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.

注：以下协议遵循由机构审查委员会（IRB）在佐治亚理工学院设置指引。 1.采血车和红血细胞分离过程<ol><li>收集40-120毫升血液捐赠者在10毫升肝素真空采血管。 4小时内收集的开始PBMC（外周血单核细胞）分离。在这段时间内，保持血液在RT。 注：O / N储存血RT还是在4℃下不建议，因为这将导致较低的PBMC产量。 </li><li>在冰冷的（4℃）无菌PBS 1：稀释全血1。 </li><li>移取10毫升的冰冷的亲水性多糖的到一个空的，无菌的50ml锥形管中。轻轻转移的亲水性多糖顶部稀释全血。 </li><li>用25毫升移液管在缓凝和吸管血液沿管的侧面非常缓慢。保证血液不与以CENTRI之前糖混合fugation因为亲水性多糖是有毒的细胞和否则，PBMC收率将会更低。 </li><li>离心血液样品30分钟，在400×g离心在4℃下。如果有的话，减少离心机的制动速度的一半最大的用于离心后更好的分离。 </li><li>抽吸掉离心血样的血浆/血小板组分。小心吸脱亲水性多糖层填充红细胞层的正上方。 </li><li>收集8-10毫升填充红细胞和稀释1：1与未消毒0.9％NaCl的盐水。 </li></ol>糖化共聚焦显微镜评价2.血块结构（模拟糖尿病凝块） <ol><li>除霜人纤维蛋白原和荧光标记的人纤维蛋白原结合物在37℃。 </li><li>吸取以下到0.5毫升刻度离心管中。 <ol><li>对于健康的，正常的纤维蛋白原血栓，混合人纤维蛋白原与10％的荧光标记的人纤维蛋白原缀合物在50mM的Tris / 100mM NaCl的缓冲液以5毫克/毫升的浓度。 </li><li>对于人工糖化血纤维蛋白原（以模拟糖尿病凝块），孵育人纤维蛋白原和10％荧光标记的纤维蛋白原结合物中的葡萄糖溶解在50mM Tris / 100mM NaCl的要得到的6.8毫克/毫升浓度5mM溶液。 </li></ol></li><li>盖采用不透明的材料，以避免暴露在光线下微离心管。孵育在37℃的水浴的管子48小时。 </li><li>使在玻璃显微镜载片的腔室由固定用的薄粘合剂在滑动的2边。 </li><li>在48小时温育期后，通过除霜FXIIIA和人α-凝血酶在RT制备试剂的血纤维蛋白的​​形成。 </li><li>稀释FXIIIA 80 U / ml的5毫米氯化钙2缓冲区。 </li><li>凝血酶稀释到4 U / ml于5毫米氯化钙2缓冲区。 <ol><li>对于正常的凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶是2.5米微克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li><li>为糖化凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶的是3.4毫克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li></ol></li><li>立即吸取30微升的血纤维蛋白溶液到由粘合剂形成的腔室。 </li><li>放置22毫米×22毫米的玻璃盖玻片为0.15毫米的2层的粘合剂的顶部的厚度。密封该开口侧用透明的粘合剂，以防止纤维蛋白凝块干燥。确保该粘合剂不与血纤蛋白凝块交互。 </li><li>允许纤维蛋白凝块聚合在21-23℃下进行共焦成像前2小时。 </li><li>采取利用共聚焦显微镜用40X / 1.30石油M27镜头采用了488纳米氩激光的凝块共聚焦显微镜图像。 </li></ol>纤维蛋白凝块的镰状细胞患者红细胞含有3.共聚焦显微镜评价<ol><li>除霜人纤维蛋白原和荧光标记的人纤维蛋白原结合物在37℃。 </li><li>吸取以下到0.5毫升刻度离心管中。 <ol><li>为健康，正常的血纤维蛋白凝块，混合人纤维蛋白原与在50mM的Tris / 100mM NaCl的缓冲液的10％荧光标记的人纤维蛋白原结合物以5毫克/毫升的浓度。 </li><li>为含有红细胞的SCD的凝块，混合人纤维蛋白原和10％的荧光标记的人纤维蛋白原结合物在50mM的Tris / 100mM NaCl的，​​使得所得的纤维蛋白原的浓度为10毫克/毫升。 </li></ol></li><li>标签使用细胞标记溶液的分离的红细胞（正常的和那些从红细胞分离协议获得镰状细胞的患者）。 <ol><li>暂停细胞以1×10 6每毫升中任何选择的无血清培养基中的密度。 </li><li>添加5微升/毫升的细胞悬浮液至细胞标记溶液。轻柔吹打拌匀平缓。 </li><lI&gt;孵育20分钟，在37℃。 </li><li>离心标记悬浮液的管在1,500rpm离心5分钟，在37℃。 </li><li>除去上清液，并轻轻重悬浮细胞在37℃的介质。 </li><li>重复清洗过程（3.3.4和3.3.5）两次以上。 </li></ol></li><li>稀释FXIIIA 80 U / ml的5毫米氯化钙2缓冲区。 </li><li>凝血酶稀释到4 U / ml于5毫米氯化钙2缓冲区。 <ol><li>对于正常的凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶的是2.5毫克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li><li>为含有红细胞的SCD的凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶的是5毫克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li></ol></li><li>吸管将10μl标记的RBCs入血纤维蛋白溶液。吸管30微升与红细胞上的显微镜玻璃纤维蛋白（参照滑动准备GLycated血块）。 </li><li>允许纤维蛋白聚合，在21-23℃下进行共焦成像前2小时。 </li><li>取使用共焦显微镜的凝块的共聚焦图像，并利用激发激光器的488纳米和633纳米的波长，以激发荧光标记的血纤维蛋白和红细胞。 </li></ol>纤溶价格在糖化血块结构4.共聚焦显微镜评价<ol><li>重复糖化血凝块（协议第2步）在糖化血栓纤维蛋白溶解率评价的共聚焦显微镜协议。 </li><li>不要封明确胶粘剂室的两端。允许纤维蛋白凝块聚合1.5小时，在21-23℃下在含有250ml水，以防止脱水密封外壳。 </li><li>聚合后，获得使用共焦显微镜的凝块的图像。 </li><li>吸管纤溶酶到腔室的开口端，并通过经由毛细管作用凝块分散纤溶酶。 <ol><li>为正常和糖化纤溶实验正常浓度，吸管25微升在纤溶酶200微克/毫升到腔室的开口。 </li><li>为糖化凝块具有减小浓度，吸管25微升，在50微克/毫升到腔室的开口。 </li></ol></li><li>使用共焦显微镜软件时间序列的特征（ 见图1）捕获的纤维蛋白溶酶裂解凝血块的实时录像。 <ol><li>点击捕获模式，然后选择“时间序列”。选择周期的数量和记录时间间隔。 </li></ol></li></ol> 5.计算纤溶价格<ol><li>通过设置的区域（2500微米2），并计算溶解血栓的一个固定区域所需的经过时间确定裂解速率。计算使用下面的等式（ 图2）的裂解速率： <img alt="式（1）" fo:content-width="2in" srC =“/文件/ ftp_upload / 52019 / 52019eq1.jpg”/&gt; </li></ol>价格纤溶6.统计分析<ol><li>分析使用统计分析软件裂解的数据。执行单向ANOVA和事后杜克-克莱默试验44,45。 </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+fibrin.">Fibrinogen and fibrin.</a> Advances in protein chemistry. 70, 247-299 (2005).</li><li>Collet, 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=Influence+of+Fibrin+Network+Conformation+and+Fibrin+Fiber+Diameter+on+Fibrinolysis+Speed+Dynamic+and+Structural+Approaches+by+Confocal+Microscopy.">Influence of Fibrin Network Conformation and Fibrin Fiber Diameter on Fibrinolysis Speed Dynamic and Structural Approaches by Confocal Microscopy.</a> Arteriosclerosis, thrombosis, and vascular biology. 20, 1354-1361 (2000).</li><li>J&#246;rneskog, G., 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=Altered+properties+of+the+fibrin+gel+structure+in+patients+with+IDDM.">Altered properties of the fibrin gel structure in patients with IDDM.</a> Diabetologia. 39, 1519-1523 (1996).</li><li>Svensson, 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=Acetylation+and+glycation+of+fibrinogen+in+vitro+occur+at+specific+lysine+residues+in+a+concentration+dependent+manner:+A+mass+spectrometric+and+isotope+labeling+study.">Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study.</a> Biochemical and Biophysical Research Communications. 421, 335-342 (2012).</li><li>Pieters, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+of+fibrinogen+in+uncontrolled+diabetic+patients+and+the+effects+of+glycaemic+control+on+fibrinogen+glycation.">Glycation of fibrinogen in uncontrolled diabetic patients and the effects of glycaemic control on fibrinogen glycation.</a> Thrombosis Research. 120, 439-446 (2007).</li><li>Baradet, T. 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.

以获得有关的疾病状态的凝血机制结构有意义的数据，以隔离参与凝血，以确定在这些条件下，蛋白质和细胞的效应的因素是很重要的。这个协议是用于在体外研究了血纤维蛋白凝块的结构在糖尿病和SCD状态的目的而开发的。 有必要了解涉及纤维蛋白形成和纤维蛋白溶解中的疾病状态，因为改变的条件下会引起高凝，动脉粥样硬化，和CVD的机制。从本实验所得到的共?...

伤到血管的内皮细胞衬里穿过止血反应，或血液凝块的形成修复。当血液渗透到细胞外基质，组织因子激活在血流中血小板，以促进凝血级联的引发。这个愈合过程中的关键机械部件是纤维蛋白基质中，血纤维蛋白纤维是高弹性的组成，并可以承受较大的力1-4。许多研究人员广泛，在过去几十年的研究5-13形成结构纤维蛋白和功能。 患者的疾病，如糖 ​​尿病和镰状细胞具有显影血栓性并发症，如心肌梗塞，缺血性心脏疾病的危险性增加，和笔画14-19。超过200万人新近每年在美国诊断为糖尿病。有两种类型的糖尿病：I型，其中身体不能产生胰岛素，和II型，其中身体变得耐胰岛素的足量的。糖尿病患者中，心血管疾病（CVD）是原因的80％与疾病20,21相关的发病率和死亡率的。 镰状细胞病（SCD）是一种遗传性血液疾病，影响超过10万人在美国22。 SCD是一个点突变的疾病，导致红血细胞成为月牙形，使其难以使细胞通过血液脉管系统23。这两种疾病状态增加显影在体内动脉粥样硬化病症的几率。其中的一个原因为，这是改变的血纤维蛋白的结构和功能在疾病状态14,24-26的结果。 在糖尿病和镰状细胞病，有高凝和诱导动脉粥样硬化和心血管疾病的低纤溶活性（℃VD）相比，患者的健康17,27,28。已知的是低纤溶促进动脉粥样硬化的进展和滋生患者过早冠状动脉疾病29复发缺血事件。在当前的手稿，我们研究在这个特定设置中的血纤维蛋白的​​物理性质的作用。纤维蛋白凝块的结构在非患病患者是由细纤维，毛孔变大，并且通常较少稠密14,24的。在健康患者增加的孔隙率和密度较小的纤维蛋白凝块已发现，以促进纤维蛋白溶解16。在hyperthrombotic条件如糖尿病和镰状细胞病，有一个增加纤维蛋白原的生产，导致纤维蛋白原浓度从2.5毫克/毫升的正常水平在健康患者30-33增加。形成在糖尿病患者的血纤维蛋白凝块已被发现是不太多孔的，更硬，有更多的分支点，并且较密时相比健康的，非直径betic患者14,24,33-35。改变的血纤维蛋白结构是发生在涉及凝块形成的蛋白质糖基化机制的结果。当葡萄糖分子结合，以赖氨酸残基上的纤维蛋白原分子，其抑制从适当交联的谷氨酰胺和赖氨酸残基33,36,37人类XIIIa因子（FXIIIA）发生非酶（不可逆的）糖基化。 血纤维蛋白网的结构分析已被广泛研究最近。特别是，研究人员利用电子显微镜和三维重建纤维网38，这两个调查血管（内皮细胞）细胞和血管外（成纤维细胞和平滑肌细胞）如何影响纤维蛋白结构39，利用粘弹性和频谱分析来分析纤维结构40，和使用纤维蛋白的结构和机械性能之间发达的相关性的实验和计算方法41 </sup&gt;。目前研究的重点是制定血块结构模拟糖尿病和镰状细胞血栓形成的条件下，并利用共聚焦显微镜检查的结构的疾病状态的凝块和功能。纤维蛋白凝块从人血纤维蛋白原，人凝血酶，和FXIIIA形成。凝块用纤维蛋白溶酶裂解。为了模拟糖尿病病症，纤维蛋白原的浓度增加温育在葡萄糖溶液中以诱导体外纤维蛋白原糖化。为了模拟镰状细胞病凝血条件，增加纤维蛋白原浓度与从患者收集以前我们组做42镰状细胞比容。这些方法被用来研究的结构和病变的条件下参与纤维蛋白凝块形成和纤维蛋白溶解功能，以及诱导CVD法的机制。基于关于这些疾病的最新信息，糖化血纤维蛋白凝块的结构是致密的具有较少一第二毛孔变小。纤维蛋白凝块与红血细胞从镰状细胞的患者（红细胞）也更致密并显示在红细胞的聚集和凝聚的纤维蛋白簇。这是一个公认的现象，即先前已43确定。也有人推测，纤维蛋白溶解率会在糖化血纤蛋白凝块有和没有减少纤溶酶相比健康，正常的血纤维蛋白显著降低。结果表明，对于糖化血纤维蛋白凝块，显著不同的裂解率的结果，观察只减少纤溶酶浓度的条件下进行。使用共焦显微镜的这种实验技术提供显著优于其它成像方法，因为细胞和蛋白质保持在它们的天然状态，这使的凝血活性的实时视频捕捉。合成诱导凝血的这种方法也更便宜，更有效的时间比获得患者样品并过滤掉个别蛋白质和酶。此外，通过使用分离的蛋白质和酶的合成凝块，该凝块被标准化，以便有没有样品之间的变异如在血浆中的其它蛋白质的结果。

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<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>
		</tr>
	</tbody>
</table>

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.

糖化纤维蛋白凝块结构共聚焦显微镜分析的正常和糖化凝块的共焦显微镜图像示于图3中 。在正常和糖化凝块的共聚焦显微镜分析表明，糖化血块与较小孔隙比正常凝块都具有和不具有凝块聚合过程中加入FXIIIA的致密。在图3A和3B中 ，存在其中在一个不太致密的结构比糖化凝块具有较高的血纤维蛋白浓度（ 图3C和3D）</str...

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.

对于检查纤维蛋白凝块结构在正常和疾病状态的实验和成像技术

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 次观看.  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.

视频: 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.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

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

科研

医学

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.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

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.

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

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.

管道在癫痫手术3D多模图像集成和计算机辅助规划

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.

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

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.

髓内锁钉股骨截骨标准化固定用于分析正常和有缺陷的骨愈合小鼠

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.

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

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.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

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.

体内Menière 病耳和正常听觉的磁共振成像对人颅神经的形态学分析

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.

心脏磁共振评估疑似心脏血栓:常规和新兴技术

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