The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.

Tüm hayvanlar Laboratuvar Hayvanları Bakım ve Kullanım 2013 Edition Kılavuzu uyarınca tedavi edilir, Görme ve Oftalmoloji (ARVO) Oftalmik ve Vizyon Araştırma Hayvanların Kullanım Tablosu Araştırma Derneği ve Kurumsal Hayvan tarafından onaylanmış Bakım ve Northwestern Üniversitesi için kullanın Komitesi. Not: Aşağıdaki prosedür bir operatör tamamen yapılabilir; görevler buna göre bölünmüş olan ancak çok daha verimli iki operatörleri ile yapılmaktadır. 1. Lazer ve Ön lazer Station hazırlayın <ol><li> Kolayca erişilebilir olabilir lazer ve yarık lamba yerleştirin. Lazer açın ve parametrelerini ayarlamak önceden belirlenmiş (örneğin 75 mikron spot büyüklüğü, 100 mW güç, 100 milisaniye süre). DİKKAT: emin olun operatör tüm hayvan ve lazer güvenlik, kişisel koruyucu ekipman ve ilgili lazer güvenlik işaretleri prosedür odası dışında görüntülenen giyer. <ol><li> Herhangi bir deneysel hayvan kullanmadan önce, bir kalibrasyon, deneysel olmayan fareyi kullanarak İdeal parametreler bulmak. Lazer parametreleri kullanılan lazer bağlıdır. İdeal parametreleri düzgün odaklı olduğunda sürekli &quot;balon&quot; oluşmasına neden olur düşük lazer güç ayarı tarafından belirlenir. Uygun &quot;balon&quot; oluşumu ile lezyonun örneğin videoyu izleyin. </li></ol></li><li> Anestezi, sıcak hayvan, doku mendil ve tüm göz damlaları (Tropikamid, Tetrakain ve yapay gözyaşı) operatöre ulaşılabilecek kolayca böylece ön lazer istasyonu hazırlayın. <ol><li> Hayvan sıcak olun olup anestezi kaynaklı hipotermi önlemek için ilk fare içine anestezik enjekte önce sıcaklık (37 ° C) düzeltmek için önceden ısıtılmış. </li></ol></li><li> Isı tutma ve lazer işlemi başladıktan sonra sıcak kalabilir, böylece sıcak fare sahne yerleştirin. </li></ol> 2. Fare Anestezi ve Pre-lazer Hazırlık <ol><li> Enjekte önceanestezi ing, hiçbir deformiteleri veya kornea netlik (örn katarakt) azaltmak anormallikleri vardır sağlamak için makroskopik göz inceleyin. </li><li> Fareyi tartılır. </li><li> Tutanak ağırlığı, cinsiyet, ve hayvan kimlik numarası. </li><li> 20 g bir fare için, 0.20 ml ksilazin / Ketamin Kokteyli enjekte; ağırlığı kullanılarak, anestezi, uygun miktarda hesaplamak 10 mg / kg ksilazin TD tribromoethanol 250 mg / kg (örneğin, 100 mg / kg ketamin hidroklorür kurum tarafından verilen ilkelere dayalı olarak kullanılmak üzere tribromoethanol OR 0.25 mi). Matematiksel hatalarını azaltmak amacıyla 1 g artışlarla ağırlığı başına önceden hesaplanmış dozajların bir tablo yazıldı. </li><li> Scruff fare ve enjekte anestezi intraperitoneal adımda 2.4 hesaplamalara dayalı. </li><li> Sıcak hayvan fare yerleştirin ve fare tamamen ayak tutam (yaklaşık 3-5 dakika) yoluyla pedal refleksini kontrol ederek anestezi kadar bekleyin. </li><li> Silin, bir doku Roll up ve Aspirat önlemek için farenin burun alanını korumakSıvı roll-off iyon. Rulo yan fare ve topikal anestezi için her bir göz içine tetrakain hidroklorür damla (yaklaşık 30 ul) yerleştirin. Çözelti etkili olması için 2 dakika bekleyin. </li><li> Pupil dilatasyonu topikal tropikamid bir damla adımı yineleyin 2.7. Seçenek olarak ise, dilatasyon fenilefrin hidroklorür (% 2.5) kullanımı. </li><li> Çözümler etkili olması için 2 dakika bekleyin; Bu süre boyunca sıcak üzerinde hayvan tutun. </li><li> Uygun zaman geçtikten sonra, hızlı bir şekilde fare sahnede fare koyun ve yarık lamba çene geri kalanı sahne yerleştirin. </li><li> Düşük ışık parlaklığına biyomikroskopta açın ve pupilla dilatasyonu derecesini kontrol edin. Öğrenci yeterince (yaklaşık 2,5-3 mm) dilate değilse, sıcak hayvana fare dönmek ve bekleyin. Alternatif olarak, tropikamid başka damla yönetmek. Göz yeterince dilate sonra, lazer prosedürüne devam edin. </li></ol> 3. Lazer Prosedürü Not: Diğer p emin olunersons odasında lazer korumalı yarık lamba göz parçasından uzakta koruyucu gözlük <ol><li> Bu ideal optik sinir görselleştirme (3.1.2 bakınız) konumlandırılmış böylece fare sahnede fare yerleşimini ayarlayın. <ol><li> Yatay yatıyor böylece diğer azından bir tarafında baş ve kuyruk, lamba ışın yarık dik, sahibine fare yönlendirmek. İdeal fare yerleştirme kapak kayma uygulandıktan sonra çok daha kolay optik sinir görselleştirme yapacaktır. </li><li> Daha yakın lazer operatörü baş bir ~ 170 ° &#39;lik açıyla yani hafifçe fare çevirin. </li><li> Kararlı ve nerede operatörün eli ince manipülasyon için yeterli alana sahip olacak olduğu bir pozisyonda yine lambayı yarık henüz mümkün olduğunca o fare kadar yakın olduğundan emin olun. </li></ol></li><li> Fare ideal yerleştirilmesinden sonra, bir 25 mm x 25 mm ebadındaki bir cam lamel yapay gözyaşı çözeltisi bir damla yerleştirin. <ol><li> Farenin opp suni gözyaşı çözümün bir damla yerleştirinosite göz - Bu sağlayacaktır göz sulu ve yardım gecikme katarakt olduğunu. </li></ol></li><li> Başparmak ve işaretçi parmakları arasında lamel köşesini tutun; konumu ve böylece cam iki parmak uçları arasında sıkılır. </li><li> Yavaşça destek ve el stabilizasyonu için hayvanın vücudu etrafında kalan üç parmaklarını sarın. Pozisyon eli böylece cam lamel kolayca farenin göze yerleştirilebilir. <ol><li> Bilek El titreme azaltmak için sağlam bir yüzey üzerinde stabilize olduğundan emin olun. </li></ol></li><li> Kararlı pozisyon elde edildikten sonra, dikkatle farenin gözün üzerine (hala yapışık yapay gözyaşı damla ile) cam lamel basın. <ol><li> Lamel lazer ışını yayılımını veya yansımasını önlemek amacıyla lazer ışını mümkün olduğunca dik olarak konumlandırılmış emin olun. Lamel kornea düzleştirmek için bir kontakt lens gibi davranır. </li></ol></li><li> Toggle unt odak yarık lamba ile ve serbest el ile bakmakil retina görülebilir. Retina görüntülenmiştir yere bağlı açık sarı / kırmızı renk olacak, farklı, kırmızı damarlar görünür olacaktır. </li><li> Yavaş ve dikkatli bir optik siniri görüntülenmesi kadar fare kafasını ve / veya lamel işlemek. Optik sinir o yayılan çok sayıda gemi ile, sarı renkli olacaktır. </li><li> Operatör optik sinirin görselleştirme onaylamıştır sonra, ışın odaklama lazeri açın. </li><li> Lazer ışını açıldıktan sonra, istenilen pozisyona (optik sinir yaklaşık 1 disk çapı) ışını odaklama lazer manevra. </li><li> Göz fundus RPE üzerinde lazer ışınını odaklanın. Uygun odak keskin ve net lazer ışını sahip sağlanır. Işını amaçlayan yarık lamba odak veya yeniden pozisyon cam lamel geçiş, oval ya da odak dışı görünüyorsa. </li><li> Hedefleme ışını RPE odaklanmıştır sonra, lazerin ayak tetiği kullanarak lazer yönetimini başlatmak. <ol><li> Intraoküler önlemek için retina damarları önlemek için emin olun oraji. </li></ol></li><li> Hemen lazer uygulamasından sonra bir kabarcığın görünüm için izleyin. Lazer atış hatları net ve hiçbir şekilde puslu olmamalıdır. <ol><li> Lazer atış kabarcığı oluşumuna yol açmaz ise, ya da darbe alan (genel ort başarılı etkisi net, keskin tanımlı sınırına kötü tanımlanmış dairesel kenarlıklı bulutlu bir görünüm) puslu görünüyor, ya da kanama lazer uygulamasından sonra görülürse, DEĞİL yapmak gelecek analizi için bu lezyonlar bulunur. </li></ol></li><li> Tekrarlayın istenen tüm CNV pozisyonları için 3.10-3.12 (genellikle 3&#39;te, 6, 9, ve optik sinir etrafında 00:00 pozisyonları) yineleyin. Lazer indüksiyonlar optik sinir yaklaşık aynı mesafede uygulanırsa, yeniden yoğunlaşma gerekli olmamalıdır. Ancak, retina bulunabilecek ışını refocusing ardışık lazer idareleri arasında gerekli olabilir fare göz ve küçük değişimlerin güçlü eğrilik. </li><li> Bir dizüstü konumu ve e sonucu rekorach lazer atış idaresi ve sonuç (başarılı, puslu, kanama vb.) göz için her uygulanan vurdu. Kullanılmadığı zaman stand-by modunda lazer yerleştirmek emin olun. </li><li> Gerekirse istikrar ve yeni bir lamel için ters elinizi kullanarak, fare, diğer göz için 3.1-3.14 tekrarlayın. </li><li> İstenen tüm lazer çekim uygulanır sonra lazer kapatın ve yarık-lamba. </li><li> Anestezi kurtarma için sıcak üzerinde lamel ve yer fareyi atın. Makroskobik herhangi bir yaralanma için göz kontrol ve sulandırılmış göz tutmak ve potansiyel gelecek katarakt gelişimini önlemek için yapay gözyaşı çözümün bir damla yerleştirin. Fare anestezi kurtarır sonra kafese geri döner. </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td></td><td> AVERTIN </td><td> AVERTIN </td><td> AVERTIN </td><td> XYL / KET </td><td> XYL / KET </td></tr><tr><td> Fare Ağırlık (g) </td><td> Doz (mg / kg) </td><td&gt; Çözelti konsantrasyonu (mg / ml) </td><td> Anestetik doz (mi) </td><td> Doz (mg / kg) </td><td> Anestetik doz (mi) </td></tr><tr><td> 15 </td><td> 250 </td><td> 20 </td><td> 0,1875 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.15 </td></tr><tr><td> 16 </td><td> 250 </td><td> 20 </td><td> 0,2 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.16 </td></tr><tr><td> 17 </td><td> 250 </td><td> 20 </td><td> 0,2125 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.17 </td></tr><tr><td> 18 </td><td> 250 </td><td> 20 </td><td> 0.225 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.18 </td></tr><tr><td> 19 </td><td> 250 </td><td> 20 </td><td> 0,2375 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.19 </td></tr><tr><td> 20 </td><td> 250 </td><td> 20 </td><td> 0.25</td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0,2 </td></tr><tr><td> 21 </td><td> 250 </td><td> 20 </td><td> 0,2625 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.21 </td></tr><tr><td> 22 </td><td> 250 </td><td> 20 </td><td> 0.275 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.22 </td></tr><tr><td> 23 </td><td> 250 </td><td> 20 </td><td> 0,2875 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.23 </td></tr><tr><td> 24 </td><td> 250 </td><td> 20 </td><td> 0.3 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.24 </td></tr><tr><td> 25 </td><td> 250 </td><td> 20 </td><td> 0.3125 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.25 </td></tr><tr><td> 26 </td><td> 250 </td><td> 20 </td><td> 0.325 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.26 </td></tr><tr><td> 27 </td><td> 250 </td><td&gt; 20 </td><td> 0,3375 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.27 </td></tr><tr><td> 28 </td><td> 250 </td><td> 20 </td><td> 0.35 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.28 </td></tr><tr><td> 29 </td><td> 250 </td><td> 20 </td><td> 0,3625 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.29 </td></tr><tr><td> 30 </td><td> 250 </td><td> 20 </td><td> 0.375 </td><td> 100 mg / kg ketamin; 10 mg / kg ksilazin </td><td> 0.3 </td></tr></tbody></table> Tablo 1: XyIKet Doz Şeması.

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+in+rodents+of+reversible+drug+induced+opacities+of+the+lens.">Occurrence in rodents of reversible drug induced opacities of the lens.</a> British J Ophthalmol. 45, 408-414 (1961).</li><li>Ridder III, W. H., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+Cataract+Development+in+Anesthetized+Mice.">Causes of Cataract Development in Anesthetized Mice.</a> Exp Eye Res. 75, 365-370 (2002).</li></ol>

The authors declare they have no competing financial interests.

Başarılı lazer indüksiyon sonrası lazer teslimat ve sonuçtaki KNV lezyon gelişimini etkileyen birden fazla faktör vardır. Bu faktörler kontrollü ve en güvenilir sonuçlar elde etmek için standardize edilmelidir. Bu faktörlerin en uygun fare seçimi (genotipi, yaş, cinsiyet ve), anestezik seçimi, ve lazer ayarları vardır. Kullanılan özel fare modeli KNV gelişme seyri üzerinde önemli bir etkiye sahip olabilir. En yaygın olarak kullanılan genotip C57BL / 6 faredir. Hayv...

Yaşa bağlı makula dejenerasyonu (AMD) 50 1-3 yaş üstü bireylerde körlüğün önde gelen nedenlerinden biridir. AMD iki formda sınıflandırılabilir: atrofik (&quot;kurutmak&quot;) AMD ve neovasküler (&quot;ıslak&quot;) AMD. Eski ikinci sızıntı, kanama ve fibrozis neden dış retina katmanlarının içine koroid anormal damarların işgali ile karakterize edilirken, retina pigment epiteli (RPE), koriokapillaris ve fotoreseptör coğrafi atrofi ile karakterize ve sonuçta bir körlük 1,2 yol açar. İki formları, neovasküler AMD görme kaybı 1 çoğunluğu oluşturuyor. Onun atrofik muadili şu anda hiçbir tıbbi tedaviler 3 kanıtlamıştır oysa Neyse ki, bu form sayısız etkili farmakolojik tedavi seçenekleri vardır. Neovasküler formu kolaylıkla bir hayvan modelinde yeniden teslim oldu edilmiştir çünkü Ayrıca, temel A&#39;ya daha yaygın olarak erişilebilir olmuşturYeni tedaviler 4 geliştirmek için altta yatan patolojik mekanizmaların keşfetmek MD araştırma. Deneysel koroid neovaskülarizasyon (KNV) ilk hayvan modeli Ryan ve arkadaşları tarafından geliştirilmiştir. insan olmayan primatlar 5. Neovasküler AMD görülene benzer anjiyojenez ile sonuçlanan bir lokal enflamatuar bir yanıt neden olduğu lazer fotokoagülasyon ile Bruch zarının Bu model bağlı kopma. Anjiyogenez sonrası lazer indüksiyon histopatolojik ilerleme modelinin geçerliliğini 6 doğruladı neovasküler AMD, taklit bulunmuştur. Insan olmayan primatlar insanlara en çok benzeyen anatomi sunuyoruz, ama ne yazık ki, kolayca genetik manipüle edilemez, bakımı pahalı ve hastalığın ilerlemesi 7 yavaş bir zaman kursumuz var. Buna karşın bir, kemirgen modelleri çok daha düşük maliyetli sürdürmek, genetik göreli kolaylıkla manipüle edilebilir, ve cou hızlı bir çok şey varhastalık ilerlemesinin rse (deney ay karşısında haftalık bir zaman ölçeğinde yapılabilir). Bu albino hayvanlarda görselleştirmek için çok zor olduğu gibi bu deneyler sadece pigmentli kemirgenlerde yapılmalıdır. İlk sonlarında 90 en 10 Campochiaro grubu tarafından geliştirilen fare lazer kaynaklı CNV modeli, son çalışmalar 11-16 çoğunda baskın bir hayvan modeli olarak büyümüştür. Nedeniyle CNV karmaşık ve hala belirsiz patogenez, lazer modeli gelecekte insan kullanımı için yeni tedavi modaliteleri değerlendirmek için anjiyogenez sürüş moleküler mekanizmaları inceleyerek kadar ıslak AMD araştırma tüm yönleriyle uygulanmıştır. Örneğin, Sakurai ve diğ. ve Espinoza-HEIDMANN ve diğ. transgenik fareler ve farmakolojik tükenme tedavileri 15, 16 kullanılarak KNV gelişimi üzerine makrofajlar etkisini araştırmak için lazer modeli kullanılır. Giani ve ark. ve Hoerster diğ. görüntüye kullanılan optik koherens tomografi (OCT) lazer kaynaklı KNV ilerlemesini karakterize ve Ekim görüntüleme 12,17 görülen bulgulara histopatolojik bulguları karşılaştırmak için bir çaba CNV. Son olarak, anti-anjiyogenik ajanlar intravitreal enjeksiyonunu içeren çalışmalar insan denemeleri için ön koşul olarak kullanılan ve neovasküler AMD 10,18,19 yönetiminde kullanılan anti-VEGF ajanların ilk nesil gelişmekte hayati önem oylandı. Deneysel CNV için alternatif modeller KNV ikna etmek için cerrahi yöntemler kullanmaktadır. Bu prosedür, pro-anjiyojenik maddeler enjekte içerir (VEGF aşırı eksprese eden örneğin, rekombinant viral vektörler, RPE hücreleri ve / veya polistiren boncuklar subretinal enjeksiyon) anjiyojenezi 8,20 neden amacıyla, neovasküler AMD görülen artış olarak VEGF ifadesini taklit etmek. Bununla birlikte, bu yöntem, neovaskülarizasyonun büyük ölçüde düşük bir oranda verir; Bu çalışmalar, bu KNV gösterdiC57 / BL6 fareleri iki ayrı fare 8,14 aynı gerginlik lazer fotokoagülasyon yönteminde görülen ~% 70 başarı oranı karşı enjeksiyonlar 31% &#39;unda görülür. Bu nedenlerden dolayı, ve insan olmayan primatlar karşı kemirgenler kullanmanın avantajları göz önüne alındığında, lazer kaynaklı KNV&#39;ların fare modeli en neovasküler AMD çalışma deneyleri 8 KNV&#39;nin standart hayvan modeli haline gelmiştir. Fare gözü ile çalışmak için bir minik, narin bir dokudur. Retina görselleştirmek için göz Manevra zordur ve ustalık elde edilene kadar çok pratik gerektirir. Bu görev hakim ve baskın olmayan el ile öğrenmiş olmalıdır gerçeği ile karmaşık. Retina görselleştirmek için gerekli ince hareketleri öğrenmiş edildikten sonra Dahası, her iki el ve lazer işletim ayak pedalı arasındaki koordinasyon önemlidir. Bu yazıda, lazer kaynaklı KNV yordam katılan fiziksel manipülasyonlar tüm öğrenme zorlukları damıtmak çalıştıoperatörler bu model ile hızlı bir başarı elde yardımcı olacak bir rehber haline edure.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>532 nm (green) argon ophthalmic laser</td>
			<td>IRIDEX</td>
			<td>GLx</td>
			<td>any ophthalmic 532 nm (green) argon laser can be used</td>
		</tr>
		<tr>
			<td>slit lamp</td>
			<td>Carl Zeiss</td>
			<td>30SL-M</td>
			<td>any slit lamp can be used as long as it is compatible with the laser</td>
		</tr>
		<tr>
			<td>tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>tert-amyl alcohol</td>
			<td>Sigma</td>
			<td>152463-1L</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>amber glass vials + septa</td>
			<td>Wheaton</td>
			<td>WH-223696</td>
			<td>tribromoethanol storage</td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>miscellaneous&#160;</td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Falcon Pharmaceuticals</td>
			<td>RXD2974251</td>
			<td>pupillary dilation</td>
		</tr>
		<tr>
			<td>0.5% tetracaine hydrochloride</td>
			<td>Alcon</td>
			<td>&#160;0065-0741-12</td>
			<td>topical anesthesia</td>
		</tr>
		<tr>
			<td>artificial tears</td>
			<td>Alcon</td>
			<td>&#160;58768-788-25</td>
			<td>hydration</td>
		</tr>
		<tr>
			<td>heat therapy pump (for animal warming)</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>used to maintain animal body temp</td>
		</tr>
		<tr>
			<td>warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510EA</td>
			<td>maintains animal body temperature</td>
		</tr>
		<tr>
			<td>30 gauge insulin needles</td>
			<td>BD</td>
			<td>328418</td>
			<td>IP anesthesia injection</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>American Weigh Scale</td>
			<td>AWS-1KG-BLK</td>
			<td>mouse weighing</td>
		</tr>
		<tr>
			<td>cover slip (25 mm x 25 mm)</td>
			<td>VWR</td>
			<td>48366089</td>
			<td>flatten cornea to visualize mouse retina</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>used for volumetric re-construction</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for image analysis</td>
		</tr>
	</tbody>
</table>

a mouse model for laser-induced choroidal neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

KNV lezyonlarının Niceleme CNV gemileri etiket immünoflüoresans boyama kullanarak düz monte choroids analizi yoluyla yapılabilir. Doku hazırlık iki en sık kullanılan yöntemler hemen endotel hücre işaretleyici ile hayvan kurban, ya da ölüm sonrası immün boyama önce perfüzyon yoluyla yapılan FITC-dekstran etiketleme vardır. Bu yöntemlerin ikisi de detaylı 13,14,21 daha önce tarif edilmiştir, sırasıyla her biri 1 ve 2 örneklerini gös...

Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

A Mouse Model for Laser-induced Choroidal Neovascularization

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Lazer kaynaklı Koroid Neovaskülarizasyonlarında A Fare Modeli

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

a-mouse-model-for-laser-induced-choroidal-neovascularization

Research

JoVE Journal

Medicine

17.4K Görüntüleme.  Northwestern University Feinberg School of Medicine. Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Video: A Mouse Model for Laser-induced Choroidal Neovascularization

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Görme Kusurları karakterize Kronik Oküler Hipertansiyon bir Lazer-kaynaklı Fare Modeli

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Tıp

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Zaman kurs Indocyanine Yeşil Anjiyografi ile Yaşa Bağlı Makula Dejenerasyonu Fare Modeli Koroid vaskülatürüne anormallikleri tespit

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Fare Kornea Neovaskülarizasyonunun bir Alkali-yanık Yaralanma Modeli

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Scarless Yara Repair Fare Fetal Cilt Modeli

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Bir Yenidoğan Fare Omurilik Sıkıştırma Yaralanması Modeli

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Puncture-Induced Iris Neovascularization as a Mouse Model of 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 for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Delici cisim kaynaklı Iris neovaskülarizasyon Rubeosis Iridis bir fare modeli olarak

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

İnsan jinjiva greft neovaskülarizasyon izleme için yeni bir yaklaşım

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Safra Kanalı TNBS infüzyonu ile Kronik Pankreatit için Bir Fare Modeli

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Zebra Balığı Larvalarında Omurilik Rejenerasyonunu İncelemek için Kontrollü Yarı Otomatik Lazer Kaynaklı Yaralanmalar

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...