啮齿动物异位心脏瓣膜移植的简化模型
Summary
该协议描述了一种简单有效的方法，用于在肾囊下移植主动脉瓣叶，以允许研究心脏瓣膜的同种异性。
Abstract
临床上迫切需要在儿童中生长的心脏瓣膜置换术。心脏瓣膜移植被提议为一种新型移植，具有提供能够体细胞生长的耐用心脏瓣膜的潜力，而不需要抗凝治疗。然而，心脏瓣膜移植的免疫生物学仍未被探索，这凸显了动物模型研究这种新型移植的必要性。已经描述了先前用于异位主动脉瓣移植到腹主动脉中的大鼠模型，尽管它们在技术上具有挑战性且成本高昂。为了应对这一挑战，在啮齿动物中开发了肾囊下移植模型，作为研究心脏瓣膜移植免疫生物学的实用且更直接的方法。在该模型中，收获单个主动脉瓣小叶并将其插入肾囊下间隙。肾脏易于接近，移植的组织安全地包含在血管化良好的囊下空间中，并且可以容纳各种组织大小。此外，由于一只大鼠可以提供三个供体主动脉瓣小叶，而单个肾脏可以为移植组织提供多个位点，因此给定研究所需的大鼠更少。这里描述了移植技术，为研究心脏瓣膜移植的移植免疫学提供了重要的一步。
Introduction
先天性心脏缺陷是人类最常见的先天性残疾，每年每1000名活产儿中就有7名受到影响1。与常规植入各种机械和生物假体瓣膜的成年患者不同，儿科患者目前没有瓣膜置换的良好选择。这些传统的植入物没有在受体儿童中生长的潜力。因此，随着儿童的成长，需要病态的再手术来将心脏瓣膜植入物更换为连续更大的版本，受影响的儿童在其一生中通常需要多达五次或更多的心脏直视手术2，3。研究表明，与年龄较大的儿童相比，婴儿免受干预或死亡的自由度明显较差，60%的心脏瓣膜假体婴儿在初始手术后3年内面临再手术或死亡4.因此，迫切需要提供一种能够在儿科患者中生长和维持功能的心脏瓣膜。
几十年来，提供不断增长的心脏瓣膜置换术的尝试一直集中在组织工程和干细胞上。然而，到目前为止，将这些瓣膜移植到诊所的尝试都没有成功5，6，7，8。为了解决这个问题，心脏瓣膜移植被提议作为一种更具创造性的手术，用于提供具有自我修复和避免血栓形成能力的不断增长的心脏瓣膜置换术。而不是移植整个心脏，只有心脏瓣膜被移植，然后与受者的孩子一起生长，类似于传统的心脏移植或Ross肺签名9，10，11。术后，受者儿童将接受免疫抑制，直到移植的瓣膜可以换成成人大小的机械假体，当不再需要瓣膜的生长时。然而，心脏瓣膜移植移植物的移植生物学仍未被探索。因此，需要动物模型来研究这种新型移植。
先前已经描述了几种大鼠模型将主动脉瓣异位移植到腹主动脉12，13，14，15，16，17，18。然而，这些模型非常棘手，通常需要训练有素的外科医生才能成功操作。此外，它们既昂贵又耗时19.开发了一种新的大鼠模型，以创建一种更简单的动物模型，用于研究心脏瓣膜移植的免疫生物学。切除单主动脉瓣小叶并将其插入肾囊下间隙。肾脏特别适合研究移植排斥反应，因为它高度血管化，可进入循环免疫细胞20，21。虽然其他几个人利用肾脏囊下模型来研究其他同种异体移植的移植生物学，如胰腺，肝脏，肾脏和角膜22，23，24，25，26，27，但这是心脏组织移植的第一个描述。这里描述了移植技术，为研究心脏瓣膜移植的移植免疫学提供了重要的一步。
Protocol
该研究根据美国国立卫生研究院实验室动物护理和使用指南，由动物研究委员会批准。 1. 动物模型信息（大鼠） 所有外科手术均使用放大倍率高达20倍的手术显微镜（见 材料表）。 根据实验需要使用同源（如刘易斯-刘易斯）或同种异体（如挪威刘易斯-布朗）菌株进行移植。 使用年龄在5-7周之间，体重为100-200克的大鼠，适合实验问题。</l…
Representative Results
为大鼠模型提供了实验设计的图形描述（图1）。此外，从供体心脏解剖的主动脉根部和准备植入的单个主动脉瓣叶也如图2所示。接下来，图3A显示了主动脉瓣叶在肾囊下植入的位置的代表性图像，并在受体大鼠内3，7和28天后（图3B-D），表明易于定位和恢复移植的组织。 <p class="…
Discussion
重要性和潜在应用
虽然机械和生物假体心脏瓣膜常规用于需要瓣膜置换术的成年患者，但这些瓣膜缺乏生长潜力，因此对于儿科患者来说并不理想。心脏瓣膜移植是一项实验性手术，旨在为患有先天性心脏病的新生儿和婴儿提供不断增长的心脏瓣膜置换术。然而，与传统心脏移植的移植免疫生物学不同，这种新型移植的移植免疫生物学仍然没有得到很好的探索。本文介绍了一种独?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
图 1 是使用 biorender.com 创建的。这项工作得到了AATS基金会TKR外科研究者计划的部分支持，南卡罗来纳州医科大学儿科系为TKR举办的儿童卓越基金，爱默生玫瑰心脏基金会对TKR的赠款，参议员保罗坎贝尔对TKR的慈善事业，NIH-NHLBI机构博士后培训补助金（T32 HL-007260）给JHK和BG， 和南卡罗来纳医科大学医学院预职的FLEX研究基金给MAH。
Materials
| 0.9% Sodium Chlordie, USP | Baxter | NDC 0338-0048-04 | |
| 4-0 Polyglactin 910 | Ethicon | J415H | |
| 7.5% Povidone-Iodine | CareFusion | 29904-004 | |
| 70% ETOH | Fisher Scientific | BP82031GAL | |
| Anesthesia induction chamber | Harvard Apparatus | 75-2030 | Air-tight inducton chamber for rats |
| Anesthesia machine | Harvard Apparatus | 75-0238 | Mobile Anesthesia System with Passive Scavenging |
| Anesthesia Mask | Harvard Apparatus | 59-8255 | Rat anesthesia mask |
| Brown Norway Rats (BN/Crl) | Charles River | Strain Code 091 | Male, 5-7 weeks, 100-200 g |
| Buprenorphine Hydrochloride, 0.3 mg/mL | PAR Pharmaceutical | NDC 42023-179-05 | 0.03 mg/kg, administered subcutaneously |
| Electric hair clippers | WAHL | 79434 | |
| Electric Heating Pad | Harvard Apparatus | 72-0492 | Maintained at 36-38 °C |
| Heparin | Sagent Pharmaceuticals | NDC 25021-400-10 | 100U/100g injection into the left atrium |
| Insulin Syringe, 1 mL | Fisher Scientific | 14-841-33 | |
| Iris forceps curved | World Precision Instruments | 15917 | |
| Iris forceps straight | World Precision Instruments | 15916 | |
| Isoflurane, USP | Piramal Critical Care | NDC 66794-017-25 | Induced at 5% isoflurance in oxygen and maintained with 3.5% isoflurane in oxygen |
| Lewis Rats (LEW/ Crl) | Charles River | Strain Code 004 | Male, 5-7 weeks, 100-200 g |
| Micro forceps | World Precision Instruments | 500233 | Dumont #5 |
| Micro scissors | World Precision Instruments | 501930 | Spring-loaded Vannas Scissors |
| Needle Driver | World Precision Instruments | 500226 | Ryder Needle Driver |
| Operating microscope | AmScope | SM-3BZ-80S | 3.5x – 90x Stereo Microscope |
| Petri Dish | Fisher Scientific | FB0875714 | |
| Petrolatum ophthalmic ointment | Dechra | NDC 17033-211-38 | |
| Skin staples | Ethicon | PXR35 | Proximate 35 |
| Sterile cotton swabs | Puritan | 25-806 1WC | |
| Sterile gauze sponges | Fisher Scientific | 22-037-902 | |
| Surgical Scissors | World Precision Instruments | 1962C | Metzenbaum Scissors |
| University of Wisconsin Buffer (Servator B) | S.A.L.F S.p.A. | 6484A1 | Stored at 4 °C |
References
- Van Der Linde, D., et al. Birth prevalence of congenital heart disease worldwide: A systematic review and meta-analysis. Journal of the American College of Cardiology. 58 (21), 2241-2247 (2011).
- Jacobs, J. P., et al. Reoperations for pediatric and congenital heart disease: An analysis of the Society of Thoracic Surgeons (STS) congenital heart surgery database. Seminars in Thoracic and Cardiovascular Surgery: Pediatric Cardiac Surgery Annual. 17 (1), 2-8 (2014).
- Syedain, Z. H., et al. Pediatric tri-tube valved conduits made from fibroblast-produced extracellular matrix evaluated over 52 weeks in growing lambs. Science Translational Medicine. 13 (585), 1-16 (2021).
- Khan, M. S., Samayoa, A. X., Chen, D. W., Petit, C. J., Fraser, C. D. Contemporary experience with surgical treatment of aortic valve disease in children. Journal of Thoracic and Cardiovascular Surgery. 146 (3), 512-521 (2013).
- Boyd, R., Parisi, F., Kalfa, D. State of the art: Tissue engineering in congenital heart surgery. Seminars in Thoracic and Cardiovascular Surgery. 31 (4), 807-817 (2019).
- Feins, E. N., Emani, S. M. Expandable valves, annuloplasty rings, shunts, and bands for growing children. Seminars in Thoracic and Cardiovascular Surgery: Pediatric Cardiac Surgery Annual. 23, 17-23 (2020).
- Lintas, V., et al. TCT-795 Human cell derived off-the-shelf tissue engineered heart valves for next generation transcatheter aortic valve replacement: a proof-of-concept study in adult sheep. Journal of the American College of Cardiology. 70 (18), 271 (2017).
- Blum, K. M., Drews, J. D., Breuer, C. K. Tissue-engineered heart valves: A call for mechanistic studies. Tissue Engineering Part B: Reviews. 24 (3), 240-253 (2018).
- Bernstein, D., et al. Cardiac growth after pediatric heart transplantation. Circulation. 85 (4), 1433-1439 (1992).
- Delmo Walter, E. M., et al. Adaptive growth and remodeling of transplanted hearts in children. Europeon Journal of Cardiothoracic Surgery. 40 (6), 1374-1383 (2011).
- Simon, P., et al. Growth of the pulmonary autograft after the Ross operation in childhood. Europeon Journal of Cardiothoracic Surgery. 19 (2), 118-121 (2001).
- Oei, F. B. S., et al. A size-matching heterotopic aortic valve implantation model in the rat. Journal of Surgical Research. 87 (2), 239-244 (1999).
- Oei, F. B. S., et al. Heart valve dysfunction resulting from cellular rejection in a novel heterotopic transplantation rat model. Transplant International. 13, (2000).
- El Khatib, H., Lupinetti, F. M. Antigenicity of fresh and cryopreserved rat valve allografts. Transplantation. 49 (4), 765-767 (1990).
- Yankah, A. C., Wottge, H. U. Allograft conduit wall calcification in a model of chronic arterial graft rejection. Journal of Cardiac Surgery. 12 (2), 86-92 (1997).
- Moustapha, A., et al. Aortic valve grafts in the rat: Evidence for rejection. Journal of Thoracic and Cardiovascualr Surgery. 114 (6), 891-902 (1997).
- Légaré, J. F., et al. Prevention of allograft heart valve failure in a rat model. Journal of Thoracic and Cardiovascualr Surgery. 122 (2), 310-317 (2001).
- Legare, J. F., Lee, T. D. G., Creaser, K., Ross, D. B., Green, M. T lymphocytes mediate leaflet destruction and allograft aortic valve failure in rats. The Annals of Thoracic Surgery. 70 (4), 1238-1245 (2000).
- Niimi, M. The technique for heterotopic cardiac transplantation in mice: Experience of 3000 operations by one surgeon. Journal of Heart and Lung Transplantation. 20 (10), 1123-1128 (2001).
- Burgin, M., et al. Kidney Subcapsular Allograft Transplants as a Model to Test Virus-Derived Chemokine-Modulating Proteins as Therapeutics. Methods in molecular biology. 2225, 257-273 (2021).
- Foglia, R. P., DiPreta, J., Donahoe, P. K., Statter, M. B. Fetal allograft survival in immunocompetent recipients is age dependent and organ specific. Annals of Surgery. 204 (4), 402-410 (1986).
- Cunha, G. R., Baskin, L. Use of sub-renal capsule transplantation in developmental biology. Differentiation. 91 (4-5), 4-9 (2016).
- Hori, J., Joyce, N., Streilein, J. W. Epithelium-deficient corneal allografts display immune privilege beneath the kidney capsule. Investigative Opthalmology & Visual Science. 41 (2), 443-452 (2000).
- Mandel, T., et al. transplantation of organ cultured fetal pig pancreas in non-obese diabetic (NOD) mice and primates (Macaca fascicularis). Xenotransplantation. 2 (3), 128-132 (1995).
- Ricordi, C., Flye, M. W., Lacy, P. E. Renal subcapsular transplantation of clusters of hepatocytes in conjunction with pancreatic islets. Transplantation. 45 (6), 1148-1150 (1988).
- Shultz, L. D., et al. Subcapsular transplantation of tissue in the kidney. Cold Spring Harbor Protocols. 2014 (7), 737-740 (2014).
- Vanden Berg, C. W., et al. Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo. Stem Cell Reports. 10 (3), 751-765 (2018).
- Mitchell, R. N., Jonas, R. A., Schoen, F. J. Pathology of explanted cryopreserved allograft heart valves: Comparison with aortic valves from orthotopic heart transplants. Journal of Thoracic and Cardiovasular Surgery. 115 (1), 118-127 (1998).
- Valante, M., et al. The aortic valve after heart transplantation. Annals of Thoracic Surgery. 60, (1995).
- O’Brien, M. F., Stafford, E. G., Gardner, M. A. H., Pohlner, P. G., McGiffin, D. C. A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies. Journal of Thoracic and Cardiovascular Surgery. 94 (6), 812-823 (1987).
- Ng, T. F., Osawa, H., Hori, J., Young, M. J., Streilein, J. W. Allogeneic neonatal neuronal retina grafts display partial immune privilege in the subcapsular space of the kidney. The Journal of Immunology. 169 (10), 5601-5606 (2002).
- Heslop, B. F., Wilson, S. E., Hardy, B. E. Antigenicity of aortic valve allografts. Annals of Surgery. 177 (3), 301-306 (1973).
- Steinmuller, D., Weiner, L. J. Evocation and persistence of transplantation immunity in rats. Transplantation. 1 (1), 97-106 (1963).
- Billingham, R. E., Brent, L., Brown, J. B., Medawar, P. B. Time of onset and duration of transplantation immunity. Plastic and Reconstructive Surgery. 24 (1), 410-413 (1959).
- Tector, A. J., Boyd, W. C., Korns, M. E. Aortic valve allograft rejection. Journal of Thoracic and Cardiovascular Surgery. 62 (4), 592-601 (1971).
- Sugimura, Y., Schmidt, A. K., Lichtenberg, A., Akhyari, P., Assmann, A. A rat model for the in vivo assessment of biological and tissue-engineered valvular and vascular grafts. Tissue Engineering Methods (Part C). 23 (12), 982-994 (2017).
Citer Cet Article
Hill, M. A., Kwon, J. H., Gerry, B., Kavarana, M., Nadig, S. N., Rajab, T. K. A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents. J. Vis. Exp. (175), e62948, doi:10.3791/62948 (2021).