罗斯手术的啮齿动物模型：全身位置的同源肺动脉移植植入
1Pediatric and Congenital Cardiac Surgery Unit, Department of Cardiac, Thoracic and Vascular Sciences and Public Health,University of Padua, 2Pediatric Cardiology Unit, Departments of Women’s and Children’s Health,University of Padua, 3Labatt Family Heart Centre, Department of Cardiovascular Surgery, The Hospital for Sick Children,University of Toronto
Summary
我们演示了如何建立肺根植入降主动脉的小鼠模型，以模拟Ross手术。该模型能够对全身位置的肺自体移植重塑进行中长期评估，这是制定治疗策略以促进其适应的基础。
Abstract
Ross主动脉瓣疾病手术由于其出色的长期结果而重新获得了新的兴趣。然而，当用作独立根部替代术时，描述了肺自体移植的可能扩张和随后的主动脉瓣反流。已经提出了几种动物模型。然而，这些通常仅限于 离体 模型或相对昂贵的大型动物模型的 体内 实验。在这项研究中，我们试图建立一种在全身位置植入肺动脉移植（PAG）的啮齿动物模型。总共包括39只成年刘易斯大鼠。安乐死后立即从供体动物身上摘取肺根（n=17）。同源受体（n = 17）和假手术（n = 5）大鼠镇静并通气。在接受组，PAG在肾下腹主动脉位置植入端到端吻合器。假手术大鼠仅接受主动脉的横切和再吻合。对动物进行连续超声检查两个月和死后组织学分析。原生位置的PAG直径中位数为3.20毫米（IQR=3.18-3.23）。随访时，PAG的中位直径在1周时为4.03 mm（IQR=3.74~4.13），1个月时为4.07 mm（IQR=3.80~4.28），2个月时为4.27 mm（IQR=3.90~4.35）（p<0.01）。1周时收缩速度峰值为220.07 mm/s（IQR=210.43-246.41），1个月时为430.88 mm/s（IQR=375.28-495.56），2个月时为373.68 mm/s（IQR=305.78-429.81），与实验结束时的假手术组（p=0.5）没有差异。组织学分析未显示任何内皮血栓形成的体征。这项研究表明，啮齿动物模型可以评估肺根对高压系统的长期适应性。系统放置的同源PAG植入代表了开发和评估新型手术技术和药物治疗的简单可行的平台，以进一步改善Ross手术的结果。
Introduction
先天性主动脉瓣狭窄是先天性心脏病的一个亚组，其特征在于左心室梗阻，其中病变位于瓣膜水平。畸形影响每1000名活产婴儿约0.04-0.38人1。
可用于校正的选项很多，每个选项都有自己的优点和缺点。对于适合进行双心室矫正的患者2，该方法可能针对瓣膜修复（经皮或手术瓣膜切开术）或其替代治疗3。当主动脉瓣被认为无法挽救时，后者是首选;然而，对于儿科患者，可用的选择是有限的。事实上，生物瓣膜由于早期钙化，不适合年轻人群中的主动脉置换术4。另一方面，机械瓣膜的退化速度要慢得多，但这些需要终身抗凝治疗5。此外，这些假体的主要局限性表现为缺乏生长潜力，这使患者容易进行额外的再干预。
儿科人群中一个有趣的治疗选择是将肺自体移植物转移到名为”Ross手术”的主动脉位置。在这种情况下，肺动脉瓣随后被同种移植物取代（图1）6。该手术可能是儿童的最佳手术选择，因为肺自体移植物保留了其生长潜力，并且没有终身抗凝治疗的风险。此外，Ross手术在年轻人中也具有很高的价值，可以避免机械或生物瓣膜，有可能成为最佳的手术解决方案。
肺自体移植术置换主动脉瓣后的效果非常好，生存率高于98%，长期结局良好7。文献研究显示，在 4 年和 12 年时，肺同种移植物的替代率分别为 93% 和 90%8。
该手术的主要局限性是自体移植物在长期内扩张的趋势，特别是当用作独立根部替代物时。这可能导致瓣膜功能不全，可能需要再次干预。事实上，迄今为止进行的最长的随访研究报告称，自体移植替代术在10年时为88%，在20年时为75%9。
在实验环境中重建Ross手术的可能性代表了研究肺自体移植适应全身压力的潜在机制的基本先决条件。过去曾提出过几种模式。然而，这些通常仅限于 离体 实验或具有相对昂贵的大型动物的 体内 动物模型。在这项研究中，我们试图建立一种啮齿动物模型，将肺动脉移植（PAG）植入在全身位置，作为独立根。
Protocol
所有程序均已获得帕多瓦大学动物护理委员会（OPBA，协议编号N°55/2017）的批准，并得到意大利卫生部的授权（授权号700/2018-PR），符合欧盟指令2010/63 / UE和意大利实验室动物护理和使用法律26/2014。 1. 动物护理和实验模型 确保所有刘易斯大鼠都是从一家公司获得的（材料表）。将老鼠养在传统设施中，免费获得食物和水。 确保受体组?…
Representative Results
本研究共纳入39只成年刘易斯大鼠：17只动物作为PAG供体，17只动物作为受体，5只作为假手术（对照组）（表1）。雄性大鼠为22只（56%），雌性为17只（44%）;后者仅用于捐助集团。 手术期间未发生致命事件，存活率为100%。在随访期间，移植组的两只动物分别在12天和51天时有致命的结果;研究结束时的存活率为91%（表1）。 受体组大…
Discussion
由于自体移植的有利特征和潜在生长，使用自体肺根置换主动脉瓣（Ross手术）是先天性主动脉瓣狭窄修复的一个有吸引力的选择10。该手术的主要局限性是主动脉新瓣膜的潜在扩张，这容易导致长期反流的发展。表征暴露于全身压力后肺动脉修饰的可能性可能是理解肺自体移植失败原因的基础。因此，我们开发了一种在啮齿动物模型中全身位置植入同源PAG的实验模型。
<p class…Disclosures
The authors have nothing to disclose.
Acknowledgements
该研究由2019年跨部门研究综合预算（BIRD）资助。
Materials
| 0.9% Sodium Chloride | Monico SpA | AIC 030805105 | Two bottles of 100 mL. The cold one (4°C) for flushing the harvesting organ; the warm one (39°C) for moistening, and rehydration of the recipient |
| 7.5% Povidone-Iodine | B Braun | AIC 032151211 | |
| Barraquer | Aesculap | FD 232R | Straight micro needle holder for the vascular anastomoses |
| Castroviejo needle holder | Not available | J 4065 | To close the animal |
| Clip applying forceps | Rudolf Medical | RU 3994-05 | For clip application |
| Cotton swabs | Johnson & Johnson Medical SpA | N/A | Supermarket product. Sterilized |
| Curved micro jeweller forceps | Rudolf Medical | RU 4240-06 | Used to pass sutures underneath the vases. |
| Depilatory cream | RB healthcare | N/A | Supermarket product |
| Electrocautery machine | LED SpA | Surton 200 | |
| Fine scissors | Rudolf Medical | RU 2422-11 | For opening the abdomen (recipient) |
| Fine-tip curved Vannas micro scissors | Aesculap | OC 497R | Only for preparing the pulmonary root, cut the lumbar vases and the 10/0 Prolene |
| Fluovac Isoflurane/Halotane Scavanger unit | Harvard Apparatus Ltd | K 017041 | Complete of anesthesia machine, anesthesia tubing, induction chamber and scavenger unit with absorbable filter |
| Gentamycin | MSD Italia Srl | AIC 020891014 | Antibiotic. Single dose, 5 mg/kg intramuscular, administered during surgery |
| Heparin | Pharmatex Italia Srl | AIC 034692044 | 500 IU into the recipient abdominal vena cava |
| I.V. Catheter | Smiths Medical Ltd | 4036 | 20G |
| Insulin Syringe, 1 mL | Fisher Scientific | 14-841-33 | To inject heparin in the harvesting animal and to flush the sectioned aorta in the recipient |
| Jeweler bipolar forceps | GIMA SpA | 30665 | 0.25 mm tip. For electrocautery of very small vases |
| Lewis rats (LEW/HanHsd) | Envigo RMS SRL, San Pietro al Natisone, Udine, Italy | 86104M | Male or female, weighing 200-250 g (pulmonary root harvesting animals) and 320-400 g (recipients) |
| Micro-Mosquito | Rudolf Medical | RU 3121-10 | In number of four, with tips covered with silicon tubing. To keep in traction the Prolene suture during anastomosis |
| Operating microscope | Leica Microsystems | M 400-E | Used with 6x, 10x and 16x in-procedure interchangeable magnifications |
| Perma-Hand silk 2-0 | Johnson & Johnson Medical SpA | C026D | To lift the aorta |
| Petrolatum ophthalmic ointment | Dechra | NDC 17033-211-38 | |
| Prolene 10-0 | Johnson & Johnson Medical SpA | W2790 | Very fine non-absorbable suture, with a BV75-3 round bodied needle, for the vascular anastomoses |
| Retractors | Not any | N/A | Two home-made retractors |
| Ring tip micro forceps | Rudolf Medical | RU 4079-14 | For delicate manipulation |
| Sevoflurane | AbbVie Srl | AIC 031841036 | Mixed with oxygen, for inhalatory anesthesia |
| Spring type micro scissors | Rudolf Medical | RU 2380-14 | Straight; 14 cm long |
| Standard aneurysm clips | Rudolf Medical | RU 3980-12 | Two clips (7.5 mm; 180 g; 1.77 N) to close the aorta |
| Sterile gauze of non-woven fabric material | Luigi Salvadori SpA | 26161V | 7.5×7.5 cm, four layers |
| Straight Doyen scissors | Rudolf Medical | RU/1428-16 | For use to the donor |
| Straight micro jeweller forceps | Rudolf Medical | RU 4240-04 | 10.5 cm long. Used throughout the anastomosis |
| Syringes | Artsana SpA | N/A | 20 mL (for the harvesting animal) and 5 mL (for the recipient). For saline flushing and dipping |
| TiCron 4-0 | Covidien | CV-331 | For closing muscles and skin |
| Tissue forceps V. Mueller | McKesson | CH 6950-009 | Used for skin and muscles |
| Tramadol | SALF SpA | AIC 044718029 | Analgesic. Single dose, 5 mg/kg intramuscular |
| Virgin silk 8-0 | Johnson & Johnson Medical SpA | W818 | For arterial branch ligation |
References
- Botto, L. D., Correa, A., Erickson, J. D. Racial and temporal variations in the prevalence of heart defects. Pediatrics. 107 (3), 32 (2001).
- Vergnat, M., et al. Aortic stenosis of the neonate: A single-center experience. The Journal of Thoracic and Cardiovascular Surgery. 157 (1), 318-326 (2019).
- Hraška, V., et al. The long-term outcome of open valvotomy for critical aortic stenosis in neonates. The Annals of Thoracic Surgery. 94 (5), 1519-1526 (2012).
- Kaza, A. K., Pigula, F. A. Are bioprosthetic valves appropriate for aortic valve replacement in young patients. Seminars in Thoracic and Cardiovascular Surgery: Pediatric Cardiac Surgery Annual. 19 (1), 63-67 (2016).
- Myers, P. O., et al. Outcomes after mechanical aortic valve replacement in children and young adults with congenital heart disease. The Journal of Thoracic and Cardiovascular Surgery. 157 (1), 329-340 (2019).
- Donald, J. S., et al. Ross operation in children: 23-year experience from a single institution. The Annals of thoracic surgery. 109 (4), 1251-1259 (2020).
- Khwaja, S., Nigro, J. J., Starnes, V. A. The Ross procedure is an ideal aortic valve replacement operation for the teen patient. Seminars in Thoracic and Cardiovascular Surgery: Pediatric Cardiac Surgery Annual. , 173-175 (2005).
- Elkins, R. C., Lane, M. M., McCue, C. Ross operation in children: late results. The Journal of Heart Valve Disease. 10 (6), 736-741 (2001).
- Chambers, J. C., Somerville, J., Stone, S., Ross, D. N. Pulmonary autograft procedure for aortic valve disease: long-term results of the pioneer series. Circulation. 96 (7), 2206-2214 (1997).
- Mazine, A., et al. Ross procedure in adults for cardiologists and cardiac surgeons: JACC state-of-the-art review. Journal of the American College of Cardiology. 72 (22), 2761-2777 (2018).
- Sengupta, P. The laboratory rat: Relating its age with humans. International Journal of Preventive Medicine. 4 (6), 624-630 (2013).
- Ashfaq, A., Leeds, H., Shen, I., Muralidaran, A. Reinforced ross operation and intermediate to long term follow up. Journal of Thoracic Disease. 12 (3), 1219-1223 (2020).
- Vida, V. L., et al. Age is a risk factor for maladaptive changes of the pulmonary root in rats exposed to increased pressure loading. Cardiovascular Pathology: The Official Journal of the Society for Cardiovascular Pathology. 21 (3), 199-205 (2012).
- Nappi, F., et al. An experimental model of the Ross operation: Development of resorbable reinforcements for pulmonary autografts. The Journal of Thoracic and Cardiovascular Surgery. 149 (4), 1134-1142 (2015).
- Vanderveken, E., et al. Mechano-biological adaptation of the pulmonary artery exposed to systemic conditions. Scientific Reports. 10 (1), 2724 (2020).
Cite This Article
Dedja, A., Cattapan, C., Di Salvo, G., Avesani, M., Sabatino, J., Guariento, A., Vida, V. A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position. J. Vis. Exp. (182), e63179, doi:10.3791/63179 (2022).