循环死亡后心脏捐献的临床前模型
1Centre de recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), 2Deparment of pharmacology and physiology, Faculty of Medicine,Université de Montréal, 3Division of Cardiovascular Surgery,Toronto General Hospital, University Health Network, 4Department of surgery, Faculty of Medicine,Université de Montréal
Summary
该协议显示了一种简单而灵活的方法,用于评估新的调理剂或策略,以提高循环死亡后心脏捐赠的可行性。
Abstract
心脏移植需求呈上升趋势;然而,由于缺乏合适的捐赠者,器官的可得性有限。循环死亡后器官捐献 (DCD) 是解决这种有限可用性的解决方案,但由于长期温暖的缺血期和组织损伤的风险,其常规使用在心脏移植中很少见。在本手稿中,我们提供了一个详细的方案,通过持续监测心脏功能,在DCD的背景下密切模仿当前的临床实践,允许评估新的心脏保护策略和干预措施,以减少缺血性再灌注损伤。
在此模型中,DCD协议通过停止通气诱导循环死亡在麻醉的Lewis大鼠中启动。当收缩压降至 30 mmHg 以下时,开始出现温暖的缺血时间。在预先设定的温暖缺血期后,心脏被冲洗与规范的性心液溶液,采购,并安装在兰根多夫前体内心脏灌注系统。在初始再灌注和稳定10分钟后,使用室内压力监测持续评估心脏修复60分钟。通过测量心脏肌钙蛋白 T 来评估心脏损伤,并且通过组织染色量化梗死大小。温暖的缺血时间可以调整和定制,以发展所需的结构和功能损伤量。这个简单的协议允许评估在心痛、初始再灌注和/或前体灌注时引入的不同心内保护调节策略。从该协议获得的发现可以复制在大型模型中,便于临床翻译。
Introduction
固体器官移植,特别是心脏移植,在全球呈上升趋势。”器官采购的标准方法是脑死亡后捐赠(DBD)。鉴于DBD的严格包容标准,只有不到40%的被录取的心被接受3,从而限制了在面对不断增长的需求和扩大器官等待名单的报价。为了解决这个问题,使用循环死亡后捐赠的器官(DCD)被认为是一个潜在的解决方案4。
然而,在DCD捐赠者中,在戒断护理后出现一个前发期,以及复苏前一段未受保护的温暖缺血期是不可避免的。循环死亡后潜在的器官损伤可能导致器官功能障碍,这解释了人们不愿意常规地采用DCD心脏移植。据报道,只有4个中心使用DCD心脏临床,与严格的标准,包括很短的温暖缺血时间和年轻的捐赠者没有慢性病理6,7。出于伦理和法律原因,在循环死亡5、8、9之前,对捐献者可以进行有限或没有心脏保护干预。因此,任何缓解缺血性再灌注(IR)损伤的缓解措施仅限于在用心电图溶液进行早期再灌注期间启动的心脏病保护疗法,并且不允许进行适当的功能评估。外体心脏灌注(EVHP)和修复DCD心脏使用专用平台已被提出作为替代解决方案,并研究由各种学者10,11,12,13.EVHP 提供了一个独特的机会,将后调理剂交付给 DCD 心脏,以改善功能恢复。然而,为了有效的临床翻译,许多技术和实际问题仍有待解决,而由于缺乏一系列灌注和功能标准来确定可移植性,情况进一步复杂化了。 8.
本文报告开发一种可重复的临床前小动物DCD协议,结合一个体外心脏灌注系统,可用于研究在采购时,在初始再灌注期间启动的器官后调节,以及/或在整个 EVHP 中。
Protocol
所有动物护理和实验规程均符合《实验室动物护理和使用指南》,并经蒙特利尔大学研究中心动物护理和使用中心机构动物护理和使用委员会批准。 1. 初步准备工作 打开水浴加热心痛输送系统(图1A)和兰根多夫前体内灌注系统(图1B)。将水温设置为 38.5 °C,溶液温度为 37°C。设置照片可以在补充图1A,B中看到。 <…
Representative Results
排泄后,血压迅速下降,以可预测的模式(图3)。预计死亡时间小于5分钟。 图 4显示了 WIT 0、10 和 15 分钟之后修复开始时的平均压力/时间曲线。收缩功能将随着时间的推移而改善。短时间的 WIT 将使收缩性恢复正常,并且形态损伤将不可检测(图 5和图 6)。 与心胸结合一?…
Discussion
这里提出的协议介绍了一个简单,方便和多功能的心脏DCD模型,提供了机会,以评估心脏功能恢复,组织损伤和使用后心形保护剂,以改善捐赠者的恢复心脏否则被丢弃移植。Ex vivo 心脏灌注系统 (EVHP) 系统经过优化,为评估心脏功能提供了一个平台,并提供独特的机会来提供和测试经过改进的解决方案,并辅以后调理药理剂保存和修复DCD心脏在小15和大动物16,17模型的心脏DCD。…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作的部分内容得到了马塞尔基金会和罗兰·戈塞林基金会和斯特凡·福米基金会的慷慨捐助的支持。尼古拉斯·诺雷克斯是FRQ-S的学者。
作者们感谢乔希·卓乐·黄、加布里埃尔·加斯康、索菲亚·吉亚西和凯瑟琳·斯卡拉布里尼在数据收集方面的支持。
Materials
| 0,9% Sodium Chloride. 1L bag | Baxter | Electrolyte solution for flushing in the modified Langendorff system. | |
| 14G 2" I.V catheter | Jelco | 4098 | To act as endotracheal tube. |
| 2,3,5-Triphenyltetrazolium chloride | Milipore-Sigma | T8877 | Vital coloration |
| 22G 1" I.V catheter | BD | 383532 | I.V catheter with extension tube that facilitates manipulation for carotid catheterization |
| Adson Dressing Fcp, 4 3/4", Serr | Skalar | 50-3147 | Additional forceps for tissue manipulation |
| Alm Self-retaining retractor 4×4 Teeth Blunt 2-3/4" | Skalar | 22-9027 | Tissue retractor used to maintain the chest open. |
| Bridge amp | ADinstruments | FE221 | Bridge amp for intracarotid blood pressure measurement |
| Calcium chloride | Milipore-Sigma | C1016 | CaCl2 anhydrous, granular, ≤7.0 mm, ≥93.0% Part of the Krebs solution |
| D-(+)-Glucose | Milipore-Sigma | G8270 | D-Glucose ≥99.5% Part of the Krebs solution |
| DIN(8) to Disposable BP Transducer | ADinstruments | MLAC06 | Adapter cable for link between bridge amp and pressure transducer |
| Disposable BP Transducer (stopcock) | ADinstruments | MLT0670 | Pressure transducer for intracarotid blood pressure measurement |
| dPBS | Gibco | 14190-144 | Electrolyte solution without calcium or magnesium. |
| Eye Dressing Fcp, Str, Serr, 4" | Skalar | 66-2740 | Additional forceps for tissue manipulation |
| Formalin solution, neutral buffered, 10% | Milipore-Sigma | HT501128 | Fixative solution |
| Heating Pad | Sunbean | 756-CN | |
| Heparin sodium 1000 UI/mL | Sandoz | For systemic anticoagulation | |
| Hydrochloric Acid 36,5 to 38,0% | Fisher scientific | A144-500 | Diluted 1:1 for pH correction |
| Ketamine | Bimeda | Anesthetic. 100 mg/ml | |
| LabChart | ADinstruments | Control software for the Powerlab polygraph, allowing off-line analyses. Version 7, with blood pressure and PV loop modules enabled | |
| Left ventricle pressure balloon | Radnoti | 170404 | In latex. Size 4. |
| Lidocaine HCl 2% solution | AstraZeneca | Antiarrhythmic for the cardioplegic solution | |
| Magnesium Chloride ACS | ACP Chemicals | M-0460 | MgCl2+6H2O ≥99.0% Part of the Krebs solution |
| Micro pressure sensor | Radnoti | 159905 | Micro pressure sensor and amplifier connected to the intraventricular balloon |
| Pacemaker | Biotronik | Reliaty | Set to generate a pulse each 200 ms for a heart rate of 300 bpm. |
| pH bench top meter | Fisher scientific | AE150 | |
| Physiological monitor | Kent Scientific | Physiosuite | For continuous monitoring of rodent temperature and saturation during the procedure |
| Plasma-Lyte A | Baxter | Electrolyte solution used as base to prepare cardioplegia | |
| Potassium Chloride | Milipore-Sigma | P4504 | KCl ≥99.0% Part of the Krebs solution |
| Potassium Chloride 2 meq/ml | Hospira | Part of the cardioplegic solution | |
| PowerLab 8/30 Polygraph | ADinstruments | Electronic polygraph | |
| Silk 2-0 | Ethicon | A305H | Suture material for Langendorff apparatus |
| Silk 5-0 | Ethicon | A302H | Suture material for carotid |
| Small animal anesthesia workstation | Hallowell EMC | 000A2770 | Small animal ventilator |
| Sodium bicarbonate | Milipore-Sigma | S5761 | NaHCO3 ≥99,5% Part of the Krebs solution |
| Sodium Chloride | Milipore-Sigma | S7653 | NaCl ≥99.5% Part of the Krebs solution |
| Sodium Hydroxide pellets | ACP chemicals | S3700 | Diluted to 5N (10 g in 50 ml) for pH correction |
| Sodium phosphate monobasic | Milipore-Sigma | S0751 | NaH2PO4 ≥99.0% Part of the Krebs solution |
| Stevens Tenotomy Sciss, Str, Delicate, SH/SH, 4 1/2" | Skalar | 22-1240 | Small scisors for atria and cava vein opening |
| Tissue slicer blades | Thomas scientific | 6727C18 | Straight carbon steel blades for tissue slicing at the end of the protocol |
| Tuberculin safety syringe with needle 25G 5/8" | CardinalHealth | 8881511235 | For heparin injection |
| Veterinary General Surgery Set | Skalar | 98-1275 | Surgery instruments including disection scisors and mosquito clamps |
| Veterinary Micro Set | Skalar | 98-1311 | Surgery instruments with microscisors used for carotid artery opening |
| Working Heart Rat/Guinea Pig/Rabbit system | Radnoti | 120101BEZ | Modular working heart system modified for the needs of the protocol. Includes all the necesary tubbing, water jacketed reservoirs and valves, including 2 and 3 way stop cock |
| Xylazine | Bayer | Sedative. 20 mg/ml |
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Citazione di questo articolo
Aceros, H., Joulali, L., Borie, M., Ribeiro, R. V. P., Badiwala, M. V., Der Sarkissian, S., Noiseux, N. Pre-clinical Model of Cardiac Donation after Circulatory Death. J. Vis. Exp. (150), e59789, doi:10.3791/59789 (2019).