightly bigger than the microscope slide</td></tr><tr><td>Periodic acid</td><td>Sigma</td><td>3951</td><td></td></tr><tr><td>Periodic acid solution</td><td></td><td></td><td>35 mM periodic acid with 12 mM sodium acetate&amp;nbsp; in H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td></tr><tr><td>Permount mounting medium&amp;nbsp;</td><td>Fischer Scientific</td><td>SP15- 100</td><td></td></tr><tr><td>Prism 9</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Prolog Gold antifade reagent with DAPI</td><td>Invitrogen&amp;nbsp;</td><td>P36935</td><td></td></tr><tr><td>Recombinant human IFN- &amp;gamma;</td><td>Peprotec</td><td>300-02</td><td></td></tr><tr><td>Recombinant human IL-1 &amp;beta;</td><td>Peprotec</td><td>200-01B</td><td></td></tr><tr><td>Recombinant human TNF-&amp;alpha;</td><td>Peprotec</td><td>300-01A</td><td></td></tr><tr><td>Schiff reagent base&amp;nbsp;</td><td>Sigma</td><td>3952016</td><td></td></tr><tr><td>Shaker Incubator (belly button shaker)</td><td>IBI Scientific&amp;nbsp;</td><td>BBUAAUV1S</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>71196</td><td></td></tr><tr><td>Steritop sterile vacuum bottle</td><td>Millipore</td><td>SCGPS05RE</td><td>Create filtered water</td></tr><tr><td>Superfrost Plus treated microscope slides&amp;nbsp;</td><td>Fischer Scientific</td><td>12-550-15</td><td>use slides from unopened box</td></tr><tr><td>Tert-butyl hydroperoxide (TBH)</td><td>Sigma</td><td>75-91-2</td><td></td></tr><tr><td>TRIZMA base</td><td>Fischer Scientific</td><td>11-101-5522</td><td>make 100 mM Tris, adjust pH to 7.8 using HCl)</td></tr><tr><td>Trypsin 1:250</td><td>Amresco</td><td>0458-50G</td><td></td></tr><tr><td>Two &amp;ldquo;brushes&amp;rdquo;</td><td></td><td></td><td>made from single black hair&amp;nbsp; taped to the end of plastic transfer pipet.&amp;nbsp; One brush with a free end. The other brush with a loop</td></tr><tr><td>Vannas Spring Scissors&amp;nbsp;</td><td>Fine Science Tools (FST)</td><td>15000-00</td><td></td></tr><tr><td>Xylene</td><td>Sigma</td><td>65351-M</td><td></td></tr><tr><td>YL trypsin solution&amp;nbsp;</td><td></td><td></td><td>3% trypsin in 0.1 M Tris (pH 7.8)</td></tr><tr><td>Zeiss LSM 710 fluorescence microscope</td><td>Zeiss Microscopy</td><td></td><td></td></tr></tbody></table>

an assay to detect protection of the retinal vasculature from diabetes-related death in mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

The successful isolation of retinal vasculature results in a flat mount of the entire network of the mouse retinal vasculature, with the architectural integrity intact (Figure 2J). Upon staining with periodic acid-schiff hematoxylin (PASH), it is possible to distinguish the two vascular cell types: endothelial cells (ECs) and pericytes (PCs) (Figure 6). The endothelial cell nuclei are elongated, lightly stained, and reside entirely within the vessel walls. Pericyte nuclei are circular, densely stained, and protrude from the capillary walls. The PASH-stained samples also reveal acellular capillaries, which lack nuclei.
The approach to induce death was guided by the following rationale. It was speculated that protection was limited, i.e., could be overwhelmed by a very strong death-inducing insult. Consequently, insults (both ischemia/oxidative stress and cytokines18) were optimized so that they would induce a readily detectable increase but yet submaximal extent of death (Figure 3 and Figure 5).
It is important to highlight that the presence of TUNEL-positive nuclei was contingent on the specific type of insult employed to trigger cell death. The ischemia/oxidative stress insult led to a tram-track pattern of cell apoptosis, as illustrated in Figure 3, whereas the cytokine insult resulted in a distinct and well-defined pattern, as depicted in Figure 5. Both patterns can be observed in retinal vessels from DR patients22, suggesting that both types of agents induce cell death in humans. Moreover, the observed morphological distinctions offer a means to evaluate if pathology was driven by oxidative stress or cytokines.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66123/66123fig01.jpg" /> 
Figure 1: Tools for isolating mouse retinal vasculature. From left to right: the mounting cassette, two single-hair brushes, an inverted transfer pipet, micro-spatula, two curved forceps, spring scissors, micro-knife, and forceps with straight tips. <a href="https://www.jove.com/files/ftp_upload/66123/66123fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66123/66123fig02.jpg" /> 
Figure 2: Schematic showing the key steps of delivering a death-inducing insult and then isolating the retinal vasculature from a mouse eye. (A)&#160;Induction of ischemia/oxidative injury, ex vivo. After enucleation, the eyeball is subjected to ischemia in the presence of TBH. (B) In vivo administration (intravitreal injection) of cytokines. (C) Cutting the eyeball into two halves. (D) Retinal enzymic digestion: removing the sclera and washing the retina in double-distilled water overnight. Incubating in YL trypsin solution at 37 &#176;C for 4 h 15 min in a well of a 24-well plate. (E) Removing the photoreceptors. (F) Removing the remaining neural and glial tissue. (G) Cleaned bowl-shaped vascular network facing upward. (H) Isolated vascular network in a 35 mm dish with a black background on the stage of a dissecting microscope. (I) Flat-mounted retinal vasculature on a slide. (J) Air-dried retinal vasculature on a slide.&#160;This figure has been modified from Li et al.18. <a href="https://www.jove.com/files/ftp_upload/66123/66123fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66123/66123fig03v2.jpg" /> 
Figure 3: Detection of apoptotic bodies within mouse retinal vessels treated ex vivo with ischemia +/- TBH. Representative images of ischemia +/- ox stress-induced apoptotic bodies in isolated retinal vessels. The heading of each column indicates the staining. (A-C) Retinal vessels isolated from eyeballs that underwent 1 h of ischemia alone. (D-F) Same as (A-C), except the 1 h insult was a combination of ischemia and oxidative stress (5 mM TBH). The red arrows point to representative TUNEL/DAPI double-positive species. (G-I) Positive control treated with DNase. Scale bar = 50 &#181;m. This figure has been modified from Li et al.18. <a href="https://www.jove.com/files/ftp_upload/66123/66123fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66123/66123fig04.jpg" /> 
Figure 4: Illustration of field selection for quantification of results. A 5 x 5 tile scan of the retinal vasculature with the merge of TUNEL and DAPI signal. The selection of six to eight fields in the far periphery surrounding the optic nerve is shown with red squares. Magnification, 200x. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66123/66123fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66123/66123fig05.jpg" /> 
Figure 5: Detection of the apoptotic bodies within mouse retinal vessels in response to intravitreal injection of cytokines (in vivo administration). Representative images of cytokine-induced apoptotic bodies in isolated retinal vessels. The heading of each column indicates the staining. (A-C) Images of the retinal vessels isolated from mice intravitreally injected with PBS. (D-F) Same as (A-C), except a cytokine cocktail was injected along with PBS. The red arrows point to representative TUNEL-positive species. Scale bars = 100 &#181;m. This figure has been modified from Li et al.18. <a href="https://www.jove.com/files/ftp_upload/66123/66123fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66123/66123fig06.jpg" /> 
Figure 6: A representative image of a PASH-stained retinal vessels. Retinal vessels of a mouse that experienced 20 weeks of STZ-induced DM were isolated as described in Figure 2, stained with PASH, and imaged while illuminated with visible light. Pericyte nuclei in capillaries tend to be more circular and densely stained (blue arrows), whereas elongated and less densely stained nuclei are diagnostic of endothelial cells (red arrows). The yellow arrow points to an acellular capillary. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66123/66123fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Author Spotlight: Understanding Retinal Vessel Resilience and Disease Progression at JoVE.com

Author Spotlight: Understanding Retinal Vessel Resilience and Disease Progression

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...

an-assay-to-detect-protection-retinal-vasculature-from-diabetes

Research

JoVE Journal

Medicine

1.0K Views.  University of Illinois at Chicago. Ons doel is om de reden achter het geleidelijke begin van diabetische retinopathie te begrijpen, die tientallen jaren na het begin van diabetes optreedt. We onderzoeken de pathogenese van diabetische retinopathie in twee fasen: verlies van bescherming en accumulatie van schade. Ons doel is om de bescherming van netvliesvaten tegen door diabetes veroorzaakte beledigingen te onderzoeken door middel van een beschermingstest.Onze beschermingstest onthult de dubbele fasen van beschermingsve...

Video: Auteur in de schijnwerpers: inzicht in de veerkracht van het netvlies en ziekteprogressie

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

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Neuroscience

Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a ...

Behavior

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Biology

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

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