作者承认悉尼大学显微镜 &amp; 微量分析研究设施 (AMMRF) 和澳大利亚显微镜 &amp; 微微量分析中心 (ACMM) 的设施和科学和技术援助。这些研究得到了澳大利亚国家卫生和医学研究理事会 (澳洲) 项目赠款161558和 APP1124011 的支持。

所有动物伦理和议定书均经悉尼地方卫生区动物福利委员会和天津医科大学动物保育与使用委员会 (IACUC) 批准。所有实验都是根据悉尼大学和天津医科大学百年研究所的指导方针/条例进行的, 1。小鼠脑内海绵状畸形的诱导模型 <ol> <li> 跨 Cdh5-CreErt2;Ccm2 fl/fl 与 Ccm2 fl/fl 和 #160 的小鼠; 用 Cdh5-CreErt2 生成凋落物;Ccm2 fl/fl 和 #160;( Ccm2 iECKO ) pup 和 littermate 控件 ( Ccm2 fl/fl )。 Cdh5-CreErt2 和 Ccm2 fl/fl 动物先前被描述为 19 。
	<li> 在100% 乙醇中溶解 4HT, 存储在-80 和 #176; C 等分30和 #181; L (4HT 浓度:10 毫克/毫升)。在使用日, 稀释 aliquoted 4HT 的玉米油 (0.5 毫克/毫升). </li>
	<li> 灌注入50和 #181; L 4HT (0.5 毫克/毫升) 到 P1 的新生幼崽, 用胰岛素注射器诱导实验性 CCM 病变. </li>
</ol> 2。微 CT 扫描的样品制备 <ol> <li> 通过二氧化碳窒息在 P8 安乐新生幼崽. </li>
	<li> 通过在 PBS 中插入一个29口径的3毫升2% 甲醛的针头, 将10毫升注射器注入老鼠心脏的心室进行内灌注. 
	警告: 甲醛有毒;穿戴适当的保护. </li>
	<li> 使用剪刀将头部与身体分离。用剪刀把所有的皮肤从头上取下, 用钳子剥开头骨, 解剖整个大脑. </li>
	<li> 在7.82x 放大倍数、1x 增益、0.6 伽玛和20毫秒曝光时间内, 将大脑的图像显微镜. </li>
	<li> 在 PBS 解决方案中一夜之间修复4% 甲醛的解剖大脑. 
	警告: 甲醛有毒;穿戴适当的保护. </li>
	<li> 在第二天, 使用镊子分离 hindbrains, 并用 PBS 溶液冲洗. </li>
	<li> 在卢戈和 #39 的碘溶液中孵育 hindbrains 48 h. </li>
	<li> 在孵化后, 短暂风干 hindbrains 以去除过量的卢戈和 #39 的碘溶液. </li>
	<li> 将卢戈和 #39 的碘染 hindbrains 在0.65 毫升离心管和密封完全与塑料石蜡膜, 以避免组织萎缩 ( 图 2 A ). </li>
	<li> 将离心管放在5毫升塑料管中, 用海绵防止它们在扫描过程中移动 ( 图 2 B )。
</ol> 3。小鼠脑内 CCM 的显微 ct 扫描 <ol> <li> 垂直装入在微 ct 系统中的铝质支架上的管中的后脑. </li>
	<li> 将扫描参数设置为540投影和2的曝光时间, 其源条件为 50 kV 和 10 W, 以获取层析数据集 (图像分辨率9.5 和 #956; m/像素). </li>
	通过微 CT 系统提供的基于硬件的投影重建软件, 对扫描的 x 射线进行自动重建, 生成16位轴向切片 (#34、TXM 和 #34; 文件) 的图像序列. </li>
</ol> 4。微 CT 数据集分析 <ol> <li> 在3维分析软件中打开重建的图像文件 (#34; TXM 和 #34; 文件), 并使用和 #34 可视化大脑; 卷呈现和 #34; 函数. </li>
	<li> 将后脑转换为所需的方向, 方法是使用 #34; 边界框和 #34; #34; 修剪平面和 #34; 函数. </li>
	<li> 在扩展模式下对转换后的图像重新取样, 并保留体素大小. </li>
	<li> 创建和 #34; 编辑新标签字段和 #34; 在转换后的图像 (4.3) 上, 仅使用灰度强度突出显示后脑. </li>
	<li> 添加突出显示的区域和运行和 #34; 标签分析和 #34; 获取整个后脑的度量值 ( 即 、总体积和面积)。在 Excel 文件中导出度量值. </li>
	<li> 使用和 #34 显示后脑; 等值渲染和 #34; 函数. </li>
	<li> 创建其他 #34; 编辑新标签字段和 #34; 在变换后的图像上, 使用灰度强度突出病灶部位. </li>
	<li> 添加突出显示的区域和运行和 #34; 标签和 #34; 分析以获得后脑内病变的测量 (即: , 总体积和面积)。在 Excel 文件中导出度量值. </li>
	<li> 使用和 #34 可视化病灶; 生成表面和 #34; #34; 曲面视图和 #34; 函数. </li>
	<li> 按所需方向拍摄后脑的图像, 或为病灶的3维可视化生成电影. </li>
</ol>

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fasudil+decreases+lesion+burden+in+a+murine+model+of+cerebral+cavernous+malformation+disease.">Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease.</a> Stroke. 43 (2), 571-574 (2012).</li><li>Maddaluno, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Gibson, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategy+for+identifying+repurposed+drugs+for+the+treatment+of+cerebral+cavernous+malformation.">Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.</a> Circulation. 131 (3), 289-299 (2015).</li><li>Choi, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+Imaging+Reveals+Mekk3+Heterozygosity+Prevents+Cerebral+Cavernous+Malformations+in+Ccm2-Deficient+Mice.">Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice.</a> PLoS One. 11 (8), 0160833 (2016).</li><li>Zhou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+cavernous+malformations+arise+from+endothelial+gain+of+MEKK3-KLF2/4+signalling.">Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.</a> Nature. 532 (7597), 122-126 (2016).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+comparative+morphology:+simple+staining+methods+allow+high-contrast+3D+imaging+of+diverse+non-mineralized+animal+tissues.">MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.</a> BMC Physiol. 9, 11 (2009).</li><li>Johnson, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+histology+of+transgenic+mouse+embryos+for+high-throughput+phenotyping.">Virtual histology of transgenic mouse embryos for high-throughput phenotyping.</a> PLoS Genet. 2 (4), 61 (2006).</li><li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Procedure+for+Rapid+Imaging+of+Adult+Mouse+Brains+with+MicroCT+Using+Iodine-Based+Contrast.">A Novel Procedure for Rapid Imaging of Adult Mouse Brains with MicroCT Using Iodine-Based Contrast.</a> PLoS One. 10 (11), 0142974 (2015).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+cerebral+cavernous+malformation+pathway+controls+vascular+stability+and+growth.">Dynamic regulation of the cerebral cavernous malformation pathway controls vascular stability and growth.</a> Dev Cell. 23 (2), 342-355 (2012).</li></ol>

作者没有什么可透露的。

CCM 是一种常见的血管畸形, 影响多达0.5% 的个人1。CCM 可以以零星或家族形式出现。患者的预后往往不清楚, 因为 CCM 病变可能破裂意外导致中风和其他神经后果。目前, 唯一的治疗方法是手术切除病灶, 伴随着高风险。
人类 CCM 条件最近被复制在动物模型7,8,9,10。这些模型为研究 CCM 疾病所涉及的机制提供了宝贵的工具。准确、有效和定量分析的方法将有助于利用这些模型的全部价值。
我们最近开发了一种显微 CT 方法, 以锇氧化为对照剂14, 在小鼠模型中成像 CCM 病变。在目前的研究中, 我们随后使用卢戈的碘溶液来增强脑组织的对比度, 以检测病灶。与锇氧化相比, 卢戈的碘溶液毒性较小, 无损, 而且价格低廉。因此, 样品可以用于其他目的, 如组织学后, 卢戈的碘溶液被冲走。
我们的显微 CT 方法可以产生足够的图像质量, 以检测在小鼠大脑中的 CCM 病变。样本准备是直接的, 不需要高度专门化的技术。此方法可以提供大约一小时扫描时间的高分辨率投影。将数据集从微 CT 扫描和3维分析软件结合在一起, 是生成3维全球脑内 CCM 病变的一种强有力的方法。相对于脑解剖的病变的定量和定性表征很容易实现。与 histology-based 3 维重建相比, 我们的方法是高度时间 cost-effective 的, 并且产生更精确的3维表示。
然而, 也有一些限制和关键步骤需要考虑。首先, 该方法需要一个微型 CT 系统, 它是一种高度专业化的设备, 可能不易获得。其次, 由于脑组织的柔软度和大小, 对新生鼠脑进行解剖是相当有挑战性的。这是非常重要的, 不损害大脑在解剖过程中, 以获得准确的测量和可视化的 CCM 病变在大脑中。最后, 对微 CT 扫描的样品制备至关重要。卢戈的碘染的大脑必须完全包装, 以避免因蒸发而导致组织萎缩。此外, 样品必须放置在微 CT 扫描安全。扫描过程中的任何移动都会改变扫描的质量, 从而导致量化和可视化。
我们只在固定的新生鼠脑中应用了我们的方法。然而, 这种方法有能力用于其他研究涉及不同的样本, 可以作为一个令人振奋的和新的工具, 其他血管畸形研究。

CCM 是薄壁, 扩张血管畸形在大脑中的流行率高达 0.5%, 在人口1。CCM 可以遗传为显性障碍, 由于功能突变的三基因之一: CCM1 (aka Krit1), CCM2, 和CCM3 (也称为PDCD10)2,3,4 ,5,6。这些基因存在于单一信号复合体中。
各种模型已经开发出模型人类 ccm 疾病和了解的 ccm 基因的下游途径, 负责 ccm7,8,9,10。最健壮的模型是有条件地删除任何一个Ccm基因与他莫昔芬诱导的Cdh5-CreERT2在 P1 在新生幼崽8,10。这些幼崽从 P6 发展到大脑中的 ccm 病变, 并有望成为临床前研究的理想模型, 以寻找治疗 ccm 疾病的机制和治疗剂。
在小鼠大脑中, CCM 病变的负担主要是用 histology-based 方法测量的, 这种方法非常耗时, 并受调查者的偏见10,11,12。采用 MRI 方法对成人小鼠模型9,13中的 CCM 损伤负荷进行评估。然而, 一个高度专业化的小动物磁共振成像仪和长时间扫描-一夜之间需要达到一个满意的分辨率, 以确定 CCM 病变。还有, MRI 是否可用于检测新生小鼠的 CCM 病变尚未报告, 分辨率可能会限制灵敏度。
我们最近开发了一种显微 CT 技术来图像和分析 CCM 病变14,15。这种高分辨率、时间和 cost-effective 的方法极大地提高了 CCM 疾病模型在机械和治疗研究中的价值。对比增强, 全贴装染色方法已用于软组织和小鼠胚胎的显微 CT 成像16,17。以前, 我们使用了 osmium-based 染色增强了在脑部14中 CCM 病变的微 CT 成像的对比度。本文采用了一种无毒、无损、低成本的试剂--碘基卢戈溶液, 增强了微 CT 成像的对比度。碘可以弥漫整个大脑和具有高亲和力的血液18。
详细的协议是在这里提出的诱导 ccm 病变的新生儿小鼠模型, 连同成像和分析的 ccm 病变的 contrast-based 微 CT。该方法对 ccm 病变体积进行了定量的全球测量, 准确地确定了小鼠脑内 ccm 病变的数目和3维位置, 大大降低了这些动物表型的成本和时间。

<table >
	
		
		
		
		
	
	<tbody>
		<tr >
			<td >4-hydroxy tamoxifen</td>
			<td >Sigma-Aldrich</td>
			<td >H6278</td>
			<td >To activate Cdh5-CreErt2</td>
		</tr>
		<tr >
			<td >Corn oil</td>
			<td >Sigma-Aldrich</td>
			<td >C8267-500ML</td>
			<td >To dilute 4-hydroxy tamoxifen</td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >Leica</td>
			<td >M205FA</td>
			<td >To take macroscopic images</td>
		</tr>
		<tr >
			<td >Lugol&#39;s Iodine solution</td>
			<td >Sigma-Aldrich</td>
			<td >L6146</td>
			<td >To stain samples for contrast micro-CT</td>
		</tr>
		<tr >
			<td >Plastic paraffin film</td>
			<td >Parafilm</td>
			<td >PM992</td>
			<td >To package samples</td>
		</tr>
		<tr >
			<td >Micro-CT</td>
			<td >Xradia</td>
			<td >MicroXCT-400</td>
			<td >Micro-CT</td>
		</tr>
		<tr >
			<td >3D rendering software</td>
			<td >FEI Visualization Science group</td>
			<td >Avizo 3D image processing software</td>
			<td >To analyse micro-CT scans</td>
		</tr>
	</tbody>
</table>

induction and micro-ct imaging of cerebral cavernous malformations in mouse model

突变在CCM1 (aka KRIT1), CCM2,或CCM3 (aka PDCD10)基因导致大脑海绵状畸形 (CCM) 在人类。在产后动物中, 用他莫昔芬引起的ccm基因缺失, 建立了小鼠 ccm 疾病模型。这些小鼠模型为研究 CCM 疾病的分子机制和治疗方法提供了宝贵的工具。准确和定量的方法来评估病变的负担和进展是必不可少的充分利用这些动物模型的价值。在这里, 我们演示了 ccm 疾病的诱导在一个小鼠模型和使用对比增强 X 射线显微计算机断层扫描 (微 CT) 方法测量 ccm 病变负荷的小鼠脑。在产后日 1 (P1), 我们使用 4-hydroxytamoxifen (4HT) 激活重组活动从 Cdh5-CreErt2 转基因, 以切割 floxed 等位基因的Ccm2。P8 对小鼠脑内 CCM 病变进行分析。在微 CT, 碘基卢戈的解决方案被用来提高对比度的脑组织。我们优化了扫描参数, 并利用了9.5 µm 的体素维数, 这导致了大约25µm 的最小特征尺寸。该分辨率足以全面准确地测量 ccm 病变体积和数量, 为小鼠脑内的 ccm 病变提供高质量的3维图谱。该方法提高了已建立的小鼠模型的价值, 研究了 CCM 和其他脑血管疾病的分子基础和潜在的疗法。

单注射4HT 在 P1 是足以诱发 CCM 病变的小脑。卢戈的碘对比微 CT 充分检测 CCM 病变, 并可以量化其体积和数量。利用优化的微 CT, 我们在Ccm2iECKO 小鼠的 hindbrains 中对 CCM 病变进行了成像。扫描的 X 射线图像重建, 以产生3维图像的鼠标的大脑, 这使得可视化整个病变的脑实质在不同的深度和方向, 和评估的结构和3的位置的病变在大脑中。重要的是, 这个图像数据集也可以用来有效和准确地量化病变的大小和病变的数字。
图 1显示了在 P8 的Ccm2iECKO (B) 的小脑中 CCM 病变的成功诱导, 但不在 littermate 控件 (Ccm2fl/fl) (A) 中。
图 2显示了用于微 ct 扫描 (aB) 的填充样本, 以及来自控制的微 ct 扫描 (Ccm2fl/fl) (C) 和Ccm2 的具有代表性的重建图像iECKO (D-E)。图 2E"在Ccm2iECKO中显示标记的病灶。
图 3显示了带有病灶的Ccm2iECKO 大脑的呈现3维图像 (a-B)。相邻表显示了一个例子, 定量输出的标记个别病灶。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56476/56476fig1.jpg"> 
图 1: 新生儿小鼠 CCM 病变的诱导.Ccm2fl/fl (A) 和Ccm2iECKO (B) 的宏观图像在产后8天。刻度线 (白色), 5 毫米.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56476/56476fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56476/56476fig2.jpg"> 
图 2: 样品制备和重构2D 图像.样品装在一个离心管 (a) 和5毫升管 (B)。绿色箭头显示示例。有代表性的图像Ccm2fl/fl (C) 和Ccm2iECKO (D-E) 大脑。(E ")Ccm2iECKO 大脑中的标记病灶。刻度线 (白色), 1 毫米.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56476/56476fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56476/56476fig3.jpg"> 
图 3: 呈现3维图像.呈现Ccm2iECKO 脑部的图像 (a) 和病灶 (B)。病灶以红色标记。右侧的表显示了一个单独标记病灶的定性输出的例子。刻度线 (白色), 1 毫米.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56476/56476fig3large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model at JoVE.com

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

本协议演示了在小鼠模型中诱导脑海绵状畸形的疾病, 并使用对比增强微计算机断层扫描来测量病变的负担。该方法提高了建立的小鼠模型的价值, 以研究脑海绵状畸形和其他血管性疾病的分子基础和潜在的疗法。

小鼠脑海绵体畸形的诱导及显微 CT 成像

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

induction-micro-ct-imaging-cerebral-cavernous-malformations-mouse

Nanyang Technological University

Research

JoVE Journal

Medicine

10.8K 次观看.  Centenary Institute. 本协议演示了在小鼠模型中诱导脑海绵状畸形的疾病, 并使用对比增强微计算机断层扫描来测量病变的负担。该方法提高了建立的小鼠模型的价值, 以研究脑海绵状畸形和其他血管性疾病的分子基础和潜在的疗法。

视频: Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

大脑中动脉闭塞的小鼠模型

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

科研

医学

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

在小鼠永久性大脑中动脉结扎术中的应用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

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 ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

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

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

同时PET / MRI成像在小鼠脑缺氧缺血

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

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

汽车测试的脑瘫新生儿小鼠模型电池

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...