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 ...

<ol>
	<li>Carmeliet, P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+mechanisms+of+nerve+and+blood+vessel+wiring.">Common mechanisms of nerve and blood vessel wiring.</a> Nature. 436, 193-200 (2005).</li><li>Eichmann, A., Thomas, J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+angiogenesis+by+a+non-canonical+Wnt-Flt1+pathway+in+myeloid+cells.">Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells.</a> Nature. 474, 511-515 (2011).</li><li>Checchin, D., Sennlaub, F., Levavasseur, E., Leduc, M., Chemtob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+microglia+in+retinal+blood+vessel+formation.">Potential role of microglia in retinal blood vessel formation.</a> Invest Ophthalmol Vis Sci. 47, 3595-3602 (2006).</li><li>Quaegebeur, A., Lange, C., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurovascular+link+in+health+and+disease:+molecular+mechanisms+and+therapeutic+implications.">The neurovascular link in health and disease: molecular mechanisms and therapeutic implications.</a> Neuron. 71, 406-424 (2011).</li><li>Yamamoto, Y., Craggs, L., Baumann, M., Kalimo, H., Kalaria, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+the+eyes+of+a+child:+understanding+retinopathy+through+ROP+the+Friedenwald+lecture.">Through the eyes of a child: understanding retinopathy through ROP the Friedenwald lecture.</a> Invest Ophthalmol Vis Sci. 49, 5177-5182 (2008).</li><li>Caballero, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+vascular+damage+can+be+repaired+by+healthy,+but+not+diabetic,+endothelial+progenitor+cells.">Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells.</a> Diabetes. 56, 960-967 (2007).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-mediated+STAT3+activation+inhibits+retinal+vascularization+by+down-regulating+local+erythropoietin+expression.">VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.</a> Am J Pathol. 180, 1243-1253 (2012).</li><li>Ritter, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+progenitors+differentiate+into+microglia+and+promote+vascular+repair+in+a+model+of+ischemic+retinopathy.">Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy.</a> J Clin Invest. 116, 3266-3276 (2006).</li><li>Saito, Y., Geisen, P., Uppal, A., Hartnett, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+NAD(P)H+oxidase+reduces+apoptosis+and+avascular+retina+in+an+animal+model+of+retinopathy+of+prematurity.">Inhibition of NAD(P)H oxidase reduces apoptosis and avascular retina in an animal model of retinopathy of prematurity.</a> Mol Vis. 13, 840-853 (2007).</li><li>Connor, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+dietary+intake+of+omega-3-polyunsaturated+fatty+acids+reduces+pathological+retinal+angiogenesis.">Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis.</a> Nat Med. 13, 868-873 (2007).</li><li>Banin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T2-TrpRS+inhibits+preretinal+neovascularization+and+enhances+physiological+vascular+regrowth+in+OIR+as+assessed+by+a+new+method+of+quantification.">T2-TrpRS inhibits preretinal neovascularization and enhances physiological vascular regrowth in OIR as assessed by a new method of quantification.</a> Invest Ophthalmol Vis Sci. 47, 2125-2134 (2006).</li><li>Dorrell, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+retinal+astrocytes+normalizes+revascularization+and+prevents+vascular+pathology+associated+with+oxygen-induced+retinopathy.">Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy.</a> Glia. 58, 43-54 (2010).</li><li>Joyal, J. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+neurons+prevent+vascular+regeneration+of+neural+tissue+by+secreting+semaphorin+3A.">Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.</a> Blood. 117, 6024-6035 (2011).</li><li>Binet, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+ER+Stress+Impedes+Myeloid-Cell-Induced+Vascular+Regeneration+through+IRE1alpha+Degradation+of+Netrin-1.">Neuronal ER Stress Impedes Myeloid-Cell-Induced Vascular Regeneration through IRE1alpha Degradation of Netrin-1.</a> Cell Metab. 17, 353-371 (2013).</li><li>Fukushima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sema3E-PlexinD1+signaling+selectively+suppresses+disoriented+angiogenesis+in+ischemic+retinopathy+in+mice.">Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice.</a> J Clin Invest. 121, 1974-1985 (2011).</li><li>Smith, L. 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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.

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		<tr height="21">
			<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>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td style="width:231px;"></td>
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			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
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			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
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			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
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			<td height="21" style="height:21px;width:639px;">Vannas Eye Scissors</td>
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			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
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			<td style="width:175px;">15915</td>
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			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
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			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
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			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
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		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
			<td rowspan="2" style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">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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Research

JoVE Journal

Neuroscience

14.3K visualizações.  McGill University.  A retina de roedores tem sido reconhecida como uma janela de acesso para o cérebro. Neste documento técnico que proporcionam um protocolo que utiliza o modelo de ratinho de retinopatia induzida por oxigénio para estudar os mecanismos que levam à insuficiência de regeneração vascular dentro do sistema nervoso central após a lesão isquémica. O sistema descrito também pode ser aproveitada para explorar estratégias para promover o crescimento de vasos sanguíneos...