Name: glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina
Uploaded: 2015-03-22T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

This paper is dedicated to my beloved wife Maren and our little Karl.

1. Preparação de Bovinos Olhos <ol><li> Obter bovina olhos imediatamente após o abate do animal. </li><li> Transportar os olhos protegidos em &quot;Sickel-solução&quot; uma forma especial, contendo NaCl 120 mM, KCl a 2 mM, 0,1 mM de MgCl2, 0,15 mM de CaCl2, 1,5 mM de NaH 2 PO 4, 13,5 mM de Na 2 HPO 4 e 5 mM de glucose no RT. </li><li> Realizar a preparação da retina em condições adaptadas escuro com uma luz vermelha ofuscante. </li><li> Retire a parte anterior do olho. Executar uma ca. incisão equatorial 4 milímetros posterior ao limbo. Posteriormente remover córnea, íris, corpo ciliar e lente em uma única peça. Mantenha as retinas em Sickel-solução. </li><li> Mecanicamente soltar os anexos vítreas para a superfície da retina e do vítreo remover da taça olho aberto. </li><li> Depois divida o olho em quatro quadrantes e perfurar rodada áreas de ca. 7 milímetros de diâmetro usando uma trefina. </li><li> Suavemente separar da retinado epitélio pigmentar e colocá-lo em um dispositivo de gravação dentro de uma caixa protegida da luz. O dispositivo de gravação é composto por uma manutenção de plástico com uma malha no meio; colocar a retina na malha e, em seguida, fixar com um anel de plástico directamente sobre os eléctrodos. NOTA: O mantenedor de plástico tem dois canais para permitir um fluxo constante do meio. </li></ol> 2. A gravação do eletrorretinograma (ERG) <ol><li> A fim de gravar o electrorretinograma, usar dois eléctrodos de prata / prata-cloreto de cada lado da retina e perfundir a retina a uma velocidade de perfusão constante de ca. 1 ml / min e temperatura constante de 37 ° C. Use a &quot;Sickel-solução&quot; saturada com oxigênio. </li><li> Antes de iniciar a medição, adaptar escuro da retina (proteger da luz durante todas as medições) e usar intervalos de estímulo de cinco min. Use um único flash xenon branco 1 Hz para a estimulação com uma intensidade definido para 6,3 mlx na superfície da retina. </li&gt; <li> Uso calibrado filtros de densidade neutra e um estímulo de luz de 10 ms controlado por um temporizador, de modo a apresentar respostas óptimas. </li><li> Para medir e processar os dados, filtrar o ERG e amplificá-lo (100 Hz filtro de alta freqüência, de 50 Hz filtro notch, 100.000 x amplificação), utilizando um RPS312RM Amplifier Grass. Tente filtrar frequências possíveis perturbadores que possam perturbar o sinal. A fim de processar os dados, use uma placa de aquisição analógico-digitais de dados em um computador desktop (PC compatível). </li><li> Após o período de adaptação ao escuro sob perfusão constante, medir a amplitude do sinal elétrico até estáveis ​​amplitudes b-ondas são gravadas. NOTA: As amplitudes são consideradas estáveis ​​se cinco medições individuais chegar a um valor médio e desviar-se menos de 10%. Um bom exemplo de medições individuais é dada na Figura 1. </li><li> Para iniciar o teste, substituir o oxigénio puro por qualquer azoto puro (número de experiências individuais, n = 5) para testar a hipóxiaou 250 um glutamato (n = 5). </li><li> Grave as respostas elétricas a cada 5 min para 45 min. </li><li> Após o período de testes, aspergir as retinas com padrão médio saturada com oxigênio por 75 min e olhar para as mudanças da amplitude da onda-b. Esta é a fase de lavagem. Medir a amplitude da onda-b da calha do uma onda para o pico da onda b. </li><li> Para investigar o efeito de hipóxia ou glutamato no potencial de fotorreceptores em condições escotópicas, suprimir a onda b pela adição de 1 mM para a solução nutriente. </li><li> Após a gravação de um potencial estável de fotorreceptores durante 30 min, realizar o procedimento como antes, expondo as retinas de 45 min para as diferentes soluções de irrigação com aspartato 1 mM. Use o mesmo período de washout (passo 2.8), como mencionado anteriormente. </li></ol> Análise 3. Dados <ol><li> A fim de avaliar estatisticamente os dados, garantir uma distribuição normal para todos os dados, por exemplo, utilizando o teste de Kolmogorov-Smirnovteste 15. </li><li> Calcula-se a redução dos a- e b-amplitudes de onda em percentagens após a fase de exposição, em comparação com a última medição antes da exposição. Comparar a redução das amplitudes-ERG após 45 min - no final do período de exposição - a ERG medido antes da aplicação. </li><li> Comparar a onda a- e b- no final da fase de lavagem para a amplitude correspondente antes de exposição a estudar a possibilidade de recuperação. </li><li> Para a análise estatística, use o software estatístico JMP software ou software SPSS. Calcular dados ao longo como a média ± desvio padrão. Estime significado pelo teste estatístico adequado. NOTA: Estes testes podem ser diferentes dependendo do escopo experimental. Neste cenário, use t-pareado de Student. </li></ol>

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The authors have nothing to disclose.

Neste estudo, um impacto significativo na amplitude da onda b após 45 minutos de hipoxia foi encontrado. Esta redução foi ainda significativo após a fase de washout. Um efeito semelhante sobre o potencial de fotorreceptores pode ser observada. Os resultados são compatíveis com outros dados publicados 16 e nos dão a oportunidade de estudar possíveis efeitos neuroprotetores após hipóxia. Depois de 45 minutos de exposição de 250 uM de glutamat...

Neuroprotection tem sido um forte campo de investigação na investigação oftalmológica nas últimas décadas. A retina é uma rede neuronal altamente sensível que depende significativamente a oxigenação e é fortemente influenciado pelo metabolismo das suas células vizinhas. As principais patologias oculares relacionadas com a lesão das células nervosas são oclusões vasculares retinianas, glaucoma e descolamento da retina. A oclusão da artéria retinal, como um exemplo para a oclusão vascular da retina, leva a uma perda súbita de visão devido a hipoxia da retina interna 1. Ela é freqüentemente associada com patologias vasculares gerais 2 e leva a uma perda visual persistente 1, com apenas 8% dos pacientes em recuperação da acuidade visual significativamente 1. Apesar de a fibrinólise arterial tem sido sugerida como uma opção de tratamento, o benefício não poderia ser mostrado em um ensaio clínico randomizado 3. Glaucoma e descolamento de retinaambos têm um aumento na concentração de glutamato 4-6. O glutamato sob condições fisiológicas é encontrado como um transmissor excitatório ao longo de todo o sistema nervoso central e retina interna a 7,8. Elevados níveis de glutamato foram encontradas não só em glaucoma e descolamento de retina 5,6, mas também na retinopatia diabética proliferativa 9. Um aumento em glutamato conduz possivelmente a excitotoxicidade e, portanto, danos das células nervosas 10. Na maioria dos casos de descolamento da retina e em alguns casos de cirurgia de retinopatia diabética proliferativa na retina (vitrectomia via pars plana) são necessários. Durante a manipulação mecânica vitrectomia, luz brilhante da fibra óptica ou tensão de cisalhamento exercida por altas taxas de fluxo de soluções de irrigação durante as operações longas exercer uma pressão adicional sobre a retina 11,12. Todas as doenças mencionadas têm em comum que a patologia está localizada ao retinuma só e representam a comunidade oftalmológica com o desafio de encontrar formas de proteger a retina como um sistema neurossensorial. O electrorretinograma (ERG) é o método padrão para a avaliação in vivo da função de fotorreceptores (uma onda) e a função da retina interna (b-ondas). O ERG é medido por eléctrodos de prata-introduzidos na córnea e os olhos estão a ser estimulado por um aumento do nível de luz para detectar defeitos em bastonetes ou cones ou na retina interna. Diferentes defeitos na retina pode ser detectada por alterações na amplitude (a força da resposta) ou a latência (o intervalo de tempo de resposta-para) do ERG. Diferentes protocolos e métodos de medição ERG (padrão-ERG, multifocal ERG-campo ou brilhante ERG) permitem uma maior diferenciação de defeitos. A técnica da retina isolado foi introduzida recentemente, tornando-se possível avaliar os efeitos sobre a retina, sem interferências de um animal, por exemplo o estudo dereações gerais 13,14. Foi o propósito deste estudo para avaliar e introduzir um modelo de estresse definido e padronizado para hipóxia e estresse glutamato na retina superfused isolado. Assim, nós estamos esperando para lançar as bases para futuros estudos sobre efeitos neuroprotetores de determinados agentes ou soluções de irrigação intra-oculares.

<table><tbody><tr><td>120 mM NaCl&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,064,041,000</td><td></td></tr><tr><td>2 mM KCl,&amp;nbsp;&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,050,010,250</td><td></td></tr><tr><td>0.1 mM MgCl2,&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>58,330,250</td><td></td></tr><tr><td>0.15 mM CaCl2</td><td>Merck Pharma, Germany</td><td>111 TA106282</td><td></td></tr><tr><td>1.5 mM NaH2PO4/13.5 mM Na2HPO4&amp;nbsp;&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,065,860,500</td><td></td></tr><tr><td>5 mM glucose</td><td>Merck Pharma, Germany</td><td>40,741,000</td><td></td></tr></tbody></table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

Neuroprotection tem sido um forte campo de investigação na investigação oftalmológica nas últimas décadas e afeta doenças como glaucoma, oclusão vascular da retina, descolamento de retina, e retinopatia diabética. Era o objeto deste estudo a introdução de um modelo de estresse padronizado para futuro teste pré-clínico terapêutico. Retina bovina foram preparadas e perfundido com uma solução saturada de oxigénio padrão, e o ERG foi gravado. Depois de gravar b-ondas estáveis, hipoxia (N 2) puro ou estresse glutamato (250 um glutamato) foi exercida por 45 min. Para investigar os efeitos sobre sozinho função de fotorreceptores, 1 mM aspartato foi adicionada para obter a-ondas. ERG-recuperação foi monitorada por 75 min. Para hipoxia, uma diminuição na amplitude da onda-um de 87,0% foi observada (p &lt;0,01) após um tempo de exposição de 45 min (36,5% de redução após o final da lavagem p = 0,03). Além disso, um decr inicialfacilidade de amplitudes de ondas de b-87.23% foi registada, que atingiu significado estatístico (p &lt;0,01, diminuição de 25,5% no final da lavagem, p = 0,03). Para 250 um glutamato, uma redução inicial de 7,8% de um amplitudes de ondas (p&gt; 0,05), seguidas por uma redução de 1,9% (p&gt; 0,05). Uma redução de 83,7% da amplitude da onda b (p &lt;0,01) foi observado; após uma lavagem de 75 min, a redução foi de 2,3% (p = 0,62). Neste estudo, um modelo de tensão é apresentada normalizada que pode ser útil para identificar possíveis efeitos neuroprotectores no futuro.

Após 1 hora de perfusão dos preparativos da retina com solução padrão saturado de oxigênio (Figura 1A e B) ERG-amplitudes mostrou estabilização e menor variação de amplitudes entre as medidas individuais. pH, pressão osmótica, temperatura, e pO 2 (excepto para o teste de hipóxia) foram mantidas constantes em todos os testes. Para isolar o sinal a partir do sinal de fotorreceptores da retina interna, aspartato 1 mM foi adicionado para a solução padrão ...

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Com este estudo, apresentamos um modelo de estresse padronizado para a retina bovina superfundido isolado para o futuro teste pré-clínico terapêutico. O efeito de qualquer hipóxia (puro N 2) ou estresse glutamato (250 mM glutamato) em função da retina representado por a- e amplitudes da onda-b foi avaliado.

Glutamato e hipóxia como um modelo de estresse para o isolado perfundido Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

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Medicine

7.8K visualizações.  University Eye Hospital Tübingen.  Com este estudo, apresentamos um modelo de estresse padronizado para a retina bovina superfundido isolado para o futuro teste pré-clínico terapêutico. O efeito de qualquer hipóxia (puro N 2) ou estresse glutamato (250 mM glutamato) em função da retina representado por a- e amplitudes da onda-b foi avaliado. 

Vídeo: Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Assessment of Vascular regeneração no SNC Usando o rato Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Neurociência

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Medicina

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Metodologia para neuromodulação Biomimetic químico de Retinas de ratos com o neurotransmissor glutamato em Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Bioengenharia

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Monitoramento de Astrocyte de reatividade e proliferação in Vitro sob condições isquêmicas

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

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, ...

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 ...

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 ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Um modelo murino de lesão isquêmica da retina induzida pela oclusão da artéria da carótida comum bilateral transitória

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Explante Retiniano da Retina de Rato Adulto como Modelo Ex Vivo para o Estudo de Doenças Neurovasculares da Retina

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...