在小鼠中建立下腹部器官和后肢的原点闭路灌注
1Department of Thoracic Surgery,The First Hospital of Jilin University, 2The Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences,University of Colorado Anschutz Medical Campus, 3Key Laboratory of Zoonoses Research, Ministry of Education,Jilin University, 4Department of Cardiology,China-Japan Union Hospital of Jilin University, 5Department of Gastrointestinal, Colorectal and Anal Surgery,China-Japan Union Hospital of Jilin University
Summary
描述小鼠下半身的原位灌注，包括膀胱、前列腺、性器官、骨骼、肌肉和足部皮肤的原位灌注。
Abstract
前活体灌注是研究分离器官（如肝脏、肾脏）功能的重要生理工具。同时，由于小鼠器官体积小，骨、膀胱、皮肤、前列腺和生殖器官的外体灌注具有挑战性或不可行。在这里，我们首次报告小鼠的原位下半身灌注回路，包括上述组织，但绕过主要清除器官（肾脏、肝脏和脾脏）。该回路通过将腹主动脉和劣质静脉冠状动脉和静脉上方以及烧灼外周血管进行开气和建立。灌注通过一个内皮泵进行，其灌注流量保持长达2小时。原位染色与荧光叶管和霍赫斯特溶液证实，微血管已成功覆盖。此小鼠模型是研究病理过程以及药物传递机制、循环肿瘤细胞进入/从肿瘤的迁移/转移以及免疫系统与扩散器官和组织相互作用的非常有用的工具。
Introduction
分离器官灌注最初是为了研究器官生理学移植1,1，2，3，,3并使人们了解器官的功能，不受其他身体系统的干扰。例如，孤立的肾脏和心脏灌注在理解血液动力学的原理和血管活性剂的作用方面非常有用，而肝脏灌注对于理解代谢功能，包括健康和患病组织,4、5、6、75中的药物46,代谢非常重要。此外，灌注研究对于了解用于移植的器官的生存能力和功能至关重要。在癌症研究中，由几组使用小鼠、大鼠和新鲜切除的人体组织8，9描述分离的肿瘤灌注9。在一些孤立的肿瘤灌注中，肿瘤被植入卵巢脂肪垫，以迫使从肠动脉10供应血管的肿瘤生长。Jain小组利用结肠腺癌的分离灌注进行开拓性研究，以了解肿瘤血流动力学和转移8,8，11，12，13。11,12,13其他创新工程外体设置包括一个96孔板为基础的灌注装置，培养初级人类多发性骨髓瘤细胞14和模块化流室工程骨髓架构和功能研究15。
除了生理学和病理学研究，器官灌注还被用来研究药物输送的原理。因此，一组人描述了孤立的大鼠肢体灌注，并研究了植入肉瘤16中脂质体积累的，而另一组则进行了解剖人体肾脏灌注，以研究纳米粒子17的内皮靶向。Ternullo等人使用一种孤立的渗透人体皮肤皮瓣作为接近体内皮肤药物渗透模型18。
尽管在大型器官和组织灌注方面取得了这些进展，但还没有关于小鼠原位灌注模型的报告：（a） 绕过肝脏、脾脏和肾脏等清除器官;（a） 旁路清除器官，如肝脏、脾脏和肾脏;（a） 旁路清除器官，如肝脏、脾脏和肾脏;（a） 旁路清除器官，如肝脏、脾脏和肾脏;（a） 旁白器官;（a） 血液移植器官;（a） 血液灌注模型。（（b） 包括骨盆器官、皮肤、肌肉、生殖器官（男性）、膀胱、前列腺和骨髓。由于这些器官体积小，供应血管，外体可灌注和建立灌注电路不可行。小鼠是癌症和免疫学研究以及药物输送中最重要的动物模型。对小小鼠器官进行功能移植的能力将允许有关药物交付给这些器官的有趣问题，包括植入骨盆的肿瘤（膀胱、前列腺、卵巢、骨髓），以及这些器官疾病的基本生理学和免疫学研究。为了解决这个缺陷，我们在小鼠身上开发了一种原位灌注回路，可以避免组织损伤，比孤立的器官灌注更适合功能研究。
Protocol
此处描述的所有方法都已获得科罗拉多大学机构动物护理和使用委员会 （IACUC） 的批准。 1. 预热灌注系统 手术前准备灌注系统，为所有水套组件（香水储液罐、湿室和盖子）启动一个 37°C 循环水浴，如图 1A中的定制配置所示。确保油管清洁，必要时更换。要限制润混量，请使用湿润室内的气泡陷阱作为润酸盐储液罐（图1…
Representative Results
我们建立了闭路灌注系统，通过腹主动脉和8-10周大小鼠的劣质静脉灌注系统，同时保持灌注缓冲液的体积小于10 mL。 图 3A 显示了使用包含 Hoechst 33342 和 DyLight 649-lectin 的 Ringer 溶液对组织进行吸收后共和图像。肌肉，骨髓，睾丸，膀胱，前列腺和脚部皮肤显示有效的核和血管染色。 图3B 显示3小时正常热灌注后器官的血氧素-eosin染色。 <p class="jove_con…
Discussion
所述电路可用于探究各种研究问题，例如不同血清成分和组织屏障在药物输送或免疫和干细胞贩运中的作用。不同的药物输送系统（如脂质体和纳米粒子）可以添加到香水中，以了解生理和生化因素在输送中的作用。灌注的持续时间可能会有所不同，具体取决于所研究的组织、科学目标和灌注的组成。我们在这里介绍灌注的结果长达2小时使用的基本灌注介质，包括林格的溶液与乳酸和白素。必须…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国家卫生和植物医院CA194058补助金对DS、斯卡格斯药学院ADR种子资助计划（DS）的支持;中国国家自然科学基金（第31771093号），吉林省国际合作项目（第2011180414085GH号），中央高校基础研究基金，JLU科技创新研究团队项目（2017-27，2019TD-36）。
Materials
| Equipment | |||
| 3.5x-90x stereo zoom microscope on boom stand with LED light | Amscope | SKU: SM-3BZ-80S | |
| Carbon dioxide, USP | Airgas healthcare | 19087-5283 | |
| Confocal microscope | NIKON | ECLIPSE Ti2 | |
| Disposable Sterile Cautery Pen with High Temp | FIAB | F7244 | |
| Moist chamber bubble trap (part 6 in Figure 1) | Harvard Apparatus | 733692 | Customized as the perfisate container; also enabled constant pressure perfusion |
| Moist chamber cover with quartz window (part 3 in Figure 1) | Harvard Apparatus | 733524 | keep the chamber's temperature |
| Moist chamber with metal tube heat exchanger | Harvard Apparatus | 732901 | Water-jacketed moist chamber with lid to maintain perfusate and mouse temperature |
| Olsen-Hegar needle holders with suture cutters | Fine Science Tools (FST) | 125014 | |
| Oxygen compressed, USP | Airgas healthcare | C2649150AE06 | |
| Roller pump (part 4 in Figure 1) | Harvard Apparatus | 730113 | deliver perfusate to cannula in the moist chamber |
| SCP plugsys servo control F/Perfusion (part 1 in Figure 1) | Harvard Apparatus | 732806 | control the purfusion speed |
| Silicone pad | Harvard Apparatus | ||
| Silicone tubing set (arrows in Figure 1) | Harvard Apparatus (TYGON) | 733456 | |
| Student standard pattern forceps | Fine Science Tools (FST) | 91100-12 | |
| Surgical Scissors | Fine Science Tools (FST) | 14001-14 | |
| Table for moist chamber | Harvard Apparatus | 734198 | |
| Thermocirculator (part 2 in Figure 1) | Harvard Apparatus | 724927 | circulating water bath for all water-jacketed components |
| Three-way stopcock (part 5 in Figure 1) | Cole-Palmer | 30600-02 | |
| Veterinary anesthesia machine | Highland | HME109 | |
| Materials | |||
| 19-G BD PrecisionGlide needle | BD | 305186 | For immobilizing the Insyte Autoguard Winged needle and scratching the cortical bone |
| 4-0 silk sutures | Keebomed-Hopemedical | 427411 | |
| 6-0 silk sutures | Keebomed-Hopemedical | 427401 | |
| Filter (0.2 µm) | ThermoFisher | 42225-CA | Filter for 5% BSA-RINGER’S |
| Permanent marker | Staedtler | 342-9 | |
| Syringe (10 mL) | Fisher Scientific | 14-823-2E | |
| Syringe (60 mL) | BD | 309653 | Filter for 5% BSA-RINGER’S |
| Reagents | |||
| 1% Evans blue ( w/v ) in phosphate-buffered saline (PBS, pH 7.5) | Sigma | 314-13-6 | |
| 10% buffered formalin | velleyvet | 36692 | |
| BALB/c mice ( 8-10 weeks old ) | Charles River | ||
| Baxter Viaflex lactate Ringer's solution | EMRN Medical Supplies Inc. | JB2324 | |
| Bovine serum albumin | Thermo Fisher | 11021-037 | |
| Cyanoacrylate glue | Krazy Glue | ||
| DyLight-649-lectin | Vector Laboratories,Inc. | ZB1214 | |
| Ethanol (70% (vol/vol)) | Pharmco | 111000190 | |
| Hoechst33342 | Life Technologies | H3570 | |
| Isoflurane | Piramal Enterprises Limited | 66794-017-25 | |
| Phosphate buffered saline | Gibco | 10010023 |
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Cite This Article
Ren, P., Yang, C., Lofchy, L. A., Wang, G., Chen, F., Simberg, D. Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice. J. Vis. Exp. (162), e60847, doi:10.3791/60847 (2020).