这项工作是由美国国立卫生研究院拨款HL87012“（HJ），HL75209”（HJ），UO1HL80711（DH，DG HJ）和世界一流大学项目“（HJ）的资金从韩国的科学，技术和教育部的支持。

1。部分结扎左侧颈内动脉<ol><li> 〜8周龄小鼠。建议至少18克的老鼠的重量。 </li><li> 30秒珠仪器灭菌消毒文书，并允许降温</li><li>诱导麻醉与氯胺酮（80mg/kg）和甲苯噻嗪（按10mg/kg）腹腔（IP）注射或替代麻醉IACUC批准。腹腔注射，注入鼠标在左侧腹部低象限。 </li><li>将鼠标放置在变暖，直至完全麻醉室。确认与脚趾捏的麻醉深度。 </li><li>将鼠标在手术阶段，在仰卧（腹朝上）位置。磁带脱颖而出爪子掌心向上和后肢爪子唯一下降的阶段。 </li><li>脱毛剂的自由主义（例如奈尔）下颌骨和胸骨之间的颈部。轻轻按摩，直到所有的头发被删除。 </li><li>清洁脱毛剂和优碘自由主义申请depilated面积。 </li><li>准备1英寸长的路段为结扎6-0丝线缝合。每个鼠标需要两片缝合。 </li><li>使用锋利的小剪刀，在颈部中间的4 - 6mm的纵切口。皮肤和底层筋膜需要削减。 </li><li>移动左腮腺从中线的左侧，并开始直言不讳地剖析恰到好处的气管。您应该能够很容易识别脉动左颈。 </li><li>按照颈尾端，直到你找到分叉。 </li><li>坦率地剖析揭露以下四个远端分支关闭左颈总动脉：颈外动脉，颈内动脉，枕动脉，甲状腺上动脉的分叉。这些船只，特别是甲状腺，可以是脆弱的。必须非常小心你的夹层。 </li><li>领带上述优越的使用6-0丝线缝合甲状腺颈外。要做到这一点，通过下面的动脉之一钳，抢了一块预切缝合与您其他钳的帮助。轻轻拉出缝线通过下面的动脉。领带坚决避免陷入结周围的任何组织。如果配合低于起飞甲状腺上动脉，你将有效地执行了一个完整的结扎，这是一个大大不同的模型的过程结束。 </li><li>配合一个使用相同的技术结的颈内动脉和枕动脉。 </li><li>验证缝合和甲状腺上动脉通畅的位置。 </li><li>近似​​的皮肤和密切皮肤使用少量的组织MEND。 </li><li>在气候变暖，直到恢复室放置老鼠。恢复通常需要30到60分钟。 </li><li>一个单剂量丁丙诺啡（为0.1mg/kg），应立即进行管理，如果使用麻醉不提供长期缓解疼痛（即吸入异氟醚），或者如果动物处于困境的第一个24小时内手术后。在我们的经验，氯胺酮剂量麻醉管理由经验丰富的外科医生进行手术时，并没有进一步的镇痛需要提供足够的镇痛。 </li></ol> 2。颈动脉超声检查<ol><li>验证是否流部分结扎引起的不安，颈动脉流有一天结扎后应检查</li><li>异氟醚麻醉诱导 - 如果需要的话，可用于麻醉诱导箱</li><li>成像阶段，肚皮朝上放置在麻醉小鼠</li><li>磁带的胳膊和腿心电图传感器成像阶段</li><li>落叶颈部由奈尔自由主义的应用程序（如果不是已经做结扎过程只需要这一步） </li><li>插入直肠温度计温度记录</li><li>应用凝胶回声颈部</li><li>在B模式开始。 </li><li>成像平面应该是90度（在探头上的旋钮应指向鼠标鼻子） </li><li>降低探测到鼠标的脖子，直到获得图像。 </li><li>操作阶段，左，右，直到气管中线结构确定</li><li>确定左颈总动脉</li><li>倾斜成像阶段，这样一个角度成像平面和颈动脉之间产生。这个角度更大，更强的多普勒信号会。 </li><li>广场脉冲多普勒左颈中。 </li><li>根据需要，使用角度校正。 </li><li>右侧颈总重复的测量。 </li><li>成功的部分颈动脉结扎会导致整体左颈总流量减少（80-90％）与右侧相比，在舒张期对主动脉口的流动逆转。 </li><li>一旦已取得令人满意的图像，关闭麻醉，擦拭ECHO OFF小鼠和自由的老鼠从磁带RES凝胶traints。将小鼠在回暖复苏室。 </li></ol> 3。从颈动脉内膜RNA分离<ol><li>牺牲小鼠CO 2吸入根据机构IACUC协议</li><li>磁带的爪子纸巾。 </li><li>剪下的鼠标从腹部到胸部上方的皮肤。 </li><li>打开腹壁下面的胸腔，用锋利的剪刀。 </li><li>用镊子提起胸骨和削减隔膜，再切了胸腔，暴露心脏。 </li><li>用剪刀剪开下腔静脉。 </li><li>灌注压力（120毫米汞柱）为2至3分钟，正常的左心室通过含有10单位/ ml肝素生理盐水，直到肺部和肝脏变得苍白。 </li><li>切开颈部皮肤，去除的脂肪，肌肉，和结缔组织，直到颈动脉暴露。 </li><li>鼠标放置在解剖显微镜下。 </li><li>穿刺孔下方在第二灌注左侧颈内动脉结扎网站。 </li><li>压力灌注，再通过左心室正常含10单位/ ml肝素生理盐水，确保左侧颈细读〜1分钟。 </li><li>用细尖镊子和小弹簧剪刀，小心地取出城郊周围的颈动脉外膜组织。要小心，不要挤压或伸展的颈动脉，在此清洗步骤。 </li><li>剪下的左侧颈内动脉，主动脉弓和颈动脉分叉处以上的结扎点之间。 </li><li>切的右颈总动脉，右锁骨下动脉分支点和颈动脉分叉处之间。 </li><li>颈动脉为35 mm培养皿中，含冰冷的HBSS。如果需要的话，请小心取出任何剩余的外膜周围组织。 </li><li>每个颈充盈每150μLQIAsol的细胞裂解液（QIAGEN），准备与29克针胰岛素注射器（3 / 10 ml注射器）。 </li><li>小心颈的一端插入针头。 </li><li>按住颈动脉和针尖与镊子，迅速（约1秒）刷新成1.5毫升管QIAsol裂解液（150μL）（内膜洗脱液）。 </li><li>冲洗颈吃剩的（媒体+外膜）曾经在HBSS中，把1.5毫升管，并在液氮快速冻结。 </li><li>内膜洗脱液和冷冻剩然后用内膜或媒体+外膜的RNA使用miRNeasy迷你试剂盒（Qiagen）根据制造商的指示隔离。 </li></ol> 4。代表性的成果： 我们部分结扎鼠标左键进行颈动脉超声检查，有一天，下面的过程。正如图1所示，成功的部分结扎减少血流速度和逆转的左颈总动脉在舒张期流量（不安流）。伴随着高脂肪食物的ApoE基因敲除（KO）的小鼠局部结扎诱导的左颈总动脉（LCA）的强劲动脉粥样硬化，但不能在对侧的右颈，两周（图2）。从颈动脉内膜收集RNA含有内皮标记基因，如PECAM - 1，而内侧平滑肌细胞标记基因，如A - SMA（图3）。 <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="图1" /> 图1：颈动脉解剖和结扎示意图： 结扎左颈总动脉的三个分支（LCA），颈外动脉（ECA），颈内动脉（ICA），枕动脉（OA）]，而留下的甲状腺上动脉（STA）的开放。 <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="图2" /> 图2：超声图像显示流速剖面，揭示部分结扎诱导LCA在舒张期流动逆转（用箭头表示）。在RCA的流量保持不变结扎后。 <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="图3" /> 图3：部分结扎和高脂肪的饮食的ApoE基因KO小鼠的生命周期，迅速 ​​引起动脉粥样硬化的ApoE基因KO小鼠部分结扎和美联储1至3周的高脂肪的饮食。从LCA的冰冻切片油红- O染色。 <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="图4" /> 图4。内膜RNA制备方法。内膜的RNA和内侧+外膜（M +）RNA假手术RCA和LCA在C57BL / 6小鼠。 PECAM - 1和α- SMA的使用作为内部控制的18S RNA的分析，半定量RT - PCR 技术（A）和定量PCR（B，C） 。条形图平均± SEM，N = 3。

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li></ol>

在此过程中，适当的识别所有常见的颈动脉分支，包括颈外动脉，颈内动脉，枕动脉，甲状腺上动脉是必不可少的。同样重要的是，以验证使用超声作为证明的流动模式。在我们经验丰富的的手中，我们的部分结扎手术的成功率超过500小鼠的超声研究为基础，超过90％。对于颈动脉内膜RNA的准备工作是很重要要注意的颈内动脉后出现的第一灌注步骤。流在这一点上左侧颈总动脉堵塞，如果有证据?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Normal Saline (0.9% sodium chloride)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>2B1323</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe (3/10 ml 29g syringe)</td>
<td>&nbsp;</td>
<td>Tyco Health care</td>
<td>8881600145</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>21-023</td>
<td>&nbsp;</td>
<tr>
<td>miRNeasy mini kit (including, QIAzol)</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>217004</td>
<td>&nbsp;</td>
<tr>
<td>Vevo 770 High-Resolution Micro-Imaging System</td>
<td>&nbsp;</td>
<td>Visualsonics</td>
<td>a.Visualsonics 770 system b.Imaging stage c.Vevo integrated rail system d.RMV scan head for mice</td>
<td>or similar system</td>
</tr>
<tr>
<td>6-0 Silk Suture, sterile</td>
<td>&nbsp;</td>
<td>Covidien</td>
<td>s-1172 c2</td>
<td>&nbsp;</td>
<tr>
<td>Small scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5675</td>
<td>or similar</td>
</tr>
<tr>
<td>Dissecting Forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5005</td>
<td>or similar</td>
</tr>
<tr>
<td>Tissue Mend II</td>
<td>&nbsp;</td>
<td>Webster Vet Supply</td>
<td>07-856-7946</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of rna isolation from carotid endothelium

尽管众所周知的密切联系，不安流连接到动脉粥样硬化的直接证据，一直缺乏。最近，我们使用修改后的版本的颈动脉部分结扎方法[1,2]表明它敏锐地诱导低，振荡流的条件下，在小鼠左颈总动脉不安流的两个关键特征。使用这个模型，我们提供了直接证据，扰乱流量确实在载脂蛋白E基因敲除小鼠动脉粥样硬化的发展快速，稳健的[3]。我们还开发了高纯度的内皮细胞RNA制备方法，从小鼠颈动脉内膜[3]。利用这种小鼠模型和方法，我们发现，部分结扎血管内皮功能障碍，导致在一个星期稳健，快速的在两个星期的动脉粥样硬化的形成在高脂血症小鼠模型以及复杂的病变，如intraplaque新生血管形成的功能，四个星期。这种快速<em&gt;在体内</em&gt;模型和内皮细胞的RNA制备方法可以用来确定底层流依赖调节血管生物学和疾病的分子机制。此外，它可用于测试各种治疗干预，针对血管内皮功能障碍和动脉粥样硬化研究的时间大大减少。

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

这描述了一个局部的颈动脉结扎手术，这会导致不安的水流条件和随后的动脉粥样硬化的发展（在两个星期）intraplaque新生血管在小鼠左颈总动脉（在四个星期内）。我们还描述了一个新颖的RNA从颈动脉内膜隔离方法，提供高纯度的内皮细胞的RNA。

一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K 次观看.  Emory University. 这描述了一个局部的颈动脉结扎手术，这会导致不安的水流条件和随后的动脉粥样硬化的发展（在两个星期）intraplaque新生血管在小鼠左颈总动脉（在四个星期内）。我们还描述了一个新颖的RNA从颈动脉内膜隔离方法，提供高纯度的内皮细胞的RNA。

视频: A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

科研

医学

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

培养一个精密的显微外科技术在大鼠颈总动脉，颈外静脉移植：介入

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成年小鼠静脉高压型号：颈总动脉，以颈外静脉吻合。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

颈动脉和颈静脉导管在有意识大鼠模型进行血流动力学措施，输液和血液采样

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

三氯化铁诱发血栓形成小鼠模型上的颈动脉和肠系膜血管

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

体内兔颈总动脉内皮的基因转移

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

颈动脉介入移植中颈静脉持久性转基因表达的家兔模型

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...