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

Summary

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.

Abstract

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 (≤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.

Introduction

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 Büchi 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.

Protocol

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

1. House C57BL/6J mice under standard conditions.

2. Preparing the required solution and infusion line

1. In a 50 mL 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.
2. 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.

3. Preparing the workspace

1. Set up a surgical/dissecting microscope under the saline bottle.
2. 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.

4. Anesthetizing the mouse

1. 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.
2. 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.

5. Dilating the iris

1. 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.

6. Anterior chamber cannulation

1. 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.
2. 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.
3. 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 by Buchi et al.².

7. Ischemic phase

1. 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.
2. 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.
3. 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.
4. Remeasure the intraocular pressure at 30 min before the procedure ends to ensure the intraocular pressure is between 90-100 mmHg.

8. Reperfusion phase

1. 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.
2. 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.

9. Post-operative care

1. Coat mouse eyes with any ophthalmic antibacterial ointment such as a bacitracin veterinary ophthalmic ointment.
2. 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.

10. Enucleating the eye³

1. 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.
2. Position the euthanized mouse on a flat, dry, and smooth surface. Rinse the eye with a few drops of saline solution.
3. 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.
4. Position the Westcott scissors to the most posterior aspect of the eye socket and sever the optic nerve.

11. Fixing the sample⁴

1. 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.
2. 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.
3. Repeat the vacuum process, sequentially placing the eye into the following solutions: 100% ethanol, 100% ethanol, 100% xylene solution, and 100% xylene solution.
4. Remove the eye and place it into a clean, empty vial. Fill the vial with 60 °C liquidized paraffin. Place the vial into a sealed 60 °C vacuum oven for 30 min and then 30 min in the 60 °C oven without vacuum.
5. Remove the eye, place it into a clean, empty vial, and fill it with 60 °C liquidized paraffin. Store the vial at 60 °C overnight.

12. Embedding the sample

1. Transfer the eye to a metal embedding mold and fill the mold with liquidized paraffin on a 60 °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 °C.

13. Sectioning the sample⁵

1. Transfer the embedded specimens to the microtome. Slowly trim the block until the eye is reached and the sections include the optic disc.
2. Cut sections as ribbons of 5 µm 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.
3. Repeat this process until there are at least 8 slides for each eye with sections centered on the optic disc.

14. Staining the sections⁵

1. Heat the slides in a 60 °C oven for 1 h to melt the paraffin. Submerge the slides in RT xylene three times, for 5 min each.
2. Submerge the slides in 100% ethanol three times, for 3 min each. Rinse the sections in tap water for 1 min.
3. Incubate the sections in hematoxylin stain for 45 s. Rinse the sections in tap water for 2 min.
4. Incubate the sections in the Bluing reagent for 1 min. Rinse the sections in tap water for 1 min.
5. Incubate the sections in eosin for 15 s.
6. 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.

15. Histological analysis⁶

1. 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.

Results

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 µm sections. The sections were stained with hematoxylin and eosin (H&E) and imaged for histological examination (Figure 1). The stained histological images showed damage and loss of cells in t...

Discussion

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. Addi...

Materials

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