Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Summary

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Abstract

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Introduction

Retinal vein occlusion (RVO) is a common retinal vascular disease that affected approximately 28 million people worldwide in 2015¹. RVO leads to vision decline and loss in working aged adults and elders, representing an ongoing sight-threatening disease estimated to increase over the proximate decade. Some of the distinct pathologies of RVO include hypoxic-ischemic injury, retinal edema, inflammation, and neuronal loss². Currently, the first line of treatment for this disorder is through the administration of vascular endothelial growth factor (VEGF) inhibitors. While anti-VEGF treatment has helped ameliorate retinal edema, many patients still face vision decline³. To further understand the pathophysiology of this disease and to test potential new lines of treatment, there is a need to constitute a functional and detailed RVO mouse model protocol for different mouse strains.

Mouse models have been developed implementing the same laser device used in human patients, paired with an imaging system scaled to the correct size for a mouse. This mouse model of RVO was first reported in 2007⁴ and further established by Ebneter and others^4,5. Eventually, the model was optimized by Fuma et al. to replicate key clinical manifestations of RVO such as retinal edema⁶. Since the model was first reported, many studies have employed it using the administration of a photosensitizer dye followed by photocoagulation of major retinal veins with a laser. However, the amount and type of the dye that is administered, laser power, and time of exposure vary significantly across studies that have used this method. These differences can often lead to increased variability in the model, making it difficult to replicate. To date, there are no published studies with specific details about potential avenues for its optimization.

This report presents a detailed methodology of the RVO mouse model in the C57BL/6J strain and a tamoxifen-inducible endothelial caspase-9 knockout (iEC Casp9KO) strain with a C57BL/6J background and of relevance to RVO pathology as a reference strain for a genetically modified mouse. A previous study had shown that non-apoptotic activation of endothelial caspase-9 instigates retinal edema and promotes neuronal death⁸. Experience using this strain helped determine and provide insight towards potential modifications to tailor the RVO mouse model, which can be applicable to other genetically modified strains.

Protocol

This protocol follows the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Rodent experiments were approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Columbia University. NOTE: All experiments used two-month-old male mice that weighed approximately 20 g. 1. Preparation and administration of tamoxifen for inducible genetic ablation of floxed genes <p cla…

Representative Results

The RVO mouse model aims to successfully achieve occlusions in the retinal veins, leading to hypoxic-ischemic injury, breakdown of the blood retinal barrier, neuronal death, and retinal edema8. Figure 1 shows a timeline of steps to ensure reproducibility, a schematic of the experimental design, and outlines steps that can be further optimized depending on the experimental questions. The three main steps that can be modified are the waiting time after rose bengal admin…

Discussion

The mouse RVO model provides an avenue to further understand RVO pathology and to test potential therapeutics. While the mouse RVO model is widely used in the field, there is a need for a current detailed protocol of the model that addresses its variability and describes the optimization of the model. Here, we provide a guide with examples from experience on what can be altered to get the most consistent results across a cohort of experimental animals and provide reliable data.

The two most es…

Materials

Carprofen	Rimadyl	NADA #141-199	keep at 4 °C
Corn Oil	Sigma-Aldrich	C8267
Fiber Patch Cable	Thor Labs	M14L02
GenTeal	Alcon	00658 06401
Ketamine Hydrochloride	Henry Schein	NDC: 11695-0702-1
Lasercheck	Coherent	1098293
Phenylephrine	Akorn	NDCL174478-201-15
Phoneix Micron IV with Meridian,  StreamPix, and OCT modules	Phoenix Technology Group
Proparacaine Hydrochloride	Akorn	NDC: 17478-263-12	keep at 4 °C
Refresh	Allergan	94170
Rose Bengal	Sigma-Aldrich	330000-5G
Tamoxifen	Sigma-Aldrich	T5648-5G	light-sensitive
Tropicamide	Akorn	NDC: 174478-102-12
Xylazine	Akorn	NDCL 59399-110-20