We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

Todos os animais utilizados neste estudo foram atendidas e tratadas de acordo com a Declaração de ARVO para o Uso de Animais em Oftalmologia e Vision Research. 1. Produção de AAV recombinante (rAAV) por transiente transfecção de células HEK-293 17,18 NOTA: Ver McClure C, Jove (2011) 19. <ol><li> Purifica-se uma preparação de plasmídeo em larga escala (pelo menos 1 mg / ml) dos plasmídeos de vector de AAV. Use 3 plasmídeos. O plasmídeo ajudante de AAV carregando os genes de replicação e da cápside sem o terminal invertida de AAV ITRs repete, a cassete de expressão do transgene transportando o ITR de AAV referida como o plasmídeo de transferência, o plasmídeo e fornecendo os genes de adenovírus ajudante E2, E4, e os genes de ARN AV referido como pHELPER. </li><li> Transfectar células 293 com estas três plasmídeos utilizando polietilenoimina (ou outro reagente de transfecção). Acima de 80% de eficiência de transfecção é necessário para uma boa viyields ral. </li><li> Purifica-se o AAV recombinante a partir de 293 de lisados ​​de células em um gradiente de iodixanol. 15%, 25%, 40%, 60% e o iodixanol é usado para obter o gradiente. Recolha 40% fracção contendo iodixanol partículas de AAV, concentrar a fracção após ultracentrifugação a 500.000 xg durante 1 h e troca de tampão contra PBS (solução salina tamponada com fosfato) com 0,001% de Pluronic. </li><li> Para determinar título genômico viral 20, realizar uma DNAse ProteinaseK digest seguido por qPCR. Os iniciadores directo e inverso, amplificar a região de 62 pb situada na ITR de AAV2. Para o plasmídeo padrão, a sonda é marcada com FAM e tem um extintor buraco negro (BHQ). </li><li> Efectuar qPCR (reacção em cadeia da polimerase quantitativa) em um volume final de 25 ul usando 340 nM de iniciador reverso ITR, 100 nM iniciador ITR para a frente, de 100 nM de sonda de AAV2 ITR, dNTPs 0,4 mM, MgCl2 2 mM, 1x tampão de Taq Platinum, 1U platina Taq e 5 template ul (padrão, amostra e sem controle template). </li><li> Parao padrão, usar o plasmídeo para a transfecção de 7 x 10 diluições em série (cada 5 ul de 2 x 09-2 outubro 3 x 10 genomas / ml resultando numa concentração final de 10 outubro - 10 abril genomas / ml). </li><li> Inicie o programa por um passo de desnaturação inicial a 95 ° C durante 15 min, seguido por 40 ciclos de desnaturação a 95 ° C em 1 min de recozimento e / extensão a 60 ° C durante 1 min. Armazenar a 4 ° C durante um armazenamento de curto prazo ou a -20 ° C durante longos períodos de armazenamento. </li></ol> 2. Injeção intravítrea de AAV <ol><li> Anestesiar ratos C57BL6J com cetamina (50 mg / kg) e xilazina (10 mg / kg) e verificar a perda do reflexo de endireitamento, o reflexo de retirada da cauda e pitada de resposta, indicando uma anestesia adequada. Dilatar alunos pela aplicação da córnea de neosynephrine 5% e 0,5% mydriaticum colírio. Use vet pomada nos olhos para evitar a secura e sob anestesia. </li><li> Passe um G disposab ultrafine 30le agulha através do equador e próxima ao limbo, na cavidade vítrea (ver Chiu K, Jove (2007) 21). Injectar 1 ul estoque contendo 1 a 4 x 10 11 pv de ShH10-GFP ou AAV5-GFP com a observação directa da agulha no centro da cavidade vítrea (Figura 1). Use um olho como um controle para experimentos de ILM. Injectar ambos os olhos para experimentos BRB. Aplicar um tratamento tópico anti-inflamatório e anti-bacteriano em olhos injetados. </li><li> Dilatar alunos com neosynephrine e mydriaticum colírio para geração de imagens. Realizar exames de fundo de olho com uma câmera de fundo de olho para seguir GFP (proteína verde fluorescente) expressão, 1 semana, 2 semanas, 1 mês e 2 meses após a injeção intravítrea (Figura 1). Sacrifício ratos por inalação de CO2 1 a 2 meses pós-injecção. </li></ol> 3. A imuno-histoquímica <ol><li> Para criosecções retina (Figura 1), dissecar olhos enucleados para remover lens e córnea, e imersão em solução de 4% de paraformaldeído durante uma hora. <ol><li> Cryoprotect os olhos previamente fixados em 10% de sacarose durante 1 hora à temperatura ambiente, 20% de sacarose para outra hora à temperatura ambiente. Cryoprotect em solução de sacarose a 30%, durante a noite a 4 ° C. Congelar e eyecups incorporar na incorporação de resina, como Cryo-matriz. Faça 10 mm criosecções e montagem em lâminas com tratamento especial para cortes de tecidos congelados e que melhorar a aderência do tecido durante a coloração. </li><li> Permeabilizar secções com Triton X100 a 0,1% em PBS pH 7,4 durante 5 min. Lavar 2x com PBS e bloqueio durante 1 h à temperatura ambiente, em PBS com 1% de BSA, 0,1% de Tween 20. </li></ol></li><li> Para flatmounts da retina (Figura 1), fixar os olhos enucleados em paraformaldeído a 4% durante 15 minutos para facilitar a dissecação. <ol><li> Em 15-30 minutos, dissecar os olhos para remover a córnea e do cristalino. Separa-se a retina a partir do epitélio pigmentado da retina (RPE) e esclera por corte em torno da ora serrata eo nervo óptico. Mergulhar em paraformaldeído a 4% durante mais 30 min para fixar a retina e a proteína fluorescente em células da retina transduzidas (fixação não deve ser superior a 24 horas). Lavar 5 min em PBS estéril, pH 7,4. </li><li> Mudar o tampão PBS para bloqueio (PBS com 1% de BSA, 2% de cabra normal ou de soro de burro, 0,5% Triton X-100), e incubar em tampão de bloqueio quer por 4 horas à temperatura ambiente ou a 4 ° C durante a noite. </li></ol></li><li> Incubar tecidos com anticorpos primários em tampão de bloqueio quer por 4 horas à temperatura ambiente ou a 4 ° C durante a noite. Lavar 3x com PBS. NOTA: Os anticorpos utilizados são os seguintes: anti-laminina (1 / 1.000) marcação dos ILM, clone anti-rodopsina 4D2 (1/500) hastes de rotulagem, anti-glutamina sintetase clone GS-6 (1 / 1.500) rotulagem células de Müller , PNA Lectin (1/40) cones de rotulagem. </li><li> Incubar tecidos com diluição 1: 500 de anticorpos secundários conjugados fluor alexa em tampão de bloqueio durante 1 h (criocortes) ou 2 hr (flatmounts) a tempe quartoratura e protegida da luz. Lavar 3x com PBS. </li><li> Fazer cortes de alívio para a retina e que flatmount numa lâmina de vidro quer com o foto-receptor ou de células ganglionares da retina (RGC) voltado para cima. Adicionar mountingmedium aquosa, aplique lamela e armazenar a 4 ° C. </li><li> Execute microscopia confocal de varredura a laser em microscópio confocal. Adquirir imagens em sequência, linha por linha, para reduzir a excitação e crosstalk emissão. Definir tamanho do passo de acordo com o teorema de amostragem de Nyquist-Shannon. Use as configurações de exposição que minimizem pixels supersaturados nas imagens finais. Processar imagens de 12 bits com FIJI, Z-seções do projeto em plano único com intensidade máxima sob função Z-projeto e finalmente converter-se ao modo de cores RGB de 8 bits. </li></ol> 4. Análise de PCR de rato As amostras de sangue <ol><li> Injectar 100 ul estoque contendo 1 a 4 10 x 11 partículas de um AAV-GFP na veia peniana de ratinhos C57BL6J anestesiados (5% de isofluranopara a indução de uma câmara de perto e 2,5% de isoflurano através de um cone de nariz durante a cirurgia), utilizando uma seringa de insulina com um ultra-fino 6 milímetros agulha. Este animal irá servir como um controlo positivo de partículas de AAV de circulação. </li><li> Amostra de sangue da cauda do rato antes da injeção e 3 horas, 24 horas, 2 dias, e 3 dias após ShH10 injeção intravítrea ou após a injeção intrapenile (Figura 1). Cerca de 10-20 ul é recolhido em tubos de heparina. Sacrifício ratos por inalação de CO2, após a conclusão do procedimento. </li><li> Extrair DNA genômico a partir de amostras de sangue, utilizando o kit de extração de sua escolha. </li><li> Efectue a amplificação por PCR de ADN genómico. Para este protocolo utilizar o seguinte par de iniciadores: GFP, sentido 5&#39;-CGACACAATCTGCCCTTTCG-3 &#39;, anti-sentido 5&#39;-CATGGACGAGCTGTACAAGGGA-3&#39;. O seguinte programa de PCR foi realizada: 95 ° C 2 min (1 ciclo); 95 ° C 45 seg, 55 ° C 1 min, 72 ° C 45 seg (30 ciclos); 72 ° C, 5 min (1 ciclo);4 ° C espera. </li></ol> 5. Método de Azul de Evans Opcional NOTA: Quantificar permeabilidade vascular medindo perda de albumina a partir de vasos sanguíneos na retina usando o método do azul de Evans 10. <ol><li> Prepare corante azul de Evans por dissolução em solução salina normal (6 mg / ml), ultra-sons durante 5 minutos num aparelho de limpeza de ultra-sons, e filtração através de um filtro de 5 | im. </li><li> Anestesiar ratos C57BL6J (isoflurano inalação, veja o passo 4.1), verifique a perda do reflexo de endireitamento, o reflexo de retirada e resposta cauda pitada indicando uma anestesia adequada, e injetar azul de Evans (45 mg / kg) através da veia peniana (Figura 1). Verifique se patas, focinho e orelhas de ratos tornam-se claramente azul seguinte Evans injeção de azul, confirmando a captação e distribuição do corante. </li><li> Anestesiar ratos C57BL6J 3 horas após a injecção do corante com cetamina (50 mg / kg) e xilazina (10 mg / kg) e verificar a perda de righting reflex indicando uma anestesia adequada. Amostra intracardial cerca de 500 ul de sangue em tubos de heparina e perfundir ratinhos durante 2 min através do ventrículo esquerdo com um tampão de citrato (0,05 M, pH 3,5; dissolve-se 7,8 g de ácido cítrico e 3,8 g de citrato de sódio em 1 L de água destilada) pré-aquecido a 37 ° C para impedir a vasoconstrição. Os ratinhos são sacrificados por perfusão. </li><li> Após a perfusão, enuclear e dissecar cuidadosamente ambos os olhos sob um microscópio cirúrgico para recolher as retinas (ver Secção 3.2). Retinas seco em um evaporador centrífugo durante a noite, pesar, e extrair o corante azul de Evans por incubação retinas com 100 ul de formamida durante 18 hr a 65 ° C com agitação (100 xg). Centrifugar as amostras da retina de 30K omega filtro em tubos 20,798.5 xg durante 2 horas a 4 ° C e as amostras de sangue centrifugar a 20,798.5 xg durante 15 min a 4 ° C. </li><li> Use os dois sobrenadantes para medir absorção. Determinar a absorvância de cada amostra subtraindo a absorvância máxima for azul de Evans a 620 nm para o mínimo de absorvência a 740 nm. Calcular a concentração azul de Evans no plasma e da retina a partir de uma curva padrão de azul de Evans em formamida. Expresse permeabilidade BRB em microlitros de azul de Evans por grama de retina seco por hora (l plasma / g de peso seco da retina / h). </li></ol> 6. Opcional Angiofluoresceinografia NOTA: O vazamento de vasos sanguíneos da retina em ratos podem ser visualizados por injeção intraperitoneal de fluoresceína. <ol><li> Anestesiar ratos C57BL6J (isoflurano inalação, veja o passo 4.1) e dilatar os alunos com colírio (neosynephrine e mydriaticum). Use vet pomada nos olhos para evitar a secura e sob anestesia. </li><li> Injectar por via intraperitoneal 0,01-0,02 ml de fluoresceína de sódio a 10% em 0,1 ml de solução salina estéril para angiofluoresceinografia. </li><li> Coletar imagens de ambos os olhos após a injeção, utilizando uma câmera de fundo de olho. Sacrifique ratos por inalação de CO2 após a proprocedimento. NOTA: 20 segundos são necessários para os vasos da retina para se tornar visível na angiografia. As imagens têm de ser capturado entre 3-10 min após a injecção máxima de fluoresceína. Os pontos de fuga (se houver) são observáveis ​​após 2,5 min. Em pontos de tempo posteriores a qualidade da imagem é perdida devido à fluoresceína de sódio difundir-se, assim, apenas as imagens vítreo, logo após obtidos são utilizados para a análise. </li></ol>

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The authors have nothing to disclose.

O BRB regula a troca de moléculas entre o sangue e da retina. A sua composição está associada com várias doenças, tais como a retinopatia diabética ou degeneração macular relacionada com a idade (AMD). Mostrámos recentemente que em um ratinho knock-out de distrofina, que exibe permeável BRB, a retina se torna mais permissiva para a entrega de genes mediada por vectores virais adeno-associados (AAV). No entanto, apesar BRB partículas de AAV permeabilidade injectado via intra-ocular permanecer confinadas ao co...

Células de Müller são o principal componente da glia da retina. Morfologicamente, a envolver o retina radialmente e sua endfeet, em contacto com o vítreo, enfrentar o ILM e componentes secretos do último. A ILM é uma membrana basal composta por cerca de dez diferentes proteínas da matriz extracelular (laminina, agrina, perlecano, nidogï¿½io, colagénio e proteoglicanos de sulfato de heparina vários). Durante o desenvolvimento, a sua presença é indispensável para a histogénese de retina, a navegação de axónios óptica, e a sobrevivência de células ganglionares 1-3. No entanto, não é essencial na ILM adulto retina e pode ser removido cirurgicamente em determinadas patologias, sem causar danos na retina 4. Em terapia génica, esta membrana torna-se uma barreira física para a transdução eficiente da retina utilizando AAV por injecção intravítrea 5. Através da vasta arborização de seus processos, as células de Müller fornecer suppo nutricional e regulamentarrt para ambos os neurónios da retina e células vasculares. Müller células estão também envolvidas na regulação da homeostase da retina, na formação e manutenção da BRB 6. Junções apertadas entre as células endoteliais dos capilares da retina, células de Müller, astrócitos e pericitos formar o BRB. BRB impede certas substâncias de entrar nas retina.In muitas doenças como a retinopatia diabética, oclusão da veia da retina e doenças respiratórias, hipóxia da retina provoca fuga através do BRB 7-9. Esta ruptura é associada com um aumento da permeabilidade vascular que conduz ao edema vasogénico, descolamento da retina e danos na retina. Células de Müller estão fortemente associados com os vasos sanguíneos e a membrana basal, desempenhando um papel importante tanto na integridade BRB e ILM. Consequentemente, marcação das células gliais de Müller é particularmente relevante para o estudo do estado físico dessas barreiras da retina. Clássicoaliado, BRB permeabilidade é medida utilizando o ensaio de azul de Evans que consiste em injecção sistémica de corante azul de Evans, que se liga de forma não covalente à albumina do plasma. Este ensaio mede a perda de albumina (proteína de tamanho intermediário, ~ 66 kDa) de vasos sanguíneos na retina (ver protocolos Seção 5) 10. Como alternativa, o vazamento vascular podem ser visualizados por angiografia fluorescência retinal atestando por vazamento de fluoresceína (molécula pequena, ~ 359 Da; consulte Protocolos Seção 6) 11. No entanto, ambos os métodos permitem a avaliação da permeabilidade BRB para pequenas moléculas e proteínas, mas eles não fornecem informações sobre a integridade ILM. Assim, para estudar BRB permeabilidade, foi utilizado um método com base de AAV que dá informação sobre a permeabilidade BRB para moléculas maiores (por exemplo, partículas de AAV, 25 nm de diâmetro). Partículas de efeito, o nosso método pode detectar a presença de AAV de transgene no sangue, o que sugere que ~ 25 nm de diâmetro fariaser capaz de se infiltrar na corrente sanguínea. Este método também proporciona informação sobre a estrutura da ILM e proteínas da matriz extracelular em condições patológicas. Duas variantes de AAV são úteis para tal estudo: AAV5 e ShH10. Subretinally injetado, AAV5 tem um tropismo natural para fotorreceptores e epitélio pigmentar da retina 12, mas não pode passar para a retina externa quando administrada no vítreo no tipo selvagem retinas com ILM intacta 5,13. ShH10 é uma variante de AAV que foi concebida especificamente para alvejar células gliais mais neurónios 14,15. ShH10 etiquetas selectivamente células de Müller em ambas as retinas saudáveis ​​e doentes com maior eficiência em retinas com barreiras comprometidos 16. Essas ferramentas virais juntamente com immuhistochemistry e análise em DNA de sangue fornecer informações sobre o estado das barreiras da retina e do seu envolvimento na doença (Figura 1).

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using adeno-associated virus as a tool to study retinal barriers in disease

Células de Müller são as principais células gliais da retina. Os finais pés formam os limites da retina nas membranas limitantes exteriores e interiores (ILM), e em conjunto com os astrócitos, células endoteliais e pericitos eles estabelecer a barreira hemato-retiniana (BRB). BRB limita o transporte de material entre a corrente sanguínea e da retina, enquanto o ILM actua como uma membrana basal que define histologicamente a fronteira entre a retina e a cavidade do vítreo. Rotular células de Müller é particularmente relevante para estudar o estado físico das barreiras da retina, uma vez que estas células são uma parte integrante da BRB e a ILM. Ambos BRB e ILM são freqüentemente alterados em doenças da retina e são responsáveis ​​por sintomas da doença. Existem vários métodos bem estabelecidos para estudar a integridade do BRB, tais como o ensaio de azul de Evans ou angiografia com fluoresceína. No entanto, estes métodos não fornecem informações sobre o grau de permeabilidade BRB to moléculas maiores, em escala nanométrica. Além disso, eles não fornecem informações sobre o estado de outras barreiras da retina, tais como a ILM. Para estudar a permeabilidade ao lado BRB ILM da retina, utilizou-se um método baseado AAV que fornece informações sobre a permeabilidade do BRB para moléculas maiores, enquanto que indica o estado das proteínas e de ILM da matriz extracelular em estados de doença. Duas variantes de AAV são úteis para tal estudo: AAV5 e ShH10. AAV5 tem um tropismo natural para os fotorreceptores, mas ela não pode passar para a retina externa, quando administrada no vítreo quando a ILM está intacto (ou seja, em retinas de tipo selvagem). ShH10 tem uma forte tropismo para células gliais e rotular selectivamente glia Müller em ambas as retinas saudáveis ​​e doentes. ShH10 prevê a entrega gene mais eficiente em retinas onde ILM está comprometida. Essas ferramentas virais juntamente com imunohistoquímica e-DNA do sangue análise lançar luz sobre o estado das barreiras da retina na doença.

Esperamos o aumento da transdução de células gliais da retina Müller ShH10 se utilizando o modelo animal mostra perturbações na estrutura da ILM (Figura 2A - B). Por exemplo, demonstramos que na ausência de DP71, alvos ShH10 especificamente, mas de forma mais eficiente Müller células gliais por injecção intravítrea, indicando um aumento da permeabilidade da ILM nesta linha de ratinhos em comparação com ratinhos de tipo selvagem 16 (Figura 2C - <...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study 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 ...

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14.9K visualizações.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. 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.

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

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

De Alta Eficiência Transdução de células de câncer de fígado por recombinante sorotipo de vírus adeno-associado 3 Vetores

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Medicina

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipagem espectral para o Estudo de Anomalias cromossômicas em humanos e camundongos com doença renal policística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Osteopático manipulativo tratamento como ferramenta adjuvante útil para Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

Retinografia colorida como uma ferramenta conveniente para estudar as respostas microvasculares a doenças cardiovasculares Fatores de Risco em estudos epidemiológicos

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

Adaptação Humanos videofluoroscópicos Andorinha Métodos de Estudo de detectar e caracterizar Disfagia em modelos de doenças Murino

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Desenvolvimento de um modelo de rato baseada alfa-sinucleína para a doença de Parkinson por meio de injecção estereotáxica de um vector viral recombinante adeno-associado

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Utilizando imagens da retina para estudar a demência

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Entrega de mediada por vírus adeno-associado de CRISPR para Gene cardíaco em ratos de edição

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

The Dyspepsia Educational Tool As a Novel Aid in Dyspepsia Management

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from this development, as it enables patient engagement in self-management. Dyspepsia is a condition thought to arise from gastric and duodenal perturbations, brain-gut axis disturbances, and dietary factors. Behavioral interventions are a major part of dyspepsia treatment, hence patient engagement and motivation through education is essential. Protocols that describe the development process of such educational tools are scarce. We provide a methodology describing development of a dyspepsia educational tool. Assessment of users' needs is the first step, followed by a literature search. The content is developed based on the main themes and entered into a content management system, to build the program. Final adjustments are made after a pilot test of the tool. The presented protocol can be used as a guide for development of a digital dyspepsia educational tool or as a tool for similar situations.

This protocol describes the development process of a digital dyspepsia educational tool. Assessment of unmet needs and literature, content development, and building of the tool are presented. The methodology can be used as a guide for future development of digital educational tools.

A ferramenta educacional da dispepsia como um auxílio novo na gerência da dispepsia

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Administração Subconjuntival de Vetores de Vírus Adenoassociados em Modelos de Pequenos Animais

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...