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, experimental paradigms that accelerate revascularization of the ischemic retina reduce pathological neovascularization 21-25,34 and thus this approach merits further investigation.&#160;
Since its introduction 20 years ago, the OIR model27 has revolutionized retinal angiogenesis research30. Here we provide an additional application for this model to study prospective strategies to modulate physiological vascular regeneration in the ischemic retina. An ideal animal model should encompass several criteria such as proximity to human patho-physiology, high reproducibility, rapid execution and ideally, low cost. The mouse model of OIR meets all these criteria and can be carried-out in less than 3 weeks.
Despite the numerous listed benefits, drawbacks include the current need to sacrifice the mouse prior to analyses and hence accurate longitudinal monitoring of an animal is not yet possible with current imaging tools. Currently, in vivo imaging techniques such as OCT or fluorescein angiography are largely unsuccessful due to the inherent optical limitation of the mouse eye (curvature radius and small size) which reduces imaging coverage39 to the most central regions of the retina which are not relevant to the early stages of the model.
When employed to study vascular regeneration as proposed in this protocol paper, all considerations that apply to studying neovascularization should also be monitored. These include recording animal weight to estimate the metabolic health of the pup, which heavily influences angiogenesis32. Accurate control of oxygen tension in the hyperoxic chamber is also critical. Variation in oxygen concentration has a direct impact on vasoobliteration, which can inevitably lead to critical data misinterpretation. It is therefore recommended to either implement continuous electronic monitoring of chamber oxygen concentration or in the least, monitor daily changes in oxygen levels and limit oxycycler door opening to maintain constant oxygen levels. Optimal processing of retinas also takes practice. Extraction, mounting and staining of the retina must be executed with care as the relatively fragile retina must be delicately handled. In addition, as for all experimentation with genetically modified animals, the genetic drift of a colony can confound results. In absence of a selective force (in a mouse colony for example), the allelic or genetic drift is a random process that can lead to large changes in population over a short period of time. Allele frequencies can change from generation to generation and result in the formation of a sub-colony40,41. These mutations are largely undetectable and it is thus important to limit the number of generations produced by the same breeder pairs in the same colony. The most efficient way to attain the highest genetic stability is to refresh &#8220;stocks&#8221; every five generations or proceed to backcrossing with a purchased mouse of identical background.
It is also recommended to test for retinal degeneration mutations (rd) 1 and 8&#160;42, at least once before starting a new line in order to avoid confounding phenotypes that can be attributed to premature photoreceptor degeneration. For transgenic animals, it is vital to ensure that both control and mutant mice are obtained from the same vendor and are necessarily on the same genetic background.
As we continue to elucidate the molecular mechanisms of blood vessel formation and growth, novel approaches to accelerate, slow, or steer nascent blood vessels are arising. A model system in which modulation of vascularization can be explored in vivo in a pathological context may provide a valuable tool to explore prospective therapeutic paradigms to counter neuronal ischemia within the CNS. Adapting the mouse OIR model to study vascular regeneration provides such a venue and will continue to be a valuable tool to further our understanding of the molecular basis for physiological and pathological angiogenesis.

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.

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			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
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			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
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			<td style="width:175px;">500338</td>
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			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
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			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
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			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
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			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
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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;
Here we present an alternative use of this model to study vascular regeneration. We will describe an example of a strategy to regenerate the ischemic retina that was recently published by our lab. In the presented study, we demonstrate that sustained neuronal ischemia activates endoplasmic reticulum (ER) stress and via one of its effector endoribonucleases IRE1&#945;, cleaves the mRNA of the classical neuronal guidance cue netrin-1. We show that intra-ocular delivery of the Netrin-1, stimulates a program of reparative angiogenesis in retinal myeloid cells and thus accelerates neural tissue revascularization after OIR25. Moreover, we provide an example of accelerated vascular regeneration using lentiviral mediated silencing of IRE1&#945;.
The described experimental paradigms can be modified to investigate the explorative treatment of choice. Importantly, the time point of intravitreal injection is determined based on the nature of the explored compound and must take into consideration the mechanism by which the investigated treatment acts. For example, to study a pharmacological compound (receptor agonist, antagonist, etc.), P14 may be selected as it corresponds to a time point where retinal revascularization is accelerating yet a rapid-acting intervention may help speed up the rate of revascularization (Figures 2 and&#160;3a). When viral vectors are employed, enough time must be allotted to allow for full expression of the passenger gene and an earlier time point may be selected to ensure full expression of the transgene or full silencing of the target gene (Figure 4). Lentiviral based vectors are particularly well suited in this regard due to their rapid expression, low inflammatory response and ease of production24,25. It is also crucial to make certain that the target gene is delivered to the appropriate retinal cell population (RGC, Endothelial cell, M&#252;ller Cell, etc.), hence the tropism of the selected viral vector must be considered. Alternatively, studying the role of a gene in vascular regeneration using transgenic or knockout animals offers the advantage of not needing to perform intra-ocular injections.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51351/51351fig1highres.jpg" width="500" /> 
Figure 1.&#160;Schematic depiction of the mouse OIR model. Mouse pups and nursing mothers are exposed to 75% O2 from P7 to P12. A ventilated oxygen chamber with steady O2 delivery and oximeter is required. During this initial period, retinal vaso-obliteration occurs. At P12, mice are returned to room air (21% O2) until P17 when maximal pathological pre-retinal tufting occurs. Neovascularization recedes over the following days. The ideal period to investigate vascular regeneration is the window between P12 and P17. Sampling and assessing several time points for extent of vaso-obliteration is key to elucidating accurate rates of revascularization.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51351/51351fig2highres.jpg" width="500" /> 
Figure 2.&#160;Time course of retinal revascularization following oxygen-induced vaso-obliteration. A) Graphic depiction of a retinal flatmounting procedure. An incision is made through the top of the cornea (black-grey dome) and the sclera (dotted red lines) and the retina is teased out. The lens can be extracted either after opening the cornea/sclera or once the sclera is removed as shown in the diagram. Hyaloid vessels are then removed and staining of retinal vessels performed. Once the staining protocol is finished, the retina is then cut into 4 equally spaced sections and mounted on a microscope slide vessel-side-up. B) Lectin-stained flatmount retinas from mice subjected to OIR. As vessels regenerate, the area of vaso-obliteration (here maximal at P12) fills in. Pathological neovascularization is maximal at P17 and appears as saturated spots in lectin stained OIR retinas. By P19-P21, retinas have fully regenerated their vascular networks. (This figure has been modified from Binet et al. Cell Metabolism 201325)
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51351/51351fig3highres.jpg" width="500" /> 
Figure 3.&#160;Example of an injectable treatment that accelerates retinal vascular regeneration: Netrin-1. A) Netrin-1 is injected at P14 and extent of vascular regrowth assessed at P17. A dose-dependent increase in vascular regeneration is observed. B) Vascular integrity can be assessed by perfusion with a low molecular weight fluorescent Dextran. If vessels show compromised barrier function, the fluorescent Dextran will leak outside the vessel (arrows). (This figure has been modified from Binet et al. Cell Metabolism 201325)
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51351/51351fig4highres.jpg" width="500" /> 
Figure 4. Example of gene silencing with lentiviral-delivered shRNAs for retinal vascular regeneration: Lv.shIRE1&#945;. To assess the ability of gene silencing of IRE1&#945; in retinal ganglion neurons to aid vascular regeneration, an intravitreal injection of a lentivirus coding for an shIRE1&#945; was injected at P3 to allow sufficient time for gene silencing when vascular growth and vaso-obliteration is assessed. The 3rd generation lentivirus employed in this example contains a modified vesicular stomatitis virus glycoprotein (VSV-G), engineered to target plasma membranes. Although these vectors effectively infect retinal ganglion cells, expression can also be noted in other local cell types. (A) This treatment did not lead to noticeable variations in oxygen-induced vaso-obliteration as determined at P12, signifying that all observed benefits on vaso-obliteration measured at later time points are due to increased vascular regeneration. (B) Inhibition of IRE1&#945; in this paradigm dramatically enhanced vascular regeneration in the regrowth phase of OIR at P17. (This figure has been modified from Binet et al. Cell Metabolism 201325) (C) Assessing a third time point of vaso-obliteration such as P14, is required to determine accurate rates of revascularization. (This figure has been modified from Joyal et al. Blood 201124) <a href="https://www.jove.com/files/ftp_upload/51351/51351fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

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). ...

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

Nanyang Technological University

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Neuroscience

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

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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. ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

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 ...