我们感谢C.公园阅读的手稿。这项工作是由NIAMS下奖号码K01AR061434和白血病和淋巴瘤协会奖学金奖（5127-09），以支持DP与健康，以CPL和DTS全国学院授予的内容完全是作者的责任，并且不一定代表美国国立卫生研究院的官方意见。

1，老鼠和预处理注:所有小鼠均保持在无病原体条件和所有的协议获得批准的机构动物护理和使用委员会（IACUC）在马萨诸塞州总医院。所有手术应在无菌条件下用高压灭菌消毒设备的Mx1-Cre重组酶 15进行。，ROSA26-loxP位一站式的loxP-EYFP（罗莎-YFP）和ROSA26-loxP位一站式loxP的tdTomato（罗莎-番茄），分别为从杰克逊实验室购买。 骨钙素-GFP小鼠由亨利科罗伦贝格博士提供。为OSPC迁移和增殖的体内定量分析， 的Mx1-Cre重组 + 罗莎-YFP +（单一颜色记者）小鼠。对于其分化更详细的跟踪到骨钙素+成熟的成骨细胞，我们用trigenic 的Mx1-Cre重组+罗莎-西红柿+ OCN-GFP +小鼠。 <ol><li>要标记的Mx1 +细胞在体内 ，在无菌PBS溶液准备聚肌胞苷酸（PIPC，2.5毫克/毫升），并注入200微升20克的Mx1-Cre重组+鼠标记者每隔一天连续10天腹腔注射一次。 </li><li>消除居民的Mx1 +造血细胞，照射小鼠9.5 Gy的单剂量。照射后，移植野生型骨髓细胞（1×10 6个细胞/鼠）静脉注射16的24小时。注意:由于的Mx1诱导的细胞包括造血干细胞和造血来源的破骨细胞，消除的Mx1 +造血细胞的提高的Mx1 +成骨细胞的成像质量和定量。 </li><li>骨髓移植后，监测动物的四到六周，以达到供体骨髓细胞的成功复育。 </li></ol> 2，莫使用准备<ol><li>腹腔注射50微升氯胺酮/甲苯​​噻嗪（100毫克/千克，甲苯噻嗪12毫克/公斤体重）的使用IACUC批准的程序，麻醉鼠标。注:效果持续时间可能氯胺酮/甲苯​​噻嗪的附加剂量进行扩展。 </li><li>判断鼠标是由缺乏应对脚趾和/或尾捏的完全麻醉。 （可选）当鼠标被麻醉，用胶带固定在鼻子的异氟醚防毒面具。调节异氟醚的水平，以确保动物是完全镇静。 </li><li>使用电动剪或小剪刀修剪的头皮头发。除去毛碎片和消毒暴露的皮肤用70％酒精棉签。适用撕胶，以防止角膜脱水。 </li></ol> 3，微骨折损伤<ol><li>后确保动物是完全麻醉的，使一个单一的倒L形切口以打开头皮的皮肤。简单地说，使第一横向切口（小于1厘米）starti从一只耳朵ng到另一个耳朵。转〜60℃，并继续朝着切开鼻子，直到〜2-3毫米远离鼻子。注意:该切口最小化疤痕组织的形成将被成像的区域的上方。 </li><li>用钳子拉向两边分开皮瓣。既额骨和矢状面和冠状缝交点应清楚暴露。 </li><li>通过用无菌PBS和温柔擦拭用无菌纱布或棉签清洁冲洗的开放面，直到所有剩余的头发被淘汰。 </li><li>产生对颅骨微裂缝，按住鼠标头部用一只手和一个30号针头与另一只手。通过插入一个30号针头的尖端轻轻压和扭转运动使微穿刺（约0.2​​毫米直径的使用&lt;1毫米深的伤口），左侧额骨矢状面和冠状缝的交叉点附近。注意:为了避免针头穿透进入大脑，从而最大限度地减少bleedi这是至关重要的纳克和组织损伤。 </li><li>切换到一个更大的针（20或25克）和扭针（约0.5毫米直径）扩大穿刺孔。 </li><li>重复步骤3.3-3.5生成第二穿刺对侧额骨。 </li><li>全歼受伤的斑点由PBS的连续下探，直到出血停止。 注:在出血骨折部位会干扰适当的成像和诱导瘢痕形成。 </li><li>适用于无菌生理盐水溶液或2％甲基纤维素的头骨上的下降，以避免干燥。注意:不要让颅骨表面干燥，这将降低图像的清晰度。使用凝胶或盐水的量足以覆盖面积是非常重要的。 </li></ol> 4，活体成像<ol><li> （可选）成像之前，一些老鼠被注射了血管染料以可视化的血管。注入眼窝用20μl非目标Qtracker 705（在50毫米硼酸盐缓冲2微米的解决方案）稀释80米微升PBS。使用胰岛素注射器，以减少试剂的浪费。如果信号不够明亮，额外金额可能是必需的。 </li><li>打开扫描仪​​。注:基于多边形的激光扫描仪允许在视频速率同步多路图像采集（每秒30帧）。视频速率扫描是活的动物成像非常重要的。 </li><li>开启多光子激光（飞秒钛:蓝宝石激光器）。波长设置为880 nm和调节功率为骨二次谐波成像（440纳米）。对于GFP和tdTomato激励，打开491 nm和561 nm的固体激光器。 </li><li>打开PMT（光电倍增管），检测器为每个信号（435±20n​​m的带通滤波器，用于产生二次谐波，一个528±19 nm的带通滤波器的GFP，和590±20对tdTomato。）（可选）使用638纳米的氦氖激光器和一个光电倍增管具有695±27.5 nm的带通滤波器来检测Qtracker 705信号。 </li><li>的PlacE在一个XYZ轴电动显微镜阶段的动物。按住鼠标中最舒服的姿势有电加热垫，以帮助维持体温（这会降低动物损失的风险）。使用磁带来保存鼠标成像过程中尽量减少运动，以保持成像区域尽可能的水平。 </li><li>适用于暖2％甲基纤维素凝胶或生理盐溶液的头骨上一滴，以避免干燥。把盖玻片上的成像区域。使用低倍率镜头（30倍水浸泡的目的与0.9 NA）扫描颅骨。 </li><li>使用XYZ轴控制器，发现颅骨的表面由从骨头检测SHG信号，并确定一个关键界标位置，如在矢状缝和冠状缝之间的交点。获取图像并记录的XYZ坐标。 </li><li>继续通过观察二次谐波和荧光信号要搜索的损伤的位置。 </li><li>当损伤部位或区域Õf息被发现，获得含SHG和荧光信号从感兴趣的细胞的最佳焦点平面的图像。保存XYZ坐标和矢状面和冠状缝的交叉点的距离来定义他们的精确位置为下轮成像。 </li><li>为了收集骨折损伤三维细胞和骨骼结构，记录图像的Z-堆栈（2 - 5微米的间隔）与〜100微米的骨内膜表面的深度。在完成损伤的一侧的成像后，重复成像过程下一损伤部位。 </li></ol> 5，岗位操作流程<ol><li>成像后，冲洗成象部位用无菌生理盐水溶液，以从头骨移除甲基纤维素凝胶。用消毒棉签，应用在表面上有少量三联抗生素软膏。覆盖皮瓣，并经手术缝合重新关闭头皮。这可以用一个低过敏性缝合线（可吸收聚乳糖苏来完成TURE）和5-0大小的缝合针。注意:好缝合技术减少疤痕形成。 </li><li>对待所有动物腹腔注射0.05-0.1毫克/公斤丁丙诺啡，每12小时48小时手术后。饲养动物在温暖的恢复室，直到他们重新获得足够的意识。后3〜5天，重新缝合，并重复活体成像的步骤来跟踪骨折愈合过程中的细胞变化。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

骨骼干细胞的调控可能是非常重要的定义更好的方法来实现骨骼再生。在细胞水平定量和序列成像一直是技术上的挑战。虽然小鼠长骨骨折模型已被广泛使用，并适合于生物力学研究17，它的深部组织的位置，不均匀断裂大小，软组织损伤，和稳定固定器的应用限制了连续的活体成像。这里，提供了一种通过共聚焦/双光子活体显微镜和鼠标颅骨​​骨折模型的组合来克服这些限制的方法。这种方法与成骨干/祖谱系跟踪显示其对实时性， 在骨折修复骨干/祖细胞的体内成像。 在此方法的一个主要的技术挑战是随着时间的推移，以取得一致的和高质量的图像。高品质的即时通讯年龄与最大成像深度依赖于荧光报告和成像区域的组织状态的亮度。此外，最大限度地减少激光曝光，以避免组织损伤和光漂白是用于长期连续成像重要。使用视频速率平台快速扫描是用于此目的的帮助。如果视图中的单个字段不能覆盖整个受伤，该地区可以通过采取蒙太奇图像覆盖大部分的骨折部位进行映射。它的Z堆栈的质量和整个成像会议期间获得的信息量之间的妥协是很重要的。例如，Z-堆栈的许多切片（ 例如 1-2微米级）和高清晰度易于进一步分析;然而，它们需要长期的激光曝光，导致更高程度的激光诱导的光漂白的。 由于连续的骨折损伤体内成像就必须皮和骨表面的重复手术开幕，颅骨表面纤维化瘢痕形成的是中断深层组织成像，并产生高背景荧光的通病。熟练的缝合技术和最低出血是至关重要的，以减少瘢痕形成。一般来说，三五时间重复成像，而不显著损失图像质量来实现。 给定重复成像，容易和准确地控制裂缝的大小和修复动力学的一致性的可能性，这种方法可以提供用于测试对骨折愈合，骨质疏松症和其他组织的再生，例如骨骼肌的治疗目标的合理的手段。

骨退行性疾病和与年龄相关的骨质流失，导致骨质疏松性骨折的高风险已成为公共卫生1的一大挑战。骨的维护是由骨形成的成骨细胞和骨吸收的破骨细胞控制。骨形成细胞的缺陷是与年龄相关的骨丢失和退行性骨疾病2,3的一个主要原因。虽然大量研究都集中在骨折愈合的改善，可靠的药物的发现治愈骨退行性疾病和扭转骨质疏松性骨折的弱点仍然是一个重要的问题。因此，研究骨形成细胞和它们的控制机制在骨再生与修复的来源提供了一种新颖的见解，以加强骨骼的再生和反向骨质流失的疾病。 基于克隆种群的鉴定已经提出了多能间质细胞在骨髓中的存在可能不同黄昏时分为成骨细胞，脂肪细胞和软骨细胞系体外4。近年来，多个研究报告称，骨骼/间充质干细胞（精原干细胞/干细胞）是成骨细胞的天然来源和对骨转换，重构，骨折修复5,6至关重要。此外，我们的谱系追踪研究显示，成熟的成骨细胞有一个意外的半衰期很短（〜60日），并通过他们的干/祖细胞在正常内环境稳定及骨折修复条件6不断补充。然而，干细胞在体内的同一性和细胞是如何起反应，例如破裂损伤，并提供骨形成细胞也不清楚。因此，开发一种方法，能够分析迁移，增殖和内源性精原干细胞/干细胞的分化在生理情况下，它是非常重要的。 骨折修复是通过复杂的阵列调节的多细胞和动态的过程细胞因子和生长因子7。对于骨折研究最流行 ​​的方法是使用具有长骨骨折的动物模型，并通过骨切片和免疫荧光技术8-10来分析骨。此修复过程可以由多个成像技术包括显微CT 11，近红外荧光的12，和化学发光成像13进行监测。然而，每个技术都有一定的局限性，出现了以监测在体内细胞水平的SSC / MSC功能没有有效的方法。最近，共聚焦/双光子活体显微镜已开发和使用，即使在单细胞分辨率来检测移植的肿瘤细胞和造血干细胞在它们的骨髓微环境的上下文中活体动物14。通过这种技术结合了一系列谱系追踪模式，我们能够定义成骨干/祖细胞可以被遗传标记的瞬态交流该粘病毒抵抗 -1（ 的Mx1）启动子和的Mx1诱导祖细胞tivation能保持大多数成熟的成骨细胞的过度时间，但不参与软骨细胞的产生在成年小鼠6。此外，我们证明了的Mx1标记OSPCs供给广大的骨折愈合6个新的成骨细胞。 这里，用骨谱系追踪模式和活体显微镜，一个协议被提供给限定在骨折修复的Mx1 +骨干/祖细胞的体内动力学。该协议提供了连续的成像技术来跟踪搬迁成骨干/祖细胞的成骨折部位和骨原扩张的早期修复过程的定量测量。这种方法可能在多个上下文，包括治疗候选人，以提高骨修复的评价是有用的。

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			<td height="63" style="height:63px;width:216px;">C57BL/6J (H-2b)</td>
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			<td style="width:195px;">67457-001-10</td>
			<td>&#160;Vial size: 10 ml (50 mg/ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine Sterile Solution</td>
			<td style="width:140px;">Lloyd Inc.</td>
			<td style="width:195px;">NADA# 139-236</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine Hl</td>
			<td style="width:140px;">BEDFORD LAB</td>
			<td style="width:195px;">NDC 55390-100-10</td>
			<td>Vial: 0.3 mg/ml, Doses: 0.05-0.1 mg/kg</td>
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			<td height="21" style="height:21px;width:216px;">DPBS, 1X</td>
			<td style="width:140px;">CORNING cellgro</td>
			<td style="width:195px;">21-031-CV</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Alcohol Prep Pads (70% Isopropyl alcohol)</td>
			<td style="width:140px;">Kendall WEBCOL</td>
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			<td style="width:195px;">14568-09</td>
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			<td height="21" style="height:21px;width:216px;">Extra fine Forceps</td>
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			<td style="width:195px;">11150-10</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">VICRYL*Plus Suture</td>
			<td style="width:140px;">Ethicon</td>
			<td style="width:195px;">VCP490G</td>
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			<td height="42" style="height:42px;width:216px;">Qtracker 705 non-targeted quantum dot</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:195px;">Q21061</td>
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			<td height="21" style="height:21px;width:216px;">Methocel 2%</td>
			<td style="width:140px;">OmmiVision</td>
			<td style="width:195px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pIpC (Polyinosinic-polycytidylic acid)&#160;&#160;</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:195px;">P0913-50MG</td>
			<td>100 &#956;l (2.5 mg/ml in PBS) for 10 g of mouse</td>
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			<td height="42" style="height:42px;width:216px;">Mai Tai Tunable Ultrafast Lasers</td>
			<td style="width:140px;">Spectra Physics</td>
			<td style="width:195px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Dual Calypso 491 + 532 nm DPSS laser</td>
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			<td style="width:195px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Radius-635 HeNe laser</td>
			<td style="width:140px;">Coherent</td>
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sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair

骨归连续且高度再生损伤后。成骨细胞的干细胞/祖细胞长期被假设为存在，但在这些细胞的体内示范最近才实现的。这里，所提供的体内成像技术来探讨内源性骨干/祖细胞的作用（OSPCs）及其在骨修复后代。使用骨系细胞追踪模型和颅盖骨诱导微裂缝的活体成像，OSPCs可以直接在受伤后的最初几天，在这在早期修复过程中的关键事件发生时观察到。损伤部位可连续拍摄显露OSPCs搬迁到伤害，增加数量和分化为成骨成骨细胞。这些方法提供了调查的干细胞内在和外在的分子调节剂对骨再生和修复的作用的​​手段。

<html>稳定的长骨骨折模型已经流行在断裂的研究。然而，长骨​​或大骨折模型导致多个组织的损伤，因此，在骨细胞功能的定量测量的限制。我们用针打孔（ 图1A-1C），开发了一种微创损伤（小于1mm直径与最小或没有侵入硬脑膜）上颅骨额骨。我们选择了颅骨额骨用于体内活微裂缝的成像的顶视图，因为这骨有一个平而薄的骨结构与骨髓，允许成像清晰受伤的骨，骨细胞和脉管系统没有其它组织的干扰（ 图1C）。我们观察到这种微创概括大断裂损伤的许多特征，包括软骨痂形成后跟矿物沉积和新骨的形成（数据未显示）。 "&gt; 顺序在的Mx1 +成骨干祖细胞的体内成像。接下来，我们测试了，如果这个方法能够在骨折愈合跟踪一个特定的成骨细胞群。以前，我们开发了trigenic Mx1/Tomato/Ocn-GFP双记者小鼠过路的Mx1-Cre重组酶的小鼠与ROSA26 -番茄记者和骨钙素-GFP小鼠（ 图2A）6。这种模式的优点是成骨干/祖细胞和成熟的成骨细胞的差异化标记。通过PIPC行政， 的Mx1 + OSPCs被明确标记由番茄表达，而成熟的成骨细胞的Mx1从诱导OSPCs分化表达番茄和GFP，但是，预先存在的Mx1从成骨细胞和成骨细胞成熟的非诱导祖细胞单独表达GFP（ 图2B）。后照射和骨髓替代，我们根儿ated的这些小鼠的额骨2微裂缝。我们确认，各骨折的面积由单个视野检测到与我们的30倍物镜。微裂缝的序列3D-活体成像显示西红柿+ OSPCs在骨折2日和扩展在第5天他们的网站搬迁。目前还没有或检测不到GFP +成骨细胞在这个时候。第12天，在断裂面附近骨原细胞的一个子集发起的成骨细胞（西红柿+ GFP +）的分化。随后，新的成骨细胞的积累和新骨形成（通过二次谐波生成进行分析，蓝）是显而易见的第21天，这表明成骨祖细胞的迁移和增殖是提供参与骨折愈合（ 图新的成骨细胞的主要机制2C）。 在骨折修复骨干/祖细胞的动力学。给TEST是否我们的方法提供了骨原号码的骨折愈合过程中一致的和定量的输出中，我们使用Mx1/YFP鼠标作为一个简单的谱系跟踪模型和跟踪的Mx1 + OSPCs在早期骨折修复。之后的Mx1 +骨髓细胞所取代野生型骨髓，我们对Mx1/YFP小鼠颅骨产生六个独立的微裂缝（2 /鼠标）。当Mx1/YFP + OSPCs被伤后追踪14天，我们始终如一地观察到，在损伤部位3日再发现少量的祖细胞。数字会连续增加7天，达到人口高峰在10天及14天（ 图3A）维持。我们通过测量YFP的信号强度（图像处理和分析与ImageJ的程序）量化的骨号码的动力学。我们重复了这个实验，一个类似的输出，这表明我们的方法（ 图3B）的一致性。 SS ="jove_content"FO:保持together.within页="总是"&gt; <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51289/51289fig1highres.jpg" src="/files/ftp_upload/51289/51289fig1.jpg" /> 鼠标鼠标额骨冠（CS）和矢状缝（SS）和连续活体成像的交叉点附近微裂缝的图1， 在小鼠体内颅骨损伤的影像学（A）示意图。（二）手术风险颅骨受伤之前。（C）对小鼠颅骨的活体成像代表微创受伤。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51289/51289fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 51289fig2.jpg"/&gt; 的trigenic Mx1/Tomato/Ocn-GFP鼠标图2。 体内跟踪OSPCs在损伤部位的（A）示意图（B）该图说明了出现在trigenic Mx1/Tomato的损伤部位可能的荧光细胞群/ OCN-GFP的鼠标。红色代表的Mx1 + OSPCs表达番茄。黄色代表的Mx1 + OSPCs分化，而 ​​绿色代表从预先存在的成骨细胞（GFP +）或的Mx1非诱导性（ 的Mx1 - ）新的成骨细胞的成骨细胞的成熟（西红柿+ GFP +）祖细胞的Mx1 + OSPCs（三）连续活体成像和成骨细胞在损伤部位。西红柿+骨原细胞和GFP +附近的Mx1/Tomato/Ocn-GFP小鼠颅骨损伤成骨细胞成像损伤（0天）后在t立即他受伤后表示次。箭头所示为的Mx1 + OSPCs（黄色）成骨细胞。蓝色，骨骼。虚线圆圈代表整个（单）骨折部位。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51289/51289fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51289/51289fig3highres.jpg" src="/files/ftp_upload/51289/51289fig3.jpg" /> 图3。骨折愈合过程中的Mx1诱导OSPCs的一致性和定量测量。（一）对Mx1/YFP小鼠颅骨三个独立的伤伤（0天）后，并在伤后指定的时间马上依次成像（B）定量测量的Mx1 +成骨干/祖细胞。 的Mx1 + OSPC动力学扩张损伤部位是由使用ImageJ YFP的信号强度测量。图显示了带6的伤害在每个实验的平均2次独立实验。蓝色，骨骼;绿色的Mx1 +骨干/祖细胞;红色，血管（Q-点）。比例尺是100微米（A）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51289/51289fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

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

在骨折愈合的骨祖细胞功能的定量测量需要高分辨率串行成像技术。这里，提供了用于使用活体显微镜和骨谱系追踪以顺序图像和量化修复骨骨折的处理内源性骨干/祖细胞的迁移，增殖和分化的协议。

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 ...

sequential-vivo-imaging-osteogenic-stemprogenitor-cells-during

Nanyang Technological University

Research

JoVE Journal

Medicine

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

10.5K 次观看.  Harvard Stem Cell Institute. 在骨折愈合的骨祖细胞功能的定量测量需要高分辨率串行成像技术。这里，提供了用于使用活体显微镜和骨谱系追踪以顺序图像和量化修复骨骨折的处理内源性骨干/祖细胞的迁移，增殖和分化的协议。 

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

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

科研

医学

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-神经导航<em&gt;在体内</em&gt;通过患者的大脑在一个自发性偏头痛

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

In vivo 19F MRI for Cell Tracking

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. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

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. ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

同时PET / MRI成像在小鼠脑缺氧缺血

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

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 ...

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 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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

颅内动脉闭塞允许经普通颈动脉修复再灌注的研究

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

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