Name: an ex vivo choroid sprouting assay of ocular microvascular angiogenesis
Uploaded: 2020-08-06T16:30:00
Description: Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

The work was supported by Grants from the Manpei Suzuki Diabetic Foundation (YT), Boston Children&#39;s Hospital OFD/BTREC/CTREC Faculty Career Development Grant, Boston Children&#39;s Hospital Ophthalmology Foundation, BCH Pilot Award, BCH Manton Center Fellowship, and Little Giraffe Foundation (ZF), The German Research Foundation (DFG; to BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH IDDRC (1U54HD090255), Massachusetts Lions Eye Foundation (LEHS).

All animal experiments described were approved by the Institutional Animal Care and Use Committee at Boston Children&#8217;s Hospital (ARCH protocol number 19-04-3913R).
1. Preparation
<ol>
	<li>Add 5 mL of Penicillin/Streptomycin (10000 U/mL) and 5 mL and 10 mL of commercially available supplements to 500 mL of complete classic medium with serum. Aliquot 50 mL of the medium initially. 
	NOTE: Do not return any medium back to the stock to avoid contamination.</li>
	<li>Put an aliquot of...

<ol>
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T., 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=Ex+vivo+model+for+studying+endothelial+tip+cells:+Revisiting+the+classical+aortic-ring+assay.">Ex vivo model for studying endothelial tip cells: Revisiting the classical aortic-ring assay.</a> Microvascular Research. 128, 103939 (2020).</li><li>Rezzola, 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=In+vitro+and+ex+vivo+retina+angiogenesis+assays.">In vitro and ex vivo retina angiogenesis assays.</a> Angiogenesis. 17 (3), 429-442 (2014).</li><li>Rezzola, 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=A+novel+ex+vivo+murine+retina+angiogenesis+(EMRA)+assay.">A novel ex vivo murine retina angiogenesis (EMRA) assay.</a> Experimental Eye Research. 112, 51-56 (2013).</li><li>Shao, Z., 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=Choroid+sprouting+assay:+an+ex+vivo+model+of+microvascular+angiogenesis.">Choroid sprouting assay: an ex vivo model of microvascular angiogenesis.</a> PLoS One. 8 (7), 69552 (2013).</li><li>Tomita, 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=Free+fatty+acid+receptor+4+activation+protects+against+choroidal+neovascularization+in+mice.">Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice.</a> Angiogenesis. 23, 385-394 (2020).</li><li>Li, J., 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=Endothelial+TWIST1+promotes+pathological+ocular+angiogenesis.">Endothelial TWIST1 promotes pathological ocular angiogenesis.</a> Investigative Ophthalmology and Vision Science. 55 (12), 8267-8277 (2014).</li><li>Liu, C. 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=Endothelial+microRNA-150+is+an+intrinsic+suppressor+of+pathologic+ocular+neovascularization.">Endothelial microRNA-150 is an intrinsic suppressor of pathologic ocular neovascularization.</a> Proceedings of the National Academy of Science U. S. A. 112 (39), 12163-12168 (2015).</li><li>Zhou, Q., 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=LncEGFL7OS+regulates+human+angiogenesis+by+interacting+with+MAX+at+the+EGFL7/miR-126+locus.">LncEGFL7OS regulates human angiogenesis by interacting with MAX at the EGFL7/miR-126 locus.</a> Elife. 8, 40470 (2019).</li><li>Kobayashi, S., Fukuta, M., Kontani, H., Yanagita, S., Kimura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+angiogenesis+of+cultured+choroidal+tissues+in+streptozotocin-diabetic+Wistar+and+spontaneously+diabetic+GK+rats.">A quantitative assay for angiogenesis of cultured choroidal tissues in streptozotocin-diabetic Wistar and spontaneously diabetic GK rats.</a> Japanese Journal of Pharmacology. 78 (4), 471-478 (1998).</li><li>Kobayashi, 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=Inhibitory+effects+of+tetrandrine+and+related+synthetic+compounds+on+angiogenesis+in+streptozotocin-diabetic+rodents.">Inhibitory effects of tetrandrine and related synthetic compounds on angiogenesis in streptozotocin-diabetic rodents.</a> Biological and Pharmaceutical Bulletin. 22 (4), 360-365 (1999).</li><li>Kobayashi, S., Shinohara, H., Tsuneki, H., Nagai, R., Horiuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N(epsilon)-(carboxymethyl)lysine+proliferated+CD34(+)+cells+from+rat+choroidal+explant+in+culture.">N(epsilon)-(carboxymethyl)lysine proliferated CD34(+) cells from rat choroidal explant in culture.</a> Biological and Pharmaceutical Bulletin. 27 (9), 1382-1387 (2004).</li><li>Kobayashi, 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=Overproduction+of+N(epsilon)-(carboxymethyl)lysine-induced+neovascularization+in+cultured+choroidal+explant+of+streptozotocin-diabetic+rat.">Overproduction of N(epsilon)-(carboxymethyl)lysine-induced neovascularization in cultured choroidal explant of streptozotocin-diabetic rat.</a> Biological and Pharmaceutical Bulletin. 27 (10), 1565-1571 (2004).</li><li>Bergers, G., Song, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pericytes+in+blood-vessel+formation+and+maintenance.">The role of pericytes in blood-vessel formation and maintenance.</a> Neuro-Oncology. 7 (4), 452-464 (2005).</li><li>Browning, A. 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The choroidal sprouting assay aids research in neovascular AMD9,10,18,19,20. Choroid explants can be isolated from mice as well as rats and humans17,21. The choroid explant includes ECs, macrophages, and pericytes17. In this assay the interaction between choroidal ECs and adjace...

Choroidal angiogenesis dysregulation is associated with neovascular age-related macular degeneration (AMD)1. The choroid is a microvascular bed present underneath the retinal pigment epithelium (RPE). It has been shown that reduced blood flow in the choroid is associated with progression of AMD2. The intricate relationship between vascular endothelium, RPE, macrophages, pericytes and other cells is responsible for the homeostasis of the tissue3,4,5. Therefore, a reproducible assay modeling choroidal microenvironment is critical for the study of neovascular AMD.
Ex vivo angiogenesis assays and in vitro endothelial cell cultures can complement studies of microvascular behavior in vivo, for testing new drugs and for studies of pathogenesis. Endothelial cells such as human retinal microvascular endothelial cells (HRMECs), Human Umbilical Vein Endothelial Cells (HUVEC), isolated primary animal brain or retinal ECs are often used in in vitro studies for ocular angiogenesis research6,7,8. HRMECs in particular have been widely used as a model of in vitro choroidal neovascularization (CNV)9 by assessing endothelial proliferation, migration, tubular formation, and vascular leakage to evaluate interventions6,10. However, ECs in culture are limited as a model of CNV because of the lack of interactions with other cell types found in the choroid and because most EC used in these assays do not originate from choroid. Mouse choroidal ECs are difficult to isolate and maintain in culture.
The aortic ring assay is widely used as a model of macro vascular proliferation. Vascular sprouts from aortic explants include ECs, pericytes and macrophages11. The aortic ring assay models large vessel angiogenesis well12,13,14. However, it has limitations as a model of choroidal neovascularization as aortic rings are a macrovascular tissue lacking the characteristic choroidal microvascular environment, and sprouts from large vessels may differ from sprouts from capillary networks involved in microvascular pathology. Recently a group published an ex-vivo retinal assay15,16. Although, it is suitable for retinal neovascular disease, it is not as appropriate for choroidal neovascularization as seen in AMD.
The choroidal sprouting assay using mouse RPE, choroid, and scleral explanted tissue was developed to better model CNV. The tissue can easily be isolated from mouse (or other species) eyes17. This assay allows reproducible evaluation of pro- and anti-angiogenic potential of pharmacologic compounds and evaluation of the role of specific pathways in choroidal neovascularization using tissue from genetically modified mice and controls18. This choroidal sprouting assay has been referenced in many subsequent publications9,10,18,19,20. Here, the method involved in the use of this assay are demonstrated.

<table>
	<tbody>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>AKORN</td>
			<td>59339-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal membrane extract (BME) Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td>10cm</td>
		</tr>
		<tr>
			<td>Complete classic medium with serum and CultureBoost</td>
			<td>Cell systems</td>
			<td>4Z0-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 Proof</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td>use for 70%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>Tissue culture plate (24-well)</td>
			<td>Olympus</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>VetaKet CIII (Ketamine)</td>
			<td>AKORN</td>
			<td>59399-114-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

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

Comparison of choroid sprouting growth per day
We dissected the choroid with sclera, embedded in BME and cultured them for 6 days (Figure 1). The choroid sprouting in C57BL/6J mice from day 3 to day 6 were examined with a microscope and quantified with SWIFT-Choroid a semi-automated quantification method in ImageJ. In a representative case, the choroidal sprouting area (the vessels extending from the explant, excluding the explant itself) was 0.38...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

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

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

This Choroid assay is highly reproducible and pertinent to Choroidal Angiogenesis research and Age related Macular Degeneration, AMD It can compliment in vivo studies of microvascular behavior. This assay can be used to screen compounds as potential treatments for neovascular AMD or to assess pathways involved in Choroidal Neovascularization using wild type and genetically modified mouse tissue. To begin, add five milliliters of penicillin, streptomycin and 10 and five milliliters of commercially available supplements to 500 milliliters of complete classic medium with serum then aliquot 50 milliliters of the medium thaw the basal membrane extract or BME overnight in a refrigerator at two to eight degrees Celsius.Put an aliquot of complete classic medium on ice. Clean the dissecting microscope, forceps and scissors with 70%ethanol. Prepare to cell culture dishes and to place one on the dissection microscope and the other on ice.Then add 10 milliliters of complete classic medium to each dish. Keep the eyes in complete classic medium on ice before dissection. Remove the connective tissue and optic nerve then use a micro scissor to circumferentially cut 0.5 millimeters posterior to the corneal Limbus.Remove the cornea Iris complex, vitreous and to the lens. Make a one millimeter incision perpendicular to the cut edge towards the optic nerve and to cut a circumferential one millimeter wide band then separate the central and peripheral regions of the complex. Use forceps to peel the retina from the RPE choroid sclera complex.Keep the peripheral choroid band in complete classic medium on ice. Isolate the other eye and repeat the process to cut a second band. Cut the circular band into five to six approximately equal square pieces.Add 30 microliters of the thawed BME into the center of each well of a 24 well tissue culture plate. Make sure that the droplet of BME forms a convex dome at the bottom of the plate without touching the edges. Place the tissue in the middle of the BME.Do not flatten the choroid X plant. Let the tissue expand within the BME. Incubate the plate at 37 degrees Celsius for 10 minutes to let the gel solidify then add 500 microliters of complete classic medium into each well.Change the classic medium every other day. Choroid sprouting can be observed after three days with a microscope. Open the choroid sprouting image with image J and check image, type and eight bit with gray scale.Then optimize the contrast by selecting image, adjust, brightness contrast and adjusting it. Use the magic wand function to outline and remove the choroid tissue which are present in the center of the sprouts. Remove the background of the image with the free selection tools.Next, go to image, adjust threshold, and use the threshold function to define the microvascular sprouts against the background in periphery. Click F2 and a summary will appear. Click save to save an image of the selected area in the same folder as the original image for future reference.After a group of samples is measured, copy the recorded data for analysis. This protocol was used to examine and quantify choroid sprouting in C 57, black six J mice from day three to day six. In a representative case, the choroidal sprouting area was 0.38, 1.47, 5.62 and 10.09 millimeters squared at days three, four, five, and six, respectively.The effects of loss of FFAR4 on choroidal vascular sprouting were evaluated using the choroid sprouting assay in FFAR4 four knockout and FFAR4 wild type mice. The sprouting area in FFAR4 knockout mice increased choroidal vascular growth compared to FFAR4 wild type at day six. Furthermore, treatment with one micromolar FFAR4 agonist reduced the choroidal sprouting area compared to untreated mice at day six.This assay allows reproducible evaluation of anti angiogenic potential of pharmacological compounds and the variation of the role of a specific pathways in Choroidal Neovascularization using genetically modified mouses tissue.

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