Yazarlar için mükemmel teknik ve videographic destek Yuliya Naumchuk (Chicago Loyola Üniversitesi) ve Agne Žiniauskaitė (Experimentica Ltd) teşekkür etmek istiyorum. Dr. Kaja'nın araştırma programı Dr John P. ve Therese E. Mulcahy donatılmış profesörlük Oftalmoloji, Chicago Loyola Üniversitesi tarafından desteklenir.

Bütün hayvanlar göre ARVO bildirimi kullanım, onaylanmış ve Finlandiya hayvan deney Yönetim Kurulu tarafından izlenen protokolleri kullanarak hayvanlar için Ophthalmic ve vizyon araştırma ve EC Direktifi 86/609/EEC hayvan deneyleri için tedavi edildi.
1. lazer kaynaklı fare KNV modeli 5
<ol><li>Hayvan macroscopically için herhangi bir anormallik gözünde bakarak kontrol edin.</li>
	<li>Fare tartın.</li>
	<li>Hesaplamak ve anestezi kullanmak için uygun bir miktarda hayvan, örneğin medetomidine (1 mg/kg), ketamin (75 mg/kg) ve distile su (% 0.9 NaCl çözüm) 1:1.5:2.5 veya ketamin (40-75 mg/kg), bir oranında karışımı ağırlık temel hazırlamak xylazine (5 mg/kg) ve 1:2. 5:1 oranında, distile su (% 0.9 NaCl çözüm); 20 g fare için 0.1 mL karışımı enjekte.</li>
	<li>Anestezi intraperitoneally enjekte et.</li>
	<li>Hayvan anestezi kadar yer fare geri içine belgili tanımlık kafes ve bekle. Fare düzgün bir pedal refleks eksikliğinden anestezi onaylayın.</li>
	<li>Lazer güvenlik kişisel koruyucu ekipman kullanımı olun.</li>
	<li>Slit lamba ve 532 nm diode lazer açın.</li>
	<li>Kafes ve Isıtma yastığını üzerinde yer fareyi kaldırın.</li>
	<li>Tropicamide bir damla Gözbebeği genişlemesi için geçerlidir. Tam (3 mm) Gözbebeği genişlemesi için 3-5 dk beklemeye.</li>
	<li>Fare slit lamba sahnesinde yerini.</li>
	<li>Bir damla göz sıvı jel bir coverslip için applanate kornea üzerine yerleştirin.</li>
	<li>Fare göz Merkezi görme sinirine kafasıyla gelecek şekilde yönlendirin.</li>
	<li>Lazer güç 100'e ayarlamak mW, süresi 100 MS ve nokta boyutu 50 µm için.</li>
	<li>Lazer ışını retina pigment epiteli (RPE) odaklanmak.</li>
	<li>Üç lazer atış bir göz retina damarları 4, 8 ve 12 yönünde konumlarını optik sinir, etrafında, ideal olarak sırasıyla kaçınarak olun. Sonra tüm çekimleri retinal kanama olmaması için lazer göz fundus inceleyin. Kontralateral göz sigara lazerle denetimi olarak hizmet vermektedir.</li>
	<li>Coverslip ve yer fare geri Isıtma yastığını atmak.</li>
	<li>PEG jel damla bir damla her iki gözde uygulanır.</li>
</ol>2. SD-Ekim 6,7
<ol><li>Fare kemirgen hizalama sahne içine yerleştirin ve baş hareketsiz.</li>
	<li>SD-Ekim (örneğin, Bioptigen/Leica Envisu R2200) göz yüz sistem objektif vivo içinde görüntüleme için X ve Y-seviyeli denetleyicilerini kullanarak hizalayın.</li>
	<li>Bruch'ın membran sonları doğrulamak için SD-Ekim taramalar: SD-Ekim bütün göz tarar kez lazerle sitelerinde başvuru satırı el ile taşımak. Bruch'ın membran sonları (bkz. şekil 1) lazerle alanlarda açıkça görünür olmalıdır.</li>
</ol>3. floresein anjiyografi 7,8,9
<ol><li>Fare ile sahibi çıkarın ve SK sisteminde (örneğin, Heidelberg Spectralis HRA2) yerleştirin.</li>
	<li>Lazer odak optik sinir izleme pencerenin ortasında başkanı ile kızılötesi yansıma modunu kullanarak göz fundus alanlarında yakmak.</li>
	<li>0.1 mL % 5 fluoresein sodyum tuz 20 gr fare subkutan veya intraperitoneally enjekte.</li>
	<li>Choroidal düzeyde odak.</li>
	<li>Fotoðraf choroidal odak seviyesinden.</li>
	<li>Retina düzeyinde yeniden odaklanın ve fotoðraf.</li>
	<li>30 s ve Yinele adımları için 3,4-3.6 bekleyin.</li>
	<li>Fare sahibinden çıkarın ve Isıtma yastığını yerleştirin.</li>
	<li>Ters anestezi tarafından α2-antagonisti medetomidine, atipamezole (0.5 mg/kg, IP) veya anestezi hayvan kurtarma için bekleyin.</li>
	<li>In vivo SD-Ekim ve SK görüntüleme imzalat hayvanlarda takip günlerini 5, 10 ve 14 üzerinde yineleyin.</li>
</ol>4. KNV sınıflandırma
<ol><li>Sınıf Bruch'ın membran OCT görüntüleri ve choroidal SK görüntüleri hemen aşağıdaki gibi gün 0 lasering sonra alınan hasar: 0 - Bruch'ın membran hasar değil 1 - Bruch'ın membran başarılı hasar</li>
	<li>Sınıf KNV varlığı gibi bir dizi retina SK resmi olarak aşağıdaki floresein sinyal dinamikleri karşılaştırarak sızıntı vardı lazerle noktalardan: 0 - retina normal görünüm 0.5 - zayıf boyama kaçağı 1.0 - Sızıntılı KNV alanları Not: OCT görüntüleme ek onay KNV veya şüpheli SK nerede OCT görüntülerde intraretinal sıvı varlığını KNV sınıflandırma öneririz kullanın.</li>
</ol>5. retina kalınlığı ölçümleri
<ol><li>Bir otomatik bölümleme yazılımı retina kalınlığı ölçümleri için kullanın. Toplam retina kalınlık, Sinir lifi tabakası RPE (sağlıklı ölçüm siteler) veya hayali bir çizgi RPE hasar (lazerle siteler) sitenin etrafına bağlanan tüm katmanlardan kalınlığı olarak kabul edilir sağlamak (Ayrıca bkz: Şekil 7).</li>
</ol>

<ol>
	<li>Dobi, E. T., Puliafito, C. A., Destro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+experimental+choroidal+neovascularization+in+the+rat.">A new model of experimental choroidal neovascularization in the rat.</a> Arch. Ophthalmol. Chic. Ill 1960. 107, 264-269 (1989).</li><li>Tobe, 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=Evolution+of+neovascularization+in+mice+with+overexpression+of+vascular+endothelial+growth+factor+in+photoreceptors.">Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.</a> Invest. Ophthalmol. Vis. Sci. 39, 180-188 (1998).</li><li>Seo, M. 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=Dramatic+inhibition+of+retinal+and+choroidal+neovascularization+by+oral+administration+of+a+kinase+inhibitor.">Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.</a> Am. J. Pathol. 154, 1743-1753 (1999).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Prog. Retin. Eye Res. 29, 500-519 (2010).</li><li>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> J Vis Exp. (106), e53502 (2015).</li><li>Giani, 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=In+vivo+evaluation+of+laser-induced+choroidal+neovascularization+using+spectral-domain+optical+coherence+tomography.">In vivo evaluation of laser-induced choroidal neovascularization using spectral-domain optical coherence tomography.</a> Invest. Ophthalmol. Vis. Sci. 52, 3880-3887 (2011).</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=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PloS One. 10, e0132643 (2015).</li><li>Sheets, K. G., 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=Neuroprotectin+D1+attenuates+laser-induced+choroidal+neovascularization+in+mouse.">Neuroprotectin D1 attenuates laser-induced choroidal neovascularization in mouse.</a> Mol. Vis. 16, 320-329 (2010).</li><li>Hoerster, 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=In-vivo+and+ex-vivo+characterization+of+laser-induced+choroidal+neovascularization+variability+in+mice.">In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice.</a> Graefes Arch. Clin. Exp. Ophthalmol. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 250, 1579-1586 (2012).</li><li>Sulaiman, R. 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+Simple+Optical+Coherence+Tomography+Quantification+Method+for+Choroidal+Neovascularization.">A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.</a> J. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. 31, 447-454 (2015).</li></ol>

Yazar Symantas Ragauskas, doktora bir çalışan (bilim) ve hissedar Teklifler araştırma hizmetleri bu makalede kullanılan preklinik KNV modeli istihdam sözleşme Experimentica Ltd. ' dir.
Eva Kielczewski bir çalışan (araştırma uygulamaları mühendis, OCT) Bu makalede kullanılan SD-Ekim sistemleri üretir Leica Mikrosistemlerin temelleri yazarıdır.
Yazar Joseph Vance'in bir çalışandır (NA OCT satış direktörü) Bu makalede kullanılan SD-Ekim sistemleri üretir Leica Mikrosistemlerin temelleri. Joseph Vance'in Başkan ve yönetim müdürü Spective, LLC da.
Yazar Simon Kaja, doktora Danışmanı Scientific Müdürü ve hissedar, Experimentica Ltd., teklifler araştırma hizmetleri, sözleşme preklinik sözleşme araştırma kuruluşu preklinik KNV modeli bu makalede kullanılan dahil olduğunu. Simon Kaja, doktora da CEO K & bilimsel P, LLC, danışmanlık firması, Yaşam Bilimleri ve Dr. John s. ve Therese E. Mulcahy donatılmış Profesör Chicago Loyola Üniversitesi, Stritch tıp Fak., Oftalmoloji olarak hizmet vermektedir. Bu düzenleme koşulları incelenmeli ve çıkar çatışması politikasına uygun olarak Chicago Loyola Üniversitesi tarafından onaylanmalıdır.
Yazar Giedrius Kalesnykas, doktora bir çalışan (CEO) ve teklifler araştırma hizmetleri bu makalede kullanılan preklinik KNV modeli istihdam sözleşme Experimentica Ltd. hissedarı olduğunu.

Multimodal görüntüleme KNV patoloji değerlendirme için değerli araçlar sunar. Burada SK, oluşan bir görüntüleme Protokolü sunulan SD-Ekim ve KNV patoloji hızlı, tekrarlanabilir ve güvenilir değerlendirilmesi için otomatik bölümleme. Lazer yönetim doğrulandı sonra Bruch'ın membran bir mola. Buna ek olarak, SD-Ekim bu aşamada da sonuçları yorumu yıkmak olası intraretinal ve subretinal kanama hemen görselleştirme izin. Retina sızıntıları floresein sinyal SK görüntülerden göre kademeli. ...

Başarılı ilk insan KNV Rodents patoloji taklit girişimi neredeyse otuz yıl önce uzun-Evans fareler1Kripton lazer ile gösterilmiştir. Bundan sonra bir Kripton lazer Bruch'ın membran en popüler fare gerginliği, C57BL/6J2,3,4kırmak için kullanıldı. KNV indüksiyon başarı oranını SK ve histolojik lekeleri ile doğrulandı. EKM gibi noninvaziv görüntüleme yöntemleri, hızlı bir gelişme alanı kemirgen Preklinik modellerin büyüme destekledi. Aynı göz içinde birden çok kez noktalarda retina morfolojik değişiklikler önemli ölçüde izleme olanağı hayvan kullanımı azaltma için katkıda bulunur ve deneysel çalışmalarda verimliliği artırır. KNV lezyonların histolojik değerlendirme oldukça basittir ve anormal damar büyüme lazer yönetim, resim alma ve alan/hacim tahmini bir görüntü analiz yazılımı kullanarak site etrafında etiketleme gerektirir. Buna ek olarak, daha karmaşık analizleri KNV patoloji ve onun yorumunu vivo içinde görüntüleme yöntemleri tanıtmak.
Burada SK, SD-Ekim, kullanarak sınıf indüksiyon, ilerleme ve gerileme KNV nispeten hızlı ve basit bir yöntem mevcut ve fare otomatik bölümleme yöntem lazer kaynaklı KNV modeli.

<table><tbody><tr><td>Medetomidine (commercial name Domitor)</td><td>Orion</td><td>Vnr 01 56 02</td><td>Anesthesia</td></tr><tr><td>Ketamine</td><td>Intervet</td><td>Vnr 51 14 85</td><td>Anesthesia</td></tr><tr><td>0,9% NaCl</td><td>B Braun</td><td>357 0340</td><td>Anesthesia</td></tr><tr><td>Xylazine (commercial name Rompun vet)</td><td>Bayer</td><td>vnr 14 89 99</td><td>Anesthesia</td></tr><tr><td>Tropicamide</td><td>Santen</td><td>Vnr 04 12 36</td><td>Mydriatic agent</td></tr><tr><td>Viscotears</td><td>Alcon</td><td>Vnr 44 54 81</td><td>Lubricant</td></tr><tr><td>Systane</td><td>Alcon</td><td>&amp;nbsp;-</td><td>Lubricant</td></tr><tr><td>5% Fluorescein sodium salt</td><td>Sigma Aldrich</td><td>F6377-100G</td><td>Fluoresent agent</td></tr><tr><td>Atipamezole (commercial name Antisedan)</td><td>Orion</td><td>Vnr 47 19 53</td><td>Anesthesia</td></tr></tbody></table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

Choroidal neovaskülarizasyon (KNV) lazer kaynaklı yaşa bağlı makula dejenerasyonu (AMD) ıslak şeklinde taklit etmek için köklü bir modeldir. Bu protokol için amacımız neovasküler süreçleri, ama oldukça multimodal boyuna elde edilebilir güçlü bilgi odaklanmak tetiklemek için değil, sadece teknik konuları lazer kaynaklı lezyonlar oluşturma üzerinden okuyucu rehberlik etmek içinde vivo takip süresi görüntüleme.
Lazer kaynaklı fare KNV modeli bir diyot lazer yönetim tarafından oluşturuldu. Multimodal vivo içinde görüntüleme teknikleri KNV indüksiyon, ilerleme ve gerileme izlemek için kullanılmıştır. İlk, spektral etki alanı optik Koherens tomografi (SD-Ekim) gerçekleştirilen hemen Bruch'ın membran bir mola doğrulamak için lasering sonra. Floresein anjiyografi (FA) kullanarak sonraki vivo içinde görüntüleme Bruch'ın membran choroidal düzeyinde alınan seri görüntüleri üzerinden başarılı hasarı doğruladı. KNV yayılması ve regresyon lasering sonra gün 5, 10 ve 14 boyuna takip SD-Ekim ve SK kullanılarak gerçekleştirildi. Basit ve güvenilir sızan KNV leasions SK görüntülerden, sınıflandırma sunulur. Otomatik bölümleme için KNV sitelerdeki retina kalınlığı ölçümü için el ile kalibre uygulama ile birlikte toplam retina kalınlık ölçüm yapılmasına izin ödem varlığı tarafsız değerlendirilmesi. Son olarak, histolojik KNV doğrulanması isolectin GS-IB4 choroidal flatmounts boyama kullanılarak gerçekleştirilir. Boyama thresholded ve isolectin-pozitif alan ImageJ ile hesaplanır.
Bu iletişim kuralı gibi hızlı, multimodal sağlar yüksek üretilen iş gibi tarama KNV patoloji gerektiren tedavi çalışmaları ve güvenilir KNV patoloji ve retina ödem sınıflandırılması özellikle yararlıdır. Buna ek olarak, yüksek çözünürlük SD-Ekim subretinal veya intraretinal sıvı birikimi gibi diğer patolojik işaretlerinden kayıt sağlayan. Ancak, bu yöntem bir olasılık el ile gerçekleştirilecek olan KNV cilt analizi SD-Ekim görüntüleri, otomatikleştirmek için sağlamaz.

Kabarcık veya subretinal hemen lasering sonra kanama her zaman görünür değil. Bu nedenle, SD-Ekim Bruch'ın membran hasar doğrulamak özellikle önemlidir. Şekil 1 lazer yönetim sonra farklı zaman noktalarda OCT görüntüleme bir örneği gösterilir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> Resim 1 : OCT tr y...

Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Vivo Multimodal görüntüleme ve fare lazer kaynaklı Choroidal neovaskülarizasyon Model analizi

Burada, boyuna vivo içinde görüntüleme kullanışlılığı lazer kaynaklı choroidal neovaskülarizasyon farelerde morfolojik değişiklikler takip içinde mevcut.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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Research

JoVE Journal

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

9.1K Görüntüleme.  Experimentica Ltd.. Burada, boyuna vivo içinde görüntüleme kullanışlılığı lazer kaynaklı choroidal neovaskülarizasyon farelerde morfolojik değişiklikler takip içinde mevcut.

Video: In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

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

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Murin Koroidinde Melanomun İmplantasyonu ve Optik Koherens Tomografi ile Değerlendirilmesi

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

Kanser Araştırmaları

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.

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 Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Fare Kornea ve Konjonktiva Canlı Görüntüleme için Özel Multifoton Mikroskop platformu

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Biyomühendislik

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Vivo Tayf alanlı optik Koherens tomografi ve lazer Ophthalmoscopy tarama Cx3cr1gfp/gfp muhabir farelerin görüntüleme

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

Mononükleer Fagositlerin Çok Parametreli Akış Sitometrik Analizi için Tüm Fare Gözlerinin Sindirimi

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

İmmünoloji ve Enfeksiyon

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupiller İki Foton Fare Retinasının In vivo Görüntülenmesi

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Nörobilim

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

İn vivo Tekrarlanabilirliği Artırmak için Fare Retinasında Vasküler Yaralanma Okumaları

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Bir Murin Arka Bacak Modelinde Arteriyogenez Sırasında Lökosit Alımını İzlemek için Multifoton İntravital Görüntüleme

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

<meta charset="utf8"></head><body>Kemirgen Retinalarındaki Yapısal Değişiklikleri Değerlendirmek için Görüntü Kılavuzluğunda Optik Koherens Tomografi

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Kemirgen Retinalarındaki Yapısal Değişiklikleri Değerlendirmek için Görüntü Kılavuzluğunda Optik Koherens Tomografi

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...