我们感谢夫人安吉拉弗氏切片塑料预埋支架以出色的技术援助。我们也感谢夫人的罗亚索乌坦和夫人安吉拉弗氏专业人士的帮助，用免疫组化染色。

1。准备和植入支架<ol><li>支架支撑（韦恩堡金属，爱尔兰卡斯尔）编织研究所纺织技术与机械工程学院，在德国亚琛工业大学（ 图1A），然后切到所需的大小。 </li><li>支架植入前，必须转 ​​移到一个2厘米的硅管，使用镊子，并放置2毫米在一个末端，称为前端（ 图1A）。 </li><li>应斜切的前端，以确保锐利尖端植入。 </li><li>在支架植入前，应大量浇水，以确保滑动。 </li></ol> 2。支架植入术<ol><li> 25-27克10-12周龄雄性C57BL / 6野生型小鼠，采用腹腔注射100毫克/千克氯胺酮和10毫克/公斤甲苯噻嗪麻醉。适当的麻醉手术前确认缺乏反射胡子运动。为了防止皮肤干燥，同时在麻醉状态下，鼠标电影的bepanthene霜遮住眼​​睛。 </li><li>剃须后颈部腹侧区和适当的消毒，正中切口1厘米的小立体显微镜下，用剪刀。分离后用无菌弯钳2脂肪酸机构，左颈总动脉中可以看出，随着脉冲气管。 </li><li> 1厘米的左颈总动脉分叉应该是免费的制备。 1节用5/0丝线左颈总动脉周围将被绑定，使用2节7/0丝线将左侧颈外动脉周围的约束，1节用7/0丝线将被绑定颈内动脉周围（ 图1B）。 </li><li>血液的流动，然后牢固地结合颈内动脉和颈外动脉的近端上的结中断，以及拉结环绕ing的左颈总动脉。该容器应该是固定的在共同和外颈动脉的方式，是在一条直线上的。 </li><li>一个小切口，颈外动脉近端结附近，使用维纳斯剪刀。导入包含支架的硅管颈外动脉，具有尖锐的端部的前面，用导丝。扩张器达到所需的位置后，硅管拉回在导向线，并允许把形状记忆扩张的支架（ 图1B）。 </li><li>颈外动脉的远侧结裹紧关闭在网站上的切口，在内部和颈总动脉的结被去掉，从而恢复血流量。 </li><li>使用3-4米歇尔缝合剪辑和米歇尔镊子皮肤切口闭合。鼠标被放置在红灯，直到完全恢复。一种镇痛药治疗是没有必要的。 </li><li>该1-3周后，可以分析斑块。要研究再内皮化，更早的结束时间点是必要的（3-4天）。我们观察到在我们的模型支架植入术，外科手术治疗后4周，特别是通过使用特定的涂料，biofunctionalize微型支架，血管生成发生在约30％的试样。这是一个后腿重塑和再生的过程，有不同的机制和另一个病理问题。要专注于血管内膜增生，支架内狭窄和/或支架植入后3周的结束时间点这些副作用将有利于混合新生血​​管的发病与再生作用机制分析。 </li></ol> 3。斑块的形成分析<ol><li>上面的结束时间点，使用腹膜内注射100毫克/千克氯胺酮和10毫克/公斤甲苯噻嗪麻醉的动物。适当的麻醉手术前确认由反射和胡须缺乏运动。 </li><li>动物被杀害，心内放血。血清的血液收集用于进一步分析。 </li><li>后打开胸腔及PBS洗涤通过心内punction的身体灌注4％多聚甲醛（PFA）溶液，进行5分钟。包含支架左颈动脉解剖，直接放置在一个4％多聚甲醛溶液，并购买塑料embbeded在至少16小时。 </li><li> 50微米厚的部分进行塑料包埋样本，用金刚石带锯。 </li><li>为了测量斑块大小，姬姆萨染色。 </li><li>内支架再内皮船只的面积率来分析血管性血友病因子（vWF），免疫组织化学。 </li></ol>

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S., 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=Differential+neointimal+response+to+coronary+artery+injury+in+pigs+and+dogs.+Implications+for+restenosis+models.">Differential neointimal response to coronary artery injury in pigs and dogs. Implications for restenosis models.</a> Arterioscler. Thromb. 14, 395-400 (1994).</li><li>Schwartz, R. S., 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=Restenosis+and+the+proportional+neointimal+response+to+coronary+artery+injury:+results+in+a+porcine+model.">Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model.</a> J. Am. Coll. Cardiol. 19, 267-274 (1992).</li><li>Ali, Z. 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=Increased+in-stent+stenosis+in+ApoE+knockout+mice:+insights+from+a+novel+mouse+model+of+balloon+angioplasty+and+stenting.">Increased in-stent stenosis in ApoE knockout mice: insights from a novel mouse model of balloon angioplasty and stenting.</a> Arterioscler. Thromb. Vasc. Biol. 27, 833-840 (2007).</li><li>Chamberlain, J., 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=A+novel+mouse+model+of+in+situ+stenting.">A novel mouse model of in situ stenting.</a> Cardiovasc. 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没有利益冲突的声明。

为了减少支架内血栓和再狭窄的风险和持续发展的新涂层药物洗脱支架，支架植入动物模型的一个简单的，简单方便的方法是必要的。小鼠动脉支架植入后的重塑和效率等药物研究的复杂机制提供了理想的系统。在鼠标支架内再狭窄的现有模型是困难的，需要较高的手术技巧和并发症如出血或瘫痪17-19意味着高风险。例如，在模型中的扩张器植入到球囊扩张的容器中，然后移植成的收件人鼠标17的颈动脉支架置入段后的供体小鼠的胸主动脉，病理机制的研究是不只能由收件人捐助物资的反应，也营养血管外膜的大规模破坏性影响。植入的不锈钢扁钢其次，死亡率高（35％），因为动脉切开腹主动脉部位的血栓或出血后腿瘫痪后升支架直接进入腹主动脉球囊扩张19。一个螺旋形的自我扩张的镍钛支架通过股动脉18到腹主动脉植入需要较高的手术技巧，主要是由于盲目指挥支架沿分支从股动脉，主动脉支架放置在合适的位置。遵循这一程序损坏股神经的高风险，因此，后腿麻痹。与这些程序相比，我们的模型支架植入鼠标并不需要较高的手术技巧。 我们的模型提供了一个简单，方便，高效的方法，来分析不同的药物涂层血管重塑的影响，支架配售的视线下，有没有风险，损害神经或其他结构。 COM复杂的分子机制，可以进行调查更容易在我们的模型小鼠颈动脉支架置入术，不仅直接访问的船只，但也由于存在不同的基因敲除小鼠品系。 作为一个限制，比较的临床过程中，我们的模型采用健康小鼠/动脉，不执行预先存在的斑块支架置入术（支架内再狭窄，但在支架内狭窄）。我们也没有进行球囊扩张支架植入前。然而，由于在这两种模型中的血管壁的大规模的伤害，赔偿性过程是相似的。不幸的是，由于金属衍生的工件， 在体内监测新内膜生长是不可能由现有的成像方法，如超声或计算机断层扫描。另一个限制因素是基于金属支架的薄切片，需要一些在金属加工的专业知识。 使用这种方法，我们能够显示，嗜中性粒细胞指示biofunctionalized的小型镍钛合金涂层支架与LL-37减少支架内再狭窄，提供了一种新的理念，以促进血管介入治疗后愈合21。 尽管有这些限制，这种模式似乎是，到现在为止，最适合的系统，从而金钱和节省时间，调查新的药物涂层支架在动脉重塑的分子事件及其对。此外，这种模式可以很容易地适应仓鼠，这是更类似于人类，让每一个治疗性假说可以验证，然后再应用到更大的动物或人，以避免不愉快的和意想不到的效果。

心血管疾病引起的动脉粥样硬化的进展，是在工业化国家死亡的首要原因。动脉粥样硬化是一个联络，炎性成纤维增生反应的血管壁的内皮损伤1，从而导致形成一个扩展的斑块该船只入管腔，影响冠状动脉血流通过。超过75％的心肌梗死的破裂导致发炎的斑块纤维帽薄2。由于这可能是致命的并发症，经皮腔内冠状动脉成形术（PTCA）与支架植入疗法在当前的医疗实践中成为第一选择。该方法允许扩张狭窄的冠状动脉的血流量，从而恢复。同时，它会导致在一定程度上损伤血管内皮细胞和血管壁3。然而，这种疗法的长期效果是有限的过多动脉remode玲和再狭窄4。 就业的支架，PTCA变得更加复杂病变的治疗有效，允许血运重建后的急性血管闭合5。该方法降低了支架内再狭窄的发生率小于10％6。除了这些好处，冠状动脉血运重建治疗的第一选择承担危及生命的支架内血栓和再狭窄的风险。 支架内血栓形成引起的去内皮的船只，然后由大量粘附的血小板和纤维蛋白的​​受伤部位。 26％的患者患有支架内血栓形成，63％死于心肌梗死7。再狭窄是指在创伤愈合过程中，涉及到血管壁的机械性损伤后新内膜增生（迁移和增殖的血管平滑肌细胞（VSMC），细胞外基质（ECM）的沉积，和重塑该船只。通常情况下，侵入性的干预成为必要dilatate严重收窄的动脉粥样硬化的血管因支架内血栓和再狭窄。 为了防止在支架内血栓形成，使用抗血栓形成药物的长期治疗是必要的。为了预防再狭窄，新一代的药物洗脱支架的洗脱抗增殖剂，如免疫抑制药物（ 例如，西罗莫司，依维莫司，佐他莫司）和抗癌药物（ 如紫杉醇）从聚合物涂层数月9,10。虽然这些药物减少新内膜形成和再狭窄，保持支架内血栓形成的危险性高，通过抑制再内皮化。 动脉损伤后，内皮室的维护是必要的，以防止血栓形成并发症。在生理条件下，人体血管内皮细胞显示一个小的成交率11。根据病理学然而，一例出现的条件下，内皮细胞的完整性受损，因此，迅速 ​​恢复周边成熟的内皮细胞和循环内皮祖细胞（EPCs）12,13。 较大的动物14-16小鼠主动脉这些复杂的分子机制的研究是一个非常困难的过程中，提供的数据有限，17-19。新型支架涂层要测试的效率，减少支架内血栓和再狭窄的新车型是势在必行。 镍钛合金代表，因为其高弹性，形状记忆效应，在患者的耐受性良好，在临床上使用裸金属支架被成功地用作支架的理想平台。这种合金有可能创造一个小型化的与外部直径为500μm，可被涂上20和植入小鼠的颈动脉支架。鼠标点击一个小型的镍钛合金支架的发展动脉arotid，允许支架植入引起的确切的分子机制的研究，并提供快速，高效地测试不同的药物涂层的影响，以防止再狭窄的可能性。此外，存在不同的基因敲除小鼠品系，是一个巨大的优势，在澄清不同的分子参与新生内膜的生长和支架内血栓形成的作用。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>nitinol-stents (self-made from nitinol-struts)</td>
			<td>Fort Wayne Metals, Castlebar, Ireland NiTi#1, superelastic, straight annealed, light oxide, diameter 500 &mu;m</td>
			<td>custom-made product</td>
			<td>Institute for Textile Technology and Mechanical Engineering</td>
		</tr>
		<tr>
			<td>silicon tube</td>
			<td>IFK Isofluor, Germany</td>
			<td>custom-made product</td>
			<td>diameter 500 &mu;m, section thickness 100 &mu;m, polytetrafluorethylene catheter</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Olympus</td>
			<td>SZ/X9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>FST, Germany</td>
			<td>91197-00</td>
			<td>standard tip curved 0.17 mm</td>
		</tr>
		<tr>
			<td>Ketamine 10%</td>
			<td>CEVA, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Medistar, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bepanthene</td>
			<td>Bayer, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST, Germany</td>
			<td>91460-11</td>
			<td>Straight</td>
		</tr>
		<tr>
			<td>Vannas scissor</td>
			<td>Aesculap, Germany</td>
			<td>OC 498 R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5/0 Silk</td>
			<td>Seraflex</td>
			<td>IC 108000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>7/0 Silk</td>
			<td>Seraflex</td>
			<td>IC 1005171Z</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>guide-wire</td>
			<td>Abbott Vascular</td>
			<td>1001782-HC</td>
			<td>0.014-inch angioplastie guide-wire</td>
		</tr>
		<tr>
			<td>Michel suture clips</td>
			<td>Aesculap, Germany</td>
			<td>BN507R</td>
			<td>7.5 x 1.75 mm</td>
		</tr>
		<tr>
			<td>Michel Forcep</td>
			<td>Aesculap, Germany</td>
			<td>BN730R</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a murine model of stent implantation in the carotid artery for the study of restenosis

尽管支架在过去几十年的发展中取得的重大进展，心血管疾病在西方国家仍然是主要的死亡原因。除了由不同的药物洗脱支架的发展所带来的好处，冠状动脉血运重建承担支架内血栓和再狭窄危及生命的风险。研究新的治疗策略缺乏适当的方法来研究支架植入术后再狭窄过程受损。在这里，我们描述了一个快速，方便的程序，它提供了一个方便的方法研究血管重塑的分子机制和不同的药物涂层的影响的可能性小鼠颈动脉支架植入术。

<ol><li>一个小型化的镍钛合金支架植入到小鼠的左颈总动脉需要25至30分钟，并示出的死亡率为10％，这主要是由于该船只在干预过程中的损害。一个更好的生存率观察小鼠的重量超过25克，支架植入术（5％）的死亡率。因此，我们选择了植入小鼠的重量在25-27克之间。手术后，小鼠从麻醉中恢复2-5分钟内没有物理损伤， 比如瘫痪，被观察到。微型计算机断层扫描（显微CT）成像进行了一个星期，支架植入后支架没有脱臼血流（ 图1C）。不幸的是，这些图像新生内膜形成的分析是不可能的，由于金属衍生的文物（ 图1D，1E）。 </li><li>我们没有看到任何船只或内皮损害的船只unstented面积的，下方的支架，通过组织学（ 图2A）和内皮细胞（ 图2B，抗小鼠CD31抗体）的特异性染色检测。更好的概要，使用双光子激光扫描显微镜（ 图2B，2C）扫描部分。 </li><li>在支架置入容器中，永久扩张15％（比动脉支架：1,15:1）小鼠的重量在25-27克之间。新生内膜形成和血栓形成可分析古典组织学染色（ 如苏木精-伊红，姬姆萨，Movat，甲苯胺蓝，马尾松Trichrom Goldner， 图3A，3B）。由于椎板外耳炎和国际是不可见的了，斑块大小的外部和管腔地区（平均斑块面积：234566±3315 2微米，平均管腔面积：12036±2662微米）的差额计算。外部圆周也测量了（意思是：1799±14微米）。为了分析细胞的组合物，部分需要要deplastified的具有特定标记的染色。对于再内皮化，我们使用了的Cy3标记的抗CD31抗体和平滑肌细胞增殖的一个FITC标记的抗α-SMA抗体（ 图3C）。再内皮管腔表面总（平均23.07±3.14％），支架植入后一周的CD-31染色阳性百分比计算。 </li></ol>当然，无限数量的特异性染色是可能的，这取决于每个实验室的经验。肌球蛋白重链，更好地表征平滑肌细胞，而且分析的浸润细胞（单核细胞，淋巴细胞），或不同的炎症性细胞因子染色分析也可以被执行，根据研究目的。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50233/50233fig1highres.jpg" sr<html> C =“/ files/ftp_upload/50233/50233fig1.jpg”/&gt; 图1的外科手术（A）的总体示意图。血液的流动被中断绑定牢固，以及由拉结周围的颈总动脉，颈内动脉和颈外动脉的近端上的结。包含该支架被引入到硅管颈外动脉在颈外动脉通过一个小切口。扩张器达到所需的位置后，硅管导丝在拉回来，并允许扩张器扩张的形状记忆。显微CT图像示出的支架的位置（B）的外科手术植入后一周。由于材料来源的文物，分析新生内膜的增长是不可能的（C，D）。 3fig2highres.jpg“SRC =”/ files/ftp_upload/50233/50233fig2.jpg“/&gt; 图2。手术过程中是不会受到该船只Unstented面积，甲苯胺蓝（A）和内皮特异性CD31染色（B，C）所示的。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50233/50233fig3highres.jpg" src="/files/ftp_upload/50233/50233fig3.jpg" /> 图3可以通过经典的组织学染色（ 如马尾松Trichrom Goldner）（A）的牌匾分析。有组织的血栓内的新生内膜的黑色染色的血纤维蛋白的沉积可以被检测到，在某些情况下，一个完整的闭塞的血管观察到（B）。使用特定标记的双重免疫荧光染色检测到的再内皮化（Cy3标记，红色）或平滑肌细胞的增殖（FITC，绿色）。用4&#39;，6 -二脒-2 -苯基吲哚（DAPI，蓝色）（C）进行复染。我们注意到，已完成的再内皮化的支架杆（左，双箭头）相比未完成管腔再内皮化（右，单箭头）。

Watch this Scientific Journal Video about A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis at JoVE.com

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

模型小鼠颈动脉支架植入术。相比其他类似的方法，这个过程是非常快速，简单，方便，在一个方便的方法来研究血管壁的反应，不同的药物洗脱支架再狭窄的分子机制提供了可能性。

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-murine-model-stent-implantation-carotid-artery-for-study

Research

JoVE Journal

Medicine

25.2K 次观看.  RWTH Aachen University. 模型小鼠颈动脉支架植入术。相比其他类似的方法，这个过程是非常快速，简单，方便，在一个方便的方法来研究血管壁的反应，不同的药物洗脱支架再狭窄的分子机制提供了可能性。

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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

人乳内动脉（IMA）移植及内支架置入术：人体模型支架内再狭窄的研究开发

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

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 Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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 ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

股动脉线损伤的小鼠模型的血管周围给药补丁植入

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

动脉再狭窄小鼠模型：股导线损伤的技术方面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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, ...