Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

Nota: Todos os procedimentos com animais foram aprovados pelo Comitê de Cuidado e Uso da Universidade do Texas Saúde Science Center San Antonio animal Institucional e foram consistentes com as diretrizes chegar. 1. A anestesia para Cortical Preparação <ol><li> Posicione o mouse em uma câmara de indução com 2-3% isoflurano misturado com o oxigênio para induzir a anestesia. Observe a diminuição da taxa de respiração como o mouse é induzida. Beliscar das garras do mouse para determinar se o mouse está pronto para passar para o cone do nariz. Nota: nível de Anestesia é um passo crítico em qualquer preparação in vivo e os cuidados devem ser tomados para não induzir a um nível que vai causar isquemia global. </li><li> Uma vez que o mouse é suficientemente anestesiados, transferir o animal para o / plataforma de imagem cirurgia e colocar o nariz do rato no cone do nariz e aplicar 1-1,2% isofluorano para manter um estado de anestesia. Certifique-se de que o mouse está deitado em uma co temperaturantrolled almofada de aquecimento para manter a temperatura corporal (37 ° C +/- 0,5 ° C) durante os procedimentos restantes. Coloque veterinário pomada sobre os olhos para evitar a secura sob anestesia. </li><li> Monitorar a fisiologia do rato utilizando um sistema de oximetria de pulso utilizando o clipe cauda ou pé fornecido com o sistema. Verifique se a taxa respiratória é mantida entre 50-65 respirações / min. Verifique se a freqüência cardíaca permanece entre 300-450 bpm e saturação de oxigênio é mantida entre 97-98% para garantir a sobrevivência a longo prazo do animal. </li><li> Quando o mouse está devidamente anestesiados, raspar o cabelo ao longo do crânio usando cortadores elétricos, remover pêlos residual e limpo, com betadine, seguido de um algodão embebido em álcool. Repita este procedimento até três vezes para garantir um ambiente cirúrgico estéril. </li></ol> 2. Procedimento Cirúrgico <ol><li> Com o couro cabeludo completamente limpo e barbeado, fazer uma incisão de 5 mm de o couro cabeludo do mouse para revelar o fissu cranialres e para localizar bregma. </li><li> Use um aplicador de algodão estéril para remover quaisquer fáscia remanescentes recobrem crânio. </li><li> Cole um anel feito de aço inoxidável personalizado (Figura 1) com adesivo de tecido para o osso que cobre o córtex parietal utilizando as coordenadas estereotáxicas de Bregma: -1 a -3 mm e lateral: 2-4 mm. Nota: A cola define tipicamente dentro de 2 minutos após a colocação do anel para o osso. </li><li> Apor o anel a um suporte estereotáxico (Figura 2) para estabilizar o rato e para impedir o movimento durante o exame. </li><li> Sob um microscópio de dissecação cirúrgica grau, perfurar lentamente uma seção 1-2 mm no crânio usando uma ferramenta dremel-like velocidade controlada (Meisinger 3,9 milímetros broca) certificando-se de manter o nível de área como ele é perfurado. Conseguir isso usando um padrão zig-zag. Para evitar a acumulação de calor, ajustar a velocidade de perfuração para baixo e faça pausas frequentes. </li><li> Quando o crânio cranial se torna luminosa da aparência, continuar o desbastedo crânio usando uma lâmina de bisturi, utilizando o mesmo padrão em zig-zag para manter o nível da superfície adelgaçada para facilitar a remoção suave de camadas finas de crânio craniana. Com a ponta da lâmina de bisturi fazer pequenos movimentos lineares com uma leve pressão para remover as camadas finas de osso de cada vez. Continue até a vasculatura é claramente visível através do microscópio de dissecção. </li><li> Se o experimentador perfura através ou quebrar o crânio durante o processo de desbaste, sacrificar o animal, devido aos danos provável que o córtex subjacente. Nota: O crânio do rato é de cerca de 300 um de espessura e é composta por duas camadas finas de um osso compacto (externa e uma camada interna) e uma camada de osso esponjoso ensanduichada entre as duas camadas de osso compacto. A camada externa de osso compacto e a maior parte do osso esponjoso são removidos dentro da área de perfuração a 5 milímetros, resultando em uma camada de 50 um aproximado de osso compacto restante (ver Figura 2B). Visualizção da vasculatura irá garantir que o crânio intactos diluído final é aproximadamente de 50 um de espessura. O crânio é, portanto, ainda presente quando diluído para esta espessura. </li></ol> 3. Microscópio Set-up <ol><li> Use um sistema de microscópio invertido (sistemas de dois fótons convencional, ou confocal), que tem um inversor objetivo. Nota: É também possível utilizar um microscópio vertical padrão. O factor limitante será o espaço entre a fase e os objectivos. Modificações na fase pode ser necessário para realizar essa configuração. </li><li> Fixe a plataforma cirúrgica / de imagem para um palco feito sob encomenda que está localizado ao lado da base do microscópio. Nota: A plataforma é feita através de uma tomada de laboratório para permitir o movimento vertical da plataforma cirúrgica / imagiologia sob a objectiva. A tomada de laboratório é montado numa placa afixada a quatro postes cilíndricos. (Ver Figura 2). </li><li> Posicione o inversor objectivo contendo uma 20Xobjectiva sobre a janela craniana. Utilizar uma fonte de luz externa para encontrar a janela craniana, olhando através da ocular do microscópio e posicionar o objectivo na área da imagiologia. Nota: A área de imagem será indicado pela presença da vasculatura. </li><li> Para objectivos à base de água, utilizar artificial fluido espinal cerebral (aCSF) (NaCl 130 mM; KCl 30 mM; 12 mM de KH 2 PO 4; 200 mM de NaHCO3; HEPES 30 mM; e glucose 100 mM) como o meio, devido à potencial fugas na cavidade craniana durante imagiologia (Figura 3). </li></ol> 4. Rosa Bengala Dye Preparação, Administração e indução do AVC <ol><li> Prepare uma nova solução / ml 20 mg de Rosa Bengala no fluido cerebrospinal artificial (aCSF); filtrar e esterilizar antes da administração. Não reutilize ou guarde o Rosa Bengala, uma vez que tem sido mista. Faça uma solução nova para cada experimento. </li><li> Dê uma injecção na veia da cauda de 0,1 ml Rosa Bengala, enquanto senlatar a janela craniana com um laser de 561 nm para assegurar a injecção adequada da solução. Nota: Rosa Bengala serão visualizados dentro de 5 segundos após a injecção na vasculatura do cérebro. O navio inteiro deve ser preenchido com Rose Bengal. </li><li> Após a injecção adequada de corante Rosa de Bengala escolher um recipiente apropriado para a trombose com base no diâmetro do vaso (40-80 mm) para assegurar a reprodutibilidade de um volume de lesão em particular. Diferenciar entre artérias e veias, olhando para a direção do fluxo sanguíneo: artérias vai passar de diâmetro maior para embarcações de menor diâmetro, veias mover do menor para embarcações de maior diâmetro. Isto é facilmente conseguido através da visualização, uma vez Rose Bengal é injetado. </li><li> Altere a definição do microscópio da seguinte forma: <ol><li> Aumente o tempo de permanência. Nota: Isto irá variar dependendo do sistema de microscópio que está sendo utilizado. </li><li> Aumentar a potência do laser a 100%. </li><li> Coletar imagens de sequência temporal em 1 frame / seg usando ovelocidade máxima de digitalização. </li></ol></li><li> Digitalizar o rato até um coágulo estável é formado no interior do vaso. Nota: Isso normalmente é alcançado dentro de 5 min de digitalização contínua (ver Figura 4). </li><li> Após a indução de formação de coágulo utilizando Rosa de Bengala, remover o rato da área de imagem para trás para o microscópio de dissecação. Retire cuidadosamente o anel de aço inoxidável do crânio craniana. Examine a janela do crânio para qualquer sangramento. Se ocorrer sangramento, encerrar o experimento. </li><li> Use 6,0 sutura monofilamento para fechar a incisão sobre o crânio. Coloque pomada antibiótica ao longo da linha de sutura para prevenir a infecção. Injectar Buprenex (0,05 mg / kg) por via subcutânea a cada 12 horas durante três dias para o manejo da dor. </li><li> Devolver o mouse para uma câmara de recuperação após a remoção do anestésico até que esteja totalmente acordado e se movendo livremente. </li><li> Devolver o mouse para uma gaiola limpa para uma investigação mais aprofundada em um momento posterior. </li></ol> 5. Imagem longitudinal sobre subsequentes Dias <ol><li> Empregam métodos a seguir para realizar imagem longitudinal em dias subsequentes pós-fototrombose. <ol><li> Anestesiar o rato como descrito na Seção 1 dos métodos. </li><li> Após a anestesia adequada re-abrir o couro cabeludo, removendo quaisquer suturas residuais para reabrir a pele que recobre o campo de imagem previamente perfurados. </li><li> Use um aplicador de algodão estéril para remover quaisquer fáscia remanescentes recobrem crânio. </li><li> Cole um anel personalizado feito de aço inoxidável (Figura 1) com adesivo tecidual ao osso que recobre o campo de imagem anterior. </li><li> Apor o anel a um suporte estereotáxico (Figura 2) para estabilizar o rato e para impedir o movimento durante o exame. </li><li> Localizar a vasculatura subjacente ao crânio previamente diluído. Use injecção na veia da cauda de FITC-dextrano para verificar a presença do coágulo induzido anteriormente. </li><li> Use 6,0 sutura monofilamento para fechar a emCision sobre o crânio. Coloque pomada antibiótica ao longo da linha de sutura para prevenir a infecção. Injectar Buprenex (0,05 mg / kg) por via subcutânea a cada 12 horas durante três dias para o manejo da dor. </li><li> Devolver o mouse para uma câmara de recuperação após a remoção do anestésico até que esteja totalmente acordado e se movendo livremente. </li></ol></li></ol> 6. A verificação do Curso de Indução (post-mortem) <ol><li> Na conclusão do estudo, verificar o volume sistólico utilizando cloreto de (TTC) Coloração de 2,3,5-trifeniltetrazólio, como mostrado na Figura 5. O método completo pode ser encontrado em 12. </li></ol>

<ol>
	<li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <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+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Annals of Neurology. 17, 497-504 (1985).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circulation Research. 40, 320-328 (1977).</li><li>Owens, A. P., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+tissue+factor+that+contribute+to+thrombosis+after+rupture+of+an+atherosclerotic+plaque.">Sources of tissue factor that contribute to thrombosis after rupture of an atherosclerotic plaque.</a> Thrombosis Research. 129, S30-S33 (2012).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics. 2, 396-409 (2005).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of stroke models in rats. , (2009).</li><li>Herz, R. C., Kasbergen, C. M., Hillen, B., Versteeg, D. H., de Wildt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion+by+an+intraluminal+thread+compromises+collateral+blood+flow.">Rat middle cerebral artery occlusion by an intraluminal thread compromises collateral blood flow.</a> Brain Research. 791, 223-228 (1998).</li><li>Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J., Pulsinelli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+brain+ischemia+in+the+rat:+methods+for+reproducible+neocortical+infarction+using+tandem+occlusion+of+the+distal+middle+cerebral+and+ipsilateral+common+carotid+arteries.">Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries.</a> Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 8, 474-485 (1988).</li><li>Zheng, W., 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=Purinergic+receptor+stimulation+reduces+cytotoxic+edema+and+brain+infarcts+in+mouse+induced+by+photothrombosis+by+energizing+glial+mitochondria.">Purinergic receptor stimulation reduces cytotoxic edema and brain infarcts in mouse induced by photothrombosis by energizing glial mitochondria.</a> PloS One. 5, e14401 (2010).</li><li>Zheng, D. M., Wewer, J., Lechleiter, J. P. 2. Y. 1. R. -. i. n. i. t. i. a. t. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IP3R-dependent+stimulation+of+astrocyte+mitochondrial+metabolism+reduces+and+partially+reverses+ischemic+neuronal+damage+in+mouse.">IP3R-dependent stimulation of astrocyte mitochondrial metabolism reduces and partially reverses ischemic neuronal damage in mouse.</a> Journal of Cerebral Blood Flow and Metabolism. 33, 600-611 (2013).</li><li>Witte, O. W., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+and+remote+effects+of+focal+cortical+infarctions:+secondary+damage+and+reactive+plasticity.">Delayed and remote effects of focal cortical infarctions: secondary damage and reactive plasticity.</a> Advances in Neurology. 73, 207-227 (1997).</li><li>Hagemann, G., Redecker, C., Neumann-Haefelin, T., Freund, H. J., Witte, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+long-term+potentiation+in+the+surround+of+experimentally+induced+focal+cortical+infarction.">Increased long-term potentiation in the surround of experimentally induced focal cortical infarction.</a> Annals of Neurology. 44, 255-258 (1998).</li><li>Kramer, 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=TTC+staining+of+damaged+brain+areas+after+MCA+occlusion+in+the+rat+does+not+constrict+quantitative+gene+and+protein+analyses.">TTC staining of damaged brain areas after MCA occlusion in the rat does not constrict quantitative gene and protein analyses.</a> Journal of Neuroscience Methods. 187, 84-89 (2010).</li><li>Blinder, P., Shih, A. Y., Rafie, C., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+basis+for+the+robust+distribution+of+blood+to+rodent+neocortex.">Topological basis for the robust distribution of blood to rodent neocortex.</a> Proceedings of the National Academy of Sciences. 107, 12670-12675 (2010).</li><li>Nishimura, N., Rosidi, N. L., Iadecola, C., Schaffer, 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=Limitations+of+collateral+flow+after+occlusion+of+a+single+cortical+penetrating+arteriole.">Limitations of collateral flow after occlusion of a single cortical penetrating arteriole.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 30, 1914-1927 (2010).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proceedings of the National Academy of Sciences. 104, 365 (2007).</li><li>Blum, 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=Memory+after+silent+stroke:+Hippocampus+and+infarcts+both+matter.">Memory after silent stroke: Hippocampus and infarcts both matter.</a> Neurology. 78, 38-46 (2012).</li><li>Heinsius, T., Bogousslavsky, J., Van Melle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+infarcts+in+the+middle+cerebral+artery+territory+Etiology+and+outcome+patterns.">Large infarcts in the middle cerebral artery territory Etiology and outcome patterns.</a> Neurology. 50, 341-350 (1998).</li><li>Wardlaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+causes+lacunar+stroke.">What causes lacunar stroke.</a> Journal of Neurology, Neurosurgery &amp; Psychiatry. 76, 617-619 (2005).</li><li>Inoue, Y., 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=Ischemic+stroke+under+anticoagulant+therapy].+Rinsho+shinkeigaku.">Ischemic stroke under anticoagulant therapy]. Rinsho shinkeigaku.</a> Clinical Neurology. 50, 455-460 (2010).</li><li>Tiannan Wang, W. C., Xie, Y., Zhang, W., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+Volume+of+the+Focal+Cerebral+Ischemic+Lesion+through+Photothrombosis.">Controlling the Volume of the Focal Cerebral Ischemic Lesion through Photothrombosis.</a> American Journal of Biomedical Sciences. 2, 33-42 (2009).</li><li>Head, B. P., Patel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+brain+protection.">Anesthetics and brain protection.</a> Current Opinion in Anaesthesiology. 20, 395-399 (2007).</li><li>Kirsch, J. R., Traystman, R. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+cerebroprotection:+experimental+aspects.">Anesthetics and cerebroprotection: experimental aspects.</a> International Anesthesiology Clinics. 34, 73-93 (1996).</li><li>Koerner, I. P., Brambrink, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+protection+by+anesthetic+agents.">Brain protection by anesthetic agents.</a> Current Opinion in Anaesthesiology. 19, 481-486 (2006).</li><li>Gelb, A. W., Bayona, N. A., Wilson, J. X., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+anesthesia+compared+to+awake+reduces+infarct+size+in+rats.">Propofol anesthesia compared to awake reduces infarct size in rats.</a> Anesthesiology. 96, 1183-1190 (2002).</li><li>Bhardwaj, A., Castro, I. A., Alkayed, N. J., Hurn, P. D., Kirsch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+choice+of+halothane+versus+propofol:+impact+on+experimental+perioperative+stroke.">Anesthetic choice of halothane versus propofol: impact on experimental perioperative stroke.</a> Stroke; A Journal Of Cerebral Circulation. 32, 1920-1925 (2001).</li><li>Barretto, R. P., Messerschmidt, B., Schnitzer, M. J. <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+fluorescence+imaging+with+high-resolution+microlenses.">In vivo fluorescence imaging with high-resolution microlenses.</a> Nature Methods. 6, 511-512 (2009).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

A capacidade de traduzir fisiopatologia curso experimental de animal para aplicação em seres humanos tem sido atormentado com o fracasso. No entanto, a utilização de modelos animais, tais como o modelo fototrombose, permite uma melhor compreensão da patofisiologia acidente vascular cerebral e a exploração de novas abordagens terapêuticas para proporcionar neuroprotecção após um acidente vascular cerebral. Pequenos acidentes vasculares cerebrais corticais e microinfartos produzidos pelo modelo photothrombotic ...

A técnica descrita permite a visualização in vivo de respostas celulares imediatamente após a indução de Rose Bengal fototrombose em um rato intacto. Rosa de Bengala (4,5,6,7-tetracloro-2 &#39;, 4&#39;, 5 &#39;, 7&#39;-tetraiodofluorescein) é um corante fotossensível utilizado para induzir apoplexia isquémica em modelos animais (ratinhos e ratos). Na sequência de uma injecção de bolus de RB através da veia da cauda e subsequente iluminação através de um crânio diluído com uma luz laser 564 nm, um trombo é induzida causando um acidente vascular cerebral fisiológico 1. O método foi originalmente descrito por Rosenblum e El-Sabban em 1977, e mais tarde foi adaptado por Watson em meados dos anos 1980 1,2. Em breve, Rosa Bengala é irradiado com luz verde de excitação (561 nm do laser no nosso caso), o qual gera a produção de espécies reactivas de oxigénio, que subsequentemente activa o factor de tecido, um iniciador da cascata de coagulação. A indução da cascata de coagulação produz uma les de isquemiaion que é patologicamente relevantes para acidente vascular cerebral clínico 3. Acidente vascular cerebral tem uma fisiopatologia complexa, devido à interação de muitos tipos de células diferentes, incluindo neurônios, glia, endotélio e do sistema imunológico. Escolhendo a melhor técnica para estudar um processo celular particular requer várias considerações. Técnicas experimentais podem repartir-se em uma das três categorias: in vitro, in vivo e in silico com cada um tendo vantagens e desvantagens Estudos in vitro têm a desvantagem principal de remover as células de seu ambiente natural e, portanto, não pode reproduzir efeitos vistos em um intacto,. animal vivo. In vivo técnicas de permitir uma maior replicação experimental de estados de doença com o aumento da importância de translação. in silico geralmente refere-se a modelação por computador de uma doença ou de um processo celular, e ao mesmo tempo cada vez mais utilizada para estudar interacções medicamentosas potenciais para exameple, qualquer informação recolhida ainda deve ser testada em células ou tecidos vivos. O modelo ideal de acidente vascular cerebral no ambiente de laboratório deve demonstrar características patológicas semelhantes aos observados na população humana. Enquanto existem características fisiológicas comuns de acidente vascular cerebral na população humana, existem também muitas diferenças, dependendo do tipo de lesão experimentado. AVC na população humana ocorre como pequenas ou grandes oclusões de vasos, lesões hemorrágicas e artéria para artéria ou cardio-embolias que resultam em volumes de infarto variadas, bem como as diferenças nos mecanismos relacionados a cada patologia. A vantagem da utilização de modelos animais de acidente vascular cerebral é a geração de enfartes reprodutíveis que imitam as características de acidente vascular cerebral humano. Os modelos mais comuns de animais de acidente vascular cerebral incluem a oclusão da artéria usando: Oclusão da artéria cerebral média (métodos de incandescência ou embólicos endovasculares) que modelos distal e o modelo MCAO fototrombose. As vantagens de umd desvantagens de cada modelo foram revistos em outro lugar (ver 4 e 5). Os modelos globais isquêmicas (MCAO), enquanto relativamente fácil de executar são menos relevantes para acidente vascular cerebral humano do que são modelos de acidente vascular cerebral focal. Além disso, estes métodos são altamente variáveis ​​na indução de lesões enfarte cerebral reprodutíveis. O modelo fototrombose é altamente reprodutível, enquanto o experimentador controla seus experimentos bem, proporcionando uma clara vantagem sobre os modelos MCAo. No entanto, devido à microvasculatura insulto o modelo tenha sido descrita para apresentar uma penumbra isquémica mínima, a área em que as células são pensados ​​para serem recuperáveis ​​6,7. Além disso, o edema vasogénico e formação de edema citotóxico também pode ser induzida a seguir a irradiação da área de imagem. Apesar destas limitações a técnica tem proporcionado uma nova visão sobre muitos processos fisiológicos seguintes acidente vascular cerebral 8, 9, 10, 11.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>330000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Anesthetic</td>
			<td>MWI Veterinary Supply</td>
			<td>088-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>1469SB</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>aCSF&#160;</td>
			<td>126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 26 mM NaHCO3 (pH 7.4).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Bioindustrial Products</td>
			<td>500-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors 14 cm</td>
			<td>Bioindustrial Products</td>
			<td>12-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont High Tech #5 style, straight</td>
			<td>Bioindustrial Products</td>
			<td>TWZ-301.22</td>
			<td></td>
		</tr>
		<tr>
			<td>LabJack 132X80</td>
			<td>Optosigma Co</td>
			<td>123-6670</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform for Labjack 8X 8</td>
			<td>Optosigma Co</td>
			<td>145-1110</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear bar holder from stereotaxic setup</td>
			<td>Stoelting/Cyborg</td>
			<td>51654</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispomed Labvent Rodent anesthesia machine</td>
			<td>DRE, Inc.</td>
			<td>15001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tech IV Isoflurane vaporizer</td>
			<td>DRE, Inc.</td>
			<td>34001</td>
			<td></td>
		</tr>
		<tr>
			<td>F Air Canister</td>
			<td>DRE, Inc</td>
			<td>80120</td>
			<td></td>
		</tr>
		<tr>
			<td>Bain circuit breathing tube</td>
			<td>DRE, Inc</td>
			<td>86111B</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent adapter for bain tube</td>
			<td>DRE, Inc</td>
			<td>891000</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 regulator for oxygen tanks</td>
			<td>DRE, Inc</td>
			<td>CE001E</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent induction chamber</td>
			<td>DRE, Inc</td>
			<td>15004C</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Silk 6-0; 18 in with P-3 needle</td>
			<td>Suture Express</td>
			<td>1639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective inverter Optical Adapter</td>
			<td>LSM technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foredom drill Dual voltage 110/120</td>
			<td>Foredom</td>
			<td>134.53</td>
			<td></td>
		</tr>
	</tbody>
</table>

rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

O objectivo deste método foi de induzir um acidente vascular cerebral isquémico em modelos animais (ratinhos e ratos) depois de uma injecção de bolus de RB através da veia da cauda e iluminação posterior de um crânio diluído com uma luz laser de 561 nm. As imagens na Figura 4 demonstram a progressão de formação de coágulo dentro de um único vaso a seguir à irradiação da área a 0, 1, 1,5 e 2 min. Antes da formação do coágulo todo o vaso é branco devido ao fluxo livre Rose Bengal. Ap...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa Bengala Fototrombose por imageamento óptico confocal<em&gt; In Vivo</em&gt;: Um Modelo de curso Veículo Individual

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

14.9K visualizações.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

Vídeo: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Não-invasivo de medição óptica do metabolismo cerebral e Hemodinâmica em Lactentes

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Medicina

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Imagem in vivo da angiogênese tumoral utilizando fluorescência confocal Videomicroscopia

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Um modelo murino de hemorragia subaracnóide

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

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 ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Fototrombose induzida por isquemia focal como um Modelo de Lesão Medular em Ratos

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualização Quantitativa de Infiltrado de Leucócitos em um Modelo Murino de Miocardite Fulminante por Microscopia de Folha de Luz

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

A abordagem de amostragem de 4 embarcações para estudos integrativos de fisiologia placentária humana<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...