This work was supported in part by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program Merit Review Award I01-BX002336, the National Institute of Health grant R56-HL095498, as well as with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT.

获得批准相应的机构动物护理和使用委员会。 1.麻醉和手术前的程序<ol><li>麻醉雄性C57BL / 6冰，8周龄，用汽化3％异氟醚和0.8升/分钟的氧气给予到丙烯酸感应腔室。 </li><li>确认缺乏反应脚趾捏足够的麻醉。通过连续吸入3％异氟烷 - 定位在动作表鼠标仰卧和定位硅氧烷掩模递送汽化2。 </li><li>腹部用化学脱毛霜去除颈部腹侧头发更低。 </li><li>之前AVF手术进行多普勒超声检查在感兴趣的领域，以记录动脉和静脉流动和血管直径的基线特征。1,2 </li><li>附加1ml注射器向地下25针和与所需药物加载注射器。弯针60度角约4从需要毫米乐一角。抓住一个弯针架在地下25针。 </li></ol> 2.手术过程<ol><li>准备切口部位有局部消毒剂和应用手术单。使用无菌手套和工具，以在整个手术保持无菌操作。 </li><li>使一个中线腹部切口用解剖刀从下肝脏边缘水平延伸到刚好耻骨之上。 </li><li>插入的牵开器以及从向右侧腹腔剔骨所有肠子。裹肠纱布用盐水浸湿。解剖连接复古腹膜和较低的结肠获得主动脉和下腔静脉的全视角膜。 </li><li>从周围组织解剖肾下主动脉和下腔静脉，对近端和远端夹紧准备。 </li><li>放置在这两个近端主动脉和在刚刚左肾静脉水平以下近端IVC单个显微剪辑。将第二显微剪辑跨两个为Distal主动脉和远侧IVC。 </li><li>把握结缔组织围绕主动脉和内侧旋转，使得主动脉的背表面稍微露出的动脉穿刺，如先前所述。1 </li><li>很快暴露穿刺部位。穿刺部位将在血管，从左肾静脉到主动脉分叉的距离的大约四分之三的尾方面。保持主动脉在一个旋转位置用左手，解剖主动脉的左侧缘，以便有充足的曝光，以允许用右手穿刺。要小心，不要主动脉和下腔静脉之间的解剖。 </li><li>保持在一个旋转位置的主动脉，并使用地下25针带有附加1ml注射器含有药物溶液通过刺穿主动脉进入下腔静脉。 （ 图1A） </li><li>注入药液 - 利用左手（100 200微升）。针可以通过扩张和薄可见下腔静脉壁当透明药液置换静脉血出下腔静脉（ 图1B，C）的。保持静止并保持在适当的位置的针15分钟。 </li><li>移除远侧显微剪辑脱夹具仅远端主动脉和远侧IVC。 </li><li>取出针再涂向上拉相邻复古腹膜组织主动脉的穿刺部位。 </li><li>取出近端显微剪辑去夹紧近端主动脉和近端IVC。在去夹紧，动脉血观察流入下腔静脉，而不是深静脉血流。保持覆盖所述穿刺孔进行1分钟。 </li><li>止血确认通过观察30秒，而没有压缩后，返回肠子到其自然的位置，并根据您批准的动物协议的连续缝合关闭的腹部。 </li></ol> 3.手术后的程序<ol><li>腹部闭合后，停止ANESThesia。应用术后护理包括镇痛和伤口护理按照机构动物护理和使用委员会建议的说明。镇痛我们使用丁丙诺啡在0.1毫克/千克intrasmuscularly以下的外科手术24小时，每12小时。 </li><li>在手术后的第一天，进行多普勒超声检查，以确认AVF通畅。此外，衡量其他船只和串行流量特性，比较从术前基线值的变化。1,2 </li></ol><img alt="图1" src="/files/ftp_upload/53905/53905fig1.jpg" /> 图1（a）执行照片AVF手术中显示管腔内交货。夹紧近端和远端主动脉，以及通过施加显微剪辑IVC。利用地下25针含附加注射器药液通过穿刺进入主动脉下腔静脉。（B）几点措施她电源图像输液前刺破下腔静脉（放大4倍）。针尖通过深色静脉血遮蔽。黄色箭头表示输液后刺破下腔静脉的墙到墙直径IVC。（C）更高的功率图（放大4倍）的。针尖（黑色箭头），可通过轻轻扩张和薄墙下腔静脉（黄色箭头）作为透明药材溶液中可以看出置换静脉血。

<ol>
	<li>Yamamoto, K., Li, X., Shu, C., Miyata, T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Aspects+of+the+Mouse+Aortocaval+Fistula.">Technical Aspects of the Mouse Aortocaval Fistula.</a> J. Vis. Exp. (77), e50449 (2013).</li><li>Yamamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+aortocaval+fistula+recapitulates+human+arteriovenous+fistula+maturation.">The mouse aortocaval fistula recapitulates human arteriovenous fistula maturation.</a> Am. J. Physiol.: Heart Circ. Physiol. 305 (12), H1718-H1725 (2013).</li><li>Escobar-Ch&#225;vez, J. J., L&#242;pez-Cervantes, M., Na&#239;k, A., Kalia, Y. N., Quintanar-Guerrero, D., Ganem-Quintanar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+thermo-reversible+pluronic+F-127+gels+in+pharmaceutical+formulations.">Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations.</a> J. Pharm. Pharm. Sci. 9 (3), 339-358 (2006).</li><li>Almeida, H., Amaral, M. H., Lob&#227;o, P., Lobo, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluronic&#174;+F-127+and+Pluronic+Lecithin+Organogel+(PLO):+main+features+and+their+applications+in+topical+and+transdermal+administration+of+drugs.">Pluronic&#174; F-127 and Pluronic Lecithin Organogel (PLO): main features and their applications in topical and transdermal administration of drugs.</a> J. Pharm. Pharm. Sci. 15 (4), 592-605 (2012).</li><li>Karper, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4+is+involved+in+human+and+mouse+vein+graft+remodeling,+and+local+gene+silencing+reduces+vein+graft+disease+in+hypercholesterolemic+APOE*3Leiden+mice.">Toll-like receptor 4 is involved in human and mouse vein graft remodeling, and local gene silencing reduces vein graft disease in hypercholesterolemic APOE*3Leiden mice.</a> Arterioscler., Thromb., Vasc. Biol. 31 (5), 1033-1040 (2011).</li><li>Redmond, E. M., Hamm, K., Cullen, J. P., Hatch, E., Cahill, P. A., Morrow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+patched-1+prevents+injury-induced+neointimal+hyperplasia.">Inhibition of patched-1 prevents injury-induced neointimal hyperplasia.</a> Arterioscler., Thromb., Vasc. Biol. 33 (8), 1960-1964 (2013).</li><li>Wong, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin+type-B+receptor+4+activation+reduces+neointimal+hyperplasia+in+human+saphenous+vein+in+vitro.">Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro.</a> J. Vasc. Surg. , (2014).</li><li>Matsumoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinating+virus+of+Japan-liposome-mediated+gene+transfer+of+endothelial+cell+nitric+oxide+synthase+inhibits+intimal+hyperplasia+of+canine+vein+grafts+under+conditions+of+poor+runoff.">Hemagglutinating virus of Japan-liposome-mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff.</a> J. Vasc. Surg. 27 (1), 135-144 (1998).</li><li>Petrofski, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+aortocoronary+saphenous+vein+grafts+in+a+large+animal+model+of+intimal+hyperplasia.">Gene delivery to aortocoronary saphenous vein grafts in a large animal model of intimal hyperplasia.</a> J. Thorac. Cardiovasc. Surg. 127 (1), 27-33 (2004).</li><li>Hata, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+phosphatidylinositol+3-kinase+signaling+reduces+intimal+hyperplasia+in+aortocoronary+saphenous+vein+grafts.">Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts.</a> J. Thorac. Cardiovasc. Surg. 129 (6), 1405-1413 (2005).</li><li>Ohta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+gene+transfer+of+endothelial+cell-nitric+oxide+synthase+suppresses+intimal+hyperplasia+of+vein+grafts+in+cholesterol-fed+rabbit:+a+limited+biological+effect+as+a+result+of+the+loss+of+medial+smooth+muscle+cells.">Intraluminal gene transfer of endothelial cell-nitric oxide synthase suppresses intimal hyperplasia of vein grafts in cholesterol-fed rabbit: a limited biological effect as a result of the loss of medial smooth muscle cells.</a> Surg. 131 (6), 644-653 (2002).</li><li>Baldwin, Z. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+vascular+remodeling+induced+by+a+brief+intraluminal+exposure+to+the+recombinant+R7020+strain+of+Herpes+simplex-1.">Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1.</a> J. Vasc. Surg. 41 (1), 115-121 (2005).</li><li>Eslami, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+in+situ+veins:+differential+effects+of+adenovirus+and+adeno-associated+viral+vectors.">Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors.</a> J. Vasc. Surg. 31 (6), 1149-1159 (2000).</li><li>Globerman, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transgene+expression+from+naked+DNA+delivered+into+an+arterio-venous+fistula+model+for+kidney+dialysis.">Efficient transgene expression from naked DNA delivered into an arterio-venous fistula model for kidney dialysis.</a> J. Gene Med. 13 (11), 611-621 (2011).</li><li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adventitial+transduction+of+lentivirus-shRNA-VEGF-A+in+arteriovenous+fistula+reduces+venous+stenosis+formation.">Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation.</a> Kidney Int. 85 (2), 289-306 (2014).</li><li>Brahmbhatt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Iex-1+in+the+pathogenesis+of+venous+neointimal+hyperplasia+associated+with+hemodialysis+arteriovenous+fistula.">The role of Iex-1 in the pathogenesis of venous neointimal hyperplasia associated with hemodialysis arteriovenous fistula.</a> PLoS ONE. 9 (7), e102542 (2014).</li><li>Karram, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cardiac+hypertrophy+by+a+controlled+reproducible+sutureless+aortocaval+shunt+in+the+mouse.">Induction of cardiac hypertrophy by a controlled reproducible sutureless aortocaval shunt in the mouse.</a> J. Investig. Surg. 18 (6), 325-334 (2005).</li><li>Perry, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+angiotensin-converting+enzyme+does+not+prevent+development+of+cardiac+hypertrophy+or+upregulation+of+angiotensin+II+in+response+to+aortocaval+fistula.">Genetic variation in angiotensin-converting enzyme does not prevent development of cardiac hypertrophy or upregulation of angiotensin II in response to aortocaval fistula.</a> Circ. 103 (7), 1012-1016 (2001).</li><li>Guzman, R. J., Krystkowiak, A., Zarins, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+and+sustained+medial+cell+activation+after+aortocaval+fistula+creation+in+mice.">Early and sustained medial cell activation after aortocaval fistula creation in mice.</a> J. Surg. Res. 108 (1), 112-121 (2002).</li><li>Alexander, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+edifoligide,+an+E2F+transcription+factor+decoy,+for+prevention+of+vein+graft+failure+following+coronary+artery+bypass+graft+surgery:+PREVENT+IV:+a+randomized+controlled+trial.">Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial.</a> JAMA. 294 (19), 2446-2454 (2005).</li><li>Khaleel, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+distention+of+the+saphenous+vein+during+preparation+results+in+increased+markers+of+inflammation:+a+potential+mechanism+for+graft+failure.">High-pressure distention of the saphenous vein during preparation results in increased markers of inflammation: a potential mechanism for graft failure.</a> Ann. thorac. surg. 93 (2), 552-558 (2012).</li></ol>

The authors have nothing to disclose.

鼠AVF模型的这种修改包含在AVF创建时腔内药物递送到静脉内皮。一个AVF通过穿刺用地下25针的红外肾主动脉和延伸穿过相对的主动脉壁进入下腔静脉穿刺，随后的药物溶液注射通过同一针创建。该解决方案是维持内静脉， 即 ，在AVF的静脉肢体，直到去夹紧。什么区别于其他鼠类AVF模型17-19这种模式是缺乏缝合或胶水可能导致管腔狭窄，急性血栓形成，并与分子分析可能的干扰。使用该刺穿主动脉同一针和IVC创建AVF也药物递送到AVF管腔的能力允许以最小的容器操纵简单递送。 有一些关键步骤和点提高该过程的成功率和一致性。公关主动脉腔静脉和oximal夹具被放置在左肾静脉下方。如果有在帧内肾IVC的上部大腰静脉，然后将这些容器下夹紧站点的修改建议通过从腰静脉逆行回到出血，以允许无需稀释药物的有效培养。主动脉腔静脉和远侧夹具被放置在髂分叉的水平，以免可能与远侧主动脉和下腔静脉的穿刺干扰。保持与弯曲缝合针夹持器的地下25针使容器穿刺期间针的最佳方向。止血是通过覆盖与相邻的复古腹膜动脉入孔来实现的。特别注意的是，以提供小心压缩，同时避免AVF闭塞和血栓形成如先前所述。1 基因转移技术的发展提供了在分子水平上，以修改手术效果的潜力。报告描述traluminal基因递送到静脉主要集中在静脉移植失败的有针对性的治疗。相比之下，这种模式允许药物输送的AVF成熟的调制。这种模式的优点包括利用小鼠模型，允许不同的鼠标遗传品系的审查。此外，15分钟腔内照射协议是很容易转移到临床手术中的设置，并可能有助于评估围手术期干预措施，防止JUXTA，吻合口狭窄，人AVF手术一般复杂。从这些初步的结果，我们表明，该方法比传统的外部路由的内皮细胞中产生更多的位点特异性基因表达。 这项研究的潜在限制是对下腔静脉扩张可变压力的作用。下腔静脉段的隔离并不在此小鼠模型中， 即 ，侧分支（腰静脉）未结扎完美。因此，很难精确地MAINT艾因孵化过程中扩张的压力水平一致。虽然IVC轻轻手动输液胀得，胀痛较高的压力可能会导致内膜增生20,21和直接平滑肌细胞受损，11导致干扰分析。它也很难完全定量药物递送的效率，因为一些药物可以输送期间通过腰静脉丢失，以及后穿过近侧IVC夹具去除和潜在通过AVF进入主动脉。最后，该方法允许一些药物的钳除去后全身被释放;药物和站点可能防范系统性释放潮红，但药物洗脱程度的量化的撤离将需要得到证实。 总之，本技术修饰鼠AVF模型较为简单，重现性和使得能够靶向内皮药物递送到小鼠AVF。在腔内jection在AVF创建时在技术上是可行的，相比外膜递送可以成功地改善内皮病毒基因表达。此AVF的药物递送模式将不仅对调节静脉AVF肢成熟的机制解剖的有益形式，同时也为AVF成熟治疗性调节。

鼠aortovenous瘘（AVF）的主动脉和下腔静脉（IVC）之间穿刺模型现在是一个既定的技术。1在该模型中，红外肾主动脉的两壁与地下25针被刺破，退出进毗邻肾下腔静脉;前主动脉入口孔被修复以简单的压缩，并且不需要缝合修复。串行随访检查采用高分辨率多普勒超声和病理分析表明，AVF有一个成熟阶段，再一个失败的阶段，扼要重述人类AVF的已知病理生理学2 探索了调节AVF成熟机制，需要用于输送治疗剂来熟化AVF内皮改进的方法。治疗剂递送至血管可以是通过血管内递送至内腔，或通过外部传递到外膜。外部递送的一个实例是玉米常用的聚醚凝胶的外膜应用。当加热到体温这种共聚物是热可逆的，并从液体到固体凝胶转化。当在体内局部施用药物在普卢兰尼克凝胶混合5,6-实现先前的研究已经表明持续的药物递送。3,4-病毒载体或siRNA与的Pluronic凝胶的外膜应用已报道是有效的血管周围递送系统，我们有还报告说，治疗由肽刺激外膜人外植隐静脉的导致内皮细胞受体蛋白的磷酸化。7 在另一方面，调查也同时使用病毒和非病毒载体腔内交付8-10犬和兔11,12车型静脉移植的。在这些报告中，静脉收获后进行基因转移体外 。伊斯拉米等报道的血管内的病毒基因递送y以颈动脉原位脉，而无需创建一个旁路13 Gloverman等。在大鼠股动脉，腹壁浅静脉瘘裸DNA的报道腔内和外膜交付14梅奥组报道在小鼠颈总动脉，颈内静脉瘘外膜的药物输送15,16然而，这些先前报告的模型需要缝合吻合创造在AVF。在这份报告中，腔内给药并同时创造AVF在小鼠中的描述，使用AVF创作的缝合线少的模型。通过使用本变形鼠AVF模型可以执行用于腔内药物输送到瘘管的静脉肢的简单方法。

<table><tbody><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td>Used as 30% solution in d-water</td></tr><tr><td>GFP antibody</td><td>NOVUS BIOLOGICALS INC</td><td>NB100-1770</td><td></td></tr><tr><td>Ad-CMV-GFP</td><td>VECTOR BIOLABS</td><td>1060</td><td></td></tr><tr><td>0.9% Sodium Chloride Irrigation, USP</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>BD PrecisionGlide Needle 25 G x 5/8</td><td>BD</td><td>305122</td><td></td></tr><tr><td>BD 1 ml Syringe Tuberculin Slip Tip</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Scalpel</td><td>Surgical Design Inc</td><td>22079707</td><td></td></tr><tr><td>6-0 ETHILON P-1 11 mm 3/8c Reverse Cutting</td><td>ETHICON INC</td><td>697G</td><td></td></tr><tr><td>Vevo 770 ultrasound machine&amp;nbsp;</td><td>Visualsonics&amp;nbsp;</td><td>20 - 60 Mhz scan head; RMV-704</td><td></td></tr><tr><td>Vascular clamp&amp;nbsp;</td><td>Roboz Surgical Instrument Co.</td><td>RS-5424</td><td></td></tr><tr><td>Clamp applying forceps&amp;nbsp;</td><td>Roboz Surgical Instrument Co.</td><td>RS-5410</td><td></td></tr></tbody></table>

intraluminal drug delivery to the mouse arteriovenous fistula endothelium

治疗剂的递送，以提高动静脉瘘（AVF）成熟可以通过腔内或外部途径给药。简单的鼠AVF模型用的药物溶液至静脉内皮同时作为瘘创建腔内施用相结合。这个模型的技术问题进行了讨论。在全身麻醉下腹部切口由和主动脉和下腔静脉（IVC）被曝光。在肾下主动脉和下腔静脉解剖夹紧。近端和远端夹紧后，穿刺部位进行曝光，将25g针头被用来穿刺主动脉并进入下腔静脉两壁。穿刺后，立即报告基因表达的病毒载体在IVC通过相同的针注入，随后温育15分钟。相比由外部给药途径腔内给药方法使能更健壮的病毒基因递送到静脉内皮。这诺夫递送l的方法将有利于研究，探索在AVF成熟内皮的作用，并能在外科手术的时间腔内给药。

在一系列33小鼠，在第一术后天存活率为97.0％; AVF通畅，如通过超声波来确定，为84.9％。 与传统的外部路由本血管内输送路线的基因转导效率进行比较。对于管腔内输送（ILD），在穿刺后，立即加入200μl腺病毒的GFP（广告-GFP）载体溶液（1×10 9 PFU / ml）的经穿刺针注入IVC，随后孵育15分钟时间。对照组小鼠用聚醚凝胶收到的Ad-GFP（1×10 9 PFU /毫升）下腔静脉的外膜交付（AD）。样品在暴露于腺病毒载体24小时后取出的。从用荧光显微镜，以及组织学检查内膜节用抗GFP进行了评估由烯面观察在IVC壁GFP和瘘管的表达抗体。 烯面观察显示在24小时（ 图2A）中的ILD治疗的小鼠与AD相比，下腔静脉壁的GFP表达更强，与周围的瘘特性增强。 GFP表达在两组永久在转染后72小时（数据未显示）。抗GFP抗体组织学分析，下腔静脉与ILD治疗的小鼠与AD相比，内膜显示更强大和特定地点的GFP表达。 （ 图2B）。为了证实这些结果，通过AD或ILD腺病毒-GFP治疗肾下IVCS而收获的印迹。仅ILD后的GFP蛋白质表达是足够的检测，在转染后24小时，这表明通过腔内路线优越病毒药物输送到下腔静脉壁。 <img alt="图2" src="/files/ftp_upload/53905/53905fig2.jpg" /> 图2.交货腺病毒GFP到AVF流明增强GFP表达。 （A）代表免疫表现在连接接收外膜交货（上）或腔内交付（底部）应用腺病毒GFP小鼠AVF内皮（24小时）的正视图。条表示25微米。（B）示出在接收由外膜输送（顶部）或管腔内输送（底部）施加腺-GFP小鼠AVF内皮（24小时）的部分代表性免疫荧光。 *表示下腔静脉管腔;条表示25微米。通过外膜或腔内航线应用于显示GFP在AVF腺病毒-GFP处理的检测（C）代表印迹。

Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

腔内药物输送到鼠标动静脉瘘内皮

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

intraluminal-drug-delivery-to-mouse-arteriovenous-fistula

Nanyang Technological University

Research

JoVE Journal

Medicine

9.3K 次观看.  Yale University. After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

视频: Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

科研

医学

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

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 ...

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

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

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

股动脉线损伤的小鼠模型的血管周围给药补丁植入

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

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型:在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

中央静脉狭窄病的Murine模型,使用具有流出狭窄病的动脉静脉瘘管病

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

血液透析通路相关手功能障碍的小鼠模型

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

血管紧张素II诱导的腹主动脉瘤小鼠模型中中心静脉导管的药物治疗及3D超声监测

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

A Modified Technique for Arteriovenous Fistula Construction in Rabbits

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

一种改良的兔动静脉瘘构建技术

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury ...