Thank David Yee for his technical assistance. The work was supported in part by the Massachusetts Lions Eye Research Foundation and the Boston University Chobanian &#38; Avedisian School of Medicine Wing Tat Lee Award.

All procedures were performed according to an animal use protocol approved by the Boston University Institutional Animal Care and Use Committee according to the NIH Guide for the Care and Use of Laboratory Animals and complies with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research.
1. Experimental animals
<ol>
	<li>House C57BL/6J mice under standard conditions.</li>
</ol>
2. Preparing the required solution and infusion line
<ol>
	<li>In a 50 mL&#160;syringe, fill with sterilized 0.9% NaCl saline solution at one per mouse. Place the syringe at a height of 120 cm above the level of the bench surface.</li>
	<li>Connect the filled syringe to an infusion line and a stopcock with a 30 G needle attached to the end of the line. Flush out all air bubbles from the line.</li>
</ol>
3. Preparing the workspace
<ol>
	<li>Set up a surgical/dissecting microscope under the saline bottle.</li>
	<li>Create a bed to stabilize the mouse during the procedure by cutting out a depression of approximately 6 cm x 3 cm in a sponge and placing it securely in a styrofoam well or other flat container.</li>
</ol>
4. Anesthetizing the mouse
<ol>
	<li>Anesthetize the mouse with a standard mixture of ketamine and xylazine at a level to achieve deep anesthetization. 
	NOTE: In this protocol, a mixture of ketamine 100 mg/kg and xylazine 10 mg/kg was injected intraperitoneally to achieve adequate anesthesia depth.</li>
	<li>Achieve adequate anesthesia when there is no paw withdrawal in response to a toe pinch and no blink reflex when the cornea is gently touched. Administer additional anesthesia if the mouse shows any signs of pain, such as hyperventilating or limb movement, while anesthetized.</li>
</ol>
5. Dilating the iris
<ol>
	<li>Dilate the iris of the eye to be perfused with 1 drop of 1% tropicamide. Allow 5 min for dilation. Apply an ophthalmic ointment such as bacitracin to the eye that will not be perfused.</li>
</ol>
6. Anterior chamber cannulation
<ol>
	<li>Securely place the anesthetized mouse in the sponge bed under the microscope. Apply saline solution to the eyes to rinse any debris or fur from the eye surface.</li>
	<li>Use a pair of non-toothed forceps and gently propose one of the eyes. 
	NOTE: An experiment will use either the left or right eyes of the mouse, but never both on the same mouse.</li>
	<li>With the infusion line closed, cannulate the anterior chamber with the needle approximately 2 mm from the limbus. Ensure the needle pierces the cornea perpendicularly to the peripheral, curved cornea surface, then slightly flattened parallel to the plane of the iris. The cornea may be manipulated to advance the needle and maintain eye control. Avoid striking the iris or lens and enlarging the corneal wound, which can result in leakage. Details are further described&#160;by Buchi et al.2.</li>
</ol>
7. Ischemic phase
<ol>
	<li>Turn the stopcock to run the infusion line. Verify that there is no leakage through the corneal wound. Ensure that there is a gradual distension of the cornea as the intraocular pressure increases. 
	NOTE: If significant leakage does not seal as the cornea distends, adequate pressure will not be maintained, and the procedure will have to be on a different mouse.</li>
	<li>Check that the intraocular pressure has elevated to 90-100 mmHg with a tonometer. Secure the infusion line with tape, carefully ensuring the needle does not change position and start leaking.</li>
	<li>Apply an ophthalmic ointment such as bacitracin to the perfused eye to prevent dryness. Place the mouse away from the microscope and underneath a heating lamp to maintain normal body temperature and monitor for the next 60 min.</li>
	<li>Remeasure the intraocular pressure at 30 min before the procedure ends to ensure the intraocular pressure is between 90-100 mmHg.</li>
</ol>
8. Reperfusion phase
<ol>
	<li>After 60 min, return the mouse to the microscope. Bring the saline solution bottle down to the level of the mouse, normalizing the intraocular pressure.</li>
	<li>Measure the intraocular pressure with the tonometry to check that it is near normal, 20 mmHg. Once the intraocular pressure is normalized, carefully remove the needle, avoiding damage to the lens or iris.</li>
</ol>
9. Post-operative care
<ol>
	<li>Coat mouse eyes with any ophthalmic antibacterial ointment such as a bacitracin veterinary ophthalmic ointment.</li>
	<li>Monitor the mouse for 1-2 h on a heated surface until they fully recover from anesthesia. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Return the mice to cages and housing once fully recovered.</li>
</ol>
10. Enucleating the eye3
<ol>
	<li>Anesthetize the mouse first with a mixture of ketamine 100 mg/kg and xylazine 10 mg/kg injected intraperitoneally and then euthanize via cervical dislocation.</li>
	<li>Position the euthanized mouse on a flat, dry, and smooth surface. Rinse the eye with a few drops of saline solution.</li>
	<li>Apply gentle pressure to the lateral canthus with the forceps until the eyeball is displaced from the socket and the optic nerve is accessible. Using Westcott scissors, cut following the orbital margins to sever the extraocular muscles.</li>
	<li>Position the Westcott scissors to the most posterior aspect of the eye socket and sever the optic nerve.</li>
</ol>
11. Fixing the sample4
<ol>
	<li>Place the enucleated eye into 4% paraformaldehyde in 0.1 M PBS in a sealed vial. After 3 days, remove the eye, place it into a vial of 70% ethanol solution, and store it overnight.</li>
	<li>Remove the eyes and place them in a vial of 95% ethanol solution. Then, place the vial in a sealed vacuum for 15 min, remove it from the vacuum, and leave it in room air for 45 min.</li>
	<li>Repeat the vacuum process, sequentially placing the eye into the following solutions: 100% ethanol, 100% ethanol, 100% xylene solution, and 100% xylene solution.</li>
	<li>Remove the eye and place it into a clean, empty vial. Fill the vial with 60 &#176;C liquidized paraffin. Place the vial into a sealed 60 &#176;C vacuum oven for 30 min and then 30 min in the 60 &#176;C oven without vacuum.</li>
	<li>Remove the eye, place it into a clean, empty vial, and fill it with 60 &#176;C liquidized paraffin. Store the vial at 60 &#176;C overnight.</li>
</ol>
12. Embedding the sample
<ol>
	<li>Transfer the eye to a metal embedding mold and fill the mold with liquidized paraffin on a 60 &#176;C hotplate, orienting the eye in the desired direction to make sections through the optic disk. Allow the mold to cool at room temperature (RT), then store at 4 &#176;C.</li>
</ol>
13. Sectioning the sample5
<ol>
	<li>Transfer the embedded specimens to the microtome. Slowly trim the block until the eye is reached and the sections include the optic disc.</li>
	<li>Cut sections as ribbons of 5 &#181;m thickness and float them on a water bath. Lift the paraffin ribbon carefully using forceps, lay ribbons of three sections onto a slide, and let it dry overnight.</li>
	<li>Repeat this process until there are at least 8 slides for each eye with sections centered on the optic disc.</li>
</ol>
14. Staining the sections5
<ol>
	<li>Heat the slides in a 60 &#176;C oven for 1 h to melt the paraffin. Submerge the slides in RT xylene three times, for 5 min each.</li>
	<li>Submerge the slides in 100% ethanol three times, for 3 min each. Rinse the sections in tap water for 1 min.</li>
	<li>Incubate the sections in hematoxylin stain for 45 s. Rinse the sections in tap water for 2 min.</li>
	<li>Incubate the sections in the Bluing reagent for 1 min. Rinse the sections in tap water for 1 min.</li>
	<li>Incubate the sections in eosin for 15 s.</li>
	<li>Rinse the sections in 95% and 100% ethanol twice for 1 min each. Rinse the sections in 100% xylene four times, for 1 min each. Then, mount the slides with mounting media.</li>
</ol>
15. Histological analysis6
<ol>
	<li>Analyze each retinal section under the microscope and record the number of retinal folds, the percentage of cell loss in the various retinal layers (ganglion cell layer, inner nuclear layer, outer nuclear layer), the presence of vitreous or subretinal hemorrhage, and the extent of retinal pigment epithelial damage. Refer to Table 1 for details on the scoring criteria.</li>
</ol>

Mouse Model for Retinal Ischemia-Reperfusion Injury to Study Retinal Pathologies

<ol>
	<li>Lee, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+induced+in+a+mouse+model+of+ischemia-reperfusion+injury+and+its+management+by+pemafibrate+treatment.">Retinal degeneration induced in a mouse model of ischemia-reperfusion injury and its management by pemafibrate treatment.</a> FASEB J. 36 (9), e22497 (2022).</li><li>Buchi, E. R., Suivaizdis, I., Fu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+retinal+ischemia+in+rats:+An+experimental+model+for+quantitative+study.">Pressure-induced retinal ischemia in rats: An experimental model for quantitative study.</a> Ophthalmologica. 203 (3), 138-147 (1991).</li><li>Aerts, J., Nys, J., Arckens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+reproducible+and+straightforward+method+to+perform+in+vivo+ocular+enucleation+in+the+mouse+after+eye+opening.">A highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening.</a> J Vis Exp. (92), e51936 (2014).</li><li>Nayagam, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+processing+eyes+implanted+with+a+retinal+prosthesis+for+localized+histopathological+analysis.">Techniques for processing eyes implanted with a retinal prosthesis for localized histopathological analysis.</a> J Vis Exp. (78), e50411 (2013).</li><li>Pang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+preparation+of+mouse+eye+sections+for+routine+histology,+immunofluorescence,+and+RNA+in+situ+hybridization+multiplexing.">Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing.</a> STAR Protoc. 2 (4), 100879 (2021).</li><li>Shome, A., Mugisho, O. O., Niederer, R. L., Rupenthal, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+grading+system+for+experimental+autoimmune+uveitis+in+mice.">Comprehensive grading system for experimental autoimmune uveitis in mice.</a> Biomedicines. 11 (7), 2022 (2023).</li><li>Ng, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-melanocyte-stimulating+hormone+maintains+retinal+homeostasis+after+ischemia/reperfusion.">Alpha-melanocyte-stimulating hormone maintains retinal homeostasis after ischemia/reperfusion.</a> Biomolecules. 14 (5), 525 (2024).</li><li>Lee, D., Kang, H., Yoon, K. Y., Chang, Y. Y., Song, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+retinal+hypoperfusion+injury+induced+by+unilateral+common+carotid+artery+occlusion.">A mouse model of retinal hypoperfusion injury induced by unilateral common carotid artery occlusion.</a> Exp Eye Res. 201, 108275 (2020).</li><li>Goit, R. K., Taylor, A. W., Lo, A. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+alpha-melanocyte-stimulating+hormone+protects+retina+after+ischemia/reperfusion+injury+in+type+I+diabetes.">Anti-inflammatory alpha-melanocyte-stimulating hormone protects retina after ischemia/reperfusion injury in type I diabetes.</a> Front Neurosci. 16, 799739 (2022).</li><li>Chen, J., Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+functional+evaluation+of+ocular+inflammatory+disease+using+the+model+of+experimental+autoimmune+uveitis.">Clinical and functional evaluation of ocular inflammatory disease using the model of experimental autoimmune uveitis.</a> Methods Mol Biol. 1899, 211-227 (2019).</li></ol>

The authors have no disclosures.

The ischemia/reperfusion model provides a reproducible method for studying the mechanisms and pathology of retinal ischemia/reperfusion injury. This model has usefulness in studying the pathology of retinal ischemia/reperfusion injury, and for identifying therapeutic targets. Several critical steps in the protocol may pose challenges and require technical skills to complete successfully. One is the actual cannulation, which can take multiple attempts to master and has a risk of damage to other structures in the eye. Additionally, leakage from the corneal wound can occur for several reasons, including improper cannulation and movement of the needle after cannulation. To minimize this risk, the protocol beds the mouse in a sponge to hold it in position right after cannulation. Once the needle and infusion line are secured to this apparatus, there is minimal unintended movement of the needle within the cannulated eye. A limitation of this model is that it is an acute ischemia/reperfusion of the whole retina, which may differ from retinopathies caused by localized or repeated ischemia/reperfusion injuries. The complexity of the retina in terms of its anatomy and pathophysiology has intricate signaling pathways in ischemic/reperfusion injury. This protocol models key components of retinal injury following an ischemia/reperfusion event that helps to promote a greater understanding of the pathology of retinal degeneration.
Other ischemia/reperfusion injury models employed, such as unilateral carotid artery occlusion8,9. However, these models have several limitations due to the technical challenges of invasive surgery, affecting multiple organs, and significant levels of systemic morbidity and mortality of the experimental animals. These limitations are mitigated by localizing the ischemia/reperfusion to the eye with an anterior chamber cannulation. Compared to other forms of anterior chamber cannulation, this model is unique in that it incorporates intraocular pressure normalization before withdrawing the needle. Upon previous histopathologic analyses, there were often significant traumatic injuries to the lens, iris, and globe that may have arisen from withdrawing the needle when the intraocular pressure was high. By lowering the infusion source down to the level of the mouse, the intraocular pressure decreased in a controlled manner to normal before withdrawing the needle. This additional step limits retinal injury and subsequent degeneration to ischemia/reperfusion injury caused by the elevated interocular pressure while minimizing the effect of the canulation and the physical manipulation of the eye and mouse. 
In addition, we have added a histopathology scoring scheme to evaluate retinal pathology. This scoring scheme was adapted from a commonly used scoring scheme for assessing the histopathology of retinas from eyes with autoimmune diseases10. There are several similarities between retinas with inflammation and retinas with ischemia/reperfusion. Commonly, hemorrhage, cell loss, and structural deformations are possible. Retinas with ischemia/reperfusion have a noticeable loss of retinal ganglion cells and a measurable rate of retinal degeneration. Since this method is not invasive, affects only the eye, and does not affect mouse survival, it is possible to collect the eyes for analysis at any time after the procedure. This allows for measuring cell loss, protein, or gene expression over time. In addition, it makes it possible to treat eyes at the time of the ischemia/reperfusion or anytime after and multiple times. The scoring of H&amp;E-stained retinal sections can be complemented with immunostaining for cytokines and growth factors or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptosis. In addition, it makes it possible, after the ischemia/reperfusion procedure, to isolate specific populations of retinal cells for proteomic and genomic studies of retinal degeneration relative to histopathology.

Ischemia/reperfusion injuries manifest in various retinal pathologies, encompassing diabetic retinopathy, glaucoma, retinal vascular occlusions, and related vaso-occlusive conditions. Given the retina's high oxygen demand, it is particularly susceptible to ischemia/reperfusion injury, a phenomenon implicated in the pathogenesis of diseases like diabetic retinopathy. This form of injury results in the demise of retinal ganglion cells (RGCs), morphological degeneration of the retina, compromised retinal function, and eventual vision impairment1. Modeling the ischemia/reperfusion is appropriate for studies on mechanisms and treatment responses in various retinal pathologies related to ischemia/reperfusion injuries.
We focused on refining a model for ischemia/reperfusion injury in the mouse eye. The anterior chamber cannulation model for pressure-induced retinal ischemia injury was first published by B&#252;chi et al. in 19912. They successfully increased the intraocular pressure to 110 mmHg for a controlled time. They found that the resulting retinal injury was consistent with findings similar to retinal and choroidal vascular occlusion. Due to its relatively simple methodology and cost-effective execution, it became a functional model for the study of retinal ischemic injury. We added the additional step of lowering the infusion source to the mouse's level before withdrawing the needle. This prevented forming a possible elevated pressure differential within the eye when the needle was removed, causing intraocular damage unrelated to the ischemia/reperfusion.
The aim was to create a controlled and replicable model for researching the mechanisms and pathology of retinal ischemia/reperfusion injury in a mouse model while minimizing procedural damage to the eye. This model offers a way to identify potential treatments and enhance our comprehension of retinal pathologies associated with vascular occlusion.

<table><tbody><tr><td>0.5% Proparacaine</td><td>Sandoz</td><td>61314-016-01</td><td></td></tr><tr><td>1% tropicamide</td><td>Sumerset Therapeutics</td><td>700069-016-01</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton Dickinson</td><td>305106</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15700</td><td></td></tr><tr><td>Bluing reagent</td><td>Fisher Scientific</td><td>&amp;nbsp;22-050-114</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Jackson Laboratories</td><td>664</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Eosin stain</td><td>Electron Microscopy Sciences</td><td>26051-11</td><td></td></tr><tr><td>Hematoxylin stain</td><td>Electron Microscopy Sciences (Gill&#039;s #2)</td><td>26030-20</td><td></td></tr><tr><td>Imager</td><td>Olympus</td><td>Q-Color 5</td><td></td></tr><tr><td>Infusion line (included in the in vivo perfusion system)</td><td>Braintree Scientific</td><td>IV4140</td><td></td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Covetrus</td><td>10004027</td><td>Zoetis NDC# 00856440301</td></tr><tr><td>Microscope&amp;nbsp;</td><td>Olympus</td><td>CX-33</td><td></td></tr><tr><td>Microtome</td><td>Microm</td><td>HM335S</td><td></td></tr><tr><td>Ophthalmic antibacterial ointment&amp;nbsp;</td><td>Henry Schein&amp;nbsp;</td><td>1410468</td><td>Baush &amp;amp; Lomb NDC# 2420879535</td></tr><tr><td>Permount Mounting Media</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Prism</td><td>GraphPad</td><td>10.3.1 for macOS</td><td>data collection, statistical anlaysis, graphs</td></tr><tr><td>Saline Solution</td><td>KD Medical Inc</td><td>50-103-1363</td><td></td></tr><tr><td>Stopcock (included in the in vivo perfusion system)</td><td>Braintree Scientific</td><td>IV4140</td><td></td></tr><tr><td>Tonometer</td><td>iCare</td><td>TA01i</td><td></td></tr><tr><td>Xylazine</td><td>Covetrus</td><td>1XYL006</td><td>Covetrus NDC# 11695402401</td></tr></tbody></table>

induction of retinal ischemia-reperfusion injury in a mouse eye model

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (&#8804;20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

To assess the pathology of the retinas after the ischemia/reperfusion, eyes were collected from one group of mice 2 days after the procedure and from another group of mice 7 days after the procedure. The enucleated eyes were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 &#181;m sections. The sections were stained with hematoxylin and eosin (H&#38;E) and imaged for histological examination (Figure 1). The stained histological images showed damage and loss of cells in the retinas seven days after I/R at a greater level compared to retinas from the Day 2 eyes (Figure 1A,B).
The retinal images of each eye were histologically scored (Table 1)7. The changes in the individual histopathological characteristic scores were seen in most layers of the Day 2 retinas (Figure 2A), with higher scores for the Day 7 retina (Figure 2B). The total scores for the Day 2 retinas were significantly higher than normal eyes, which have an average histopathology score of 0 (Figure 2C). The histopathology scores of the Day 7 retinas were significantly higher than the Day 2 scores (Figure 2C). The results demonstrate the progression in retinal degeneration over time following ischemia/reperfusion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67030/67030fig01.jpg" /> 
Figure 1: Representative histology. The retinal sections were stained with H&#38;E for histopathology analysis. The eyes of two different mice were collected 2 or 7 days after the ischemia/reperfusion procedures. The images presented are from the same distance from the optic nerve. Abbreviations:&#160;RGC: retinal ganglion cells, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, RPE: retinal pigment epithelial cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67030/67030fig01large.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/67030/67030fig02.jpg" /> 
Figure 2: Histopathology scores. Stained retinal sections were analyzed using the histopathological score criteria (Table 1). The scores present are for each mouse A) 2 or 7 days after the ischemia/reperfusion procedure. Eight mice were assayed each time, and the scores were based on the image for the whole retinal section. C. The total scores per mouse in each group. Abbreviations:&#160;RGC: retinal ganglion cells, INL: inner nuclear layer, ONL: outer nuclear layer, RPE: retinal pigment epithelial cells. &#8734; P &#8804; 0.005 from a mean score of 0 for normal retinas, ** P &#8804; 0.01 between the groups. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67030/67030fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Histopathology Characteristic</td>
			<td>Score Range</td>
		</tr>
		<tr>
			<td>1. Retinal folds</td>
			<td>0: None</td>
		</tr>
		<tr>
			<td></td>
			<td>1: One to two folds</td>
		</tr>
		<tr>
			<td></td>
			<td>2: Three to four folds</td>
		</tr>
		<tr>
			<td></td>
			<td>3: Five to six folds</td>
		</tr>
		<tr>
			<td></td>
			<td>4: Seven or more folds</td>
		</tr>
		<tr>
			<td>Cell Loss:</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Ganglion Cell Layer</td>
			<td>0: &#60;1%</td>
		</tr>
		<tr>
			<td>3. Inner Nuclear Layer</td>
			<td>1: 1-24%</td>
		</tr>
		<tr>
			<td>4. Outer Nuclear Layer</td>
			<td>2: 25-49%</td>
		</tr>
		<tr>
			<td>5. Photoreceptor Dropout</td>
			<td>3: 50-74%</td>
		</tr>
		<tr>
			<td></td>
			<td>4: 75-100%</td>
		</tr>
		<tr>
			<td>Hemorrhage</td>
			<td></td>
		</tr>
		<tr>
			<td>6. Vitreous</td>
			<td>0: None</td>
		</tr>
		<tr>
			<td>7. Subretinal</td>
			<td>1: Trace</td>
		</tr>
		<tr>
			<td></td>
			<td>2: Mild (&#60;5% area)</td>
		</tr>
		<tr>
			<td></td>
			<td>3: Moderate (6 - 10% area)</td>
		</tr>
		<tr>
			<td></td>
			<td>4: Severe (&#62;10% area)</td>
		</tr>
		<tr>
			<td>8. RPE Damage</td>
			<td>0: None</td>
		</tr>
		<tr>
			<td></td>
			<td>1: Trace/Mild</td>
		</tr>
		<tr>
			<td></td>
			<td>2: Moderate</td>
		</tr>
		<tr>
			<td></td>
			<td>3: Severe</td>
		</tr>
		<tr>
			<td></td>
			<td>4: Very severe</td>
		</tr>
	</tbody>
</table>
Table 1: Histopathological score criteria. aH&#38;E-stained retinal sections after the ischemia/reperfusion procedure are scored at 100x for each of the 8 characteristics at their severity level. The maximum score is 32. Scores between groups need to be statistically analyzed using non-parametric ANOVA.

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, ...

induction-of-retinal-ischemia-reperfusion-injury-in-a-mouse-eye-model

Nanyang Technological University

Research

JoVE Journal

Biology

270 Views.  Boston University Chobanian & Avedisian School of Medicine. This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

Video: Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Medicine

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Neuroscience

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

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Chapters in this video

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice