我们要感谢纳丁·海恩和阿伦德Heerschap的宝贵援助。这项工作得到了再生医学的荷兰研究所（NIRM，FES0908），欧盟第七框架计划ENCITE（医学-F5-2008-201842）和TargetBraIn（医学-F2-2012-279017），从大众汽车基金会的资助（支持的I/83 443），荷兰科学研究组织（VENI 700.10.409和VIDI 917.76.363），ERC（高级格兰特269019）和Radboud大学Nijmegen医学中心（AGIKO-2008-2-4）。

注:所有的实验和程序涉及动物，必须进行按照相关的道德准则和符合标准的动物护理和人性化的要求。 1。细胞标记（与共同孵育标准协议） <ol><li>添加19女性标签8未成熟DC在4 mg/10 6细胞浓度（2毫升培养基）。轻轻摇晃混合。 </li><li>孵育的细胞与标签的成熟和收获的DC前3天。 </li><li>收集区议会，并在PBS冲洗至少3倍去除多余的标签。计数细胞。 </li><li>重新悬浮于小体积的PBS注射或进一步测试。 </li></ol>注:该协议是有关这些特定的DC和标签。该程序进行了优化，在其他出版物8和包含在这里仅仅是步骤的样品准备图像，因为该协议的重点是成像。 ħENCE，要么需要在文献中被发现，或由研究人员优化了协议分离，培养和特定细胞类型的标签。那已经在文献中使用的所有其他19女性标签摘要6其他地方提出。 2。 19 F /电池使用19 F-NMR测定<ol><li>放置标记细胞的公知的数（通常为大于0.5×10 6，或一个数字，导致足够的信号）中的NMR管中。 </li><li>加10微升的5％三氟乙酸（TFA）。如果TFA的谐振频率是不合适的，由于带标签的重叠，使用另一种水溶性化合物，优选与单个19女性共振。 </li><li>将样品放置在一个NMR波谱仪，并获得19女性光谱。确保该带宽足以同时为TFA（参考）和代理。 </li></ol>注:TR应该是适用于F足够长基准的丝凯蒂松弛和样品旋转（大约5时间T 1弛豫管内的最长Ŧ1组分时，通常是TR〜5-8秒）。有些光谱仪需要D 2 O中的一个频率锁定信号。一个标准的19女性谱就足够了。广泛的平均或更高的单元号将被要求如果标签的细胞摄取不足或如果细胞数量低。频谱应参考和有关标签峰之间居中，除非更正作出解释部分的激励。 <ol start="4"><li>计算的相对面积下的基准和样品（或样品的主峰）的峰，然后计算19女性的/细胞的样品中的平均数目。 </li></ol>注意:整个过程必须重复并取平均值广泛，足以达到统计学意义。这也可以直接使用光谱在MRI扫描仪完成的。 ħH但是，请注意，此过程对于大量的细胞，而不是细胞之间的变异性揭示了只有平均19女性负载。检查细胞摄取的均匀性要求在19女性剂的荧光性成分的存在下，使细胞摄取可使用显微镜或流式细胞仪进行分析。 3。注意事项实验设计 - 细胞注射由于19女性 MRI对细胞示踪，动物模型，细胞将大量本地化比较差的敏感性是必要的成功的体内成像。典型的灵敏度值范围为1000〜100000 cells/voxel/1小时扫描时间6，在10 11 -10 13个氟原子的范围内的小区负载。 成像会话的数量和频率，必须精心策划，因为这会影响麻醉的选择。需要注意的是异氟醚可能不适合初学者E的19女性 MRI检查，如果异氟醚的19女性共振频率接近，所使用的标记化合物的。异氟醚可能仍然是合适的，如果在成像会话是足够短以防止显著积聚。几种可供选择的麻醉剂在文献10,11已被使用，一个例子是包含在成像协议。麻醉剂，可能需要对不同小鼠品系和疾病模型进行优化。 4。外部19女性参考设计<ol><li>使用含有定量12 19 F信号的已知量的外部参考。选择的基准的信号强度为大致比得上从受试者的预期。 </li></ol>请注意:一般来说，它是最简单的，如果参考化合物是相同的单元格标签，以匹配松弛参数。如果没有空间定位分光序列或序列被使用，那么交流渣浆泵具有不同的化学位移可能是必要的。 <ol start="2"><li>修改的位置，大小和参考的形状根据需要，依赖于感兴趣区域（ROI）的区域。注意:它一般是最容易使用的管具有均匀的横截面为基准。 </li></ol> 5。成像和鼠标准备<ol><li>稀释市售氯胺酮和甲苯噻嗪1点10分体积/体积生理盐水，得到10毫克/毫升氯胺酮原液和2毫克/毫升的甲苯噻嗪。 </li><li>开启加热系统在扫描仪（通常为暖空气），以确保孔将热情鼠标成像之前。 </li><li>注射100毫克/千克氯胺酮和10毫克/公斤甲苯噻嗪腹腔开始麻醉。注:这些剂量已经过测试，CD1（ICR）和NU-Foxn1 NU 8周龄雄性小鼠。其它菌株可能需要稍微不同的剂量，这应该在一个单独的实验进行优化。此剂量将保持鼠标麻醉的回合45分钟。注意:麻醉的深度通常是足够的，如果动物没有显示出反应到脚夹紧。 </li><li>麻醉后，将动物在动物摇篮和固定，以防止在MRI期间的运动。如果需要，定位在外部参考旁边的动物。两个参考和投资回报率（注射的细胞的预期位置）应在线圈的中心。动物应该被连接到温度和呼吸控制进行扫描期间生理监测。 </li><li>覆盖鼠标用无菌眼药膏的眼睛，以防止在长时间成像会干燥。 </li><li>如果成像时间超过40分钟，对于注入额外氯胺酮/甲苯​​噻嗪准备额外的导管前的动物可能会醒来。位置两个独立的皮下导管与氯胺酮和甲苯噻嗪的工作方案。使用此导管，让鼠标的第一个40分钟后，一个额外的2.5毫克氯胺酮每20分钟。如果需要的话，还通过注入额外0.5毫克甲苯噻嗪穿过导管的初始注射后延长麻醉，100分钟。在所有情况下，实验的时间不应超过3小时，可能除了根据终端麻醉。 </li><li>确保鼠标在第一次注射后直接升温，并保持在37℃的体温。有了这个协议，&lt;80％的血液氧合气氛下呼吸的空气检测。以防止副作用的降低血液氧合作用，使用100％的氧气（0.3升/分钟）。体温和呼吸，应在整个实验，直到动物的全面恢复控制。请注意，自主呼吸速率下氯胺酮/甲苯​​噻嗪是非常高的（200-250/min）。在呼吸模式，而不是呼吸率的凹凸，可以显示一个更高的剂量是必要的，以保持麻醉的一个合适的状态。鼠标会自发地唤醒在20-30分钟AFTEŘ麻药的最后剂量。 </li></ol>注意:对于小区的跟踪，这是必要的图像相同的动物不止一次。在这种情况下，成像会议应安排并考虑到有必要从学院的制度和动物使用指南间隙麻醉时间。 6。成像 1小时调整和成像 <ol><li>定位动物扫描仪内，使得ROI是在等角点使用低分辨率，解剖用不同的切片方向的扫描（航向）。 </li><li>使用常规的垫补协议上1小时 ， 如线圈内的整个体积在全球垫片。 </li><li>调整参考脉冲增益（RG）， 即 ，发射功率，以dB为1毫秒的厄米90°射频脉冲测量，使用由供应商提供的调整扫描。 </li></ol>注:此项调整将随RF线圈的类型，并为19女性 MRI必不可少的，因为在19 F频率的信号通常太低，直接调整。作为一个结果，对RG的估计值，必须从在1 H信号的测量进行。用于与表面线圈的RF传输，通过1 H RG应通过所关注的部分进行调整，以平行于线圈的平面上，为卷线圈与同质乙1更新时，调整的精确设置是不太重要的。 <ol start="4"><li>只有当两个1 H和19女性 RF传输是用相同的（单或双调谐）的RF线圈的19女性基准脉冲的增益调整下列协议的工作。对于这样的成像设置，线圈面（B 1场发射），通过1 H应该正比于19女性线圈配置文件，当一个固定的RG被应用， 即乙1,1 H = C *乙1,19 F 用道具ortionality常数C。 <ol><li>要确认线圈分布是成比例的特定设置，获得一个1 H和19女性翻转角映射（见乙1篇修改下面的部分）上管高度集中19楼如三氟乙酸水溶液（1:1体积/ v）的使用字段的视图相同的（视场），预先（ 图3）。 </li><li>使用相同的RG，检查两个地图都一样，除了全球性因素C。 </li><li>一旦1小时 RG是确定的，注意这个数字下降。 </li></ol></li><li>进行常规的1小时 MRI扫描作为19 F成像的解剖参考。调整系统的细胞标记的19 F频率和开展19女性 MR平扫。理想情况下，19女性 MRI扫描都会有，现场，观点和切片选择为1小时 MRI相同，但通常以较低的分辨率。动物可以尽快意马起死回生NG就完成了。图像处理然后可以离线进行。 </li><li>通过关系DB 19F =分贝1H确定19女性 RG -20 * 10日志（三）。估计19女性试剂频率在一个单独的体外实验。 </li></ol>注意: 在体内 ，确切的频率可以从实验中略有不同的实验，由于不同的匀场的条件。为防止化学位移伪影的精确频率，因此应在每次实验用19 F刘健，确定可能的话。对于非常低的19 F信号一种典型的扫描将是全局的（脉冲采集）光谱（TR = 200毫秒，NEX = 3000元，TA = 10分钟，BW = 50千赫）。 NEX，从而助教，可显着降低高19 F信号。在19女性剂频率是从频谱傅立叶变换和相位校正后厘定。 NEX指激发的数量，TA是采集时间和BW是带宽。 注:在11.7T/16厘米的动物专用扫描仪典型的成像参数9是:使用快速自旋回波（TSE）序列与TR /有效回波时间（TE EFF）的解剖1小时成像扫描= 2,200 msec/42.8毫秒，每个激励8相呼应，NEX = 2，连续10个1毫米厚的片，FOV = 1.92 x1.92厘米2，128×128矩阵， 即 150×150×1000微米3，TA = 1分钟，BW = 50的分辨率kHz和线性相位编码方案。购入具有相同的序列和匹配的几何形状19女性图像，但在较低的面内分辨率和较低的带宽（NEX = 256，48×480的矩阵， 即 400×400×1000的分辨率微米3，TA = 57分钟，BW = 10千赫）。 <ol start="7"><li>乙1篇修改 使用RF线圈不均匀乙1档， 如表面线圈，可以阻碍19 F数据量化，因为信号取决于不仅对19 F浓度也对来自线圈的距离。获得A B 1地图上的1 H通道，以正确的追溯19 F数据。这就要求1 H和19女性线圈配置文件是相同的，除了比例系数C，并有足够的1小时，背景信号中的19 F。地区提供一个示例协议用 ​​于校正的19女性自旋回波序列的是使用单调谐的Tx / Rx表面线圈调谐到两个1 H和19女性提出，并用比例乙1型材13。 <ol><li>收购与两个翻转角法14的1 H翻转角图。获取一个梯度回波扫描，很长TR（&gt; 3次1 HT 1）略低于90°估计翻转角度。关闭线圈。收购与RG第二梯度回波扫描=分贝1H -6， 即两次翻转第一次扫描的角度。 </li><li>计算根据1 H翻转角α，在体素（X，Y，Z）通过所述第一和第二梯度回波扫描的信号强度（SI）:α（X，Y，Z）=科斯（SI GE，1（ X，Y，Z）/ 2SI GE，2（X，Y，Z））14。在19女性翻盖角度是相同的（除了系数1 /℃）由于比例乙1档。根据对等15，19 F信号强度从自旋回波序列（激发翻转角α，重新调整翻转角度2α）在信号接收衰减的法拉第原理是ATT 接收 （X，Y，Z）〜α（X，Y ，Z）。由于不完善的激励衰减ATT 的Tx（X，Y，Z）〜罪3（α（X，Y，Z））16。整体的衰减被写成ATT = attRx * attTx =α*罪3（α）/ 90°。注意，这是归一化为1的情况下完美90°/ 180°激发/再聚焦翻转角。在B 1修正19 F图像的信号强度SI 19F，更正是通过SI 19F，更正 （X，Y，Z）= SI 19F（X，Y，Z）/ ATT（X，Y，Z）计算。 </li><li>以从不同的实验进行比较的信号强度，将一个参考管中的视场限定19 F浓度，并归一化的SI 19F，防锈向在参考平均信号。 </li></ol></li></ol>注:其他1 HB 1 /翻转角映射方法可以用来和不同的19女性脉冲序列需要ATT TX，19F的修改。任何种类的B1修正方案将细胞定量分析过程中引入的附加误差。如果准确的定量细胞是主要的前提下，RF线圈与同质B1的配置文件可以被用来规避这部分的协议。 7。影像处理<ol><li>制作1 H和19女性叠加图像<ol><li>导出的图像数据为最后的1 H和从扫描仪19女性大小的图像。 </li><li>打开图像处理程序的文件，如ImageJ的（免费软件，美国国立卫生研究院）。 </li><li>进行图像必要的调整（裁剪，亮度，对比度）保持调整相同的所有图像。在19女性图像通常将需要放大的，以较高的分辨率，以匹配相应的1小时的影像。 </li><li>呈现假色的19女性图像（选择不同的查找表，根据图像中j中的"图像"菜单），然后以本地化信号叠加上对应的1小时的影像。 </li></ol></li><li> 19女性量化<ol><li>选择一个19女性图像（图像大小）有关的细胞和参考可见。 </li><li>在程序中，如图像Ĵ，绘制的ROI在相关细胞中，受（噪声）以外的参考和地区。 </li><li>使用该命令分析然后测量（或干脆按Ctrl + M）来计算这些区域内的平均像素强度，它们的面积。设S（细胞）指的是平均像素强度在ROI过的细胞。乘上体积（=面积×切片厚度）来计算总的信号。 </li><li>计算出的SNR，例如使用公式SNR = 0.65个S（细胞）/σ，其中σ是像素强度的样品，该样品不含任何化学位移伪影（通常在背景空气）以外的ROI的标准偏差17。如果SNR低于5时，校正由于噪声的信号可能是必要的，并如别处11 10说明可以进行。 </li><li>否则，计算19女性的总量乘以切片厚度（计算量）由在参考19 F中的浓度在该切片的基准的区域负责在ROI超过参考信号，这是已知的，并且假定是均匀的。 </li><li>通过总信号在ROI在细胞中，通过总信号除以在参考来计算细胞的19 F中的量乘以该值。 </li><li>除以该数乘以值19 F /细胞，将其计算出的第2计算单元的数量。 </li></ol></li></ol>

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Magn. Reson. Med. , (2009).</li><li>Srinivas, M., Morel, P. A., Ernst, L. A., Laidlaw, D. H., Ahrens, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine-19+MRI+for+visualization+and+quantification+of+cell+migration+in+a+diabetes+model.">Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model.</a> Magn. Reson. Med. 58, 725-734 (2007).</li><li>Mangala Srinivas, E. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+labeling+and+quantification+for+nuclear+magnetic+resonance+techniques.">Cellular labeling and quantification for nuclear magnetic resonance techniques.</a> US patent. , (2007).</li><li>Boehm-Sturm, P., Pracht, E. D., Aswendt, M., Henn, N., Hoehn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the International Society for Magnetic Resonance in Medicine. , (2012).</li><li>Insko, E. K., Bolinger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+Radiofrequency+Field.">Mapping of the Radiofrequency Field.</a> J. Magn. Res. A. 103, 82-85 (1993).</li><li>Haacke, E. M., Brown, R. W., Thompson, M. 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> Magnetic resonance imaging: physical principles and sequence design. , (1999).</li><li>Scheffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pictorial+description+of+steady-states+in+rapid+magnetic+resonance+imaging.">A pictorial description of steady-states in rapid magnetic resonance imaging.</a> Concepts Magn. Res. 11, 291-304 (1999).</li><li>Firbank, M. J., Coulthard, A., Harrison, R. M., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+two+methods+for+measuring+the+signal+to+noise+ratio+on+MR+images.">A comparison of two methods for measuring the signal to noise ratio on MR images.</a> Phys. Med. Biol. 44, 261-264 (1999).</li><li>de Chickera, S. N., 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=Labelling+dendritic+cells+with+SPIO+has+implications+for+their+subsequent+in+vivo+migration+as+assessed+with+cellular+MRI.">Labelling dendritic cells with SPIO has implications for their subsequent in vivo migration as assessed with cellular MRI.</a> Contrast Media Mol. Imaging. 6, 314-327 (2011).</li></ol>

作者没有冲突的利益披露。

这里所概述的协议描述了用于在体内 19女性 MRI小区追踪的一般方法。尽管所描述的共培养方法，有用于与19女性剂标记细胞数不同的协议。然而，共培养是最常用的，并且可以通过加入转染试剂6的进一步优化，例如 。实际的细胞标记协议将取决于细胞类型。唯一密钥标记的步骤在这里描述的协议。通常，标签的制备和加入到细胞中的选定时间范围从几个小?...

体内细胞跟踪是用于细胞疗法1的优化和监测至关重要。由于其非侵入性的性质，成像提供了极好的机会来监测细胞在体内 。磁共振成像（MRI）是独立的电离辐射，并且允许一个优越的成像分辨率和固有软组织的对比。基于MRI的小区追踪已经用于临床跟踪的树突状细胞中的黑色素瘤患者2。传统的临床MRI进行的1小时核，目前在用流动清水组织中。另外，也可以进行MRI上的其他活性核，如13 C，19 F和23的Na。然而，只有19女性 MRI已成功应用于体内细胞示踪因为它提供了1后H的最高灵敏度在组织中缺乏的MRI检测的内源性19女性允许高信号选择性的检测外源性19 F对比剂，并允许直接从图像数据的氟浓度进行定量。有关19女性 MRI的详细讨论，请参见3-5。与19女性 MRI检查的一个关键问题是需要开发和优化适合19女性电池标签，虽然有几个标签已被开发，以实现多式联运代理6的趋势。 我们在这里描述的协议是由我们的团体7-9，其中所描述的体内定量19女性基于MRI的细胞跟踪10,11的第一篇文章的基础上的研究。使用19女性 MRI小区追踪的一般方法概述于图1中 。我们描述了一个通用协议的树突状细胞（DCs）使用一个特制的氟碳造影剂8的标记和成像。成像协议是普遍适用于不同细胞类型，标签和动物模型。该这里描述的细胞类型和动物模型应该只作为例子，并且因此我们不提供对细胞的分离和标识细节，而是着眼于成像协议。修改将是必要为每个标签，细胞类型，动物模型和成像设置，并且这些可以在文献中找到，或可能需要由研究者进行优化。一些常见的修改都包括在讨论。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>MFCD00131855</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TFA</td>
			<td>Sigma-Aldrich</td>
			<td>76-05-1 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine (Ketavet)</td>
			<td>Pfizer</td>
			<td>778-551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>QN05 cm92</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophtosan </td>
			<td>Produlab Pharma</td>
			<td>2702</td>
			<td>eye ointment</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material name </td>
		 </tr>
		 <tr>
			<td>MRI scanner</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>NMR spectrometer</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

in vivo 19f mri for cell tracking

在体内 19女性 MRI检查可定量细胞追踪，而无需使用电离辐射。它是一个可以应用到人类的非侵入性技术。在这里，我们描述了一种通用协议，用于细胞标记，成像和图像处理。该技术适用于各种类型的细胞和动物模型中，虽然在这里，我们专注于一个典型的小鼠模型来跟踪小鼠的免疫细胞。细胞标记的最重要的问题进行了描述，因为这些是适用于所有型号。同样的，关键的成像参数都列出来，虽然细节将根据MRI系统和个人设置的不同而不同。最后，我们有定量的图像处理协议。变化对于这一点，并且协议的其他部分，被评估的讨论部分。根据此处所描述的具体步骤，用户将需要适应的协议为每个特定的细胞类型，细胞标记，动物模型和成像设置。注意，在protocol还可以适于人用的，只要临床限制得到满足。

这里，我们表明，涉及19 F-标记的细胞归巢至引流淋巴结的转移协议的典型结果。 图2示出了使用TFA参考10 6标记细胞的19 F NMR光谱。成像设置进行在协议（ 图3）中所述。对于体内成像，我们使用了一个参考由同一标签的用于细胞放置在鼠标的脚之间的密封圆柱体。一位代表处理后的图像示于图4。通过应用在7.2节中描述的协议，...

Watch this Scientific Journal Video about In vivo 19F MRI for Cell Tracking at JoVE.com

In vivo 19F MRI for Cell Tracking

我们描述了一种通用协议，用于在小鼠模型中具有活体外标记的细胞用MRI 体内细胞的跟踪。包含一个典型的协议，用于细胞标记，图像采集处理和量化。

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Research

JoVE Journal

Medicine

In vivo 19F MRI for Cell Tracking

14.6K 次观看.  Radboud University Medical Center. 我们描述了一种通用协议，用于在小鼠模型中具有活体外标记的细胞用MRI 体内细胞的跟踪。包含一个典型的协议，用于细胞标记，图像采集处理和量化。 

视频: In vivo 19F MRI for Cell Tracking

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

科研

医学

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

脑血管反应性通过气体吸入挑战MRI映射

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Non-fluoroscopic Catheter Tracking for Fluoroscopy Reduction in Interventional Electrophysiology

A technological platform (MediGuide) has been recently introduced for non-fluoroscopic catheter tracking. In several studies, we have demonstrated that the application of this non-fluoroscopic catheter visualization system (NFCV) reduces fluoroscopy time and dose by 90-95% in a variety of electrophysiology (EP) procedures. This can be of relevance not only to the patients, but also to the nurses and physicians working in the EP lab. Furthermore, in a subset of indications such as supraventricular tachycardias, NFCV enables a fully non-fluoroscopic procedure and allows the lab staff to work without wearing lead aprons. With this protocol, we demonstrate that even complex procedures such as ablations of atrial fibrillation, that are typically associated with fluoroscopy times of &#62;30 min in conventional settings, can safely be performed with a reduction of &#62;90% in fluoroscopy exposure by the additional use of NFCV.

Radiation exposure is an underestimated risk in complex ablation procedures. Here, we describe a protocol to significantly decrease fluoroscopy time and dosage for both the patient and the lab staff by using a novel non-fluoroscopic catheter visualization system.

非透视导管跟踪透视减少介入电

A technological platform (MediGuide) has been recently introduced for non-fluoroscopic catheter tracking. In several studies, we have demonstrated that ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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 ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...