经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究
1Institute for Cardiovascular and Metabolic Research, School of Biological Sciences,University of Reading, 2Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria,Universidad de Murcia, 3Institute of Virology and Immunology (IVI), 4Departamento de Anatomía y Anatomía Comparada, Facultad de Veterinaria,Universidad de Murcia, 5Unidad de Trasplante Hematopoyético y Terapia Celular, Departamento de Hematología, Hospital Universitario Virgen de la Arrixaca, IMIB,Universidad de Murcia
Summary
在心脏再生医学新的治疗策略需要广泛和详细的研究在大型前动物模型, 才可以考虑使用在人类。在这里, 我们演示了经皮超声心动图引导心肌注射技术在兔, 这是有价值的假说测试这种新疗法的功效。
Abstract
细胞和基因治疗是令人振奋的和有希望的策略, 以心脏再生的目的在设置心力衰竭与减少射血分数 (HFrEF)。在人们可以考虑使用和实施之前, 在大型动物模型中需要进行广泛的临床前研究, 以评估 injectate (如干细胞) 一旦被送到心肌的安全性、有效性和命运。小啮齿类动物模型提供好处 (例如, 成本效益, 可为基因操纵);然而, 鉴于这些模型的固有局限性, 这些发现很少转化为临床。相反, 大型动物模型, 如兔子, 有优势 (例如, 与人类和其他大型动物相比, 类似的心脏电生理), 同时保持一个良好的成本效益平衡。在这里, 我们演示了如何执行经皮超声心动图引导心肌注射技术, 这是微创, 安全, 耐受性好, 并非常有效的靶向交付 injectates, 包括细胞,在兔子模型的心肌内的几个位置。为了实现这一技术, 我们也利用了广泛使用的临床超声心动图系统。在实践了这里描述的协议之后, 一个具有基本的超声知识的研究人员将会胜任在实验中常规使用的这一多才多艺和微创技术的表现, 目的是假设测试家兔模型的心脏再生疗法的能力。一旦能力达到, 整个程序可以在25分钟内完成麻醉兔。
Introduction
细胞和基因治疗是令人兴奋和不断发展的策略, 以再生/修复损伤心肌 HFrEF。有几项研究比较了不同的细胞传递途径的有效性 (例如、细胞保留率), 这一结果始终如一地证明了在冠状动脉或静脉通路上的优越性,1,2,3,4,5. 因此, 对受损心肌干细胞治疗的转化模型研究的很大比例, 并不令人惊讶, 在一个开放的胸部手术中直接观察到的 injectate 通过在一个直视下进行,6,7.但是, 这种方法有几个限制, 包括程序的侵入性, 这会带来围手术期死亡的风险 (通常是报告的)8。此外, 直接视野下的一个人也不能排除无意中射入心室腔的可能性。在临床实践中, 在开胸手术中, 在冠状动脉旁路移植术 (搭桥) 过程中, 可以采用一种合适的治疗细胞分娩方法, 即例如。然而, 这种方法可能不适合在全球非缺血性心肌病 (如, HFrEF 继发于蒽诱发的心肌病 (AICM)) 的细胞分娩。
毫无疑问, 缺血性心脏病 (IHD) 是最常见的原因 HFrEF (约 66%)9,10; 然而, 非缺血性心肌病, 包括 AICM, 仍然影响了相当比例的患者与 HFrEF (33%)9.事实上, 最近在临床肿瘤学的进展已经导致超过1000万的癌症幸存者在美国单独11, 与一个类似的数字在欧洲的估计, 符合整体趋势, 以改善癌症患者的生存率12 ,13。因此, 探索新疗法的好处, 如干细胞移植治疗非缺血性心肌病, 以及法的有效和微创途径的干细胞传递是至关重要的, 鉴于越来越多的病人受抗癌药物心脏的影响。
值得注意的是, 假设测试的研究采用干细胞疗法, 目的是修复/再生受伤的心肌, 经常涉及使用小啮齿动物 (例如, 老鼠和老鼠)。这些模型通常需要昂贵的高频超声系统来评价心肌功能, 通常配有线性阵列传感器, 它们有一些固有的相关限制 (例如, 混响)14。然而, 其他模型, 如家兔, 代表一个大型的前临床模型, 有一定的优势, 假设测试的干细胞治疗在 HFrEF。因此, 与老鼠和老鼠相比, 兔子保持一个 Ca+2传输系统和细胞电生理学类似的人和其他大型动物 (如如, 狗和猪)15,16,17 ,18,19。另一个优势, 是他们的可的心脏超声成像使用相对廉价和广泛可用的临床超声心动图系统配备了相对高频率相控阵传感器,例如, 12 MHz, 如常用于新生儿和儿科心脏病。这些系统允许优秀的超声心动图成像技术的状态, 他们利用谐波成像的优越性20。此外, 广泛的假设测试的潜力心脏再生疗法 (如, 干细胞治疗), 其安全性, 疗效, 心肌的潜力, 以及评估的命运, injectate 一旦交付到心肌, 是强制性的, 才可以考虑为人类使用, 他们需要使用大型的前临床动物模型, 如兔17,19。在这里, 我们描述了一种微创技术, 通过经皮超声心动图引导下的细胞分娩的临床超声心动图系统, 这是针对干细胞移植治疗非缺血性心肌病20.我们还描述了印度墨水的好处 (InI, 也称为中国墨水) 作为一个超声造影剂和原位示踪的 injectate 在兔心脏。
Protocol
本文所述的实验得到了西班牙穆尔西亚大学伦理研究委员会的批准, 并按照欧洲委员会 2010/63/欧盟的指令进行。所描述的步骤是在标准操作协议下执行的, 这些程序是工作计划的一部分, 没有仅为拍摄所附视频而执行。 1. 细胞和哺乳动物表达载体的制备 注意: 在这里, 我们简要描述了一个细胞系的制备和转染的协议 (人类胚胎肾脏 293 (HEK-293));但是, 应优化单?…
Representative Results
经皮超声心动图引导下与 InI 对照: 使用上述协议, 一旦针尖的最佳定位通过超声心动图和注射启动, 壁 hyperechogenicity 观察在交付的 InI (10% 伏/v 在 PBS) (图 2E), 以及后不久, 到目标区域 (图 2F)。当他紧接着被安乐死和心脏移除后, 在心脏外部检查时很容易看到有 InI 的沉积?…
Discussion
主要目的是开发一种微创技术, 可用于将干细胞输送到家兔的心肌 (一个大型的临床前动物模型)17,18, 同时利用相对低廉的成像系统在许多临床和研究中心随时可用。在这里, 我们表明, 使用临床超声心动图系统, 并辅以 InI, 一个广泛可用的代理, 既具有原位跟踪能力和回声的性质, 成功的经皮超声造影引导下, 是非常有效的把 injectate 送到兔子心脏的…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢希拉 Monfort, 布伦达. 马丁内斯, 卡洛斯 Micó, 阿尔贝托ñ oz 和曼努埃尔-莫林在收集数据时提供了出色的支持, 而卡洛斯布埃诺提供了 EGFP (+) HEK-293 细胞。这项工作得到了部分支持: 基金会 Séneca、75,000 de Ciencia Tecnología、西班牙穆尔西亚 Región (JT) (赠款号: 11935/PI/09);红 de Terapia 细胞, ISCIII-潜艇gral.Redes, VI PN i + D + i 2008-2011 (格兰特号。RD12/0019/0001) (JMM), 由欧洲联盟 (菲德) (JMM) 的结构筹资共同出资;并且, 英国的读书大学 (AG, GB) (中央资助)。资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。
Materials
| HD11 XE Ultrasound System | Philips | 10670267 | Echocardiography system. |
| S12-4 | Philips | B01YgG | 4-12 MHz phase array transducer |
| Ultrasound Transmision Gel (Aquasone) | Parket laboratories Inc | N 01-08 | |
| Vasovet 24G | Braun | REF 381212 | over-the-needle catheter |
| Omnifix-F 1 ml syringe | Braun | 9161406V | |
| Imalgene (Ketamine) | Merial | RN 9767 | Veterinary prescription is necessary |
| Domtor (Medetomidine) | Esteve | CN 570686.3 | Veterinary prescription is necessary |
| Heating Pad | |||
| Faber-Castel TG1 | Faber-Castel | 16 33 99 | India (China) Ink |
| Holter Syneflash | Ela medical | SF0003044S | 24 h Holter ECG system. |
| Electrodes Blue Sensor® | Ambu (NUMED) | VLC-00-S | Holter ECG electrodes. |
| Microtome | Leica Biosystems | RM2155 | |
| Microscope | Olimpus | CO11 | |
| ABC Vector Elite | Vector Laboratories | PK-6200 | Avidin Biotin Complex Kit. |
| Chicken anti-GFP antibody | Invitrogen | A10262 | Primary antibody. |
| Biotinylated goat-anti-chicken IgG Antibody | Vector Laboratories | BA-9010 | Secondary Antibody. |
| 3,30-diaminobenzidine tetrahydrochloride (DAB) | DAKO (Agilent) | S3000 | |
| Fluorescence Microscope | Carl Zeiss MicroImaging |
Zeiss AX10 Axioskop | |
| Holter ECG | Elamedical | Syneflash SF0003044S | |
| Dulbecco’s modified Eagle medium (DMEM) | Fisher Scientific | 11965084 | |
| 10% fetal calf serum (FCS) | Fisher Scientific | 11573397 | |
| 0.05% Trypsin-Ethylenediaminetetraacetic acid (EDTA) | Fisher Scientific | 25300054 | |
| Lipofectamine 2000 (Lipid transfection reagent) | Fisher Scientific | 11668019 | |
| Reduced serum medium (Opti-MEM) | Fisher Scientific | 31985070 | |
| Hygromycin B | Calbiochem (MERCK) | 400051 | |
| Xylene (histological) | Fisher Scientific | X3S-4 | |
| Hydrogen Peroxide Solution (H2O2) | Sigma | H1009 | |
| Pronase | Fisher Scientific | 53-702-250KU |
Referências
- Hou, D., et al. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery: implications for current clinical trials. Circulation. 112, I150-I156 (2005).
- Freyman, T., et al. A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. Eur Heart J. 27, 1114-1122 (2006).
- Perin, E. C., et al. Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction. J Mol Cell Cardiol. 44, 486-495 (2008).
- Dib, N., Khawaja, H., Varner, S., McCarthy, M., Campbell, A. Cell therapy for cardiovascular disease: a comparison of methods of delivery. J Cardiovasc Transl Res. 4, 177-181 (2011).
- Li, S. H., et al. Tracking cardiac engraftment and distribution of implanted bone marrow cells: Comparing intra-aortic, intravenous, and intramyocardial delivery. J Thorac Cardiovasc Surg. 137, 1225-1233 (2009).
- Shiba, Y., et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature. 489, 322-325 (2012).
- Chong, J. J., et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature. 510, 273-277 (2014).
- Lu, C., et al. Autologous bone marrow cell transplantation improves left ventricular function in rabbit hearts with cardiomyopathy via myocardial regeneration-unrelated mechanisms. Heart vessels. 21, 180-187 (2006).
- McMurray, J. J., et al. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC . Eur J Heart Fail. 14, 803-869 (2012).
- Sueta, C. A. The life cycle of the heart failure patient. Curr Cardiol Rev. 11, 2-3 (2015).
- Carver, J. R., et al. American Society of Clinical Oncology clinical evidence review on the ongoing care of adult cancer survivors: cardiac and pulmonary late effects. J Clin Oncol. 25, 3991-4008 (2007).
- Verdecchia, A., et al. Recent cancer survival in Europe: a 2000-02 period analysis of EUROCARE-4 data. Lancet Oncol. 8, 784-796 (2007).
- De Angelis, R., et al. Cancer survival in Europe 1999-2007 by country and age: results of EUROCARE–5-a population-based study. Lancet Oncol. 15, 23-34 (2014).
- Abu-Zidan, F. M., Hefny, A. F., Corr, P. Clinical ultrasound physics. J Emerg Trauma Shock. 4, 501-503 (2011).
- Del, M. F., Mynett, J. R., Sugden, P. H., Poole-Wilson, P. A., Harding, S. E. Subcellular mechanism of the species difference in the contractile response of ventricular myocytes to endothelin-1. Cardioscience. 4, 185-191 (1993).
- Pogwizd, S. M., Bers, D. M. Rabbit models of heart disease. Drug Discov Today Dis Mod. 5, 185-193 (2008).
- Gandolfi, F., et al. Large animal models for cardiac stem cell therapies. Theriogenology. 75, 1416-1425 (2011).
- Harding, J., Roberts, R. M., Mirochnitchenko, O. Large animal models for stem cell therapy. Stem Cell Res Ther. 4, 23 (2013).
- Chong, J. J., Murry, C. E. Cardiac regeneration using pluripotent stem cells–progression to large animal models. Stem Cell Res. 13, 654-665 (2014).
- Talavera, J., et al. An Upgrade on the Rabbit Model of Anthracycline-Induced Cardiomyopathy: Shorter Protocol, Reduced Mortality, and Higher Incidence of Overt Dilated Cardiomyopathy. BioMed Res Int. 2015, 465342 (2015).
- Bueno, C., et al. Human adult periodontal ligament-derived cells integrate and differentiate after implantation into the adult mammalian brain. Cell Transplant. 22, 2017-2028 (2013).
- Sahn, D. J., DeMaria, A., Kisslo, J., Weyman, A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation. 58, 1072-1083 (1978).
- Thomas, W. P., et al. Recommendations for standards in transthoracic two-dimensional echocardiography in the dog and cat. Echocardiography Committee of the Specialty of Cardiology, American College of Veterinary Internal Medicine. J Vet Intern Med. 7, 247-252 (1993).
- Lang, R. M., et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. Eur Heart J Cardiovasc Imaging. 16, 233-270 (2015).
- Feldman, A. T., Wolfe, D. Tissue processing and hematoxylin and eosin staining. Methods Mol Biol. 1180, 31-43 (2014).
- Howat, W. J., Wilson, B. A. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods. 70, 12-19 (2014).
- Cohen, A. H. Masson’s trichrome stain in the evaluation of renal biopsies. An appraisal. Am J Clin Pathol. 65, 631-643 (1976).
- Corti, R., et al. Real time magnetic resonance guided endomyocardial local delivery. Heart. 91, 348-353 (2005).
- Springer, M. L., et al. Closed-chest cell injections into mouse myocardium guided by high-resolution echocardiography. Am J Physiol Heart Circ Physiol. 289, H1307-H1314 (2005).
- Aoki, M., et al. Efficient in vivo gene transfer into the heart in the rat myocardial infarction model using the HVJ (Hemagglutinating Virus of Japan)–liposome method. J Mol Cell Cardiol. 29, 949-959 (1997).
- Guzman, R. J., Lemarchand, P., Crystal, R. G., Epstein, S. E., Finkel, T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circ Res. 73, 1202-1207 (1993).
- Magovern, C. J., et al. Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector. Ann Thorac Surg. 62, 425-433 (1996).
- Suzuki, K., et al. Role of interleukin-1beta in acute inflammation and graft death after cell transplantation to the heart. Circulation. 110, II219-II224 (2004).
- Fukushima, S., et al. Direct intramyocardial but not intracoronary injection of bone marrow cells induces ventricular arrhythmias in a rat chronic ischemic heart failure model. Circulation. 115, 2254-2261 (2007).
- Vela, D., Maximilian Buja, L., Miller, L. W., Taylor, D. A., Willerson, J. T. . Stem Cell and Gene Therapy for Cardiovascular Disease. , 13-23 (2016).
- Fargas, A., Roma, J., Gratacos, M., Roig, M. Distribution and effects of a single intramuscular injection of India ink in mice. Ann Anat. 185, 183-187 (2003).
- Dib, N., et al. Recommendations for successful training on methods of delivery of biologics for cardiac regeneration: a report of the International Society for Cardiovascular Translational Research. JACC Cardiovasc Interv. 3, 265-275 (2010).
- Mu, Y., Cao, G., Zeng, Q., Li, Y. Transplantation of induced bone marrow mesenchymal stem cells improves the cardiac function of rabbits with dilated cardiomyopathy via upregulation of vascular endothelial growth factor and its receptors. Exp Biol Med (Maywood). 236, 1100-1107 (2011).
- Giraldo, A., et al. Percutaneous intramyocardial injection of amniotic membrane-derived mesenchymal stem cells improves ventricular function and survival in non-ischaemic cardiomyopathy in rabbits. Eur Heart J. 36, 149 (2015).
- Giraldo, A., et al. Allogeneic amniotic membrane-derived mesenchymal stem cell therapy is cardioprotective, restores myocardial function, and improves survival in a model of anthracycline-induced cardiomyopathy. Eur J Heart Fail. 19, 594 (2017).
- Prendiville, T. W., et al. Ultrasound-guided transthoracic intramyocardial injection in mice. J Vis Exp. , e51566 (2014).
- Laakmann, S., et al. Minimally invasive closed-chest ultrasound-guided substance delivery into the pericardial space in mice. Naunyn Schmiedebergs Arch Pharmacol. 386, 227-238 (2013).
- Hasenfuss, G. Animal models of human cardiovascular disease, heart failure and hypertrophy. Cardiovasc Res. 39, 60-76 (1998).
- Ponikowski, P., et al. Depressed heart rate variability as an independent predictor of death in chronic congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol. 79, 1645-1650 (1997).
- Nolan, J., et al. Prospective study of heart rate variability and mortality in chronic heart failure: results of the United Kingdom heart failure evaluation and assessment of risk trial (UK-heart). Circulation. 98, 1510-1516 (1998).
- Galinier, M., et al. Depressed low frequency power of heart rate variability as an independent predictor of sudden death in chronic heart failure. Eur Heart J. 21, 475-482 (2000).
- Sheng, C. C., Zhou, L., Hao, J. Current stem cell delivery methods for myocardial repair. BioMed Res Int. 2013, 547902 (2013).
- Kim, R. J., et al. The use of contrast-enhanced magnetic resonance imaging to identify reversible myocardial dysfunction. N Engl J Med. 343, 1445-1453 (2000).
- Perin, E. C., et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation. 107, 2294-2302 (2003).
Citar este artigo
Giraldo, A., Talavera López, J., Fernandez-Del-Palacio, M. J., García-Nicolás, O., Seva, J., Brooks, G., Moraleda, J. M. Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model. J. Vis. Exp. (131), e56699, doi:10.3791/56699 (2018).