N.E.B由国家环境卫生科学研究所（2018年5T32ES007126-35）和美国心脏协会博士前奖学金（20PRE35120321）提供的培训补助金支持。E.S..M.B是一名KL2学者，部分得到UNC临床和转化科学奖-K12学者计划（KL2TR002490，2018）和国家心肺血液研究所（K01HL145354）的支持。作者感谢联合国军国署显微镜服务实验室的巴勃罗·阿里尔博士协助LSFM。光片荧光显微镜在显微镜服务实验室进行。病理学和实验室医学部显微镜服务实验室部分由P30 CA016086癌症中心向UNC Lineberger综合癌症中心提供核心支持赠款。

这里描述的所有方法都得到了北卡罗来纳大学教堂山分校机构动物护理和使用委员会（IACUC）的批准。
1. 术前程序
<ol><li>消毒手术器械。手术前自动切除所有手术器械。如果在同一天进行多次手术，则使用干珠消毒器在手术之间对仪器进行消毒。</li>
	<li>在25%的普鲁罗尼-127凝胶（在无菌蒸馏水中稀释）中准备治疗。</li>
	<li>设置一个2F Fogarty气球导管到充气器，并将导管的气球端放在一个充满盐水溶液的1mL注射器中。</li>
	<li>通过将老鼠放在一个室中，用5%的异氟兰来诱导麻醉。<ol>
<li>把老鼠从房间里移开，记录老鼠的体重。使用剪发器在腹颈区域剃毛皮。</li>
		<li>将大鼠用5%的异氟化液放回腔室，以确保麻醉的感应。</li>
	</ol>
</li>
	<li>将大鼠苏平放在手术平台上，将脸部插入鼻锥，使大鼠脸朝向外科医生。<ol>
<li>将吸入麻醉降低到1.5%异氟化。通过四只脚的脚趾捏反射来验证麻醉的深度。</li>
		<li>把四条腿都贴到手术台上。</li>
	</ol>
</li>
	<li>打开热灯。</li>
	<li>在皮下注射阿托平（0.01毫克/千克），以减少气道分泌物。</li>
	<li>在皮下注射卡普罗芬（5毫克/千克），用于预防性疼痛管理。如果抗炎药物在实验中不能接受，请参阅步骤 3.2.2 和 3.2.3。</li>
	<li>使用无菌棉签将润滑剂眼药膏涂抹在双眼上，以防止角膜在手术过程中干燥。</li>
	<li>在 70% 乙醇之间交替的圆形运动中将颈部拭子三次，然后从剃光区域中心向外挥动贝塔丁，对切口部位进行消毒。</li>
	<li>渗透切口线 s.c. 与 0.25% 布皮瓦卡因。</li>
	<li>在处理无菌手术器械和用品之前戴上无菌手术手套。</li>
	<li>将所有自动切除的手术器械放在无菌手术纸上。</li>
	<li>切三个独立的 1 英寸链无菌 7 - 0 丙烯缝合。</li>
	<li>将棉签和纱布放在手术纸上。</li>
	<li>用无菌手术单将大鼠拖曳，只暴露未消毒的颈部区域。</li>
	<li>在表中再切一个小开口，露出部分鼻锥。这将是在受伤时录下气球导管的地点。</li>
</ol>2. 操作程序
<ol><li>在整个外科手术过程中，通过监测呼吸速率（速度应一致且视为正常）以及每 15 分钟用脚趾捏一次来评估麻醉深度。如果呼吸率增加或对脚趾捏有反应，则暂停手术操作，增加的异体高达2.5%。</li>
	<li>暴露常见的胡萝卜动脉（CCA）。<ol>
<li>在老鼠的下颚骨骼之间做一个肤浅的，直的，纵向的领口切口。切口长度约为1.5-2厘米。</li>
		<li>通过皮肤下的结缔组织切口，直到肌肉层暴露。将唾液腺置换到皮肤下方，以进入肌肉组织。</li>
		<li>通过在肌肉层和结缔组织之间插入封闭的剪刀，轻轻打开剪刀，同时向上拉皮，将结缔组织与肌肉分开。</li>
	</ol></li>
	<li>沿着气管的左侧纵向解剖两种可见的肌肉（骨质激素和胸肌类），直到观察到与两种表面肌肉垂直的第三块肌肉（omohyoid）。</li>
	<li>使用钳子创建一个窗口，将这种垂直肌肉（omohyoid）与在气管上运行的纵向肌肉（斯特诺希奥德）分开。轻轻进行这种分离，以防止气管的钝性创伤。</li>
	<li>到达垂直肌肉下方的钳子，切开两个纵向肌肉并暴露CCA。</li>
	<li>剖析CCA。<ol>
<li>在内部胡萝卜动脉 （ICA） 和外部胡萝卜动脉 （ECA） 暴露之前，在分叉附近剖析 CCA。</li>
		<li>解剖优越的甲状腺动脉（STA），它从ECA分支。</li>
		<li>使用预切割的丙烯缝合线，在各自的分叉附近对 STA 和 ECA 进行诉讼。将大部分缝合线留在结的一侧，然后用弯曲的血吸管抓住每个缝合线。</li>
		<li>完成对ICA的解剖，到达ICA下面和周围的钳子，并使用非粉碎血管夹来实现分离控制。与ICA一起夹住腹膜动脉。</li>
		<li>解剖CCA的近似分叉，确保将迷走神经与CCA分离。</li>
		<li>到达CCA下面和周围的钳子，并使用非粉碎血管夹来实现近似控制。将夹子从分叉中放置至少 5 毫米。</li>
	</ol></li>
	<li>执行气球伤害。<ol>
<li>操纵每个被勒住的动脉分支的弯曲的血站，以暴露 ECA 和上级分支之间的分裂。</li>
		<li>在分叉处轻轻解剖组织，然后使用微切除剪刀在 ECA 和上级分支之间进行动脉切除术切口。</li>
		<li>使用棉签将所有血液从CCA中挤出并清理动脉切除位点。</li>
		<li>将未起浮的气球导管插入动脉切除术，并推进到CCA，直到气球的近端经过分叉。</li>
		<li>将导管粘在鼻锥上，这样气球就不会在通货膨胀时从动脉中滑出。</li>
		<li>慢慢地将气球膨胀到5个压力气氛，并在CCA中离开5分钟，以诱发动脉损伤。确保整个 5 分钟的压力保持不变。</li>
		<li>5分钟后，放气气球，并通过动脉切除术轻轻地从CCA中取出。</li>
		<li>轻轻挤压CCA的夹子，冲洗CCA。不要取下夹子。</li>
		<li>将 ECA 近体与动脉切除术分离，然后从 CCA 和 ICA 中取出夹子，以恢复通过 CCA 到 ICA 的血液流动。确保动脉切除术周围没有明显的出血，并且 CCA 正在脉动。</li>
	</ol></li>
	<li>沿受伤的CCA单独应用100μL的治疗性或多面性凝胶车辆。这样做，将 50 μL 涂在 CCA 的左侧，然后在 CCA 的右侧应用 50 μL，以确保受伤的动脉均匀涂层。</li>
	<li>关闭伤口部位。<ol>
<li>切割多余的丙烯缝合线。</li>
		<li>沿结缔组织使用中断的 4-0 或 6-0 vicryl 层关闭伤口。</li>
		<li>完成关闭伤口使用运行4-0尼龙缝合沿皮肤。</li>
	</ol></li>
</ol>3. 术后程序
<ol><li>将大鼠单独放在一个干净的笼子里，将一半的笼子放在加热灯下，并进行监测，直到老鼠恢复足够的意识以维持体面休养。将老鼠关在单独的笼子里，直到动物完全警觉和移动，然后再转移到原来的笼子里。</li>
	<li>在接下来的三天里每天监测老鼠，然后每周监测三次，直到安乐死。安乐死使用异氟过量，然后双边胸切除术如下所述。<ol>
<li>利用国家动物替代精炼和减少研究中心 （NC3R） 鬼脸量表来识别术后疼痛水平。如果任何动物似乎正在经历疼痛或出现任何神经折衷，立即牺牲。</li>
		<li>对于未接受卡洛芬的动物，在手术前24小时通过手术后48小时在饮用水中施用对乙酰氨基酚6毫克/mL。对乙酰氨基酚提供镇痛药，抗炎效果最小。</li>
		<li>或者，其他镇痛策略，具有最小的抗炎效果，可以使用，如丁丙诺啡或丁丙诺啡延长释放。咨询贵机构的兽医团队。</li>
	</ol>
</li>
</ol>4. 组织收获和成像
<ol><li>手术两周后，通过过量麻醉（5%是异体）对大鼠实施安乐死。或者，在更早的时间点对大鼠实施安乐死，以分析动脉损伤反应的各个方面。<ol>
<li>一旦呼吸停止，作为安乐死的次要方法进行双边胸切除术。</li>
	</ol></li>
	<li>通过腹部进行横向切口，然后向上切开，穿过隔膜和肋骨，露出胸腔。</li>
	<li>渗透和修复动脉。<ol>
<li>通过左心室插入附着在引力注水固定系统的 18 G 管子。通过标记注水系统相对于台面的高度（120 厘米高程，相当于 91 ± 3 mmHg），保持大鼠之间的等效压力。</li>
		<li>使用弯曲的止血剂将管子与心室夹在一起。</li>
		<li>在右中庭切开，打开血管回路，然后用PBS开始灌注，然后是2-4%的甲醛（每个约250毫脱）。</li>
		<li>准备在牺牲当天或最多在牺牲前一天晚上稀释在PBS中的甲醛。如果在牺牲当天准备，请确保准甲醛在开始输液前冷却到室温。将甲醛储存在4°C。</li>
	</ol></li>
	<li>固定后，提取左和右胡萝卜素动脉，并在4°C下储存2小时，在2-4%的甲醛。</li>
	<li>将动脉转移到30%的蔗糖，并在4°C过夜。</li>
	<li>16-24小时后，将动脉嵌入最佳切割温度（OCT）化合物中，并冻结OCT嵌入动脉块。<ol>
<li>条件动脉在OCT在室温下10分钟。将动脉平行放置在充满 OCT 的低温的平面上，标记动脉分叉所面对的低温的一侧。液氮中快速冻结。</li>
		<li>长期存放冷冻块-80°C。</li>
	</ol></li>
	<li>使用冷冻统计器的部分冻结块。<ol>
<li>每张幻灯片收集 6 个 5μm 厚的动脉横截面，幻灯片 1 从分叉开始。</li>
		<li>部分冻结块，直到增生不再可见（约100张幻灯片）。</li>
	</ol></li>
	<li>血红素 + 欧辛 （H&E） 污渍幻灯片18<ol>
<li>从分叉开始，每十张幻灯片中就有一张被弄脏，从而找到受伤区域（例如，滑梯 1、10、20、30、40、50、60、70、80、90 和 100）。</li>
		<li>在受伤部位周围染色额外的幻灯片，以找到具有峰值遮挡的幻灯片（例如，幻灯片 20、30 和 40 具有可见的增生，因此染色幻灯片 15、25、35 和 45）。</li>
		<li>在峰值遮挡幻灯片前后（例如，在幻灯片 35 处发现峰值遮挡，然后对幻灯片 25、45 等）进行污渍和量化幻灯片，每个大鼠总共有 3-10 张幻灯片。</li>
	</ol></li>
	<li>对于光片荧光显微镜成像，在固定步骤4.4后，将动脉在4°C下过夜。<ol>
<li>探针动脉与1:500稀释兔子抗CD31原发抗体在稀释（pH 7.4）3天。然后用1:500稀释抗兔亚历克萨氟647次次抗体对抗动脉2天19。</li>
		<li>使用 iDISCO+20清除动脉。</li>
		<li>使用光片荧光显微镜21对动脉进行成像。使用软件渲染图像（例如，伊马里斯）19.</li>
	</ol></li>
	<li>量化新金质增生。如果可能，以盲目的方式执行量化。<ol>
<li>使用 ImageJ 软件在上面确定的 3-10 张幻灯片（步骤 4.8.3）上跟踪动脉的内脏、内部弹性拉米纳 （IEL） 和外部弹性拉米纳 （EEL） 的周长。</li>
		<li>量化 ImageJ 中每个跟踪区域的区域并导出这些值。起义痕迹产生流明区域，IEL微量产生IEL区域，鳗鱼痕迹产生鳗鱼区域。</li>
		<li>平均从3-10张幻灯片获得的值，以获得每只大鼠胡萝卜动脉的平均伤害（百分比遮挡，刺激:介质（I:M）比，新皮质增生）。 新金质增生 = IEL 区域 - 卢门区域 <img alt="Equation 1" src="/files/ftp_upload/60473/60473eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/60473/60473eq2.jpg">
</li>
	</ol></li>
</ol>

<ol>
	<li>American Heart Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+Disease:+A+Costly+Burden+for+America,+Projections+Through+2035.">Cardiovascular Disease: A Costly Burden for America, Projections Through 2035.</a> American Heart Association CVD Burden Report. , (2017).</li><li>Singh, R. B., Mengi, S. A., Xu, Y. J., Arneja, A. S., Dhalla, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+atherosclerosis:+A+multifactorial+process.">Pathogenesis of atherosclerosis: A multifactorial process.</a> Experimental and Clinical Cardiology. 7 (1), 40-53 (2002).</li><li>Clowes, A. W., Reidy, M. A., Clowes, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+stenosis+after+arterial+injury.">Mechanisms of stenosis after arterial injury.</a> Laboratory Investigation. 49 (2), 208-215 (1983).</li><li>Clowes, A. W., Reidy, M. A., Clowes, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+cellular+proliferation+after+arterial+injury.+I.+Smooth+muscle+growth+in+the+absence+of+endothelium.">Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium.</a> Laboratory Investigation. 49 (3), 327-333 (1983).</li><li>Sartore, 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=Contribution+of+adventitial+fibroblasts+to+neointima+formation+and+vascular+remodeling:+from+innocent+bystander+to+active+participant.">Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant.</a> Circulation Research. 89 (12), 1111-1121 (2001).</li><li>Tanaka, K., 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=Circulating+progenitor+cells+contribute+to+neointimal+formation+in+nonirradiated+chimeric+mice.">Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice.</a> The FASEB Journal. 22 (2), 428-436 (2008).</li><li>Henry, M., 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=Carotid+angioplasty+and+stenting+under+protection.+Techniques,+results+and+limitations.">Carotid angioplasty and stenting under protection. Techniques, results and limitations.</a> The Journal of Cardiovascular Surgery. 47 (5), 519-546 (2006).</li><li>Kounis, N. G., 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=Thrombotic+responses+to+coronary+stents,+bioresorbable+scaffolds+and+the+Kounis+hypersensitivity-associated+acute+thrombotic+syndrome.">Thrombotic responses to coronary stents, bioresorbable scaffolds and the Kounis hypersensitivity-associated acute thrombotic syndrome.</a> Journal of Thoracic Disease. 9 (4), 1155-1164 (2017).</li><li>Jackson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+restenosis.">Animal models of restenosis.</a> Trends in Cardiovascular Medicine. 4 (3), 122-130 (1994).</li><li>Shears, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+inhibition+of+intimal+hyperplasia+by+adenovirus-mediated+inducible+nitric+oxide+synthase+gene+transfer+to+rats+and+pigs+in+vivo.">Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo.</a> Journal of the American College of Surgeons. 187 (3), 295-306 (1998).</li><li>Takayama, 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=A+murine+model+of+arterial+restenosis:+technical+aspects+of+femoral+wire+injury.">A murine model of arterial restenosis: technical aspects of femoral wire injury.</a> Journal of Visualized Experiments. (97), (2015).</li><li>Zhang, L. N., Parkinson, J. F., Haskell, C., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+intimal+hyperplasia+learned+from+a+murine+carotid+artery+ligation+model.">Mechanisms of intimal hyperplasia learned from a murine carotid artery ligation model.</a> Current Vascular Pharmacology. 6 (1), 37-43 (2008).</li><li>Jahnke, 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=Characterization+of+a+new+double-injury+restenosis+model+in+the+rat+aorta.">Characterization of a new double-injury restenosis model in the rat aorta.</a> Journal of Endovascular Therapy. 12 (3), 318-331 (2005).</li><li>Gregory, E. K., 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=Periadventitial+atRA+citrate-based+polyester+membranes+reduce+neointimal+hyperplasia+and+restenosis+after+carotid+injury+in+rats.">Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats.</a> American Journal of Physiology-Heart and Circulatory Physiology. 307 (10), 1419-1429 (2014).</li><li>Buglak, N. E., Jiang, W., Bahnson, E. S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinnamic+aldehyde+inhibits+vascular+smooth+muscle+cell+proliferation+and+neointimal+hyperplasia+in+Zucker+Diabetic+Fatty+rats.">Cinnamic aldehyde inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in Zucker Diabetic Fatty rats.</a> Redox Biology. 19, 166-178 (2018).</li><li>Bahnson, E. 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=Long-term+effect+of+PROLI/NO+on+cellular+proliferation+and+phenotype+after+arterial+injury.">Long-term effect of PROLI/NO on cellular proliferation and phenotype after arterial injury.</a> Free Radical Biology and Medicine. 90, 272-286 (2016).</li><li>Gilbert, J. C. W., Davies, M. C., Hadgraft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+Pluronic+F127+in+aqueous+solution+studied+using+fluorescent+probes.">The behaviour of Pluronic F127 in aqueous solution studied using fluorescent probes.</a> International Journal of Pharmaceutics. 40 (1-2), 93-99 (1987).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+morphometric+analyses+for+rat+carotid+balloon+injury+model.">Histological and morphometric analyses for rat carotid balloon injury model.</a> Methods in Molecular Medicine. 139, 31-66 (2007).</li><li>Buglak, N. E., 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=Light+Sheet+Fluorescence+Microscopy+as+a+New+Method+for+Unbiased+Three-Dimensional+Analysis+of+Vascular+Injury.">Light Sheet Fluorescence Microscopy as a New Method for Unbiased Three-Dimensional Analysis of Vascular Injury.</a> Cardiovascular Research. , (2020).</li><li>Renier, N., 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=iDISCO:+a+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Ariel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UltraMicroscope II - A User Guide. , (2018).</li><li>Touchard, A. G., Schwartz, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+restenosis+models:+challenges+and+successes.">Preclinical restenosis models: challenges and successes.</a> Toxicologic Pathology. 34 (1), 11-18 (2006).</li><li>Xiangdong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+atherosclerosis+research:+a+review.">Animal models for the atherosclerosis research: a review.</a> Protein Cell. 2 (3), 189-201 (2011).</li><li>Chen, H., Li, D., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Rat+Models+for+Atherosclerosis.">Novel Rat Models for Atherosclerosis.</a> Journal of Cardiology and Cardiovascular Sceinces. 2 (2), 29-33 (2018).</li><li>Xing, D., Nozell, S., Chen, Y. F., Hage, F., Oparil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+mechanisms+of+vascular+protection.">Estrogen and mechanisms of vascular protection.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 29 (3), 289-295 (2009).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods in Molecular Medicine. 139, 1-30 (2007).</li><li>Pellet-Many, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilins+1+and+2+mediate+neointimal+hyperplasia+and+re-endothelialization+following+arterial+injury.">Neuropilins 1 and 2 mediate neointimal hyperplasia and re-endothelialization following arterial injury.</a> Cardiovascular Research. 108 (2), 288-298 (2015).</li><li>Wu, B., 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=Perivascular+delivery+of+resolvin+D1+inhibits+neointimal+hyperplasia+in+a+rat+model+of+arterial+injury.">Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury.</a> Journal of Vascular Surgery. 65 (1), 207-217 (2017).</li><li>Tan, J., Yang, L., Liu, C., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-26a+targets+MAPK6+to+inhibit+smooth+muscle+cell+proliferation+and+vein+graft+neointimal+hyperplasia.">MicroRNA-26a targets MAPK6 to inhibit smooth muscle cell proliferation and vein graft neointimal hyperplasia.</a> Scientific Reports. 7, 46602 (2017).</li><li>Pearce, C. G., 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=Beneficial+effect+of+a+short-acting+NO+donor+for+the+prevention+of+neointimal+hyperplasia.">Beneficial effect of a short-acting NO donor for the prevention of neointimal hyperplasia.</a> Free Radical Biology and Medicine. 44 (1), 73-81 (2008).</li><li>Cao, 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=S100B+promotes+injury-induced+vascular+remodeling+through+modulating+smooth+muscle+phenotype.">S100B promotes injury-induced vascular remodeling through modulating smooth muscle phenotype.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1863 (11), 2772-2782 (2017).</li><li>Madigan, M., Entabi, F., Zuckerbraun, B., Loughran, P., Tzeng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+inhaled+carbon+monoxide+mediates+the+regression+of+established+neointimal+lesions.">Delayed inhaled carbon monoxide mediates the regression of established neointimal lesions.</a> Journal of Vascular Surgery. 61 (4), 1026-1033 (2015).</li><li>Khurana, R., 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=Angiogenesis-dependent+and+independent+phases+of+intimal+hyperplasia.">Angiogenesis-dependent and independent phases of intimal hyperplasia.</a> Circulation. 110 (16), 2436-2443 (2004).</li><li>Tsihlis, N. D., Vavra, A. K., Martinez, J., Lee, V. R., Kibbe, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+is+less+effective+at+inhibiting+neointimal+hyperplasia+in+spontaneously+hypertensive+rats.">Nitric oxide is less effective at inhibiting neointimal hyperplasia in spontaneously hypertensive rats.</a> Nitric Oxide. 35, 165-174 (2013).</li><li>Chen, 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=Inhibition+of+neointimal+hyperplasia+in+the+rat+carotid+artery+injury+model+by+a+HMGB1+inhibitor.">Inhibition of neointimal hyperplasia in the rat carotid artery injury model by a HMGB1 inhibitor.</a> Atherosclerosis. 224 (2), 332-339 (2012).</li><li>Mano, T., Luo, Z., Malendowicz, S. L., Evans, T., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+GATA-6+downregulation+promotes+smooth+muscle+differentiation+and+inhibits+intimal+hyperplasia+in+balloon-injured+rat+carotid+artery.">Reversal of GATA-6 downregulation promotes smooth muscle differentiation and inhibits intimal hyperplasia in balloon-injured rat carotid artery.</a> Circulation Research. 84 (6), 647-654 (1999).</li><li>Becher, 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=Three-Dimensional+Imaging+Provides+Detailed+Atherosclerotic+Plaque+Morphology+and+Reveals+Angiogenesis+after+Carotid+Artery+Ligation.">Three-Dimensional Imaging Provides Detailed Atherosclerotic Plaque Morphology and Reveals Angiogenesis after Carotid Artery Ligation.</a> Circulation Research. 126 (5), 619-632 (2020).</li></ol>

作者声明，本手稿的出版不存在利益冲突。

大鼠胡萝卜素动脉气球损伤是最广泛使用和研究的恢复动物模型之一。原来的气球损伤模型3和修改的压力控制段损伤变异10都告诉了许多方面，动脉损伤的反应也发生在人类身上，少数限制是富含纤维素的血栓很少发展，局部炎症是最小的相比，其他伤害模型，如在高胆固醇兔子或猪模型9，22。老鼠也可以在不同的?...

心血管疾病（CVD）仍然是全球的主要死因。动脉粥样硬化是大多数 CVD 相关发病率和死亡率的根本原因。动脉粥样硬化是动脉内斑块的积累，导致流明变窄，妨碍器官和脱血组织2的适当血液注入。治疗严重动脉粥样硬化的临床干预措施包括带支架或不放置的气球血管成形术。这种干预包括将气球导管推进到斑块的部位，并充气气球将斑块压缩到动脉壁上，扩大发光区域。然而，这一程序损害了动脉，导致动脉损伤反应3。这种损伤反应的长时间激活导致动脉恢复，或重新缩小，辅之以新皮质增生和血管重塑。在血管成形术中，刺激层被内皮细胞破坏，导致立即血小板吸收和局部炎症。局部信号诱导血管平滑肌肉细胞（VSMC）和冒险成纤维细胞的表型变化。这导致VSMC和成纤维细胞向内迁移和扩散到流明，导致新金体增生4，5。循环的祖细胞和免疫细胞也有助于恢复体积6。在适用的情况下，药物疏松支架（DES）是目前抑制重生7的标准。然而，DES抑制动脉再内皮化，从而创造一个亲血栓形成的环境，可能导致后期支架血栓形成8。因此，动物模型对于了解再造病的病理生理学，以及开发更好的治疗策略以延长再血管化程序的疗效都是不可或缺的。
几个大小动物模型9被用于研究这种病理学。其中包括动脉发光侧的气球损伤3、10或线伤11，以及动脉周围的部分粘合12或袖口放置13。气球和电线损伤都使动脉内皮层凹陷，模仿血管成形术后的临床情况。特别是，气球损伤模型使用与临床环境类似的工具（即气球导管）。气球损伤最好在大鼠模型中执行，因为大鼠动脉适合市售气球导管。在这里，我们描述了一个压力控制的段动脉损伤，一个成熟的，修改版本的老鼠胡萝卜动脉气球损伤。这种压力控制的方法密切模仿临床血管成形术，并允许可重复的新生儿增生形成两周后受伤14，15。此外，这种压力控制的动脉损伤导致完全内皮层恢复2周后手术16。这直接对比了最初的气球损伤模型，由Clowes描述，其中内皮层永远不会回到全覆盖3。
手术后，可以通过多种方法对受伤的动脉进行治疗或针对受伤动脉。此处描述的方法使用嵌入在多音压凝胶溶液中的小分子的间接应用。具体来说，我们应用100μM肉碱醛溶液在25%的普鲁罗尼-F127凝胶到动脉受伤后立即抑制新皮质增生形成15。多音速-F127是一种无毒、可热可逆的凝胶，能够以受控的方式在当地输送药物。同时，动脉损伤是局部的，因此地方管理允许测试一个积极的原则，同时尽量减少偏离目标的影响。然而，使用这种方法进行治疗的有效交付将取决于所使用的小分子或生物的化学成分。

<table border="0" cellpadding="0" cellspacing="0" style="width:776px;" width="776">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">1 mL Syringe</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:119px;">14955450</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Syringe with needle</td>
			<td style="width:148px;">BD</td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 French Fogarty Balloon Embolectomy Catheter</td>
			<td style="width:148px;">Edwards LifeSciences</td>
			<td style="width:119px;">120602F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Ethilon (Nylon) Suture</td>
			<td style="width:148px;">Ethicon Inc</td>
			<td style="width:119px;">662H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl Suture</td>
			<td style="width:148px;">Ethicon Inc</td>
			<td style="width:119px;">J214H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-0 Prolene Suture</td>
			<td style="width:148px;">Ethicon Inc</td>
			<td style="width:119px;">8800H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethyl alcohol</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Anti-Rabbit Alexa Fluor 647</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A21245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atropine Sulfate</td>
			<td style="width:148px;">Vedco Inc</td>
			<td style="width:119px;"></td>
			<td>for veterinary use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton Swabs</td>
			<td style="width:148px;">Puritan</td>
			<td style="width:119px;">806-WC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved Hemostats</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">13009-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Curved Forceps</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">11203-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Scissors</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">14090-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gauze</td>
			<td style="width:148px;">Covidien</td>
			<td style="width:119px;">2252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">IHC-Tek Diluent (pH 7.4)</td>
			<td style="width:148px;">IHC World</td>
			<td style="width:119px;">IW-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insufflator</td>
			<td style="width:148px;">Merit Medical</td>
			<td style="width:119px;">IN4130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodine solution</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lubricating Eye Ointment</td>
			<td style="width:148px;">Dechra</td>
			<td style="width:119px;"></td>
			<td>for veterinary use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo Scissors</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">14010-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Serrefines</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">18055-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microdissection Scissors</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">15004-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Serrefine Clamp Applying Forceps</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">18057-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle Holder</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">12003-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pluronic-127 (diluted in sterile water)</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2443</td>
			<td style="width:183px;">25% prepared</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Rabbit Anti-CD31</td>
			<td style="width:148px;">Abcam</td>
			<td style="width:119px;">ab28364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retractor</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td style="width:183px;">Bent paper clips work well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rimadyl (Carprofen)</td>
			<td style="width:148px;">Zoetis Inc</td>
			<td style="width:119px;"></td>
			<td>for veterinary use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline solution</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Forceps</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">11006-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Drape</td>
			<td style="width:148px;">Dynarex</td>
			<td style="width:119px;">4410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-Pins</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat carotid artery pressure-controlled segmental balloon injury with periadventitial therapeutic application

心血管疾病仍然是全世界死亡和残疾的主要原因，部分原因是动脉粥样硬化。动脉粥样硬化斑块缩小了动脉的光泽表面区域，从而减少了流向器官和分膜组织的充足血液。临床上，重新血管化程序，如气球血管成形术有或没有支架放置旨在恢复血液流动。虽然这些程序通过减少斑块负担来重建血液流动，但它们会损坏血管壁，从而启动动脉愈合反应。长期愈合反应导致动脉恢复，或重新缩小，最终限制这些血管化程序的长期成功。因此，前科动物模型是分析驱动恢复的病理生理机制的组成部分，为测试新的治疗策略提供了机会。与大型动物模型相比，Murine 模型更便宜、更容易操作。气球或电线损伤是穆林模型中常用的两种伤害模式。气球损伤模型特别模仿临床血管成形术，对动脉造成足够的损伤，以发展恢复性。在这里，我们描述了手术细节的性能和组理分析修改，压力控制大鼠胡萝卜动脉气球损伤模型。此外，此协议还突出了治疗药物的局部近心应用如何用于抑制新皮质增生。最后，我们提出光片荧光显微镜作为成像和可视化动脉损伤的三维新方法。

图 1显示了用于执行此手术的所有材料和手术工具。两周受伤的动脉横截面的血氧素和欧辛 （H&E） 染色允许新脑增生的清晰可视化。图 2显示了健康、受伤和经过治疗的动脉的 H&E 染色动脉横截面的代表性图像。图2还概述了如何使用广泛使用的图像处理软件ImageJ来量化受伤动脉的新生儿增生水平。使用此方法，可追踪到新膜的周?...

Watch this Scientific Journal Video about A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application at JoVE.com

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

大鼠胡萝卜动脉气球损伤模仿临床血管成形术，以恢复动脉粥样硬化血管的血液流动。该模型通过松开动脉壁和破坏内皮细胞的刺激层来诱导动脉损伤反应，最终导致重塑和刺激性高塑性反应。

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

a-rat-carotid-artery-pressure-controlled-segmental-balloon-injury

Research

JoVE Journal

Medicine

4.9K 次观看.  University of North Carolina at Chapel Hill. 大鼠胡萝卜动脉气球损伤模仿临床血管成形术，以恢复动脉粥样硬化血管的血液流动。该模型通过松开动脉壁和破坏内皮细胞的刺激层来诱导动脉损伤反应，最终导致重塑和刺激性高塑性反应。

视频: A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

压力控制通气诱导小鼠急性肺损伤

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

科研

医学

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 ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

血管球囊损伤和腔内管理在大鼠颈动脉

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

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, ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

大鼠颈动脉球囊损伤模型试验抗血管重建治疗

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

家兔加速动脉粥样硬化模型的研究--髂动脉球囊损伤的方法学观察

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

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 ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

大鼠颈总动脉的相位对比磁共振成像

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

氯化铁诱导的犬颈动脉血栓形成: 一种大的血管损伤动物模型

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...