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.

HINWEIS: Das folgende Protokoll befolgt die vom Institutional Review Board (IRB) an der Georgia Tech Richtlinien. 1. Blutentnahme und Rotes Blutkörperchen Isolierungsverfahren <ol><li> Sammeln Sie 40 bis 120 ml Blut von Spendern in 10 ml Heparin-Vacutainer-Röhrchen. Starten PBMC (periphere Blut mononukleären Zellen) Isolierung innerhalb von 4 Stunden der Sammlung. Während dieser Zeit halten Blut bei RT. HINWEIS: O / N Lagerung von Blut in RT oder bei 4 ° C wird nicht empfohlen, da dies zu niedrigeren PBMC Ausbeute führen. </li><li> Verdünnen Sie Vollblut 1: 1 in eiskaltem (4 ° C) sterile PBS. </li><li> Je 10 ml eiskaltem hydrophile Polysaccharid in eine leere, sterile 50 ml konischen Röhrchen. Verdünntem Vollblut übertragen Sie leicht über der hydrophilen Polysaccharid. </li><li> Verwenden Sie eine 25 ml Pipette in Zeiteinstellung und Pipetten Blut an der Seite des Rohres sehr langsam. Sicherzustellen, dass das Blut nicht mit der Saccharid vor Centri vermischtfugation weil das hydrophile Polysaccharid Zellen toxisch, und ansonsten wird die PBMC Ausbeute niedriger sein. </li><li> Zentrifuge Blutproben für 30 min bei 400 × g bei 4 ° C. Falls verfügbar, verringern die Bremsgeschwindigkeit der Zentrifuge auf die Hälfte der maximalen zur besseren Trennung nach der Zentrifugation. </li><li> Absaugen des Plasmas / Thrombozytenfraktion der zentrifugierten Blutprobe. Sorgfältig absaugen die hydrophile Polysaccharid Schicht direkt über der RBC-Schicht verpackt. </li><li> Sammeln Sie 8-10 ml gepacktes Erythrozyten und verdünnt 1: 1 mit unsterilen 0,9% NaCl Kochsalzlösung. </li></ol> 2. konfokale Mikroskopie Bewertung der Glykiertes Clot Strukturen (Simulated Diabetische Blutgerinnsel) <ol><li> Abzutauen Humanfibrinogen und fluoreszierend markierten Fibrinogen-Konjugat bei 37 ° C. </li><li> Pipettieren Sie die folgenden zu einem 0,5 ml-Messmikrozentrifugenröhrchen. <ol><li> Für die gesunde, normale Fibrinogen gerinnt, mischen, Fibrinogen mit 10% fluoreszenzmarkierten FibrinogenKonjugat in einem 50 mM Tris / 100 mM NaCl-Puffer bei einer Konzentration von 5 mg / ml. </li><li> Künstlich glykiertes Fibrinogen (diabetische Gerinnsel zu simulieren), Inkubation Humanfibrinogen und 10% fluoreszierend markierten Fibrinogen-Konjugat in einer 5 mM Lösung von Glucose in 50 mM Tris / 100 mM NaCl bei einer resultierenden Konzentration von 6,8 mg / ml. </li></ol></li><li> Umfassen die Mikrozentrifugenröhrchen mit einem undurchsichtigen Material, um Belichtung zu vermeiden. Die Röhrchen in ein 37 ° C Wasserbad für 48 Stunden inkubieren. </li><li> Machen Sie eine Kammer auf einen Glasobjektträger durch Aufbringen eines dünnen Klebe auf 2 Seiten des Schlittens. </li><li> Nach 48 Stunden Inkubationszeit vorzubereiten Reagenzien für die Fibrinbildung durch Auftauen FXIIIa und Menschen alpha Thrombin bei RT. </li><li> Verdünne FXIIIa bis 80 U / ml in 5 mM CaCl 2 Puffer. </li><li> Verdünnte Thrombin bis 4 U / ml in 5 mM CaCl 2 Puffer. <ol><li> Für normale gerinnt, sicherzustellen, dass die Endkonzentrationen an Fibrinogen, Faktor XIIIa und Thrombin sind 2,5 mg / ml, 20 U / ml, und 1 U / ml in einem Volumen von 50 ul. </li><li> Für die glykierte gerinnt, sicherzustellen, dass die Endkonzentrationen an Fibrinogen, Faktor XIIIa und Thrombin sind 3,4 mg / ml, 20 U / ml, und 1 U / ml in einem Volumen von 50 ul. </li></ol></li><li> Unmittelbar Je 30 ul der Fibrin-Lösung in die durch den Kleber gebildete Kammer. </li><li> Legen Sie eine 22 mm x 22 mm Deckglas mit einer Dicke von 0,15 mm auf der Oberseite der zwei Klebstoffschichten. Verschließen Sie die offenen Seiten mit klaren Klebstoff, um das Fibringerinnsel austrocknet. Sicherzustellen, dass der Klebstoff nicht mit dem Fibringerinnsel interagieren. </li><li> Lassen Sie die Fibringerinnsel zu 21-23 ° C für 2 Stunden, bevor die konfokale Abbildung zu polymerisieren. </li><li> Nehmen Sie die konfokale Mikroskopie Bilder der Gerinnsel mit einem konfokalen Mikroskop mit einem 40x / 1,30 Oil M27 Objektiv mit einem 488 nm Argon-Laser. </li></ol> 3. konfokale Mikroskopie Bewertung von Fibringerinnseln haltigen Erythrozyten von Sichelzellanämie Patienten <ol><li> Abzutauen Humanfibrinogen und fluoreszierend markierten Fibrinogen-Konjugat bei 37 ° C. </li><li> Pipettieren Sie die folgenden zu einem 0,5 ml-Messmikrozentrifugenröhrchen. <ol><li> Für den gesunden, normal Fibringerinnsels mischen menschlichem Fibrinogen mit 10% fluoreszierend markierten Fibrinogen-Konjugat in einem 50 mM Tris / 100 mM NaCl-Puffer bei einer Konzentration von 5 mg / ml. </li><li> Für die Gerinnsel enthaltenden SCD RBCs mischen Humanfibrinogen und 10% fluoreszierend markierten Fibrinogen-Konjugat in einem 50 mM Tris / 100 mM NaCl, daß die resultierende Konzentration der Fibrinogen 10 mg / ml. </li></ol></li><li> Beschriften Sie die isolierten Erythrozyten (sowohl normale als auch jene von Sichelzellpatienten von der RBC-Isolierungsprotokoll erhalten wird) mit einem Zell-Markierungslösung. <ol><li> Suspend-Zellen bei einer Dichte von 1 x 10 6 pro ml in jeder gewählten serumfreien Kulturmedium. </li><li> Mit 5 ul / ml der Zellsuspension in Zellmarkierungslösung. Gut mischen durch vorsichtiges Pipettieren vorsichtig. </li><li&gt; Inkubieren für 20 min bei 37 ° C. </li><li> Zentrifugieren der markierten Suspension Röhrchen bei 1.500 UpM für 5 min bei 37 ° C. </li><li> Entfernen Sie den Überstand und sanft wieder die Zellen in 37 ° C Medium. </li><li> Wiederholen Sie den Waschvorgang (3.3.4 und 3.3.5) zwei weitere Male. </li></ol></li><li> Verdünne FXIIIa bis 80 U / ml in 5 mM CaCl 2 Puffer. </li><li> Verdünnte Thrombin bis 4 U / ml in 5 mM CaCl 2 Puffer. <ol><li> Für normale gerinnt, sicherzustellen, dass die Endkonzentrationen an Fibrinogen, Faktor XIIIa und Thrombin sind 2,5 mg / ml, 20 U / ml, und 1 U / ml in einem Volumen von 50 ul. </li><li> Für die Gerinnsel enthaltenden SCD RBCs, sicherzustellen, dass die Endkonzentrationen an Fibrinogen, Faktor XIIIa und Thrombin werden 5 mg / ml, 20 U / ml, und 1 U / ml in einem Volumen von 50 ul. </li></ol></li><li> Je 10 ul der markierten roten Blutkörperchen in das Fibrin-Lösung. Pipette 30 ul des Fibrin mit Erythrozyten auf das Mikroskop Glas (siehe Vorbereitung gl schiebenycated Blutgerinnsel). </li><li> Ermöglichen das Fibrin auf 21-23 ° C für 2 Stunden, bevor die konfokale Abbildung zu polymerisieren. </li><li> Nehmen konfokale Bilder der Gerinnsel mit dem konfokalen Mikroskop, und nutzen Anregungslaser mit Wellenlängen von 488 nm und 633 nm, um die Fluoreszenz-markierten Fibrin und Erythrozyten anregen. </li></ol> 4. konfokale Mikroskopie Bewertung der Fibrinolyse Preise in Glykiertes Clot Strukturen <ol><li> Wiederholen der Konfokalmikroskopie Protokoll von glykiertem Blutgerinnsel (Protocol Schritt 2) für die Fibrinolyse Rate Auswertung in glykierten Gerinnsel. </li><li> Verschließen Sie nicht die Enden der Kammer mit klaren Klebstoff. Lassen Sie die Fibringerinnsel für 1,5 h bei 21-23 ° C in einem geschlossenen Gehäuse, das 250 ml Wasser, um Austrocknung zu verhindern polymerisieren. </li><li> Nach der Polymerisation erhalten Bilder der Gerinnsel mit dem konfokalen Mikroskop. </li><li> Pipette Plasmin in das offene Ende der Kammer und verteilen das Plasmin durch das Gerinnsel durch Kapillarwirkung. <ol><li> Fürder normale und der glykierten Fibrinolyse Experimente mit normalen Konzentrationen, Pipetten 25 ul mit 200 ug / ml Plasmin in die Öffnung der Kammer. </li><li> Für die glykierte Gerinnseln mit reduzierten Konzentrationen Pipette 25 ul bei 50 ug / ml in die Öffnung der Kammer. </li></ol></li><li> Verwenden Sie den Zeitreihen-Funktion (siehe Abbildung 1) in der konfokalen Mikroskop-Software, um Echtzeit-Videoaufnahmen von der Plasmin Lyse des Gerinnsels zu erfassen. <ol><li> Klicken Erfassungsmodus und wählen Sie &quot;Time Series&quot;. Wählen Sie die Anzahl der Zyklen und die Aufnahmezeitintervall. </li></ol></li></ol> 5. Berechnung Fibrinolyse Preise <ol><li> Bestimmen Sie die Lyse Rate, indem Sie einen Bereich (2500 &amp; mgr; m 2) und der Berechnung der verstrichenen Zeit erforderlich ist, um einen festen Bereich der Gerinnsel aufzulösen. Errechnen Lyserate Verwendung der folgenden Gleichung (2): <img alt="Gleichung 1" fo:content-width="2in" src = &quot;/ files / ftp_upload / 52.019 / 52019eq1.jpg&quot; /&gt; </li></ol> 6. Statistische Analyse der Fibrinolyse Preise <ol><li> Analysieren Sie die Lyse-Daten mit Hilfe statistischer Analyse-Software. Führen Sie eine one-way ANOVA und post hoc Tukey-Kramer-Test 44,45. </li></ol>

<ol>
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C., Haselgrove, J. C., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+fibrin+clot+networks+from+stereoscopic+intermediate+voltage+electron+microscope+images+and+analysis+of+branching.">Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching.</a> Biophysical journal. 68, 1551-1560 (1995).</li><li>Campbell, R. A., Overmyer, K. A., Selzman, C. H., Sheridan, B. C., Wolberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+extravascular+and+intravascular+cells+to+fibrin+network+formation,+structure,+and+stability.">Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability.</a> Blood. 114, 4886-4896 (2009).</li><li>Curtis, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.

Um aussagekräftige Daten über die Struktur des Gerinnungsmechanismen bei Krankheitszuständen zu erhalten, ist es wichtig, die Faktoren der Blutgerinnung beteiligt sind, um die Effekte der Proteine ​​und Zellen unter diesen Bedingungen zu bestimmen, zu isolieren. Dieses Protokoll wurde zum Zweck der Untersuchung der Struktur des Fibringerinnsels bei diabetischen und SCD Zustände in vitro entwickelt. Es ist notwendig, um die Mechanismen in der Fibrinbildung und Fibrinolyse bei...

Schädigung eines Blutgefäßes Endothelauskleidung durch die hämostatische Reaktion oder die Bildung eines Blutgerinnsels repariert. Wenn Blut dringt in die extrazelluläre Matrix, Gewebefaktoren aktiviert Blutplättchen in den Blutstrom, die Einleitung der Gerinnungskaskade zu erleichtern. Der Schlüssel mechanische Komponente dieser Heilungsprozess ist die Fibrinmatrix, von Fibrin Fasern, die hochelastisch sind zusammengesetzt und kann große Kräfte 1-4 zu erhalten. Viele Forscher haben die Bildung Struktur und Funktion von Fibrin ausführlich in den vergangenen Jahrzehnten 5-13 untersucht. Patienten mit Krankheiten, wie Diabetes mellitus und Sichelzellen ein erhöhtes Risiko der Entwicklung von thrombotischen Komplikationen wie Herzinfarkte, ischämische Herzerkrankungen und Schlaganfälle 14-19. Über 2 Millionen Menschen neu mit Diabetes mellitus diagnostiziert jedes Jahr in den Vereinigten Staaten. Es gibt zwei Arten von Diabetes: Typ I, wo dieKörper nicht ausreichende Mengen an Insulin und Typ II, wo der Körper wird resistent gegen Insulin zu produzieren. Unter Diabetikern, kardiovaskulärer Erkrankung (CVD) ist die Ursache für die 80% der Morbidität und Mortalität, die mit der Krankheit assoziiert 20,21. Sichelzellanämie (SCD) ist eine genetisch bedingte Blutgerinnungsstörung, die mehr als 100.000 Menschen in den Vereinigten Staaten 22 wirkt. SCD ist eine Punkt-Mutation Krankheit, die roten Blutkörperchen bewirkt, halbmondförmige geworden, dass es schwierig für die Zellen durch die Blutgefäßsystem 23 übergeben. Beide Krankheitszuständen erhöht die Chancen der Entwicklung atherothrombotischer Bedingungen im Körper. Einer der Gründe dafür ist, auf einer veränderten Fibrin Struktur und Funktion in erkrankten Zuständen 14,24-26. In beiden Diabetes und Sichelzellanämie, gibt es Hyperkoagulation und Hypofibrinolyse Aktivität, die Atherothrombose und kardiovaskulären Erkrankungen induziert (CVD) im Vergleich zu gesunden Patienten 17,27,28. Es ist bekannt, dass Hypofibrinolyse fördert Atherosklerose Progression und erzeugt rezidivierende ischämische Ereignisse bei Patienten mit vorzeitigem Koronararterienerkrankung 29. In der aktuellen Manuskript untersuchten wir die Rolle von Fibrin physikalischen Eigenschaften in diesem besonderen Ambiente. Fibringerinnsel Strukturen in nicht erkrankten Patienten werden von dünnen Fasern, größere Poren, und in der Regel weniger dicht 14,24 zusammen. Die erhöhte Porosität und weniger dicht Fibringerinnseln in gesunden Patienten wurde gefunden, dass die Fibrinolyse 16 erleichtern. In hyperthrombotischen Bedingungen wie diabetische und Sichelzellanämie, gibt es eine Erhöhung in der Produktion von Fibrinogen, wodurch der Fibrinogenkonzentration um vom Normalniveau von 2,5 mg / ml in gesunden Patienten 30-33 erhöhen. Fibringerinnseln in diabetischen Patienten gebildet haben sich als weniger porös, steifer, mehr Verzweigungspunkte, und dichter im Vergleich zu gesunden, nicht-diaBetic Patienten 14,24,33-35. Die veränderte Fibrinstruktur ist ein Ergebnis der Glykierung Mechanismen, die in den Proteinen der Gerinnselbildung beteiligt auftreten. Nichtenzymatische (irreversibel) Glykosylierung tritt auf, wenn Glukose-Moleküle binden an Lysinresten auf der Fibrinogen-Molekül, das die menschliche Faktor XIIIa (FXIIIa) ordnungsgemäß Vernetzungs Glutamin und Lysin-Reste 33,36,37 hemmt. Die Strukturanalyse von Fibrin-Netzwerken wurde ausgiebig vor kurzem untersucht. Insbesondere haben Forscher Elektronenmikroskopie und 3D-Rekonstruktion von Fibrin-Netzwerken 38, untersucht, wie auch intravaskuläre (endotheliale Zellen) und extravaskuläre (Fibroblasten und glatte Muskelzellen) Zellen beeinflussen Fibrinstruktur 39, verwendet viskoelastischen und Spektralanalyse zu Fibrin Strukturen 40 analysiert und verwendet entwickelten Korrelationen zwischen Fibrin Struktur und mechanischen Eigenschaften mit experimentellen und theoretischen Ansätze 41 </sup&gt;. Der Fokus der vorliegenden Studie war es Gerinnsel Strukturen unter simulierten diabetischen und Sichelzellen Thrombose Bedingungen formulieren und konfokale Mikroskopie zur Untersuchung der Struktur und Funktion der Blutgerinnsel in Krankheitszuständen verwendet werden. Fibringerinnsel wurden aus menschlichem Fibrinogen, humanes Thrombin und FXIIIa gebildet. Die Gerinnsel wurden mit Plasmin lysiert. Diabetischer Bedingungen zu simulieren, wurde erhöhte Konzentration an Fibrinogen in der Glukoselösung inkubiert, um eine in vitro Glykierung Fibrinogen induzieren. Um Sichelzellanämie Gerinnungsbedingungen zu simulieren, wurden erhöhte Fibrinogenkonzentrationen mit Sichelzellen Hämatokrit von Patienten gesammelt, wie zuvor von unserer Gruppe 42 getan gemischt. Diese Verfahren wurden verwendet, um die Struktur und die Funktionen in Fibringerinnselbildung und Fibrinolyse bei Krankheitszuständen beteiligt sind, sowie die Mechanismen, die CVD induzieren zu untersuchen. Basierend auf den aktuellen Informationen über diese Krankheiten waren die glykosylierten Fibringerinnsel Strukturen dichter mit weniger einnd kleineren Poren. Die Fibringerinnsel mit roten Blutzellen von Sichelzellpatienten (RBCs) wurden ebenfalls dichter und angezeigt Aggregation der Erythrozyten und agglomeriert Fibrin Clustern. Dies ist ein gut etabliertes Phänomen, das zuvor 43 festgestellt hat. Es wurde auch vermutet, dass die Fibrinolyse Quote wäre in glykosylierten Fibringerinnsel mit und ohne reduzierte Plasmin im Vergleich zu gesunden, normalen Fibrin deutlich geringer. Die Ergebnisse zeigten, dass für glykosyliertes Fibringerinnseln, signifikant Lyserate Ergebnisse wurden nur unter den Bedingungen einer reduzierten Plasminkonzentration beobachtet. Dieses experimentelle Technik der Verwendung der konfokalen Mikroskopie bietet signifikante Vorteile gegenüber anderen bildgebenden Verfahren, weil die Zellen und Proteine ​​in ihrem nativen Zustand, der die Erfassung von Echtzeitvideo der Gerinnungsaktivität ermöglicht bleibt. Diese Methode synthetisch induzierenden Blutgerinnung ist auch billiger und zeiteffizienter als Erhalt von Patientenproben und Ausfiltern einzelneProteine ​​und Enzyme. Ferner ergeben sich mit getrennten Proteine ​​und Enzyme, um Gerinnsel zu synthetisieren, die Gerinnsel wurden standardisiert, so dass es nicht die Variabilität zwischen den Proben als Folge von anderen Proteinen im Plasma.

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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</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.

Konfokale Mikroskopie Analyse der Glykiertes Fibringerinnsel Strukturen Konfokales Mikroskopiebilder von normalen und glykiertem Blutgerinnsel sind in Abbildung 3 dargestellt. Die konfokale Mikroskopie-Analyse der normalen und glykiertem Blutgerinnsel zeigt, dass glykiertes Blutgerinnsel sind dichter mit kleineren Poren als die normalen Gerinnsel sowohl mit als auch ohne Zusatz von FXIIIa während der Gerinnsel Polymerisation. In 3A und 3B ist</stron...

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.

Experimentelle und Bildgebende Verfahren für die Prüfung Fibringerinnsel Strukturen in normalen und erkrankten Staaten

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

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11.3K Aufrufe.  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.

Serie: 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.

Behandlung von osteochondralen Defekten in des Kaninchens Kniegelenk durch Implantation von allogenen mesenchymalen Stammzellen in Fibringerinnseln

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

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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 Hochauflösende<em&gt; In vivo</em&gt; Bildtechnik, um die Dynamik der intrakranielle Strukturen zu Tumorwachstum und Therapeutics Studieren

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.

Eine Pipeline für 3D Multimodalität Bildintegration und Computergestützte Planung in der Epilepsie-Chirurgie

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.

Visualisierung von Gefäß- und Parenchymregeneration nach 70% Teilhepatektomie in normalen Mäusen

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.

Eine intramedulläre Verriegelungsnagel für Standardisierte Fixierung von Femur Osteotomien zur Analyse von normalen und Defekte Knochenheilung bei Mäusen

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

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

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

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

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

In vivo Verfolgung von Ödementwicklung und mikrovaskulärer Pathologie in einem Modell der experimentellen zerebralen Malaria mit Magnetresonanztomographie

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.

Quantitative Visualisierung von Leukozyten-Infiltrat in einem Murin-Modell der Fulminant-Myokarditis durch Licht-Blatt-Mikroskopie

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 Morphometrische Analyse der menschlichen Hirnnerven mittels Kernspintomographie in der Menière Krankheit Ohren und Normal hörende Ohren

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.

Herz-Magnetresonanz zur Beurteilung von vermutetem Herzthrombus: Konventionelle und aufkommende Techniken

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