这项工作是由杰克逊实验室癌症中心的试点格兰特和美国缅因州癌症基金会的支持。

1。协议<ol><li>麻醉的小鼠氯胺酮和甲苯噻嗪，剂量为0.1毫克氯胺酮和0.01毫克甲苯噻嗪每1克的机身重量。卡洛芬（0.005毫克每1克的机身重量）用于镇痛，术前进行管理。 </li><li>所有的外科手术工具，包括牙科钻头，是在高压釜中灭菌的蒸汽。如果批处理以进行手术，手术器械的前端必须重新灭菌用玻璃珠灭菌器（精细科学工具FST 250）之前，每个后续的手术。 </li><li>一旦鼠标已经达到了手术麻醉平面的，没有一个响应于一个脚趾捏评定，它被放置在化妆棉的加热垫，然后放置在〜37℃下保持体温在手术过程中麻醉深度监测。 </li><li>皮草是从一个大致呈三角形面积的背头〜3 mm的尾侧鼻孔和尾部延伸向行政长官rvical椎骨。眼药膏的眼睛，以保护他们免受脱水和刺激。 </li><li>的鼠标放置在腹侧recumbancy的皮肤消毒手术碘，70％乙醇和无菌拭子。用细剪刀，一个0.8×1.0厘米的皮瓣覆盖的头骨的正面和顶骨的背侧皮肤被删除。 </li><li>本地骨膜和暴露在伤口的周边组织的局部止痛剂，0.5％的利多卡因，被施加。骨膜除去，用手术刀刀片轻轻刮头骨，这将有助于氰基丙烯酸酯胶，坚持到骨头。 </li><li>皮质区的头骨的标志上列出的兴趣。感兴趣的区域不应该设在颅骨缝合线，以避免潜在的大血管的损害。 </li><li>一层薄薄的氰基丙烯酸酯类粘合剂被施加到伤口上边距，serosanguinous流体，以防止渗漏，除了在颅骨感兴趣的区域。 </li><li>在我们的协议中使用的光活化的牙科水泥密封的头骨。牙科用粘固剂的应用之前，所有暴露颅骨除了用于与变薄的区域用的引物，并随后用粘接剂的光活化的牙科用粘固剂套件。一旦干燥粘接剂，光活化的牙科用粘固剂被施加到覆盖颅骨。为了随后的外科手术和成像稳定的鼠标的头部，无菌＃00-90六角螺母嵌入的牙科用粘固剂在从区域的距离以变薄。光激活的牙科水泥的使用，而不必急于完成的整个过程，让我们准备的头骨。一旦在颅骨上的牙科用粘固剂制备完成时，牙科用粘固剂的固化与可见光。 </li><li>的牙科用粘固剂固化后，头状从立体定向装置持有人使用＃00-90螺钉鼠标头被安装到上的六角螺母。 </li><li>一个博士运算室温，0.9％无菌盐水中被施加到颅骨区域以变薄。在立体显微镜的帮助下，一个高速的微钻头用于薄（通常约4-5毫米的直径）在感兴趣区域的圆形区域的颅骨。钻井和应用生理盐水钻取区域变薄过程中间歇地进行，以避免热损伤相关的脑组织。盐水吸收热量，还有助于软化骨骼。 </li><li>小鼠颅骨包括两个薄层密质骨，中间夹了一层厚厚的海绵状骨。紧凑的骨和大部分的海绵骨的外部层中除去使用的钻头。 </li><li>后的松质骨的大部分被除去，在海绵骨内的其余空腔可以看到在解剖显微镜下，表明钻探接近的内部致密骨层。在这个阶段，颅骨厚度仍然应该超过50微米。颅骨变薄继续仔细，得到一个非常薄的（〜20微米）和光滑的制备。 </li><li>一旦实现所需的头骨厚度（20微米），颅骨抛光用一个特制的硅鞭钻石膏（6-15微米）约10分钟。钻石膏抛光的目的有两个。首先，它进一步变薄颅骨的减薄颅骨上不施加压力，从而避免意外损坏底层脑组织。其次，它作为一个初始抛光步骤，用于减薄颅骨细粒的氧化锡被用于进一步的颅骨抛光之前。 </li><li>继钻石膏抛光，减薄的颅骨抛光用氧化锡为约10分钟。一旦头骨是抛光，氧化锡用生理盐水冲走直到出现减薄颅骨干净。 </li><li>在这一步，如果颅窗口是用于纵向体内成像，清洁的薄颅骨区域干燥。一小滴明确腈基丙烯酸酯胶施加到密封颅窗。减薄的颅骨和盖玻片之间的腈基丙烯酸酯胶的层应尽可能地薄。 </li><li>如果注射剂是创建一个端口颅窗后要执行的，在此步骤中，使用26号注射器针头的抛光薄膜颅骨窗口的一侧上创建一个小的开口。此开口用于GBM细胞注射。 </li><li>的无菌玻璃吸移管的前端开口的直径约为50μm的拉使用的移液管的牵拉。此吸移管将用于GBM细胞注射，被加载到显微操作。吸液管是装在一个30度角，使GBM细胞注射部位会离最终的成像站点。在解剖显微镜下的前端的移液管除去。 GBM的细胞悬浮液通过吸进移液管使用的注射器泵前加载。 </li><li>一旦被加载与GBM细胞的注射针，将鼠标移回下个Ë显微镜。降低对注射针小口颅窗和，最后插入大脑通过使用一台显微开幕。 1微升GBM细胞悬浮液被注入到大脑从大脑表面（速度：0.1微升/分钟，持续时间：10分钟）的100-200微米。 </li><li>干燥的头骨，明确的氰基丙烯酸酯胶一小滴被施加到干燥的颅骨，和3毫米大小排名第1盖玻片一块粘接薄和抛光的头骨区域。盖玻片之间的空间中与相邻的牙科用粘固剂是氰基丙烯酸酯胶密封。 </li><li>手术后，小鼠皮下注射1毫升温水无菌生理盐水，补充热量，以维持体温，直到完全从麻醉中恢复。 </li><li>成像，老鼠是在0.1毫克的氯胺酮和甲苯噻嗪每1克体重0.01毫克的剂量用氯胺酮和甲苯噻嗪麻醉。小鼠固定使用一个自定义的立体框架在显微镜下。如果每天重复的活体成像，异氟烷将是一个更好的选择，因为老鼠会出现重复使用氯胺酮的依赖。 </li></ol> 2。代表性的成果一个成功的端口颅窗手术治疗颅窗保持清醒的几个星期到几个月。 图1显示了一个端口颅窗下方的血管应用后向散射光。这些的血管图像被重复成像定位相同的大脑区域的地标。为了形象化的血管内皮细胞和GBM细胞，我们穿过B6.Cg-TG（TEK-CRE）1Ywa / J小鼠血管内皮细胞，其中标注的TM9 B6.Cg-GT（ROSA）26Sor“”（CAG-tdTomato ）HZE / J小鼠，和生成的红色荧光蛋白tdTomato的标记用血管内皮细胞的小鼠。然后，我们进行这些英里的端口开颅手术过程策，并注入到他们的大脑的GFP标记的GBM细胞通过一个小口的颅窗的一侧上。然后，我们在体内进行双光子成像纵向上的小鼠注射。的激光激发波长为910 nm，这导致在激发GFP和tdTomato。双光子成像与两个探测器同时绿色/红色荧光检测能力进行一个定制的双光子激光扫描显微镜。 图2显示了脑胶质瘤开始从附近的血管注入GBM细胞在体内的例子。的端口颅窗也是一个很好的选择，长期时间的推移， 在体内成像。 图3显示了从小鼠的皮质部分没有端口的手术，鼠标端口，手术后7天，和鼠标7天的端口手术后，GFAP染色生理盐水注射。很明显，从图像中的鼠标没有手术和鼠标PORTS手术不会有胶质细胞增生，而注射生理盐水后，胶质细胞增生的小鼠在脑组织中观察到，在大脑中，由于移液渗透和生理盐水注射。这一结果表明，胶质细胞增生不会发生的颅窗下的端口，提供独立的验证颅窗这里所描述的端口不会导致胶质细胞增生，通常与“开放性颅骨”慢性颅窗。 图4显示了高清晰度树突棘的端口窗口创建后从手术当天（0天）相同的鼠标和32天（32日），显示的端口窗口也是一个很好的选择， 在体内成像的高分辨率的成像。 <img alt="图1" src="/files/ftp_upload/4201/4201fig1.jpg" /> 图1。抛光和增强薄头骨的颅窗采用反向散射光下的血管可视化。数指吨<html> GBM细胞注射完成后立即采取手术当天开始他的天数，手术后的图像。比例尺：0.5毫米。 <a href="http://www.jove.com/files/ftp_upload/4201/4201fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4201/4201fig2.jpg" /> 图2。注入的的GBM细胞和血管在体内的可视化。小鼠被用于运载TEK-Cre和ROSA26 CAG-tdTomato的GBM细胞注射。血管内皮细胞标记与tdTomato（红色），与GFP（绿色）和GBM细胞标记。图像被收购GBM细胞注射后24小时开始。共10-12相邻的视场被组装彼此相邻的，与所识别的注入GBM细胞的总场的中心，以产生最终的图像。天手术后的天数。比例尺：100微米。HREF =“http://www.jove.com/files/ftp_upload/4201/4201fig2large.jpg”目标=“_blank”&gt;点击这里查看大图。 <img alt="图3" src="/files/ftp_upload/4201/4201fig3.jpg" /> 图3。胶质细胞增生不会发生以下端口的颅窗手术。上面板：低倍率下的小鼠大脑冠状切面。左面板：鼠标没有任何脑神经窗口手术，没有GFAP激活的脑组织中观察到。中间面板：鼠标与A端口的大脑的右侧产生的颅窗;胶质原纤维酸性蛋白激活的脑组织中观察到。右侧面板：鼠标端口与颅窗和生理盐水注射侧的端口窗口。 GFAP的可观察到激活的端口“窗口的侧面上，但不包括在完整的脑组织的一侧。比例尺：1毫米。下面板：更高功率的图像领域的脑组织，在上部面板的图像的白框的指示。比例尺：100微米。 <aHREF =“http://www.jove.com/files/ftp_upload/4201/4201fig3large.jpg”目标=“_blank”&gt;点击这里查看大图。 <img alt="图4" src="/files/ftp_upload/4201/4201fig4.jpg" /> 图4。从THY-1 YFPH小鼠的树突棘的高分辨率成像。图像被收购的端口手术的当天，端口手术后32天。这是明显的，从图像，大多数棘上存在的天0顷清晰可见32天以下端口手术。比例尺：2微米。

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没有利益冲突的声明。

一个成功的端口颅窗的关键是减薄和抛光。虽然最初的细化，可以迅速执行，护理应采取措施，确保在大面积同质的头骨变薄。我们通常涂上一层薄薄的生理盐水到颅骨，然后薄颅骨一通的时间，使得生理盐水溶液蒸发后不久的微钻头已经越过颅骨一次。这让我们缓慢而均匀薄头骨。在显微镜下是一个稳定的手也很关键。一旦，颅骨变薄接近预期的厚度，我们通常使用金刚石研磨膏打磨变薄颅骨1...

<table border="1"><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> DMEM/F12（1:1），丙酮酸钠</td><td>热科学Hyclone公司</td><td> SH3026101 </td><td></td></tr><tr><td> B-27血清游离补充（50X） </td><td> Invitrogen公司</td><td> 17504-044 </td><td></td></tr><tr><td> GlutaMax-1补充</td><td> Invitrogen公司</td><td> 35050061 </td><td></td></tr><tr><td>青霉素 - 链霉素溶液</td><td>热科学Hyclone公司</td><td> SV30010 </td><td></td></tr><tr><td> Accutase酶细胞支队中</td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> T25培养瓶，通气帽绿色</td><td> SARSTEDT </td><td> 83.1810.502 </td><td></td></tr><tr><td> 70％的酒精</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td> 10％聚维酮碘溶液外用</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td>氯胺酮盐酸</td><td>巴特勒动物保健供应</td><td> NDC＃11695-0550-1 </td><td></td></tr><tr><td>二甲苯胺噻嗪</td><td> Akorn公司</td><td> NADA＃139-236 </td><td></td></tr><tr><td>卡洛芬</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td>眼药膏</td><td> Dechra兽医产品</td><td> 17033-211-38 </td><td></td></tr><tr><td> 0.5％盐酸利多卡因</td><td>里程公司</td><td> NDC 0517-0625-25 </td><td></td></tr><tr><td>氰基丙烯酸酯胶</td><td>汉高公司</td><td> 46551 </td><td></td></tr><tr><td>无菌棉签</td><td> Fisher Scientific则</td><td> 23-400-114 </td><td></td></tr><tr><td>贴钻</td><td>小工具供应</td><td> BBE60 </td><td></td></tr><tr><td>氧化锡</td><td> LORTONE公司</td><td> 591-038 </td><td></td></tr><tr><td>班轮债券2V </td><td>可乐丽医疗公司</td><td> 1921-KA </td><td></td></tr><tr><td>可乐丽菲露AP-X </td><td>可乐丽医疗公司</td><td> 1721-KA </td><td></td></tr><tr><td>盐水</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td>盖玻片</td><td>华纳仪器</td><td> 64-0720 </td><td></td></tr><tr><td>六角螺母</td><td>小零件， </td><td> HNX-0090-C </td><td></td></tr><tr><td>注射泵</td><td> Syringepump.com </td><td> NE-1000 </td><td></td></tr><tr><td>双光子成像系统</td><td>自定义内置（任何商业系统工作） </td><td></td><td></td></tr></table>

imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window

神经胶质瘤是最致命形式的人类癌症之一。最有效的神经胶质瘤治疗，手术日期，然后通过辐射处理，为患者提供只有温和的好处，因为大多数患者没有生存超过5年诊断后，由于神经胶质瘤复发1,2。在人脑肿瘤中的癌干细胞的发现带来了希望有新的治疗策略的发展产生巨大的影响脑胶质瘤3。癌症干细胞是指通过他们的自我更新和分化能力，被认为是唯一的肿瘤细胞有能力启动新的肿瘤4。神经胶质瘤复发放射治疗后产生的电阻（GSCS）治疗脑胶质瘤干细胞被认为是5-10。 在体内 ，的GSCS是居住在血管周围是很重要的利基，为保持其干细胞样特性11-14 。中央的organization的GSC利基的血管内皮细胞12。现有证据表明GSCS及其与血管内皮细胞的肿瘤的发展是重要的，和拣选GSCS及其与内皮细胞作为重要的治疗目标胶质瘤。实验确定他们的能力，引发新的肿瘤，在原位肝移植15的存在GSCS。这通常是通过注入GBM细胞的特定数目的从人类肿瘤中分离到的严重的免疫缺陷小鼠的大脑，或鼠标GBM细胞到同源宿主小鼠的大脑。检测肿瘤的生长足够的时间，让GSCS注入GBM细胞之间引起新的肿瘤，通常几个星期或几个月后进行。因此，现有的检测不允许从单一的GSCS 体内肿瘤的发生过程中的重要病理检查。因此，基本我nsights到GSCS和它们的相互作用，与血管内皮细胞的肿瘤发生的早期阶段的具体作用缺乏。这种见解是开发新的治疗脑胶质瘤的策略至关重要，并且将有很大的影响，对防止脑胶质瘤复发的患者。在这里，我们已经适应了端口颅窗口程序16和体内双光子显微镜，让可视化注入的的GBM细胞在活老鼠的大脑肿瘤的发生。我们的技术铺平了道路，为未来努力澄清的关键信号传导机制之间的GSCS和血管内皮细胞在神经胶质瘤开始。

Watch this Scientific Journal Video about Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window at JoVE.com

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

通过结合抛光和增强的薄的头骨（端口）的颅窗和胶质母细胞瘤（GBM）细胞注射，我们可以观察到活老鼠大脑中的纵向注入GBM细胞胶质瘤的萌生和扩展。

成像神经胶质瘤启动<em在体内</em&gt;通过抛光和增强的薄膜头骨的颅窗

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

imaging-glioma-initiation-vivo-through-polished-reinforced-thin-skull

Research

JoVE Journal

Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

13.0K 次观看.  The Jackson Laboratory. 通过结合抛光和增强的薄的头骨（端口）的颅窗和胶质母细胞瘤（GBM）细胞注射，我们可以观察到活老鼠大脑中的纵向注入GBM细胞胶质瘤的萌生和扩展。

视频: Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

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 ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

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&#039;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 ...

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

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

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

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

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

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

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 ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

艺术颅超声成像在新生儿的状态

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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 ...

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 ...