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 ....

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 solu....

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 the re.......

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

List of materials used in this article

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