PS holds a Canada Research Chair in Retinal Cell Biology and the Alcon Research Institute New Investigator Award. This work was supported by grants from the Canadian Institutes of Health Research (221478), the Canadian Diabetes Association (OG-3-11-3329-PS), the Natural Sciences and Engineering Research Council of Canada (418637) and The Foundation Fighting Blindness Canada. Support was also provided by the Reseau de Recherche en Sant&#233; de la Vision du Qu&#233;bec.

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)
<ol>
	<li>Record date of birth of mouse pups as P0.</li>
	<li>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 3rd generation lentiviruses24,25,33 are recommended.</li>
	<li>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&#160;exposure.&#160;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 strains30,29.&#160;</li>
	<li>At P12, remove mice from the oxygen chamber and return animals to ambient O2.</li>
</ol>
2. Intravitreal Injection for Delivery of Compounds to the Inner Retina (When Assessing Effects of a Pharmacological Compound or Recombinant Protein)
<ol>
	<li>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.</li>
	<li>Place the mouse on its belly.</li>
	<li>Using a sterile 10 &#181;l syringe fitted with a beveled pulled-glass needle, perform an injection of a maximal volume of 1 &#181;l of solution containing the compound being investigated or vehicle (physiological saline) at the posterior limbus of the eye, with a 45&#176; angle avoiding the lens. Note: The pulled glass-needle is attached to the syringe using a drop of epoxy-resin.</li>
	<li>Apply a drop of lubricant ophthalmic ointment (ideally with antibiotic) with a swab to the mouse&#8217;s eye.</li>
	<li>Return the mouse back to the cage with fostering mother.&#160;Mice are then carefully monitored until recovered and fully ambulatory.</li>
</ol>
3. Assessment of Vessel Perfusion and Barrier Function (Integrity) by Fluorescein Angiography
<ol>
	<li>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.</li>
	<li>Once anesthetized, weigh the mouse.</li>
	<li>Make a midline abdomen incision with dissecting scissors.&#160;Note: Dissecting instruments should be regularly checked and sharpened.</li>
	<li>Cut ribs laterally and raise the ribcage with the aid of forceps.&#160;Note: It is necessary to cut as laterally as possible to avoid damage to the heart.</li>
	<li>After removing peripheral tissue from the heart, clamp the descending aorta with hemostatic forceps.</li>
	<li>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&#8217;t excess 4 min.</li>
	<li>Decapitate mice 2&#160;min&#160;after injection with operating scissors.</li>
</ol>
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.
<ol>
	<li>Tilt the mouse head and place it on its side.</li>
	<li>Remove skin and eyelids covering the eye using dissecting scissors.</li>
	<li>Place curved forceps below the eye and gently pull it up until the optic nerve is severed.</li>
	<li>Turn the mouse&#8217;s head onto its other side and perform the same steps (steps&#160;4.2 and 4.3).</li>
	<li>To ensure better penetration of fixative, puncture a hole in the anterior chamber of the eye using 30 G needle.</li>
	<li>Transfer eyes to a tube containing 4% paraformaldehyde (PFA) and fix for 1 hr at room temperature.</li>
	<li>Remove PFA and wash eyes 4 times with a solution of ice-cold PBS.</li>
</ol>
5. Retinal Dissection
<ol>
	<li>Place mouse eyes in a Petri dish containing cold PBS and perform dissection of the retinas under a stereomicroscope.</li>
	<li>Remove extra fat/tissue surrounding the eye with micro-dissection scissors.</li>
	<li>Cut off the cornea with micro-dissection scissors.</li>
	<li>Using two pairs of forceps, minutely peel the sclera away from the periphery towards the optic nerve and discard.</li>
	<li>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.</li>
	<li>Detach the hyaloid vessels from the inner side of the retina using small brushes (size 0) and forceps.</li>
	<li>Remove bundles of hyaloid vessels connected to the optic disc using forceps.</li>
	<li>Transfer dissected retinas to 2 ml microcentrifuge tubes containing PBS and place on ice prior to starting the staining procedure.</li>
</ol>
6. Retinal Vascular Staining
<ol>
	<li>Incubate dissected retinas overnight with gentle shaking at 4 &#176;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.</li>
	<li>On the following day, remove staining solution and wash retinas 3x&#160;in PBS for 10 min at room temperature.</li>
</ol>
7. Preparation of Retinal Flatmounts
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
8. Imaging and Quantification of Vasoobliteration (VO) and Neovascularization (NV) as Previously Described31
<ol>
	<li>Take images of whole-mounted retinas with an epi-fluorescence microscope at a magnification of 10X.</li>
	<li>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.</li>
	<li>Determine extent of VO by dividing the number of pixels in the avascular area by the number of pixels in the total retinal area.</li>
	<li>Determine extent of NV by dividing the number of pixels of NV by the number of pixels in the total retinal area as described31.</li>
</ol>

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The authors have nothing to disclose.

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 function35-38. Hence to counter early injury, reinstating regional micro-circulation during the immediate/early segment of disease may prove beneficial. In an ocular context, experime...

Throughout CNS development, nerves, immune cells and blood vessels establish remarkably coupled networks to ensure adequate tissue perfusion and allow transmission of sensory information1-5. 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&#8217;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 prematurity12,13 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 supply14-16. 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&#160;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 &#8220;physiological-like&#8221; vascular regrowth in ischemic retinopathies has been elegantly demonstrated through introduction of endothelial precursor cells17, inhibition of M&#252;ller cell-expressed VEGF induced downregulation of other angiogenic factors18, injection of myeloid progenitors19, inhibition of NADPH oxidase induced apoptosis20, increasing dietary &#969;-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 angiogenesis24,25. 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 neovascularization19,23,24,26, 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&#160;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&#160;(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 neovascularization29-31.
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 colleagues30,31. 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.

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

assessment of vascular regeneration in the cns using the mouse retina

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&#8217;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.

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 diseases27,29,30. Findings obtained using this model can be loosely extrapolated to ischemic retinopathies such as proliferative diabetic retinopathy and retinopathy of prematurity30.&#160;
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Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

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.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

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14.3K Views.  McGill University. 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.

Video: Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Medicine

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Summary

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Chapters in this video

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis