Name: observation of the ciliary movement of choroid plexus epithelial cells ex vivo
Uploaded: 2015-07-13T15:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

This work was supported by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (T.I.) and Grants-in-Aid for Scientific Research (C) from MEXT (S.T. and K.N).

Os protocolos e uso de animais de laboratório foram aprovados pelos cuidados com os animais e uso comitês institucionais da Universidade de Yamanashi e Universidade de Waseda. Cuidados com os animais foi realizada de acordo com as diretrizes institucionais. 1. Preparação CPEC <ol><li> Preparar os seguintes aparelhos e materiais: um microscópio estéreo, de um modo preferido capaz de transmitir luz a partir do fundo; um par de fórceps Dumont relojoeiro (# 3 ou # 4), esterilizada por chama, tesouras operacionais em linha reta, esterilizada por chama; dois pratos de 10 centímetros de plástico estéreis contendo 20 ml de gelo-frio Leibovitz L-15 de forma; Pratos com fundo de vidro 35 milímetros contendo 2 ml RT Leibovitz meio L-15; Num copo de 100 ml contendo 70% de etanol; filhotes de rato recém-nascidos. </li><li> Resumidamente mergulhe um rato neonatal em 70% de etanol e eutanásia rapidamente por decapitação usando a tesoura operacionais. </li><li> Coloque a cabeça imediatamente em gelo frio Leibovitz L-15, na forma estéril 10 centímetros dish. </li><li> Remover a pele dos calvária usando o par de pinças de relojoeiro, abriu o crânio para expor o cérebro, e depois cortar os nervos cranianos para isolar todo o cérebro. </li><li> Transferir o cérebro para uma nova placa contendo gelada Leibovitz L-15 médio e observar ao microscópio estéreo, certificando-se que o cérebro está completamente imerso no meio. </li><li> Defina o aspecto dorsal do cérebro voltado para cima, e orientar o cérebro para que os bulbos olfatórios residir na posição de três horas (para pessoas destras). Segure o cérebro suavemente com a pinça na mão esquerda. </li><li> Usando a pinça de dissecação fina (Dumont # 3 ou # 4) na mão direita, corte do corpo caloso e debaixo do parênquima conectando os hemisférios cerebrais, ao longo da fissura longitudinal do cérebro. </li><li> Com cuidado, empurre os hemisférios cerebrais longe para os lados laterais e expor a fissura transversa do cérebro. </li><li> Separe os hemisférios por beliscar fora the parênquima entre o hemisfério e tálamo. </li><li> Retire cuidadosamente o plexo coróide ventriculares laterais que é anexado para o lado lateral do hipocampo pelo Affixa lâmina. </li><li> Transferir o plexo coróide isoladas para o prato fundo de vidro 35 milímetros contendo fresco Leibovitz meio L-15, e sobrepor o peso máximo (Lista de Materiais) com cuidado para segurar o tecido no lugar. </li></ol> 2. Imagens ao vivo da CPEC Cilia <ol><li> Confirmar ultravioleta adequada (UV) e filtro (s) inferida (IR) de corte para bloquear luz inferior a 400 nm e acima de 700 nm, e que a densidade neutro (ND) os filtros (25% e 6%), são inseridos no caminho da luz do microscópio invertido. </li><li> Ajuste o foco da lente objetiva aproximadamente por olho, e então ajustar o condensador de modo que o centro e foco para estar de acordo com a iluminação Köhler. Inserir um contraste de interferência diferencial adequado (DIC) prisma, um elemento DIC, bem como elementos do analisador e polarizador na licaminho lutar para estar de acordo com a ótica DIC. </li><li> Ajustar o contraste de vista pela posição do prisma DIC modo que a estrutura da superfície do tecido é mais reconhecível. Se todos os cílios das células alvo são móveis, uma visão clara dos cílios móveis não podem ser obtidos por olho por causa do seu movimento. </li><li> Altere o caminho de luz para a câmera de vídeo e remova os filtros ND para aumentar o poder de luz. </li><li> Use a câmera no modo de focagem para ajustar o campo de visão e foco. Durante focagem, em que as imagens de vídeo são exibidas em tempo real no monitor, uma visão clara dos cílios móveis não está disponível. </li><li> Use a câmera em 200 Hz com um tempo de exposição de 0,1 ms para o período desejado (segundos a minutos). Após a aquisição da pilha de imagens, quadros únicos irá exibir estruturas ciliares claras. Se bordas ciliares são borradas, aumentar a taxa de quadros ou use um tempo de exposição mais curto. </li><li> Grave o movimento de CPEC cílios dentro de 25-60 minutos após a eutanásia em LeibovitzMeio L-15. </li></ol> 3. Análise do Movimento ciliar <ol><li> Controlar manualmente batendo padrões de cada cílio no monitor do computador. Marcar posições de ponta ciliar em cada quadro com o ponteiro do mouse, que são montados para informação trajetória de cada cílio. Ou analisar a informação trajetória utilizando o mesmo software ou exportar para outras aplicações mais gerais para uma análise mais aprofundada. A eficiência deste passo de análise é descrito na discussão. </li><li> Classifique as trajetórias em dois modos de movimento, volta-e-vem de rotação, ou por olho. </li><li> Calcula-se a frequência de batimento ciliar (CBF), utilizando a seguinte fórmula: [CBF = (número de quadros por segundo) / (número médio de imagens para um único batimento)] 14, que pode ser obtido a partir de um diagrama de ponta movimento ciliar (Figura 3 ). Repetir este cálculo para múltiplos ciclos de batimento ciliar, porque outra cílios na mesma célula pode interferir com o movimento de cada cilium, resultando em irregularidade. </li><li> Para analisar a uniformidade angular do batimento ciliar eixos dentro de uma única célula, definem o ângulo θ batida para cada trajectória (Figura 4). Para trajetórias de vai-e-vem, se encaixam as posições da ponta dos cílios para uma linha reta, e definir θ como o ângulo da linha faz com eixo x. Para trajectórias de rotação, encaixam as posições de uma elipse, e definir θ como o ângulo do eixo maior da elipse faz com eixo x. Detalhes da instalação são descritos na secção de resultados representativos. </li><li> Para obter uma descrição quantitativa de cada trajetória, calcular generalizada proporção AR. Resumidamente, rodar a trajectória por - θ e definir AR como a relação entre as larguras da distribuição ao longo - x e y -axes (Figura 4B). Detalhes são mostrados na seção Resultados Representante, ea interpretação, Significado ea limitação do parâmetro são descritos na discussão. </li></ol> 4. Preparação de Amostras para SEM Nota: SEM é um importante método para avaliar o estado dos cílios em CPECs de uma forma abrangente. Para preparar as amostras para SEM, um procedimento padrão reportada anteriormente 15 é empregue com ligeiras modificações. <ol><li> Antes de dissecação do tecido a partir do cérebro, preparar o fixador em um frasco de vidro de 5 ml com uma tampa de polietileno. O fixador é constituído por 2% de paraformaldeído, glutaraldeído a 2,5% (metade da solução de Karnovsky 16) em tampão fosfato 0,1 M, pH 7,4. </li><li> Dissecar o tecido do cérebro, tal como descrito no passo 1. </li><li> Resumidamente enxaguar o tecido isolado com solução salina equilibrada de Hank (HBSS) numa nova placa e, em seguida, fixar o tecido no fixador no frasco de vidro, durante 1 h à temperatura ambiente. Use pipetas de transferência descartáveis ​​e manipular os espécimes gently. Depois de enxaguar em HBSS, o tecido torna-se pegajosa. <ol><li> Para transferir as amostras para a solução fixadora, lentamente expulsar uma pequena quantidade de solução que continha o tecido a partir da pipeta de transferência como uma gota e adicionar ao fixador. </li></ol></li><li> Após a fixação, descartar o fixador e lavar o tecido com tampão fosfato de três vezes. </li><li> Mergulha-se o tecido numa solução de sacarose a 10% para lavar os aldeídos restantes. Para assegurar a eliminação total de aldeídos, imergir as amostras na solução durante 10 min e, em seguida, repetir duas vezes com sacarose a 10% fresco. Este passo é importante para garantir a boa pós-fixação em etapas subseqüentes. </li><li> Mergulha-se o tecido numa solução de 1% de tetróxido de ósmio em tampão de fosfato durante 30 min e, em seguida, colocar em gelo para pós-fixação. Julgar o grau de osmification pela cor da amostra: quando os aldeídos são completamente removidas, a amostra é preto. </li><li> Lave as amostras pós-fixadas de tecido extensivamente com duplo Distilled água várias vezes. </li><li> Desidratar as amostras por imersão em concentrações graduadas de etanol, geralmente 65%, 75%, 85%, 95%, 99%, e 100%, durante 10 min cada. Obtenção de etanol anidro, colocando peneiras moleculares em etanol 99,5% de uma garrafa recém-adquirido. Repita desidratação com etanol anidro três vezes. </li><li> Coloque as amostras desidratadas em acetato de isoamilo, um reagente para a substituição ponto crítico de secagem, durante 10 min. Repita esta etapa duas vezes. Este reagente e evapora-se rapidamente a amostra pode tornar-se seca, resultando na destruição pela tensão superficial. Portanto, não secar a amostra completamente. </li><li> Após a última troca de acetato de isoamilo, remover a maior parte do solvente, imediatamente embrulhar o frasco de vidro aberta com uma folha de alumínio e coloque o frasco em gelo seco. Utilizando uma agulha ou uma pinça fina, fazer vários orifícios na folha que cobre a boca do frasco, de modo que o dióxido de carbono líquido flui facilmente para dentro do frasco no secador de ponto crítico. Prossiga para a próxima etapao mais rápido possível. </li><li> Nesta etapa, minimizar a transição de acetato de isoamilo na câmara do secador, mas não deixe que a amostra secar completamente antes de secar ponto crítico. Além disso, não deixe a amostra em gelo seco para um desnecessariamente muito tempo para evitar a formação de geada no frasco. </li><li> Transferir os frascos de vidro envolto com folha de alumínio contendo as amostras de tecido em secador de ponto crítico a que assegura a estrutura da superfície do tecido permanece intacta, enquanto a remoção da água contida no tecido. Informações pormenorizadas sobre a operação do secador de ponto crítico pode ser obtido a partir de instruções do fabricante. </li><li> Lidar com as amostras cuidadosamente usando um palito para minimizar danos mecânicos. As amostras de tecidos secos resultantes são frágeis. Monte as amostras em bases metálicas e revestimento com ouro-paládio usando uma pulverização catódica de íons. </li></ol> 5. Observação por SEM <ol><li> Observe por SEM e gravar imagens com uma câmera digitalequipado com microscópio eletrônico de varredura. </li><li> Transferir os dados de imagem digitais para um PC para análise. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes. Este trabalho tem como objetivo relatar metodologia detalhada para observar a motilidade ciliar em tecidos isolados do plexo coróide. Novidades científicas têm sido relatados em estudos anteriores 1,8.

Perspectivas deste método Embora a técnica aqui descrita não proporciona uma análise mais detalhada dos cílios do que os métodos previamente publicados, o significado desta técnica reside na simplicidade do sistema e relação custo-eficácia, que pode ser facilmente aplicado a qualquer tipo de rastreio de motilidade ciliar ex vivo. Em particular, TI Workbench fornece uma interface simples e fácil de usar que permite aos pesquisadores observar e analisar a motilidade ciliar ...

Cilia are hair-like projections on the surface of most vertebrate cells, which have attracted attention by medical researchers because of a class of diseases termed ciliopathies2&#8211;4. Despite the ubiquitous expression of the organelle, a wide variety of ciliary functions have been reported, including motility and biosensing. For example, motile cilia on the mucoepithelial surface transport mucus5 and epithelial debris to the outlet of tracts, thereby preventing disease by clearing the surface of epithelia. Moreover, during early developmental periods and embryonic stages, cilia regulate the proliferation of stem cells6, and are involved in the determination of left&#8211;right asymmetry of the vertebrate body7. 
Choroid plexus epithelial cells (CPECs) are derivatives of neuroepithelial cells that cover the surface of the choroid plexus tissue in the brain, which play important roles in maintaining homeostasis of the intracranial environment by production of cerebrospinal fluid (CSF). It has been previously demonstrated that CPECs have multiple non-motile cilia that regulate the production of CSF through G-protein-coupled receptors that are specifically concentrated on the cilia8. Although these cilia had been regarded as quiescent non-motile cilia, it was discovered that some CPEC cilia exhibit transient motility during the neonatal period1. This finding was quite important because it revealed that so-called non-motile cilia are not necessarily immotile from the beginning of development and might display transient motility during specific time windows, possibly in response to specific physiological demands and functions9. To precisely describe the motile nature of CPEC cilia, it is necessary to develop an ex vivo observation system that encompasses analysis of the kinetic profiles unique to CPEC cilia. 
With respect to motility, although several technical reports have described observations of the motile cilia of the tracheal epithelium5,10, motile single-cell flagella11, so-called conventional motile cilia12, and nodal cilia13, detailed analytical methods applicable to relatively undulated structures such as the choroid plexus have not been well documented so far. Moreover, a high time resolution is required to analyze the ciliary movement of CPECs, in which expensive high-speed cameras are indispensable. To circumvent this necessity and simplify monitoring the ciliary motility of various cell types, a low cost, high-speed camera has been introduced, and an easily accessible and convenient method to record the motility of motile cilia, especially to describe the speed and pattern of motion of each cilium, has been developed1. Moreover, original image analysis software &#8220;TI Workbench&#8221; has been used here to facilitate detailed analysis of motility. Collectively, this method provides a new concise strategy to analyze ciliary motion together with correlative scanning electron microscopy (SEM), which can be adopted in a wide range of cilium research.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Required reagents&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>ethanol</td><td>Wako Chemicals</td><td>057-00456</td><td></td></tr><tr><td>Leibovitz L-15 medium</td><td>Life Technologies</td><td>11415-064</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for SEM preparation&lt;/strong&gt;</td></tr><tr><td>ethanol</td><td>Wako Chemicals</td><td>057-00456</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>Life Technologies</td><td>14170112</td><td></td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>Merck</td><td>1040051000</td><td></td></tr><tr><td>glutaraldehyde&amp;nbsp;</td><td>Nacalai tesque</td><td>17003-05</td><td></td></tr><tr><td>isoamyl acetate</td><td>Nacalai tesque</td><td>02710-95</td><td></td></tr><tr><td>Molecular Sieves 4A 1/8&amp;nbsp;</td><td>Wako Chemicals</td><td>130-08655</td><td>for preparation of anhydrous ethanol</td></tr><tr><td>phosphate buffer saline (PBS)</td><td>Sigma-Aldrich</td><td>D1408</td><td></td></tr><tr><td>phosphate buffer, 0.1 M</td><td></td><td></td><td>To make 100 ml, mix 19.0 ml of 0.1&amp;nbsp;M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; and 81.0 ml of 0.1&amp;nbsp;M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>monosodium phosphate (dihydrate)</td><td>Nacalai tesque</td><td>31718-15</td><td></td></tr><tr><td>disodium phosphate (anhydrous)</td><td>Nacalai tesque</td><td>31801-05</td><td></td></tr><tr><td>suclose</td><td>Nacalai tesque</td><td>30406-25</td><td></td></tr><tr><td>osmium tetroxide</td><td>Nisshin EM</td><td>300</td><td></td></tr><tr><td>dry ice</td><td></td><td></td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Tools and materials for dissection&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for both live imaging and SEM preparation&lt;/strong&gt;</td></tr><tr><td>stereo microscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>flat paper towel</td><td></td><td></td><td></td></tr><tr><td>&amp;Phi;10 cm plastic dish</td><td></td><td></td><td></td></tr><tr><td>100 ml beaker</td><td></td><td></td><td></td></tr><tr><td>straight operating scissors</td><td>Sansyo</td><td>S-2B</td><td></td></tr><tr><td>watchmaker forceps</td><td>Dumont</td><td>No.DU-3 or -4, INOX</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>glass bottom dish</td><td>Matsunami Glass</td><td>D110300</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for SEM preparation&lt;/strong&gt;</td></tr><tr><td>alminum foil</td><td></td><td></td><td></td></tr><tr><td>5 ml glass vial with a polyethylene cap</td><td>Nichiden Rika-Glass</td><td>PS-5A</td><td></td></tr><tr><td>transfer pipette</td><td>Samco Scientific</td><td>SM251-1S</td><td>for specimen tranfer</td></tr><tr><td>toothpick</td><td></td><td></td><td>for specimen transfer</td></tr><tr><td>ion sputter with gold-palladium</td><td>Hitachi</td><td>E-1030</td><td></td></tr><tr><td>critical point dryer</td><td>Hitachi</td><td>HCP-2</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Microscopic equipment and materials&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>100 W mercury lump housing and power supply</td><td>Olympus</td><td>U-ULH and BH2-RFL-T3</td><td></td></tr><tr><td>100 W mercury lamp</td><td>Ushio</td><td>USH103D</td><td></td></tr><tr><td>DIC condenser, n.a. 0.55</td><td>Olympus</td><td>IX-LWUCD</td><td></td></tr><tr><td>electrrical shutter</td><td>Vincent Associates</td><td>VS35S22M1R3-24 and VMM-D1</td><td>manual shutter can be used.</td></tr><tr><td>band-pass filter (400-700&amp;nbsp;nm, &amp;Phi;45&amp;nbsp;mm)</td><td>Koshin Kagaku</td><td>C10-110621-1</td><td></td></tr><tr><td>ND filter (&amp;Phi;45 mm)</td><td>Olympus</td><td>45ND6, 45ND25</td><td>combination of 25% and 6% ND filters are used</td></tr><tr><td>objective lens (water immersion) with DIC element</td><td>Olympus</td><td>UApo 40XW/340, n.a., 1.15 with IX-DPAO40</td><td></td></tr><tr><td>high-speed video camera</td><td>Allied Vision Technologies</td><td>GE680</td><td>&amp;#8805;200 Hz frame rate and 1 msec expose time</td></tr><tr><td>image acquisition / analysis software</td><td>in-hous software</td><td>TI Workbench</td><td>capable of acquisition at high frame rates.</td></tr><tr><td>PC for camera control / analysis</td><td>Apple</td><td>Mac Pro</td><td></td></tr><tr><td>vibration isolation table</td><td>Meiritsu Seiki</td><td>AD0806</td><td></td></tr><tr><td>weight for tissue</td><td>Warner Instruments</td><td>slice anchor kits</td><td>It can be made with nylon mesh glued to a U-shape squashed &amp;Phi;0.5&amp;nbsp;mm platinum wire.</td></tr><tr><td>cover glass (alternative weight for tissue)</td><td>Matsunami</td><td>made-to-order</td><td>A cover glass can be used as a tissue weight.</td></tr><tr><td colspan="4">&lt;strong&gt;for SEM&lt;/strong&gt;</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>scanning electron microscope</td><td>JEOL</td><td>JSM-6510</td><td></td></tr></tbody></table>

observation of the ciliary movement of choroid plexus epithelial cells ex vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Uma visão geral do fluxo de trabalho é mostrado na Figura 1, incluindo imagens dos dispositivos. Observações de movimento ao vivo de CPECs Filme 1 mostra um filme de CPECs isoladas de um rato perinatal, e Filme 2 mostra uma visão expandida das imagens em filme 1. Deve-se notar que as pontas ciliares individuais são menos claras nas imagens fixas comparados com os dos filmes. A Figur...

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Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Observação do movimento ciliar de células epiteliais do plexo coróide<em&gt; Ex Vivo</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

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Neuroscience

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

12.1K visualizações.  Waseda University. In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Vídeo: Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Cultivo de células epiteliais de pigmento Retinal em um modelo Ex Vivo da membrana de Bruch humano envelhecido

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Biologia do Desenvolvimento

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imagens ao vivo da ependimária Cilia nas laterais ventrículos do cérebro do rato

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Neurociência

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

O método ex vivo para High Resolution Imaging de Motilidade Cilia em Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Biologia

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Isolamento e Cultura de Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Cauterização em círculo completo do plexo vascular limbal no glaucoma induzido cirurgicamente em roedores

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

Microdissecção e Visualização por Microscopia Eletrônica de Varredura de Montagem Completa do Plexo Coroide de Camundongos

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Um Ensaio Ex Vivo Choroid de Angiogênese Microvascular Ocular

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo

Source: Tomita, Y., et al. <a href="https://app.jove.com/v/61677">An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis.</a> J. Vis. Exp. (2020)
This video demonstrates the development of an ex vivo model of choroidal sprouting to study ocular microvascular angiogenesis. The assay involves culturing mouse eye explants containing the choroid tissue attached to the sclera and retinal pigment epithelium under specific conditions that promote choroidal microvascular sprouting.

Ensaio de brotamento de coróide para estudar a proliferação microvascular ocular ex vivo

Source: Tomita, Y., et al. An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis. J. Vis. Exp. (2020)
This video demonstrates the ...

JoVE Encyclopedia of Experiments

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Técnicas de Ensaio

Ensaios baseados em células

Isolating Immune Cells from Mouse Choroid Plexuses

Source: Dominguez-Belloso, et al. <a href="https://app.jove.com/v/63487">Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure of isolating immune cells from mouse choroid plexuses. This procedure is used in neuroimmunology research to understand how immune cells in the choroid plexuses impact brain health and various neurological diseases.

Isolando células imunes de plexos coróides de camundongos

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. ...

Observação do movimento ciliar de células epiteliais do plexo coróide

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