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 arterioles and small vessels⁸. In addition, impaired vascular barrier function is thought to contribute multiple sclerosis⁹ and amyotrophic lateral sclerosis¹⁰.

Of direct relevance to the retinal model described in this protocol, blinding diseases such as diabetic retinopathy¹¹ and retinopathy of prematurity^12,13 are characterized by a phase of early vascular degeneration. The ensuing ischemic stress on the neurovascular retina triggers a second phase of excessive and pathological neovascularization that likely originates as a compensatory response to re-instate oxygen and energy supply^14-16. An attractive strategy to overcome the ischemic stress that is central to disease progression is to restore functional vascular networks specifically in the ischemic zones of the neuro-retina (Figures 2 and 3). Provoking a controlled angiogenic response may come across as counter-intuitive for a condition in which anti-angiogenic treatments such as anti-VEGFs are considered as adapted treatments. Yet, evidence for the validity of this approach is mounting. For example, enhancing “physiological-like” vascular regrowth in ischemic retinopathies has been elegantly demonstrated through introduction of endothelial precursor cells¹⁷, inhibition of Müller cell-expressed VEGF induced downregulation of other angiogenic factors¹⁸, injection of myeloid progenitors¹⁹, inhibition of NADPH oxidase induced apoptosis²⁰, increasing dietary ω-3 polyunsaturated fatty acid intake²¹, treatment with a carboxyl-terminal fragment of tryptophan tRNA synthetase²², and direct administration of VEGF or FGF-2 for protection of glial cells²³. Moreover, we have demonstrated that modulating classical neuronal guidance cues such as Semaphorins or Netrins in ischemic retinopathies accelerates vascular regeneration of healthy vessels within the retina and consequently reduces pathological angiogenesis^24,25. Of direct clinical relevance, several of the aforementioned animal studies provide evidence that promoting vascular regeneration during the early ischemic phase of retinopathies can significantly reduce sight-threatening pre-retinal neovascularization^19,23,24,26, likely through the reduction of ischemic burden.

Devising therapeutic strategies that stimulate regeneration of functional vessels remains a significant challenge for vascular biologists. Here we describe an experimental system that employs the mouse model of oxygen-induced retinopathy (OIR) to explore strategies to modulate vascular regrowth within the retina. Developed by Smith et al. in 1994²⁷, this model serves as a proxy for human proliferative retinopathies and consists of exposing P7 mouse pups to 75% O₂ until P12 and subsequently re-introducing the pups to ambient room O₂-tension (Figure 1). This paradigm loosely mimics a scenario where a premature infant is ventilated with O₂. The exposure of mouse pups to hyperoxia provokes degeneration of retinal capillaries and microvasculature, and yields a reproducible area of vaso-obliteration (VO) typically assessed upon exit from O₂ at P12, although maximal VO area is reached at 48 hr (P9) after exposure to O₂ ²⁸. In the mouse, the avascular VO zones spontaneously regenerate over the course of the week following re-introduction to room air and eventually VO zones are completely re-vascularized (Figure 2). Re-introduction to room air of mice subjected to OIR also provokes pre-retinal neovascularization (NV) (maximal at P17) that is typically assessed to determine the efficacy of anti-angiogenic treatment paradigms. In its purest form, the OIR model provides a highly reproducible and quantifiable tool to assess oxygen-induced vascular degeneration and determine the extent of destructive pre-retinal neovascularization^29-31.

Various explorative treatment paradigms that modulate CNS vascular regeneration can be investigated using the OIR model including use of pharmacological compounds, gene therapy, gene deletion and more. The propensity of a given approach to influence vascular regrowth is assessed step-wise in the window between P12 (maximal VO after exit from hyperoxia) and P17 (maximal NV). Evaluation of treatment outcome on pathological NV can be rapidly and easily determined in parallel and has been thoroughly described by Stahl and colleagues^30,31. Here we provide a simple step-by-step procedure to investigate the modulation of physiological revascularization within the neural retina by pharmacological compounds, prospective therapeutics, viral vectors or to study the influence of candidate genes in transgenic or knockout mice.

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 recommended to use littermates as control (for genetically modified mice as well as mice receiving experimental treatments). When assessing effects of a viral vector, one must consider tropism of the virus and allow sufficient time for full expression of virally-delivered transgenes. Rapidly expressing viral vectors such as 3^rd generation lentiviruses^24,25,33 are recommended.
3. Place mouse pups at P7 (C57BL/6 or desired strain) and a CD1 fostering mother into an oxygen chamber set at 75% O₂ for 5 days²⁷. Environmental humidity and temperature were held constant throughout O₂ exposure. Note: Research facilities equipped with a central source of O₂ are ideal and limit the cumbersome replacement of empty O₂ tanks. If working with transgenic or knockout mice, it is important to ensure that control and experimental mice are acquired from the same vendor to limit genetic drifts within the same strains^30,29. 
4. At P12, remove mice from the oxygen chamber and return animals to ambient O₂.

2. Intravitreal Injection for Delivery of Compounds to the Inner Retina (When Assessing Effects of a Pharmacological Compound or Recombinant Protein)

1. At P14, anesthetize mice with 2% isoflurane in oxygen 2 L/min (or animal protection committee-approved anesthetic of choice). In order to verify the effectiveness of the anesthesia, sequentially pinch the tail, rear foot and ear with forceps.
2. Place the mouse on its belly.
3. Using a sterile 10 µl syringe fitted with a beveled pulled-glass needle, perform an injection of a maximal volume of 1 µl of solution containing the compound being investigated or vehicle (physiological saline) at the posterior limbus of the eye, with a 45° angle avoiding the lens. Note: The pulled glass-needle is attached to the syringe using a drop of epoxy-resin.
4. Apply a drop of lubricant ophthalmic ointment (ideally with antibiotic) with a swab to the mouse’s eye.
5. Return the mouse back to the cage with fostering mother. Mice are then carefully monitored until recovered and fully ambulatory.

3. Assessment of Vessel Perfusion and Barrier Function (Integrity) by Fluorescein Angiography

1. Anesthetize mice with 2% isoflurane in oxygen 2 L/min (or animal protection committee-approved anesthetic of choice). In order to verify the effectiveness of the anesthesia, sequentially pinch the tail, rear foot and ear with forceps. Note: This is typically performed at P17 when regeneration is assessed. Also carry-out the analysis at P19 and P21 to determine if vascular integrity is preserved over time.
2. Once anesthetized, weigh the mouse.
3. Make a midline abdomen incision with dissecting scissors. Note: Dissecting instruments should be regularly checked and sharpened.
4. Cut ribs laterally and raise the ribcage with the aid of forceps. Note: It is necessary to cut as laterally as possible to avoid damage to the heart.
5. After removing peripheral tissue from the heart, clamp the descending aorta with hemostatic forceps.
6. Slowly inject fluorescein-dextran to the left ventricle using a 25 G needle. Note: If vascular barrier function is investigated, 70 kDa fluorescein-dextran is employed as it will leak out of vessels when vessel integrity is compromised. If the investigator wants to cast blood vessels, 2 MDa fluorescein-dextran is used. Critical steps: 1) To ensure a homogenous repartition, centrifuge fluorescein-dextran and inject the supernatant, 2) In order to prevent vessel constriction, inject warmed fluorescein-dextran solution, 3) Its circulation time shouldn’t excess 4 min.
7. Decapitate mice 2 min after injection with operating scissors.

4. Enucleation and Eye Fixation

Note: When assessing rates of vascular regeneration, first collect retinas at P12 and additionally at P14 and P17. Increase the number of sampled time points for more accurate determination of rates of revascularization²⁴.

1. Tilt the mouse head and place it on its side.
2. Remove skin and eyelids covering the eye using dissecting scissors.
3. Place curved forceps below the eye and gently pull it up until the optic nerve is severed.
4. Turn the mouse’s head onto its other side and perform the same steps (steps 4.2 and 4.3).
5. To ensure better penetration of fixative, puncture a hole in the anterior chamber of the eye using 30 G needle.
6. Transfer eyes to a tube containing 4% paraformaldehyde (PFA) and fix for 1 hr at room temperature.
7. Remove PFA and wash eyes 4 times with a solution of ice-cold PBS.

5. Retinal Dissection

1. Place mouse eyes in a Petri dish containing cold PBS and perform dissection of the retinas under a stereomicroscope.
2. Remove extra fat/tissue surrounding the eye with micro-dissection scissors.
3. Cut off the cornea with micro-dissection scissors.
4. Using two pairs of forceps, minutely peel the sclera away from the periphery towards the optic nerve and discard.
5. Pinch the lens (whitish ball beneath the cornea) with forceps and extract it from the eye cup. Use one pair of forceps as a support, and the other to grip and carefully raise and remove the lens.
6. Detach the hyaloid vessels from the inner side of the retina using small brushes (size 0) and forceps.
7. Remove bundles of hyaloid vessels connected to the optic disc using forceps.
8. Transfer dissected retinas to 2 ml microcentrifuge tubes containing PBS and place on ice prior to starting the staining procedure.

6. Retinal Vascular Staining

1. Incubate dissected retinas overnight with gentle shaking at 4 °C in a solution of fluorescently coupled-isolectin B4 (rhodamine-lectin or other) in PBS containing 1 mM CaCl₂ (a 1:100 dilution of a 2 mg/ml isolectin B4 solution is recommended). During the entire staining procedure, cover tubes with aluminum foil or an opaque foil to protect from light.
2. On the following day, remove staining solution and wash retinas 3x in PBS for 10 min at room temperature.

7. Preparation of Retinal Flatmounts

1. Transfer retinas, photoreceptor-side down, onto a microscope slide and make four deep equidistant radial incisions using a surgical scalpel to divide the retina into four equal-sized quadrants. During the incisions, brace the retina with a brush so that it does not move.
2. Using two brushes soaked in PBS, carefully flatten the quadrants photoreceptor side-down and immerse the retina in mounting medium to prevent photo-bleaching. Then carefully place a coverslip on the surface of the mounted retina without applying pressure and making sure that air bubbles do not accumulate under the cover slip.

8. Imaging and Quantification of Vasoobliteration (VO) and Neovascularization (NV) as Previously Described³¹

1. Take images of whole-mounted retinas with an epi-fluorescence microscope at a magnification of 10X.
2. Open the retinal image in photo editing software, stitch together and measure the total retinal area, and avascular area. Area can be expressed in pixels.
3. Determine extent of VO by dividing the number of pixels in the avascular area by the number of pixels in the total retinal area.
4. Determine extent of NV by dividing the number of pixels of NV by the number of pixels in the total retinal area as described³¹.

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. 

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

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