The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTA: O protocolo a seguir segue as diretrizes estabelecidas pelo Conselho de Revisão Institucional (IRB) no Georgia Tech. 1. Coleta de Sangue e Blood Red isolamento de células Procedimento <ol><li> Recolha 40-120 ml de sangue de doadores em 10 ml tubos Vacutainer contendo heparina. Comece PBMC (célula mononucleated sangue periférico) isolamento dentro de 4 horas da coleta. Durante este tempo, manter o sangue à temperatura ambiente. NOTA: O armazenamento / N de sangue na RT ou em 4 ° C não é recomendada como isso irá resultar em menor rendimento PBMC. </li><li> Dilui-se todo o sangue 1: 1 em arrefecido em gelo (4 ° C) PBS estéril. </li><li> Pipetar 10 ml de gelado polissacarídeo hidrofílico em um, estéril tubo de 50 ml vazio. Suavemente transferir sangue total diluído em cima do polissacárido hidrófilo. </li><li> Usar uma pipeta de 25 ml no ajuste lento e sangue pipeta ao longo do lado do tubo muito lentamente. Certifique-se de que o sangue não é misturado com o sacárido antes centrifugation porque o polissacárido hidrófilo é tóxico para as células e de outro modo, o rendimento de PBMC será menor. </li><li> Centrifugar as amostras de sangue durante 30 minutos a 400 xg, a 4 ° C. Se disponíveis, reduzir a velocidade do travão da centrífuga para metade do máximo para uma melhor separação, após a centrifugação. </li><li> Aspirar fora da fracção de plasma / plaquetas da amostra de sangue centrifugado. Aspirar cuidadosamente a camada hidrofílica polissacarídeo diretamente acima da camada RBC lotado. </li><li> Recolha 8-10 ml de concentrado de eritrócitos e diluir 1: 1 com unsterile 0,9% NaCl salina. </li></ol> 2. microscopia confocal de Avaliação de Estruturas glicada Clot (simulado diabéticos coágulos) <ol><li> Descongelar o fibrinogénio humano marcado com fluorescência e conjugado de fibrinogénio humano a 37 ° C. </li><li> Pipeta a seguinte em um 0,5 ml graduada tubo de microcentrífuga. <ol><li> Para os saudáveis, coágulos normais de fibrinogênio, misturar fibrinogénio humano com 10% fluorescente etiquetado fibrinogénio humanoconjugado em tampão Tris 50 mM / NaCl 100 mM a uma concentração de 5 mg / ml. </li><li> Para o fibrinogénio glicada artificialmente (para simular a formação de coágulos diabéticos), incubar fibrinogénio humano e 10% de conjugado marcado com fluorescência fibrinogénio numa solução 5 mM de glucose dissolvida em mM Tris / NaCl 100 mM para 50 a consequente concentração de 6,8 mg / ml. </li></ol></li><li> Cubra os tubos micro-centrífuga, usando um material opaco para evitar a exposição à luz. Incubar os tubos num banho de água a 37 C ° durante 48 h. </li><li> Faça uma câmara em uma lâmina de microscópio de vidro por aposição de um adesivo fino em 2 lados do slide. </li><li> Após o período de incubação de 48 horas, para preparar reagentes e a formação de fibrina por trombina e descongelação FXIIIa alfa humano à TA. </li><li> Diluir FXIIIa a 80 U / ml em tampão de CaCl 2 5 mM. </li><li> Dilui-se a trombina a 4 U / ml em tampão de CaCl 2 5 mM. <ol><li> Para coágulos normais, garantir que as concentrações finais de fibrinogênio, FXIIIa, e trombina são 2,5 mg / ml, 20 U / ml, e 1 U / ml, respectivamente, em um volume de 50 ul. </li><li> Para os coágulos glicada, assegurar que as concentrações finais de fibrinogénio, FXIIIa, e trombina são de 3,4 mg / ml, 20 U / ml, e 1 U / ml, respectivamente, a um volume de 50 ul. </li></ol></li><li> Imediatamente pipeta 30 ul da solução de fibrina para dentro da câmara formada pelo adesivo. </li><li> Coloque um 22 milímetros x 22 mm deslizamento tampa de vidro com espessura de 0,15 mm no topo das duas camadas de adesivo. Selar os lados abertos com adesivo transparente para evitar que o coágulo de fibrina de secar. Certifique-se de que o adesivo não interage com o coágulo de fibrina. </li><li> Permitir que os coágulos de fibrina para polimerizar a 21-23 ° C por 2 horas antes de imagem confocal. </li><li> Tome imagens de microscopia confocal dos coágulos usando um microscópio confocal com uma lente de 40x / 1,30 Oil M27 com 488 nm laser de argônio. </li></ol> 3. microscopia confocal Avaliação de fibrina coágulos que contêm apenas hemácias de foice Pacientes Celulares <ol><li> Descongelar o fibrinogénio humano marcado com fluorescência e conjugado de fibrinogénio humano a 37 ° C. </li><li> Pipeta a seguinte em um 0,5 ml graduada tubo de microcentrífuga. <ol><li> Para o saudável, coágulo de fibrina normais, misturar fibrinogénio humano com 10% de conjugado marcado com fluorescência fibrinogénio humano num tampão mM de NaCl 50 mM de Tris / 100 a uma concentração de 5 mg / ml. </li><li> Para os coágulos que contêm eritrócitos SCD, misturar fibrinogénio humano e 10% de conjugado marcado com fluorescência fibrinogénio humano em tampão Tris 50 mM / NaCl 100 mM, de tal modo que a concentração resultante do fibrinogénio é de 10 mg / ml. </li></ol></li><li> Rotular as hemácias isoladas (normais e as de doentes falciformes obtidos a partir do protocolo de isolamento RBC), utilizando uma solução de marcação celular. <ol><li> Suspender as células a uma densidade de 1 x 10 6 por ml em qualquer meio de cultura isento de soro escolhido. </li><li> Adicionam-se 5 ul / ml de suspensão de células para a solução de marcação celular. Misture bem por pipetagem suave suavemente. </li><li&gt; Incubar durante 20 min a 37 ° C. </li><li> Centrifugar os tubos de suspensão rotulados a 1.500 rpm durante 5 min a 37 ° C. </li><li> Remover o sobrenadante e re-suspender suavemente as células em meio de 37 ° C. </li><li> Repita o procedimento de lavagem (3.3.4 e 3.3.5) mais duas vezes. </li></ol></li><li> Diluir FXIIIa a 80 U / ml em tampão de CaCl 2 5 mM. </li><li> Dilui-se a trombina a 4 U / ml em tampão de CaCl 2 5 mM. <ol><li> Para a formação de coágulos normais, assegurar que as concentrações finais de fibrinogénio, FXIIIa, e trombina são de 2,5 mg / ml, 20 U / ml, e 1 U / ml, respectivamente, a um volume de 50 ul. </li><li> Para os coágulos que contêm eritrócitos SCD, assegurar que as concentrações finais de fibrinogénio, FXIIIa, e trombina são de 5 mg / ml, 20 U / ml, e 1 U / ml, respectivamente, a um volume de 50 ul. </li></ol></li><li> Pipetar 10 ul de hemácias marcadas para a solução de fibrina. Pipeta 30 ul da fibrina com hemácias no vidro do microscópio (consulte a deslizar preparação para glcoágulos ycated). </li><li> Permitir que a fibrina para polimerizar a 21-23 ° C durante 2 h antes de imagem confocal. </li><li> Tome imagens confocal dos coágulos usando o microscópio confocal, e utilizar lasers de excitação com comprimentos de onda de 488 nm e 633 nm para excitar a fibrina marcado por fluorescência e hemácias. </li></ol> 4. Avaliação da microscopia confocal Fibrinolysis Tarifas em glicada Estruturas Clot <ol><li> Repita o protocolo de microscopia confocal de coágulos glicada (Protocolo Etapa 2) para a avaliação da taxa de fibrinólise em coágulos glicada. </li><li> Não selar as extremidades da câmara com adesivo transparente. Permitir que os coágulos de fibrina que polimerizam durante 1,5 h a 21-23 ° C num invólucro selado contendo 250 ml de água para evitar a desidratação. </li><li> Após a polimerização, obter imagens dos coágulos usando o microscópio confocal. </li><li> Pipeta plasmina dentro da extremidade aberta da câmara e dispersar a plasmina através do coágulo através da acção capilar. <ol><li> Paraas experiências normais e fibrinólise glicada com concentrações normais, de pipeta 25 ul a 200 ug / ml de plasmina para a abertura da câmara. </li><li> Para os coágulos glicada com concentrações reduzidas, pipeta 25 ul a 50 ug / ml para a abertura da câmara. </li></ol></li><li> Use o recurso de série de tempo (veja a Figura 1) no software microscópio confocal para capturar em tempo real imagens de vídeo do plasmina lise do coágulo. <ol><li> Clique em modo de aquisição e, em seguida, selecione &quot;Séries Temporais&quot;. Selecione o número de ciclos e do intervalo de tempo de gravação. </li></ol></li></ol> 5. Cálculo Fibrinólise Rates <ol><li> Determinar a taxa de lise, definindo uma área (2.500 mm 2) e calculando o tempo decorrido necessário para dissolver uma área fixa do coágulo. Calcula-se a taxa de lise utilizando a seguinte equação (Figura 2): <img alt="Equação 1" fo:content-width="2in" src = &quot;/ files / ftp_upload / 52019 / 52019eq1.jpg&quot; /&gt; </li></ol> 6. Análise Estatística da fibrinólise Rates <ol><li> Analisar os dados de lise usando o software de análise estatística. Realize uma análise de variância one-way e um post hoc teste de Tukey-Kramer 44,45. </li></ol>

<ol>
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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=A+study+of+microstructural+templating+in+fibrin-thrombin+gel+networks+by+spectral+and+viscoelastic+analysis.">A study of microstructural templating in fibrin-thrombin gel networks by spectral and viscoelastic analysis.</a> Soft Matter. 9, 4883-4889 (2013).</li><li>Kim, E., 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=Correlation+between+fibrin+network+structure+and+mechanical+properties:+an+experimental+and+computational+analysis.">Correlation between fibrin network structure and mechanical properties: an experimental and computational analysis.</a> Soft Matter. 7, 4983-4992 (2011).</li><li>Keegan, P. M., Surapaneni, S., Platt, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+disease+activates+peripheral+blood+mononuclear+cells+to+induce+cathepsins+k+and+v+activity+in+endothelial+cells.">Sickle cell disease activates peripheral blood mononuclear cells to induce cathepsins k and v activity in endothelial cells.</a> Anemia. 2012, 201781 (2012).</li><li>Allison, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+sickle-cell+haemoglobin.">Properties of sickle-cell haemoglobin.</a> Biochemical Journal. 65, 212 (1957).</li><li>Kuehl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of experiments : statistical principles of research design and analysis. , (2000).</li><li>Lindman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of variance in experimental designs. , (1992).</li><li>Pan, W., Galkin, O., Filobelo, L., Nagel, R. L., Vekilov, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastable+Mesoscopic+Clusters+in+Solutions+of+Sickle-Cell+Hemoglobin.">Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin.</a> Biophysical Journal. 92, 267-277 (2007).</li><li>Wootton, D. M., Popel, A. S., Alevriadou, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+the+dissolution+of+mural+fibrin+clots+by+tissue-type+plasminogen+activator.">An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.</a> Biotechnology and bioengineering. 77, 405-419 (2002).</li><li>Sazonova, I. 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=127+Mathematic+model+of+fibrin+clot+lysis+by+plasmin.">127 Mathematic model of fibrin clot lysis by plasmin.</a> Fibrinolysis and Proteolysis. 12, 45 (1998).</li></ol>

The authors have nothing to disclose.

Para obter dados significativos sobre a estrutura de mecanismos de coagulação em estados de doença, é importante isolar os factores implicados na coagulação para determinar os efeitos das proteínas e células nestas condições. Este protocolo foi desenvolvido para os efeitos da análise da estrutura de coágulo de fibrina em estados diabéticos e DF in vitro. É necessário compreender os mecanismos envolvidos na formação de fibrina e fibrinólise em estados de doença, de...

Lesão de revestimento endotelial de um vaso sanguíneo é reparada através da resposta hemostática, ou a formação de um coágulo de sangue. Quando o sangue penetra na matriz extracelular, os factores de tecidos activar as plaquetas do sangue que facilitam a iniciação da cascata de coagulação. O componente mecânico chave deste processo de cicatrização é a matriz de fibrina, composto de fibras de fibrina que são altamente elástico, e pode suportar grandes forças de 1-4. Muitos pesquisadores têm estudado a estrutura de formação e função de fibrina extensivamente nas últimas décadas 13/05. Pacientes com doenças como diabetes mellitus e falciforme têm um risco aumentado de desenvolvimento de complicações trombóticas, tais como infarto do miocárdio, doença isquêmica do coração e acidentes vasculares cerebrais 14-19. Mais de 2 milhões de pessoas são diagnosticadas com diabetes mellitus a cada ano nos Estados Unidos. Existem dois tipos de diabetes: Tipo I, onde ocorpo não consegue produzir quantidades adequadas de insulina, e Tipo II, em que o corpo se torna resistente à insulina. Nos pacientes com diabetes, doença cardiovascular (DCV) é a causa de 80% da morbidade e mortalidade associadas com a doença 20,21. A doença falciforme (SCD) é uma desordem genética do sangue que afeta mais de 100 mil pessoas nos Estados Unidos 22. SCD é uma doença ponto de mutação que faz com que as células vermelhas do sangue para se tornar em forma de crescente, fazendo com que seja difícil para as células que passam através da vasculatura sanguínea 23. Ambos os estados de doença aumentar as possibilidades de desenvolver condições aterotrombóticas no corpo. Uma das razões para isso é o resultado da estrutura da fibrina alterada e função em estados doentes 14,24-26. Em ambas as diabetes e doença das células falciformes, há um estado de hipercoagulabilidade e hipofibrinólise actividade que induz a aterotrombose e doença cardiovascular (CVD), em comparação com pacientes saudáveis ​​17,27,28. Sabe-se que hipofibrinólise promove a progressão da aterosclerose e gera eventos isquêmicos recorrentes para os pacientes com doença arterial coronariana prematura 29. No manuscrito atual, nós investigamos o papel das propriedades físicas de fibrina neste cenário particular. Estruturas de coágulos de fibrina em pacientes não-doentes são compostas por fibras finas, poros de maiores dimensões, e geralmente menos densa 14,24. O aumento da porosidade e coágulos de fibrina menos densas em pacientes saudáveis, foram encontrados para facilitar a fibrinólise 16. Em condições hyperthrombotic tais como a doença de células falciformes e diabética, há um aumento na produção de fibrinogénio, fazendo com que a concentração de fibrinogénio a partir de aumentar os níveis normais de 2,5 mg / ml em pacientes saudáveis ​​30-33. Coágulos de fibrina formados em pacientes diabéticos têm sido encontrados para ser menos poroso, mais rígida, têm mais pontos de ramificação, e mais denso quando comparado com saudável, não-diâmpacientes Bética 14,24,33-35. A estrutura de fibrina é alterada um resultado de glicação de mecanismos que ocorrem nas proteínas envolvidas na formação do coágulo. Não enzimática (irreversível) glicação ocorre quando as moléculas de glicose se ligar a resíduos de lisina na molécula de f ibrinogénio, que inibe XIIIa factor humano (FXIIIa) de glutamina e lisina apropriadamente de reticulação 33,36,37. A análise estrutural de redes de fibrina tem sido extensivamente estudado recentemente. Em particular, os pesquisadores utilizaram microscopia eletrônica e reconstrução 3D de redes de fibrina 38, investigou como ambas as células e extravasculares (fibroblastos e do músculo liso) intravascular (células endoteliais) afetam a estrutura da fibrina 39, viscoelástico utilizados e análise espectral para analisar estruturas de fibrina 40, e correlações desenvolvidos entre a estrutura da fibrina e propriedades mecânicas usando experimental e computacional se aproxima 41 </sup&gt;. O foco do estudo foi a formulação de estruturas coágulo em condições trombose célula diabética e foice simulados e usar microscopia confocal para análise da estrutura e função dos coágulos nos estados doentes. Coágulos de fibrina foram formados a partir de fibrinogénio humano, trombina humana, e FXIIIa. Os coágulos foram lisadas utilizando plasmina. Para simular condições diabéticas, o aumento da concentração de fibrinogénio foi incubada em solução de glicose para induzir in vitro fibrinogénio glicação. Para simular a doença de células falciformes condições de coagulação, o aumento da concentração de fibrinogênio foram misturados com hematócrito falciforme coletadas de pacientes como feito anteriormente pelo nosso grupo 42. Estes métodos foram usados ​​para examinar a estrutura e as funções envolvidas na formação do coágulo de fibrina e fibrinólise sob condições de doença, bem como os mecanismos que induzem a DCV. Com base em informações atuais sobre essas doenças, as estruturas do coágulo de fibrina glicada foram mais denso com menos umnd poros menores. Os coágulos de fibrina com hemácias de pacientes com anemia falciforme (hemácias) também eram mais densas e exibido agregação das hemácias e aglomerados de fibrina aglomeradas. Este é um fenômeno bem estabelecido que tenha sido determinado anteriormente 43. Também foi levantada a hipótese de que a taxa de fibrinólise seria significativamente menor nos coágulos de fibrina glicada com e sem plasmina reduzida em comparação com saudável, normal, de fibrina. Os resultados mostraram que, para a formação de coágulos de fibrina glicada, resultados significativamente diferentes taxas de lise foram observados apenas sob condições de baixa concentração de plasmina. Esta técnica experimental utilizando microscopia confocal oferece vantagens significativas sobre outros métodos de imagem, porque as células e proteínas permanecem no seu estado nativo, que permite a captação de vídeo em tempo real da actividade de coagulação. Este método de coagulação sinteticamente indutor também é mais barato e mais eficiente do que o tempo de obtenção de amostras de pacientes e filtrar indivíduoproteínas e enzimas. Além disso, utilizando proteínas e enzimas separados para sintetizar a formação de coágulos, os coágulos foram normalizados de modo que não existe variabilidade entre as amostras, como resultado de outras proteínas do plasma.

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			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;width:315px;">PBS</td>
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			<td style="width:119px;">10010031</td>
			<td></td>
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			<td height="21" style="height:21px;width:315px;">Ficoll-Paque (hydrophilic polysaccharide)</td>
			<td style="width:287px;">GE Healthcare</td>
			<td style="width:119px;">45-001-749</td>
			<td></td>
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			<td height="21" style="height:21px;">10 ml heparinized vacutainer tubes</td>
			<td style="width:287px;">BD Biosciences</td>
			<td style="width:119px;">366643</td>
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			<td height="21" style="height:21px;width:315px;">Human Fibrinogen</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
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			<td height="21" style="height:21px;">Alexa Fluor 488 human fibrinogen conjugate</td>
			<td style="width:287px;">Molecular Probes</td>
			<td style="width:119px;">F13191</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL graduated microcentrifuge tube</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-120</td>
			<td></td>
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			<td height="21" style="height:21px;width:315px;">Glucose powder</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">15023-02</td>
			<td></td>
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			<td height="21" style="height:21px;width:315px;">FXIIIa</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
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			<td height="21" style="height:21px;">VIS Confocal Microscope</td>
			<td style="width:287px;">Zeiss LSM 510</td>
			<td style="width:119px;">LSM 510</td>
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			<td height="21" style="height:21px;width:315px;">50 mM Tris</td>
			<td style="width:287px;">Lonza</td>
			<td style="width:119px;">S50-642</td>
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			<td height="25" style="height:25px;width:315px;">Calcium Chloride (CaCl2)</td>
			<td style="width:287px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiD cell-labeling solution</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">L7781</td>
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			<td height="21" style="height:21px;width:315px;">Plasmin</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
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			<td height="21" style="height:21px;width:315px;">Sodium Chloride (5 M NaCl)</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">AM9759</td>
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			<td height="42" style="height:42px;width:315px;">Statistical Modeling Software</td>
			<td>IBM</td>
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experimental and imaging techniques for examining fibrin clot structures in normal and diseased states

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

Microscopia Confocal Análise de glicada fibrina Coágulo Estruturas As imagens de microscopia confocal de coágulos normais e glicadas são apresentados na Figura 3. A análise por microscopia confocal dos coágulos normais e glicada revela que a formação de coágulos glicada são mais densos com poros mais pequenos do que os coágulos normais ambos com e sem a adição de FXIIIa coágulo durante a polimerização. Na Figura 3A e 3B, não...

Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Técnicas Experimentais e de imagem para o exame de Estruturas do coágulo de fibrina em normais e doentes Unidos

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

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11.3K visualizações.  Georgia Institute of Technology & Emory University School of Medicine. In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Vídeo: Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

Tratamento de defeitos osteocondrais no Joelho do Coelho por Implantação de alogênico Células-Tronco Mesenquimais em coágulos de fibrina

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Medicina

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Implantação de vidro Medular do Windows em camundongos adultos com Experimental Auto-imune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

A Pipeline para 3D Multimodality Integração imagem e Planejamento assistida por computador em Cirurgia de Epilepsia

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

Visualização dos Vascular e parênquima regeneração após 70% hepatectomia parcial em ratos normais

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Uma haste intramedular de bloqueio para Padronizado Fixação do fêmur Osteotomias para analisar normal e defeituosa cicatrização óssea em ratos

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

In Vivo Tracking of Edema Development and Microvascular Pathology em um Modelo de Malária Cerebral Experimental Usando Ressonância Magnética

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

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 ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Na Vivo Análise morfométrica de humano nervos cranianos, utilizando imagens de ressonância magnética da doença doença ouvidos e orelhas de audição Normal

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

Ressonância magnética cardíaca para avaliação de suspeita de trombo cardíaco: técnicas convencionais e emergentes

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...