小鱼肾注射液循环系统中微粒的简易有效管理与可视化
1Institute of Biology at Irkutsk State University, 2Institute of Biology at Karelian Research Centre of Russian Academy of Sciences, 3Baikal Research Centre, 4Institute of Materials Research and Engineering, Agency for Science, Technology, and Research (A*STAR), 5University of Oulu, Optoelectronics and Measurement Techniques Laboratory
Summary
本文阐述了快速、微创荧光微粒注入小鱼循环系统的原理和鱼血中微粒的体内可视化。
Abstract
将微粒径粒子系统地管理到生物体中, 可用于血管可视化、药物和疫苗的提供、转基因细胞的植入和微小的光学传感器。然而, 静脉注射 microinjections 的小动物, 主要用于生物和兽医实验室, 是非常困难的, 需要训练有素的人员。在此, 我们展示了一种健壮和有效的方法, 引入微粒进入成年斑马鱼 (斑马斑马) 的循环系统, 注入到鱼肾。为了对微血管中引入的微粒进行可视化, 我们提出了一种简单的鱼鳃活体成像技术。在体内监测斑马鱼的血液 pH 值是使用注射微胶囊荧光探针, SNARF-1, 以证明其中一个可能的应用的描述技术。本文详细介绍了 pH 敏感染料的封装, 并说明了该微胶囊在荧光信号活体记录中快速注射和可视化的原理。所提出的注射方法的特点是低死亡率 (0-20%) 和高效率 (70-90% 成功), 并易于使用常用设备。所有被描述的规程可以执行对其他小鱼种类, 例如孔雀鱼和鳉。
Introduction
在药物和疫苗的提供1、血管可视化2、转基因细胞植入3和微型光学传感器植入等领域, 微粒径颗粒的管理是一项重要任务。4,5. 然而, 微型微粒进入小实验动物的血管系统的植入程序是困难的, 特别是对于微妙的水生生物。对于像斑马鱼这样的热门研究标本, 建议使用视频协议来澄清这些程序。
心内和毛细血管 microinjections 需要训练有素的人员和独特的显微外科设施, 以提供 microobjects 到斑马鱼的血液。此前,3是一种简便有效的全细胞管理方法。然而, 在我们的经验中, 由于眼毛管网络的面积很小, 这项技术需要大量的实践才能达到预期的效果。
在此, 我们描述了一种方法, 通过手动注射直接注入到富含毛细血管和肾血管的成年斑马鱼的肾脏组织中, 以强力有效的微粒注入循环系统。该技术是基于视频协议的细胞移植到斑马鱼肾脏6, 但创伤和耗时的显微手术步骤被淘汰。该方法具有低死亡率 (0-20%) 和高效率 (70-90% 成功) 的特点, 易于使用常用设备。
建议的协议的一个重要部分是可视化的植入微粒 (如果它们是荧光或彩色) 在鳃毛细血管, 这允许验证注射质量, 一个粗略的相对评估的数量注入粒子, 并检测光谱信号的生理测量直接从循环血液。作为描述技术的可能应用的一个例子, 我们展示了在活体测量斑马鱼血液 pH 值使用微胶囊化荧光探针, SNARF-1, 最初建议在 Borvinskaya 等.20175。
Protocol
所有实验程序都是按照欧盟 2010/63/欧盟关于动物实验的指令进行的, 并经伊尔库茨克州立大学生物研究所动物学科研究委员会批准。 1. 微胶囊的制备 注: 携带荧光染料的微胶囊采用相反带电聚电解质7、8的逐层组装制备。所有的程序都是在室温下进行的。 为了合成多孔碳酸钙3 microcores 包围荧光?…
Representative Results
得到的结果来自三个主要类别的协议: 荧光染料的封装形成荧光微粒 (图 1), 肾脏注射微囊的进一步可视化鳃毛细血管 (图 2 和 3), 最后,在体内光谱记录 SNARF-1 荧光监测血液 pH 值 (图 4)。 逐层方法使用模板碳酸钙3芯的涂?…
Discussion
为了证明微粒注入斑马鱼肾脏, 我们使用了半渗透性微胶囊加载的指示染料。因此, 该协议包含使用相反带电聚电解质7、8、15、16、17 的逐层组装制备微囊的指示 ,18 (图 1A)。这项技术的优点是它易于使用现有的实验室设备。根?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者非常感谢伯登 Osadchiy 和叶甫 Protasov (伊尔库茨克州立大学) 在编写视频协议方面的帮助。这项研究得到了俄罗斯科学基金会 (#15 14-10008) 和俄罗斯基础研究基金会 (#15-29-01003) 的支持。
Materials
| SNARF-1-dextran, 70000 MW | Thermo Fisher Scientific | D3304 | Fluorescent probe. Any other appropriate polymer-bound fluorescent dye can be used as a microcapsule filler |
| Albumin-fluorescein isothiocyanate conjugate (FITC-BSA) | SIGMA | A9771 | Fluorescent probe |
| Rhodamine B isothiocyanate-Dextran (RITC-dextran) | SIGMA | R9379 | Fluorescent probe |
| Calcium chloride | SIGMA | C1016 | CaCO3 templates formation |
| Sodium carbonate | SIGMA | S7795 | CaCO3 templates formation |
| Poly(allylamine hydrochloride), MW 50000 (PAH) | SIGMA | 283215 | Cationic polymer |
| Poly(sodium 4-styrenesulfonate), MW 70000 (PSS) | SIGMA | 243051 | Anionic polymer |
| Poly-L-lysine [20 kDa] grafted with polyethylene glycol [5 kDa], g = 3.0 to 4.5 (PLL-g-PEG) | SuSoS | PLL(20)-g[3.5]-PEG(5) | Final polymer to increase the biocompatibility of microcapsules |
| Sodium chloride | SIGMA | S8776 | To dissolve applied polymers |
| Water Purification System | Millipore | SIMSV0000 | To prepare deionized water |
| Magnetic stirrer | Stegler | For CaCO3 templates formation | |
| Eppendorf Research plus pipette, 1000 µL | Eppendorf | Dosing solutions | |
| Eppendorf Research plus pipette, 10 µL | Eppendorf | Dosing solutions | |
| Pipette tips, volume range 200 to 1000 µL | F.L. Medical | 28093 | Dosing solutions |
| Pipette tips, volume range 0.1-10 μL | Eppendorf | Z640069 | Dosing solutions |
| Mini-centrifuge Microspin 12, High-speed | BioSan | For microcapsule centrifugation-washing procedure | |
| Microcentrifuge tubes, 2 mL | Eppendorf | Z666513 | Microcapsule synthesis and storage |
| Shaker Intelli-mixer RM-1L | ELMY Ltd. | To reduce microcapsule aggregation | |
| Ultrasonic cleaner | To reduce microcapsule aggregation | ||
| Head phones | To protect ears from ultrasound | ||
| Ethylenediaminetetraacetic acid | SIGMA | EDS | To dissolve the CaCO3 templates |
| Monosodium phosphate | SIGMA | S9638 | Preparation of pH buffers |
| Disodium phosphate | SIGMA | S9390 | Preparation of pH buffers |
| Sodium hydroxide | SIGMA | S8045 | To adjust the pH of the EDTA solution and buffers |
| Thermostat chamber | To dry microcapsules on glass slide | ||
| Hemocytometer blood cell count chamber | To investigate the size distribution and concentration of the prepared microcapsules | ||
| Fluorescent microscope Mikmed 2 | LOMO | In vivo visualization of microcapsules in fish blood | |
| Set of fluorescent filters for SNARF-1 (should be chosen depending on the microscope model; example is provided) | Chroma | 79010 | Visualization of microcapsules with fluorescent probes |
| Fiber spectrometer QE Pro | Ocean Optics | Calibration of microcapsules under microscope | |
| Optical fiber QP400-2-VIS NIR, 400 μm, 2 m | Ocean Optics | To connect spectrometer with microscope port | |
| Collimator F280SMA-A | Thorlabs | To connect spectrometer with microscope port | |
| Glass microscope slide | Fisherbrand | 12-550-A3 | Calibration of microcapsules under microscope |
| Coverslips, 22 x 22 mm | Pearl | MS-SLIDCV | Calibration of microcapsules under microscope |
| Glass microcapillaries Intra MARK, 10 µL | Blaubrand | BR708709 | To collect fish blood |
| Clove oil | SIGMA | C8392 | Fish anesthesia |
| Lancet No 11 | Apexmed international B.V. | P00588 | To cut the fish tail and release the steel needle from the tip of insulin autoinjector |
| Heparin, 5000 U/mL | Calbiochem | L6510-BC | For treating all surfaces that come in contact with fish blood during fish blood collection |
| Seven 2 Go Pro pH-meter with a microelectrode | Mettler Toledo | To determine fish blood pH | |
| Insulin pen needles Micro-Fine Plus, 0.25 x 5 mm | Becton, Dickinson and Company | For injection procedure. Any thin needle (Ø 0.33 mm or less) is appropriate | |
| Glass capillaries, 1 x 75 mm | Hirschmann Laborgeräte GmbH & Co | 9201075 | For injection procedure |
| Gas torch | To solder steel needle to glass capillary | ||
| Microinjector IM-9B | NARISHIGE | For precise dosing of microcapsules suspension | |
| Petri dishes, 60 mm x 15 mm, polystyrene | SIGMA | P5481 | For manipulations with fish under anesthesia |
| Plastic spoon | For manipulations with fish under anesthesia | ||
| Damp sponge | For manipulations with fish under anesthesia | ||
| Dissection scissors | Thermo Scientific | 31212 | To remove the gill cover from the fish head |
| Pasteur pipette, 3.5 mL | BRAND | Z331767 | To moisten fish gills |
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Cite This Article
Borvinskaya, E., Gurkov, A., Shchapova, E., Karnaukhov, D., Sadovoy, A., Meglinski, I., Timofeyev, M. Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection. J. Vis. Exp. (136), e57491, doi:10.3791/57491 (2018).