Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
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Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

Todos os protocolos foram realizados em conformidade com as políticas do Comitê animal Institucional Cuidado e Uso quando aplicável. 1. Preparação <ol><li> Preparar os meios de estabilização com 75% de Dulbecco Modified Eagle Médium (DMEM), 25% de Hanks Solução Salina Equilibrada (SSHE). Misture bem, alíquota e armazenar a -20 ° C até à sua utilização. </li><li> Prepara-se o tampão fosfato salino (PBS) e esterilizar em autoclave. Guardar a solução à temperatura ambiente. Aquece-se a PBS para 37 ° C antes da experiência (5 ml por poço). </li><li> Preparar 10%, 20% e 30% de sacarose em PBS. Filtrar todas as soluções individualmente mais separados 0,22 filtros para esterilização. Salvar as soluções a 4 ° C. </li><li> Preparar a solução de estoque de histamina em PBS a uma concentração de 90 mM. Esterilizar a solução por filtração através de um filtro de 0,22 um. Alíquotas e guardar a solução a -80 ° C. Evitar ciclos múltiplos de congelação-e-descongelação. </li><li> Adicione fresco paraformaldeído a 4% (PFA) em PBS antes da experiência. Colocá-lo em gelo. Cuidado: Mantenha PFA em um exaustor e usar equipamento de protecção adequado. </li><li> Descongelar o meio de estabilização (20 ml por retina). Adicionar penicilina-estreptomicina a uma concentração final de 100 U / mL. parte quente do meio (15 ml por retina) a 37 ° C na incubadora antes da experiência. Deixar o resto do meio em gelo. </li><li> Coloque a HBSS (10 ml por retina) em gelo. </li></ol> 2. Retinal Cultura organotípicas <ol><li> Transferir as amostras de um capuz de cultura de tecidos. Abra a viseira cortando ao redor da lente. Usando um pincel, retire suavemente o humor vítreo, sem puxar a retina. Suavemente descolar a retina a partir do bordo de corte com uma escova para expor o disco óptico. Severa do nervo óptico com uma navalha e solte a retina. </li><li> Transferir a retina em uma placa de Petri e lavar cuidadosamente a retina uma vez usando fria HBSS. Colocar a amostra no HBSS na ice. Dissecar como muitos retinas, conforme necessário, repetindo Etapa 2.1 e este passo. </li><li> Cortar a retina ao meio simetricamente com uma navalha. Suavemente transferir amostras para uma placa de 6 cavidades com 3 mL de meios de estabilização por poço. Permitir o equilíbrio durante 30 minutos a 37 ° C numa incubadora com atmosfera contendo 5% de CO 2. </li><li> Preparar os seguintes reagentes durante a incubação no Passo 2.3: Diluir solução-mãe de histamina para a concentração desejada (450 uM) em meio morno. Dissolve-LXA4 (armazenada sob atmosfera de árgon) para o meio a uma concentração final de 250 ng / ml. Nota: Quando se mede o efeito de LXA4, uma metade de um único retina é usado só por histamina, enquanto que a outra metade é usado para a histamina com LXA4. </li><li> Aspirar o meio e adicionar estabilização cuidadosamente o meio preparado a cada poço (3 ml por cavidade). Incubar as amostras a 37 ° C durante 1 hora numa incubadora com atmosfera contendo 5% de CO 2. </li><li> Lavar oamostras de uma vez em água morna, PBS estéril. </li></ol> 3. Prepare as amostras para imunocoloração <ol><li> Aspirar PBS a partir das placas. Adicionar PFA a 4% (3 ml por cavidade) e incuba-se durante 15 min à temperatura ambiente. </li><li> aspirar rapidamente a PFA. Lavar as amostras de uma só vez utilizando PBS. Transferir as amostras a 10% de sacarose (5 ml) por poço e incubar durante 2 a 4 horas a 4 ° C. </li><li> Transferir as amostras para uma sacarose a 30% (5 ml por poço) e incubar durante a noite a 4 ° C. Misturar uma parte do meio de congelamento comercial, com duas partes de 20% de sacarose para tornar o meio de congelação de trabalho. Deixe que a 4 ° C durante a noite ou mais tempo até que as bolhas de ar desaparecer. </li><li> Transferir as amostras para o meio de congelação de trabalho e permitir o equilíbrio durante 5 min à temperatura ambiente. Apare cada retina em retângulos (cerca de 3 mm por 5 mm). </li><li> transferir cuidadosamente as amostras para o meio de congelação de trabalho contido num recipiente cilíndrico (cerca de 1 cm de raio x 2 cm de altura) devzada da folha de alumínio. Assegure-se que as fatias de retina são verticais (orientada para proporcionar um corte transversal da retina após o corte) e congelá-los em azoto líquido. Guardar os blocos a -80 ° C até à sua utilização. </li><li> Seção cada bloco em um criostato em 14 mm fatias grossas e montar as secções em lâminas de vidro. Armazenar as lâminas a -80 ° C até à sua utilização. </li></ol> 4. A imunocoloração <ol><li> Lavam-se as secções com tampão de fosfato de 5% de sacarose (SPB, 5% de sacarose em PBS, pH 7,4) uma vez. Bloquear os locais de ligação não específicos por incubação das secções em tampão de bloqueio (5% SPB com 10% de soro normal de cabra e 0,5% de Triton X-100) durante 1 h à temperatura ambiente. </li><li> Incubam-se as secções em lâminas () com o anticorpo primário apropriado (GFAP ou MAP2, diluído a 1: 500 em tampão de bloqueio) durante 1 h. Aspirar a solução. Lavar três vezes em 5% SPB. Para a coloração da IgG endógena, ignore este passo. </li><li> Incubar as secções com o secon correspondentedary anticorpos (IgG anti-ratinho / anti-coelho de burro conjugado Cyanine3 / FITC, diluída a 1: 800 no tampão de bloqueio) durante 1 h. </li><li> Lavam-se as secções cuidadosamente com 5% SPB três vezes. Preparar as amostras para microscopia por montá-los no meio de montagem contendo DAPI e cobrindo com lamelas. </li><li> Usando um laser microscópio confocal, capturar imagens através de uma lente de 20X sob parâmetros idênticos, como a intensidade do laser, o valor ganho, tempo de permanência, etc. entre os grupos. Definir os comprimentos de onda de excitação / emissão para o canal azul (para detectar DAPI) como 408/447 nm, para o canal vermelho (para detectar Cyanine3) como 561/785 nm e para o canal verde (para detectar FITC) como 488/525 nm . </li></ol> Análise 5. Imagem <ol><li> Quantificar percentagem de vasos sanguíneos com vazamentos de acordo com os seguintes critérios 12: Inclua apenas os vasos sanguíneos com os núcleos das células endoteliais dentro do plano de corte na contagem; considerar um leaky vasos sanguíneos se mostra agradient de imunocoloração em torno do navio. </li><li> Para determinar o nível de expressão, selecione regiões de interesse e medir o valor de cinza média usando o software de análise de imagem. </li><li> Para medir a largura do processo, escolher uma área de secção em que os processos são distintos (a camada externa nuclear, ONL, por exemplo). Em seguida, escolha um campo óptico, onde tais processos são visíveis e contínua. Defina a barra de escala de acordo com a configuração microscópica, desenhar uma linha em todo o processo e realizar a medição. </li><li> Para analisar a continuidade do processo, selecione regiões de interesse, aplicar o filtro chamado de variação que melhora as bordas da imagem, substituindo cada pixel com sua variância vizinhança, ajustar automaticamente o contraste e brilho, contar as linhas e normalizar a contagem pela área. </li><li> Calcule significância estatística usando teste t de Student de duas caudas. *, P &lt;0,05; **, P &lt;0,01; ***, P &lt;0,001. Pmuito o resultado como média ± SEM. </li></ol>

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The authors have nothing to disclose.

In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

Um crescente corpo de evidências suportam a existência de barreira retiniana sanguínea (BRB) 1-5 e a sua semelhança com a barreira hemato-encefálica (BHE) 6,7. Compromisso da BHE foi firmemente ligado causalmente ou como um marcador diagnóstico para doenças neurodegenerativas crónicas tais como a doença de Alzheimer (AD) e 8,9 agudas condições tais como delírio 10. percepções mecanicistas para estas patologias e descobertas para alvos potenciais da droga são geralmente prejudicada pela acessibilidade e rede limitada complexidade do cérebro. Alternativas tais como imagiologia in vivo 11, cérebro cultura organotípica 12, as culturas de células primárias 13,14 e sistemas de co-cultura com 15 ter sido gerado. No entanto, a maioria destes modelos requerem instrumentos especiais, períodos experimentais longos ou múltiplos marcadores para identificar células. semelhanças estruturais e funcionais entre a certificação e BRB, bem como uma correlação entre o dysfunctions de os dois têm sido argumentado 16-19. Além disso, um acesso mais fácil, tipos celulares bem definidos e uma estrutura em camadas da retina têm permitido bem caracterizado como uma janela para o cérebro. As identidades estruturais e funcionais da BHE e BRB continuam a ser comparado em pormenor. No entanto, as patologias da retina, principalmente a quebra BRB, também foram fortemente associada com a progressão de várias doenças, incluindo a diabetes e 18-19 21,22 AD. Assim, é de interesse para estabelecer um sistema de disfunção BRB não só para delinear o mecanismo, mas também para pesquisar potenciais drogas. Neste relatório, um protocolo que permite a disfunção BRB usando uma cultura aguda de retina simples é desenvolvida e apresentada. O aumento da permeabilidade da BHE e alterações patológicas ad como foram estabelecidas em uma cultura organotípica cérebro incubadas com histamina, um mediador pró-inflamatório 12. Portanto, no sistema apresentado, a histamina foi Applied para a cultura ex vivo para induzir disfunção da retina BRB. Retinas de várias espécies, como Mus musculus e Bos Taurus, foram testados. Devido à sua disponibilidade comercial e semelhança com o tecido humano, globos oculares suínos frescos foram utilizados para fornecer os dados aqui relatados. Após incubação com histamina e / ou outras drogas, as retinas foram processadas para avaliação por imunocoloração para várias proteínas de 12, tais como a imunoglobulina G (IgG), um dos principais componentes do sangue; proteína ácida fibrilar glial (GFAP), um marcador conhecido para a activação da glia; e proteína associada a microtúbulos 2 (MAP2), uma proteína do citoesqueleto específica para neurónios essencial para a montagem dos microtúbulos. Além disso, a estrutura em camadas da retina permite uma análise detalhada dos processos de células de Muller e células ganglionares, tais como alterações na sua largura e continuidade. Assim, vários parâmetros adicionais estão disponíveis para avaliar as consequências daBRB violação numa fase inicial e para avaliar os efeitos de reversão de potenciais tratamentos também. Neste protocolo, os efeitos potenciais de inversão de drogas crivados são avaliadas a partir de três pontos de vista: o vazamento dos vasos sanguíneos (BVS), a activação das células da glia e os danos-resposta de células neuronais. Vários métodos de quantificação são utilizados, por exemplo, o nível de expressão mostrado pela intensidade da imunocoloração, medição da largura de um processo e de continuidade de processos neuronais mostrados por um filtro de reforço. Para melhor ilustrar o método e para ajudar a interpretar os resultados, da lipoxina A4 (LXA4), um composto sintetizado endogenamente em resposta a uma lesão inflamatória e atenuando a disfunção endotelial 23, foi escolhida para fins de demonstração.

<table><tbody><tr><td>DMEM</td><td>Life Technologies&amp;nbsp;</td><td>11965-092</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies&amp;nbsp;</td><td>14170-112</td><td></td></tr><tr><td>Sucrose</td><td>J.T.Baker</td><td>4072-05</td><td></td></tr><tr><td>Histamine&amp;nbsp;</td><td>Sigma</td><td>H7125-1G</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>PFA</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Freezing Media&amp;nbsp;</td><td>Triangle Biomedical Sciences</td><td>TFM-5</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Rockland</td><td>D104-00-0050</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>GFAP Antibody</td><td>Millipore</td><td>AB5804</td><td></td></tr><tr><td>MAP2 Antibody</td><td>EMD Millipore</td><td>MAB3418</td><td></td></tr><tr><td>FITC conjugated Donkey anti-rabbit IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>711-095-152</td><td></td></tr><tr><td>Cy3 conjugated Donkey anti-mouse IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>715-165-150</td><td></td></tr><tr><td>mounting medium containing DAPI</td><td>Vector Laboratories, Inc.</td><td>H-1200</td><td></td></tr><tr><td>Laser Confocal Microscope</td><td>Nikon</td><td>Eclipse Ti microscope</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>1.45s</td><td></td></tr></tbody></table>

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.

Apresentamos, um sistema de baixo custo em tempo eficiente e fácil de usar para avaliar potenciais tratamentos que podem proteger contra a violação BRB induzida pela histamina. IgG é limitada dentro dos vasos na retina controle (Figura 1A), mas vaza dos vasos sanguíneos após a exposição de histamina (Figura 1B), confirmando que o modelo seja estabelecida com sucesso. LXA4 foi escolhido...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

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

Um baixo custo, o sistema easy-to-use e poderoso é estabelecida para avaliar potenciais tratamentos que possam amenizar violação sangue barreira retina induzida pela histamina. vazamento dos vasos sanguíneos, a ativação de células Müller e a continuidade dos processos neuronais são utilizados para avaliar a resposta a danos e sua reversão com uma droga potencial, lipoxina 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 ...

an-acute-retinal-model-for-evaluating-blood-retinal-barrier-breach

Research

JoVE Journal

Medicine

7.1K visualizações.  Rowan University.  Um baixo custo, o sistema easy-to-use e poderoso é estabelecida para avaliar potenciais tratamentos que possam amenizar violação sangue barreira retina induzida pela histamina. vazamento dos vasos sanguíneos, a ativação de células Müller e a continuidade dos processos neuronais são utilizados para avaliar a resposta a danos e sua reversão com uma droga potencial, lipoxina A4. 

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Modelos experimentais para estudo da fisiologia epitélio pigmentar da retina e Fisiopatologia

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neurociência

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Modelo de ratos de sangue-cérebro Rompimento de barreira para permitir alvejado Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Medicina

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 ...

Gerando degeneração retiniana para investigar estratégias de reparo celular em Xenopus

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

Autor em destaque: explorando os mecanismos de regeneração da retina no sapo Xenopus 

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Gerando modelos de lesão retiniana em girinos Xenopus

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

Biologia do Desenvolvimento

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Um modelo de mouse Retinal isquemia-reperfusão Através elevação da pressão intra-ocular

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

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 ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

Avaliação fisiofisiológica da retina em um modelo de rato

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Ensaio de Transcitose de Células Endoteliais como Modelo In Vitro para Avaliar a Permeabilidade da Barreira Interna Sangue-Retina

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

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

Resumo

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