Este trabalho foi apoiado por concessões do und Ernst Berta Grimmke Stiftung e da Deutsche Ophthalmologische Gesellschaft (cão).

Os procedimentos experimentais deste estudo foram aprovados pelo Animal conta Comissão de Renânia-Palatinado, Alemanha. Cuidados com animais em conformidade com as orientações institucionais e a associação para pesquisa em visão e oftalmologia (ARVO) instrução para o uso de animais em oftalmologia e visão pesquisa. Animais foram tratados de acordo com a directiva da União Europeia 2010/63/UE para experiências em animais. Masculinos camundongos C57BL/6J (The Jackson Laboratory, Porto da barra, ME, EUA), com 3-4 meses de idade foram utilizados para os experimentos. Os animais estavam alojados em condições normais (temperatura de 23 ± 2 ° C, umidade gama 55 ± 10% e 12 h ciclos de claro/escuro) e tinham acesso ao mouse padrão chow e água ad libitum.
1. isolamento da Retina do rato
<ol><li>Coloque os instrumentos de dissecção na mesa de preparação e verificar se todos os instrumentos são completos para permitir uma rápida preparação.</li>
	<li>Sacrifica o mouse por inalação de CO2 .</li>
	<li>Decapitar o mouse com uma tesoura de aço, corte sagitally o crânio em duas metades, usando o mesmo par de tesouras e retire a pele e o cérebro usando tesouras de olho (Figura 1).</li>
	<li>Corte o osso do crânio para deixar somente o osso orbital usando tesouras de olho.</li>
	<li>Transferi a órbita em um prato de dissecação com gelada tampão Krebs-Henseleit.</li>
	<li>Mantenha o prato de dissecação no gelo ou altere o buffer de cada 10 min.</li>
	<li>Corte o osso orbital usando tesouras de olho e remova cuidadosamente o globo de olho, juntamente com o tecido orbital usando pinças de precisão 4 tipo e estudante Vannas para primavera (Figura 2).</li>
	<li>Remova cuidadosamente a glândula Harderian, tecido conjuntivo e os músculos extraoculares usando pinças de precisão 5 tipo e Vannas para Capsulotomia, tomando cuidado para não danificar o principal ramo da artéria oftálmica.</li>
	<li>Ligate orbitais ramos da artéria oftálmica e, eventualmente, a extremidade proximal do principal ramo da artéria oftálmica usando suturas de Nylon 10-0 (Figura 3).</li>
	<li>Transferi o globo de olho em uma placa de Petri contendo etanol a 70% para 10 s usando tipo 4 pinças de precisão. Coloque o prato de dissecação do globo do olho e substituir o buffer com solução de Krebs fresca. A córnea aparecerá esbranquiçada após exposição ao etanol.</li>
	<li>Perfurar a córnea periférica com uma agulha de 30 calibre e dissecar a córnea usando Vannas para Capsulotomia. Cortar o tecido da íris suavemente, coloque uma ponta afiada das Vannas para Capsulotomia entre a coroide e retina e cortou a coroide e a esclera. Deixe algum tecido scleral em torno do nervo óptico (Figura 4).</li>
</ol>2. montagem da Retina na câmara de perfusão
Nota: A câmara de perfusão utilizada para experimentos de arteríola retinal é feito em casa. Consiste em um reservatório transparente com um tubo dentro e vazão (Figura 5). Uma extremidade de um tubo de silicone está colada ao fundo da câmara com adesivo de histoacryl e a outra extremidade anexado a uma torneira de três vias. Conecte uma seringa com solução tampão de Krebs fresco para a torneira de três vias e preencher o tubo com buffer.
<ol><li>Leve uma micropipeta de vidro com um porta-agulha e empurre-a para a extremidade do tubo na parte inferior da câmara de perfusão. Então quebre a ponta da micropipeta com tesouras tipo 4 para obter uma gorjeta de 100 µm de diâmetro.</li>
	<li>Concentre-se na ponta da micropipeta a e eliminá-la através da seringa conectada ao tubo. Cuide-se que o capilar não é obstruído por detritos. Em caso de oclusão, removê-lo, lave o tubo e inserir mais uma micropipeta.</li>
	<li>Encha a câmara de perfusão com frio tampão Krebs e colocar a câmara sob um microscópio estéreo.</li>
	<li>Coloque uma plataforma de plástico transparente para a câmara de perfusão. Esta plataforma tem um recuo de 1,8 mm de largura para o nervo óptico e artéria oftálmica e é colocada em dois fios de aço de diâmetro de 1,6 mm. Em seguida, coloque um anel de aço inoxidável de diâmetro 2,8 mm e 4,0 mm diâmetro externo para a plataforma.</li>
	<li>Transferi a retina com o nervo óptico e artéria oftálmica anexada da câmara de dissecação na câmara de perfusão com extremo cuidado, usando uma colher. Este passo é importante para evitar a potencial alongamento dos tecidos, como pode ser prejudicial para o experimento.</li>
	<li>Cortar a parte suturada da extremidade proximal da artéria oftálmica, coloque duas alças pré-formadas de sutura de nylon 10-0 para a micropipeta e Canule a artéria oftálmica com a micropipeta. Amarrar a artéria para a micropipeta (Figura 6) e coloque a retina para a plataforma transparente usando pinças-multa-ponto.</li>
	<li>Delicadamente te abrir a capsula de lente anterior com duas pinças de ponta fina, retire a lente e cortar a capsula de lente inteira usando micro-tesouras.</li>
	<li>Posteriormente, fazer quatro incisões radiais na retina metade a distância entre a pars plana para o nervo óptico e coloque um anel de aço inoxidável na retina para corrigi-lo para o fundo. A retina está agora pronta para o experimento (Figura 7).</li>
</ol>3. preparação de arteríolas da retina para o experimento
<ol><li>Coloque a câmara de perfusão sob um microscópio de luz. O objetivo é uma lente de objetiva de imersão de água X 100.</li>
	<li>Conectar as duas tubulações para em - e out - flow para uma bomba pericíclica e circulam na câmara com tampão Krebs oxigenado com 95% O2 e carbonatadas com 5% CO2 a 37 ° C. Use uma taxa de fluxo entre 100 e 120 mL/min.</li>
	<li>Conecte o tubo de silicone, que é conectado com a micropipeta, um reservatório do silicone mangueira através de uma torneira de três vias. Em seguida, encher o reservatório com tampão Krebs para uma altura correspondente a 50 mm Hg, mas não abra a torneira de três vias ainda.</li>
	<li>Olhe através dos objectivos do microscópio e foco na superfície da retina. Busca de vasos sanguíneos ou células vermelhas do sangue. É possível que os vasos sanguíneos são completamente recolhidos, às vezes apenas que algumas células vermelhas do sangue são visíveis.</li>
	<li>Quando um vaso sanguíneo encontra-se, concentre-se nele e abra a torneira de três vias. O diâmetro do vaso deve aumentar imediatamente e os glóbulos vermelhos também mover centrifugamente, se for uma arteríola, ou centripetamente em caso de uma Vénula. As arteríolas são menores em diâmetro, em comparação com vênulas da mesma ordem.</li>
	<li>Uma vez que encontra-se um vaso sanguíneo com uma parede bem visível, pode ser empregado para experiências após um período de equilíbrio de 30 min (Figura 8). Durante o período de equilíbrio, as arteríolas desenvolvem apenas fraco tom intrínseco, resultando em menos de 10% de redução de diâmetro luminal.</li>
</ol>4. realizando o experimento
<ol><li>Teste a viabilidade dos navios com o tromboxano mimética 9,11-dideoxy-9, 11α-methanoepoxy prostaglandina F2α (U46619), adicionando-o para a solução de banho. Este agente contrai as arteríolas retina do rato por cerca de 50% do diâmetro de repouso em concentrações ≥ 10-6 M.</li>
	<li>Uma vez que o navio está confirmado viável, lave o agonista fora da banheira pela bomba de circulação e fornecer fresco tampão Krebs para o banho.</li>
</ol>

<ol>
	<li>Toda, N., Nakanishi-Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+ocular+blood+flow,+glaucoma,+and+diabetic+retinopathy.">Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy.</a> Prog Retin Eye Res. 26 (3), 205-238 (2007).</li><li>Schuster, A. K., Fischer, J. E., Vossmerbaeumer, C., Vossmerbaeumer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography-based+retinal+vessel+analysis+for+the+evaluation+of+hypertensive+vasculopathy.">Optical coherence tomography-based retinal vessel analysis for the evaluation of hypertensive vasculopathy.</a> Acta Ophthalmol. 93 (2), e148-e153 (2015).</li><li>Cherecheanu, A. P., Garhofer, G., Schmidl, D., Werkmeister, R., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+perfusion+pressure+and+ocular+blood+flow+in+glaucoma.">Ocular perfusion pressure and ocular blood flow in glaucoma.</a> Curr Opin Pharmacol. 13 (1), 36-42 (2013).</li><li>Faraci, F. M., Sigmund, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+biology+in+genetically+altered+mice+:+smaller+vessels,+bigger+insight.">Vascular biology in genetically altered mice : smaller vessels, bigger insight.</a> Circ Res. 85 (12), 1214-1225 (1999).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Retinal+Blood+Flow+Using+Fluorescently+Labeled+Red+Blood+Cells.">Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells.</a> eNeuro. 2 (2), (2015).</li><li>Guevara-Torres, A., Joseph, A., Schallek, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label+free+measurement+of+retinal+blood+cell+flux,+velocity,+hematocrit+and+capillary+width+in+the+living+mouse+eye.">Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.</a> Biomed Opt Express. 7 (10), 4229-4249 (2016).</li><li>Schallek, J., Geng, Y., Nguyen, H., Williams, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+topography+of+retinal+pericytes+in+the+living+mouse+retina+using+in+vivo+adaptive+optics+imaging+and+ex+vivo+characterization.">Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.</a> Invest Ophthalmol Vis Sci. 54 (13), 8237-8250 (2013).</li><li>Gericke, 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=Identification+of+the+muscarinic+acetylcholine+receptor+subtype+mediating+cholinergic+vasodilation+in+murine+retinal+arterioles.">Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles.</a> Invest Ophthalmol Vis Sci. 52 (10), 7479-7484 (2011).</li><li>Gericke, 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=Functional+role+of+alpha1-adrenoceptor+subtypes+in+murine+ophthalmic+arteries.">Functional role of alpha1-adrenoceptor subtypes in murine ophthalmic arteries.</a> Invest Ophthalmol Vis Sci. 52 (7), 4795-4799 (2011).</li><li>Bohmer, 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=The+alpha(1)B+-adrenoceptor+subtype+mediates+adrenergic+vasoconstriction+in+mouse+retinal+arterioles+with+damaged+endothelium.">The alpha(1)B -adrenoceptor subtype mediates adrenergic vasoconstriction in mouse retinal arterioles with damaged endothelium.</a> Br J Pharmacol. 171 (16), 3858-3867 (2014).</li></ol>

Os autores têm sem interesses financeiros concorrentes.

A medição das respostas vasculares na retina do rato é um desafio devido ao pequeno tamanho de vasos da retina. Com a técnica apresentada, arteríolas retina são visualizadas por microscopia de luz transmitida. Isso é possível, porque a retina isolada é translúcida. A vantagem da técnica é a alta resolução óptica. A resolução espacial calculada é 11 px/µm. No entanto, a resolução real para este sistema óptico que utiliza a luz branca é entre 200 e 300 nm, que é explicado pelo limite de difração d...

Distúrbios na perfusão da retina têm sido implicados na patogênese de várias doenças oculares, como retinopatia diabética, retinopatia hipertensiva e glaucoma1,2,3. Assim, estudos que visam medir a reatividade vascular na retina são importantes para compreender a fisiopatologia dessas doenças, e desenvolver novo tratamento se aproxima.
Devido à possibilidade de manipulação genética no genoma murino, o mouse tornou-se um modelo animal utilizado para estudos do sistema cardiovascular4. No entanto, por causa do pequeno tamanho dos vasos sanguíneos da retina (≤ 30 µm), medição de reatividade vascular na retina do rato é um desafio. Por exemplo, stereomicroscopic técnicas para a medição na vivo são limitadas em sua resolução óptica e, portanto, só permitem exatamente detectar alterações no fluxo de diâmetro ou sangue no sangue pequeno de menos de 30 µm de diâmetro ≤ quando equipado com dispositivos adicionais de sofisticados, tais como um microscópio confocal usando corantes fluorescentes ou a óptica adaptativa digitalização luz oftalmoscópio5,6. Além disso, a interpretação das medições na vivo vista identificar locais sinalização mecanismos nos vasos da retina podem ser confundidos por anestésicos, altera na pressão arterial sistêmica e a influência dos vasos sanguíneos de retrobulbar.
Portanto, nós desenvolvemos um método para medir respostas de rato dos vasos sanguíneos da retina com alta resolução óptica ex vivo. A técnica apresentada neste documento permite visualização das arteríolas retina através de transmitidas microscopia de luz. Esse método, que também pode ser usado em ratos, fornece acesso às vantagens do gene segmentação tecnologia na investigação vascular ocular.

<table><tbody><tr><td>Steel Scissors</td><td>Carl Roth GmbH</td><td>3576.1</td><td>1x 140 mm</td></tr><tr><td>Eye Scissors</td><td>Geuder</td><td>G-19390</td><td>1x straight, 10.5 cm</td></tr><tr><td>Precision tweezers, straight with fine tips</td><td>Carl Roth GmbH</td><td>LH68.1</td><td>2x type 4</td></tr><tr><td>Precision tweezers, straight with extra fine tips</td><td>Carl Roth GmbH</td><td>LH53.1</td><td>2x type 5</td></tr><tr><td>Vannas capsulotomy scissors</td><td>Geuder</td><td>19760</td><td>1x straight, 77 mm</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91501-09</td><td>1x curved,</td></tr><tr><td>&lt;em&gt;Barraquer&lt;/em&gt; Needle Holder</td><td>Geuder</td><td>G-17500</td><td>1x curved, 120 mm</td></tr><tr><td>Needle</td><td>Becton, Dickinson and Company</td><td>305128</td><td>1x 30 G</td></tr><tr><td>Glass Capillaries (for producing micropipettes)</td><td>Drummond Scientific Company</td><td>9-000-1211</td><td>1x (1.2 x 0.8 mm; outer/inner diameter)</td></tr><tr><td>Nylon Suture</td><td>Alcon</td><td>198001</td><td>1x 10-0</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>153066</td><td>1x 35 mm diameter</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>172931</td><td>1x 100 mm diameter</td></tr><tr><td>Discofix C</td><td>Braun</td><td>16500C</td><td>10 cm</td></tr><tr><td>Histoacryl adhesive</td><td>B. Braun Surgical, S.A.</td><td>1050052</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pericyclic pump&amp;nbsp; (CYCLO II)</td><td>Carl Roth GmbH</td><td>EP76.1</td><td>1x</td></tr><tr><td>Vertical Pipette Puller Model 700C</td><td>David Kopf Instruments</td><td></td><td>1x</td></tr><tr><td>Microscope (Vanox-T AH-2)</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Water immersion objective LUMPlanFL, 1.0 NA</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Digital camera (TK-C1381)</td><td>JVC</td><td></td><td>1x</td></tr><tr><td>Perfusion chamber</td><td>self-made</td><td></td><td>1x</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Drugs and Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth GmbH</td><td>K928.4</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>5239.1</td><td></td></tr><tr><td>Kalium chloride (KCl)</td><td>Carl Roth GmbH</td><td>6781.1</td><td></td></tr><tr><td>Kalium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>3904.2</td><td></td></tr><tr><td>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>261.2</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Carl Roth GmbH</td><td>9265.2</td><td></td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>0965.3</td><td></td></tr><tr><td>&amp;alpha;-(D)-(+)- Glucose monohydrate</td><td>Carl Roth GmbH</td><td>6780.1</td><td></td></tr><tr><td>9,11-dideoxy-9&amp;alpha;,11&amp;alpha;-methanoepoxy prostaglandin F2&amp;alpha; (U-46619)</td><td>Cayman Chemical</td><td>16450</td><td></td></tr><tr><td>Acetylcholine chloride</td><td>Sigma-Aldrich</td><td>A6625-25G</td><td></td></tr></tbody></table>

preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo

Insuficiência vascular e alterações na perfusão retiniana normal estão entre os fatores principais para a patogênese de várias doenças oculares visão em risco, tais como retinopatia diabética, retinopatia hipertensiva e, possivelmente, glaucoma. Portanto, preparações microvasculares da retina são ferramentas cruciais para estudos fisiológicos e farmacológicos delinear os mecanismos fisiopatológicos subjacentes e a concepção terapias para as doenças. Apesar da ampla utilização de modelos de rato em pesquisa oftálmica, estudos sobre a reatividade vascular da retina são escassos nesta espécie. Das principais razões para esta discrepância é os desafiante procedimentos de isolamento devido ao tamanho pequeno destes vasos sanguíneos da retina, que é ~ ≤ 30 µm de diâmetro luminal. Para contornar o problema de isolamento direto destes microvessels da retina para estudos funcionais, estabelecemos uma técnica de isolamento e preparação que permite ex vivo estudos de vasoactivity da retina do rato sob condições fisiológicas perto . Embora as preparações experimentais presentes irão se referir especificamente as arteríolas retina do rato, esta metodologia prontamente pode ser empregada para microvessels de ratos.

U-46619 produziu respostas vasoconstritor dependente da concentração em arteríolas da retina de ratos do selvagem-tipo do fundo C57Bl/6J. Em uma concentração de 10-6 M, diminuição do diâmetro luminal foi ≈50% do diâmetro de descanso. Figura 9A mostra uma curva concentração-resposta representativa de uma arteríola retinal. Em arteríolas preconstricted com U46619, cumulativa administração de acetilcolina evocados dependente da concen...

Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Muitas doenças oculares visão em risco estão associadas com microvessels da retina disfuncional. Portanto, a medição das respostas arteríola retinal é importante para investigar os mecanismos fisiopatológicos subjacentes. Este artigo descreve um protocolo detalhado para isolamento de rato arteríola retinal e preparação para avaliar os efeitos de substâncias vasoativas no diâmetro vascular.

Etapas de preparação para medição de reatividade no Mouse arteríolas retina Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

preparation-steps-for-measurement-reactivity-mouse-retinal-arterioles

Research

JoVE Journal

Medicine

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

7.1K visualizações.  University Medical Center of the Johannes Gutenberg University Mainz. Muitas doenças oculares visão em risco estão associadas com microvessels da retina disfuncional. Portanto, a medição das respostas arteríola retinal é importante para investigar os mecanismos fisiopatológicos subjacentes. Este artigo descreve um protocolo detalhado para isolamento de rato arteríola retinal e preparação para avaliar os efeitos de substâncias vasoativas no diâmetro vascular.

Vídeo: Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Usando vírus adeno-associado como uma ferramenta para o estudo da retina Barreiras na Doença

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Medicina

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Um Modelo aguda da retina para a Avaliação sanguíneos da retina barreira Descumprimento e potenciais fármacos para o tratamento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Determinação da respiração mitocondrial e glicolise em amostras de tecido retinal ex vivo

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Neurociência

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

Modelo de Retinopatia Induzida por Oxigênio para Doenças Isquêmicas da Retina em Roedores

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Quantificação de Parâmetros Vasculares em Retinas inteiras de Camundongos com Retinopatias Não Proliferativas e Proliferativas

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

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 ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

Medindo o diâmetro do vaso retiniano a partir de imagens de angiografia fluorescente de camundongo

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Isolamento de arteríolas retina para estudos de fisiologia celular Ex Vivo

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Biologia

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Medição do metabolismo energético, no tecido da retina explantado, usando análise de fluxo extracelular

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Monitoramento do crescimento dinâmico dos vasos retinais no modelo de rato de retinopatia induzido pelo oxigênio

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...