一个<em>离体</em>方法培养大鼠肠系膜微血管网络的时间推移成像
Summary
Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.
Abstract
血管生成，定义为新的血管从预先存在的血管的生长，包括内皮细胞，周细胞，平滑肌细胞，免疫细胞，并与淋巴管和神经的协调。多小区，多系统的相互作用必要的血管生成在生理相关环境的调查。因此，虽然使用体外细胞培养模型提供了机械的见解，一个共同的批评是它们不概括与微血管网络相关联的复杂性。该协议的目的是证明之前和在培养的大鼠肠系膜组织血管生成的刺激后，使完整的微血管网络的时移进行比较的能力。培养组织含有维持其层次微血管网络。免疫标记证实了内皮细胞，平滑肌细胞，周细胞，血管和淋巴管的存在。在一个ddition，与BSI-凝集素标记的组织使前后血清或生长因子刺激由毛细血管发芽和血管密度后，本地网络区域的延时比较。相比于普通细胞培养模型，这种方法提供了在生理学相关微血管网络内皮细胞谱系研究和组织特异性血管生成药物评价的工具。
Introduction
微血管网络的增长和重塑是用于组织功能共同点，伤口愈合，和多个病症和一个关键过程是血管生成，定义为新血管从已有的1，2的生长。组织工程新船或设计基于血管生成疗法，了解参与血管生成的细胞动力学的重要性是至关重要的。然而，这个过程是复杂的。它可以在一个微血管网络内的特定位置而变化，并且涉及多种细胞类型（ 即内皮细胞，平滑肌细胞，周细胞，巨噬细胞，干细胞）和多个系统（淋巴网络和神经网络）。虽然在体外模型已作出了巨大贡献，以检查参与血管生成3种不同的细胞之间的关系，它们的生理相关性可由于兵卫被削弱 – [R复杂性有限，事实上，他们没有真实地反映体内的情况。为了克服这些限制，三维培养系统3， 离体组织模型4中 ，微流体系统5，6，和计算模型7已经被开发并引入在最近几年。然而，仍然需要具有时间推移能力的模型来研究在完整微血管网络体外血管生成。血管生成研究建立新的延时模式与复杂性的水平将提供一个宝贵的工具来了解调节血管生成的基本机制，以提高治疗。
使血管跨越一个完整的微血管网络体外调查一个潜在的模型大鼠肠系膜养殖模式> 8。在最近的工作中，我们已经表明，血液和淋巴微血管网络保持培养后存活。更重要的是，大鼠肠系膜培养模型可用于研究官能周细胞 – 内皮细胞相互作用，血液和淋巴管内皮细胞的连接，和延时成像。本文的目的是为我们的时间推移成像方法的协议。我们的代表性的结果文件证明血管生成的刺激与血清后保持存活，并提供使用用于量化组织特定的血管生成反应以及内皮细胞追踪研究该方法的实施例的多种细胞类型。
Protocol
所有的动物实验和程序由美国杜兰大学的机构动物护理和使用委员会（IACUC）的批准。 1.手术过程设置高压灭菌仪器，手术用品，并在手术前文化用品。每只大鼠手术耗材包括：1悬垂性好，悬垂1与预切孔（0.5英寸×1.5英寸）的中心，纱布垫，1个吸水焊。手术器械包括：1手术刀用10号刀片，2双镊子和一对细剪刀。培养用品包括：1的悬垂性，1镊子，并准备用的聚碳酸酯…
Representative Results
后在培养3天后，组织被标记有活/死活力/细胞毒性试剂盒以证明在大鼠肠系膜培养模型（ 图2A）微血管的生存能力。大部分存在于肠系膜细胞保持在血管内皮细胞，根据他们在微血管段位置确定的文化活力。内皮细胞的增殖也被外源凝集素/ BrdU标记（ 图2D）确认。平滑肌细胞和周细胞的存在沿血管被证实与NG2标记（ 图2B）。标记为LYVE…
Discussion
该协议记录了使用大鼠肠系膜文化模型作为微血管网络增长的时间推移成像的体外工具的方法。在我们的实验室以前的工作已经建立了使用我们的1模型）血管生成8,2）淋巴管8，3）周细胞-内皮细胞相互作用8，和4）抗血管生成药物测试的9。对于在多个时间点培养的大鼠肠系膜组织成像的能力提供了用于评价组织特异性…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.
Materials
| Drape | Cardinal Health | 4012 | 12”x12” Bio-Shield Regular Sterilization Wraps |
| Scalpel Handle | Roboz Surgical Instrument | RS-9843 | Scalpel Handle, #3; Solid; 4" Length |
| Sterile Surgical Blade | Cincinnati Surgical | 0110 | Stainless Steel; Size 10 |
| Culture Dish (60mm) | Thermo Scientific | 130181 | 10/Sleeve |
| Graefe Forcep (curved tweezers) | Roboz Surgical Instrument | RS-5135 | Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4" Length |
| Graefe Forcep (straight tweezers) | Roboz Surgical Instrument | RS-5130 | Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4" Length |
| Noyes Micro Scissor | Roboz Surgical Instrument | RS-5677 | Noyes Micro Dissecting Spring Scissors; Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2" Overall Length |
| Gauze Pads | FisherBrand | 13-761-52 | Non-Sterile Cotton Gauze Sponges; 4"x4" 12-Ply |
| Cotton-Tippled Applicators | FisherBrand | 23-400-124 | 6" Length; Wooden Shaft; Single Use Only |
| 6-Well Plate | Fisher Scientific | 08-772-49 | Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic |
| Sterile Syring 5ml | Fisher Scientific | 14-829-45 | Luer-Lok Tip |
| Sterile Bowl | Medical Action Industries Inc. | 01232 | 32 oz. Peel Pouch; Blue; Sterile Single Use |
| 6-Well Plate Inserts (CellCrown Inserts) | SIGMA | Z681792-3EA | 6-Well Plate Inserts; Non-Sterile |
| Polycarbonate Filter Membrane | SIGMA | TMTP04700 | Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0 µm, 47 mm, White Plain |
| Name | Company | Catalog Number | Comments/Description |
| Beuthanasia | Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply) | MWI #: 011168 | Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium |
| Ketamine | Fort Dodge Animal Health (Ordered from MWI Veterinary Supply) | MWI #: 000680 | Kateset 100 mg/ml |
| Xylazine | LLOYD. Inc. (Ordered from MWI Veterinary Supply) | MWI #: 000680 | Anased 100 mg/ml |
| Saline | Baxter | 2F7122 | |
| PBS | Invitrogen | 14040-133 | |
| MEM | Invitrogen | 11095080 | |
| PenStrep | Invitrogen | 15140-122 | |
| FBS | Invitrogen | 16000-044 | |
| BSA | Jackson ImmunoResearch | 001-000-162 | |
| Saponin | SIGMA | S7900-100G | |
| Isopropyl Alcohol | Fisher Scientific | S25372 | |
| Povidone-Iodine | Operand | 82-226 | |
| Hydrochloric Acid | SIGMA | 320331 | |
| Methanol | Fisher Scientific | 67-56-1 | |
| Glycerol | Fisher Scientific | 56-81-5 | |
| FITC-conjugated Lectin | SIGMA | L9381-2MG | |
| Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody | SIGMA | AB5320 | |
| PECAM (CD31) Antibody | BD Biosciences | 555026 | |
| LYVE-1 Antibody | AngioBio Co. | 11-034 | |
| Goat Anti-Rabbit Cy2-conjugated Antibody | Jackson ImmunoResearch | 111-585-144 | |
| Goat Anti-Mouse Cy3-conjugated Antibody | Jackson ImmunoResearch | 115-227-003 | |
| Streptavidin Cy3-conjugated Antibody | Jackson ImmunoResearch | 016-160-084 | |
| Live/Dead Viability/Cytotoxicity Kit | Invitrogen | L3224 | |
| Normal Goat Serum | Jackson ImmunoResearch | 005-000-121 | |
| 5-Bromo-2'-Deoxyuridine | SIGMA | B5002 | |
| Monoclonal Mouse Anti-Bromodeoxyuridine Clone Bu20a | Dako | M074401-8 | |
| Mouse Anti-Rat CD11b | AbD Serotec | MCA275R |
References
- Carmeliet, P., Jain, R. K. Angiogenesis in cancer and other diseases. Nature. 407 (6801), 249-257 (2000).
- Carmeliet, P. Angiogenesis in life, disease and medicine. Nature. 438 (7070), 932-936 (2005).
- Kaunas, R., Kang, H., Bayless, K. J. Synergistic Regulation of Angiogenic Sprouting by Biochemical Factors and Wall Shear Stress. Cell Mol Bioeng. 4 (4), 547-559 (2011).
- Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protoc. 5 (9), 1518-1534 (2010).
- Song, J. W., Munn, L. L. Fluid forces control endothelial sprouting. Proc Natl Acad Sci U S A. 108 (37), 15342-15347 (2011).
- Chan, J. M., et al. Engineering of in vitro 3D capillary beds by self-directed angiogenic sprouting. PLoS One. 7 (12), e50582 (2012).
- Peirce, S. M., Mac Gabhann, F., Bautch, V. L. Integration of experimental and computational approaches to sprouting angiogenesis. Curr Opin Hematol. 19 (3), 184-191 (2012).
- Stapor, P. C., Azimi, M. S., Ahsan, T., Murfee, W. L. An angiogenesis model for investigating multicellular interactions across intact microvascular networks. Am J Physiol Heart Circ Physiol. 304 (2), H235-H245 (2013).
- Azimi, M. S., et al. An ex vivo model for anti-angiogenic drug testing on intact microvascular networks. PLoS One. 10 (3), e0119227 (2015).
- Nicosia, R. F., Ottinetti, A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab Invest. 63 (1), 115-122 (1990).
- Hutter-Schmid, B., Kniewallner, K. M., Humpel, C. Organotypic brain slice cultures as a model to study angiogenesis of brain vessels. Front Cell Dev Biol. 3, 52 (2015).
- Sawamiphak, S., Ritter, M., Acker-Palmer, A. Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses. Nat Protoc. 5 (10), 1659-1665 (2010).
- Unoki, N., Murakami, T., Ogino, K., Nukada, M., Yoshimura, N. Time-lapse imaging of retinal angiogenesis reveals decreased development and progression of neovascular sprouting by anecortave desacetate. Invest Ophthalmol Vis Sci. 51 (5), 2347-2355 (2010).
- Norrby, K. In vivo models of angiogenesis. J Cell Mol Med. 10 (3), 588-612 (2006).
- Kelly-Goss, M. R., Sweat, R. S., Azimi, M. S., Murfee, W. L. Vascular islands during microvascular regression and regrowth in adult networks. Front Physiol. 4, 108 (2013).
- Kelly-Goss, M. R., et al. Cell proliferation along vascular islands during microvascular network growth. BMC Physiol. 12, 7 (2012).
- Skalak, T. C., Price, R. J. The role of mechanical stresses in microvascular remodeling. Microcirculation. 3 (2), 143-165 (1996).
- Kadohama, T., Nishimura, K., Hoshino, Y., Sasajima, T., Sumpio, B. E. Effects of different types of fluid shear stress on endothelial cell proliferation and survival. J Cell Physiol. 212 (1), 244-251 (2007).
- Milkiewicz, M., Brown, M. D., Egginton, S., Hudlicka, O. Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo. Microcirculation. 8 (4), 229-241 (2001).
- Corliss, B. A., Azimi, M. S., Munson, J. M., Peirce, S. M., Murfee, W. L. Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis. Microcirculation. 23 (2), 95-121 (2016).
Tags
Keywords:
Ex VivoTime-lapse ImagingRat Mesenteric Microvascular NetworksMicrovascular RemodelingEndothelial Cell InteractionsBlood Lymphatic Endothelial Cell ConnectionsAnti-angiogenic Drug EffectsPlexiglass PlatformAnesthetized Adult Male RatAbdominal IncisionMesenteric WindowsSterile SalinePBSCell Culture PlatePolycarbonate Filter Membrane
Cite This Article
Azimi, M. S., Motherwell, J. M., Murfee, W. L. An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks. J. Vis. Exp. (120), e55183, doi:10.3791/55183 (2017).