使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜
1Department of Physiology and Biophysics,Dalhousie University, 2Department of Microbiology and Immunology,Dalhousie University, 3Department of Neuroscience,Dalhousie University, 4Department of Anesthesia, Pain Management, and Perioperative Medicine,Dalhousie University, 5Live Cell Imaging Center,University of Calgary, 6Department of Physiology and Pharmacology,University of Calgary, 7Department of Biochemistry and Molecular Biology,University of Calgary, 8Department of Pharmacology,Dalhousie University
Summary
活体荧光显微镜可用于实时研究白细胞 – 内皮相互作用和毛细血管灌注。该协议描述了使用真空稳定的肺成像系统在肺微循环中对这些参数进行成像和量化的方法。
Abstract
白细胞-内皮相互作用的活体成像为活体动物免疫介导的疾病提供了有价值的见解。急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）和其他体内 呼吸系统疾病 的研究由于肺部的可及性和固有的运动伪影有限而很困难。尽管如此，已经制定了各种方法来克服这些挑战。该协议描述了一种用于玻璃体内荧光显微镜的方法，用于在ALI的实验模型中研究肺微循环中的实时白细胞 – 内皮相互作用。使用 体内 肺成像系统和3D打印的活体内显微镜平台来保护麻醉小鼠并稳定肺部，同时最大限度地减少混杂的肺损伤。制备后，使用宽视场荧光显微镜研究白细胞粘附，白细胞滚动和毛细管功能。虽然这里介绍的方案侧重于炎症性肺病急性模型中的成像，但它也可能适用于研究肺部的其他病理和生理过程。
Introduction
活体显微镜（IVM）是一种有用的成像工具，用于可视化和研究 体内各种生物物理过程。肺 在体内 成像是非常具有挑战性的，因为它的封闭位置，其组织的脆弱性以及呼吸和心跳引起的运动伪影1，2。已经开发了各种活体显微镜（IVM）装置，用于肺微循环中白细胞 – 内皮相互作用的实时成像，以克服这些挑战。这些方法基于手术暴露和稳定肺部以进行成像。
动物通常通过外科手术为肺病媒体移植做准备。首先，对动物进行插管和通气，这允许手术切除胸窗并随后进行干预以稳定肺部以进行成像。一种技术涉及将实质粘附在玻璃盖玻片3上，该过程可能会对成像组织造成重大的物理创伤。更先进的是利用真空系统在玻璃窗4下稳定肺部。这种设置有助于通过分布在大面积区域的可逆真空将肺表面松散地粘附到盖玻片上，并扩大肺，同时仍然限制x，y和z尺寸4的运动。真空通过围绕装置成像区域的通道均匀施加，并将组织拉入面向成像级盖玻片4的浅锥形区域。通过这个观察窗，可以使用各种光学成像方式研究肺微循环。
肺病媒体能对多种微循环参数进行定量成像。这些包括诸如白细胞轨迹速度和长度5，红细胞流速6和氧合7，肿瘤转移8，免疫细胞亚群9，10，11的区别，微粒的可视化12，肺泡动力学13，14，血管通透性15和毛细血管功能16的测量.这里的重点是白细胞募集和毛细血管功能。肺微循环中白细胞募集的开始涉及白细胞和内皮细胞之间的瞬时滚动相互作用和牢固的粘附相互作用，两者在炎症条件下均增加16，17。通常，滚动通过操作员定义的参考线的白细胞数量来量化，而粘附则通过内皮16上不动的白细胞数量来量化。毛细血管功能也可能在炎症状态下受到影响，通常导致灌注减少。这可以归因于几个因素，包括红细胞变形性的降低18和内皮细胞诱导的NO合酶的杂交表达导致病理性分流19。通常，测量每个区域灌注毛细血管的聚集长度，并将其报告为功能性毛细血管密度（FCD）。
实时研究肺部白细胞募集需要用荧光染料或荧光标记抗体标记生物靶标20。或者，各种转基因小鼠菌株如溶菌酶M-green荧光蛋白（LysM-GFP）小鼠可用于对特异性免疫细胞亚群（如嗜中性粒细胞21，22）进行成像。然后可以使用宽视场荧光显微镜，共聚焦显微镜或多光子显微镜观察荧光标记的白细胞。这些技术通过利用特定的激发波长和检测发射的荧光来实现对比度，同时阻断激发波长的检测，从而突出显示标记的物体。
关于小鼠肺白细胞滚动、粘附和功能毛细血管密度量化的现有研究主要依赖于手动视频分析。这是通过开源软件（如斐济6，23）、专有软件（如CapImage12）或定制图像处理系统24实现的。相反，各种专有软件平台（例如，NIS Element，Imaris，Volocity，MetaMorph）可以自动测量各种其他生理参数，包括前面提到的许多5，6，7，8，9，10，11，12，13，15。
关于使用肺病媒的急性肺损伤 （ALI） 和急性呼吸窘迫综合征 （ARDS） 的病理学，已经做出了重要的观察结果。ARDS的特征在于肺部的一系列病理生理过程，包括由内皮和上皮屏障25功能障碍引起的肺水肿和肺泡损伤。使用小鼠模型，已经发现脓毒症诱导的ALI与肺环境中免疫细胞运输的显着有害变化有关26。发现招募到脓毒症诱导的ALI小鼠毛细血管的嗜中性粒细胞阻碍微循环，从而增加ALI26的缺氧。此外，IVM已被用于深入了解ARDS27发作后的潜在修复机制。肺病媒综合防治也是了解各种阻塞性肺疾病病理生理学变化的宝贵工具。例如，囊性纤维化（CF）和慢性阻塞性肺疾病（COPD）等疾病中粘液运输的可视化促进了对粘液清除的新型和现有治疗方法的研究28。这些条件下的白细胞运输也进行了分析17.
该协议扩展了Lamm等人最初描述的方法，以 使用常规荧光显微镜研究白细胞 – 内皮相互作用。所描述的程序采用 体内 肺成像系统，其中包括16.5 cm x 12.7cm金属底座，显微操作器和真空成像窗口（图1）。该系统安装在20 cm x 23.5 cm的3D打印平台（补充文件1）中，为通风机管和加热垫提供安全连接。该方法为小鼠肺微循环 在体内提供了可重复和可量化的成像。详细解释了手术准备的重要方面以及真空稳定肺成像系统的正确利用。最后，利用ALI实验模型对改变的白细胞滚动、白细胞粘连和与炎症相关的毛细血管灌注进行有代表性的成像和分析。使用该方案应有助于进一步重要研究急性疾病状态下肺部微循环的病理生理学变化。
Protocol
这里描述的所有程序都是在达尔豪斯大学实验动物委员会（UCLA）的事先批准下进行的。 1. 准备工作 肺部成像系统：为了准备窗口，在外圈顶部施用一层薄薄的真空润滑脂，同时避免真空通道的污染。将干净的8毫米玻璃盖玻片放在窗户上，然后轻轻按下以形成密封。 宽视场荧光显微镜：使用配备20x/0.40长工作距离物镜和帧速率为25 FPS的黑白电荷耦?…
Representative Results
为了说明通过该方案可以获得的结果，在成像前6小时使用鼻内细菌脂多糖（LPS）滴注模型诱导急性肺损伤（ALI）。简而言之，用异氟醚麻醉小鼠（n = 3），并将来自铜 绿假单胞菌 在无菌盐水（10mg / mL）中的LPS小液滴以5mg / kg的剂量移液到左肾上腺素中。将其与幼稚小鼠（n = 3;无鼻内给药）进行比较。 在影像学检查后，成功的手术准备可以通过几个因素来识别。肺应相?…
Discussion
这里介绍的协议需要练习并注意几个关键步骤。首先，在开始插管和手术之前准备成像窗口很重要。使用最少量的真空润滑脂涂覆成像窗口的外环，涂上盖玻片，并用一滴蒸馏水测试吸力。提前准备可以防止暴露的肺在设置过程中变干。虽然可以用温盐水冲洗，但这样做可能会损害脆弱的肺组织。
插管后，在将鼠标转移到IVM平台时，插管可能偶尔会移位。为了防止这种情况?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢Pina Colarusso博士，他在编辑和修订本手稿方面提供了丰富的专业知识。
Materials
| 1 mL BD Luer Slip Tip Syringe sterile, single use | Becton, Dickinson and Company | 309659 | 1 mL syringe |
| ADSON Dressing Forceps, Tip width 0.6 mm, teeth length 11.5 mm, 12 cm | RWD Life Science Co. | F12002-12 | Blunt forceps |
| Albumin-Fluorescein Isothiocyanate | Sigma-Aldrich | A9771-1G | FITC-albumin |
| Alcohol Swab Isopropyl Alcohol 70% v/v | Canadian Custom Packaging Company | 80002455 | Alcohol wipe |
| AVDC110 Advanced Digital Video Converter | Canopus | 00631069602029 | Digital video converter |
| B/W – CCD – Camera | Horn Imaging | BC-71 | Camera |
| Bovie Deluxe High Temperature Cautery Kit | Fine Science Tools | 18010-00 | Cauterizer |
| C57BL/6 Mice | Charles River Laboratories International | C57BL/6NCrl | C57BL/6 Mice |
| Cotton Tipped Applicators | Puritan | 806-WC | Cotton applicator |
| CS-8R 8mm Round Glass Coverslip | Warner Instruments | 64-0701 | Glass coverslip |
| Digital Pressure Gauge | ITM Instruments Inc. | DG2551L0NAM02L0IM&V | Digital Pressure Gauge |
| Dr Mom Slimline Stainless LED Otoscope | Dr. Mom Otoscopes | 1001 | Otoscope |
| Ethyl Alchohol 95% Vol | Commercial Alcohols | P016EA95 | 95% ethanol |
| Fine Scissors – Martensitic Stainless Steel | Fine Science Tools | 14094-11 | Scissors |
| Fisherbrand Colored Labeling Tape | Fisher Scientific | 1590110 | Labeling tape |
| Gast DOA-P704-AA High-Capacity Vacuum Pump | Cole-Parmer Canada Company | ZA-07061-40 | Vacuum pump |
| Hartman Hemostats | Fine Science Tools | 13003-10 | Hemostatic forceps |
| High Vacuum Grease | Dow Corning | DC976VF | Vacuum grease |
| Isoflurane USP | Fresenius Kabi | CP0406V2 | Isoflurane |
| LIDOcaine HCl Injection 1% 50 mg/5 mL | Teligent Canada | 0121AD01 | Lidocaine HCl 1% |
| Lung SurgiBoard | Luxidea, Inc. | IMCH-0001 | Designed for intravital microscopy of the lung |
| Mineral Oil | Teva Canada | 00485802 | Mineral oil |
| Mouse Endotracheal Intubation Kit | Kent Scientific Corporation | ETI-MSE | Intubation stand, anesthesia mask, 20 G endotracheal cannula, fibre optic cable |
| MST49 Fluorescence Microscope | Leica Microsystems | 10 450 022 | Fluorescence Microscope |
| N Plan L 20x/0.40 Long Working Distance Microscope Objective | Leica Microsystems | 566035 | 20x objective |
| Non-Woven Sponges 2" x 2" | AMD-Ritmed | A2101-CH | Gauze |
| Optixcare Eye Lube Plus | Aventix | 5914322 | Tear gel |
| Original Prusa i3 MK3S+ 3D Printer | Prusa Research | PRI-MK3S-KIT-ORG-PEI | 3D printer |
| Oxygen, Compressed | Linde Canada Inc. | Oxygen | |
| PrecisionGlide Needle 30 G x 1/2 (0.3 mm x 13 mm) | Becton, Dickinson and Company | 305106 | 30 G needle |
| Pyrex 5340-2L 5340 Filtering Flasks, 2000 mL | Cole-Parmer Canada Company | 5340-2L | Vacuum flask |
| Rhodamine 6 G | Sigma-Aldrich | 252433 | Rhodamine 6G |
| Secure Soft Cloth Medical Tape – 3" | Primed | PM5-630709 | Cloth tape |
| Silastic Medical Grade Tubing .040 in. ID x .085 in. OD | Dow Corning | 602-205 | 1.0 mm I.D. polyethylene tubing |
| Somnosuite Low-Flow Anesthesia System | Kent Scientific Corporation | SS-01, SS-04-module | Small rodent ventilator, Low-flow anesthesia system, Heating pad, Rectal temperature probe, Pulse oximeter |
| Tissue Forceps, 12.5cm long, Curved, 1 x 2 Teeth | World Precision Instruments | 501216 | Toothed forceps |
| Transpore Medical Tape, 1527-1, 1 in x 10 yd (2.5 cm x 9.1 m) | 3M | 7000002795 | Medical tape |
| Tubing,Clear,3/8 in Inside Dia. | Grainger Canada | USSZUSA-HT3314 | 1.0 cm I.D. polyethylene tubing |
| Whatman 6720-5002 50 mm In-Line Filters, PTFE, 0.2 µm | Cole-Parmer Canada Company | 6720-5002 | Inline 0.2µm filter |
Riferimenti
- Alizadeh-Tabrizi, N., Hall, S., Lehmann, C. Intravital imaging of pulmonary immune response in inflammation and infection. Frontiers in Cell and Developmental Biology. 8, 620471 (2021).
- Gaertner, M., et al. Toward a comprehensive interpretation of intravital microscopy images in studies of lung tissue dynamics. Journal of Biomedical Optics. 20 (6), 066009 (2015).
- Kreisel, D., et al. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proceedings of the National Academy of Sciences of the United States of America. 107 (42), 18073-18078 (2010).
- Looney, M., et al. Stabilized imaging of immune surveillance in the mouse lung. Nature Methods. 8 (2), 91-96 (2011).
- Bennewitz, M. F., Watkins, S. C., Sundda, P. Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vasoocclusion in unchallenged sickle cell disease mice. IntraVital. 3 (2), 29748 (2014).
- Blueschke, G., et al. Automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats. Journal of Visualized Experiments: JoVE. (93), e51630 (2014).
- Tabuchi, A., et al. Precapillary oxygenation contributes relevantly to gas exchange in the intact lung. American Journal of Respiratory and Critical Care Medicine. 188 (4), 474-481 (2013).
- Rodriguez-Tirado, C., et al. Long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window. Journal of Visualized Experiments: JoVE. (116), e54603 (2016).
- Fiole, D., et al. Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts. Infection and Immunity. 82 (2), 864-872 (2014).
- Thanabalasuriar, A., Neupane, A. S., Wang, J., Krummel, M. F., Kubes, P. iNKT cell emigration out of the lung vasculature requires neutrophils and monocyte-derived dendritic cells in inflammation. Cell Reports. 16 (12), 3260-3272 (2016).
- Neupane, A. S., et al. Patrolling alveolar macrophages conceal bacteria from the immune system to maintain homeostasis. Cell. 183 (1), 110-125 (2020).
- Tschernig, T., et al. Direct visualisation of microparticles in the living lung. Experimental and Toxicologic Pathology. 65 (6), 883-886 (2013).
- Mertens, M., et al. Alveolar dynamics in acute lung injury: Heterogeneous distension rather than cyclic opening and collapse. Critical Care Medicine. 37 (9), 2604-2611 (2009).
- Matuszak, J., Tabuchi, A., Kuebler, W. M. Ventilation and perfusion at the alveolar level: Insights from lung intravital microscopy. Frontiers in Physiology. 11, 291 (2020).
- Margraf, A., et al. 6% Hydroxyethyl starch (HES 130/0.4) diminishes glycocalyx degradation and decreases vascular permeability during systemic and pulmonary inflammation in mice. Critical Care. 22 (1), 1-12 (2018).
- Roller, J., et al. Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte-endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice. Inflammation Research. 62 (3), 275-282 (2012).
- Marques, P., et al. Cigarette smoke increases endothelial CXCL16-leukocyte CXCR6 adhesion in vitro and in vivo. Potential consequences in chronic obstructive pulmonary disease. Frontiers in Immunology. 8, 1766 (2017).
- Condon, M. R., Kim, J. E., Deitch, E. A., Machiedo, G. W., Spolarics, Z. Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis. American Journal of Physiology-Heart and Circulatory Physiology. 284 (6), 2177-2184 (2003).
- Trzeciak, S., et al. Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: Relationship to hemodynamics, oxygen transport, and survival. Annals of Emergency Medicine. 49 (1), 88-98 (2007).
- Kim, Y. M., Jeong, S., Choe, Y. H., Hyun, Y. M. Two-photon intravital imaging of leukocyte migration during inflammation in the respiratory system. Acute and Critical Care. 34 (2), 101-107 (2019).
- Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood. 96 (2), 719-726 (2000).
- Orthgiess, J., et al. Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM-Cre mice in myeloid cell research. European Journal of Immunology. 46 (6), 1529-1532 (2016).
- Schindelin, J., et al. Fiji: An open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
- Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. Intravital microscopy of the murine pulmonary microcirculation. Journal of Applied Physiology. 104 (2), 338-346 (2008).
- Butt, Y., Kurdowska, A., Allen, T. C. Acute lung injury: A clinical and molecular review. Archives of Pathology and Laboratory Medicine. 140 (4), 345-350 (2016).
- Park, I., et al. Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury. European Respiratory Journal. 53 (3), 1800786 (2019).
- Kim, J. K., et al. In vivo imaging of tracheal epithelial cells in mice during airway regeneration. American Journal of Respiratory Cell and Molecular Biology. 47 (6), 864-868 (2012).
- Pieper, M., Schulz-Hildebrandt, H., Mall, M. A., Hüttmann, G., König, P. Intravital microscopic optical coherence tomography imaging to assess mucus-mobilizing interventions for muco-obstructive lung disease in mice. American Journal of Physiology – Lung Cellular and Molecular Physiology. 318 (3), 518-524 (2020).
- Lamm, W. J. E., Bernard, S. L., Wiltz, W., Wagner, J., Glenny, R. W. Intravital microscopic observations of 15-µm microspheres lodging in the pulmonary microcirculation. Journal of Applied Physiology. 98 (6), 2242-2248 (2005).
- Entenberg, D., et al. A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nature Methods. 15 (1), 73-80 (2018).
- Amato, M. B. P., et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. New England Journal of Medicine. 338 (6), 347-354 (2009).
- Looney, M. R., Bhattacharya, J. Live imaging of the lung. Annual Review of Physiology. 76, 431-445 (2013).
Citazione di questo articolo
Hall, S., Faridi, S., Euodia, I., Tanner, S., Chojnacki, A. K., Patel, K. D., Zhou, J., Lehmann, C. Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System. J. Vis. Exp. (182), e63733, doi:10.3791/63733 (2022).