我们感谢 AdVec, 公司允许使用 HDAd 试剂, 朱莉娅 Feyk 为行政协助, 和比较医学兽医服务部的外科咨询和支持。这项工作得到了 HL114541 和小约翰. 洛克慈善信托基金的支持。

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</ol>

作者没有什么可透露的。

外科技术的某些方面值得特别注意。通过仔细解剖, 充分暴露和动员颈总动脉, 将促进基因转移和 arteriotomy 修复。然而, 在解剖过程中, 应尽量减少对颈动脉的直接操纵, 以防止血管痉挛。此外, 在动脉旁的任何出血应以纱布轻压停止, 淤血血液应立即清除, 用生理盐水冲洗区域。同样重要的是避免损害迷走神经, 这是平行于颈总动脉。修剪计划 arteriotomy 区域中的膜将有助于操作人员执行干净和功能 arteriotomy 并修复 arteriotomy。最后, 必须确定和安全结扎的颈总动脉分支, 以防止从颈动脉腔漏出的载体。
还有一些关键方面的载体输液和 arteriotomy 修复。在载体输液过程中, 扩张颈动脉对生理或稍大口径的作用是很重要的, 以实现有效的转导。在修复 arteriotomy 时, 缝合线应尽可能接近 arteriotomy 的边缘, 并且要干净地进入和退出腔体, 而不是通过容器壁轴向传递。如果只把血管壁的外层拉在一起, 是因为缝线通过血管壁通过轴向通过, 而不是通过壁径向穿过墙壁进入腔内, 那么在内膜中就会留下空隙, 血栓形成的风险会增加。如果缝合线的间距太大, 或者如果缝合线过紧, 在 arteriotomy 部位会产生组织褶皱。这些褶皱破坏正常的层流血流, 也增加血栓的风险。保持一致的流动特性对于实验动物模型 (g.)、动脉粥样硬化) 是很重要的, 因为改变的流动可能有助于疾病进程30。
新的操作员经常在收获时遇到血栓动脉。腔内血栓形成是一种破坏性的并发症, 使动脉无法作为实验样本。为了防止血栓形成, 我们在基因转移之前例行地管理 IV 肝素。如上文所述, arteriotomy 的仔细关闭也防止血栓形成。肝素剂量可以增加, 以防止血栓形成, 或阿司匹林可以给术后。然而, 较高剂量的肝素也会增加出血并发症的可能性, 阿司匹林可能干扰实验终点, 特别是如果炎症正在研究中。因此, 最好把重点放在改进的外科技术上, 作为预防血栓形成的手段。
如果操作过于剧烈, 颈动脉可能会痉挛, 这也可能导致血栓形成, 减少流量。如果遇到血管痉挛, 可以通过局部罂粟碱的应用来缓解。当血管的收成计划超过 2-3 天后转导, 和血栓形成是一个关注, 经皮超声可用于无创性评估血管通畅。血栓动脉的发现可能导致安乐死, 以节省住房费用。其他动物也可以迅速登记, 以填充实验组。与此方法相关的围术后和干预后死亡率应 < 1% (i. e), 与在正常家兔上进行的其他手术相关的死亡率不同, 这与死亡有关31。
在操作者对手术协议的技术方面感到满意后, 需要验证, 使用上文第节中所述的具有代表性结果的方法, 进行有效的基因转移到内皮细胞的能力。如果出现可重现基因转移的问题, 罪魁祸首可能在两个领域之一。当血管被隔离, 血液从腔内冲洗后, 去除 DMEM 洗涤缓冲器, 使注入的载体溶液在转导过程中不被稀释是很重要的。另一个重要的方面是扩张血管的生理或稍大口径的媒介输液。因为, 根据我们的经验, 在20分钟的输注过程中, 未完全扩张或不保持扩张的颈动脉有很低的转基因表达, 有可能是动脉扩张到生理口径改善转导效率。然而, 我们从未有系统地研究过这一点。在生理水平以上增加输液压力有可能提高转导效率, 同时也能使血管介质中细胞传导更高水平。然而, 血管内皮屏障的破坏可能会损害血管并增加血栓形成的风险。如果血管在整个20分钟的向量潜伏期内不保持扩张, 则可能是该载体从颈总动脉的分支处漏出。在解剖时要小心, 以识别和结扎所有分支, 以防止从颈动脉腔漏出的媒介。
这种方法有几个与兔子使用有关的限制。兔子比其他大动物 (狗, 猪, 绵羊) 更便宜的房子和饲料;然而, 购买、住宿和喂养兔子的费用比老鼠和老鼠要大得多。手术室设施和对兔子手术的管理要求也远比啮齿目动物更广泛。此外, 有效执行手术基因转移协议需要大量的技术专长。这些专门知识可以由没有正式外科训练的操作员获得。我们小组中至少有2人 (包括本手稿的主要作者) 已经学会了手术技巧并有效地应用了这些技术。然而, 在操作员能够开始生成高质量数据之前, 需要细致的训练和对操作员的基因转移效率和重现性 (如代表性结果部分所述) 进行仔细的验证。
转基因表达的禁闭几乎完全与内皮与此方法29,32,33,34,35,36,37是有用的, 它允许调查内皮细胞中的转基因效应, 以及测量内皮细胞中顺式作用的 DNA 调控序列的活性。少量的血管外膜或内侧细胞可能转基因;然而, 这种方法无法有效地传感器 nonendothelial 细胞, 阻碍了该方法在其他类型的血管壁细胞 (如平滑肌细胞和巨噬细胞) 的研究中的应用。虽然转基因内皮细胞分泌蛋白的过度表达可以允许调查这些蛋白质对其他血管细胞类型的影响, 但非分泌蛋白在其他血管细胞类型中的作用 (e. g. 受体或涉及信号传导的蛋白质) 不能用这种方法来研究。
作为检测动脉壁靶向基因治疗的手段, 该方法与其他临床前方法有两种主要的不同。首先, 该方法利用兔子模型进行基因治疗的体内测试, 而不是使用更常用的啮齿动物模型8,12,15,38,39。使用家兔可以评估转基因的表达和转基因产品在 outbred 动物模型中的生物学作用, 这比近亲交配啮齿动物更能代表人类遗传多样性。该模型可用于评估正常家兔动脉或兔动脉中的基因治疗, 其动脉病理与病变的人动脉相似。兔动脉比啮齿动物动脉更接近人动脉, 并提供比啮齿动物动脉更大的分析组织。第二个主要区别是, 该方法采用手术方法将转基因载体传递到血管内皮。体细胞基因治疗往往是系统地提供, 最频繁的目标是肝脏的目的是改变血浆水平的转基因蛋白25,40。该方法以血管内皮为靶点, 在局部提供治疗性转基因产品, 其峰值浓度集中在动脉壁内, 正是血管疾病治疗所需要的。通过在本地提供转基因, 该方法也消除了转基因产品的系统性副作用。其他组正在开发方法, 使用肽, 抗体, 或其他衣壳修改, 以系统地注入的媒介, 以健康或病变的内皮, 提供局部血管基因治疗41,42,43. 然而, 这些目标方法仍在开发中, 而且由于大量的系统转导而变得复杂, 特别是在肝脏42、43、44。我们的手术方法提供了一个精确和有效的介绍转基因血管内皮与最小, 如果任何转导在其他地方。1该方法还是一种更方便、更高效、更高的全平均 (与细菌线转基因或系统注射内皮靶向载体相比), 用于检测大血管中 DNA 调控序列的活性。内皮, 用于检测动脉壁的转基因蛋白功能, 以及用于检测血管壁靶向基因治疗。
这种方法允许调查任何转基因的生物学作用, 当它在大动脉内皮表达。如果修改以表达功能损失的试剂, 如显性负受体或短发夹 RNA, 它将允许调查内源性内皮蛋白和信号通路的作用。该方法还可用于测量大动脉内皮细胞中任何顺作用 DNA 序列的转录活性5,6,7。此外, 该方法允许检测任何类型的基因转移载体, 以提高效率和安全性时, 在当地提供给内皮, 并将揭示的能力, 以实现持久的转基因表达。最后, 该方法允许开发和测试的载体和转基因的组合, 旨在提供血管壁靶向基因治疗。该方法可以作为筛选工具, 以确定可能的治疗基因-未来-将交付到所有大动脉内皮 (或所有病变的大动脉) 通过经皮注射血管壁靶向载体。由于人类对腺病毒5型45的预先存在免疫, 在临床应用中使用腺病毒型5的载体将具有挑战性。腺病毒5衣壳的工程, 使用替代腺病毒血清型46, 或使用不免疫的载体, 如 AAV 可能需要把血管基因治疗的临床。然而, 作为一个实验工具, 我们不知道任何媒介, 可以竞争与帮助者依赖腺病毒5在实现有效和持久的转基因表达血管1。

该方法的目的是在兔颈总动脉内皮中引入一种转基因。引入转基因可以评估转基因产品在正常动脉和兔模型的人类动脉疾病的生物学作用。在疾病模型中过度表达转基因可以揭示转基因 (及其蛋白产品) 是否显示为治疗剂1,2,3,4。纳入转基因表达式卡带中的顺式调节元素, 可以评估这些元素在动脉内皮体内5,6中的活动。了解特定的顺行调节元素的活动, 可用于设计更主动的表达盒, 并探讨在大动脉内皮细胞的基因调节机制在体内7。
家兔是人类血管生理学和疾病各个方面的重要模型。家兔与人类共有许多血管特征。例如, 基线血液学值, 止血调节和血管纵向张力是相似的兔子和人类的8。兔血管疾病模型复制了许多人类疾病的关键特征, 包括: 动脉瘤 (类似的几何和流特征)9, 血管痉挛 (类似的反应血管内治疗)10,11, 和动脉粥样硬化 (内膜斑块具有类似的功能, 包括一个核心富含脂质, 巨噬细胞, 和平滑的肌肉单元在纤维帽)12,13。因此, 兔子模型已经发展为许多血管疾病, 如血栓, 血管痉挛, 动脉瘤, 糖尿病, 血管移植狭窄, 动脉粥样硬化8,13,14, 15,16。
对于研究人员在动物模型中选择血管生理学和疾病, 兔有几个优点。与啮齿类动物相比, 较大的家兔血管允许手术操作更容易, 血管内装置的使用, 以及大量的组织进行定量测量。兔子比啮齿目动物的 phylogenetically 更接近灵长类动物17, outbred 兔的遗传多样性更接近人类的遗传变异。遗传多样性对临床前研究特别重要, 因为它们的性质-目的是开发可应用于基因多样化的人类群体的治疗方法。与许多, 如果不是所有其他模型物种, 兔子基因是容易克隆或合成, 因为兔子基因组已测序高覆盖率 (7.48x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway？db=oryCun2]。与其他大型动物模型 (如狗, 猪, 或绵羊) 相比, 兔子购买和房子相对便宜, 他们更容易繁殖和处理。家兔特定的血管疾病模型各有其自身的优势和不足, 作为人类疾病的模型, 超出了本手稿的范围8,12,18。研究人员应审查这些优点和缺点, 以确定是否兔子是回答特定实验问题的最佳模型。
将脱氧核糖核酸 (DNA) 调控序列引入内皮细胞在体内可以在复杂的生理环境中对这些序列的活动进行调查。体外对转染内皮细胞的研究有助于对 DNA 调控序列进行初步评估;但是, 当研究重复在体内5,19,20时, 组织培养模型中的表达水平有时不会重现。体外系统也可用于探索蛋白质信号和内皮生理学的基本途径以及培养的血管细胞之间的沟通;然而, 在体内系统6、20中, 最好研究由相邻血管细胞或免疫系统复杂群体影响的更复杂的通路或调节网络。本文介绍的方法提供了一个平台, 探讨在血管内皮细胞内的转基因表达调控, 无论是否有疾病。体内系统还允许对生理和病理细胞串扰进行调查, 并确定免疫系统对基因表达式6的调节作用。
细菌线转基因 (尤其是小鼠) 是一种将转基因表达定向到内皮细胞的替代方法。这种方法可以提供终生的转基因表达, 以特定的启动子或监管区域介导的内皮靶向21,22。然而, 转基因小鼠的世代是费时和昂贵的, 必须经常测试一些转基因线, 以确保转基因的靶向所需的细胞类型和取得适当的转基因表达水平, 和实验结果在小鼠系统可能是应变依赖性。具有内皮靶向转基因的小鼠转基因模型有许多优点: 没有必要对每一个实验动物进行手术, 以达到转基因, 实验鼠可以与许多其他可用的转基因小鼠一起繁殖。为了测试基因和表型的相互作用, 有广泛的选择抗体, 与小鼠的蛋白质反应, 促进表征的表型。然而, 通过细菌线将转基因定向到内皮细胞, 通常会导致整个血管的转基因表达,22使得很难确定转基因产品的作用地点。当转基因产品分泌时尤其如此, 因为在整个血管的血管内皮细胞分泌的转基因产品可能在动物体内的任意数量的部位有生物活性。虽然本手稿中描述的方法需要技术专长和专门设施, 但它的耗时和成本比开发内皮特异的转基因小鼠线要少得多。它允许在大动脉段的内皮细胞中有选择地评估蛋白质的功能, 并且它允许使用对侧的颈动脉作为配对的控制 (消除系统因素, 可以改变在实验动物-例如, 血压或胆固醇水平-作为不受控制的变量)。
基因疗法是治疗血管疾病, 特别是慢性病的一种有希望的方法, 因为单一的应用可以提供持续或可能终生表达的治疗基因23。基因治疗在体细胞基因转移动物模型中的治疗前景已被探索, 通常针对肝脏24,25, 这是一个相对容易的目标, 因为许多血液传播的病毒载体是 hepatotropic 的。然而, 为了对血管疾病产生影响, 针对肝脏的基因治疗必须实现蛋白质的系统性过度表达。这通常需要大剂量的向量, 这可能是有毒的, 甚至是致命的26。此外, 蛋白质系统水平的提高增加了非靶向副作用的风险, 这可能使实验结果的解释复杂化甚至模糊不清。本手稿所描述的针对血管内皮的局部基因治疗可以避免系统性的副作用, 因为注入的载体没有广泛传播到转基因动脉段之外, 而且可以实现局部血管效应, 而无需系统血浆蛋白水平的变化。27此外, 传感器动脉段所需的载体数量远低于实现强健肝传导的需要。随着时间的推移, 来自肝脏的转基因表达被报告为下降, 可能是由于细胞更替, 如果要维持高水平的转基因表达, 需要反复用药。28相比之下, 内皮细胞的低更替率为喂食家兔至少48周提供稳定表达, 在胆固醇喂养家兔的动脉粥样硬化性病变中至少有24周。1,27
为了确定这种基因转移到兔颈总动脉内皮的方法是否合适, 应在特定研究目标的背景下考虑其优缺点 (表 1)。该方法的优点包括: outbred 兔比近交系小鼠更好地代表人类遗传多样性 (对前期工作很重要);兔子提供更大的血管, 便于操作和更多的组织进行分析;该方法能较快地实现转基因小鼠的血管内皮靶向转基因表达;矢量剂量可以很容易地调整为模型可变水平的转基因表达;可对大动脉内皮的特定过程进行研究;局部血管转基因允许同一动物的相对颈动脉作为一种控制, 将系统因素排除为不受控制的变量。缺点包括: 需要特殊设施和专门知识;更少的基因改良的背景, 以实验是在兔子比在小鼠;对兔子和老鼠蛋白的抗体的选择较少 (对于转基因蛋白和其他抗原的 immunodetection, 这在解释实验结果中可能是重要的)。

<table><tbody><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3mL syringe with 24G needle</td><td>Becton Dickinson</td><td>309571</td><td>2x for gene transfer surgery; 3x for harvest surgery</td></tr><tr><td>1mL syringe with 27G needle</td><td>Becton Dickinson</td><td>309623</td><td>6x for gene transfer surgery; 1x for harvest surgery</td></tr><tr><td>20mL syringe, luer lock</td><td>Nipro Medical Corp</td><td>JD+20L</td><td></td></tr><tr><td>Catheters, 24G x 3/4&quot;</td><td>Terumo Medical Products</td><td>SROX2419V</td><td></td></tr><tr><td>19G needle</td><td>Becton Dickinson</td><td>305187</td><td>Gene transfer surgery only</td></tr><tr><td>21G needle</td><td>Becton Dickinson</td><td>305165</td><td>For 20 mL syringe of saline</td></tr><tr><td>Gauze 4&quot; x 4&quot;</td><td>Dynarex</td><td>3242</td><td>~10-15 per surgery</td></tr><tr><td>3-0 silk suture</td><td>Covidien Ltd.</td><td>S-244</td><td></td></tr><tr><td>5-0 silk suture</td><td>Covidien Ltd.</td><td>S-182</td><td>Gene transfer surgery only</td></tr><tr><td>7-0 polypropylene suture</td><td>CP Medical</td><td>8648P</td><td>Gene transfer surgery only</td></tr><tr><td>5-0 polyglycolic acid suture</td><td>CP Medical</td><td>421A</td><td>Gene transfer surgery only</td></tr><tr><td>3-0 polyglycolic acid suture</td><td>CP Medical</td><td>398A</td><td>Gene transfer surgery only</td></tr><tr><td>Alcohol swabs</td><td>Covidien Ltd.</td><td>6818</td><td>For placement of I.V. line</td></tr><tr><td>Catheter plug</td><td>Vetoquinol</td><td>411498</td><td>Gene transfer surgery only</td></tr><tr><td>Ketamine HCl, 100 mg/mL</td><td>Vedco Inc.</td><td>05098916106</td><td></td></tr><tr><td>Xylazine, 100 mg/mL</td><td>Akorn Inc.</td><td>4821</td><td></td></tr><tr><td>Lidocaine HCl, 2%</td><td>Pfizer</td><td>00409427702</td><td></td></tr><tr><td>Bupivacaine HCl, 0.5%</td><td>Pfizer</td><td>00409161050</td><td></td></tr><tr><td>Beuthanasia D-Special</td><td>Intervet Inc.</td><td>NDC 00061047305</td><td>Harvest surgery only</td></tr><tr><td>Buprenorphine HCl, 0.3 mg/mL&amp;nbsp;</td><td>Patterson Veterinary</td><td>12496075705</td><td>Gene transfer surgery only</td></tr><tr><td>Saline IV bag, 0.9% sodium chloride</td><td>Baxter</td><td>2B1309</td><td>2x for gene transfer surgery; can use vial of sterile saline in place of one</td></tr><tr><td>Heparin&amp;nbsp; (5000 U/mL)</td><td>APP Pharmaceuticals</td><td>NDC 63323-047-10</td><td>Gene transfer surgery only</td></tr><tr><td>Fentanyl patch, 25 mcg/hr&amp;nbsp;</td><td>Apotex Corp.</td><td>NDC 60505-7006-2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Gene transfer vector</td><td></td><td></td><td>Dilute 350 &amp;micro;L per artery; 2 x 10&lt;sup&gt;11&lt;/sup&gt; vp/mL for adenovirus; gene transfer surgery only</td></tr><tr><td>&lt;strong&gt;Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metzenbaum needle holder 7&quot; straight</td><td>Roboz</td><td>RS-7900</td><td>Gene transfer surgery only</td></tr><tr><td>Operating scissors 6.5&quot; straight blunt/blunt</td><td>Roboz</td><td>RS-6828</td><td></td></tr><tr><td>Needle holder /w suture scissors</td><td>Miltex</td><td>8-14-IMC</td><td>Gene transfer surgery only</td></tr><tr><td>Castroviejo scissors</td><td>Roboz</td><td>RS-5658</td><td></td></tr><tr><td>Castroviejo needle holder, 5.75&quot; straight with lock</td><td>Roboz</td><td>RS-6412</td><td>Gene transfer surgery only</td></tr><tr><td>Stevens scissors 4.25&quot; curved blunt/blunt</td><td>Roboz</td><td>RS-5943</td><td></td></tr><tr><td>Alm retractor 4&quot; 4X4 5mm blunt prongs</td><td>Roboz</td><td>RS-6514</td><td>2x</td></tr><tr><td>Backhaus towel clamp 3.5&quot;</td><td>Roboz</td><td></td><td>4x</td></tr><tr><td>Micro clip setting forceps 4.75&quot;</td><td>Roboz</td><td>RS-6496</td><td>Gene transfer surgery only</td></tr><tr><td>Micro vascular clips, 11 mm</td><td>Roboz</td><td></td><td>2x for gene transfer surgery only</td></tr><tr><td>Surg-I-Loop</td><td>Scanlan International</td><td>1001-81M</td><td>5 cm length</td></tr><tr><td>Bonaccolto forceps, 4&amp;rdquo; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td><td>Roboz</td><td>RS-5210</td><td></td></tr><tr><td>Dumont #3 forceps Inox tip size .17 X .10mm</td><td>Roboz</td><td>RS-5042</td><td></td></tr><tr><td>Graefe forceps, 4&amp;rdquo; (10 cm) long serrated straight, 0.8mm tip</td><td>Roboz</td><td>RS-5280</td><td></td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; straight, 1.3mm tips</td><td>Roboz</td><td>RS-7110</td><td>2x</td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; curved, 1.3mm tips</td><td>Roboz</td><td>RS-7111</td><td></td></tr><tr><td>Jacobson mosquito forceps 5&quot; curved extra delicate, 0.9 mm tips</td><td>Roboz</td><td>RS-7117</td><td></td></tr><tr><td>Kantrowitz forceps, 7.25&quot; 90 degree delicate, 1.7 mm tips</td><td>Roboz</td><td>RS-7305</td><td></td></tr><tr><td>Tissue forceps 5&quot;, 1X2 teeth, 2 mm tip width</td><td>Roboz</td><td>RS-8162</td><td></td></tr><tr><td>Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td><td>Fine Science Tools</td><td>11092-12</td><td>2x</td></tr><tr><td>Adson forceps, 12 cm, serrated, straight</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Veterinary electrosurgery handpiece and electrode</td><td>MACAN Manufacturing</td><td>HPAC-1; R-F11</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Suite Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Circulating warm water blanket and pump</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Forced air warming unit</td><td>3M</td><td>Bair Hugger Model 505</td><td>Gene transfer surgery only</td></tr><tr><td>IV infusion pump</td><td>Heska</td><td>Vet IV 2.2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane vaporizer and scavenger</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Veterinary multi-parameter monitor</td><td>Surgivet</td><td>Surgivet Advisor</td><td></td></tr><tr><td>Veterinary electrosurgery unit</td><td>MACAN Manufacturing</td><td>MV-9</td><td></td></tr><tr><td>Surgical microscope</td><td>D.F. Vasconcellos</td><td>M900</td><td>Needs ~16x magnification</td></tr></tbody></table>

in vivo gene transfer to the rabbit common carotid artery endothelium

该方法的目的是将转基因技术引入到兔颈总动脉孤立段的内皮细胞中。该方法实现了局灶性内皮选择性转基因, 从而使研究者能够确定内皮表达转基因的生物学作用, 并定量测定大动脉 DNA 序列的体内转录活性。内皮细胞。该方法采用手术隔离兔颈总动脉和 arteriotomy, 将转基因表达的病毒载体传递到动脉腔内。在腔内的载体的短潜伏期, 与随后的流明内容的愿望, 足以实现有效和持久的基因在内皮细胞的表达, 没有可检测的转导或表达以外的孤立的动脉段。该方法允许对正常动脉和人体血管疾病模型中的转基因产品的生物活性进行评估, 同时避免通过将基因传递定向到其他站点 (e. g) 而引起的系统性影响。肝脏) 或通过另一种途径, 通过生殖线转基因向内皮细胞提供遗传构造。这种方法的应用受限于需要熟练的外科医生和麻醉, 设备齐全的手术室, 购买和养兔的费用, 以及在基因转移载体的建设和使用方面的专门知识的需要。该方法获得的结果包括: 与转基因有关的动脉结构、细胞构成、细胞外基质或血管功能的改变;增加或减少动脉炎症;血管细胞凋亡的变化;疾病的进展, 迟缓, 或退化, 如内膜增生或动脉粥样硬化。该方法还允许测量本机和合成 DNA 调控序列在内皮细胞中改变转基因表达的能力, 提供的结果包括: 转基因 mRNA 的水平、转基因蛋白的水平和转基因的水平。酶活性。

为了实现这种方法的信心, 初步实验是必要的, 以确定该操作者获得有效和可再生的基因转移, 以转基因表达主要在腔内内皮细胞。根据我们的经验, 这是最容易评估使用的向量, 表达β-乳糖酶。5-溴-4-氯-3-哚β D-galactopyranoside (X-加仑) 染色的颈总段清除后3天的载体输液, 以及测量β-乳糖酶 mRNA (信使 RNA) 与定量反向转录聚合酶链反应 (qRT PCR), 将揭示效率, 重现性和转基因细胞的位置。对于研究转基因表达的影响或测量顺式转录元素活性的实验, 我们通常将转基因 mRNA 作为基因转移的水平和重现性的初步指标来衡量。
我们实验室的一个新操作员试图通过传感兔颈总动脉与腺病毒载体 (2 x 1011 vp/mL) 来建立对这种方法的熟练程度, 表达β-乳糖酶转基因, 由巨细胞病驱动 (CMV)。促进。转基因动脉3天后被收割, 并被横切成段。各自的段然后被切开开放轴向或左作为原封圆环。所有的片段都是 X-加仑染色的离心管。经轴向切开的段被放置在水平面上, 腔面表面最大地暴露, 使用针脚以防止段卷曲。En 面部腔内表面的图像显示 X-gal (图 3A和3B) 的强健内皮染色。完整的颈动脉环内腔表面的轴向图像显示 X-gal 仅在腔内表面 (图 3C和3D) 染色。经轴向打开的段被加工成石蜡切片, 用苏木精和血红或核快红色进行反染色。尽管血管外膜层中有少量染色(图 3E和3F), 但图像在内皮中主要显示 X-gal 染色。血管外膜细胞的传导通过 arteriotomy 部位的渗漏或通过小枝的渗漏通过侧支结扎的部位而发生。29
在一个单独的实验中, 一个新的操作员试图建立熟练程度达到可再生的基因表达水平, 作为试验的前奏, 旨在调查转基因的生物活动。家兔颈总动脉的转基因与帮助依赖腺病毒 (2 x 1011 vp/毫升), 其中包括一个蛋白 A I (HDAdApoAI) 或 IL-10 (HDAdIL10) 转基因, 都在控制的巨细胞病毒启动子。转基因动脉在转导后3天内被收割, 横截成段。RNA 是从血管片段提取, 和 IL-10 mRNA 表达的 qRT PCR 量化, 与规范化的甘油 3-磷酸脱氢酶 (GAPDH) mrna 在同一提取物 (图 4)。在 HDAdIL10-transduced 动脉中, 只有1的6动脉有一个非常低, 但可检测到的蛋白 I mRNA 信号。转基因与 HDAdApoAI 血管转基因的血管, 其平均表达为700倍。HDAdApoAI 转基因动脉内内源性 IL-10 mRNA 水平较低, HDAdIL10-transduced 动脉平均表达增加6倍。值得注意的是, 在转导效率和转基因表达方面有相当可观的动脉内和间的变异性, 如图 3和4中所示。我们发现这种变化, 即使有经验丰富的运营商。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56982/56982fig1.jpg"> 图 1.颈总动脉 Arteriotomy.细钳用于掌握颈动脉膜, 并将上行牵引应用于动脉。这种机动扩大了血管腔, 并产生一个垂直的表面, 其中插入弯曲的19G 针, 从而减少了刺穿动脉后壁的风险。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56982/56982fig2.jpg"> 图 2.颈总 arteriotomy 闭合。arteriotomy 用7-0 聚丙烯缝合闭合, 使用 X 型。针和缝合的第一个通行证进入 arteriotomy (站点 1) 右下角的动脉腔, 并退出左下角的流明 (站点 2)。针和缝线, 然后穿过 arteriotomy 和重新进入流明在右上 (网站 3)。然后, 针头从左上方的腔口流出 (4 点)。在缝合两端的温和牵引关闭 arteriotomy。缝合结束 (从站点1和站点4离开) 被栓与2平方结。灰色圆圈表示穿过容器壁的缝线的位置。实心蓝线表示在血管壁外缝合的区域。点蓝线表示缝线在腔内的区域。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56982/56982fig3.jpg"> 图 3.有效的内皮转基因表达.兔颈总动脉转基因与腺病毒载体表达β乳糖酶转基因和收获3天后。转基因动脉段的 X-加仑染色, 无论是完整的环或后被打开的轴向切割。(A-B)颈动脉环的腔内表面图像, 轴向切开。(C-D)对完整的颈动脉环的腔内空间进行轴向查看。(E-F)X-加仑染色, 石蜡嵌入颈动脉段切片和反染色的任何 (E) 苏木精和血红素或 (F) 核快红色。刻度条 = 100 µm。I = 内膜;M = 媒体;和 A = 膜。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56982/56982fig4.jpg"> 图 4.转基因 mRNA 表达的量化.家兔颈总动脉的转基因与帮助依赖腺病毒表达的是一 HDAdApoAI 或 IL-10 (HDAdIL10) 控制下的巨细胞病毒启动子。3天后, 动脉被收割。(A) IL-10 和 (B) 的 mrna 表达式由 qRT PCR 量化, 规范化为 GAPDH mRNA 在同一动脉, 并表示为任意单位 (AU)。条形指示平均值;P值来自秩和测试。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>优势</td>
			<td>缺点</td>
		</tr>
<tr>
<td rowspan="5">与啮齿动物模型相比</td>
			<td>更接近 phylogenetically 灵长类动物</td>
			<td>更昂贵的购买和房子</td>
		</tr>
<tr>
<td>更大的遗传多样性, 缓解临床翻译</td>
			<td>更难繁殖和处理</td>
		</tr>
<tr>
<td>较大的血管允许更容易的手术操作, 并提供更多的组织进行定量分析</td>
			<td>更广泛的监管要求</td>
		</tr>
<tr>
<td>允许使用为人类设计的血管内装置</td>
			<td>更少的基因改造背景</td>
		</tr>
<tr>
<td></td>
			<td>对兔蛋白抗体的不广泛选择</td>
		</tr>
<tr>
<td rowspan="2">与较大的动物模型相比</td>
			<td>相对便宜购买和房子</td>
			<td>可能比其他大型动物模型对某些血管疾病的临床相关程度更低</td>
		</tr>
<tr>
<td>易于繁殖和处理</td>
			<td></td>
		</tr>
<tr>
<td rowspan="5">与细菌线转基因相比</td>
			<td>仅在大动脉中表达的转基因;转基因在兴趣点的作用可以确定</td>
			<td>方法在非内皮细胞中的应用是困难的</td>
		</tr>
<tr>
<td>可将对侧颈动脉作为配对对照;消除系统参数 (e. g, 血压, 胆固醇水平) 作为不受控制的变量</td>
			<td>手术室和外科专业需要;核心设施可能不可用</td>
		</tr>
<tr>
<td>提高大血管内皮细胞 DNA 调控序列活性的高通量</td>
			<td>转基因不能表达在大多数血管床上</td>
		</tr>
<tr>
<td>可能更快, 成本更低</td>
			<td></td>
		</tr>
<tr>
<td>系统暴露于转基因蛋白质不太可能-最小化目标效应</td>
			<td></td>
		</tr>
<tr>
<td rowspan="5">与系统基因治疗方法相比 (e。经外周静脉注射的肝转导)</td>
			<td>更稳定的转基因表达比全身 (肝) 基因治疗</td>
			<td>载体传递需要手术干预</td>
		</tr>
<tr>
<td>在动脉壁上表达的转基因, 允许本地提供高水平的转基因蛋白</td>
			<td>手术室和外科专业需要</td>
		</tr>
<tr>
<td>系统暴露于转基因蛋白质不太可能-最小化目标效应</td>
			<td>治疗限于专门针对干预的动脉;不治疗系统性因素 (e. g. 脂类)</td>
		</tr>
<tr>
<td>可将对侧颈动脉作为配对对照;消除系统参数 (如血压、胆固醇水平) 为不受控制的变量</td>
			<td></td>
		</tr>
<tr>
<td>需要更低的矢量剂量</td>
			<td></td>
		</tr>
</tbody></table>表1。兔颈总动脉内皮选择性基因转移模型的优缺点。

Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

该方法将转基因技术引入兔颈动脉内皮细胞。转基因的引入可以评估转基因产品在正常动脉或疾病模型中的生物学作用。该方法对测定 DNA 调控序列的活性也有很重要的意义。

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 ...

in-vivo-gene-transfer-to-the-rabbit-common-carotid-artery-endothelium

Nanyang Technological University

Research

JoVE Journal

Medicine

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

9.3K 次观看.  University of Washington. 该方法将转基因技术引入兔颈动脉内皮细胞。转基因的引入可以评估转基因产品在正常动脉或疾病模型中的生物学作用。该方法对测定 DNA 调控序列的活性也有很重要的意义。

视频: In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

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

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

科研

医学

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

缺血性心力衰竭在临床前模型的转基因

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

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 ...

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 ...

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, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

糖尿病家兔欣德肢体缺血的临床前模型

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

通过 Cuff 技术将组织工程血管移植物植入小鼠颈动脉

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

生物工程

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model

Source: Nishio, H., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
This video demonstrates the interposition of the rabbit carotid artery with the jugular vein to induce venous intimal hyperplasia by subjecting the veins to arterial pressure. This model will help investigate molecular and genetic interventions to attenuate the progression of intimal hyperplasia after revascularization surgeries.

颈动脉和颈静脉插入:一种在兔模型中诱导静脉内膜增生的技术

Source: Nishio, H., et al. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under ...