This work was supported by research grants from the National Institutes of Health (EY022383 and EY022683; EJD) and Core grant (P30EY001765), Imaging and Microscopy Core Module.

Ethics Statement: All procedures were performed in accordance with the guidelines set forth by the Johns Hopkins University Institutional Animal Care and Use Committee.
Note: Mice used during filming are C57BL/6 mice from Jackson, although other rodent strains or species may also be used. When using other strains or species, be aware that anesthesia dosages and injury timeline may vary. It is important to adapt I/R conditions to accommodate strain, species, and experimental variations.
<p ...

<ol>
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Retinal I/R&#160;injury by elevated IOP has proven its utility in modeling cellular damage and dysfunction, particularly neurodegeneration, in the rodent retinal neurovascular unit. This procedure provides a robust control tissue and is easily accessible in terms of technical sophistication. It has been noted in this and other I/R injury models that increasing the pressure and duration of ischemia may increase injury severity24. For this reason, some practitioners have elected to use ischemic pressures and dur...

Retinal ischemia-reperfusion (I/R) characterizes many human retinal pathologies, including glaucoma, diabetic retinopathy, and retinal vascular occlusions1. In retinal I/R, reduced blood flow (ischemia) in the retinal vasculature creates a state of retinal hypersensitivity to oxygen and other nutrients, precipitating severe oxidative and inflammatory damage when circulation is subsequently reinstated (reperfusion)2. The neural retina appears particularly vulnerable to these changes, with retinal neurodegeneration being perhaps the most salient feature of I/R-induced damage. Presented here is a protocol for modeling retinal I/R injury in the mouse. This technique enables researchers to examine potential mechanisms and treatment strategies for human diseases of the retinal neurovascular unit.
Pioneered in 1952 by surgeons seeking to understand the neurodegenerative consequences of surgical anemia3, rodent retinal I/R by elevated intraocular pressure (IOP) was reestablished in 1991 for the purpose of standardizing neurodegenerative endpoints after ischemic insult4. Using the drip of a saline reservoir to raise IOP above systolic blood pressure, these studies demonstrated that pressurized ocular cannulation was sufficient to suspend the retinal circulation and thereby initiate neuronal degeneration. More recent efforts using retinal I/R by elevated IOP have begun to elaborate the mechanisms underlying I/R-induced retinal neurodegeneration5-12. Multiple groups have reported additional pathologic changes including inflammation13,14, vascular permeability15,16, and capillary degeneration14,17. Taken together, these studies have established retinal I/R injury by elevated IOP as a model of retinal neurovascular disease more generally.
Characterizing the mechanisms of I/R injury is essential for the study of vascular disease. Retinal I/R injury by elevated IOP is one of many hypoxia-induced injury models, including I/R injuries in lung18, heart19, brain20, liver21, kidney22, and intestine23. These models have been paramount in advancing our understanding of vascular illness and its clinical remedies. By extending the investigation of I/R processes to ocular tissues, retinal I/R injury by elevated IOP helps to paint a more comprehensive picture of these related conditions.
Corresponding closely with clinical neurodegenerative conditions in retina, retinal I/R injury by elevated IOP presents a valuable tool for researchers interested in exploring ischemic pathogenesis. The protocol described herein is targeted, tractable, and accessible. It is complemented well by endpoints in neuronal degeneration, such as quantification of retinal neurons, measurement of retinal thickness, and electrophysiological recording of retinal neuron function. This model has proven its utility in advancing neurovascular inquiry, and it shows promise in earning status as a foundational protocol in visual medicine research.

<table border="0" cellpadding="0" cellspacing="0" width="544">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Heparin Sodium Injection, USP</td>
			<td style="width:136px;">Abraxis Pharmaceutical Products</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">1000 USP/mL</td>
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			<td height="43" style="height:43px;width:136px;">BSS Sterile Irrigating Solution</td>
			<td style="width:136px;">Alcon Laboratories, Inc.</td>
			<td style="width:136px;">9007754-0212</td>
			<td style="width:136px;">500 mL</td>
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			<td height="43" style="height:43px;width:136px;">SC-2kg Digital Pocket Scale</td>
			<td style="width:136px;">American Weigh Scales, Inc.</td>
			<td style="width:136px;">SC-2kg</td>
			<td style="width:136px;"></td>
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			<td height="64" style="height:64px;width:136px;">Tropicamide Ophthalmic Solution USP 1%</td>
			<td style="width:136px;">Bausch + Lomb</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">1% (10 mg/mL)</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:136px;">Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5%</td>
			<td style="width:136px;">Bausch + Lomb</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">0.5% (5 mg/mL)</td>
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			<td height="64" style="height:64px;width:136px;">INTRAMEDIC Polyethylene Tubing</td>
			<td style="width:136px;">Becton Dickinson and Company</td>
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			<td height="64" style="height:64px;width:136px;">30G1/2 PrecisionGlide Needles</td>
			<td style="width:136px;">Benton Dickinson and Company</td>
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			<td height="85" style="height:85px;width:136px;">BC 1mL TB Syringe, Slim Tip with Intradermal Bevel Needle, 26G x 3/8</td>
			<td style="width:136px;">Benton Dickinson and Company</td>
			<td align="right" style="width:136px;">309625</td>
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			<td height="43" style="height:43px;width:136px;">BD 60mL Syringe Luer-Lok Tip</td>
			<td style="width:136px;">Benton Dickinson and Company</td>
			<td align="right" style="width:136px;">309653</td>
			<td style="width:136px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Zeiss OPMI Visu 200/S8 Microscope</td>
			<td style="width:136px;">Carl Zeiss AG</td>
			<td style="width:136px;">000000-1179-101</td>
			<td style="width:136px;"></td>
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			<td height="43" style="height:43px;width:136px;">Sterile Syringe Filter</td>
			<td style="width:136px;">Corning Inc.</td>
			<td style="width:136px;">CLS431224</td>
			<td style="width:136px;">0.20 &#181;m</td>
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			<td height="43" style="height:43px;width:136px;">Durasorb Underpads</td>
			<td style="width:136px;">Covidien</td>
			<td align="right" style="width:136px;">1038</td>
			<td style="width:136px;">23 x 24 inches</td>
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			<td height="22" style="height:22px;width:136px;">Alcohol Prep</td>
			<td style="width:136px;">Covidien</td>
			<td align="right" style="width:136px;">6818</td>
			<td style="width:136px;">2 Ply, Medium</td>
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			<td height="43" style="height:43px;width:136px;">Student Dumont #5 Forceps</td>
			<td style="width:136px;">Fine Science Tools</td>
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			<td height="43" style="height:43px;width:136px;">Hartman Hemostats</td>
			<td style="width:136px;">Fine Science Tools</td>
			<td style="width:136px;">13002-10</td>
			<td style="width:136px;"></td>
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			<td height="85" style="height:85px;width:136px;">Primary Set, Macrobore, Prepierced Y-Site, 80 Inch</td>
			<td style="width:136px;">Hospira</td>
			<td style="width:136px;">12672-28</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Phosphate Buffered Saline pH 7.4 (1X)</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:136px;">10010-049</td>
			<td style="width:136px;">500 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Distilled water</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:136px;">15230-204</td>
			<td style="width:136px;">500 mL</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">C57BL/6J Mice</td>
			<td style="width:136px;">The Jackson Laboratory</td>
			<td align="right" style="width:136px;">664</td>
			<td style="width:136px;"></td>
		</tr>
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			<td height="64" style="height:64px;width:136px;">AnaSed Injection: Xylazine Sterile Solution</td>
			<td style="width:136px;">LLOYD, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">20 mg/mL</td>
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			<td height="64" style="height:64px;width:136px;">Lubricating Jelly, Water Soluble Bacteriostatic</td>
			<td style="width:136px;">MediChoice</td>
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			<td height="106" style="height:106px;width:136px;">NAMIC Angiographic Pressure Monitoring Manifold</td>
			<td style="width:136px;">Navilyst Medical, Inc.</td>
			<td align="right" style="width:136px;">70039355</td>
			<td style="width:136px;">5-Valve Manifold with Seven Female Ports</td>
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			<td height="148" style="height:148px;width:136px;">Goniosoft, Hypromellose 2.5% Ophthalmic Demulcent Solution: Hydroxypropyl Methylcellulose</td>
			<td style="width:136px;">OCuSOFT, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">2.5% (25 mg/mL)</td>
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			<td height="64" style="height:64px;width:136px;">Ketaset CIII: Ketamine Hydrochloride</td>
			<td style="width:136px;">Pfizer, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">100 mg/mL</td>
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			<td height="85" style="height:85px;width:136px;">Trans-Pal I.V. Stand&#160;</td>
			<td style="width:136px;">Pryor Products</td>
			<td align="right" style="width:136px;">372</td>
			<td style="width:136px;">Furnished with a home-constructed 60-cm stainless steel extension</td>
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			<td height="85" style="height:85px;width:136px;">Acepromazine: Acepromazine Maleate Injection, USP</td>
			<td style="width:136px;">Vet One</td>
			<td style="width:136px;"></td>
			<td style="width:136px;">10 mg/mL</td>
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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.

The neurodegenerative effects of retinal I/R by elevated IOP are commonly evaluated using two standard approaches. NeuN immunolabeling of neuronal nuclei has revealed significant neuronal cell loss following I/R insult (Figure 1). Briefly, eyes enucleated 7 days after I/R were fixed in paraformaldehyde, labeled with the neuronal cell marker NeuN, and whole-mounted. Images were captured using confocal microscopy, and cells labeled with NeuN were quantified by counting...

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

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, ...

The overall goal of this technique, is to provide an experimental model of Retinal Ischemia-Reperfusion Injury, and it's pathologic consequences. This method is commonly used to investigate the mechanisms underlying Retinal Ischemia-Reperfusion, as well as to evaluate potential therapeutic approaches for treating associated pathologies. The main advantage of this technique is that it creates an injury highly targeted to ocular structures, while causing minimal damage to surrounding tissues.The usefulness of this technique extends towards the understanding and three month of multiple manifestations of Retinal Ischemia-Reperfusion Injury, including:inflammation, oxidative stress, and stare dysfunction in the retinal neurovascular unit. Video demonstration of this method is critical because the surgical techniques for cannula placement may be difficult to reproduce without a visual guide. Before beginning the procedure, warm an empty cage on the table.Then insert the sharp end of the primary set pre-pierced Y-site tubing into a bottle of 0.1%Heparin sodium, and hang the bottom of Heparin sodium from an IV pole extension. Snap open the air filter cap on the tubing. Then elevate the Heparin sodium bottle to 163 centimeters from the tabletop to the peak of the sodium Heparin drip, to produce a Heparin sodium flow pressure of 120 mililiters of mercury.Open the primary set pre-pierced Y-site tubing, to allow the Heparin sodium to fill the tubing, and connect the primary set pre-pierced Y-site tubing to the five valve manifold. Then, using Luer male to Luer male tube fittings, connect 30 gauge half-inch needles to the five port manifold. Insert each of the needles into its own ten inch segment of 30 gauge tubing.Then, using hemostats, break new 30 gauge half inch needle tips and insert their blunt ends into open ends of the 30 gauge tubing. Next, use tape to arrange the needle tubes such that the tubes connected to the inner ports are positioned on top of the tubes connected to the outer ports to prevent tangling of the tubing during the Anterior Chamber Cannulation. Now, turn on the 0.1%Heparin sodium flow to the five valve manifold and to each individual port for two to three minutes to remove any air bubbles.Then, turn off all of the ports on the five valve manifold. To perform the cannulation, after administering the anesthesia, place all of the mice into the warm cage on the surgery table, confirming the achievement of deep anesthetization by lack of response to toe pinch after five to ten minutes. Then administer one drop of Tropicamide for pupil dilation, and one drop of Proparacaine for local anesthesia into each of the animals'eyes, and arrange the mice in the order of anesthetization, so that the first mouse anesthetized will be the first mouse to be cannulated.While waiting for the eye drops to take effect, prepare four inch straight pieces of tape, pulling tightly on the tape before tearing, and setting aside one piece of tape per animal. Note that failing to pull tightly on the tape, will result in curling of the tape. When the first mouse is ready, place the animal under a surgical microscope.Then, turn on the first 0.1%Heparin sodium port on the five valve manifold, and use a pair of forceps to gently proptose one eye. Next, insert the 30 gauge cannula needle into the anterior chamber approximately halfway between the zonular fibers and the apex of the cornea. Taking care to avoid penetrating the conrea a second time.Note that a gentle twisting motion is used to insert the cannula deeply into the anterior chamber. Now, press the 30 gauge tubing against the tabletop to minimize any movement of the inserted cannula while reaching for a piece of tape, and use the tape to secure the tubing to the table. Once the tube has been secured, record the start time of the surgery.Use the microscope to verify that there is no leakage, and that the Ischemia-Reperfusion eye is larger than the contralateral eye, to establish the presence of ocular distension. The apply Hypromellose to both eyes every 30 minutes, until the end of the experiment to lubricate the cornea, and to seal any micro leaks. Should an animal stir during the procedure, lift the tail and administer a Ketamine booster intraperitoneally.90 minutes after cannulating the first animal, gently pull the cannula from the anterior chamber, and lubricate both eyes with lubricating jelly. As subsequent cannulae are removed, place each mouse back into the cage on the surgical table, until the animals are fully recovered. After all of the cannulae have been removed, disinfect them with alcohol wipes.Then use a 60 milliliter syringe filled with air to expel the Heparin sodium from the five valve manifold, 30 gauge needles, 30 gauge tubing, and cannulae. Next, rinse all of the instruments with a 60 milliliter syringe filled with distilled water. Finally, expel the distilled water from all of the materials with 60 milliliters of air, and store the disinfected and rinsed equipment for later use.NeuN immunolabeling of neuronal nuclei reveal significant nuronal cell loss, following Ischemia-Reperfusion insult, by confocal microscopic imaging, seven days after Ischemia-Reperfusion Injury. Indeed, manual quantification of the NeuN positive cells in control, and Ischemia-Reperfused eyes, demonstrates a decrease in the gangelion cell layer neuron counts after injury, indicating the presence of Ischemia-Reperfusion induced cell death. Lower a-wave and b-wave amplitudes documented by electroretinography also signify the occurrence of Ischemia-Reperfusion induced impairments in retinal neuronal function compared to control, non-injured eyes.Once mastered, this technique can be performed on ten animals in approximately five hours, including the final two hours spent waiting for the mice to recover from anesthesia. While attempting this procedure, it's important to maintaim deep anesthesia. As an effective sedation will prevent displacement of the cannula by animal movement.Following this procedure, other methods, like electroretinography can be performed to answer additional questions about retinal function. This technique has enabled researchers in the ophthalmology neuroscience and vascular biology fields to explore retinal neurovasuclar pathogenesis, and therapeutic intervention in rodents. After watching this video, you should have a good understanding of how to create a targeted retinal injury, though the cannulation of the ocular anterior chamber.Don't forget, that working with surgical instruments can be extremely hazardous, and that precautions, such as using an effective sharps disposal, should always be taken while performing this procedure.

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14.6K visualizações.  Johns Hopkins University. The overall goal of this technique, is to provide an experimental model of Retinal Ischemia-Reperfusion Injury, and it's pathologic consequences. This method is commonly used to investigate the mechanisms underlying Retinal Ischemia-Reperfusion, as well as to evaluate potential therapeutic approaches for treating associated pathologies. The main advantage of this technique is that it creates an injury highly targeted to ocular structures, while causing minimal damage to surrounding tissues.