Name: in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model
Uploaded: 2018-01-21T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

Os autores gostaria de agradecer a Yuliya Naumchuk (Loyola University Chicago) e Žiniauskaitė Agne (Experimentica Ltd.), pelo excelente apoio técnico e videográfico. Programa de pesquisa do Dr. Kaja é suportado pelo Dr. John P. e Therese E. Mulcahy dotado Professorship em oftalmologia na Loyola University Chicago.

Todos os animais foram tratados em conformidade com a instrução de ARVO para o uso de animais em oftalmologia e Vision Research e a CE Directiva 86/609/CEE para experiências em animais, usando protocolos aprovados e monitorados pelo Conselho da Finlândia experimento Animal.
1. induzida por laser rato CNV modelo 5
<ol><li>Inspecione os olhos do animal macroscopicamente para qualquer anormalidade.</li>
	<li>Pese o mouse.</li>
	<li>Calcular e preparar uma quantidade apropriada de anestésicos para utilizar, com base no peso do animal, por exemplo, uma mistura de medetomidina (1 mg/kg), ketamina (75 mg/kg) e água destilada (solução de NaCl 0,9%) na proporção de 1:1.5:2.5, ou cetamina (40-75 mg/kg), xilazina (5 mg/kg) e água destilada (solução de NaCl 0,9%) na proporção de 1:2. 5:1; para um mouse de 20g, injete 0,1 mL da mistura.</li>
	<li>Injete o anestésico intraperitonealmente.</li>
	<li>Posicione o mouse volta para a gaiola e espere até o animal é anestesiado. Confirme que o mouse é devidamente anestesiado pela falta de um pedal reflexo.</li>
	<li>Garantir o uso de equipamento de proteção pessoal da segurança do laser.</li>
	<li>Ligue a lâmpada de fenda e um laser de díodo 532 nm.</li>
	<li>Remova o rato da gaiola e coloque sobre a almofada de aquecimento.</li>
	<li>Aplique uma gota de tropicamida para dilatação da pupila. Espere 3-5 min para dilatação pupilar completo (3 mm).</li>
	<li>Posicione o mouse no palco da lâmpada de fenda.</li>
	<li>Coloque uma gota de gel líquido thoughtful sobre uma lamela de applanate da córnea.</li>
	<li>Oriente o olho do rato com a cabeça do nervo óptico no centro.</li>
	<li>Definir a potência do laser a 100 mW, a duração de 100 ms e o tamanho de ponto para 50 µm.</li>
	<li>Focar o feixe de laser o epithelium retinal do pigment (RPE).</li>
	<li>Fazer três tiros de laser em um olho, evitando os vasos da retina idealmente na 4, 8 e 12:00 posições em torno do nervo óptico, respectivamente. Inspecione o fundo do olho, depois todos tiros por ausência de hemorragia retiniana a laser. O olho contralateral serve como um controle de não-com laser.</li>
	<li>Descarte o mouse lamela e lugar volta sobre a almofada de aquecimento.</li>
	<li>Aplique uma gota de gotas de gel de PEG em ambos os olhos.</li>
</ol>2. SD-OCT 6,7
<ol><li>Coloque o mouse para a fase de alinhamento de roedores e imobilizar a cabeça.</li>
	<li>Alinhe a lente do sistema SD-OCT (por exemplo, Bioptigen/Leica Envisu R2200) para enfrentar o olho para a imagem latente com na vivo usando controladores de X - e Y-estágio.</li>
	<li>Realizar exames de SD-OCT para verificar se a quebra da membrana de Bruch: uma vez que o SD-OCT examina o olho inteiro, Mova manualmente a linha de referência nos sites lasered. Quebras de membrana de Bruch devem ser claramente visíveis em áreas lasered (ver Figura 1).</li>
</ol>3. fluoresceína angiografia 7,8,9
<ol><li>Remover o rato com o titular e coloque-o sobre o sistema de FA (por exemplo, Heidelberg Spectralis HRA2).</li>
	<li>Foco para laser queimar áreas do fundo do olho usando o modo de reflectância de infravermelho com a cabeça do nervo óptico, no meio da janela de visualização.</li>
	<li>Injete 0,1 mL de sal de sódio 5% fluoresceína para um rato de 20 g por via subcutânea ou intraperitonealmente.</li>
	<li>Concentre-se no nível da coroide.</li>
	<li>Leve uma imagem do nível de foco da coroide.</li>
	<li>Re-focar a nível da retina e uma imagem.</li>
	<li>Espere por 30 s e repita etapas 3.4-3.6.</li>
	<li>Remova o mouse do suporte e coloque-o sobre a almofada de aquecimento.</li>
	<li>Reverta anestesia por α2-antagonista de medetomidina, atipamezole (0,5 mg/kg, i.p.) ou espera para animal recuperação da anestesia.</li>
	<li>Repita em vivo SD-OCT e FA imagiologia em animais anestesiados sobre os acompanhamento dias 5, 10 e 14.</li>
</ol>4. CNV classificação
<ol><li>Classe os danos da membrana de Bruch da OCT imagens e imagens de FA da coroide tomadas imediatamente após o lasering no dia 0 como seguindo: 0 - membrana de Bruch não foi danificado 1 - sucesso dano da membrana de Bruch</li>
	<li>Classe a presença de CNV de lasered manchas que tinha vazamento, como observado, comparando a dinâmica do sinal de fluoresceína em uma série de imagens da retina de FA como seguindo: 0 - aparência normal da retina 0,5 - fraco coloração de escapamento 1.0 - áreas CNV gotejantes Nota: Use a imagem da OCT para confirmação adicional de CNV ou em FA questionável onde a presença de fluido intraretinal em imagens OCT sugeriria CNV classificação.</li>
</ol>5. medições de espessura retina
<ol><li>Use um software automatizado de segmentação para medições de espessura da retina. Certifique-se de que a espessura total da retina é considerada, como a espessura de todas as camadas da camada de fibras nervosas RPE (sites de medição saudável), ou para uma linha imaginária ligando RPE em torno do local do dano (lasered sites) (ver também Figura 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>

O autor Symantas Ragauskas, pH.d. é um funcionário (cientista) e acionista do Ltd. de Experimentica que oferece contrato de serviços de pesquisa empregando o modelo CNV pré-clínicos usado neste artigo.
O autor Eva Kielczewski é um empregado (engenheiro de aplicações de pesquisa, OCT) da Leica Microsystems que produz sistemas de SD-OCT usados neste artigo.
O autor Joseph Vance é um empregado (at OCT Sales Director) da Leica Microsystems que produz sistemas de SD-OCT usados neste artigo. Joseph Vance também é presidente e diretor executivo da leção, LLC.
O autor Simon Kaja, pH.d. é consultor Chief Scientific Officer e acionista da Experimentica Ltd., uma contrato pré-clínicos pesquisa organização que oferece contrato de serviços de pesquisa, incl. modelo de CNV pré-clínicos usado neste artigo. Simon Kaja, pH.d. é também CEO da K & P científica, LLC, das Ciências da vida, consultoria e atua como o Dr. John P. e Therese E. Mulcahy dotado Professor em oftalmologia na Loyola University Chicago Stritch School of Medicine. Os termos desse acordo foram revistos e aprovados pela Loyola University Chicago, em conformidade com sua política de conflito de interesses.
O autor Giedrius Kalesnykas, pH.d. é um funcionário (CEO) e acionista do Ltd. de Experimentica que oferece contrato de serviços de pesquisa empregando o modelo CNV pré-clínicos usado neste artigo.

Imagiologia multimodal oferece valiosas ferramentas para avaliação de patologia CNV. Aqui apresentamos um protocolo de imagem consistindo de FA, SD-OCT e segmentação automática para a avaliação rápida, reprodutível e confiável de patologia CNV. Uma ruptura da membrana de Bruch após administração do laser foi confirmada. Além disso, o uso de SD-OCT nesta fase também permitiu a visualização imediata dos possíveis hemorragias intraretinal e subretinal, que pode confundir a interpretação dos resultados. V...

A primeira tentativa bem sucedida para imitar a patologia humana CNV em roedores foi demonstrada há quase três décadas com um laser de krypton em Long-Evans ratos1. Depois disso, um laser de krypton foi usado para invadir a cepa de rato mais popular, C57BL/6J2,3,4a membrana de Bruch. A taxa de sucesso da indução CNV foi verificada com FA e manchas histológicas. Um rápido desenvolvimento de modalidades de imagem não-invasivos, como OCT, estimulou o crescimento do campo de roedores modelos pré-clínicos. A capacidade de monitorar mudanças morfológicas na retina em múltiplos pontos de tempo no mesmo olho significativamente contribui para a redução do uso de animais e aumenta a eficiência em estudos experimentais. Avaliação histológica das lesões CNV é bastante simples e exige a rotulagem de crescimento vascular anormal ao redor do local de administração do laser, aquisição de imagem e estimativa de área/volume usando um software de análise de imagem. Em contraste, na vivo modalidades de imagem apresentar análises mais complexas de patologia CNV e sua interpretação.
Aqui nós apresentamos um método simples e relativamente rápido para indução de grau, progressão e regressão de CNV usando FA, SD-OCT, e o método de segmentação automatizada no mouse laser-induzida CNV modelo.

<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

Induzida por laser a neovascularização de coroide (CNV) é um modelo bem estabelecido para imitar a forma úmida da degeneração macular relacionada à idade (DMRI). Neste protocolo, nosso objetivo é orientar o leitor não simplesmente através das considerações técnicas de gerar lesões induzida por laser para desencadear processos neovascular, mas prefiro focar a informação poderosa que pode ser obtida multimodal longitudinal vivo em de imagem durante todo o período de seguimento.
O mouse laser-induzida modelo CNV foi gerado por uma administração de laser de diodo. Multimodal no vivo técnicas de imagem foram utilizadas para monitorar CNV indução, progressão e regressão. Primeiro, a tomografia de coerência óptica de domínio espectral (SD-OCT) foi realizada imediatamente após o lasering para verificar uma ruptura da membrana de Bruch. Subsequentes na vivo imagens usando angiografia fluoresceína (FA) confirmaram sucesso dano da membrana de Bruch da série imagens adquiridas a nível da coroide. Acompanhamento longitudinal da proliferação de CNV e regressão nos dias 5, 10 e 14 após o lasering foi realizado usando SD-OCT e FA. Simples e confiável de classificação de leasions de CNV com vazamento de imagens de FA é apresentado. Segmentação automatizada para medição da espessura total da retina, combinada com a aplicação manual calibre para medição da espessura da retina em sites CNV, permitir uma avaliação imparcial da presença de edema. Finalmente, verificação histológica da CNV é executada usando isolectin GS-IB4 mancha na flatmounts da coroide. A coloração é thresholded, e a área de isolectin-positivo é calculada com o ImageJ.
Este protocolo é especialmente útil em estudos de terapêutica que exigem alta taxa de transferência-como triagem de patologia CNV como rápido, permite multimodal e classificação confiável de edema de patologia e retinal CNV. Além disso, a alta resolução SD-OCT permite a gravação de outras características patológicas, tais como o acúmulo de fluido subretinal ou intraretinal. No entanto, este método não oferece a possibilidade de automatizar a análise de volume CNV de imagens SD-OCT, que tem de ser executada manualmente.

Uma bolha ou apresentam sangramento imediatamente após lasering nem sempre é visível. Portanto, o SD-OCT é particularmente importante para verificar os danos da membrana de Bruch. A Figura 1 mostra um exemplo de imagem OCT em pontos diferentes de tempo após a administração do laser.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> Figura...

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

Na Vivo Imagiologia multimodal e análise do modelo de neovascularização coroide de induzida por Laser de Mouse

Aqui, apresentamos a utilidade da imagem longitudinal na vivo na sequência de alterações morfológicas da neovascularização de coroide do laser-induzida em camundongos.

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

in-vivo-multimodal-imaging-analysis-mouse-laser-induced-choroidal

Research

JoVE Journal

Neuroscience

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

9.1K visualizações.  Experimentica Ltd.. Aqui, apresentamos a utilidade da imagem longitudinal na vivo na sequência de alterações morfológicas da neovascularização de coroide do laser-induzida em camundongos.

Vídeo: 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.

Um modelo de rato induzida por laser de crônica Ocular Hypertension para Caracterizar defeitos visuais

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

Medicina

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.

Implantação e Avaliação do Melanoma na Coroide Murina via Tomografia de Coerência Óptica

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

Pesquisa do câncer

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.

Um modelo de rato para induzida por laser coróide Neovascularização

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.

Uma plataforma personalizada de microscopia multifotn para imagens ao vivo de córnea de rato e conjuntiva

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

Bioengenharia

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.

Na Vivo Imagem de Cx3cr1gfp/gfp repórter ratos com a tomografia de coerência óptica espectral-domínio e digitalização Laser Oftalmoscopia

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.

Digestão de olhos inteiros de rato para análise citométrica de fluxo multi-parameter de fagocitos mononucleares

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

Imunologia e Infecção

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.

Imagem transpupilar de dois fótons in vivo da retina do rato

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

Neurociência

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Otimização do Modelo de Oclusão de Veia da Retina em Camundongo para Limitar a Variabilidade

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

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).

In vivo Leituras de lesões vasculares na retina de camundongos para promover a reprodutibilidade

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.

Imagem Intravital Multifóton para Monitoramento do Recrutamento de Leucócitos durante a Arteriogênese em Modelo de Membro Posterior Murino

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