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Medicine

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

Published: July 14th, 2016

DOI:

10.3791/54065

1Department of Ophthalmology, School of Medicine, Johns Hopkins University

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, 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 anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

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....

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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.

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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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Retinal I/R 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.......

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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.

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Name Company Catalog Number Comments
Heparin Sodium Injection, USP Abraxis Pharmaceutical Products 1000 USP/mL
BSS Sterile Irrigating Solution Alcon Laboratories, Inc. 9007754-0212 500 mL
SC-2kg Digital Pocket Scale American Weigh Scales, Inc. SC-2kg
Tropicamide Ophthalmic Solution USP 1% Bausch + Lomb 1% (10 mg/mL)
Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% Bausch + Lomb 0.5% (5 mg/mL)
INTRAMEDIC Polyethylene Tubing Becton Dickinson and Company 427400 Inner diameter: 427400
30G1/2 PrecisionGlide Needles Benton Dickinson and Company 305106
BC 1mL TB Syringe, Slim Tip with Intradermal Bevel Needle, 26G x 3/8 Benton Dickinson and Company 309625
BD 60mL Syringe Luer-Lok Tip Benton Dickinson and Company 309653
Zeiss OPMI Visu 200/S8 Microscope Carl Zeiss AG 000000-1179-101
Sterile Syringe Filter Corning Inc. CLS431224 0.20 µm
Durasorb Underpads Covidien 1038 23 x 24 inches
Alcohol Prep Covidien 6818 2 Ply, Medium
Student Dumont #5 Forceps Fine Science Tools 91150-20
Hartman Hemostats Fine Science Tools 13002-10
Primary Set, Macrobore, Prepierced Y-Site, 80 Inch Hospira 12672-28
Phosphate Buffered Saline pH 7.4 (1X) Invitrogen 10010-049 500 mL
Distilled water Invitrogen 15230-204 500 mL
C57BL/6J Mice The Jackson Laboratory 664
AnaSed Injection: Xylazine Sterile Solution LLOYD, Inc. 20 mg/mL
Lubricating Jelly, Water Soluble Bacteriostatic MediChoice 3-Gram Packet
NAMIC Angiographic Pressure Monitoring Manifold Navilyst Medical, Inc. 70039355 5-Valve Manifold with Seven Female Ports
Goniosoft, Hypromellose 2.5% Ophthalmic Demulcent Solution: Hydroxypropyl Methylcellulose OCuSOFT, Inc. 2.5% (25 mg/mL)
Ketaset CIII: Ketamine Hydrochloride Pfizer, Inc. 100 mg/mL
Trans-Pal I.V. Stand  Pryor Products 372 Furnished with a home-constructed 60-cm stainless steel extension
Acepromazine: Acepromazine Maleate Injection, USP Vet One 10 mg/mL
V-Top Surgery Table/Adjustable Hydraulic VSSI 100-4041-21
Tube Fitting Luer Male to Luer Male Warner Instruments 64-1579

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