Este trabalho foi financiado pelo Instituto Karolinska KID-financiamento Autoridade de Protecção, sueco Radiação, Instituto Karolinska Eye Research Foundation, Gun och Bertil Stohnes Stiftelse, Olho St. Erik Hospital Research Foundation, Ögonfonden, Konung Gustav V: s och Drottning Viktorias Frimurarstiftelse.

1. A exposição à radiação ultravioleta <ol><li> 15 min antes da exposição, anestesiar uma ratazana Sprague-Dawley do sexo feminino, com uma mistura de 90 mg / kg de Ketalar (cetamina), e 10 mg / kg de Rompun (xilazina) por injecção intraperitoneal. </li><li> Colocar o animal em um limitador de rato e apertar as correias até a imobilização do rato sem provocar um aperto do tronco 13. </li><li> Instilar Mydriacyl (tropicamida), 10 mg / ml, em ambos os olhos dos ratos para induzir midríase. </li><li> Colocar o animal para que um olho está posicionado de encontro a um estreito feixe de RUV a 300 nm 14 nm com 10 largura total a metade do valor máximo e proteger o olho contralateral com fita adesiva preta. </li><li> Exponha o rato unilateralmente limiar sub-dose de 1 kJ / m 2 RUV-B a 300 nm para 15 min 15. </li></ol> 2. Dissecação <ol><li> Após pré-determinado intervalo de pós-exposição (1, 5, 24 e 120 horas), o sacrifício do rato de seis semanas de idade (peso &lt;200 g) por carbon asfixia e dióxido de deslocamento das vértebras cervicais. </li><li> Enuclear olhos. Depois disso, coloque a córnea do olho para baixo, lado posterior para cima. Em seguida, o perfurador de um furo através da aplicação de um mínimo de uma cânula de calibre 27 e empurre tangencialmente à esclerótica para evitar danificar a lente que é muito próximo da esclera. Em seguida, usar um par de tesouras de cirurgia oftálmica para cortar circunferencialmente logo atrás do limbo até que a porção posterior da esclerótica pode ser levantada. Depois, levante a lente com uma pinça sem corte curvas. </li><li> Remover restos do corpo ciliar do equador do cristalino sob um microscópio, mantendo a lente na solução de sal equilibrada de não mais do que 5-10 minutos. </li></ol> 3. RT-PCR quantitativa <ol><li> Colocar uma lente em uma mistura de 350 ul de tampão de lise RA1 (NucleoSpin ARN II de ARN total isolado kit) e 3,5 ul de β-mercaptoetanol em 2 ml de tubo Eppendorf. </li><li> Manter a lente nesta mistura durante 30 min à temperatura ambiente. </li><li> Homogeneizar a lente comuma agulha até que apenas o núcleo duro do cristalino é deixada entre a lente (córtex e da cápsula da lente são lisadas em tampão de lise). Remover o núcleo a partir da mistura. </li><li> Armazenar as amostras imediatamente a -70 ° C. </li><li> Descongelar as amostras e seguir o protocolo &quot;purificação RNA total a partir de células cultivadas e tecidos&quot; (NucleoSpin RNA II RNA total isolamento kit). </li><li> Armazenar as amostras de ARN a -70 ° C. </li><li> Para verificar a remoção suficiente de DNA de cada amostra, execute uma PCR sob as seguintes condições (passo 1: 95 ° C durante 2 min; passo 2 durante 40 ciclos de: 95 ° C durante 20 segundos, 55 ° C durante 20 seg, 72 ° C durante 20 segundos; passo 3: 72 ° C durante 7 min) com os iniciadores de ADN específicos de p53, para a frente 5&#39;-ACCCTCTGACCTTTTTCCCA-3 &#39;e inverso 5&#39;-TGCTGGGATCTTAGGCACTC-3&#39; e a Taq DNA polimerase (dNTPack), de acordo com a fabricação protocolo. O produto de PCR esperado é 243 pares de bases. </li><li> Executar uma electroforese em gel de agarose 1,5% para detectar ADN do produto de PCR específico de 243 pares de bases. Para preparar1,5% de agarose gel em tampão TBE ter 3,75 g de agarose e resolvê-lo em 250 ml de tampão TBE. Adicionar brometo de etídio (0,5 mg / ml) em 500 ul de 500 ml de solução a 1,5% de gel de agarose. Aquece-se a mistura num forno de microondas owen e agitar para resolver a agarose. </li></ol> Nenhuma das amostras tem de revelar quaisquer produtos de PCR específicos de DNA em electroforese em gel de agarose a 1,5%. <ol start="9"><li> Medir a concentração de amostras de RNA (1 ul) e a razão entre a absorvância da amostra a 260 nm (RNA) e 280 nm (de proteína) em um espectrofotômetro ND-1000 NanoDrop. Se o RNA relação à proteína de RNA absorvância da amostra é de 2,0 ou mais, em seguida, a amostra de RNA é puro. Se a razão de absorvância da amostra de RNA é menor do que 2,0, refazer o RNA etapa de purificação de 3,5. </li><li> Tomar um volume de amostra de ARN correspondente a 1 ug e sintetizar cDNA após a 1 ª Strand Kit protocolo de síntese de ADNc (1 st Strand Kit de síntese de ADNc por RT-PCR). </li><li> Armazenar as amostras de cDNA a -20 ° C(Por um período de um ou mais anos, armazenar a -70 ° C). </li><li> Executar quantitativa PCR em tempo real em iCycler única cor MyiQ Real Time sistema de detecção de PCR. Carga triplicados em placa de 96 poços com 1 ul da amostra de cDNA, TaqMan Gene Expression Mix Master, TaqMan Gene Expression Assay por caspase 3, de acordo com as instruções para a fabricação. Carga triplicados em outra placa de 96 poços com 1 ul da amostra de cDNA, TaqMan Gene Expression Mix Master, TaqMan Gene Expression Assay por 18s, de acordo com as instruções para a fabricação (TaqMan Gene Expression Assay Protocol). Diluir em série de uma fracção de cDNA a partir de três escolhidos aleatoriamente não expostas lentes. Executar diluições em série juntamente com o cDNA a partir de amostras em uma placa de 96 poços. </li><li> Utilizar o método de curva padrão para a quantificação dos resultados. O número de ciclos a fluorescência limiar é utilizado como a medida em software MyiQ. Uma curva padrão de expressar o número de ciclos de limiar como uma função da concentração relativa de calibrador é eslecido para as diluições em série em cada uma das placas. cDNA a partir de três amostras não tratadas foi de lente usada para calibração. Relacionar o número limite de ciclos para cada amostra com a curva padrão para se obter a concentração relativa da amostra de cDNA medido. Finalmente, obter o nível de expressão dos genes alvo através da relação entre a concentração relativa do alvo de cDNA para os 18s de controlo interno de cDNA. </li></ol> 4. A coloração imuno-histoquímica <ol><li> Fixação <ol><li> Dissecar o olho em PBS (Tampão fosfato salino, pH 7,4). </li><li> Colocar o olho em um tubo cheio com paraformaldeído a 4% (PFA) em frio de gelo durante 20 min. Prepare PFA 4% no dia anterior. Armazenar a 4 ° C até à sua utilização. PFA é fresco por cerca de uma semana. </li><li> Remover PFA com uma micropipeta. Em seguida, lava-se com PBS arrefecido em gelo durante 20 min. </li><li> Colocar o olho em sacarose a 30%, a +4 ° C durante a noite. </li><li> Colocar o olho em um copo cheio de temperatura de corte ideal (outubro) e médio (Tissue-Tek). Colocar o olho em posição adequada para o corte. </li><li> Congelar em OCT médio sobre gelo seco. </li><li> Armazenar a -70 ° C até à sua utilização. </li></ol></li><li> Seccionamento <ol><li> Posicione o olho para crio-corte em um plano apropriado. </li><li> Corte três 5 mm de espessura médio-sagital secções de uma porção central de cada uma das lentes. Descartar pelo menos seis trechos entre seções seqüenciais para evitar manchar os mesmos núcleos em diferentes seções. </li><li> Uma vez que a seção tenha sido cortada, coloque delicadamente um slide em cima dela. A seção deve, então, ficar com o slide. </li><li> Deixar as lâminas secar ao ar antes da utilização. As lâminas podem ser armazenadas a -20 ° C até serem necessárias. </li></ol></li><li> Fluorescência imunohistoquímica <ol><li> Deixe as amostras atingir a temperatura ambiente (5-10 min). </li><li> Desenhar bordas em torno dos espécimes com uma caneta PAP-(Invitrogen). </li><li> Deixe que a fronteira seco durante algum tempo (10 min). </li><li> Rotular as lâminas de microscópio. </li><li> Rehidratar em 1 × PBS durante 15 min. </li><li> Permeabilizar a solução padrão para bloco de 30 min. </li><li> Adicionar anticorpo primário (caspase-3 de coelho, policlonal de anticorpos clivados Asp175 9661; tecnologia de sinalização celular, Inc) diluída 1:3.000 em solução bloco padrão. </li><li> Armazenar numa câmara humidificada a 4 ° C durante a noite. </li><li> Lavar com PBS por pipetagem (3 x 5 min) ou por imersão num banho de grandes dimensões (&gt; 15 min, 1-2 alterações). </li><li> Adicionar anticorpo secundário (anticorpo de coelho anti secundária com um espectro de absorção / emissão específico) diluído 1:300 em solução de bloqueio padrão. </li><li> Lavar com PBS por pipetagem (3 x 5 min) ou por imersão num banho de grandes dimensões (&gt; 15 min, 1-2 alterações). </li><li> Limpe PAP pen-manchas. </li><li> Armazenar numa câmara húmida à temperatura ambiente durante 3 hr. Evitar a exposição à luz. </li><li> Adicionar uma gota de Vectashield e colocar uma lamela sobre a lâmina. Evite fazer bolhas. </li><li> Deixe secar a camada Vectashield (~ 20 min). </li><li> Mantenha seções in no escuro até a análise. </li><li> Todas as secções de controlo são tratados, na ausência de anticorpo primário. </li><li> Olhe para os resultados dentro de algumas horas sob um microscópio de fluorescência. </li><li> Conte núcleos de células epiteliais &quot;de um arco lateral nuclear para outro lado arco nuclear de cada lente. Aplicar o filtro padrão azul para ver todos os núcleos lente epitelial em azul e um filtro padrão que se encaixa a emissão do anticorpo secundário para ver os núcleos caspase-3 positiva em verde. </li><li> Registar o número de todos os núcleos lente epiteliais e o número de núcleos positivos. Conte as células três vezes para cada seção. </li></ol></li></ol>

<ol>
	<li>Brian, G., Taylor, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cataract+blindness:+challenge+for+the+21+st+century.">Cataract blindness: challenge for the 21 st century.</a> Bulletin of the World Health Organization. 79, 249-256 (2001).</li><li>Jose, J. G., Pitts, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength+dependency+of+cataracts+in+albino+mice+following+chronic+exposure.">Wavelength dependency of cataracts in albino mice following chronic exposure.</a> Experimental Eye Research. 41, 545-563 (1985).</li><li>S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+cataract+in+the+rat+after+exposure+to+radiation+in+the+300+nm+wavelength+region.+A+study+of+the+macro-,+micro-+and+ultrastructure.">Acute cataract in the rat after exposure to radiation in the 300 nm wavelength region. A study of the macro-, micro- and ultrastructure.</a> Acta Ophthalmol. (Copenh). 66, 141-152 (1988).</li><li>Meyer, L., Dong, X., Wegener, A., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+dependent+cataractogenesis+and+Maximum+Tolerable+Dose+(MTD+2.3:16)+for+UVR+-+B+induced+cataract+in+C57BL/6J+mice.">Dose dependent cataractogenesis and Maximum Tolerable Dose (MTD 2.3:16) for UVR - B induced cataract in C57BL/6J mice.</a> Experimental Eye Research. 86, 282-289 (2008).</li><li>McCarty, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lifetime+ocular+exposure+to+UV-B:+the+Melbourne+visual+impairment+project.">Assessment of lifetime ocular exposure to UV-B: the Melbourne visual impairment project.</a> Developments in Ophthalmology. 27, 9-13 (1997).</li><li>Sasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+cortical+cataract+in+subjects+of+diverse+races+and+latitude.">Localization of cortical cataract in subjects of diverse races and latitude.</a> Invest. Ophthalmol. Vis. Sci. 44, 4210-4214 (2003).</li><li>S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cataract+induced+by+ultraviolet+radiation.">Experimental cataract induced by ultraviolet radiation.</a> Acta Ophthalmol. (Copenh. 68, 1-77 (1990).</li><li>Galichanin, K., L&#246;fgren, S., Bergmanson, J., S&#246;derberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+damage+in+the+lens+after+in+vivo+close+to+threshold+exposure+to+UV-B+radiation:+cytomorphological+study+of+apoptosis.">Evolution of damage in the lens after in vivo close to threshold exposure to UV-B radiation: cytomorphological study of apoptosis.</a> Exp. Eye Res. 91, 369-377 (2010).</li><li>McCarty, C. A., Taylor, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+epidemiologic+evidence+linking+ultraviolet+radiation+and+cataracts.">A review of the epidemiologic evidence linking ultraviolet radiation and cataracts.</a> Dev. Ophthalmol. 35, 21-31 (2002).</li><li>Li, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+epithelial+cell+apoptosis+appears+to+be+a+common+cellular+basis+for+non-congenital+cataract+development+in+humans+and+animals.">Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals.</a> Journal of Cell Biology. 130, 169-181 (1995).</li><li>Michael, R., Vrensen, G., van Marle, J., Gan, L., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+rat+lens+after+in+vivo+threshold+dose+ultraviolet+irradiation.">Apoptosis in the rat lens after in vivo threshold dose ultraviolet irradiation.</a> Investigative Ophthalmology and Visual Science. 13, 2681-2687 (1998).</li><li>Ayala, M. N., Strid, H., Jacobsson, U., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+Expression+and+Apoptosis+In+The+Lens+After+Ultraviolet+Radiation+Exposure.">p53 Expression and Apoptosis In The Lens After Ultraviolet Radiation Exposure.</a> Investigative ophthalmology of visual science. 48, 4187-4191 (2007).</li><li>Galichanin, K., Wang, J., Lofgren, S., Soderberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+universal+rat+restrainer+for+ophthalmic+research.">A new universal rat restrainer for ophthalmic research.</a> Acta Ophthalmol. 89 (1), (2011).</li><li>Ayala, M. N., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cataract+after+repeated+exposure+to+ultraviolet+radiation.">In vivo cataract after repeated exposure to ultraviolet radiation.</a> Experimental Eye Research. 70, 451-456 (2000).</li><li>S&#246;derberg, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+ultraviolet+radiation+exposure+to+the+lens+expressed+by+maximum+tolerable+dose+(MTD).">Toxicity of ultraviolet radiation exposure to the lens expressed by maximum tolerable dose (MTD).</a> Developments in Ophthalmology. 35, 70-75 (2002).</li><li>Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+repair+of+cataract+induced+by+ultraviolet+radiation.">Development and repair of cataract induced by ultraviolet radiation.</a> Ophthalmic Research. 32, 1-45 (2000).</li><li>L&#246;fgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cataract from ultraviolet radiation. , (2001).</li><li>Ayala, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influence of exposure patterns and oxidation in UVR induced Cataract. , (2005).</li><li>Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safety limit estimation for cataract induced by ultraviolet radiation. , (2005).</li><li>L&#246;fgren, S., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+age+and+sex+in+ultraviolet+radiation+cataract+in+the+rat.">Impact of age and sex in ultraviolet radiation cataract in the rat.</a> Investigative Ophthalmology and Visual Science. 44, 1629-1633 (2003).</li><li>Ayala, M. N., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+exposure+time+for+UV+radiation-induced+cataract.">Influence of exposure time for UV radiation-induced cataract.</a> Investigative Ophthalmology and Visual Science. 41, 3539-3543 (2000).</li></ol>

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<html> Este artigo descreve métodos que permitem estudar eventos moleculares que ocorrem durante a catarata induzida UVR-B. Considerando, que a maioria das informações disponíveis para in vivo UVR catarata induzida foi derivada a partir de experiências sobre albino Sprague-Dawley 7, 16, 17, 18, ​​19, decidimos usar o rato Sprague-Dawley albinos, no presente estudo. Idade dos ratos foi de seis semanas de idade. O gênero foi escolhido para ser fêmea, porque, em co...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Ketalar </td><td> Pfizer </td><td> 150086 </td><td> 50 mg / ml </td></tr><tr><td> Rompun </td><td> Bayer </td><td> 022545 </td><td> 20 mg / ml </td></tr><tr><td> Simplex Oculentum </td><td> Apoteket, Suécia </td><td> 336164 </td><td> 5 g </td></tr><tr><td> Mydriacyl </td><td> Alcon </td><td> 00352 </td><td> 10 mg / ml </td></tr><tr><td> Solução salina balanceada </td><td> Alcon </td><td> 0007950055 </td><td></td></tr><tr><td> 3,5 ul β-mercaptoetanol </td><td></td><td></td><td></td></tr><tr><td> NucleoSpin ARN II de isolamento de ARN total kit </td><td> Macherey-Nagel GmbH &amp; Co, Duren, Alemanha </td><td> 740.955,50 </td><td></td></tr><tr><td> p53 iniciadores de ADN específicos </td><td> biomers.net GmbH </td><td> Feitos </td><td></td></tr><tr><td> Taq DNA polimerase, dNTPack </td><td> Roche </td><td> 04 728 866 001 </td><td></td></tr><tr><td> Agarose </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Solução de brometo de etídio 0,5 mg / ml </td><td> Sigma </td><td> E1385 </td><td></td></tr><tr><td> Nano Drop-ND-1000 espectrofotômetro </td><td> Produtos Nanodrop </td><td></td><td></td></tr><tr><td> 1 st Strand kit de síntese de ADNc por RT-PCR (AMV) </td><td> Roche Diagnostics GmbH </td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ única cor em tempo real sistema de detecção de PCR </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr> &lt;tr&gt; <td> Placa de 96 poços </td><td> Sarstedt </td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan Gene Expression Master Mix </td><td> Applied Biosystems </td><td> 4369016 </td><td></td></tr><tr><td> TaqMan Gene Expression Assay da caspase 3 </td><td> Applied Biosystems </td><td> Rn00563902_m1 </td><td></td></tr><tr><td> Ensaio TaqMan Gene Expression para maiores de 18 </td><td> Applied Biosystems </td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ software </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr><tr><td> Caspase-3 clivada (Asp175) </td><td> Tecnologia de sinalização celular </td><td> 9661 </td><td></td></tr><tr><td> Anti IgG de coelho </td><td> Abcam </td><td> Ab6798 </td><td></td></tr><tr><td> Sacarose </td><td> Sigma Aldrich </td><td> 84097 </td><td></td></tr&gt; <tr><td> Vectashield </td><td> Vector Laboratories </td><td></td><td></td></tr><tr><td> Microscópio Universal Axioplan 2 Imagem </td><td> Carl Zeiss </td><td></td><td></td></tr><tr><td> Paraformaldeído </td><td> Sigma Aldrich </td><td> 441244 </td><td></td></tr><tr><td> Tampão TBE 10x </td><td> Promega </td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

A catarata é a principal causa de cegueira no mundo 1. A Organização Mundial de Saúde define catarata como uma turvação da lente do olho, o que impede a transferência da luz. A catarata é uma doença multi-factorial associada com diabetes, o tabagismo, radiação ultravioleta (RUV), o álcool, a radiação ionizante, esteróides e hipertensão. Há evidências experimentais 2-4 e epidemiológica forte que 5,6 UVR provoca catarata. Foi desenvolvido um modelo animal para a RUV B catarata induzida em ambos com 7 anestesiados e não anestesiados animais 8. A única cura para a catarata é a cirurgia, mas este tratamento não é acessível a todos. Estimou-se que o atraso de aparecimento da catarata por 10 anos poderia reduzir a necessidade de cirurgia de catarata em 50% 9. Para atrasar a incidência de catarata, que é necessária para compreender os mecanismos de formação de catarata e encontrar estratégia de prevenção eficazgias. Entre os mecanismos para o desenvolvimento de cataratas, a apoptose desempenha um papel crucial na iniciação da catarata em humanos e animais 10. Nosso foco tem sido recentemente apoptose na lente como o mecanismo para o desenvolvimento de cataratas 8,11,12. Prevê-se que uma melhor compreensão do efeito da radiação UV sobre a via de apoptose proporcionará possibilidades para a descoberta de novos fármacos para prevenir a catarata. Neste artigo, nós descrevemos como catarata pode ser induzida experimentalmente pela exposição in vivo a RUV-B. Além disso RT-PCR e imuno-histoquímica estão apresentados como ferramentas para o estudo dos mecanismos moleculares de catarata induzida por radiação UV-B.

As várias fontes de variação nas medidas foram estimadas com uma análise de variância, e verificou-se que, considerando três medidas por animal da variância para as medições foi da ordem de 15% do que para os animais. Assim, tendo em vista a análise da lente inteira, não é possível aumentar a precisão. Testes ortogonal elucidado um contraste estatisticamente significativo para a caspase-3 de mensagem entre 120 intervalo de latência hr vs intervalos mais curtos de latência. <e...

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Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

A catarata é a principal causa de cegueira no mundo. Radiação solar ultravioleta (UVR) é o principal fator de risco para o desenvolvimento de catarata. Um modelo animal de longe RUV-B catarata induzida foi desenvolvido. Neste artigo vamos descrever os métodos de investigação de formação de catarata: a exposição a raios ultravioletas, RT-PCR e imuno-histoquímica.

Caracterização de mecanismos moleculares de<em&gt; Em vivo</em&gt; RUV catarata induzida

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

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Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K visualizações.  Karolinska Institutet. A catarata é a principal causa de cegueira no mundo. Radiação solar ultravioleta (UVR) é o principal fator de risco para o desenvolvimento de catarata. Um modelo animal de longe RUV-B catarata induzida foi desenvolvido. Neste artigo vamos descrever os métodos de investigação de formação de catarata: a exposição a raios ultravioletas, RT-PCR e imuno-histoquímica.

Vídeo: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared fluorescência de imagem intravascular (NIRF) Molecular da placa inflamatória, uma Abordagem Multimodal para imagem da Aterosclerose

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Medicina

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Um In vivo Modelo de Contração de roedores induzida por lesões e não-invasivos acompanhamento da recuperação

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI para Rastreamento celular

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Profiling Molecular do microambiente do tumor invasivo em um modelo 3-Dimensional de células de câncer colorretal e<em&gt; Ex vivo</em&gt; Os fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Induzida pela quimioterapia Vascular Toxicidade - em tempo real<em&gt; In vivo</em&gt; Imagem de Impairment Vessel

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Criação e Caracterização da UTI e CAUTI em um modelo de rato

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo de deteção de Mitophagy em células humanas, C. Eleganse os ratos

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Um modelo de murina induzida por doxorubicina de cardiomiopatia dilatada em Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Geração e Caracterização do Infarto do Miocárdio Ventricular Direito Induzido pela Ligação Permanente da Artéria Coronária Direita em Camundongos

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...