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

Arbejdet blev støttet af Grants fra Manpei Suzuki Diabetic Foundation (YT), Boston Children's Hospital OFD/BTREC/CTREC Faculty Career Development Grant, Boston Children's Hospital Oftalmology Foundation, BCH Pilot Award, BCH Manton Center Fellowship og Little Giraffe Foundation (ZF), The German Research Foundation (DFG; til BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH IDDRC (1U54HD090255), Massachusetts Lions Eye Foundation (LEHS).

Alle beskrevne dyreforsøg blev godkendt af Udvalget for Institutionel Dyrepleje og -anvendelse på Boston Children's Hospital (ARCH-protokolnummer 19-04-3913R).
1. Forberedelse
<ol><li>Der tilsættes 5 ml Penicillin/Streptomycin (10000 U/ml) og 5 ml og 10 ml kommercielt tilgængelige kosttilskud til 500 ml komplet klassisk medium med serum. Aliquot 50 ml af mediet i første omgang. BEMÆRK: Tilbagelever ikke noget medium til lageret for at undgå kontaminering.</li>
	<li>Sæt en aliquot ...

<ol>
	<li>Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+the+pathogenesis+of+age-related+macular+degeneration.">Current concepts in the pathogenesis of age-related macular degeneration.</a> Arch Ophthalmol. 122 (4), 598-614 (2004).</li><li>Pemp, B., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+blood+flow+in+diabetes+and+age-related+macular+degeneration.">Ocular blood flow in diabetes and age-related macular degeneration.</a> Canadian Journal of Ophthalmology. 43 (3), 295-301 (2008).</li><li>Murakami, Y., Ishikawa, K., Nakao, S., Sonoda, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+response+in+retinal+homeostasis+and+inflammatory+disorders.">Innate immune response in retinal homeostasis and inflammatory disorders.</a> Progress in Retinal and Eye Research. 74, 100778 (2020).</li><li>Fu, 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=Dyslipidemia+in+retinal+metabolic+disorders.">Dyslipidemia in retinal metabolic disorders.</a> EMBO Molecular Medicine. 11 (10), 10473 (2019).</li><li>Daruich, 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=Mechanisms+of+macular+edema:+Beyond+the+surface.">Mechanisms of macular edema: Beyond the surface.</a> Progress in Retinal and Eye Research. 63, 20-68 (2018).</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=Long-Acting+FGF21+Inhibits+Retinal+Vascular+Leakage+in+In+Vivo+and+In+Vitro+Models.">Long-Acting FGF21 Inhibits Retinal Vascular Leakage in In Vivo and In Vitro Models.</a> International Journal of Molecular Sciences. 21 (4), 21041188 (2020).</li><li>Maisto, 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=ARPE-19-derived+VEGF-containing+exosomes+promote+neovascularization+in+HUVEC:+the+role+of+the+melanocortin+receptor+5.">ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5.</a> Cell Cycle. 18 (4), 413-424 (2019).</li><li>Mazzoni, 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=The+Wnt+Inhibitor+Apcdd1+Coordinates+Vascular+Remodeling+and+Barrier+Maturation+of+Retinal+Blood+Vessels.">The Wnt Inhibitor Apcdd1 Coordinates Vascular Remodeling and Barrier Maturation of Retinal Blood Vessels.</a> Neuron. 96 (5), 1055-1069 (2017).</li><li>Fu, 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=Adiponectin+Mediates+Dietary+Omega-3+Long-Chain+Polyunsaturated+Fatty+Acid+Protection+Against+Choroidal+Neovascularization+in+Mice.">Adiponectin Mediates Dietary Omega-3 Long-Chain Polyunsaturated Fatty Acid Protection Against Choroidal Neovascularization in Mice.</a> Investigative Ophthalmology and Visual Sciences. 58 (10), 3862-3870 (2017).</li><li>Gong, 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=Cytochrome+P450+Oxidase+2C+Inhibition+Adds+to+omega-3+Long-Chain+Polyunsaturated+Fatty+Acids+Protection+Against+Retinal+and+Choroidal+Neovascularization.">Cytochrome P450 Oxidase 2C Inhibition Adds to omega-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 36 (9), 1919-1927 (2016).</li><li>Nicosia, R. F., Zorzi, P., Ligresti, G., Morishita, A., Aplin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+angiogenesis+by+different+cell+types+in+the+aorta+ring+model.">Paracrine regulation of angiogenesis by different cell types in the aorta ring model.</a> International Journal of Developmental Biology. 55 (4-5), 447-453 (2011).</li><li>Bellacen, K., Lewis, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+ring+assay.">Aortic ring assay.</a> Journal of Visulaized Experiments. (33), e1564 (2009).</li><li>Masson, V. V., 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=Mouse+Aortic+Ring+Assay:+A+New+Approach+of+the+Molecular+Genetics+of+Angiogenesis.">Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis.</a> Biological Procedures Online. 4, 24-31 (2002).</li><li>Katakia, Y. 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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Den choroidal spirende assay hjælpemidler forskning i neovaskulær AMD9,10,18,19,20. Choroid explants kan isoleres fra mus samt rotter og mennesker17,21. Choroid explant omfatter EC'er, makrofager og pericytes17. I denne analyse samspillet mellem choroidal ECs og tilstødende c...

Choroidal angiogenese dysregulation er forbundet med neovaskulær aldersrelateret makuladegeneration (AMD)1. Den choroid er en mikrovaskulær seng til stede under retinale pigment epitel (RPE). Det er blevet påvist, at nedsat blodgennemstrømning i choroid er forbundet med progression af AMD2. Det indviklede forhold mellem vaskulær endotel, RPE, makrofager, pericytter og andre celler er ansvarlig for homøostaseafvævet3,4,5. Derfor er en reproducerbar analyse modellering choroidal mikromiljø er afgørende for studiet af neovaskulær AMD.
Ex vivo angiogenese analyser og in vitro endotel cellekulturer kan supplere undersøgelser af mikrovaskulær adfærd in vivo, til test af nye lægemidler og for undersøgelser af patogenese. Endotelceller såsom humane retinale mikrovaskulære endotelceller (HRMEC), Human Umbilical Vein Enothelial Cells (HUVEC), isolerede primære dyrs hjerne eller retinale ECs anvendes ofte i in vitro-undersøgelser til okulær angiogenese forskning6,7,8. Især HRMEC'er har været meget anvendt som model for in vitro choroidal neovaskulærisation (CNV)9 ved at vurdere endotelspredning, migration, rørformet dannelse og vaskulær lækage til vurdering afinterventioner 6,10. EC'er i kultur er imidlertid begrænset som en model af CNV på grund af manglende interaktioner med andre celletyper, der findes i choroid, og fordi de fleste EF, der anvendes i disse analyser, ikke stammer fra choroid. Mus choroidal ECs er vanskelige at isolere og vedligeholde i kulturen.
Den aorta ring assay er meget udbredt som en model af makro vaskulær spredning. Vaskulære spirer fra aorta explants omfatter ECs, pericytes og makrofager11. Den aorta ring assay modeller store fartøj angiogenese godt12,13,14. Men det har begrænsninger som en model af choroidal neovascularization som aorta ringe er en makrovaskulær væv mangler den karakteristiske choroidal mikrovaskulære miljø, og spirer fra store fartøjer kan afvige fra spirer fra kapillære netværk, der er involveret i mikrovaskulær patologi. For nylig offentliggjorde en gruppe en ex-vivo retinal analyse15,16. Selv om det er egnet til retinal neovaskulær sygdom, er det ikke så hensigtsmæssigt for choroidal neovascularization som set i AMD.
Den choroidal spirende assay ved hjælp af mus RPE, choroid, og scleral explanted væv blev udviklet til bedre model CNV. Vævet kan nemt isoleres fra mus (eller andre arter) øjne17. Denne analyse gør det muligt at reproducere pro- og anti-angiogene potentiale af farmakologiske forbindelser og evaluere den rolle, som specifikke veje i choroidal neovaskulærisering ved hjælp af væv fra genetisk modificerede mus ogkontrol 18. Denne choroidal spirende analyse er blevet refereret i mange efterfølgende publikationer9,10,18,19,20. Her er den metode, der er involveret i brugen af denne analyse er påvist.

<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

Patologisk choroidal angiogenese, en fremtrædende funktion af aldersrelateret makuladegeneration, fører til synsnedsættelse og blindhed. Endotelcelle (EF) spredningsanalyser ved hjælp af humane retinale mikrovaskulære endotelceller (HRMEC' er) eller isolerede primære nethinde-EC'er anvendes i vid udstrækning in vitro-modeller til undersøgelse af retinal angiogenese. Men, isolere ren murine retinale endotelceller er teknisk udfordrende og retinale ECs kan have forskellige spredning svar end choroidal endotelceller og forskellige celle / celle interaktioner. En meget reproducerbar ex vivo choroidal spiring assay som en model af choroidal mikrovaskulær spredning blev udviklet. Denne model omfatter samspillet mellem choroid vaskulatur (EF, makrofager, pericytter) og retinale pigment epitel (RPE). Muse-RPE/choroid/scleral explants isoleres og inkuberes i vækstfaktorredrimeret basalmembranekstrakt (BME) (dag 0). Medium ændres hver anden dag og choroid spiring kvantificeres på dag 6. Billederne af individuelle choroid explant er taget med en omvendt fase mikroskop og spiring område kvantificeres ved hjælp af en semi-automatiseret makro plug-in til ImageJ software udviklet i dette laboratorium. Denne reproducerbare ex vivo choroidal spiringsanalyse kan anvendes til at vurdere forbindelser til potentiel behandling og til mikrovaskulær sygdomsforskning for at vurdere veje, der er involveret i choroidal mikrokarprolifeion ved hjælp af vildtype og genetisk modificeret musevæv.

Sammenligning af choroid spirende vækst per dag
Vi dissekeret choroid med sclera, indlejret i BME og dyrkede dem i 6 dage(Figur 1). Choroid spiring i C57BL/6J mus fra dag 3 til dag 6 blev undersøgt med et mikroskop og kvantificeret med SWIFT-Choroid en semi-automatiseret kvantificering metode i ImageJ. I et repræsentativt tilfælde var det choroidalspiringsområde (de fartøjer, der strækker sig fra explanten, bortset fra selve explanten) 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

Denne protokol præsenterer choroid spiring assay, en ex vivo model af mikrovaskulær spredning. Denne analyse kan bruges til at vurdere veje involveret i prolifererende choroidal mikro fartøjer og vurdere narkotika behandlinger ved hjælp af vilde type og genetisk modificerede musevæv.

En Ex Vivo Choroid Spirende Assay af okulær mikrovaskulær angiogenese

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.

Research

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The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

C-FOS Protein Immunohistologisk Detection: Et nyttigt værktøj som en markør for Central involverede veje i specifikke fysiologiske reaktioner<em&gt; In vivo</em&gt; og<em&gt; Ex vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

En ex vivo vævskultur model af brusk remodeling i kvæg knæ Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Glomerular udvækst som en Ex Vivo Assay at analysere veje involveret i Parietal Epitelial Cell Aktivering

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Enkelt Myofiber Kultur Assay til vurdering af voksne muskelstamcellefunktionalitet Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...