Name: puncture-induced iris neovascularization as a mouse model of rubeosis iridis
Uploaded: 2018-03-08T15:00:00
Description: Watch this Scientific Journal Video about Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis at JoVE.com

Forfatterne takke Linnea Tankred og Diana Rydholm for husdyrhold.

BALB/c mus unger af begge køn blev brugt i overensstemmelse med erklæringen om brug af dyr i Ophthalmologic og Vision forskning, og protokollerne, der blev godkendt af Stockholms udvalg for etiske Animal forskning. Mus blev opstaldet i kuld, sammen med den ammende mor, med en 12-timers dag/nat cyklus, fri adgang til mad og vand, og overvåges dagligt.
Bemærk: For den kirurgiske procedure, mus blev holdt under anæstesi med flygtige isofluran. Okulær salver er afskrækket under okulær proc...

<ol>
	<li>Morrison, J. C., Van Buskirk, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+collateral+circulation+in+the+primate+eye.">Anterior collateral circulation in the primate eye.</a> Ophthalmology. 90 (6), 707-715 (1983).</li><li>Dreyfuss, J. L., Giordano, R. J., Regatieri, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+Angiogenesis.">Ocular Angiogenesis.</a> J Ophthalmol. 2015, 892043 (2015).</li><li>Breuss, J. M., Uhrin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-initiated+angiogenesis+and+the+uPA/uPAR+system.">VEGF-initiated angiogenesis and the uPA/uPAR system.</a> Cell Adh Migr. 6 (6), 535-615 (2012).</li><li>Rodrigues, G. 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=Neovascular+glaucoma:+a+review.">Neovascular glaucoma: a review.</a> Int J Retina Vitreous. 2, 26 (2016).</li><li>Stahl, 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=The+mouse+retina+as+an+angiogenesis+model.">The mouse retina as an angiogenesis model.</a> Invest Ophth Vis Sci. 51 (6), 2813-2826 (2010).</li><li>DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+wound+repair:+when+enough+is+enough.">Angiogenesis and wound repair: when enough is enough.</a> J Leukocy Biol. 100 (5), 979-984 (2016).</li><li>Eming, S., Brachvogel, B., Odorisio, T., Koch, M. <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:+Wound+healing+as+a+model.">Regulation of angiogenesis: Wound healing as a model.</a> Prog Histochem Cytoc. 42 (3), 115-170 (2007).</li><li>Beaujean, O., Locri, F., Aronsson, M., Kvanta, A., Andr&#233;, H. <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+in+vivo+model+of+puncture-induced+iris+neovascularization.">A novel in vivo model of puncture-induced iris neovascularization.</a> PloS One. 12 (6), e0180235 (2017).</li><li>Zudaire, E., Gambardella, L., Kurcz, C., Vermeren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Computational+Tool+for+Quantitative+Analysis+of+Vascular+Networks.">A Computational Tool for Quantitative Analysis of Vascular Networks.</a> PloS One. 6 (11), (2011).</li><li>Dorrell, M., Uusitalo-Jarvinen, H., Aguilar, E., Friedlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+neovascularization:+basic+mechanisms+and+therapeutic+advances.">Ocular neovascularization: basic mechanisms and therapeutic advances.</a> Surv Ophthalmol. 52 Suppl 1, S3-S19 (2007).</li><li>Kvanta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+angiogenesis:+the+role+of+growth+factors.">Ocular angiogenesis: the role of growth factors.</a> Acta Ophthalmol. 84 (3), 282-288 (2006).</li><li>Salman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasilicone+Bevacizumab+Injection+for+Iris+Neovascularization+after+Vitrectomy+for+Proliferative+Diabetic+Retinopathy.">Intrasilicone Bevacizumab Injection for Iris Neovascularization after Vitrectomy for Proliferative Diabetic Retinopathy.</a> Ophthalmic Res. 49 (1), 20-24 (2013).</li><li>Jeong, Y. C., Hwang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiology+and+Features+of+Eyes+with+Rubeosis+Iridis+among+Korean+Patients:+A+Population-Based+Single+Center+Study.">Etiology and Features of Eyes with Rubeosis Iridis among Korean Patients: A Population-Based Single Center Study.</a> PloS One. 11 (8), e0160662 (2016).</li><li>Speier, S., Nyqvist, D., K&#246;hler, M., Caicedo, A., Leibiger, I. B., Berggren, P. -. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye.">Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye.</a> Nat Protoc. 3 (8), 1278-1286 (2008).</li><li>Stefansson, E., Landers, M. B., Wolbarsht, M. L., Klintworth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neovascularization+of+the+Iris+-+an+Experimental-Model+in+Cats.">Neovascularization of the Iris - an Experimental-Model in Cats.</a> Invest Ophth Vis Sci. 25 (3), 361-364 (1984).</li><li>Nork, T. M., Tso, M., Duvall, J., Hayreh, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Mechanisms+of+Iris+Neovascularization+Secondary+to+Retinal+Vein+Occlusion.">Cellular Mechanisms of Iris Neovascularization Secondary to Retinal Vein Occlusion.</a> Arch Ophthalmol-Chic. 107 (4), 581-586 (1989).</li><li>Hjelmeland, L. M., Stewart, M. W., Li, J. W., Toth, C. A., Burns, M. S., Landers, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Experimental-Model+of+Ectropion+Uveae+and+Iris+Neovascularization+in+the+Cat.">An Experimental-Model of Ectropion Uveae and Iris Neovascularization in the Cat.</a> Invest Ophth Vis Sci. 33 (5), 1796-1803 (1992).</li><li>Tolentino, M. J., Miller, J. W., Gragoudas, E. S., Chatzistefanou, K., Ferrara, N., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+is+sufficient+to+produce+iris+neovascularization+and+neovascular+glaucoma+in+a+nonhuman+primate.">Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate.</a> Arch Ophthalmol-Chic. 114 (8), 964-970 (1996).</li><li>Paula, 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=Rabbit+Rubeosis+Iridis+Induced+by+Intravitreal+Latex-derived+Angiogenic+Fraction.">Rabbit Rubeosis Iridis Induced by Intravitreal Latex-derived Angiogenic Fraction.</a> Curr Eye Res. 36 (9), 857-859 (2011).</li><li>Takei, A., Ekstr&#246;m, 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=Gene+Transfer+of+Prolyl+Hydroxylase+Domain+2+Inhibits+Hypoxia-inducible+Angiogenesis+in+a+Model+of+Choroidal+Neovascularization.">Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization.</a> Sci Rep. 7, 42546 (2017).</li></ol>

I denne protokol præsenteres en roman metode til induktion af iris vaskulære svar af uveal punktering. Punktering udløser sår helbredende mekanismer og fremmer vaskulære svar i iris10,11. Dette er i overensstemmelse med okulær patologier såsom PDR og NVG, hvor forværret angiogene svar fra nethinden i det bageste segment af øjet kulminerer i den kliniske tilstand af rubeosis iridis, en øget vascularization af iris12 ,<...

Iris, corpus ciliare og årehinden, består den uvea, som er den mest vaskulariserede væv af øjet. Iris Vaskulaturen er afgørende for opretholdelsen af homøostase i det forreste kammer i øjet. Som følge af rigelige Anastomotiske forbindelser mellem arterier og vener leverer iris blodkar næringsstoffer og forsyning af ilt ikke kun iris, sig selv, men det hele forreste segment for øje1.
Dannelsen af nye blodkar, eller angiogenese fra allerede eksisterende, er grundlæggende fysiologiske processer, såsom udvikling og sårheling2. Angiogenese er reguleret fint af en lang række kanoniske faktorer, såsom vaskulær endotel vækstfaktor (VEGF) og plasminogen aktivator inhibitor (PAI), samt flere provokerende faktorer, og en ubalance af disse faktorer kan føre til patologiske angiogenese3.
I øjet er neovascularization årsag til sight-truende sygdomme, såsom proliferativ diabetisk retinopati (PDR) og neovaskulær glaukom (NVG). I disse okulær sygdomme, den fokale neovascularization findes almindeligt i retinal væv, men ubalance i inflammatoriske og angiogene faktorer i begge posterior og anterior okulære afdelinger i øjet har været forbundet med rubeosis iridis, den Klinisk udtryk for iris patologiske neoangiogenesis4. Disse patologier angive voksen iris evne til at gennemgå angiogenese. I mus, okulær Vaskulaturen er umodne efter fødslen og fortsætter modning postpartum. Denne ejendommelighed af udvikling udnyttes i musemodel af ilt-induceret retinopati, en model, der nøje efterligner den kliniske tilstand af retinopati prematurity5. Derudover angiogenese og inflammation spiller en central rolle i sårheling mekanismer6, og sårheling, selv har været forbundet med angiogenese modeller7.
I denne undersøgelse beskriver vi en model af punktering-induceret iris neovascularization. Uveal punkteringer er udført nær den ydre grænse for limbus, som inducerer iris neovascularization ved at udløse sårheling system. På grund af gennemsigtigheden af hornhinden, iris Vaskulaturen kan være analyseret nemt i vivo af noninvasive metoder. Punkteret øjnene præsentere en stigning af vaskulære seng i iris, som har været forbundet med en stigning af plasminogen aktivering og inflammatoriske markører8. Den præsenterede model har stort potentiale som et nyt værktøj til at studere angiogenese og screening angiogene forbindelser, og giver mulighed for direkte i vivo visualisering af angiogene processer.

<table>
	<tbody>
		<tr>
			<td>Bonn eye scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>23060</td>
			<td></td>
		</tr>
		<tr>
			<td>Clayman-Vannas curved scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>E3383 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Clayman-Vannas straight scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>E3383 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective adapter for camera</td>
			<td>Handcrafted</td>
			<td>N/A</td>
			<td>Or any system that allows adapting a camera to the microscope</td>
		</tr>
		<tr>
			<td>Heating Pad 100-110 watts</td>
			<td>Non Applicable</td>
			<td>N/A</td>
			<td>Available in pet/veterinarian stores</td>
		</tr>
		<tr>
			<td>Hypodermic 30g beveled needle</td>
			<td>KDM GmBH germany</td>
			<td>911914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iphone 4S</td>
			<td>Apple</td>
			<td>Non Applicable</td>
			<td>Or other high resolution image acquistion device</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>KDG 9623</td>
			<td></td>
		</tr>
		<tr>
			<td>McPherson tying forceps</td>
			<td>Bausch &#38; Lomb</td>
			<td>E1815 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tying forceps</td>
			<td>Bausch &#38; Lomb</td>
			<td>63140</td>
			<td></td>
		</tr>
		<tr>
			<td>Minims tetracaine hydrochloride</td>
			<td>Bausch &#38; Lomb</td>
			<td>N/A</td>
			<td>1 % (w/v) Eye Drops</td>
		</tr>
		<tr>
			<td>Neutral-buffered formalin</td>
			<td>Bioreagens</td>
			<td>0018-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline solution</td>
			<td>Fresenius Kabi</td>
			<td>210352</td>
			<td>0.9 % (w/v) NaCl in injectable water</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>Balanced and buffered PBS pH 7.4</td>
		</tr>
		<tr>
			<td>Petri dish 10 cm</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 3 cm</td>
			<td>Starstedt</td>
			<td>83.3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Seal Tube 2.0 mL</td>
			<td>Starstedt</td>
			<td>72.685.200</td>
			<td>Or any eppendorf style tubes</td>
		</tr>
		<tr>
			<td>TC plate 96-well</td>
			<td>Starstedt</td>
			<td>83.3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette 3.5 mL</td>
			<td>Starstedt</td>
			<td>86.1171</td>
			<td>Or any other Pasteur pipette style</td>
		</tr>
		<tr>
			<td>Univentor 400 anesthesia unit</td>
			<td>Univentor Limited</td>
			<td>N/A</td>
			<td>Or equivalent flow regulator with induction chamber and mask for volatile anesthesia</td>
		</tr>
		<tr>
			<td>Wild M650 surgical microscope</td>
			<td>Wild Heerbrugg</td>
			<td>N/A</td>
			<td>Or other surgical or magnifying stereoscope</td>
		</tr>
	</tbody>
</table>

puncture-induced iris neovascularization as a mouse model of rubeosis iridis

Vi beskriver en model af punktering-induceret iris neovascularization som en generel model for noninvasiv evaluering af angiogenese. Modellen er også relevante for målretning neovaskulær glaukom, en sight-truende komplikation af diabetisk retinopati. Denne metode er baseret på induktion af iris vaskulære svar af en række selvstændige forsegling uveal punkteringer på BALB/c mus og drager fordel af postpartum modningen af musen okulær Vaskulaturen. Musen pups gennemgå uveal punkteringer fra postnatal dag 12,5, når hvalpene naturligvis åbne deres øjne indtil postnatal dag 24,5. På grund af gennemsigtigheden af hornhinden, kan iris Vaskulaturen analyseres nemt gennem tiden af noninvasive i vivo metoder. Derudover kan BALB/c mus halvgennemsigtig iris være flatmounted for detaljerede immunohistologic analyse med minimal uspecifikke baggrund farvning. I denne model, angiogenese er primært drevet af de inflammatoriske og plasminogen aktivering systemer. Punktering-induceret modellen er først til at fremkalde iris neovascularization i små gnavere, og har fordelen, at direkte noninvasive i vivo analyse af angiogene proces. Modellen kan desuden kombineres med angiogene modulerende stoffer, der fremhæver dens potentiale i studiet af angiogenese med en i vivo perspektiv.

Albino BALB/c mus unger på P12.5 blev udsat for uveal punkteringer, gentaget hver fjerde dag (eksperimentel dag 0, 4, 8, 12), indtil P24.5. På P27.5, mus blev aflivet og iris dissekeret omhyggeligt (eksperimentel dag 15). Billeder af mus øjne blev taget med et kamera, der er knyttet til en kirurgisk Stereoskopet før hver punktering serie i hver eksperimenterende dag at vurdere noninvasive evaluering af iris vaskulære svar. Uveal punkteringer fremkalde en vaskulær svar fra iris ved a...

Watch this Scientific Journal Video about Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis at JoVE.com

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

Iris neovascularization, en almindelig komplikation af iskæmisk retinal sygdom, kan føre til sight-truende neovaskulær glaukom. Her, beskriver vi en murine protokol for at fremkalde eksperimentelle iris neovascularization, der kan bruges til noninvasive evaluering af angiogenese-modulerende stoffer.

Punktering-induceret Iris Neovascularization som en musemodel af Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

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