This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

O protocolo a seguir está em conformidade com os Animal Care Institucional e Comitê de Uso orientações Thomas Jefferson University e com a Declaração de ARVO para o Uso de Animais em Vision Research. 1. Configuração e Preparação de Lentes de Cultura Ex Vivo de Feridas <ol><li> Coloque três pratos de 100 mm de petri em um estéril, capela de fluxo laminar. Encha duas das placas de Petri até meio com tampão Tris / dextrose (tampão TD; 140 mM de NaCl, 5 mM de KCl, 0,7 mM de Na 2 PO 4, 5 mM de D-glucose, 8,25 mM de Tris base, pH a 7,4 com HCl) à TA , deixando a terceira vazio. Meios de cultura de pré-aquecer (Media suplementado com 199, 1% de L-glutamina e 1% de penicilina / estreptomicina) a 37 ° C. Nota: O padrão de cura de feridas é o meio de cultura isento de soro, como ocorre in vivo; no entanto, as culturas de reparação da ferida podem ser cultivadas com sucesso em condições de meios definidos, que incluem soro ou outros factores. </li><li> Remover fértil d embrionárioay 15 clara de ovo pintainho leghorn da incubadora (realizada em 37,7 ° C com agitação suave) </li><li> Coloque o ovo selecionado na capela de fluxo laminar e limpo fora do shell com etanol a 70% a partir de um frasco de lavagem. Realizar todos os procedimentos a seguir em condições de assepsia na câmara de fluxo laminar, usando soluções e instrumentos estéreis. </li><li> Craque conteúdo dos ovos e colocar no vazio 100 milímetros placa de Petri. Decapitar embrião usando uma pinça e tesouras padrão finas. Coloque a cabeça de embrião de pinto em uma placa de Petri contendo tampão de TD e descartar adequadamente o restante do embrião. Opcionalmente manter as cabeças de embrião de pinto em tampão de TD por um curto período de tempo, não mais do que 15 min. </li><li> Coloque a cabeça de embrião de galinha em uma tampa de placa de Petri. Utilizando uma pinça de alta precisão, remova a lente junto com seu humor vítreo do olho ligados na seguinte sequência. Comprima a parte posterior do olho com a pinça para criar uma pequena abertura na parte posterior do olho. </li><li> Em seguida, segure o humor vítreo with fórceps e puxar suavemente no vítreo com um movimento de rolamento, o vítreo com a lente vai ser ligado desalojado do olho. Lugar da lente / vítreo na placa de Petri contendo tampão restante TD. Permitir que as lentes permaneçam em tampão TD durante o período de 30 min. </li><li> Mover a lente para uma nova tampa de placa de petri sob um microscópio de dissecação. Deste ponto em diante executar todas as etapas sob um microscópio de dissecação. Com uma pinça de alta precisão cuidadosamente afastar qualquer corpo ciliar (células pigmentadas) que foram desalojados com a lente usando a ponta da pinça, tomando cuidado para não danificar o tecido da lente. Nota: A remoção do corpo ciliar assegura que os tipos de células que não são endógenos às lentes não estão incluídos na cultura da reparação da ferida. </li><li> Separa-se a lente do humor vítreo com uma pinça de alta precisão por beliscar fora do corpo vítreo de sua associação com a cápsula posterior do cristalino. </li><li> Usando alta precisão forceps transferir a lente em uma pequena gotade tampão de TD (cerca de 200 ul) num prato de cultura de tecidos 35 milímetros. </li></ol> 2. realização de cirurgia de catarata Mock <ol><li> Orientar a lente na gota de tampão de TD no prato de 35 mm com o aspecto anterior da lente voltada para cima. Nota: A anterior da lente pode ser facilmente identificados pela presença de um anel denso no tecido que regista a fronteira entre a região anterior e equatorial do epitélio da lente. Em contraste, há uma ausência de marcas sobre a parte posterior da cápsula da lente para que as células das fibras da lente estão ligados. </li><li> Usando duas pinças de alta precisão fazer uma pequena incisão (aproximadamente 850μm) no centro da cápsula anterior da lente, a espessura da membrana basal que circunda a lente de tecido, e a sua associada epitélio anterior do cristalino, por agarrar o tecido com uma pinça em cada mão e puxando suavemente em direções opostas. </li><li> Remover a massa celular da fibra, que constitui a maior parte do tecido da lente, dislodging-lo de suas ligações com o epitélio da lente e circundante cápsula do cristalino por hydro-eluição (uma abordagem usada em cirurgia de catarata clássico, modelado na Figura 1A). </li><li> Encher uma seringa de 1 mL com uma ponta de agulha de 27,5 L com 300 ul de tampão de TD. Inserir a ponta da agulha dentro da incisão feita na cápsula anterior do cristalino, e cerca de metade para a lente. </li><li> Pressione levemente a seringa injetar o tampão TD na massa celular fibra lente. Injectar entre 50 e 200 ul TD, e nunca mais de 300 ul. Observe a massa celular fibra soltando-se do epitélio e lente cápsula. </li><li> Utilizando uma pinça de alta precisão, retire a massa celular fibra soltos da lente através do local da incisão anterior. Nota: Este procedimento deixa a cápsula posterior do cristalino membrana basal ao qual as células de fibra tinha sido anexado desnudada de células, e um epitélio da lente ferido apenas adjacente a este site. </li></ol> 3. Preparing o Lens Ferido para Ex Cultura Vivo <ol><li> Nivelar o saco capsular da lente, que resulta da cirurgia da catarata descrito acima no prato de cultura, o lado das células para cima, fazendo cinco cortes na face anterior do saco capsular. </li><li> Corte perpendicular ao local da incisão original através do equador do cristalino. Achate as resultantes cinco &quot;retalhos&quot; da cápsula do cristalino com epitélio anexado no lado da cultura cápsula prato para baixo, celular voltado para cima. Note-se a lente ex vivo feridos agora deve assumir uma forma em estrela ou flowerlike (ver Figura 1B). </li><li> Para fixar a cápsula para o prato, pressione suavemente para baixo com a pinça em cada ponto da estrela. Isso fará com que um pequeno recuo para as cinco dicas mais fora do explante e resultar em anexo sustentado para o prato. Nota: É possível danificar a cápsula durante este processo e por isso é importante para assegurar que a cápsula se o prato mais próximo possível das pontas das flaPS quanto possível, bem como para tornar a menor quantidade de pontos de fixação quanto possível (em geral duas, máximo de três por retalho). </li><li> Remover o tampão de TD no prato de 35 mm e substituí-la com 1,5 ml de meio de pré-aquecido. Cobrir o prato de 35mm com a sua tampa e lugar na incubadora (37 ° C, 5% CO 2). </li></ol> 4. Separação da Zona de Migração Central (CMZ), onde Re-epitelização da área ferida do Posterior Capsule Ocorre, do anexo original Zone (OAZ) de lente células epiteliais, para análise quantitativa. Nota: As células começam a mover-se para a região de CMZ imediatamente em resposta à lesão. Por um dia em cultura células suficientes estão migrando através do CMZ para análise molecular e bioquímica, após a separação da CMZ eo OAZ por micro-dissecção 23. Este protocolo envolve a remoção de uma aba (OAZ) de cada vez a partir da área ferida da cápsula. <ol><li> Observar uma demarcatião, claramente visíveis ao microscópio de dissecação, e entre o OAZ CMZ (ver Figura 2A). Usando duas pinças de alta precisão, agarrar com as duas pinças na extremidade da linha OAZ / CMZ, apenas um lado para o outro, em ambos os lados da linha (ver figura 2A, B, seta). </li><li> Usando uma mão / um fórceps no lado da CMZ continuar a sustentar a cultura feridos, enquanto com a outra mão / pinça, puxe o OAZ ao longo da linha OAZ / CMZ. O CMZ facilmente se separa do OAZ ao longo desta linha. Continue ao longo desta linha em torno de toda a cultura até que as duas regiões são completamente separados. </li><li> Estude as fracções separadas OAZ e CMZ para a análise molecular, tais como 24 ou bioquímica análise de RNA-Seq como western blot ou co-imunoprecipitação 23,25. </li></ol>

<ol>
	<li>Walker, J. L., 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=Unique+precursors+for+the+mesenchymal+cells+involved+in+injury+response+and+fibrosis.">Unique precursors for the mesenchymal cells involved in injury response and fibrosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 13730-13735 (2010).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nature reviews. Molecular cell biology. 10, 445-457 (2009).</li><li>Riahi, R., Yang, Y., Zhang, D. D., Wong, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+wound-healing+assays+for+probing+collective+cell+migration.">Advances in wound-healing assays for probing collective cell migration.</a> Journal of laboratory automation. 17, 59-65 (2012).</li><li>Khalil, A. A., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+leader+cells+in+collective+cell+migration.">Determinants of leader cells in collective cell migration.</a> Integrative biology : quantitative biosciences from nano to macro. 2, 568-574 (2010).</li><li>Walker, J. L., Wolff, I. M., Zhang, L., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+SRC+kinases+signals+induction+of+posterior+capsule+opacification.">Activation of SRC kinases signals induction of posterior capsule opacification.</a> Investigative ophthalmology &amp; visual science. 48, 2214-2223 (2007).</li><li>Sta Iglesia, D. D., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+basement+membrane+after+corneal+debridement.">Disruption of the basement membrane after corneal debridement.</a> Investigative ophthalmology &amp; visual science. 41, 1045-1053 (2000).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Brown, M., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+the+study+of+recurrent+corneal+epithelial+erosions:+alpha9beta1+integrin+implicated+in+progression+of+the+disease.">A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease.</a> Investigative ophthalmology &amp; visual science. 45, 1775-1788 (2004).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+the+basement+membrane+enhances+corneal+wound+healing.">Removal of the basement membrane enhances corneal wound healing.</a> Experimental eye research. 93, 927-936 (2011).</li><li>Stepp, M. A., 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=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Experimental eye research. 121, 178-193 (2014).</li><li>Kuwabara, T., Perkins, D. G., Cogan, 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=Sliding+of+the+epithelium+in+experimental+corneal+wounds.">Sliding of the epithelium in experimental corneal wounds.</a> Investigative ophthalmology. 15, 4-14 (1976).</li><li>Sherrard, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+endothelium+in+vivo:+its+response+to+mild+trauma.">The corneal endothelium in vivo: its response to mild trauma.</a> Experimental eye research. 22, 347-357 (1976).</li><li>Stramer, B. M., Zieske, J. D., Jung, J. C., Austin, J. S., Fini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+controlling+the+fibrotic+repair+phenotype+in+cornea:+implications+for+surgical+outcomes.">Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes.</a> Investigative ophthalmology &amp; visual science. 44, 4237-4246 (2003).</li><li>Escamez, M. J., 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=An+in+vivo+model+of+wound+healing+in+genetically+modified+skin-humanized+mice.">An in vivo model of wound healing in genetically modified skin-humanized mice.</a> The Journal of investigative dermatology. 123, 1182-1191 (2004).</li><li>Werner, S., Breeden, M., Hubner, G., Greenhalgh, D. G., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+keratinocyte+growth+factor+expression+is+reduced+and+delayed+during+wound+healing+in+the+genetically+diabetic+mouse.">Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse.</a> The Journal of investigative dermatology. 103, 469-473 (1994).</li><li>Tarin, D., Croft, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+of+wound+healing+in+mouse+skin.+II.+Dermo-epidermal+interrelationships.">Ultrastructural studies of wound healing in mouse skin. II. Dermo-epidermal interrelationships.</a> Journal of anatomy. 106, 79-91 (1970).</li><li>Croft, C. B., Tarin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+of+wound+healing+in+mouse+skin+I.+Epithelial+behaviour.">Ultrastructural studies of wound healing in mouse skin I. Epithelial behaviour.</a> Journal of anatomy. 106, 63-77 (1970).</li><li>Winstanley, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+reaction+in+the+healing+of+excised+cutaneous+wounds+in+the+dog.">The epithelial reaction in the healing of excised cutaneous wounds in the dog.</a> Journal of comparative pathology. 85, 61-75 (1975).</li><li>Wormstone, I. M., Wride, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ocular+lens:+a+classic+model+for+development,+physiology+and+disease.">The ocular lens: a classic model for development, physiology and disease.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 366, 1190-1192 (2011).</li><li>Danysh, B. P., Duncan, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+capsule.">The lens capsule.</a> Experimental eye research. 88, 151-164 (2009).</li><li>Awasthi, N., Guo, S., Wagner, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+capsular+opacification:+a+problem+reduced+but+not+yet+eradicated.">Posterior capsular opacification: a problem reduced but not yet eradicated.</a> Archives of ophthalmology. 127, 555-562 (2009).</li><li>Walker, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+attempts+to+reduce+posterior+capsule+opacification+after+cataract+surgery--a+review.">Pharmacological attempts to reduce posterior capsule opacification after cataract surgery--a review.</a> Clinical &amp; experimental ophthalmology. 36, 883-890 (2008).</li><li>Schmidbauer, J. M., 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=Posterior+capsule+opacification.">Posterior capsule opacification.</a> International ophthalmology clinics. 41, 109-131 (2001).</li><li>Menko, A. S., 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=A+central+role+for+vimentin+in+regulating+repair+function+during+healing+of+the+lens+epithelium.">A central role for vimentin in regulating repair function during healing of the lens epithelium.</a> Molecular biology of the cell. 25, 776-790 (2014).</li><li>Chauss, D., 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=Differentiation+state-specific+mitochondrial+dynamic+regulatory+networks+are+revealed+by+global+transcriptional+analysis+of+the+developing+chicken+lens.">Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens.</a> G3 (Bethesda). 4, 1515-1527 (2014).</li><li>Leonard, M., Zhang, L., Bleaken, B. M., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+N-Cadherin+linked+c-Src+and+fyn+kinases+in+lens+development.">Distinct roles for N-Cadherin linked c-Src and fyn kinases in lens development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 242, 469-484 (2013).</li><li>Sieg, D. J., 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=FAK+integrates+growth-factor+and+integrin+signals+to+promote+cell+migration.">FAK integrates growth-factor and integrin signals to promote cell migration.</a> Nature cell biology. 2, 249-256 (2000).</li><li>Sieg, D. J., Hauck, C. R., Schlaepfer, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Required+role+of+focal+adhesion+kinase+(FAK)+for+integrin-stimulated+cell+migration.">Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration.</a> Journal of cell science. 112 (Pt 16), 2677-2691 (1999).</li><li>Hauck, C. R., Hsia, D. A., Schlaepfer, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+focal+adhesion+kinase--a+regulator+of+cell+migration+and+invasion).">The focal adhesion kinase--a regulator of cell migration and invasion).</a> IUBMB life. 53, 115-119 (2002).</li><li>Zhao, X., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+kinase+and+its+signaling+pathways+in+cell+migration+and+angiogenesis.">Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis.</a> Advanced drug delivery reviews. 63, 610-615 (2011).</li><li>Menko, A. S., Bleaken, B. M., Walker, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional-specific+alterations+in+cell-cell+junctions,+cytoskeletal+networks+and+myosin-mediated+mechanical+cues+coordinate+collectivity+of+movement+of+epithelial+cells+in+response+to+injury.">Regional-specific alterations in cell-cell junctions, cytoskeletal networks and myosin-mediated mechanical cues coordinate collectivity of movement of epithelial cells in response to injury.</a> Experimental cell research. 322, 133-148 (2014).</li><li>Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing--aiming+for+perfect+skin+regeneration.">Wound healing--aiming for perfect skin regeneration.</a> Science. 276, 75-81 (1997).</li><li>Ferguson, M. W., O'Kane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scar-free+healing:+from+embryonic+mechanisms+to+adult+therapeutic+intervention.">Scar-free healing: from embryonic mechanisms to adult therapeutic intervention.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 359, 839-850 (2004).</li><li>Redd, M. J., Cooper, L., Wood, W., Stramer, B., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+inflammation:+embryos+reveal+the+way+to+perfect+repair.">Wound healing and inflammation: embryos reveal the way to perfect repair.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 359, 777-784 (2004).</li><li>Nodder, S., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+embryos:+a+review.">Wound healing in embryos: a review.</a> Anatomy and embryology. 195, 215-228 (1997).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li></ol>

The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

A, a cirurgia de catarata simulada clinicamente relevante, ex vivo epitelial modelo cicatrização de feridas aqui descrito foi desenvolvido para fornecer uma ferramenta para investigar os mecanismos que regulam a reparação de tecidos epiteliais, em resposta a uma lesão. Os principais recursos que foram destinados para na criação deste modelo incluiu uma condições) que prevêem que de perto replicados a resposta in vivo para ferindo em uma configuração de cultura, 2) facilidade de modular os elementos reguladores de reparação, e 3) capacidade de imagem do processo de reparação, na sua totalidade, em tempo real. O desafio, portanto, era criar um modelo de cultura em que foi possível estudar e manipular, reparação epitelial no microambiente nativo das células. A disponibilidade deste modelo ferida-reparação abre novas possibilidades para identificar os sinais de sinalização endógenas de matriz proteínas, citocinas e quimiocinas, que regulam o processo de reparação. Além disso, o modelo é ideal para examinar como umn epitélio é capaz de mover-se como uma folha colectivo para re-epithelialize área da ferida 2,3, e para determinar a linhagem de células mesenquimais líder no bordo da ferida que funcionará em colectivo dirigir a migração do epitélio ferido 4. Este modelo também fornece uma plataforma com a qual identificar terapêuticas que possam promover a cicatrização de feridas eficaz e evitar que a reparação de feridas aberrante 5. Há já uma série de modelos ferida de reparação disponíveis, tanto em cultura e in vivo, que forneceram a maioria do que se sabe sobre o processo de reparação ferida hoje. Em modelos animais de lesão, tais como a córnea 6-12 e 13-17 da pele, há a oportunidade para estudar a resposta do tecido do ferimento no contexto de todos os mediadores de reparo que podem estar envolvidos no processo, incluindo contribuições do vasculatura e no sistema nervoso. No entanto, existem limitações para manipular a expericondições mentais in vivo, e ainda não é possível realizar estudos de imagem da resposta de reparação in vivo, de forma contínua ao longo do tempo. Em contraste, a maioria dos modelos de cultura in vitro de reparação de feridas, tais como a ferida zero, pode ser facilmente manipulada e seguido ao longo do tempo, mas falta o contexto ambiental de estudar a cicatrização de feridas no tecido in vivo. Enquanto ex vivo em modelos oferecem a vantagem de estudar o processo de reparação da lesão de forma contínua ao longo do tempo no contexto da microambiente das células juntamente com a capacidade para modular os reguladores moleculares de reparação em qualquer ponto de tempo no processo, existem poucos modelos que se encaixam estes parâmetros. Aqui é descrito um procedimento para gerar altamente reprodutível ex vivo epitelial culturas que reproduzem a resposta de um tecido epitelial a um ferimento fisiológico cura. Usando a lente de embrião de galinha como uma fonte de tecido, um ex vivo mock cirurgia de catarata é realizada. A lente é um tecido ideal para usar para esses estudos, uma vez que é auto-suficiente dentro de uma cápsula de membrana basal espessa, avascular, não inervados, e livre de qualquer estroma associada 18,19. Na doença humana, a cirurgia da catarata aborda a perda de visão devido a opacificação da lente, e envolve a remoção da massa celular da fibra da lente, o qual compreende a maior parte da lente. Seguindo a visão cirurgia de catarata é restaurada através da inserção de uma lente intra-ocular artificial. O procedimento de cirurgia de catarata, através da remoção das células da fibra, induz uma resposta a uma lesão no epitélio do cristalino adjacente, que responde por re-epitelialização da área posterior da cápsula da lente que tinha sido ocupada pelas células de fibra. Na cirurgia de cataratas, como na maioria das respostas de reparação de ferida, há por vezes ocorre um resultado de fibrose aberrante para a resposta de cicatrização das feridas, associado com o aparecimento dos miofibroblastos, em que a lente é conhecido como posterior Capsule Opacificação 20-22. Para gerar o modelo de cicatrização de feridas cirurgia de catarata, um procedimento de cirurgia de catarata é imitado em lentes removidas do olho de embrião de pinto para produzir uma lesão fisiológica. Remoção microcirúrgico da fibra lente células resulta em uma área de ferida circular muito consistente rodeado pelas células epiteliais da lente. Esta população de células permanece firmemente ligado à cápsula membrana basal da lente e é ferido pelo procedimento cirúrgico. As células epiteliais migrar para a área desnudo da membrana basal endógena para curar a ferida, liderada por uma população de células mesenquimais rico em vimentina conhecidas no processo de reparação como células líder um. Com este modelo, a resposta de um epitélio de lesão pode ser visualizada facilmente seguido e com o tempo no contexto do microambiente das células. As células são facilmente acessíveis a alteração da expressão ou activação de moléculas que se espera que desempenham um papel na reparação de feridas. Um poderoso recurso do thé o modelo é a capacidade de isolar e estudar alterações específicas do migração no âmbito da cicatrização de feridas. A capacidade para preparar um grande número de envelhecidos ex vivo de cura de feridas culturas combinadas para estudos é outra vantagem deste modelo. Assim, este sistema modelo proporciona uma oportunidade única de provocar uma separação mecanismos de reparo de feridas e terapêutica de teste para o seu efeito sobre o processo de cicatrização de feridas. O modelo de cirurgia de catarata ex vivo simulada deverá ter ampla aplicabilidade, fornecendo um recurso crítico para o estudo de mecanismos de reparação do prejuízo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Ex Vivo Modelo criado para estudar o processo de cicatrização da ferida no microambiente das células nativas Para investigar os mecanismos envolvidos na regulação da cicatrização da ferida de um epitélio em microambiente das células nativas, um modelo de cirurgia de catarata clinicamente relevante ex vivo simulada foi criado. Este modelo é criada a partir de tecido da lente que oferece muitas vantagens, devido às suas propriedades intrínsecas: 1) A lente é um ?...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Estabelecimento de um clinicamente relevantes<em&gt; Ex Vivo</em&gt; Mock Cataract Surgery Modelo para investigar Epithelial Wound Repair em um microambiente Native

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Research

JoVE Journal

Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

6.5K visualizações.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

Vídeo: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Captura de Reparo de Tecidos em Zebrafish larvas com a Time-lapse Brightfield lupa estereoscópica

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Biologia do Desenvolvimento

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epicárdio Conseqüência Cultura Ensaio e<em&gt; Ex Vivo</em&gt; Avaliação da migração celular Epicárdica derivado

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adenoviral terapia genética para Diabetic Ceratopatia: Efeitos sobre a cicatrização de feridas e Stem Cell marcador expressão em humanos Corneas Organ-cultivadas e células epiteliais Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Um Sanduíche papel de filtro submerso por longo prazo<em&gt; Ex Ovo</em&gt; Imagem Time-lapse de galinha precoce embriões

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Tratamento de drogas e<em&gt; In Vivo</em&gt; Imagem de Osteoblast-osteoclastos Interações em um modelo de Medaka Peixe Osteoporose

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Isolamento rápido de BMPR-IB + adiposo-derivados de células estromais para uso em um Defeito Modelo Cura calota craniana

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

Um dispositivo para executar a migração celular / cicatrização de feridas em uma placa de 96 poços

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Estabelecimento de Zebrafish Larval como um modelo Animal para investigar Trypanosoma cruzi motilidade In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Imuno-fluorescente rotulagem de microtúbulos e proteínas Centrosomal em tecido Intestinal Ex Vivo e 3D em Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

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&#039;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 ...