一种新的小鼠模型主动脉瘤腔内修复
1INSERM U698 Cardiovascular Remodelling,Hôpital X. Bichat, AP-HP, Paris, 2Bio-Ingénierie des Polymères Cardiovasculaires (BPC),Institut Galilée – Université Paris 13, Paris, France, 3Service de Chirurgie Vasculaire, Hôpital Henri Mondor, AP-HP,Université Paris-Est Creteil, 4Ecole de chirurgie de l’assistance publique des hôpitaux de Paris, 5Service de Chirurgie Cardiaque et Vasculaire, Hôpital Européen Georges Pompidou, AP-HP,Université René Descartes
Summary
动脉瘤的组织学和生化修改后移植物排斥目前仍不清楚。我们描述了一种新的模式移植物植入对小鼠动脉瘤。通过血栓并没有持久性循环血液流接口，和新的血管内的生物材料评价的分析，这种模式将有助于更好地了解血管内动脉瘤排除病理学。
Abstract
血管内动脉瘤排除是经过验证的技术，以防止动脉瘤破裂。长期业绩突出的腹主动脉瘤（AAA）的病理生理技术的局限性和新的方面。有没有腹主动脉瘤移植物排斥模型便宜和可重复性，这将使治疗前后的AAA级深调查。我们在此描述了如何诱导，然后一个覆盖冠状动脉stentgraft的腹主动脉瘤的大鼠排除。众所周知的弹性蛋白酶诱导AAA模型首次报道于1990年1大鼠，小鼠2然后描述。弹性蛋白的降解导致的主动脉扩张的腹壁和管腔内血栓的炎性浸润，配合人力AAA。腔内隔绝小覆盖stentgraft，然后执行，不包括血液循环和动脉瘤血栓之间的任何互动。适当的的排斥和stentgraft通畅是通过血管造影证实之前安乐死，认为左侧颈动脉。部分的弹性蛋白酶扩散的控制，使得每个动物的动脉瘤形状不同。这是很难创建一个动脉瘤，这将允许一个适当长度的主动脉在动脉瘤一个简单的stentgraft的引入以下，并有足够的近端和远端颈部，以防止内漏。大量的失败可能会导致有时会导致主动脉撕裂的痛苦和烦恼十字绣stentgraft介绍，与后运算主动脉血栓形成和血管内皮损伤。到大鼠植入stentgraft前给予阿司匹林，降低故障率没有大的出血。钳位时间激活嗜中性粒细胞，血管内皮细胞和血小板，并且可能会干扰生物分析。
Introduction
腔内的技能进步，因为第一腔内主动脉瘤修复术（EVAR）于1991年3帕罗迪，几乎所有腹主动脉瘤的一半现在都被视为EVAR 4。不像手术，EVAR叶血栓，但不包括从全身的血液。动脉瘤的特点是血红蛋白丰富的多层次的血栓，薄薄的媒体，和fibrosed和外膜发炎。三十年前已被证明的关键作用，在动脉瘤的进展蛋白水解5 6，7：血栓的动脉瘤囊与主动脉壁薄，弹性蛋白的裂解，在媒体上的平滑肌细胞的低密度，和高水平的免疫炎症外膜反应8,9。这些变化表明，蛋白水解酶的活性发生血栓，而不是直接在主动脉壁内。
此外，血栓管腔层是含有丰富的凝集的红细胞策LLS释放血红蛋白。它们主要涉及纤维蛋白形成，血小板活化和诱导凝血酶形成。最后，血栓引起的t-PA和纤溶酶原潴留，从而参与纤溶及招聘白细胞，中性粒细胞为主。这是更重要的12倍比在血液流10中的血栓。有关他们的存在是具有速率高型基质金属蛋白酶（MMP-8），MMP-9和弹性蛋白酶：他们释放颗粒丝氨酸蛋白酶和亲氧化剂降解纤维矩阵，并最终主动脉壁破裂9,11 -13。
选修瘤的干预旨在防止破裂。 EVAR叶瘤壁血栓完好。因此，动脉瘤灌注主动脉抵押品（腰动脉，肠系膜下动脉…），被命名为内漏，是一个特定的血管内介入治疗并发症的14,15，有时会导致破裂前夕低压内漏Ń。此外，在某些患者中，有动脉瘤直径16没有增加。循环血液和动脉瘤壁之间的相互作用维持上述管腔内血栓的生物活性。因此，V型内漏，定义为没有确定内漏瘤囊扩张，可以解释为瘤内血栓的酶的活性。
无论是放射成像（18FDG-PET扫描，血小板活化显像，铁OXYDE对比度MRI17），也不外周血采样（MMP-9，血小板衍生微粒，纤维蛋白溶酶/抗纤维蛋白溶酶复合物）用于直接探索人类主动脉瘤腔内影响评估生化途径。
在我们的知识，也没有任何重复性好，价格便宜的实验排除瘤动物模型。这一个允许前，后动脉瘤排除生物和组织学的修改加以探讨。
Protocol
方法已获批准由比沙德勃雷伦理委员会（N°2012-15/698-0074），该协议。 第一弹性蛋白酶灌注模型已在1990年1由Anidjar描述：腹主动脉瘤是由弹性蛋白酶灌注诱导雄性Wistar大鼠，年龄在8至9周，250-300克。 2至4周后，进行重新剖腹手术，直径为3毫米的覆盖冠状动脉stentgraft的用于排除动脉瘤。嫁接，插入通过远端aortotomy，部署下视力放大。两个星期后，前?…
Representative Results
瘤固定在4％多聚甲醛溶液中24小时，然后进入的磷酸盐缓冲盐溶液，直到固定在树脂中的集团。我们使用HPS（苏木精Phloxin赛峰）（ 图1）染色，示出愈合增殖细胞周围的PTFE（ 图2）。然后，间充质细胞，其特征在于由α-肌动蛋白染色（ 图3）。 图1?…
Discussion
AAA两种型号都已经在我们的实验室：异种移植19和弹性蛋白酶诱导模型。1弹性蛋白酶模型是最相似的人主动脉瘤：一个大圆周腔内的血栓，然后排除动脉瘤腔内修复。
内漏是一个平常的并发症EVAR 14,15。很多问题依然未解：，AAA破裂已与低内漏，有时无瘤囊增大16。内漏是一个很平常的EVAR术后并发症，维持血液循环细胞和动脉瘤血栓之间的接?…
Declarações
The authors have nothing to disclose.
Acknowledgements
笔者想感谢恭Guesdon（雅培）的礼物Stentgrafts的。
Materials
| Animals | |||
| Wistar rats | aged from 8 to 9 weeks. Weight range from 350 to 400 g | ||
| Instruments | |||
| Isoflurane anesthesic system | 4,5% at the beginning, then 2% | ||
| Penthobarbital | Ceva sante animale, Libourne, France | ||
| Dissection stereomicroscope | |||
| Steriles gloves | |||
| Microsurgical Steriles instruments | Moria, Antony, France | Needle holder, Forceps, Scissors,Gilbert approximator o 3 Micro clamps | |
| N°40 Silk string | |||
| Prolen 9-0 and 10-0 | Ethicon, Johnson & Johnson, Auneau, France | ||
| Heat-tapered polyethylene tubing | PE 10 | ||
| Syringe infusion pump | |||
| Porcine pancreatic elastase | Sigma, St. Louis, Mo., USA | 550 μl of salin is mixed with 175 μl of elastase | |
| Indeflator | |||
| 9 to 16 mm length, 3 mm diameter coronary PTFE covered stentgraft | Abbott, Abbott Park, Illinois, USA | ||
| C-Arm OEC 9800 | GE Medical Systems, Milwaukee, WI | ||
| Aspegic 1000 mg | Aspirin, Sanofi-Aventis, Paris, France | ||
| Reagent | |||
| Aerane isoflurane 100ML | Baxter | Aerane | |
| Penthobarbital | Centravet | 053pen203 | |
| Silk Suture 4-0 | Fine Science | 18020-40 | |
| Microsurgical steriles instruments | Moria | 9980-9983-9987-204/A-204/D-8148-4877A-4878A | |
| Prolen 9-0 and 10-0 | Ethicon | W2829 – NS2850 | |
| Heat tapered Polyethylene tubing : PE 10 | Bioseb | MRE-010 | |
| Infusion pump | World precision instrument | AL-1000 | |
| Porcine pancreatic elastase | Sigma | E1250-100MG | |
| Indeflator | Longreal | KY025 25 100 | |
| Aspegic 1000mg (Aspirin) | Sanofi-Aventis | 3400931898191 | |
| C-Arm OEC 9800 | General electric. | OEC 9800 | |
| Stentgraft : Jostent | Abbott | 210CG1230 210CG1630 |
Referências
- Anidjar, S., et al. Elastase-induced experimental aneurysms in rats. Circulation. 82, 973-981 (1990).
- Azuma, J., Asagami, T., Dalman, R., Tsao, P. S. Creation of murine experimental abdominal aortic aneurysms with elastase. J. Vis. Exp. (29), e1280 (2009).
- Parodi, J. C., Palmaz, J. C., Barone, H. D. Transfemoral intraluminal graft implantation for abdominal aortic aneurysms. Ann. Vasc. Surg. 5, 491-499 (1991).
- Lees, T., Stansby, G. . The nationalvascular database report, 2009. , (2012).
- Sakalihasan, N., Limet, R., Defawe, O. D. Abdominal aortic aneurysm. Lancet. 365, 1577-1589 (2005).
- Busuttil, R. W., Abou-Zamzam, A. M., Machleder, H. I. Collagenase activity of the human aorta. A comparison of patients with and without abdominal aortic aneurysms. Arch. Surg. 115, 1373-1378 (1980).
- Michel, J. B. Contrasting outcomes of atheroma evolution: intimal accumulation versus medial destruction. Arterioscler. Thromb. Vasc. Biol. 21, 1389-1392 (2001).
- Vorp, D. A., et al. Association of intraluminal thrombus in abdominal aortic aneurysm with local hypoxia and wall weakening. J. Vasc. Surg. 34, 291-299 (2001).
- Kazi, M., et al. Influence of intraluminal thrombus on structural and cellular composition of abdominal aortic aneurysm wall. J. Vasc. Surg. 38, 1283-1292 (2003).
- Kuijper, P. H., et al. Neutrophil adhesion to fibrinogen and fibrin under flow conditions is diminished by activation and L-selectin shedding. Blood. 89, 2131-2138 (1997).
- Touat, Z., et al. Renewal of mural thrombus releases plasma markers and is involved in aortic abdominal aneurysm evolution. Am. J. Pathol. 168, 1022-1030 (2006).
- Fontaine, V., et al. Role of leukocyte elastase in preventing cellular re-colonization of the mural thrombus. Am. J. Pathol. 164, 2077-2087 (2004).
- Sakalihasan, N., Delvenne, P., Nusgens, B. V., Limet, R., Lapiere, C. M. Activated forms of MMP2 and MMP9 in abdominal aortic aneurysms. J. Vasc. Surg. 24, 127-133 (1996).
- Alsac, J. M., et al. The significance of endoleaks in thoracic endovascular aneurysm repair. Ann. Vasc. Surg. 25, 345-351 (2011).
- Buth, J., Harris, P. L., van Marrewijk, C., Fransen, G. The significance and management of different types of endoleaks. Semin. Vasc. Surg. 16, 95-102 (2003).
- Fransen, G. A., et al. Rupture of infra-renal aortic aneurysm after endovascular repair: a series from EUROSTAR registry. Eur. J. Vasc. Endovasc. Surg. 26, 487-493 (2003).
- van der Vaart, M. G., et al. Application of PET/SPECT imaging in vascular disease. Eur. J. Vasc. Endovasc. Surg. 35, 507-513 (2008).
- Close, B., et al. Recommendations for euthanasia of experimental animals: Part 2. DGXT of the European Commission. Lab. Anim. 31, 1-32 (1997).
- Allaire, E., Guettier, C., Bruneval, P., Plissonnier, D., Michel, J. B. Cell-free arterial grafts: morphologic characteristics of aortic isografts, allografts, and xenografts in rats. J. Vasc. Surg. 19, 446-456 (1994).
- Gawenda, M., Jaschke, G., Winter, S., Wassmer, G., Brunkwall, J. Endotension as a result of pressure transmission through the graft following endovascular aneurysm repair–an in vitro study. Eur. J. Vasc. Endovasc. Surg. 26, 501-505 (2003).
- Delbosc, S., et al. Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats. PLoS One. 6, e18679 (2011).
- Trollope, A., Moxon, J. V., Moran, C. S., Golledge, J. Animal models of abdominal aortic aneurysm and their role in furthering management of human disease. Cardiovasc Pathol. 20, 114-123 (2011).
- Yamaguchi, T., et al. Factors influencing mortality in the rat elastase-induced-aneurysm model. J. Surg. Res. 94, 81-83 (2000).
- von Bruhl, M. L., et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J. Exp. Med. 209, 819-835 (2012).
- Smith, P. D. Neutrophil activation and mediators of inflammation in chronic venous insufficiency. J. Vasc. Res. 36, 24-36 (1999).
- Zaragoza, C., et al. Animal models of cardiovascular diseases. J. Biomed. Biotechnol. 2011, 497841 (2011).
- Kuivaniemi, H., Elmore, J. R. Opportunities in abdominal aortic aneurysm research: epidemiology, genetics, and pathophysiology. Ann. Vasc. Surg. 26 (12), 862-870 (2012).
- Schanzer, A., et al. Predictors of abdominal aortic aneurysm sac enlargement after endovascular repair. Circulation. 123, 2848-2855 (2011).
Citar este artigo
Rouer, M., Meilhac, O., Delbosc, S., Louedec, L., Pavon-Djavid, G., Cross, J., Legagneux, J., Bouilliant-Linet, M., Michel, J., Alsac, J. A New Murine Model of Endovascular Aortic Aneurysm Repair. J. Vis. Exp. (77), e50740, doi:10.3791/50740 (2013).