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. 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.
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.
1. Prepare the Anesthesia Cocktail
2. Prepare the Anesthesia Booster
3. Prepare the Surgical Suite
4. Prepare the Balanced Salt Solution with Heparin Sodium
5. Set up the IV Pole
6. Set up the Sodium Heparin Drip
7. Prepare the Mice for Surgery
8. Cannulate the Anterior Chamber
9. Monitor Anesthesia
10. Remove the Cannula from the Anterior Chamber
11. Clean the Equipment
12. Return All Mice to Their Home Cages
13. Perform Retinal Assessment
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 counting11. Decreases in ganglion cell layer neuron counts indicate I/R-induced cell death.
Impairments in retinal neuron function have been documented using electroretinography (Figure 2). Briefly, seven days after I/R, scotopic electroretinograms were recorded at multiple flash intensities. Amplitudes of the a- and b-waves were quantified using image analysis software12. Here, lower a- and b-wave amplitudes signify I/R-induced impairments in retinal neuronal function. In these and other I/R endpoints, the non-I/R eye serves as a robust negative control for I/R-induced damage.
Figure 1. Retinal I/R Induces Neuronal Cell Death in the Ganglion Cell Layer (GCL). Representative retinal images from control and I/R eyes are shown with scale bars denoting 100 µm. (A) The number of NeuN-positive cells in the GCL is significantly reduced in I/R eyes as compared to controls (B). n = 9, error bar: standard error, ***p < 0.0001 Please click here to view a larger version of this figure.
Figure 2. Retinal I/R Impairs Retinal Neuron Function. Decreased a- and b-wave amplitudes indicate impaired membrane physiology in neuronal cells following I/R. n = 6, error bar: standard error, *p < 0.05 Please click here to view a larger version of this figure.
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 durations differing from those presented here4,6-10,12. Therefore retinal I/R injury by elevated IOP offers an advantage over alternative retinal I/R techniques in that it allows one to adjust surgical parameters to accommodate one's particular experimental goals.
Nonetheless, alternative techniques have been employed for inducing retinal I/R injury in rodents. Ligation of the optic nerve bundle25,26 or the central retinal artery alone27 has been used to arrest retinal blood flow temporarily. Similar strategies for systemic conditions have required ligation of the cerebral artery28 or the cephalic artery29 to reduce blood flow without fully obstructing it. One rarer methodology involves circumferentially compressing the retinal globe using a thread that is weighted on both ends30. While such strategies have successfully contributed to an understanding of hypoxia-induced neurovascular changes in the retina, the technique described herein offers several advantages over these alternatives. Necessitating minimal non-retinal damage only to the cornea, I/R by elevated IOP provides a more specifically targeted retinal injury than is afforded by ligation techniques and so may be more useful for researchers interested in retina-specific disease. Also, elevated IOP methods are more tractable than ligation or compression models, such that the IOP method allows quick attainment of retinal ischemia as well as subsequent reperfusion. Finally, elevated IOP protocols require minimal surgical and technological sophistication and so may be more widely accessible than their alternatives.
Retinal I/R injury by elevated IOP is not without its challenges. Cannulation of the anterior chamber requires manual dexterity, and care must be taken to preserve the integrity of the iris, lens, and cornea. Caution is also advised after insertion of the cannula, as the cannula may be pulled from the anterior chamber while the 30-gauge tubing is secured with tape.
Other critical processes in this protocol include maintaining a warm body temperature for anesthetized animals, administering booster anesthesia in a timely manner, and sustaining lubrication of the cornea using hypromellose. It is also important to note mouse stress or sickness behaviors (for example, lack of grooming, hunching, etc.) prior to surgery, as these extra-surgical variables may influence drug potency and mortality. By attending to these issues, one may achieve a highly regulated and reproducible model for retinal I/R injury.
It should also be acknowledged that retinal I/R injury by elevated IOP is only a model, and caution is advised when extrapolating findings to specific diseases, particularly chronic conditions. While differing from these diseases in time frame and etiological origin, however, retinal I/R injury by elevated IOP may nonetheless provide a sound platform for evaluating mechanisms of retinal degeneration and recovery.
The retina is composed of multiform cells and signaling processes, and a comprehensive story of retinal dysregulation remains to be elucidated. The current literature demonstrates the utility of retinal I/R injury by elevated IOP not only in examining retinal degenerative processes6-8,10,12, but also in identifying targets for therapeutic prevention and intervention7,9,11,12,14. In addition, there is growing evidence to support the utility of retinal I/R injury by elevated IOP in non-neuronal endpoints such as retinal inflammation, vascular degeneration, and leakage14,15,17. Given its anatomical specificity, experimental tractability, and technical accessibility, retinal I/R injury by elevated IOP promises to maintain a leading role in pursuing these inquiries.
The authors have nothing to disclose.
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.
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 |