Name: in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy
Uploaded: 2017-11-11T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

Este trabalho foi financiado por um subsídio da Swiss National Science Foundation (SNSF; #320030_156019). Os autores receberam apoio financeiros Heidelberg Engineering GmBH, Alemanha.

em todos os procedimentos, BALB/c adulto masculinos e femininos ratos que expressam gfp sob o promotor da Cx3cr1 foram usados 24. Os ratos foram tratados de acordo com a instrução de ARVO para o uso de animais em oftalmologia e visão pesquisa e todos os procedimentos foram aprovados do governo suíço, de acordo com os regulamentos federais suíços em matéria de bem-estar Animal. Os ratos foram anestesiados por uma injeção subcutânea de cloridrato de medetomidina (0,75 mg/kg) e ...

<ol>
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O presente artigo demonstra um protocolo para a aquisição de B-retina e a imagem latente de gfp microglia positiva distribuição na retina do rato na mesma sessão de imagens. SD-OCT e SLO são cada vez mais usados em modelos animais de doença da retina para fornecer informações de alterações da retina ao longo do tempo,10,14,17,18,21. Com este protoco...

Na oftalmologia experimental, exame de patologia retiniana geralmente é avaliada usando técnicas histológicas. No entanto, histologia requer a eutanásia de animais e pode causar alteração para as propriedades reais do tecido. SD-OCT e SLO são rotineiramente usados em oftalmologia clínica para fins de diagnóstico e de acompanhamento de diversas doenças da retina como edema macular diabético9, neuropatia óptica isquêmica anterior10ou retinite pigmentosa11 . SD-OCT e SLO são técnicas não-invasivas que geram imagens de alta resolução da retina, que são visualizadas através da pupila dilatada sem mais intervenção. SD-OCT fornece informações da estrutura da retina e da espessura da retina através da recolha de dados Retrodispersão para criar imagens transversais da retina, enquanto SLO coleta dados de fluorescência para produzir imagens estereoscópicas de alto contraste da retina. Hoje em dia, ambas as técnicas são cada vez mais utilizadas em oftalmologia experimental usando pequenos roedores12,13,14,15 (ou mesmo zebrafish16,17) e pode fornece tanto informações qualitativas e quantitativas12,17,18,19,20,21.
Acumulação de fluorophores endógena como lipofuscins ou a formação de drusen na retina pode ser visualizada por SLO como sinal fluorescente auto. Esta característica torna SLO uma técnica valiosa para diagnóstico e monitoramento de doenças da retina como a degeneração macular relacionada à idade ou retinite pigmentosa22,23. Na oftalmologia experimental, fluorescência de auto imagem (AF) pode ser usada para a detecção de tipos de células específicas em linhas de rato de repórter. Por exemplo, ratos heterozigotos para a expressão de gfp sob o promotor de Cx3cr124 são vantajosos para visualização no vivo das pilhas microglial na retina normal e para a investigação de micróglia/macrófago dinâmica na doença da retina,21. Microglia são os macrófagos residentes da retina, que desempenham um papel fundamental na homeostase do tecido e reparação dos tecidos após lesão1,25,26. Ativação da microglia na retina tem sido relatada em degeneração, sugerindo um papel dessas células na doença da retina,2,3,4,5, , isquemia e lesão da retina 6.
O objectivo do presente protocolo é descrever um método relativamente simples para a imagem da retina e medição de espessura da retina usando SD-OCT e para visualização de células de gfp microglia positivo no rato retina utilizando Cx3cr1gfp/gfp SLO (sistema Heidelberg Spectralis HRA + OCT). Este protocolo pode ser utilizado para medições de imagem e espessura da retina saudável ou doente em várias linhas de rato. Além disso, as análises morfométricas podem ser realizadas para a identificação e quantificação de números microglia e ativação da microglia na retina usando SLO21. As células da micróglia são associadas com doenças degenerativas no sistema nervoso central (CNS), incluindo a retina27,28,29. Assim, combinando os dois métodos utilizados no presente protocolo, pode ser feita correlação de distribuição microglia e degeneração da retina, que pode facilitar o monitoramento severidade da doença ou a eficácia da terapêutica se aproxima na vivo.

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

Tomografia de coerência óptica de domínio espectral (SD-OCT) e digitalização Oftalmoscopia do laser (SLO) são usados extensivamente em oftalmologia experimental. No presente protocolo, camundongos expressando verde proteína fluorescente (gfp) sob o promotor da Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) foram usados para imagem microglia células na vivo na retina. Microglia são macrófagos residentes da retina e têm sido implicados em várias doenças da retina1,2,3,4,5,6. Este protocolo fornece uma abordagem detalhada para geração de B-retina, com SD-OCT e imagem latente de distribuição de célula microglia em camundongos Cx3cr1gfp/gfp com SLO na vivo, usando um sistema de plataforma de imagem oftálmica. O protocolo pode ser usado em várias linhas de rato de repórter. No entanto, existem algumas limitações do protocolo aqui apresentado. Em primeiro lugar, ambos SLO e SD-OCT, quando usado no modo de alta resolução, coletar dados com alta resolução axial, mas a resolução lateral são mais baixos (3,5 µm e 6 µm, respectivamente). Além disso, o nível de saturação e foco em SLO é altamente dependente da seleção do parâmetro e o alinhamento correcto do olho. Além disso, usar dispositivos projetados para pacientes humanos em ratos é desafiador, devido a maior potência óptica total do olho do rato comparado ao olho humano; Isso pode levar a lateral ampliação imprecisões7, que também são dependentes a ampliação pela lente do mouse entre outros. No entanto, apesar de que a tomografia axial posição é dependente da ampliação lateral, as medições de SD-OCT axiais são precisos8.

Usando o protocolo aqui apresentado, SD-OCT verifica e SLO imagens foram obtidas a partir de Cx3cr1gfp/gfp ratos na mesma sessão de imagens. Figura 3 inclui representante SD-OCT único exames obtidos com um 30 ° ou uma lente de 55 ° (Figura 3A) e imagens representativas de SLO obtidos com um 55 ° ou uma lente de 102 °, onde gfp positivas microglia células são visualizadas. Maior refletividade da coroid...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

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

Este protocolo descreve técnicas como de alta resolução de imagem como tomografia de coerência óptica de domínio espectral e digitalização laser Oftalmoscopia pode ser utilizado em pequenos roedores, usando um sistema de plataforma de imagem oftálmica, para obter informações sobre espessura da retina e microglial celular distribuição, respectivamente.

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 ...

Research

JoVE Journal

Medicine

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

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Análise de biofilme oral de Palatal Expansores por fluorescência de hibridização in situ e microscopia confocal a laser Scanning

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

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Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Implantação intracraniana com 3D subseqüentes In Vivo Bioluminescent imagem de Gliomas murino

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

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Doppler Tomografia de Coerência Óptica de circulação retiniana

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Segmentação e medição de volumes de gordura em modelos de obesidade em roedores usando raios-X Tomografia Computadorizada

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

Geração e quantificação 3-Dimensional de Arterial lesões em ratos por meio de tomografia óptica de Projeção

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

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In Vivo Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Imagem não invasivo e Análise de Isquemia Cerebral em Ratos de Vida Usando Positron Emission Tomography com<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

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Combined In vivo Optical and Ãƒâ€šÃ‚ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Avaliação da viabilidade de gordura humana injecção em ratinhos nu com Micro-Tomografia Computadorizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Validação de nanocorpo e Antibody Based<em&gt; In Vivo</em&gt; Experimentos Tumor Xenoenxerto NIRF-imagem em ratos usando<em&gt; Ex Vivo</em&gt; Citometria de fluxo e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...