Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Summary

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Abstract

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer’s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

Introduction

Throughout CNS development, nerves, immune cells and blood vessels establish remarkably coupled networks to ensure adequate tissue perfusion and allow transmission of sensory information^1-5. The breakdown of vascular systems results in insufficient tissue oxygenation and compromised metabolic supply and is increasingly recognized as an important contributor to the pathogenesis of neurodegenerative diseases⁶. Vascular dropout and the deterioration of the neurovascular unit within the brain, for example, is associated with vascular dementia, vascular lesions of the white matter of the brain⁷ and Alzheimer’s disease with stenosis of....

Protocol

Ethics statement: All animal experimentation adheres the animal care guidelines established by the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the Canadian Council of Animal Care.

1. Oxygen Induced Retinopathy (OIR)

1. Record date of birth of mouse pups as P0.
2. Record all weights of animals upon entry into O₂ to ensure an adequate weight range. Note: For C57BL/6 mice at P17, body weight should range between 5 and 7.5 g for maximal NV³². In order to maintain environmental consistency, it is recommend....

Results

The OIR model is widely used to study oxygen-induced vascular degeneration and ischemia-induced pathological neovascularization in the retina and has been instrumental in the development of currently employed anti-angiogenic treatments for ocular diseases^27,29,30. Findings obtained using this model can be loosely extrapolated to ischemic retinopathies such as proliferative diabetic retinopathy and retinopathy of prematurity³⁰. 

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Discussion

What is the most effective way to stimulate growth of new healthy vessels in ischemic nervous tissue? Is it therapeutically valid to interfere with and accelerate naturally occurring vascular regrowth? In neuro-ischemic pathologies such as ischemic retinopathies or stroke, vascular degeneration is associated with reduced neuronal function^35-38. Hence to counter early injury, reinstating regional micro-circulation during the immediate/early segment of disease may prove beneficial. In an ocular context, experime.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57Bl/6 mice (Other strains may be used; angiogenic response varies from one strain to the other)
CD1 nursing mothers	Vendor of choice
Operating Scissors straight	World Precision Instruments	14192
Dissecting Scissors straight	World Precision Instruments	14393
Vannas Eye Scissors	Harvard Apparatus	72-8483
Iris Forceps, curved, serrated	World Precision Instruments	15915
Brushes 362R size 0	Dynasty
Dumont Forceps #3; straight	World Precision Instruments	500338
Surgical Blade, size 10	Bard-Parker	371110
Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I	Vector Laboratories, Inc	RL-1102
Microscope slides	VWR	16004-368
Fluoromount G	Electron Microscopy Sciences	17984-25
Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope	Zeiss
Leica MZ9.5 Stereomicroscope	Leica
Fluorescein isothicyanate-dextran, 70000	Sigma-Aldrich	46945