这项工作已经由国家癌症研究所的资助以R01 CA120368，以R01 CA109558（国民党，缩减工时，护送队，BWP），RO1的CA132750（兆焦耳，BWP）和K25中CA138578（佛罗里达州）的资助，加拿大健康研究的博士后奖学金获奖研究院（国民党）。部分资金由高级研究技术（蒙特利尔，QC）的荧光断层成像系统的发展。

1。动物的制备<ol><li>麻醉裸鼠腹腔内注射氯胺酮 - 甲苯噻嗪（100毫克/千克10毫克/公斤的IP）（查尔斯河，威尔明顿，马萨诸塞州）。 </li><li>鼠标放置在立体框架，使一个切口，将头皮上的头骨左侧，使用18号针头，创建头骨2毫米到1毫米直径的孔从中央线和2毫米的背后前囟门。 </li><li> 5μL磷酸盐缓冲溶液注入5×10 5的U251人类神经胶质瘤细胞（请在达特茅斯学院，汉诺威，NH博士马克以色列提供）到左侧大脑半球表面以下约2毫米的深度大脑。使用一个Hamilton微量注射器18钝截至细胞植入27号针头插入针从头骨的外表面3毫米的一角，然后退出1毫米到创建一个细胞的口袋。 </li><li>缝合切口部位，并允许重新covery从手术。 </li><li>等待〜14天，使肿瘤生长前成像。 </li></ol> 2。荧光体层摄影术系统标定<ol><li>鼠标成像的日子，启动系统和激光器和光探测器，允许约20分钟热身，以避免在系统的灵敏度漂移。 </li><li>放置一个100°×4°在直接成像龙门中心工程线扩散器（Thorlabs，牛顿，新泽西州），正常的激励激光皮秒脉冲80MHz的多模635 nm激光二极管（PicoQuant光子北美国公司，西田，马）。扩散器的角度调整，以最大限度地发挥所有五个光收集渠道检测到的信号量。提供一个完整的描述成像几何别处11,14,15。 </li><li>将外径2中性密度过滤器在前面的所有荧光检测的光电倍增管（PMT）和外径1中性密度过滤器（Thorlabs，牛顿，新泽西州）（THorlabs，牛顿，新泽西州），在前面所有透过率检测的光电倍增管。收集100时空传播的激光脉冲型材（TPSF），每个1的整合时间。 </li><li>正常化的激光参考每个TPSF，正确的时空漂移在激光参考，超过平均每个探测器的所有迭代。这些的平均TPSFs是探测器特定的仪器响应函数（IRF）的光学图像重建中使用。 </li></ol> 3。成像议定书“ <ol><li>麻醉与异氟醚2％的氧气（1升/分钟）的鼠标。 </li><li>注入100μL磷酸盐缓冲液，腹腔，12小时前1 nanomole的IRDye 800CW-EGF的Alexa Fluor 647和1 nanomole针对表皮生长因子受体的过度表达，在肿瘤成像。 </li><li>鼠标放置到玻璃纤维成像床支持，安排的鼠标，所以它的鼻子仍然在提供异氟醚麻醉锥。 </li><li>恩确保鼠标定位： 即在床上适当的荧光断层成像系统固定在床上时鼠标是在的成像龙门近似中心。有关鼠标的激发激光180°旋转，确保激光的焦点照亮中心鼠标一点，大约从激光在各个角度的角度来看，这种定位可以指导。 </li><li>一旦定位，仔细转移成像床和鼠标的微型电脑（探索轨迹，GE医疗，伦敦，ON）扫描仪，并收集在鼠标的整个头的分辨率为93微米的各向同性的解剖信息。 </li><li>可视化CT图像堆栈和选择（S）与荧光断层成像系统成像切片。 </li><li>仔细转让荧光断层成像系统成像床和鼠标。选择源位置的编号，以收集有关鼠标的数据，为每个成像SLICE（32），积分时间为每个TPSF测量（1秒），为每个源的位置（10）的迭代的数量，并从步骤3.6 CT图像堆栈中的地位和所需的影像片的数量。括号中的数字是每个成像产生每成像片〜5分钟的数据采集参数的典型值。 </li><li>放置在前面的荧光检测的光电倍增管的三重缺口过滤器（色度科技公司，波纹管瀑布，VT），以限制任何达到荧光探测器的激光，外径2中性密度滤光片的透过率检测的光电倍增管前，以避免饱和这些探测器。 </li><li>运行数据采集软件，收集在每个定义的源，探测器位置和荧光激发波长（635 nm和755 nm的激发的Alexa Fluor 647和IRDye 800CW-EGF的示踪剂，分别）为每个和透光TPSFs的。为每一套TPSFs收集，监视和记录的激光强度参考的PMT通道。 </li></ol> 4。图像重建<ol><li>确定鼠标的外表面和成像床支撑杆从CT图像的位置，并创建口罩，分别涵盖了鼠标的束缚，成像棒。 </li><li>使用鼠标面具产生了使用的NIRFAST软件19动物的有限元网格。 </li><li>本地化基于微型电脑和荧光的空间登记统筹20网格表面上的荧光断层成像系统的源和探测器的位置。 </li><li>卸下光学数据源或探测器的位置成像床支撑杆的位置交互点。 </li><li>规范化数据收集在每个源探测器的位置由激光参考，正确的时间漂移​​在激光参考，正确的过滤器的灵敏度，这是由实验测试确定<html>在购买时间15 </li><li>每个源探测器位置的数据的出生比率（荧光分为透光）和繁殖与动物统一的光学性质的有限元网格上的透光率的正向模型的仿真。这样做，以减轻相关的错误源或探测器组织耦合21，校准数据模型22，和数据调整等方面的数据模型不匹配23,24。 </li><li>构建一个数据向量组成的出生比例收集到的数据在两个波长的规模差异。比例因子的选择，以最大限度地提高EGFR结合对比。执行时域与校准的区别使用TPSF的数据作为输入的每个检测通道的图像重建，并建立荧光增强针对性示踪15地图。 </li></ol> 5。代表结果“&gt;荧光重建（ 图1b确定脑胶质瘤的质量中心共同注册的鼠标在图1b 1 U251细胞原位脑胶质瘤肿瘤与头部CT的解剖图像叠加荧光重建的一个例子。 ）CT和MRI图像质量对比增强磁共振成像（ 图1a）确定的肿瘤中心内1毫米。共登记基于一个共同的信息化改造。 <img src="/files/ftp_upload/4050/4050fig1.jpg" alt="图1" /> 图1。对比度增强（钆）鼠标头部的磁共振成像（一）。鼠标用的U251人脑胶质瘤细胞株接种原位。吸收超过正常脑造影剂，肿瘤的位置，可以看到在左侧大脑半球（右图像）和白色箭头指示。 “都有对应Ğ计算机断层扫描图像（从同一位置上的鼠标头），（二）与表皮生长因子有针对性的荧光减去不相关的荧光重建覆盖描绘。荧光的单位是在逆毫米和涉及到的结合针对性的荧光量子效率和乘以其浓度的吸收系数。

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没有利益冲突的声明。

荧光断层扫描（FT）是一个敏感，电离辐射自由分子成像方式的基础上通过生物组织的可见光和近红外光传输。在FT的利益大部分都集中在小动物实验模型1和重要的研究领域之一，一直是癌症的生物标志物的表达和26分子疗法的研究，以加速药物发现和开发的潜力。目前，有两个相互竞争的办法FT系统设计。最常见的设计是基于冷却的电荷耦合器件（CCD）摄像机荧光检测4-9。这种设计提供了一个高密度的测量，最大限度地组织取样，因为每个像素的CCD相机可以检测光线已走遍组织的一个独特的路径。然而，CCD相机有一个有限的动态范围和读出噪声限制了他们的最终灵敏度。第二个设计，避免了潜在的限制<html> CCD相机检测的tions采用高度敏感的单光子计数技术的基础上，这种探测器使用光电倍增管或雪崩光电二极管10-13的影响。这些更敏感的检测方法的缺点是，每个探测器可以只收集在一个单一的点光，因此，要实现组织密度采样，要么许多探测器，必须使用（这是非常昂贵），或许多预测成像具有相同探测器（可以是耗时）。小动物的FT组织取样的最佳水平，而没有商定，对案件逐案的基础上可能会有所不同，它同意，单光子计数仪器更适合探索金融时报“的敏感性限制条款它能够探测到低浓度的分子标记。在这项研究中，我们提供了一种方法，为开展金融时报“使用单光子计数检测仪器定位在小鼠的肿瘤。 耳鼻喉科“&gt;有涉及到生产与时间相关单光子计数金融时报”的强大的数据集的四个关键步骤。首先是一个合适的和简单的校准过程中的应用。在提出的方法，每个检测通道各自的敏感性占收集计量基准的激发光传输线扩散器通过旨在引导光等于每个探测器的分数15。此外，在实验中检测光不断校准激光参考，双方强度和平均时间，这可能会随着时间的推移而波动11,15激光参考通道的运作，第二个关键步骤是“金融时报数据的准确收集和解剖成像制导荧光重建共登记单没有提供解剖信息;因此，为了创造一个光传输模型可以用来重建的劳在试样表面荧光检测的标本内的荧光来源的阳离子，解剖标本FT系统必须准确知道。在我们的系统中，被收购的解剖信息与空间坐标空间已注册的FT系统15,20微型计算机断层扫描系统。第三个关键的一步是确保最佳曝光（ 即总光子探测时间为每个激光投影）受聘在每个源探测器的位置。这是很重要的原因有两个：第一，要确保有足够的信号与噪声在每个检测位置和第二，以避免探测器饱和，这可能会损坏检测单位。为了实现在每个探测器位置的最佳曝光，自动曝光控制，基本上从二，低信号暴露14三角测量的最佳曝光。第四个关键该方法的步骤是引用传输激发光量的荧光数据收集。此参照通常被称为出生比率，并提供金融时报“的许多好处，主要是减缓的数据模型不匹配的错误23,24之一。该系统的设计，同时检测窜，在每个检测通道分为2个独立的光电倍增管的光激发光荧光和传播。通过这样做，我们避免出生比率的准确性议案的任何影响。 它一方面与一个强大的数据集时域数据，图像重建涉及逆问题求解的有限元网格表达： D = JX 其中d是一个向量为N×M为N源，探测器的预测和 m TPSF时间门元素; J是一个 N x M--升灵敏度矩阵（或雅可比），L的网格节点; x是在每个节点上的荧光光学性质的载体，具有尺寸 L d是校准实验和 J期间收集的数据是模拟采用有限元解决方案。到时域扩散近似的荧光运输25。时间维的 J也探测器特定的仪器响应函数的卷积。X荧光地图利益的代表，并用Tikhonov正则化15 1的Levenberg-Marqardt非负最小二乘法解决。 这里的方法提出，它描述了一个程序化荧光标记的使用高度敏感的光子计数荧光检测小鼠肿瘤的能力，有潜力，推动金融时报的限制。在先前的研究中，采用这种潜在在较大的小鼠动物模型，如大鼠，以及在现有鼠标中小型标本系统设计，提高了灵敏度，方法，结果表明17。这种方法的直接应用，将小动物肿瘤模型体内一种高通量的手段，以评估药物疗效监测的生物标志物的表达。该系统在多个波长的荧光激发和检测的能力允许多个荧光标记物的同时检测。额外的荧光标记物提供了一种手段，审问的病理多个方面，同时，或可用于在这项研究中，双记者的方法测量在体内结合的潜力，受体密度的标记，如聘请更多的定量成像方法26,27。

<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>目录编号</td><td>评论（可选） </td></tr><tr><td> IRDye 800CW - 表皮生长因子</td><td>李-COR公司Biosciences公司</td><td> 926-08446 </td><td></td></tr><tr><td>的Alexa Fluor 647，琥珀酰亚胺酯</td><td>生命科技</td><td> A20106 </td><td>与水反应，以尽量减少非特异性结合</td></tr></tbody></table>

computed tomography-guided time-domain diffuse fluorescence tomography in small animals for localization of cancer biomarkers

小动物荧光分子成像（FMI）可以是一个强大的工具，临床前药物发现和开发研究1。然而，由组织生色团（如血红蛋白，水，脂肪，黑色素）的光吸收通常限制光信号的传播，通过厚度大于2几毫米。相比其他可见光波长，组织吸收红光和近红外（近红外）的光吸收显着降低和非弹性散射占主导地位的光组织的互动机制。荧光剂的吸收和发射光在近红外范围（600-1000 nm）的，相对近期的发展带动了成像系统和光的传播模式，可以实现在3小动物全身三维成像的发展。 尽管在这一领域的巨大进步，弥漫性荧光体层摄影术的病态性质仍然是一个重要的的稳定，相反​​复苏和空间分辨率图像重建技术，在FMI的小动物的最佳途径的问题还有待商定。大多数的研究小组已投资在电荷耦合器件（CCD）为基础的系统，提供丰富的组织取样，但最理想的敏感性4-9，而我们组和10-13少数人的追求非常高的灵敏度探测器的基础上的系统，允许组织密度采样，此时要实现成本低成像吞吐量。在这里，我们证明申请单光子荧光断层成像系统的检测技术，本地化癌细胞在小鼠模型脑损伤的方法。 荧光断层成像（FT）系统采用单光子计数用光电倍增管（PMT）和信息丰富的时域光检测在非接触式构象11。这提供了一个同步的山坳激发和发射光传输的经文，包括激发自动曝光控制14，激光参照，与小动物计算机断层扫描系统（微型电脑）15，共登记。裸鼠模型被用于成像。原位接种动物与人脑胶质瘤细胞株（U251细胞）在左侧大脑半球，2周后成像。肿瘤注射荧光示踪剂荧光，IRDye 800CW表皮生长因子（Li-COR Biosciences公司，林肯，NE）的有针对性的表皮生长因子受体，细胞膜蛋白已知U251肿瘤行过度和许多其他癌症18。第二，没有针对性的荧光示踪的Alexa Fluor 647（Life Technologies公司，大岛，NY）也被注入帐户针对性示踪剂的摄取提供量化示踪结合和受体的可用性/密度的一种手段，非受体介导的效果27。一个CT引导下，TIME域算法，用于重建的两个荧光示踪剂的位置（ 即 ，肿瘤的位置）在小鼠的大脑和他们的能力，本地化的肿瘤对比增强磁共振成像验证。 虽然荧光成像在脑胶质瘤的小鼠模型表明，在这部影片中提出的方法可以扩展到不同的肿瘤模型中的各种小动物模型可能上升到老鼠的大小17。

Watch this Scientific Journal Video about Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers at JoVE.com

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

弥漫性荧光体层摄影术提供了一个相对较低的成本和潜在的高通量的方法，以临床<em&gt;在体内</em&gt;肿瘤显像。计算机断层扫描引导非接触式时域使用荧光针对肿瘤标志物的表皮生长因子受体在小鼠脑胶质瘤模型系统的光学数据收集，校准和图像重建方法。

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

computed-tomography-guided-time-domain-diffuse-fluorescence

Research

JoVE Journal

Medicine

12.2K 次观看.  Dartmouth College. 弥漫性荧光体层摄影术提供了一个相对较低的成本和潜在的高通量的方法，以临床在体内肿瘤显像。计算机断层扫描引导非接触式时域使用荧光针对肿瘤标志物的表皮生长因子受体在小鼠脑胶质瘤模型系统的光学数据收集，校准和图像重建方法。

视频: Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

科研

医学

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

一次性充填机对治疗药物的肺吹入小动物

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

Cone Beam Intraoperative Computed Tomography-based Image Guidance for Minimally Invasive Transforaminal Interbody Fusion

Transforaminal lumbar interbody fusion (TLIF) is commonly used for the treatment of spinal stenosis, degenerative disc disease, and spondylolisthesis. Minimally invasive surgery (MIS) approaches have been applied to this technique with an associated decrease in estimated blood loss (EBL), length of hospital stay, and infection rates, while preserving outcomes with traditional open surgery. Previous MIS TLIF techniques involve significant fluoroscopy that subjects the patient, surgeon, and operating room staff to non-trivial levels of radiation exposure, particularly for complex multi-level procedures. We present a technique that utilizes an intraoperative computed tomography (CT) scan to aid in placement of pedicle screws, followed by traditional fluoroscopy for confirmation of cage placement. Patients are positioned in the standard fashion and a reference arc is placed in the posterior superior iliac spine (PSIS) followed by intraoperative CT scan. This allows for image-guidance-based placement of pedicle screws through a one-inch skin incision on each side. Unlike traditional MIS-TLIF that requires significant fluoroscopic imaging during this stage, the operation can now be performed without any additional radiation exposure to the patient or operating room staff. After completion of the facetectomy and discectomy, final TLIF cage placement is confirmed with fluoroscopy. This technique has the potential to decrease operative time and minimize total radiation exposure.

The purpose of this article is to provide image-guidance for minimally invasive transforaminal interbody fusion.

锥形束术中计算机断层扫描图像指南,用于微创跨体间融合

Transforaminal lumbar interbody fusion (TLIF) is commonly used for the treatment of spinal stenosis, degenerative disc disease, and spondylolisthesis. ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

非门控断层扫描的冠状动脉钙化识别

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

小型动物正电子发射断层扫描/计算机断层扫描中的连续血液采样支持动脉输入函数的测量

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Four-Dimensional Computed Tomography-Guided Valve Sizing for Transcatheter Pulmonary Valve Replacement

The measurements of the right ventricle (RV) and pulmonary artery (PA), for selecting the optimal prosthesis size for transcatheter pulmonary valve replacement (TPVR), vary considerably. Three-dimensional (3D) computed tomography (CT) imaging for device size prediction is insufficient to assess the displacement of the right ventricular outflow tract (RVOT) and PA, which could increase the risk of stent misplacement and paravalvular leak. The aim of this study is to provide a dynamic model to visualize and quantify the anatomy of the RVOT to PA over the entire cardiac cycle by four-dimensional (4D) cardiac CT reconstruction to obtain an accurate quantitative evaluation of the required valve size. In this pilot study, cardiac CT from sheep J was chosen to illustrate the procedures. 3D cardiac CT was imported into 3D reconstruction software to build a 4D sequence which was divided into eleven frames over the cardiac cycle to visualize the deformation of the heart. Diameter, cross-sectional area, and circumference of five imaging planes at the main PA, sinotubular junction, sinus, basal plane of the pulmonary valve (BPV), and RVOT were measured at each frame in 4D straightened models prior to valve implantation to predict the valve size. Meanwhile, dynamic changes in the RV volume were also measured to evaluate right ventricular ejection fraction (RVEF). 3D measurements at the end of the diastole were obtained for comparison with the 4D measurements. In sheep J, 4D CT measurements from the straightened model resulted in the same choice of valve size for TPVR (30 mm) as 3D measurements. The RVEF of sheep J from pre-CT was 62.1 %. In contrast with 3D CT, the straightened 4D reconstruction model not only enabled accurate prediction for valve size selection for TPVR but also provided an ideal virtual reality, thus presenting a promising method for TPVR and the innovation of TPVR devices.

This study assessed a new methodology with a straightened model generated from the four-dimensional cardiac computed tomography sequence to obtain the desired measurements for valve sizing in the application of transcatheter pulmonary valve replacement.

用于经导管肺动脉瓣置换术的四维计算机断层扫描引导的瓣膜尺寸

The measurements of the right ventricle (RV) and pulmonary artery (PA), for selecting the optimal prosthesis size for transcatheter pulmonary valve ...