此处，我们介绍一种在常温下异位移植心脏后的评估方案 离体 大鼠模型中的保存
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本文内容
勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
心脏移植是治疗终末期心力衰竭最有效的疗法。尽管治疗手段和干预措施不断进步，等待移植的心力衰竭患者数量仍在持续增加。常温 离体 保存技术已被确立为与传统静态冷保存技术相媲美的方法。该技术的主要优势在于供体心脏可在生理条件下保存长达12小时。此外，该技术可在循环死亡后对供体心脏进行复苏，并实施必要的药理学干预，以改善移植后的供体心脏功能。已有多种动物模型被建立，以优化常温机械灌注 离体 保存技术并消除与保存相关的并发症。尽管与小型动物模型相比，大型动物模型更容易操作，但其成本较高且存在挑战性。我们介绍一种大鼠的常温 离体 供体心脏保存后进行异位腹部移植。该模型成本相对较低，可由单个实验人员完成。
引言
心脏移植仍是治疗难治性心力衰竭的唯一有效疗法1,2,3,4。尽管需要心脏移植的患者数量持续增加，但供体器官的可获得性并未相应提高5。为应对这一问题，研究人员已开发出多种新型供心保存方法，旨在改善相关挑战并提高供体器官的可获得性6,7,8,9。
使用器官维护系统（OCS）设备进行常温离体心脏灌注（NESHP）已作为一种临床干预手段出现1,3。该技术被认为是传统静态冷保存（SCS）方法的一种合适替代方案2,9。NESHP能有效缩短供体器官的冷缺血时间，降低代谢需求，并在器官运输....
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方案
本研究的所有动物实验均经全南国立大学医院实验动物研究中心伦理委员会批准（批准号：CNU IACUC - H - 2022-36）。实验所用雄性Sprague-Dawley大鼠（350-450 g）的饲养和护理均遵循《实验动物护理与使用指南》的规定。大鼠饲养于温度可控的环境中，光照周期为12小时明/12小时暗，自由摄取标准饲料和饮水。
1. 准备
注意：单个实验人员即可完成所有实验操作。
- 组装Langendorff装置，包括氧合器、泵和灌流管路，在手术前完成（图2用20 mL生理盐水填充灌注回路，并循环至回路被自体血液充分预充。
注意：此步骤的目的是预热体外循环管路。 - 将心脏停跳液管路连接至循环回路 通过 连接至主动脉插管的三通阀，并准备注射泵以进行最终的心肌停搏液灌注。
注意：确保从灌注回路和心脏停搏液管路中排除所有气泡。 - 将温度传感器置于将要存放供体心脏的储液器内，使循环系统的温度维持在 37 °C。
- 手术准备
- 为每只供体和受体大鼠分别准备一套独立的无菌显微器械和材料。
- 准备供体手术器械包：手术剪一把、显微镊一把、锐利的蚊式钳一把、5-0 丝线缝合....
- 为每只供体和受体大鼠分别准备一套独立的无菌显微器械和材料。
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结果
图1 展示了在小型动物模型中使用的实验设计。图2 显示了改良的朗根多夫灌注装置，该装置包含一个小型动物氧合器。图3 展示了异位腹部移植的吻合顺序。
图4 显示用于评估心脏存活性的参数 离体 灌注，例如乳酸、钾和平均主动脉压。在本研究中，采用常温 原位以外 保存使六例成功病例的总缺血时间缩短至46.2 ± 4.7分钟，而总离体时间为166.2 ± 4.7分钟（图5）。供体心脏的摘取及移植前准备 原位以外 灌注和异位移植耗时5.8 ± 1.3分钟，如图所示 图5手术的总体成功率为70%，六例成功病例的平均吻合时间为38.4 ± 3.4分钟。在所有实验中，植入后心率均显著下降，但随.......
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讨论
我们建立该模型的重点是复制常温下的人类心脏移植。非射血模型是离体环境中保存供体心脏的常用技术16。尽管射血模型在评估离体灌注期间的心脏功能方面具有诸多优势17，但并不适用于异位移植模型。在异位移植中，植入的供体心脏需要克服受体循环系统中宿主心脏产生的收缩期后负荷压力，从而导致供体心脏功能受限，并在评估中产生低估18。因此，在异位移植中，非射血模型更具优势。在非射血模型中，供体心脏虽被灌注，但不支持受体的循环，从而显著限制了对心脏功能的评估。当功能评估受限时，组织形态学和分子学评价（如组织学染色和印迹分析）可用于检查供体心脏的状态。此外，可利用正电子发射断层扫描（PET）或磁共振成像（MRI）等先进技术评估代谢标志物19。该模型可用于在移植前测试药物和基因干预措施的长期有效性。
许多研究团队已开发出一种常温ex situ（离体）保存模型，并已.......
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致谢
本工作由全南国立大学医院生物医学研究所资助项目B2021-0991以及韩国国家研究基金会项目NRF-2020R1F1A1073921资助
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材料
| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 主动排风系统（AES） | Smiths medical | PC-6769-51A | 用于清除二氧化碳和过量异氟烷 |
| 麻醉机 | Smiths medical | PC-8801-01A | 混合异氟烷与氧气并输送至动物 |
| B20 患者监护仪 | GE medical systems | B20 | 用于监测平均主动脉压和体温 |
| 恒温监测系统 | Harvard apparatus | 55-7020 | 用于监测并维持动物体温 |
| Micro-1 大鼠氧合器 | 东莞科威医疗器械有限公司 | Micro-MO | 用于Langendorff循环中的气体交换 |
| 微穿刺导入器套装 | COOK medical | G48007 | 通过腹主动脉向主动脉弓输送停跳液 |
| 显微镜 | Amscope | MU1403 | 用于放大手术视野（受体） |
| 手术放大镜 | SurgiTel | L2S09 | 用于放大手术视野（供体） |
| 注射泵 | AMP all | SP-8800 | 用于输送停跳液 |
| Transonic 流量传感器 | Transonic | ME3PXL-M5 | 灌注回路流量传感器 |
| Transonic 管路流量模块 | Transonic | TS410 | 流量采集系统 |
| Watson - Marlow 泵 | Harvard apparatus | 010.6131.DAO | 用于灌注液循环的蠕动泵 |
| WBC-1510A | JEIO TECH | E03056D | 加热浴槽 |
| SD大鼠（Sprague-Dawley rats） | 韩国三塔科生物有限公司，韩国乌山市 | ||
| 药物 | |||
| BioHAnce 凝胶滴眼液 | SENTRIX Animal care | 眼部湿润软膏 | |
| 头孢唑林 | JW pharmaceutical | 用于预防性治疗 | |
| Custodiol 器官保存液 | DR, FRANZ KOHLER CHEMIE GMBH | 用于心脏获取 | |
| 双氯芬酸 | 明文制药有限公司 | 用于疼痛控制 | |
| 肝素 | JW pharmaceutical | 抗凝剂 | |
| 胰岛素 | JW pharmaceutical | 激素治疗 | |
| 生理盐水 | JW pharmaceutical | 用于补液治疗 |
参考文献
- Langmuur, S. J. J., et al. Normothermic ex-situ heart perfusion with the organ care system for cardiac transplantation: A meta-analysis. Transplantation. 106 (9), 1745-1753 (2022).
- Ardehali, A., et al.
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重印与许可
勘误
Formal Correction: Erratum: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation
Posted by JoVE Editors on 8/28/2023. Citeable Link.
An erratum was issued for: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation. The Protocol section was updated.
Section 4 of the Protocol was updated from:
4. Implantation
- Preparation of recipient
- Begin the recipient preparation 30 min before the cessation of ex situ perfusion.
- Anesthetize the recipient animal using the same method as mentioned in step 2.2.
- Place the rat in a supine position on the heating pad and insert the temperature probe into the rectum to maintain the body temperature at 37 °C.
- Apply eye lubricant, shave the pubic to the epigastric area, and cleanse the area with an iodine-based scrub and 70% alcohol.
- Medications
- Inject 2 mL of warm saline subcutaneously to compensate for the fluid lost during the surgery. Inject 200 IU of heparin subcutaneously.
- Administer antibiotic prophylaxis by injecting 10 mg/kg cefazolin dissolved in 0.3 mL of saline subcutaneously or intramuscularly.
- Administer pain control by injecting 20 mg/kg of diclofenac subcutaneously.
- Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure.
- Prevent dehydration by wrapping the abdominal organs with warm and wet gauze. Intermittingly spread warm saline with a 50 mL syringe during the surgery.
- Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs to expose the Abd. A. and IVC. Prepare the Abd. A and IVC for anastomosis and systematically implant the donor heart, in accordance with Figure 3 or previously documented methods15.
NOTE: Do not separate the Abd. A. and IVC.- Assuming vascular anastomosis to be placed infrarenal, prepare a sufficient portion of the aorta and IVC for clamping.
- Perform blunt preparation using cotton swabs or sharp-serrated forceps to remove the fats and fascia around the vessels.
- Place 5-0 silk ligatures to the mesenteric branches and both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures. Remember to spare the testicular arteries and veins and do not clamp them.
- Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Be sure to switch off the heating pad before placing the clamps, as excess heating can exacerbate limb ischemia.
- Puncture the aorta using a 27 G needle and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta (Asc. A), which is approximately 5 mm.
- Make a longitudinal incision on the IVC in the same way as the aortotomy, but make it 3 mm closer to the caudal side compared to the aorta incision.
- Starting the anastomoses, placed the donor heart on the right side of the recipient's abdomen and attach the donor Asc. A to the recipient's Abd. A with one simple interrupted stitch (9-0 polypropylene) at the cranial corner of the longitudinal incision.
- Move the heart to the left side of the recipient abdomen and perform anastomosis of the donor's Asc. A with the recipient's Abd. A using a running 9-0 polypropylene suture.
- Fixate the donor pulmonary artery to the IVC with two interrupted sutures (9-0 polypropylene) at the caudal and cranial corners of the longitudinal incision.
- Perform the first half of the venous anastomosis from the intraluminal side of the vessel and complete the second half from the extraluminal side of the vessel. Before tightening the knots, flush the field with saline to prevent air embolism.
- De-airing and de-clamping
- Remove the mesenteric vein clamp first after completing the anastomosis to allow the right side of the heart to fill with venous blood.
- Remove the air in the coronary circuit and Asc. A. by applying retrograde coronary perfusion for several seconds.
- Place a piece of gauze on both sides of the vessels and remove the caudal clamp and the cranial clamp.
- Apply gentle compression with cotton swabs for 1-2 min. After ensuring adequate hemostasis, remove the swabs and wash the anastomoses with warm saline.
NOTE: The heart should begin beating within the first minute of reperfusion. If the recipient rat's body temperature is below 35 °C, the heart rhythm will normalize after the temperature reaches 36 °C.
- Replace the abdominal organs in a meander-like manner and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.
to:
4. Implantation
- Preparation of recipient
- Begin the recipient preparation 30 min before the cessation of ex situ perfusion.
- Anesthetize the recipient animal using the same method as mentioned in step 2.2.
- Place the rat in a supine position on the heating pad and insert the temperature probe into the rectum to maintain the body temperature at 37 °C.
- Apply eye lubricant, shave the pubic to the epigastric area, and cleanse the area with an iodine-based scrub and 70% alcohol.
- Medications
- Inject 2 mL of warm saline subcutaneously to compensate for the fluid lost during the surgery. Inject 200 IU of heparin subcutaneously.
- Administer antibiotic prophylaxis by injecting 10 mg/kg cefazolin dissolved in 0.3 mL of saline subcutaneously or intramuscularly.
- Administer pain control by injecting 20 mg/kg of diclofenac subcutaneously.
- Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure.
- Prevent dehydration by wrapping the abdominal organs with warm and wet gauze. Intermittingly spread warm saline with a 50 mL syringe during the surgery.
- Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs to expose the Abd. A. and IVC. Prepare the Abd. A and IVC for anastomosis and systematically implant the donor heart, in accordance with Figure 3 or previously documented methods15.
NOTE: Do not separate the Abd. A. and IVC.- Assuming vascular anastomosis to be placed infrarenal, prepare a sufficient portion of the aorta and IVC for clamping.
- Perform blunt preparation using cotton swabs or sharp-serrated forceps to remove the fats and fascia around the vessels.
- Place 5-0 silk ligatures to the mesenteric branches and both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures. Remember to spare the testicular arteries and veins and do not clamp them.
- Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Switch off the heating pad before placing the clamps, as excess heating can exacerbate limb ischemia. Ensure to switch on the heating pad after de-clamping the vessels to avoid hypothermia.
- Puncture the aorta using a 27 G needle and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta (Asc. A), which is approximately 5 mm.
- Make a longitudinal incision on the IVC in the same way as the aortotomy, but make it 3 mm closer to the caudal side compared to the aorta incision.
- Starting the anastomoses, placed the donor heart on the right side of the recipient's abdomen and attach the donor Asc. A to the recipient's Abd. A with one simple interrupted stitch (9-0 polypropylene) at the cranial corner of the longitudinal incision.
- Move the heart to the left side of the recipient abdomen and perform anastomosis of the donor's Asc. A with the recipient's Abd. A using a running 9-0 polypropylene suture.
- Fixate the donor pulmonary artery to the IVC with two interrupted sutures (9-0 polypropylene) at the caudal and cranial corners of the longitudinal incision.
- Perform the first half of the venous anastomosis from the intraluminal side of the vessel and complete the second half from the extraluminal side of the vessel. Before tightening the knots, flush the field with saline to prevent air embolism.
- De-airing and de-clamping
- Remove the mesenteric vein clamp first after completing the anastomosis to allow the right side of the heart to fill with venous blood.
- Remove the air in the coronary circuit and Asc. A. by applying retrograde coronary perfusion for several seconds.
- Place a piece of gauze on both sides of the vessels and remove the caudal clamp and the cranial clamp.
- Apply gentle compression with cotton swabs for 1-2 min. After ensuring adequate hemostasis, remove the swabs and wash the anastomoses with warm saline.
NOTE: The heart should begin beating within the first minute of reperfusion. If the recipient rat's body temperature is below 35 °C, the heart rhythm will normalize after the temperature reaches 36 °C.
- Replace the abdominal organs in a meander-like manner and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.
- After the surgery, place the anesthetized animal on a clean area over a heating pad until the body temperature reaches 37°C.
NOTE: Do not initiate the postoperative examinations till the body temperature reaches 37°C. Maintain anesthesia at 2-2.5% isoflurane until the end of the experiments. - Monitor ECG of the transplanted donor heart for 3 h. Then, excise the heart under deep anesthesia for histological studies.
NOTE: Confirm anesthesia depth via lack of pedal reflex before excising the heart. The surgical procedure and the ECG monitoring take less than 6 h. Diclofenac, administered perioperatively (step 4.2.3.), enables pain management for the entire duration of this procedure. The analgesia regimen can be adjusted per the institutional animal use guidelines.
