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>

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

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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

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

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

从金属支架表面使用的腺病毒载体栓通过水解的交联剂血管基因转移

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

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 ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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 ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

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 ...

Transvenous Embolization of Carotid Cavernous Fistula through Inferior Petrosal Sinus with Detachable Coils and Ethylene Vinyl Alcohol Copolymer

Carotid cavernous fistula (CCF) is a rare disease caused by abnormal communications between the internal carotid artery (direct fistula) or meningeal branches of the external carotid artery (indirect fistula) and the cavernous sinus (CS). Trauma is the most common cause of CCF. The clinical presentation of CCF is closely related to the venous drainage pattern. Orbital and neuro-ophthalmological symptoms are the most common clinical presentation of CCF with drainage through the superior ophthalmic vein (SOV). Endovascular embolization by arterial or venous approaches is the most common management of CCF. Transvenous embolization using detachable coils and ethylene-vinyl alcohol copolymer (EVOH) is an alternative method for the treatment of CCF. Endovascular embolization offers different options to treat CCF by minimally invasive approach decreasing morbidity and residual fistulas. The purpose of this article is to report our treatment experiences via the inferior petrosal sinus (IPS), and immediate-term outcomes of endovascular embolization of CCF by using detachable coils and EVOH.

The purpose of the article is to present our experience in endovascular treatment via the inferior petrosal sinus of the carotid cavernous fistula with detachable coils and ethylene-vinyl alcohol copolymer.

Carotid cavernous fistula (CCF) is a rare disease caused by abnormal communications between the internal carotid artery (direct fistula) or meningeal ...

Complete and Partial Resuscitative Endovascular Balloon Occlusion of the Aorta for Hemorrhagic Shock

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for hemorrhage control. With the advent of purpose-built devices, REBOA has become increasingly common in civilian trauma and acute care settings. Currently available REBOA catheters were designed as complete aortic occlusion devices. However, the therapeutic window for complete aortic occlusion is time-limited due to ischemia-reperfusion injury. The partial procedure allows blood flow past the level of occlusion while maintaining targeted proximal pressure, which has been shown to reduce distal ischemia and adjunctive resuscitation requirements in preclinical studies with prolonged occlusion times as compared to traditional complete occlusion.
pREBOA-PRO is the first catheter designed to enable partial and complete aortic occlusion and is currently in limited market release at seven Level I trauma centers in North America. This paper will focus on procedural considerations for REBOA, including patient selection criteria and a comparison of complete and partial aortic occlusion in a simulator, along with highlighting critical steps to improve clinical outcomes. Additionally, this paper reviews a contrast-enhanced CT scan from a trauma patient that shows distal perfusion after 2 h of partial aortic occlusion using this newly designed catheter and discusses representative results from the limited market release to highlight the profound effect of technological innovation on outcomes in vascular emergencies.

A commercial catheter was designed to facilitate true partial resuscitative endovascular balloon occlusion of the aorta (REBOA) and address the complications associated with complete aortic occlusion. Initial clinical reports indicate that partial REBOA improves transition to reperfusion, reduction of distal ischemia, and extension of safe occlusion time compared to complete occlusion.

完全和部分复苏性主动脉血管内球囊闭塞治疗出血性休克

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.