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.

REMARQUE: Le protocole suivant est conforme aux orientations fixées par le Conseil d&#39;examen institutionnel (IRB) à Georgia Tech. 1. Collecte de sang et de globules rouges Isolation procédure <ol><li> Recueillir 40 à 120 ml de sang provenant de donneurs dans 10 ml tubes Vacutainer héparinisés. Lancer PBMC (cellules mononucléées du sang périphérique) isolement au sein de quatre heures de la collecte. Pendant ce temps, maintenir le sang à température ambiante. REMARQUE: O / N stockage du sang en RT ou 4 ° C ne est pas recommandé car cela se traduira par un rendement plus faible CMSP. </li><li> Diluer sang total 1: 1 dans de la glace-froid (4 ° C) PBS stérile. </li><li> Pipette 10 ml de glace-froid polysaccharide hydrophile en un, 50 ml tube conique stérile vide. Transférer doucement sang total dilué sur le dessus du polysaccharide hydrophile. </li><li> Utiliser une pipette de 25 ml dans le sang et à prise lente de la pipette le long du côté du tube très lentement. Assurez-vous que le sang ne est pas mélangé avec le saccharide avant Centrifugation parce que le polysaccharide hydrophile est toxique pour les cellules et les autres, le rendement PBMC sera plus faible. </li><li> Centrifuger des échantillons de sang pour 30 min à 400 g à 4 ° C. Le cas échéant, réduire la vitesse de frein de la centrifugeuse à la moitié du maximum pour une meilleure séparation après centrifugation. </li><li> Aspirer la fraction plasma / plaquettes de l&#39;échantillon de sang centrifugé. Aspirer soigneusement la couche de polysaccharide hydrophile directement au-dessus de la couche de RBC emballé. </li><li> Recueillir 8-10 ml de globules rouges emballés et diluer 1: 1 avec 0,9% de NaCl non stérile saline. </li></ol> 2. confocale Microscopie évaluation de glyquée Structures Clot (simulées diabétiques caillots) <ol><li> Dégivrage fibrinogène humain marqué par fluorescence et de fibrinogène humain conjugué à 37 ° C. </li><li> Pipetter le suivant dans un 0,5 ml tube gradué à centrifuger. <ol><li> Pour les caillots de fibrinogène, normaux en bonne santé, mélanger fibrinogène humain avec 10% marqué par fluorescence fibrinogène humainconjugué dans un tampon Tris 50 mM / NaCl 100 mM à une concentration de 5 mg / ml. </li><li> Pour fibrinogène glyquée artificiellement (pour simuler les caillots diabétiques), incuber fibrinogène humain et de 10% de fibrinogène marqué par fluorescence conjugué dans une solution à 5 mM de glucose dissous dans Tris / NaCl 100 mM 50 pour une concentration résultante de 6,8 mg / ml. </li></ol></li><li> Couvrir les tubes micro-centrifugeuse en utilisant un matériau opaque pour éviter l&#39;exposition à la lumière. Incuber les tubes dans un bain d&#39;eau à 37 ° pendant 48 heures. </li><li> Faites une chambre sur une lame de microscope en verre par l&#39;apposition d&#39;une mince adhésif sur 2 côtés de la lame. </li><li> Après la période d&#39;incubation de 48 heures, préparer des réactifs pour la formation de fibrine par la thrombine et la décongélation FXIIIa alpha humain à TA. </li><li> Diluer FXIIIa à 80 U / ml dans un tampon 5 mM de CaCl 2. </li><li> Diluer la thrombine à 4 U / ml dans un tampon 5 mM de CaCl 2. <ol><li> Pour caillots normaux, se assurer que les concentrations finales de fibrinogène, FXIIIa, et la thrombine sont de 2,5 mg / ml, 20 U / ml et 1 U / ml, respectivement à un volume de 50 ul. </li><li> Pour les caillots glyquées, en sorte que les concentrations finales de fibrinogène, FXIIIa, et la thrombine sont 3,4 mg / ml, 20 U / ml, et 1 U / ml, respectivement à un volume de 50 ul. </li></ol></li><li> Immédiatement pipette 30 ul de la solution de fibrine dans la chambre formée par l&#39;adhésif. </li><li> Placez un 22 mm x 22 mm lamelle de verre d&#39;une épaisseur de 0,15 mm sur le dessus des deux couches d&#39;adhésif. Sceller les côtés ouverts avec adhésif transparent pour empêcher le caillot de fibrine de se dessécher. Se assurer que l&#39;adhésif ne interagit pas avec le caillot de fibrine. </li><li> Autoriser les caillots de fibrine polymériser à 21-23 ° C pendant 2 heures avant l&#39;imagerie confocale. </li><li> Prendre des images de microscopie confocale des caillots utilisant un microscope confocal avec un objectif 40X / 1,30 huile M27 avec un laser à argon 488 nm. </li></ol> 3. microscopie confocale évaluation des caillots de fibrine contenant des hématies de patients Sickle Cell <ol><li> Dégivrage fibrinogène humain marqué par fluorescence et de fibrinogène humain conjugué à 37 ° C. </li><li> Pipetter le suivant dans un 0,5 ml tube gradué à centrifuger. <ol><li> Pour la bonne santé, un caillot de fibrine normale, mélanger le fibrinogène humain avec 10% de conjugué marqué par fluorescence du fibrinogène humain dans un tampon Tris 50 mM / NaCl 100 mM à une concentration de 5 mg / ml. </li><li> Pour les caillots contenant des globules rouges SCD, mélanger le fibrinogène humain et de 10% de conjugué marqué par fluorescence du fibrinogène humain dans un tampon Tris 50 mM / NaCl 100 mM de sorte que la concentration résultante du fibrinogène est de 10 mg / ml. </li></ol></li><li> Étiqueter les globules rouges isolés (à la fois normales et ceux provenant de patients drépanocytaires obtenues à partir du protocole d&#39;isolement RBC) en utilisant une solution cellulaire étiquetage. <ol><li> Suspendre les cellules à une densité de 1 x 10 6 par ml dans un milieu de culture sans sérum choisi. </li><li> Ajouter 5 ul / ml de la suspension cellulaire à la solution de cellules étiquetage. Mélangez bien par pipetage doux doucement. </li><li&gt; incuber pendant 20 min à 37 ° C. </li><li> Centrifuger les tubes de suspension marqués à 1500 rpm pendant 5 min à 37 ° C. </li><li> Retirer délicatement le surnageant et remettre en suspension les cellules dans 37 ° C moyenne. </li><li> Répétez la procédure de lavage (3.3.4 et 3.3.5) deux fois de plus. </li></ol></li><li> Diluer FXIIIa à 80 U / ml dans un tampon 5 mM de CaCl 2. </li><li> Diluer la thrombine à 4 U / ml dans un tampon 5 mM de CaCl 2. <ol><li> Pour caillots normaux, en sorte que les concentrations finales de fibrinogène, FXIIIa, et la thrombine sont de 2,5 mg / ml, 20 U / ml, et 1 U / ml, respectivement à un volume de 50 ul. </li><li> Pour les caillots contenant SCD globules rouges, en sorte que les concentrations finales de fibrinogène, FXIIIa, et la thrombine sont de 5 mg / ml, 20 U / ml, et 1 U / ml, respectivement à un volume de 50 ul. </li></ol></li><li> Introduire à la pipette 10 ul des globules rouges marqués dans la solution de fibrine. Pipette 30 ul de la fibrine avec des hématies sur le verre de microscope (voir glisser préparation glycated caillots). </li><li> Laisser la fibrine polymériser à 21-23 ° C pendant 2 heures avant l&#39;imagerie confocale. </li><li> Prenez images confocales des caillots à l&#39;aide du microscope confocal, et d&#39;utiliser des lasers d&#39;excitation avec des longueurs d&#39;onde de 488 nm et 633 nm pour exciter la fibrine marqué par fluorescence et de globules rouges. </li></ol> 4. microscopie confocale évaluation de la fibrinolyse Tarifs en glyquée Structures Clot <ol><li> Répétez le protocole de microscopie confocale de caillots glyquée (Protocole Step 2) pour l&#39;évaluation du taux de la fibrinolyse dans caillots glyquée. </li><li> Ne pas fermer les extrémités de la chambre avec un adhésif clair. Laisser les caillots de fibrine à polymériser pendant 1,5 heures à 21-23 ° C dans une enceinte étanche contenant 250 ml d&#39;eau pour éviter la déshydratation. </li><li> Après polymérisation, d&#39;obtenir des images des caillots à l&#39;aide du microscope confocal. </li><li> Pipette plasmine dans l&#39;extrémité ouverte de la chambre et disperser la plasmine par l&#39;intermédiaire du caillot par action capillaire. <ol><li> Pourles expériences de la fibrinolyse normales et avec des concentrations normales glyquées, pipette 25 ul à 200 pg / ml de plasmine dans l&#39;ouverture de la chambre. </li><li> Pour les caillots glyquées, avec des concentrations réduites pipette 25 ul à 50 ug / ml dans l&#39;ouverture de la chambre. </li></ol></li><li> Utilisez la fonction de séries chronologiques (voir la figure 1) dans le logiciel de microscope confocal à capturer en temps réel des séquences vidéo de la plasmine lyse du caillot. <ol><li> Cliquez sur le mode d&#39;acquisition, puis sélectionnez «Time Series&quot;. Sélectionnez le nombre de cycles et l&#39;intervalle de temps d&#39;enregistrement. </li></ol></li></ol> 5. Calcul de la fibrinolyse Tarifs <ol><li> Déterminer le taux de lyse en définissant un espace (2500 um 2) et en calculant le temps écoulé nécessaire pour dissoudre une zone fixe du caillot. Calculer le taux de lyse à l&#39;aide de l&#39;équation suivante (figure 2): <img alt="Equation 1" fo:content-width="2in" src = &quot;/ files / ftp_upload / 52019 / 52019eq1.jpg&quot; /&gt; </li></ol> 6. Analyse statistique de la fibrinolyse Tarifs <ol><li> Analyser les données de lyse en utilisant un logiciel d&#39;analyse statistique. Effectuer une analyse de variance à une voie et un post hoc de Tukey-Kramer 44,45 essai. </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.

Pour obtenir des données significatives sur la structure des mécanismes de coagulation dans des états pathologiques, il est important d&#39;isoler les facteurs impliqués dans la coagulation pour déterminer les effets des protéines et des cellules dans ces conditions. Ce protocole a été développé pour les fins de l&#39;enquête sur la structure du caillot de fibrine dans les états diabétiques et SCD in vitro. Il est nécessaire de comprendre les mécanismes impliqués dan...

Des traumatismes de la paroi endothéliale d&#39;un vaisseau sanguin est réparé par la réponse hémostatique, ou la formation d&#39;un caillot de sang. Lorsque le sang pénètre dans la matrice extracellulaire, les facteurs tissulaires activer les plaquettes dans le flux de sang qui facilitent l&#39;initiation de la cascade de coagulation. L&#39;élément mécanique essentiel de ce processus de guérison est la matrice de fibrine, composé de fibres de fibrine qui sont hautement élastique, et peut supporter des forces importantes 1-4. De nombreux chercheurs ont étudié la structure de la formation, et la fonction de la fibrine abondamment dans les dernières décennies 5-13. Les patients atteints de maladies telles que le diabète sucré et la drépanocytose ont un risque accru de développer des complications thrombotiques telles que l&#39;infarctus du myocarde, la cardiopathie ischémique, les accidents vasculaires cérébraux et 14-19. Plus de 2 millions de personnes sont nouvellement diagnostiquées avec le diabète sucré chaque année aux États-Unis. Il existe deux types de diabète: type I, où lecorps ne parvient pas à produire des quantités adéquates d&#39;insuline, et Type II, où le corps devient résistant à l&#39;insuline. Chez les patients diabétiques, les maladies cardiovasculaires (CVD) est la cause de 80% de la morbidité et de la mortalité associée à la maladie 20,21. La drépanocytose (SCD) est une maladie génétique du sang qui touche plus de 100 000 personnes aux États-Unis 22. SCD est une maladie point mutation qui provoque des globules rouges à devenir en forme de croissant, ce qui rend difficile pour les cellules de passer à travers le système vasculaire sanguin 23. Ces deux états pathologiques augmenter les chances de développer des maladies athérothrombotiques dans le corps. Une des raisons à cela est le résultat de la structure de fibrine modifié et la fonction dans les Etats malades 14,24-26. Dans les deux diabète et la drépanocytose, il est hypercoagulation et l&#39;activité de hypofibrinolyse qui induit l&#39;athérothrombose et les maladies cardiovasculaires (CVD) par rapport à des patients sains 17,27,28. Il est connu que hypofibrinolyse favorise la progression de l&#39;athérosclérose et engendre des événements ischémiques récurrents pour les patients atteints de maladie coronarienne prématurée 29. Dans le manuscrit en cours, nous avons étudié le rôle des propriétés physiques fibrine dans ce contexte particulier. Structures de caillot de fibrine chez les patients non malades sont composés de fibres fines et des pores plus grands, et généralement moins dense 14,24. La porosité accrue et des caillots de fibrine moins denses chez les patients en bonne santé ont été trouvées 16 pour faciliter la fibrinolyse. Dans des conditions hyperthrombotic telles que la maladie diabétique et la faucille cellule, il existe une augmentation de la production de fibrinogène, ce qui provoque la concentration de fibrinogène à augmenter à partir de niveaux normaux de 2,5 mg / ml dans 30 à 33 patients en bonne santé. Les caillots de fibrine formés chez les patients diabétiques se sont révélés être moins poreux, plus rigide, plus de points de ramification, et plus dense en comparaison avec sain, non-diapatients Bétique 14,24,33-35. La structure de fibrine modifiée est le résultat de mécanismes de glycation qui se produisent dans les protéines impliquées dans la formation du caillot. Non enzymatique (irréversible) glycation se produit lorsque les molécules de glucose se lient à des résidus de lysine sur la molécule de fibrinogène, qui inhibe le facteur XIIIa humaine (FXIIIa) à partir de résidus lysine et glutamine correctement reticulation 33,36,37. L&#39;analyse structurale des réseaux de fibrine a été largement étudié ces derniers temps. En particulier, les chercheurs ont utilisé la microscopie électronique et reconstruction 3D de réseaux de fibrine 38, a étudié comment les deux (endotheliales) Les piles et extravasculaires (fibroblastes et de muscle lisse) intravasculaires cellules influent sur ​​la structure de fibrine 39, viscoélastique utilisé et l&#39;analyse spectrale à analyser les structures de fibrine 40, et corrélations développés entre la structure de fibrine et les propriétés mécaniques utilisant des approches expérimentales et informatiques 41 </sup&gt;. L&#39;objectif de l&#39;étude était de formuler des structures de caillot dans des conditions de thrombose cellulaire diabétique et la faucille simulées et d&#39;utiliser la microscopie confocale pour l&#39;examen de la structure et la fonction des caillots dans les États malades. Les caillots de fibrine ont été formés à partir de fibrinogène humain, de la thrombine humaine, et FXIIIa. Les caillots ont été lysées à l&#39;aide de plasmine. Pour simuler les conditions diabétiques, augmentation de la concentration de fibrinogène a été incubé dans une solution de glucose pour induire in vitro fibrinogène glycation. Pour simuler les conditions de coagulation drépanocytaires, une augmentation des concentrations de fibrinogène ont été mélangés avec hématocrite falciforme recueillies auprès de patients comme précédemment par notre groupe 42. Ces méthodes ont été utilisées pour examiner la structure et les fonctions impliquées dans la formation de caillot de fibrine et de la fibrinolyse dans des conditions malades, ainsi que les mécanismes qui induisent les maladies cardiovasculaires. Basé sur l&#39;information courante sur ces maladies, les structures de fibrine caillot glyquée étaient plus dense avec moins d&#39;unnd pores plus petits. Les caillots de fibrine avec les globules rouges provenant de patients atteints de drépanocytose (RBC) étaient également plus dense et affichées agrégation des globules rouges et des grappes agglomérées de fibrine. Ce est un phénomène bien établi qui a été précédemment déterminé 43. On a également émis l&#39;hypothèse que le taux de la fibrinolyse est sensiblement inférieure à la formation de caillots de fibrine avec ou sans glyquées réduite par rapport à la plasmine en bonne santé, la fibrine normal. Les résultats ont montré que, pour la formation de caillots de fibrine glyquées, des résultats sensiblement différents de taux de lyse n&#39;a été observée que dans des conditions de concentration de la plasmine réduite. Cette technique expérimentale de l&#39;utilisation de la microscopie confocale offre des avantages significatifs par rapport à d&#39;autres méthodes d&#39;imagerie parce que les cellules et les protéines restent dans leur état natif, ce qui permet la capture de vidéo en temps réel de l&#39;activité de coagulation. Cette méthode de coagulation synthétiquement induction est également moins coûteux et plus efficace du temps que l&#39;obtention d&#39;échantillons de patients et le filtrage out individueldes protéines et des enzymes. En outre, en utilisant des protéines et des enzymes séparés de synthétiser des caillots, les caillots ont été normalisées de sorte qu&#39;il n&#39;y avait pas variabilité entre les échantillons à la suite d&#39;autres protéines dans le plasma.

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			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">PBS</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
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		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="25">
			<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Plasmin</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
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		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Statistical Modeling Software</td>
			<td>IBM</td>
			<td style="width:119px;">SPSS Statistics 22</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.

Microscopie confocale Analyse des glyquée fibrine Structures Clot Les images de microscopie confocale de caillots normaux et glyquée sont présentés dans la figure 3. Confocale analyse au microscope des caillots normaux et glyquée révèle que caillots glyquée sont plus denses avec des pores plus petits que les caillots normaux à la fois avec et sans l&#39;ajout de FXIIIa cours de la polymérisation caillot. Sur la figure 3A et 3B, il ...

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.

Techniques expérimentales et d&#39;imagerie pour examen caillot de fibrine Structures en normaux et malades Unis

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

experimental-imaging-techniques-for-examining-fibrin-clot-structures

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Cardiovascular Drugs: Anticoagulants and Antianginal Agents

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11.3K vues.  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.

Vidéo: 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.

Traitement des défauts ostéochondraux à genou la commune du lapin par implantation de cellules allogéniques souches mésenchymateuses dans caillots de fibrine

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

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Médecine

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 haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

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.

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.

Un pipeline pour la 3D multimodalité Intégration image et assistée par ordinateur Planification en chirurgie de l&#39;épilepsie

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.

Visualisation des vasculaire et parenchymateuse Régénération après 70% hépatectomie partielle chez des souris normales

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.

Un clou intramédullaire verrouillage pour Standardized Fixation de Fémur Ostéotomies pour l&#39;analyse normale et défectueuse des os de guérison chez la souris

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.

Le suivi in vivo du développement de l&#39;œdème et de la pathologie microvasculaire dans un modèle de paludisme cérébral expérimental utilisant l&#39;imagerie par résonance magnétique

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.

Visualisation quantitative de l&#39;infiltration de leucocytes dans un modèle murin de myocardite fulminante par microscopie à feuilles lumineuses

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.

In Vivo Analyse morphométrique des humains nerfs crâniens à l’aide de l’imagerie par résonance magnétique oreilles maladie de Menière et des oreilles de l’audition normale

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.

Résonance magnétique cardiaque pour l'évaluation du thrombus cardiaque présumé : techniques conventionnelles et émergentes

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