This work was funded by the Wellcome Trust and the British Heart Foundation.

All steps are performed at room temperature, unless otherwise indicated.
1. Enucleation and Fixation of Postnatal Eyes
<ol>
	<li>Place humanely killed mouse pup on its side, remove the skin covering the eye with scissors.</li>
	<li>Use scissors and forceps to enucleate the eye.</li>
	<li>Place scissors below the enucleated eye, cut the optic nerve and surrounding tissues and lift out the eye. Transfer to a 24-well plate containing 4% paraformaldehyde (PFA) made up in 2x PBS at room temperature.</li>
	<li>Allow each eye to fix for 10 - 15 min, then transfer to cold 2x PBS on ice for 5 - 10 min. It is best to stagger enucleations and subsequent fixation to ensure the degree of fixation is equal for each eye.</li>
</ol>
2. Dissection of Postnatal Murine Retinas
<ol>
	<li>Transfer eyes into a Petri dish containing 2x PBS using a plastic Pasteur pipette that has been cut to widen the bore and place under a dissecting microscope.</li>
	<li>Remove any fat around the eye using forceps and scissors.</li>
	<li>Pierce the edge of the cornea with sharp scissors and cut around the cornea and iris and discard.</li>
	<li>Insert forceps between retina and sclera and remove the sclera taking care not to tear the retina. The choroid layer can also be removed, but if there is a risk of damage to the retina occurring during this step, the choroid layer can be left on as it should not significantly affect the quality of the immunostaining.</li>
	<li>Remove the lens and the vitreous humour using forceps.</li>
	<li>Remove the hyaloids vessels by gathering them together in the center of the eye with fine forceps then quickly pull away from the center of the retina (alternatively snip at base of hyaloid vessels with fine scissors).</li>
	<li>Rinse the cup shaped retina with PBS in a clean Petri dish.</li>
	<li>Make 4 to 5 radial incisions reaching approximately 2/3 of the radius of the retina using spring scissors to create a 'petal' shape.</li>
	<li>Draw off PBS using a Pasteur pipette to flatten the retina, and then remove any excess PBS with a small piece of absorbent paper.</li>
	<li>Slowly pipette cold (-20 &deg;C) methanol drop by drop onto surface of retina until totally covered and then flood with additional methanol. This will help to fix the retinas and will facilitate permeabilization. The retinas will turn white.</li>
	<li>Transfer to a 2 ml tube using a plastic Pasteur pipette that has been cut to widen the bore.</li>
	<li>Leave retina in cold methanol for at least 20 min before proceeding with immunostaining. Of the antibodies used here, only VE-Cadherin cannot be used successfully on methanol fixed retinas. In this case retinas need to proceed straight to immunostaining after step 9.</li>
	<li>If required, retinas can be stored in methanol at -20 &deg;C for up to several months prior to staining.</li>
</ol>
3. Immuno/isolectin Staining of the Retinal Vasculature
<ol>
	<li>Carefully remove/pour off methanol and gently rinse retinas briefly in PBS (at this stage the retina is still in the same 2 ml tube from step 11 above).</li>
	<li>Remove PBS and cover retinas with 100 &mu;l of Perm/Block solution (PBS + 0.3% Triton + 0.2% BSA) + 5% of appropriate serum (see Table 1) and shake gently for 1 hr.</li>
	<li>Remove Perm/Block and incubate retinas with gentle shaking overnight at 4 &deg;C with 100 &mu;l of selected primary antibodies (see Table 1) and/or IsolectinB4, all prepared by diluting in Perm/Block.</li>
	<li>Remove antibody/isolectin and wash retinas 4 x 10 min in PBS + 0.3% Triton (PBSTX).</li>
	<li>In some cases, e.g. following staining with IsolectinB4-Alexa488 or a directly conjugated primary antibody, a secondary antibody is not required and retinas can be mounted directly (step 7). When an unconjugated primary antibody has been used in step 3, then 100 &mu;l of the appropriate fluorescent secondary antibody (diluted 1/200 in PBSTX) is added and left to incubate overnight at 4 &deg;C or for 4 hr at room temperature.</li>
	<li>Remove secondary antibody and wash retinas 4 x 15 min in 2 ml PBSTX.</li>
	<li>Transfer retinas onto slides using a wide bore plastic Pasteur pipette and remove excess PBSTX with absorbant tissue. Mount retinas by adding 50 &mu;l of Prolong mountant onto a coverslip and gently position over the retina.</li>
	<li>Transfer slides to fridge overnight at 4 &deg;C, ensuring that they are flat and protected from light, to allow the Prolong mountant to set.</li>
	<li>Image retina using a confocal microscope or an epifluorescence microscope.</li>
</ol>

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473 (7347), 298-307 (2011).</li><li>Staton, C. 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=Current+methods+for+assaying+angiogenesis+in+vitro+and+in+vivo.">Current methods for assaying angiogenesis in vitro and in vivo.</a> Int J. Exp. Pathol. 85 (5), 233-248 (2004).</li><li>Goodwin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+of+angiogenesis+for+assessment+of+angiogenic+and+anti-angiogenic+agents.">In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.</a> Microvasc. Res. 74 (2-3), 172-183 (2007).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev. Biol. 248 (2), 307-318 (2002).</li><li>Fruttiger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+retinal+vasculature.">Development of the retinal vasculature.</a> Angiogenesis. 10 (2), 77-88 (2007).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat. Protoc. 5 (10), 1659-1665 (1038).</li><li>Mahajan, V. B., Skeie, J. M., Assefnia, A. H., Mahajan, M., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+eye+enucleation+for+remote+high-throughput+phenotyping.">Mouse eye enucleation for remote high-throughput phenotyping.</a> J. Vis. Exp. (57), e3184 (2011).</li><li>Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+the+neonatal+vasculature+and+analysis+of+retinal+angiogenesis+in+mice.">Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.</a> Nat. Protoc. 5 (9), 1518-1534 (2010).</li><li>Allinson, K. R., Lee, H. S., Fruttiger, M., McCarty, J., Arthur, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+expression+of+TGFbeta+type+II+receptor+is+required+to+maintain+vascular+integrity+during+postnatal+development+of+the+central+nervous+system.">Endothelial expression of TGFbeta type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.</a> PLoS One. 7 (9), e39336 (2012).</li><li>Mahmoud, 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=Pathogenesis+of+arteriovenous+malformations+in+the+absence+of+endoglin.">Pathogenesis of arteriovenous malformations in the absence of endoglin.</a> Circ. Res. 106 (8), 1425-1433 (2010).</li><li>Lebrin, 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=Thalidomide+stimulates+vessel+maturation+and+reduces+epistaxis+in+individuals+with+hereditary+hemorrhagic+telangiectasia.">Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia.</a> Nat. Med. 16 (4), 420-428 (2010).</li><li>Pi, L., 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=Role+of+connective+tissue+growth+factor+in+the+retinal+vasculature+during+development+and+ischemia.">Role of connective tissue growth factor in the retinal vasculature during development and ischemia.</a> Invest. Ophthalmol. Vis. Sci. 52 (12), 8701-8710 (2011).</li><li>Powner, M. B., 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=Visualization+of+gene+expression+in+whole+mouse+retina+by+in+situ+hybridization.">Visualization of gene expression in whole mouse retina by in situ hybridization.</a> Nat. Protoc. 7 (6), 1086-1096 (2012).</li></ol>

The authors declare that they have no competing financial interests.

The method presented here offers a simple and efficient way to obtain images of stained whole mount preparations of neonatal mouse retinas, making it an easily quantifiable and physiologically relevant model of angiogenesis. Initially, the mouse eye can be quite difficult to dissect due to its small size and globe shape, so some practice and good dissection tools are needed for reliable results. Dissection of eyes prepared in 2x concentration of PBS is important because this causes a small amount of water loss from the e...

Angiogenesis is a complex developmental process defined by the formation of new blood vessels from a pre-existing vessel network and is regulated by several signalling pathways. The most prominent of these is the VEGF pathway. VEGF is released by ischemic cells and leads to the initiation of angiogenic sprouting in neighboring blood vessels. The leading endothelial cell from a new vascular sprout is a 'tip' cell, which produces filopodia that reach out towards the source of VEGF, and is followed by proliferating 'stalk' endothelial cells. In this way, activated endothelial cells migrate and proliferate towards an avascular region where they form new vascular tubes. In order to stabilize these tubes to support blood flow, endothelial cells interact with pericytes and smooth muscle cells, which surround the new blood vessels 1. Angiogenesis is essential for vascularization of the embryo and growing tissues. However, it also contributes to many pathological disorders such as cancer; whereas defects in angiogenesis are associated with ischemic diseases. The development of effective drug treatments to regulate angiogenesis as a means of treating disease is therefore of great interest. For example, effective anti-angiogenic factors will reduce cancer growth, whilst pro-angiogenic strategies can be used to treat ischemic disease. Thus, models of angiogenesis are essential to better understand the regulation of new vessel formation and for developing new therapeutic strategies to enhance or decrease vascular growth.
A fundamental element of angiogenesis research is the choice of an appropriate vascular model. Many in vitro models have been designed to reproduce the basic steps of angiogenesis such as endothelial cell migration, proliferation and survival 2,3 . A frequently used in vitro assay is the formation of a branched network of endothelial cells on a Matrigel matrix, although this is not specific for endothelial cells, nor does it usually incorporate the support of pericytes or smooth muscle cells. Assessment of ex vivo sprouting angiogenesis using mouse organ culture such as the embryoid body assay, the aortic ring assay and the fetal metatarsal assay allows the analysis of angiogenesis of endothelial cells in the context of additional supporting cells. However, the main limitations of these assays include the lack of blood flow and the regression of vessels over time, giving a limited window for analysis. Therefore, to understand the regulation of angiogenesis more accurately, in vivo models are required such as those developed in mouse using subcutaneously implanted sponges or matrigel plugs, as well as the hind limb ischemia model. However, these models are challenging to analyze due to the complex 3D architecture of the neovessels. In contrast, the zebrafish embryo has proved an excellent model for visualizing angiogenesis in the whole organism 4. However, the mouse is evolutionarily much more closely related to human than zebrafish, making the mouse a preferred model in many cases. Consequently angiogenesis of the postnatal mouse retina represents a well characterized method of choice for angiogenesis research. It not only provides a physiological setting, but also a 2D vascular plexus that can be readily visualized using appropriate fluorescent stains. The architecture of the vessel network, endothelial proliferation, sprouting, perivascular cell recruitment and vessel remodelling can all be investigated using this technique.
In the neonatal mouse retina, development of retinal blood vessels occurs in the first week of postnatal life. Mice, unlike humans, are born blind and the retinas only become vascularized after birth. Retinal vessels arise from the center of the retina close to the optic nerve at postnatal day 0 (P0), and develop by angiogenesis to form a highly organized vascular plexus that reaches the retinal periphery by approximately P8 5. Blood vessels develop in a characteristic manner with the capillary plexus gradually growing outward from the optic disc toward the periphery to generate a regular alternating pattern of arteries and veins with an intervening capillary network. The retinal vasculature provides an ideal model to study angiogenesis as the vessels can be easily identified as they grow in what is essentially a 2D plane, thus making it relatively easy to examine the retinal vasculature in flat-mount preparations 5. A method for isolating the mouse neonatal retina for analysis of endothelial tip cell responses over several hours ex vivo has been reported 6. Alternatively, a more detailed investigation of the whole retinal vasculature and associated vascular markers requires immunostaining of fixed retina specimens at different stages of development.
Here we provide a detailed description of a reliable and technically straight forward method that we use for whole mount staining of the murine retinal vascular plexus, from the initial enucleation of the postnatal eye (see also 7) through the staining protocols to final imaging under the microscope.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>Plastic pipettes 3 ml</td><td>Scientific Laboratory Supplies</td><td>PIP4210</td><td>Remove tip with scissors to create a wide bore</td></tr><tr><td>BD Falcon 24-well plates</td><td>Scientific Laboratory Supplies</td><td>353226</td><td></td></tr><tr><td>Select Petri dishes TV 90 mm</td><td>Scientific Laboratory Supplies</td><td>SLS2002</td><td></td></tr><tr><td>Histobond slides</td><td>VWR</td><td>631-0624</td><td></td></tr><tr><td>Coverslips 22 x 22 mm</td><td>VWR</td><td>631-1336</td><td></td></tr><tr><td>Dumont Tweezers #5</td><td>World precision instruments</td><td>14095</td><td></td></tr><tr><td>Spring scissors 10.5 cm long, with straight 8 mm blades</td><td>World precision instruments</td><td>501235</td><td>Used for enucleation</td></tr><tr><td>Vannas scissors 8.5 cm long, with straight 7 mm blades, and 0.025 x 0.015 mm superfine tips</td><td>World precision instruments</td><td>500086</td><td>Used for dissecting retina</td></tr><tr><td>Superfine vannas scissors 8 cm long with straight 3 mm blades, and 0.015 x 0.015 mm superfine tips</td><td>World precision instruments</td><td>501778</td><td>Used for dissecting retina</td></tr><tr><td>crystal clear&amp;#8217; tubes 2 ml</td><td>Starlab</td><td>E1420-2000</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>S0876</td><td>PBS reagent</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5655</td><td>PBS reagent</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td>PBS reagent</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>Make up in 2x PBS and store at -20 &amp;#176;C</td></tr><tr><td>BSA</td><td>Sigma</td><td>A7638</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma</td><td>D9663</td><td></td></tr><tr><td>Goat serum</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>Prolong Gold anti-fade reagent</td><td>Invitrogen</td><td>P36930</td><td>Mounting reagent</td></tr><tr><td>Isolectin GS-IB4-alexa 594</td><td>Invitrogen</td><td>I21413</td><td>Use at 1:200 dilution</td></tr><tr><td>Isolectin GS-IB4-alexa 488</td><td>Invitrogen</td><td>I21411</td><td>Use at 1:200 dilution</td></tr><tr><td colspan="4">&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td></tr><tr><td>Anti-NG2 (rabbit polyclonal )</td><td>Millipore</td><td>AB5320</td><td>Detects pericytes</td></tr><tr><td>Anti-GFAP (clone GA5, mouse monoclonal)</td><td>Millipore</td><td>MAB360</td><td>Detects astrocytes</td></tr><tr><td>Anti-Desmin (clone Y66, rabbit monoclonal)</td><td>Millipore</td><td>04-585</td><td>Detects pericytes</td></tr><tr><td>Anti-Collagen IV (rabbit polyclonal )</td><td>Chemicon</td><td>AB756P</td><td>Detects vascular basement membrane</td></tr><tr><td>Anti-alpha smooth muscle actin-Cy3 (clone 1A4, mouse monoclonal)</td><td>Sigma</td><td>C6198</td><td>Detects vascular smooth muscle cells.</td></tr><tr><td>Anti-Endoglin (clone MJ7/18, rat monoclonal)</td><td>BD Pharmingen</td><td>550546</td><td>Detects endothelial cells</td></tr><tr><td>Anti-CD31 (clone MEC13.3, rat monoclonal)</td><td>BD Pharmingen</td><td>553370</td><td>Detects endothelial cells</td></tr><tr><td>Anti-VE-Cadherin (clone 11D4.1, rat monoclonal)</td><td>BD Pharmingen</td><td>550548</td><td>Detects endothelial cell junctions</td></tr><tr><td>Anti-Alk1 (goat polyclonal)</td><td>R&amp;amp;D Systems</td><td>AF770</td><td>Detects endothelial cells</td></tr><tr><td colspan="4">&lt;strong&gt;Secondary Antibodies&lt;/strong&gt;</td></tr><tr><td>Goat anti-rabbit-Alexa594</td><td>Invitrogen</td><td>A11012</td><td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &amp;#176;C.</td></tr><tr><td>Goat anti-rat-Alexa488</td><td>Invitrogen</td><td>A11006</td><td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &amp;#176;C.</td></tr><tr><td>Goat anti-rat-Alexa594</td><td>Invitrogen</td><td>A11007</td><td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &amp;#176;C.</td></tr><tr><td>Goat anti-mouse-Alexa488</td><td>Invitrogen</td><td>A11029</td><td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &amp;#176;C.</td></tr><tr><td>Donkey anti-goat-Alexa594</td><td>Invitrogen</td><td>A11058</td><td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &amp;#176;C.</td></tr><tr><td colspan="4">&lt;strong&gt;Microscopes&lt;/strong&gt;</td></tr><tr><td>Dissection microscope</td><td>Zeiss</td><td>Stemi SV6</td><td></td></tr><tr><td>Camera</td><td>Zeiss</td><td>Axiocam HRc</td><td>Camera for dissection microscope</td></tr><tr><td>Epifluorescent Microscope</td><td>Zeiss</td><td>Axioimager with Apotome</td><td></td></tr><tr><td>Camera</td><td>Zeiss</td><td>Axiocam HRm</td><td>Camera for Axioimager</td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>A1R</td><td></td></tr></tbody></table>

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.

After dissection the retina should look like a flat flower (Figure 1). By using the protocol, outlined above, endothelial cells can be seen using isolectin B4-Alexa488 staining and supporting vascular muscle cells are visualized using anti-alpha smooth muscle actin (Figure 2). The low power view using a 5X objective in Figure 2 allows an overview of the vascular organization of the retina. Also, by using a different combination of antibodies given in the protocol above, ...

Watch this Scientific Journal Video about Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo at JoVE.com

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.

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. ...

whole-mount-immunofluorescent-staining-neonatal-mouse-retina-to

Research

JoVE Journal

Neuroscience

45.5K Views.  Newcastle University. 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.

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

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Medicine

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Immunology and Infection

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

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 ...

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, ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

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

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Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model