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。

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 a...

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 border="0" cellpadding="0" cellspacing="0" style="width:888px;" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Disposables</td>
			<td style="width:181px;"></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">3mL syringe with 24G needle</td>
			<td>Becton Dickinson</td>
			<td>309571</td>
			<td style="width:199px;">2x for gene transfer surgery; 3x for harvest surgery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">1mL syringe with 27G needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td style="width:199px;">6x for gene transfer surgery; 1x for harvest surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">20mL syringe, luer lock</td>
			<td>Nipro Medical Corp</td>
			<td>JD+20L</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheters, 24G x 3/4&#34;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">19G needle</td>
			<td>Becton Dickinson</td>
			<td>305187</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">21G needle</td>
			<td>Becton Dickinson</td>
			<td>305165</td>
			<td style="width:199px;">For 20 mL syringe of saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Gauze 4&#34; x 4&#34;</td>
			<td>Dynarex</td>
			<td style="width:149px;">3242</td>
			<td style="width:199px;">~10-15 per surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-244</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-182</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8648P</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>398A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alcohol swabs</td>
			<td>Covidien Ltd.</td>
			<td>6818</td>
			<td style="width:199px;">For placement of I.V. line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheter plug</td>
			<td>Vetoquinol</td>
			<td>411498</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Ketamine HCl, 100 mg/mL</td>
			<td>Vedco Inc.</td>
			<td>05098916106</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Xylazine, 100 mg/mL</td>
			<td>Akorn Inc.</td>
			<td>4821</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Lidocaine HCl, 2%</td>
			<td>Pfizer</td>
			<td>00409427702</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Bupivacaine HCl, 0.5%</td>
			<td>Pfizer</td>
			<td>00409161050</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Beuthanasia D-Special</td>
			<td>Intervet Inc.</td>
			<td>NDC 00061047305</td>
			<td style="width:199px;">Harvest surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Buprenorphine HCl, 0.3 mg/mL&#160;</td>
			<td>Patterson Veterinary</td>
			<td>12496075705</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Saline IV bag, 0.9% sodium chloride</td>
			<td>Baxter</td>
			<td>2B1309</td>
			<td style="width:199px;">2x for gene transfer surgery; can use vial of sterile saline in place of one</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Heparin&#160; (5000 U/mL)</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-047-10</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Fentanyl patch, 25 mcg/hr&#160;</td>
			<td>Apotex Corp.</td>
			<td>NDC 60505-7006-2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:359px;">Gene transfer vector</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;">Dilute 350 &#181;L per artery; 2 x 1011 vp/mL for adenovirus; gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Instruments</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Metzenbaum needle holder 7&#34; straight</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7900</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Operating scissors 6.5&#34; straight blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6828</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Needle holder /w suture scissors</td>
			<td>Miltex</td>
			<td style="width:149px;">8-14-IMC</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo needle holder, 5.75&#34; straight with lock</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6412</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Stevens scissors 4.25&#34; curved blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5943</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alm retractor 4&#34; 4X4 5mm blunt prongs</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6514</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Backhaus towel clamp 3.5&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">4x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Micro clip setting forceps 4.75&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6496</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Micro vascular clips, 11 mm</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">2x for gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surg-I-Loop</td>
			<td>Scanlan International</td>
			<td style="width:149px;">1001-81M</td>
			<td style="width:199px;">5 cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Bonaccolto forceps, 4&#8221; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5210</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Dumont #3 forceps Inox tip size .17 X .10mm</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5042</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Graefe forceps, 4&#8221; (10 cm) long serrated straight, 0.8mm tip</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5280</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; straight, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7110</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; curved, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7111</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Jacobson mosquito forceps 5&#34; curved extra delicate, 0.9 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7117</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Kantrowitz forceps, 7.25&#34; 90 degree delicate, 1.7 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7305</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Tissue forceps 5&#34;, 1X2 teeth, 2 mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-8162</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11092-12</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Adson forceps, 12 cm, serrated, straight</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11006-12</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery handpiece and electrode</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">HPAC-1; R-F11</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Suite Equipment</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Circulating warm water blanket and pump</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Forced air warming unit</td>
			<td>3M</td>
			<td style="width:149px;">Bair Hugger Model 505</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">IV infusion pump</td>
			<td>Heska</td>
			<td style="width:149px;">Vet IV 2.2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane vaporizer and scavenger</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary multi-parameter monitor</td>
			<td>Surgivet</td>
			<td style="width:149px;">Surgivet Advisor</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery unit</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">MV-9</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical microscope</td>
			<td style="width:181px;">D.F. Vasconcellos</td>
			<td style="width:149px;">M900</td>
			<td style="width:199px;">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...

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

Research

JoVE Journal

Medicine

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

9.0K 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

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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

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

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

培养一个精密的显微外科技术在大鼠颈总动脉，颈外静脉移植:介入

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

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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

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

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

颈动脉支架植入再狭窄的研究的小鼠模型

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

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

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

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

成年小鼠静脉高压型号:颈总动脉，以颈外静脉吻合。

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

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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, ...