这项工作是由NIH / NCI资助人数1R01CA158372-01（齐）和新药物国家重点项目资助编号009ZX09102-205（齐）部分支持。由朱迪Racadio博士提供写作服务以及是由辛辛那提大学血液学和肿瘤学系。 Vontz核心成像实验室（VCIL）中医药学院在辛辛那提大学。

动物使用的伦理陈述。所有的动物研究批准了辛辛那提大学的机构动物护理和使用委员会（IACUC协议号：11-05-05-02）和辛辛那提儿童医院研究基金会（动物福利保障号码A3108-01）。所有涉及小鼠实验其次辛辛那提和辛辛那提儿童医院研究基金会的大学动物护理指引。 1，准备动物模型 注意：下面列出三种不同的动物模型中我们先前的研究已被用于： <ol><li>原位脑瘤鼠标：使用女/女无胸腺雌性小鼠已颅内注射人U87-ΔEGFR-Luc细胞。这些小鼠建立一个积极的肿瘤，显示人脑胶质瘤的典型特征。 </li><li>基因工程的脑肿瘤小鼠模型13：品种MUT3（GFAP-CRE; Nf1loxP / +; TRP53-/ +）雄性小鼠Trp53loxP/loxP; PtenloxP / loxP位女性产生Mut6小鼠（GFAP-CRE; Nf1loxP / +; Trp53-/loxP; PtenloxP / +）。通过繁殖雄性MUT3小鼠雌性B6CBAF1 / J小鼠保持MUT3小鼠B6CBAF1 / J品种。基因型P9和P12之间的小鼠和收获自己的组织后确认基因型。 </li><li> K / BXN关节炎：已经通过腹膜内给药与KRN x NOD F1小鼠150微升血清使用C57BL/6J小鼠。这些小鼠患上关节炎24至48小时以下的血清注射。关节炎小鼠的成像是在第7天进行血清给药后，一个时间点小鼠表现出明显的宏观关节炎。小鼠应使用于下一步骤中所述的标准进行评价。 <ol><li>评价采用关节炎指数宏观评分系统如下老鼠宏观关节炎：0 =未检测到关节炎，1 =肿胀和/或爪的发红或1位，2 =涉及两个关节，3 = 3关节国际富豪VED，和4 =整个爪和数字的严重的关节炎。关节炎的评分系统是用来确定受影响的关节的数目和关节炎的小鼠爪的严重性。甚至小鼠具有最高可能的关节炎得分很少表现出不动的迹象。然而，关节炎是监视3x/week和小鼠中过量的疼痛（如从肿爪子严重不动，抑制食物和水的消耗）处死。 </li><li>注意：流体注入四到小鼠尾静脉中已无菌维持整个实验。无尘，无菌的，一次性注射器和小瓶用于研究溶液的配制和管理。 </li></ol></li></ol> 2，制备荧光标记SAPC-DOPS纳米囊泡的<ol><li> SAPC蛋白生产：重组SAPC蛋白质与人类的确切顺序SAPC产于大肠杆菌大肠杆菌细胞如先前修改4所述。SAPC用乙醇沉淀接着用高效液相色谱法纯化。冷冻干燥后，干燥SAPC被使用，其浓度通过它的重量来确定。 </li><li>混合SAPC蛋白如前所述7,10,11。混合DOPS（0.18毫克）和CVM（0.03毫克）在玻璃管中并使用氮气以蒸发脂溶剂。 </li><li>加SAPC蛋白粉（0.32毫克）到混合物中，在1ml的PBS缓冲液和浴超声处理暂停干燥混合物约15分钟，如先前所描述7,10,11。然后通过悬浮液通过在Sephadex G25柱（PD-10），以除去游离的CVM染料。激发和最终产品， 即 SAPC-DOPS-CVM纳米囊泡，的发射最大值是653 nm和677 nm处，分别为。 </li></ol> 3。成像<ol><li>使用脑肿瘤和关节炎的小鼠模型（在上面步骤1中描述的）来测试MAROI系统。麻醉小鼠的2％异氟醚来实现。 1-2％异氟醚是mainta独董用于成像过程的持续时间。暖空气被连续地和轻轻输送到成像室，用于成像的持续时间。小珠的无菌人工泪液软膏施加到小鼠的每一只眼睛，以覆盖和润滑眼睛。把小鼠放入MARS系统通过将小鼠在其脊柱仰卧位置开始朝 ​​向摄像头（ 图1）。校准MARS 380°支撑膜和使用该软件的旋转在布鲁​​克MI协议标签定位鼠标。获得小鼠基线图像之前SAPC-DOPS-CVM施用在下面描述的方式。 </li><li>注入200μLSAPC-DOPS-CVM的静脉注射入小鼠尾静脉中。管理控制老鼠和关节炎或脑肿瘤的小鼠。 </li><li>图像小鼠24小时后喷射，并再次在7-9天注射后通过取荧光（25秒曝光时间）和X-射线（10秒曝光时间）上法师在10°增量超过380度的过程中，创造了轻微的重叠，以确保有在旋转数据集没有间隙。用布鲁克MI软件，叠加到荧光X射线图像进行解剖定位。 </li></ol> 4，图像分析<ol><li>绘制一个矩形ROI涵盖视发病部位（肿瘤和关节炎）的（FOV）的字段的宽度。的投资回报率必须足够大，以在FOV内保持该疾病的特征为在380°旋转的过程中，动物的移动。对于脑肿瘤小鼠（原位及转基因模型），利用每个肿瘤模型相同的矩形ROI和它的三（3）各自的对照组小鼠的所有时间点（基线，24小时，9天）。矩形ROI每个鼠标的定位被保留在所有的时间点，利用解剖标志在每个动物的对应的X射线图像。确定在肿瘤模型中的解剖学界标（s）必须也可用于PLACe对每一个模式的各个控件相同的矩形的ROI。确定在X射线图像，允许一致的投资回报率放置解剖标志包括颅底和颧弓的后方面。它们可视化在右侧和左侧头骨在后前（PA）的图像。 </li><li>后自动背景扣除，确定每个图像平均荧光强度。荧光图像转换为使用布鲁克指数成像软件光子/秒/毫米2。绘制荧光值作为摄像角度的函数，并应用作为误差棒从使用Excel或其它图形软件的对照小鼠中获得的平均荧光值的标准偏差。 </li></ol>

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没有什么可以透露。

准确地确定位置和实体瘤和炎性病灶的大小在风湿性疾病的关键是要实现充分的治疗和随访病情恶化或缓解。虽然宝贵，当前的成像策略（X射线，核磁共振，超声，X射线计算机断层扫描）提供疾病状态不完整的评估。例如，关节炎的关节损伤通常是由X射线，它提供了对骨结构，但不能对软组织的炎症和破坏，在疾病的早期阶段的特征信息进行评估。这里介绍的方法MAROI在一个集成的，非侵入性的和更简单的平台，它也允许在患病组织或器官的完整三维映射和重建结合了X射线和复杂的软组织成像模态（ 例如 MRI或超声）的优点小动物如小鼠。 该方法以选择性亲和力的优势Øf SAPC-DOPS纳米囊泡为暴露的磷脂酰丝氨酸残基，其大量存在于癌症和炎症细胞的膜。此绑定的决定因素是SAPC，促融合溶酶体蛋白与阴离子磷脂如磷脂酰丝氨酸7,10,11很强的亲和力。当缀合至荧光探针（CVM），全身注射SAPC-DOPS可以通过荧光成像被跟踪到肿瘤和关节炎的站点。 我们的方法的局限性是与它的灵敏度，这在目前它的使用限制，以小动物，如小鼠成像。与其它成像方法，最佳的荧光信号的信噪比是由肿瘤的大小或关节炎的程度限制，而成像的组织或器官中具有高背景（自发荧光），如耳（脑成像），肠时可能会受到影响/屎（腹部成像）和熊掌（后肢成像）。在这方面，我们发现，一个遥远的红色染料，如南亚亲志愿组织更好光谱分离和分辨率在体内设置比在可见光范围内的其他的荧光探针。 其它缺陷包括动物的运动潜力在成像过程中，无论是在麻醉和验尸 （ 尸僵 ）。后肢的定位，特别是，往往难以稳定，以避免旋转过程中移动。在目前的状态的技术也耗费时间，与扫描时间只要60分钟才能完成一个完整的旋转，并获得高品质的图像。 该MAROI方法提出了一些优势比其他成像方式。在从38（或更多）不同的角度的能力，图像病变组织的荧光允许从一个单一的平面评估它时，可能会受到阻碍的可视化;这是在动物研究中有价值的，因为它可以帮助最大限度地减少所造成的影像在不恰当的角度假阴性的数量。由承的英透视图像和荧光图像，对病变部位的精确解剖定位可以被确定。最后，现场（in vivo）中成像的可能性允许要执行纵向研究。

整个动物成像已成为在动物病理生理学的研究的强有力的工具。其中当前成像系统中，MS FX PRO使研究人员能够准确地形象化荧光标记的（或发光）在活小鼠的化合物和/或组织，并且同时获得X-射线图像。与最近推出的多模态动物旋转系统（MARS）鼠标一个完整的，自动化的旋转，以在特定的角度1同时捕获荧光/发光和X射线图像的实现。图像采集可以进行编程，这样连续的图像序列可以在特定的，增量的角度小至1度被捕获。这允许一个来识别动物的最佳取向， 即得 。在该内部产生的荧光/发光信号与系统的检测装置之间的距离是最短的。这，反过来，有利于动物的用于随后的成像精确定位本身在纵向研究ssions。 在这份报告中，我们描述了一个多角度旋转光学成像（MAROI）系统， 在体内定量荧光标记强度的实施。 MAROI信号曲线分析可以在对荧光信号的分布精确地对应患病部位或感兴趣的生物学过程的直接相关性的纵向研究中使用。 本系统是用来监测荧光标记SAPC-DOPS纳米囊泡通过原位和自发性肿瘤的吸收，以及由关节炎病灶，在活的小鼠;它提供了从动物的完整的旋转覆盖多光谱派生和多数据集。中当前可用于体内成像的许多荧光探针，那些发射的近红外和远红外光谱区域赋予与皮肤和组织中的最低干扰，并提供了最高的渗透和图像清晰度辨率。我们使用CellVue褐色（CVM）2,3，远红荧光细胞连接器（防爆647/Em 667），标记SAPC-DOPS（SAPC-DOPS-CVM）4-12。

<table border="0" cellpadding="0" cellspacing="0" width="1246">
	<colgroup>
		<col /> 
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Dulbecco&#39;s modified eagle medium</td>
			<td style="width:423px;">Gibco (Grand Island, NY)</td>
			<td style="width:307px;">11965</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Fetal Bovine Serum</td>
			<td style="width:423px;">Gibco (Grand Island, NY)</td>
			<td style="width:307px;">16000077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Penicillin-streptomycin</td>
			<td style="width:423px;">Hyclone (Logan, Utah)</td>
			<td style="width:307px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Dioleoylphosphatidylserine</td>
			<td style="width:423px;">Avanti Polar Lipids (Alabaster, AL)</td>
			<td style="width:307px;">840035C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">CellVue Maroon</td>
			<td style="width:423px;">Molecular Targeting Technologies, Inc. (Exton, PA)</td>
			<td style="width:307px;">C-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sephadex G25 column PD-10</td>
			<td style="width:423px;">Amersham Pharmacia Biotech, (Piscataway, NJ)</td>
			<td style="width:307px;">17-0851-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:351px;">New Standard Stereotaxic for Rats and Mice</td>
			<td style="width:423px;">Harvard Apparatus (Holliston, MA)</td>
			<td style="width:307px;">726335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Bransonic Ultrasonic Cleaners Model 1510</td>
			<td style="width:423px;">Branson Ultrasonics (Danbury,CT)</td>
			<td style="width:307px;">CPN-952-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Multi-spectral FX system</td>
			<td>Bruker Corporation (Billerica, MA)</td>
			<td style="width:307px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-angle Rotational Optical Imaging Device</td>
			<td>Bruker Corporation (Billerica, MA)</td>
			<td style="width:307px;"> </td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo optical imaging of brain tumors and arthritis using fluorescent sapc-dops nanovesicles

我们描述了一种多角度旋转的光学成像（MAROI）系统，用于在体内监测标记有荧光标记物的生理病理过程。小鼠模型（脑肿瘤和关节炎）被用来评价这种方法的实用性。鞘脂激活蛋白C（SAPC） - dioleoylphosphatidylserine（DOPS）纳米囊泡标记CellVue褐色（CVM）荧光静脉给药。然后，将动物放置在体内成像系统的旋转支架（MARS）。图片是在10°以上的步骤380℃收购。感兴趣区域（ROI）的矩形区域被放置在整个图像宽度在模型发病部位。内的投资回报率，并为每一个图像，平均荧光强度为背景扣除后计算出来的。在所研究的小鼠模型中，被标记的纳米囊泡被接在两个原位和转基因脑肿瘤，并在关节炎位点（脚趾和脚踝）。多角度IMA的曲线分析GE的ROI中确定的角度具有最高信号。因此，对于每个成像疾病部位的最佳角度进行了表征。适用于荧光化合物的成像MAROI方法是一种非侵入性的，经济的和精确的工具，用于在体内量化中所描述的小鼠模型中的疾病状态的分析。

我们在这里展示了标有一个远红染料（CVM）SAPC-DOPS纳米囊泡特别积聚在原位和自发小鼠脑肿瘤，以及在K / BXN小鼠的关节炎关节。从投资回报率在小鼠完整旋转放置在每一个患病部位获得的串行荧光/透视图像经受MAROI曲线分析，结果发现具有最高荧光强度的最佳成像角度。 使用MARS系统的主要目的是确定荧光的最佳角度，让最精确的测量可以采取。使用小鼠脑肿瘤或关节炎的三个实验的代表性结果示。使用SAPC-DOPS-CVM和MARS系统（ 图1），观察肿瘤或炎症由于关节炎最佳的图像的角度是确定的。荧光图像，后跟一个透视采集，购入每10°在鼠标的380°旋转。荧光图像被叠加在对应的X射线图像进行图像显示和旋转动画的产生。 结果从原位脑肿瘤模型显示在图2。代表原位荷瘤小鼠（Ortho1）的荧光图像示于图2A中 。此动物的最佳图像角是10°，在该荧光光子强度是最大的（ 图2B）的位置。测量是在注射前与SAPC-DOPS-CVM（基线）和24小时注射后。对照组小鼠（肿瘤免费）收到了类似的待遇。 图3示出了从遗传工程脑肿瘤小鼠模型中比较的数据。荧光图像和光子的测量注射SAPC-DOPS-CVM（基线），然后采取和24小时（ 图3A和&lt;STRONG&gt; 3B）和注射后9天（ 图3C）。这些图表明，在荷瘤动物（肿瘤Mut49）的最佳成像角是20°24小时后喷射，但变化到10°后第9天注射。这表明，荧光信号改变与形态变化，这可能反映了肿瘤生长相关。 如表1所示，本MAROI方法清楚地表明，对于突起以增加旋转远离最佳成像角的荧光信号降低。在脑肿瘤中的荧光信号中的7％，平均下降而获得，如果在动物的身体取向是±10°的最佳成像角度偏移。平均21％的跌幅在荧光信号测量在±20°。因此，从最佳角度相对较小的偏移量可能会导致显著信号衰减。利用图像定位MAROI技术将允许英伟stigators产生更一致和可靠的数据。 该MAROI方法终于被用来评估关节炎关节由SAPC-DOPS-CVM注射后SAPC-DOPS-CVM 24小时的目标。这种动物拿下3有三个关节炎关节。脚趾和关节炎小鼠的脚踝的荧光图像示于图4A和4B。相应的光子的测量在10℃的旋转的时间间隔被绘制在图4C和4D。找到的鞋头和脚踝的最佳成像角分别为140°和120°。 总之，MAROI系统的荧光SAPC-DOPS纳米囊泡的组合表示一种非侵入性，准确且高灵敏度的策略，实时成像，这允许肿瘤和关节炎的进展在小动物的定量研究。获取一个360°多式联运成像数据集的可能性大大即兴ES数据分析和解释，如什么是可以实现的使用单一角度成像技术相比。 <img alt="表1" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187table1highres.jpg" src="/files/ftp_upload/51187/51187table1.jpg" width="600px" /> 表1。优化成像角度对各小鼠模型。的最大光子荧光（FLR最佳角度）的角度和标准的解剖角度（透视）之间的差异可以看出。当这两个角度变得越来越不同，所测得的信号显著变化。 <a href="https://www.jove.com/files/ftp_upload/51187/51187table1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig1highres.jpg" src="/files/ftp_upload/51187/51187fig1.jpg" width="600px"/&gt; 图1：多角度旋转光学成像（MAROI）设备。 <a href="https://www.jove.com/files/ftp_upload/51187/51187fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig2highres.jpg" src="/files/ftp_upload/51187/51187fig2.jpg" width="600px" /> 图2。荧光信号与图像的角度在原位脑肿瘤小鼠模型。峰的荧光信号在10°的最佳图像角（A）。图片。蓝色框显示用于量化辐射光子的投资回报率。（B）。图图像的角度对光子发射。代表原位荷瘤小鼠（Ortho1）作图对来自相同的ROI三个非肿瘤小鼠平均荧光值。测量是在基线（注射前）和24小时后注射液Ñ​​。误差线表示标准偏差。 <a href="https://www.jove.com/files/ftp_upload/51187/51187fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig3highres.jpg" src="/files/ftp_upload/51187/51187fig3.jpg" width="600px" /> 图3。荧光信号与在遗传工程小鼠模型的自发性脑肿瘤图像的角度。从投资回报率1（顶部蓝色框）在20°的最佳图像的角度。（B）和（C）的峰值荧光信号（A）的图像。从投资回报率1影像角度与光子发射图。值代表自发性脑荷瘤小鼠，肿瘤及物49，是由三个非肿瘤小鼠对绘制平均值。测量是在基线（注射前）和24小时（B）和9日（三）注射后。错误B<html> ARS表示标准偏差。 <a href="https://www.jove.com/files/ftp_upload/51187/51187fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig4highres.jpg" src="/files/ftp_upload/51187/51187fig4.jpg" width="600px" /> 图4。荧光信号与图像的角度在鼠标跟趾和踝关节的关节炎。 （A）和（B）。显示的峰值荧光信号的脚趾（A）和踝关节（B），内中的红色框所示的ROI（C）角。图图像与平均光子发射的脚趾。峰值光子发射可以在140°的角度看到的角度（D）的关系曲线意味着光子发射的脚踝。;最大强度发生在120°的角度。目标=“_blank”&gt;点击这里查看大图。

Watch this Scientific Journal Video about In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles at JoVE.com

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

我们描述了一种多角度旋转的光学成像（MAROI）系统用于体内定量荧光标记由鞘脂激活蛋白C（SAPC） - dioleoylphosphatidylserine（DOPS），纳米囊泡输送的。用人癌症和关节炎的小鼠模型，我们将演示如何在MAROI信号曲线分析可以用于精确制图和疾病过程的生物学特性。

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

in-vivo-optical-imaging-brain-tumors-arthritis-using-fluorescent-sapc

Research

JoVE Journal

Medicine

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

11.2K 次观看.  University of Cincinnati College of Medicine. 我们描述了一种多角度旋转的光学成像（MAROI）系统用于体内定量荧光标记由鞘脂激活蛋白C（SAPC） - dioleoylphosphatidylserine（DOPS），纳米囊泡输送的。用人癌症和关节炎的小鼠模型，我们将演示如何在MAROI信号曲线分析可以用于精确制图和疾病过程的生物学特性。 

视频: In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral symptoms displayed by individuals with autism. While structural neuroimaging findings have provided some insights into brain abnormalities in autism, the consistency of such findings is questionable. Functional neuroimaging, on the other hand, has been more fruitful in this regard because autism is a disorder of dynamic processing and allows examination of communication between cortical networks, which appears to be where the underlying problem occurs in autism. Functional connectivity is defined as the temporal correlation of spatially separate neurological events1. Findings from a number of recent fMRI studies have supported the idea that there is weaker coordination between different parts of the brain that should be working together to accomplish complex social or language problems2,3,4,5,6. One of the mysteries of autism is the coexistence of deficits in several domains along with relatively intact, sometimes enhanced, abilities. Such complex manifestation of autism calls for a global and comprehensive examination of the disorder at the neural level. A compelling recent account of the brain functioning in autism, the cortical underconnectivity theory,2,7 provides an integrating framework for the neurobiological bases of autism. The cortical underconnectivity theory of autism suggests that any language, social, or psychological function that is dependent on the integration of multiple brain regions is susceptible to disruption as the processing demand increases. In autism, the underfunctioning of integrative circuitry in the brain may cause widespread underconnectivity. In other words, people with autism may interpret information in a piecemeal fashion at the expense of the whole. Since cortical underconnectivity among brain regions, especially the frontal cortex and more posterior areas 3,6, has now been relatively well established, we can begin to further understand brain connectivity as a critical component of autism symptomatology.
A logical next step in this direction is to examine the anatomical connections that may mediate the functional connections mentioned above. Diffusion Tensor Imaging (DTI) is a relatively novel neuroimaging technique that helps probe the diffusion of water in the brain to infer the integrity of white matter fibers. In this technique, water diffusion in the brain is examined in several directions using diffusion gradients. While functional connectivity provides information about the synchronization of brain activation across different brain areas during a task or during rest, DTI helps in understanding the underlying axonal organization which may facilitate the cross-talk among brain areas. This paper will describe these techniques as valuable tools in understanding the brain in autism and the challenges involved in this line of research.

Neuroimaging techniques, such as functional MRI and Diffusion Tensor Imaging have become increasingly useful in characterizing the cognitive and neural deficits in autism. An examination of brain connectivity in autism at a network level along with adaptations for scanning children with developmental disabilities is presented.

探讨孤独症的脑功能磁共振成像和弥散张量成像

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

在体内的巨噬细胞成像采用MR靶向造影剂化脓性关节炎的纵向评价

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;：单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

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 Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...