笔者想感谢他的技术援助与显微CT成像，爱德华TH叶MD，手术援助，丽贝卡·巴托博士MD安德森小动物成像设备的基思·米歇尔，社论援助。部分支持这项工作是由美国心脏协会。

1。渗透泵灌注<ol><li>准备泵时使用无菌技术（ 如手套，层流罩）。从包装中取出泵和流量调节剂。称量空泵和记录的重量（mg）。 </li><li>慢慢地注入泵，避免产生气泡，1cc的注射器用一个小钝头27G填充管。当溶液达到泵的顶部，停止填充。 </li><li>擦去多余的解决方案和权衡填充泵。适合于大多数水溶液，重量以mg微升的体积相同。在空对填充重量的差异应该是90％以上的规定的平均填充量。如果没有，疏散用注射器泵和笔芯。如果您遇到技术困难，请参阅泵说明故障排除部分http://www.alzet.com/products/guide_to_use/filling.html </li><li>使用一双镊子打破白色法兰流moderat从的小心，不要弯曲或粉碎管。附加P50导管主持人任一端，占地约3-4毫米。用注射器填充的导管和连接的调节剂，留下的注射器安装在导管的末端部分。 </li><li>将进入泵的流量调节剂，，直到导管与泵的表面齐平。然后，取出注射器从导管的前端。将泵在无菌的0.9％的生理盐水或PBS溶液，在37℃的培养箱至少6小时（最好是O / N）黄金植入到老鼠的前级泵。泵输送率随型号（型号1002 = 0.25微升/小时为14天）。 </li></ol> 2。创建单侧肢体缺血的鼠标<ol><li>麻醉小鼠用异氟醚集在2-4％之间，在100％O 2，流速为0.4升/分钟，并除去毛（最好是在手术前一天）上的后肢和背部区域与depilatoRY霜。 </li><li>手术前，所有的外科手术工具消毒。鼠标放置在感应麻醉室。使用相同的麻醉第2.1节中描述的设置。 </li><li>等待，直到鼠标是对外界刺激无反应，然后将其放置在一个配备提供了源源不断的异氟醚（1.5-2.0％）的鼻锥，俯卧位走上手术台。 </li><li>切口部位注入麻药（布比卡因，0.25％（&lt;1毫升/公斤））的推荐剂量为治疗皮肤用聚维酮碘溶液，然后用70％乙醇。做一个4-6毫米的切口的背面（上面侧的脊柱，在肩胛区域）。用细镊子分离皮下膜，然后转动鼠标仰卧位治疗皮肤用聚维酮碘溶液，然后用70％乙醇和0.8-1厘米的切口，用显微手术剪刀向大腿内侧膝盖以上，后股动脉路径。 </li><li>隧道了躯干的小的鞘管（6 - 法国）从大腿内侧切口的背面侧。 </li><li>鼠标放置在俯卧位。删除引物的渗透泵/从PBS溶液中的导管和引导导管，通过鞘管，在大腿上。 </li><li>泵内部的背面的位置，并关闭后切口用6-0 prolene线缝合（治疗皮肤用聚维酮碘的溶液，然后用70％乙醇）。在仰卧位将鼠标背部;除去鞘管，离开导管通过切口暴露在大腿上。用外科手术胶带固定腿。 </li><li>分离皮下脂肪组织揭示了血管。为了更好的可视化的血管，用牵开器打开切口。 </li><li>皮尔斯股骨膜轻轻地用细尖头镊子访问维管束鞘。从股静脉和神经腹股沟附近的股动脉解剖和分离，照顾不造成前逐次出血。 </li><li>放置两个8-0 prolene线相邻的缝合线中断股动脉近端股骨分叉（股深腹壁动脉起源）2-3毫米。 </li><li>切导管到适当的长度，将尖到大腿前外侧区域，确保用6-0 prolene线缝合的浅筋膜，以确保靶向的物质。关闭腿切口缝合用6-0运行。管理最终皮下注射镇痛（丁丙诺啡0.1毫克/千克），治疗皮肤用聚维酮碘溶液，然后用70％乙醇和鼠标回到笼子里。在恢复过程中使用的加热灯。 </li><li>经过30分钟的恢复期，准备鼠标激光多普勒成像技术确认创建后肢缺血。 </li><li>术后第5天，每天两次监测动物。将观察到的动物的流动性来分析疼痛;在笼子里的废弃物将作为印度语ATOR吃。此外，在监视期间确定如果动物经历疼痛或不适，并给予丁丙诺啡（为0.1mg/kg），皮下注射，每天两次。 </li></ol> 3。成像试样制备<ol><li>在2.1节所列麻醉鼠标。鼠标传输走上手术台，在仰卧位。注入1,000 IU的肝素皮下注射，以防止在血管凝血。当鼠标被安装到鼻锥，大到足以暴露的胸腔和下腹部做一个切口。腹部器官移动的动物的左侧以暴露腹主动脉（IAA）。小心地分离主动脉，下腔静脉。 </li><li>结扎近端IAA，导管插入过程中，以减少出血。然后，在IAA上做一个小切口。插入一个27½克钝针（附到P10导管）的IAA用6-0缝线固定。 </li><li>填补两个20cc的注射器用肝素化生理盐水，10％中性福尔马林缓冲液中（NBF）。对比不透X线的硅橡胶复合填写一个5毫升的注射器。混合三种成分MV化合物：MV-MV稀释剂：固化剂比例为1:1.25:0.5工作。由于混合物（微滤）只有20分钟的工作时间代理聚合之前，值得推荐，直到正确的不添加的MV固化剂，注射到血管之前。 </li><li>在右心房和管理作出削减了过量的异氟醚诱导安乐死和多余的液体，以便排水。将生理盐水充式注射器的针尖，并开始冲洗5分钟脉管。确保所有的血液会被冲出来的船只，关闭异氟醚和断开的nosecone开始冲洗一次。接下来，将在福尔马林充式注射器上文详述的（3.2）的导管和压力灌注5分钟。最后，灌注注射器，与对照剂，慢慢进入鼠标5分钟。 </li><li>可选的脉管用照相机拍摄的微滤，以确保只有动脉结构灌注。 </li><li>鼠标放置在一个容器充满10％NBF和储存于4°CO / N为造影剂聚合。 24小时后，去除皮肤上躯干的下半部分（低级腹主动脉结扎远端）通过使用清扫工具，更新10％NBF，鼠标放置到容器4天以上，在RT。 </li><li>解剖对侧缺血后肢，并将标本中甲酸溶液（加州EX II）的48小时便利脱钙骨。然后，在流水下冲洗标本1小时，返回到10％的NBF进行Faxitron（X射线检测系统使用，以确保骨脱钙）和显微CT成像。 </li></ol>

<ol>
	<li>Epstein, S. E., Fuchs, S., Zhou, Y. F., Baffour, R., Kornowski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+enhancing+collateral+development+by+administration+of+growth+factors:+basic+principles,+early+results+and+potential+hazards.">Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.</a> Cardiovasc. Res. 49, 532-542 (2001).</li><li>Esaki, J., Marui, A., Tabata, Y., Komeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+systems+of+angiogenic+growth+factors+for+cardiovascular+diseases.">Controlled release systems of angiogenic growth factors for cardiovascular diseases.</a> Expert Opin. Drug Deliv. 4, 635-649 (2007).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, 1267-1279 (2002).</li><li>Golomb, B. A., Dang, T. T., Criqui, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+arterial+disease:+morbidity+and+mortality+implications.">Peripheral arterial disease: morbidity and mortality implications.</a> Circulation. 114, 688-699 (2006).</li><li>Hiatt, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+therapy+for+peripheral+arterial+disease+and+claudication.">Pharmacologic therapy for peripheral arterial disease and claudication.</a> J. Vasc. Surg. 36, 1283-1291 (2002).</li><li>Hirsch, A. 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=Peripheral+arterial+disease+detection,+awareness,+and+treatment+in+primary+care.">Peripheral arterial disease detection, awareness, and treatment in primary care.</a> JAMA. 286, 1317-1324 (2001).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nat. Med. 9, 713-725 (2003).</li><li>Hirsch, A. 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=ACC/AHA+Guidelines+for+the+Management+of+Patients+with+Peripheral+Arterial+Disease+(lower+extremity,+renal,+mesenteric,+and+abdominal+aortic):+a+collaborative+report+from+the+American+Associations+for+Vascular+Surgery/Society+for+Vascular+Surgery,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+for+Vascular+Medicine+and+Biology,+Society+of+Interventional+Radiology,+and+the+ACC/AHA+Task+Force+on+Practice+Guidelines+(writing+committee+to+develop+guidelines+for+the+management+of+patients+with+peripheral+arterial+disease)--summary+of+recommendations.">ACC/AHA Guidelines for the Management of Patients with Peripheral Arterial Disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (writing committee to develop guidelines for the management of patients with peripheral arterial disease)--summary of recommendations.</a> J. Vasc. Interv. Radiol. 17, 1383-1397 (2006).</li><li>Zagorchev, L., Mulligan-Kehoe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+vessels+in+mouse+models+of+disease.">Molecular imaging of vessels in mouse models of disease.</a> Eur. J. Radiol. 70, 305-311 (2009).</li><li>Losordo, D. W., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+and+vasculogenesis+for+ischemic+disease.+Part+I:+angiogenic+cytokines.">Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines.</a> Circulation. 109, 2487-2491 (2004).</li><li>Waters, R. E., Terjung, R. L., Peters, K. G., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+human+peripheral+arterial+occlusive+disease:+implications+for+investigation+of+therapeutic+agents.">Preclinical models of human peripheral arterial occlusive disease: implications for investigation of therapeutic agents.</a> J. Appl. Physiol. 97, 773-780 (2004).</li></ol>

作者宣称没有利益冲突。所有动物的程序进行，按照实验动物的护理和使用指引，并获批准的机构动物护理和使用委员会（IACUC）在得克萨斯大学健康科学中心在休斯敦大学。

在这里，我们提出了一个方法渗透药物/物质后肢缺血小鼠模型的交付。此外，我们描述了一个铸造技术，其中我们已经使用微滤产生三维重建血管网的分析。 的水平或缺血严重程度不同，根据由动脉结扎/切除。我们创建了一个双重结扎股动脉近端股深和腹壁动脉的起源，这种方法可以产生更严重的缺血后肢远端。手术后一定要留意动物，并提供由当地镇痛药缓解疼痛。此外...

外周动脉疾病（PAD）是动脉粥样硬化的疾病，它引起在腿部4供血不足。它影响到8万至12万美国人，目前的医疗只能提供有限的救济5,6。改善腿部血液循环的新的治疗药物，不仅会抑制疾病的进展，但也提高生活质量。 PAD的发病率较高，50岁以上的人，所以当地的药物治疗是一种较为理想的治疗方式，因为减少的肾脏和肝功能经常出现在老年患者，可降低全身用药的药物代谢和增加副作用。 因此，我们创建了一个鼠标垫检查是否减轻局部给药制剂通过促进血管生成和血管重塑后肢缺血模型。具体而言，我们使用一个插管的渗透泵连续提供的治疗剂缺血小鼠大腿肌肉。使用我们的输送系统，我们可以在本地环境中，以保持最佳的药物浓度，这种方法允许适当的药物活性，避免了可能出现的全身性副作用，克服有限的局部药物接入与全身用药的缺点。此外，评估局部给药制剂是否促进血运重建，我们采用先进的铸造和高清晰度成像技术，使微血管变化的定量分析。总的来说，使用相结合的方法，在这个视频文章是有用的临床前研究，以帮助了解药物诱导的心肌血运重建术在PAD患者7-9。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

外周动脉疾病的动物模型的临床前研究，在测试旨在刺激微循环的治疗药物的疗效起着至关重要的作用。发送方法的选择对这些药物的给药途径是非常重要的，因为这些药物的1,2深刻地影响着生物活性和疗效。在这篇文章中，我们将演示如何在本地使用插管的渗透泵服用一种物质，在缺血后肢。此泵可输送的配发的时间内将固定体积的水溶液，连续。我们也提出我们的普通股动脉，股深的起源和腹壁动脉，左后肢近端结扎单侧肢体缺血诱导的小鼠模型。最后，我们描述了在体内的插管和微滤，不透射线的聚硅氧烷铸件剂的腹主动脉和灌注的后肢血管结扎。微滤灌注填满整个血管床（动脉和静脉），因为我们已经退出结扎大血管导管，代理可以保留在血管未来体外成像小标本采用显微CT 3 。

动画演示渗透泵插入和后肢缺血手术在协议中详细说明。 图1显示了激光多普勒图像后肢灌注，缺血确认。与微滤血管铸型后，一个三维显微CT图像的血管网显示，微滤可以有效地填补船舶（ 图2A），但不连续性可能会发生（ 图2B）由于各种程序的因素（ 例如 ，气泡，缺乏压力，粘度，微滤）。 动画传奇<ol><li>鼠标放置在俯卧位?...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

我们在这里展示的<em&gt;在体内</em&gt;插入渗透泵恒定的局部给药和后肢缺血小鼠模型中创建。此外，后肢血管灌流微滤，硅不透X线剂，准备为微型计算机断层扫描（微CT）成像。

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Research

JoVE Journal

Medicine

12.2K 次观看.  The Texas Heart Institute at St. Luke's Episcopal Hospital. 我们在这里展示的在体内插入渗透泵恒定的局部给药和后肢缺血小鼠模型中创建。此外，后肢血管灌流微滤，硅不透X线剂，准备为微型计算机断层扫描（微CT）成像。

视频: Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

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

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

腔内药物输送到鼠标动静脉瘘内皮

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

用于慢性病大型哺乳动物心脏模型对比增强显影显微计算机断层扫描的组织制备技术

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.