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: Il seguente protocollo aderisce alle linee guida stabilite dal Institutional Review Board (IRB) presso la Georgia Tech. 1. Raccolta di sangue e Blood Red isolamento delle cellule Procedura <ol><li> Raccogliere 40-120 ml di sangue da donatori in 10 ml vacutainer eparina. Inizia PBMC (cellule mononucleate del sangue periferico) isolamento entro 4 ore di raccolta. Durante questo tempo, mantenere il sangue a RT. NOTA: O / N stoccaggio di sangue in RT o 4 ° C non è raccomandato in quanto questo si tradurrà in minori rendimenti PBMC. </li><li> Diluire sangue intero 1: 1 in ghiaccio freddo (4 ° C) PBS sterile. </li><li> Pipettare 10 ml di ghiacciata polisaccaride idrofila in un sterile 50 ml tubetto vuoto, conico. Trasferire delicatamente sangue intero diluito in cima al polisaccaride idrofila. </li><li> Utilizzare una pipetta 25 ml in presa lenta e sangue pipetta lungo il lato del tubo molto lentamente. Assicurarsi che il sangue non viene miscelato con il saccaride prima Centrifugation perché il polisaccaride idrofila è tossico per le cellule e non, la resa PBMC sarà inferiore. </li><li> Centrifugare i campioni di sangue per 30 min a 400 xg a 4 ° C. Se disponibile, ridurre la velocità del freno della centrifuga alla metà del massimo per una migliore separazione dopo centrifugazione. </li><li> Aspirare off la frazione di plasma / piastrine del campione di sangue centrifugato. Aspirare accuratamente lo strato polisaccaride idrofila direttamente sopra il livello RBC imballato. </li><li> Raccogliere 8-10 ml di eritrociti concentrati e diluire 1: 1 con il non sterile 0,9% NaCl soluzione salina. </li></ol> 2. Microscopia confocale Valutazione di glicata Strutture Clot (simulati Diabetici Clots) <ol><li> Scongelare fibrinogeno umano e fluorescente coniugato fibrinogeno umano a 37 ° C. </li><li> Pipettare il seguente in un 0,5 ml graduato provetta. <ol><li> Per i sani, normali coaguli fibrinogeno, mescolare fibrinogeno umano con il 10% fluorescente fibrinogeno umanoconiugato in tampone 50 mM Tris / 100 mM NaCl ad una concentrazione di 5 mg / ml. </li><li> Per fibrinogeno artificialmente glicata (per simulare coaguli diabetici), incubare fibrinogeno umano e il 10% fluorescente fibrinogeno coniugato in una soluzione 5 mM di glucosio sciolto in 50 mM Tris / NaCl 100 mM per una concentrazione risultante di 6,8 mg / ml. </li></ol></li><li> Coprire le provette micro-centrifuga con un materiale opaco per evitare l&#39;esposizione alla luce. Incubare le provette in un bagno d&#39;acqua a 37 ° per 48 ore. </li><li> Fare una camera su un vetrino da microscopio in vetro apponendo un adesivo sottile su 2 lati della diapositiva. </li><li> Dopo il periodo di incubazione 48 ore, preparare i reagenti per la formazione di fibrina da sbrinamento FXIIIa e trombina alfa umano RT. </li><li> Diluire FXIIIa a 80 U / ml in 5 CaCl mM tampone 2. </li><li> Diluire trombina a 4 U / ml in 5 mM CaCl 2 tampone. <ol><li> Per coaguli normali, assicurarsi che le concentrazioni finali di fibrinogeno, FXIIIa, e trombina sono 2,5 mg / ml, 20 U / ml e 1 U / ml, rispettivamente, in un volume di 50 microlitri. </li><li> Per i coaguli glicata, accertarsi che le concentrazioni finali di fibrinogeno, FXIIIa, e trombina sono 3,4 mg / ml, 20 U / ml, e 1 U / ml, rispettivamente, ad un volume di 50 ml. </li></ol></li><li> Subito pipettare 30 microlitri della soluzione di fibrina nella camera formata dal collante. </li><li> Inserire un x 22 mm vetrino di vetro 22 mm con spessore di 0,15 mm su cima alle 2 strati di adesivo. Sigillare i lati aperti con adesivo trasparente per evitare che il coagulo di fibrina si secchi. Assicurarsi che l&#39;adesivo non interagisce con il coagulo di fibrina. </li><li> Lasciare i coaguli di fibrina polimerizzare a 21-23 ° C per 2 ore prima di imaging confocale. </li><li> Prendere immagini di microscopia confocale dei coaguli utilizzando un microscopio confocale con un obiettivo 40X / 1.30 Olio M27 con 488 nm Argon laser. </li></ol> 3. Microscopia confocale Valutazione della fibrina Clots contenenti globuli rossi di pazienti Sickle Cellulari <ol><li> Scongelare fibrinogeno umano e fluorescente coniugato fibrinogeno umano a 37 ° C. </li><li> Pipettare il seguente in un 0,5 ml graduato provetta. <ol><li> Per il sano, normale coagulo di fibrina, fibrinogeno umano miscelare con il 10% fluorescente coniugato fibrinogeno umano in tampone 50 mM Tris / 100 mM NaCl ad una concentrazione di 5 mg / ml. </li><li> Per i coaguli contenenti SCD eritrociti, mescolare fibrinogeno umano e il 10% fluorescente coniugato fibrinogeno umano in 50 mM Tris / NaCl 100 mM tale che la concentrazione risultante del fibrinogeno è di 10 mg / ml. </li></ol></li><li> Etichettare i globuli rossi isolati (sia normali e quelli di pazienti con anemia falciforme ottenuti dal protocollo di isolamento RBC) utilizzando una soluzione di cellule di etichettatura. <ol><li> Sospendere le cellule ad una densità di 1 x 10 6 per ml in qualsiasi mezzo di coltura privo di siero prescelto. </li><li> Aggiungere 5 microlitri / ml di sospensione cellulare alla soluzione cellulare etichettatura. Mescolare bene delicatamente pipettando delicatamente. </li><li&gt; incubare per 20 minuti a 37 ° C. </li><li> Centrifugare le provette etichettate sospensione a 1500 rpm per 5 min a 37 ° C. </li><li> Rimuovere il surnatante e delicatamente risospendere le cellule in 37 ° C di media. </li><li> Ripetere la procedura di lavaggio (3.3.4 e 3.3.5) altre due volte. </li></ol></li><li> Diluire FXIIIa a 80 U / ml in 5 CaCl mM tampone 2. </li><li> Diluire trombina a 4 U / ml in 5 mM CaCl 2 tampone. <ol><li> Per coaguli normali, assicurarsi che le concentrazioni finali di fibrinogeno, FXIIIa, e trombina sono 2,5 mg / ml, 20 U / ml, e 1 U / ml, rispettivamente, ad un volume di 50 ml. </li><li> Per i coaguli contenenti SCD globuli rossi, accertarsi che le concentrazioni finali di fibrinogeno, FXIIIa, e trombina sono 5 mg / ml, 20 U / ml, e 1 U / ml, rispettivamente, ad un volume di 50 ml. </li></ol></li><li> Pipettare 10 ml di globuli rossi etichettati nella soluzione di fibrina. Pipettare 30 ml di fibrina con globuli rossi sul vetro del microscopio (vedere scorrere la preparazione per glcoaguli ycated). </li><li> Lasciare fibrina polimerizzare a 21-23 ° C per 2 ore prima di imaging confocale. </li><li> Prendere immagini confocale dei coaguli utilizzando il microscopio confocale, e utilizzare laser con lunghezza d&#39;onda di eccitazione di 488 nm e 633 nm per eccitare fibrina fluorescente e globuli rossi. </li></ol> 4. Microscopia confocale Valutazione della fibrinolisi tariffe in glicata Strutture Clot <ol><li> Ripetere il protocollo di microscopia confocale di coaguli glicata (Protocollo Fase 2) per la valutazione del tasso di fibrinolisi in grumi glicata. </li><li> Non sigillare le estremità della camera con adesivo trasparente. Lasciare i coaguli di fibrina a polimerizzare per 1,5 ore a 21-23 ° C in un contenitore sigillato contenente 250 ml di acqua per evitare la disidratazione. </li><li> Dopo la polimerizzazione, ottenere immagini dei coaguli utilizzando il microscopio confocale. </li><li> Pipettare plasmina nell&#39;estremità aperta della camera e disperdere la plasmina attraverso il coagulo tramite azione capillare. <ol><li> Pernormali e glicata esperimenti fibrinolisi con concentrazioni normali, pipette 25 ml a 200 mg / ml di plasmin nella apertura della camera. </li><li> Per i coaguli glicata con concentrazioni ridotte, pipetta 25 microlitri a 50 ug / ml nella apertura della camera. </li></ol></li><li> Utilizzare la funzione serie temporale (vedi Figura 1) nel software microscopio confocale a catturare sequenze video in tempo reale della plasmina lisi del coagulo. <ol><li> Fare clic su modalità di acquisizione e quindi selezionare &quot;Serie Time.&quot; Seleziona il numero di cicli e l&#39;intervallo di tempo di registrazione. </li></ol></li></ol> 5. Calcolo fibrinolisi Tariffe <ol><li> Determinare il tasso lisi impostando un&#39;area (2,500 micron 2) e calcolando il tempo trascorso necessario per sciogliere la superficie fissa del coagulo. Calcolare il tasso di lisi utilizzando la seguente equazione (figura 2): <img alt="Equazione 1" fo:content-width="2in" src = &quot;/ files / ftp_upload / 52019 / 52019eq1.jpg&quot; /&gt; </li></ol> 6. Analisi statistica della fibrinolisi Tariffe <ol><li> Analizzare i dati di lisi utilizzando software di analisi statistica. Eseguire un ANOVA a senso unico e un post hoc test di Tukey-Kramer 44,45. </li></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.

Per ottenere dati significativi sulla struttura dei meccanismi di coagulazione in stati di malattia, è importante isolare i fattori coinvolti nella coagulazione per determinare gli effetti delle proteine ​​e cellule in queste condizioni. Questo protocollo è stato sviluppato per i fini della valutazione della struttura del coagulo di fibrina in stati diabetici e SCD in vitro. E &#39;necessario comprendere i meccanismi coinvolti nella formazione di fibrina e fibrinolisi in stati...

Lesioni a rivestimento endoteliale di un vaso sanguigno viene riparato attraverso la risposta emostatico, o la formazione di un coagulo di sangue. Quando il sangue permea nella matrice extracellulare, fattori tissutali attivano piastrine nel sangue che facilitano l&#39;iniziazione della cascata coagulativa. Il componente chiave meccanica di questo processo di guarigione è la matrice di fibrina, composto da fibre di fibrina che sono altamente elastica, e può sostenere le grandi forze 1-4. Molti ricercatori hanno studiato la struttura di formazione, e la funzione di fibrina ampiamente negli ultimi decenni 5-13. I pazienti con malattie come il diabete mellito e falciforme hanno un aumentato rischio di sviluppare complicanze trombotiche, come infarto miocardico, cardiopatia ischemica, e colpi 14-19. Oltre 2 milioni di persone sono di nuova diagnosi di diabete mellito ogni anno negli Stati Uniti. Esistono due tipi di diabete: tipo I, dove lacorpo non riesce a produrre quantità sufficienti di insulina, e tipo II, dove il corpo diventa resistente all&#39;insulina. Tra i pazienti diabetici, malattie cardiovascolari (CVD) è la causa per l&#39;80% della morbilità e la mortalità associate con la malattia 20,21. Anemia falciforme (SCD) è una malattia genetica del sangue che colpisce più di 100.000 persone negli Stati Uniti 22. SCD è una malattia mutazioni puntiformi che induce le cellule rosse del sangue per diventare a forma di mezzaluna, il che rende difficile per le cellule di passare attraverso il sistema vascolare del sangue 23. Entrambi questi stati di malattia aumentano le probabilità di sviluppare condizioni aterotrombotici nel corpo. Uno dei motivi per questo è il risultato di strutture di fibrina alterata e funzione negli stati malati 14,24-26. In entrambi diabete e anemia falciforme, c&#39;è ipercoagulazione e l&#39;attività hypofibrinolysis che induce aterotrombosi e malattie cardiovascolari (CVD) rispetto ai pazienti sani 17,27,28. E &#39;noto che hypofibrinolysis promuove la progressione dell&#39;aterosclerosi e genera eventi ischemici ricorrenti per i pazienti con malattia coronarica precoce 29. Nel manoscritto attuale, abbiamo studiato il ruolo di fibrina proprietà fisiche in questo ambiente particolare. Strutture coagulo di fibrina nei pazienti non malati sono composti da fibre sottili, pori più grandi, e in genere meno densa 14,24. La maggiore porosità e coaguli di fibrina meno fitta in pazienti sani sono stati trovati per facilitare fibrinolisi 16. In condizioni hyperthrombotic quali malattia a cellule falciformi diabetica e, vi è un aumento della produzione di fibrinogeno, causando la concentrazione di fibrinogeno aumentare da livelli normali di 2,5 mg / ml in pazienti sani 30-33. Coaguli di fibrina formata in pazienti diabetici sono stati trovati per essere meno porosa, più rigida, avere più punti di ramificazione, e più densa rispetto a sano, non diapazienti Betica 14,24,33-35. La struttura di fibrina alterata è il risultato di meccanismi di glicazione che si verificano nelle proteine ​​coinvolte nella formazione del coagulo. Non enzimatica (irreversibile) glicazione si verifica quando le molecole di glucosio si legano a residui di lisina sulla molecola fibrinogeno, che inibisce il fattore XIIIa umana (FXIIIa) da glutamina e lisina residui opportunamente incrociati collegano 33,36,37. L&#39;analisi strutturale delle reti di fibrina è stato ampiamente studiato di recente. In particolare, i ricercatori hanno utilizzato la microscopia elettronica e la ricostruzione 3D delle reti di fibrina, 38 indagati come sia intravascolari (endoteliali) cellule e extravascolari (fibroblasti e muscolari lisce) cellule influenzano la struttura di fibrina 39, viscoelastico utilizzati e l&#39;analisi spettrale per analizzare le strutture di fibrina 40, e correlazioni sviluppati tra struttura di fibrina e le proprietà meccaniche utilizzando approcci sperimentali e computazionali 41 </sup&gt;. Il focus di questo studio è stato quello di formulare strutture coagulo in condizioni trombosi cellulare diabetico e falce simulati e di utilizzare la microscopia confocale per l&#39;esame della struttura e funzione dei coaguli in stati malati. Coaguli di fibrina sono formati da fibrinogeno umano, trombina umana, e FXIIIa. I coaguli sono stati lisati con plasmina. Per simulare le condizioni di diabetici, aumento della concentrazione di fibrinogeno è stato incubato in soluzione di glucosio di indurre in vitro fibrinogeno glicazione. Per simulare malattie cella condizioni coagulazione falce, un aumento delle concentrazioni di fibrinogeno sono stati mescolati con ematocrito falciforme raccolti da pazienti, come fatto in precedenza dal nostro gruppo 42. Questi metodi sono stati usati per esaminare la struttura e le funzioni coinvolte nella formazione di coaguli di fibrina fibrinolisi e in condizioni malate, nonché i meccanismi che inducono CVD. Sulla base delle informazioni attuali su queste malattie, le strutture di coagulo di fibrina glicata erano più denso con meno di unnd piccoli pori. I coaguli di fibrina con globuli rossi di pazienti con anemia falciforme (RBC) erano anche più denso e visualizzati aggregazione dei globuli rossi e di cluster di fibrina agglomerati. Questo è un fenomeno ben noto che è stato determinato in precedenza 43. E &#39;stato anche ipotizzato che il tasso di fibrinolisi sarebbe significativamente più bassa nel coaguli di fibrina glicata con e senza riduzione plasmina rispetto ai sani, di fibrina normale. I risultati hanno mostrato che per coaguli di fibrina glicate, risultati molto diversi tassi lisi stati osservati solo in condizioni di concentrazione di plasmina ridotta. Questa tecnica sperimentale mediante microscopia confocale offre vantaggi significativi rispetto ad altri metodi di imaging perché le cellule e proteine ​​rimangono nel loro stato nativo, che permette la cattura di video in tempo reale dell&#39;attività coagulazione. Questo metodo di coagulazione sinteticamente Induzione è anche più economico e più tempo efficiente di ottenere campioni di pazienti e filtrando individualeproteine ​​ed enzimi. Inoltre, utilizzando proteine ​​ed enzimi separati per sintetizzare coaguli, i coaguli sono stati standardizzati in modo che non ci fosse la variabilità tra i campioni a causa di altre proteine ​​del 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>
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		<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>
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		<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>
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		<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>
		</tr>
		<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>
			<td></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 confocale Analisi di glicata fibrina Strutture Clot Le immagini microscopia confocale di coaguli normali e glicate sono presentati in Figura 3. Microscopia confocale dei coaguli normali e glicate rivela che coaguli glicate sono più densi con pori inferiori ai coaguli normali con e senza l&#39;aggiunta di FXIIIa durante la polimerizzazione coagulo. Nella Figura 3A e 3B, vi è una concentrazione di fibrina inferiore che crea una s...

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.

Tecniche sperimentali e di imaging per l&#39;esame Strutture coagulo di fibrina in normali e patologiche Uniti

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 Views.  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.

Video: 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.

Il trattamento dei difetti osteocondrali del ginocchio congiunta del Coniglio con impianto di cellule staminali mesenchimali allogeniche in coaguli di fibrina

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

Ricerca

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.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

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.

Una pipeline per il 3D multimodalità integrazione di immagini e progettazione assistita da computer in chirurgia dell&#39;epilessia

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.

Visualizzazione dei Vascolare e parenchimale rigenerazione dopo il 70% epatectomia parziale in topi normali

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 chiodo endomidollare di bloccaggio per standardizzata Fissazione del femore osteotomie per analizzare normale e difettoso guarigione ossea nei topi

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.

Tracciamento in vivo di sviluppo dell&#39;edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l&#39;utilizzo di immagini a risonanza magnetica

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.

Visualizzazione quantitativa di leucociti infiltrati in un modello di murina della miocardite fulminante mediante microscopia a foglio chiaro

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 Analisi morfometrica dei nervi cranici umano usando la formazione immagine a risonanza magnetica in orecchie di malattia di Menière e orecchie udito 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.

Risonanza magnetica cardiaca per la valutazione di un trombo cardiaco sospetto: tecniche convenzionali ed emergenti

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