使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估
Summary
受管制的肠道上皮屏障功能和免疫反应是炎症性肠病的标志，由于缺乏生理模型，这些特征仍然调查不力。在这里，我们描述了一个鼠标肠道循环模型，它采用了血管化和外化良好的肠道部分，以研究粘膜渗透性和白细胞在体内的招募。
Abstract
肠道粘膜由一层上皮细胞排列，形成动态屏障，允许球细胞传输营养物质和水，同时防止发光细菌和外源物质的通过。这一层的破坏导致对发光成分的渗透性和免疫细胞的招募增加，这两者都是肠道病理状态的标志，包括炎症性肠病（IBD）。
由于缺乏允许定量分析的实验性体内方法，对多态核嗜中性粒细胞（PMN）上皮屏障功能和转位迁移（TEpM）的机制不完全了解。在这里，我们描述了一个强大的木质实验模型，它采用了外化肠道部分的肠或近结肠。外化肠道环 （iLoop） 完全血管化，与通常用于研究上皮细胞单层的渗透性和 PMN 迁移的前体内室方法相比，具有生理优势。
我们详细演示了该模型的两种应用：（1） 通过检测血清中的荧光标记的脱氧转体在血清中检测肠道渗透性（2） 在肠道上皮中引入化疗法后对迁移的 PMN 进行定量评估。我们证明了这个模型的可行性，并提供了利用iLoop在小鼠缺乏上皮紧密结相关蛋白质JAM-A相比对照的结果。JAM-A 已被证明在炎症反应期间调节上皮屏障功能以及 PMN TEPM。我们使用 iLoop 的结果证实了以前的研究，并强调了 JAM-A 在调节肠道渗透性和 PMN TEPM 在体温平衡和疾病期间在体内的重要性。
iLoop模型为肠道平衡和炎症的活体研究提供了高度标准化的方法，将显著提高对IBD等疾病中肠道屏障功能和粘膜炎症的认识。
Introduction
肠道粘膜包括单层柱状肠道上皮细胞（IECs）、底层拉米纳丙膜免疫细胞和肌肉粘膜。除了它在营养物质吸收中的作用外，肠道上皮是保护身体内部免受发光共生细菌、病原体和膳食抗原侵害的物理屏障。此外，IEC 和拉米纳丙膜免疫细胞协调免疫反应，根据上下文和刺激诱导耐受性或反应。据报道，上皮屏障的破坏可以先于病理粘膜炎症的发生，并导致炎症性肠病（IBD），包括溃疡性结肠炎和克罗恩病1，2，3，4，5，6，7。溃疡性结肠炎患者出现多态核嗜中性粒细胞（PMN）的过度转导（TEPM），形成隐秘脓肿，这一发现与疾病8、9的严重程度有关。虽然上皮屏障功能受损和免疫反应过度是IBD的标志，但缺乏对肠道渗透性和免疫细胞招募进行肠道粘膜的定量评估的体内检测实验。
最常见的方法用于研究肠道上皮渗透性和PMN TEPM采用前体内室为基础的方法，使用IEC单层培养的半渗透多孔膜插入10，11，12。上皮屏障的完整性通过测量转位电阻 （TEER） 或荧光素异氰酸素 （FITC） 标记的脱氧叶素的准细胞通量从尖顶到基底隔间13、14、15进行监测。同样，PMN TEPM 通常针对下腔16中添加的化疗吸引剂进行研究。PMN 被放置在上室，在潜伏期后，已迁移到基底舱的 PMN 被收集和量化。虽然这些方法是有用的，易于执行和非常可重复的，它们显然是减少的方法，并不一定代表在体内条件的准确反映。
在小鼠中，研究肠道准细胞渗透性的常见检测方法是通过对FITC-dextran的口服测定和随后对血清13、17中FITC-德克斯特兰外观的测量。这种检测的缺点是，它代表了对胃肠道整体屏障完整性的评估，而不是对区域肠道贡献的评估。此外，Evans Blue 常用于评估18号体内的血管渗漏，还用于评估小鼠和19、20、21的肠道粘膜渗透性。肠道粘膜中埃文斯蓝的量化需要从一夜之间在形态酰胺中孵化的组织中提取。因此，同一组织不能用于研究肠道上皮渗透和嗜中性粒细胞渗透。
在这里，我们强调一个简单的协议，减少动物的数量，需要收集可重复的数据，结肠粘膜渗透性和白细胞在体内的转世迁移。因此，我们建议使用在血清中易于检测到的FITC-dextrans，而不损害肠道循环的完整性，这些肠环可以收获作进一步分析。值得注意的是，肠道利口环已用于各种物种（包括老鼠，大鼠，兔子，小牛）研究细菌感染（如沙门氏菌，李斯特菌单细胞基因和大肠杆菌）22，23，24，25以及肠道渗透性26：然而，据我们所知，没有研究调查PMN TEPM在肠道的特定区域的机制，如伊利姆或结肠，通常涉及IBD。
在这里，我们描述了小鼠肠道循环（iLoop）模型，这是一个强大和可靠的显微外科活体方法，采用了良好的血管化和外化肠道部分的肠或近结肠。iLoop 模型在生理上相关，允许评估麻醉下活鼠的肠道屏障完整性和 PMN TEPM。我们演示了两个应用：1） 在 iLoop 2 的血清水平 4 kDa FITC-dextran 后，在 iLoop 2） 中对 iLoop 流明中转移的 PMN 进行量化，在注射强力化学微量微量剂 Leukotriene B4 （LTB4）27后进行。此外，利用iLoop模型与Jam-a-空小鼠或老鼠怀有选择性损失的JAM-A在IEC（维林克里：与对照小鼠相比，我们能够证实先前的研究，这些研究报告对紧结相关蛋白质JAM-A对肠道渗透性和嗜中性粒细胞转运有重大贡献，15、28、29、30、31。
iLoop 模型是一种功能和生理上非常全的方法，可用于证实体外测定。此外，这是一个多功能的实验模型，允许研究各种试剂，可以注射到循环流明，包括化疗因子，细胞因子，细菌病原体，毒素，抗体和治疗。
Protocol
所有动物实验都是根据国家卫生研究院的指导方针和政策进行的，并经密歇根大学动物护理与使用机构委员会批准。 1. 术前准备 注：这种方法是利用C57BL/6遗传背景的成年小鼠产生的，年龄在8-12周。所有小鼠都处于严格的特定无病原体条件下，并可获得正常的周和水。结果使用C57BL/6，果酱-空小鼠（Jam-a-/-）或在IEC（维林-克里）上有?…
Representative Results
图1和图2分别描绘了ileal循环和pcLoop模型的示意图。 解剖图片显示了手术的关键步骤，包括肠道部分（图1B和图2B）的外部化，确定一个适当的位置，使血液供应的最小干扰（图1C和图2C）和清洁后，可以充满试剂溶液的iLoop切?…
Discussion
对IBD等病理条件下肠道屏障功能调节不良和免疫细胞招募的机制不完全了解。在这里，我们详细介绍了一个强大的体内粘液模型，它采用了肠道或近端结肠的血管化外化肠道部分，并允许评估肠道渗透性、中性粒细胞迁移研究以及其他应用。
iLoop 是在活体动物身上进行的非恢复性手术。麻醉必须在实验过程中持续监测，并强制评估饱和深度。最关键的步骤包括 （1） iLoop 的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢维尔茨堡大学的斯文·弗莱明博士为建立近亲结肠循环模型做出了贡献，肖恩·沃森对老鼠群落的管理做出了贡献，奇特拉·穆拉利达兰帮助获取了iLoop模型的照片。这项工作得到了德国研究基金会/DFG（BO 5776/2-1）的支持，包括KB、R01DK079392、R01DK072564和R01DK061379到C.A.P.
Materials
| Equipment and Material | |||
| BD Alcohol Swabs | BD | 326895 | |
| BD PrecisionGlide Needle, 25G X 5/8" | BD | 305122 | |
| BD PrecisionGlide Needle, 30G X 1/2" | BD | 305106 | |
| BD 1ml Tuberculin Syringe Without Needle | BD | 309659 | |
| 15ml Centrifuge Tube | Corning | 14-959-53A | |
| Corning 96-Well Solid Black Polystyrene Microplate | FisherScientific | 07-200-592 | |
| Corning Non-treated Culture Dish, 10cm | MilliporeSigma | CLS430588 | |
| Cotton Tip Applicator (cotton swab), 6", sterile | FisherScientific | 25806 2WC | |
| Dynarex Cotton Filled Gauze Sponges, Non-Sterile, 2" x 2" | Medex | 3249-1 | |
| EZ-7000 anesthesia vaporizer (Classic System, including heating units) | E-Z Systems | EZ-7000 | |
| Falcon Centrifuge Tube 50ml | VWR | 21008-940 | |
| Fisherbrand Colored Labeling Tape | FisherScientific | 15-901-10R | |
| Halsey Needle Holder (needle holder) | FST | 12001-13 | |
| Kimwipes, small (tissue wipe) | FisherScientific | 06-666 | |
| 1.7ml Microcentrifuge Tubes | Thomas Scientific | c2170 | |
| Micro Tube 1.3ml Z (serum clot activator tube) | Sarstedt | 41.1501.105 | |
| Moria Fine Scissors | FST | 14370-22 | |
| 5ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap (35 µm nylon mesh) | Falcon | 352235 | |
| Puralube Vet Ointment, Sterile Ocular Lubricant | Dechra | 12920060 | |
| Ring Forceps (blunt tissue forceps) | FST | 11103-09 | |
| Roboz Surgical 4-0 Silk Black Braided, 100 YD | FisherScientific | NC9452680 | |
| Semken Forceps (anatomical forceps) | FST | 1108-13 | |
| Sofsilk Nonabsorbable Coated Black Suture Braided Silk Size 3-0, 18", Needle 19mm length 3/8 circle reverse cutting | HenrySchein | SS694 | |
| Student Fine Forceps, Angled | FST | 91110-10 | |
| 10ml Syringe PP/PE without needle | Millipore Sigma | Z248029 | |
| 96 Well Cell Culture Plate | Corning | 3799 | |
| Yellow Feeding Tubes for Rodents 20G x 30 mm | Instech | FTP-20-30 | |
| Solutions and Buffers | |||
| Accugene 0.5M EDTA | Lonza | 51201 | |
| Ammonium-Chloride-Potassium (ACK) Lysing Buffer | BioWhittaker | 10-548E | |
| Hanks' Balanced Salt Solution | Corning | 21-023-CV | |
| Phosphate-Buffered Saline without Calcium and Magnesium | Corning | 21-040-CV | |
| Reagents | |||
| Alexa Fluor 647 Anti-Mouse Ly-6G Antibody (1A8) | BioLegend | 127610 | |
| CD11b Monoclonal Antibody, PE, eBioscience (M1/70) | ThermoFisher | 12-0112-81 | |
| CountBright Absolute Counting Beads | Invitrogen | C36950 | |
| Dithiotreitol | FisherScientific | BP172-5 | |
| Fetal Bovine Serum, heat inactivated | R&D Systems | 511550 | |
| Fluorescein Isothiocyanate-Dextran, average molecular weight 4.000 | Sigma | 60842-46-8 | |
| Isoflurane | Halocarbon | 12164-002-25 | |
| Leukotriene B4 | Millipore Sigma | 71160-24-2 | |
| PerCP Rat Anti-Mouse CD45 (30-F11) | BD Pharmingen | 557235 | |
| Purified Rat Anti-Mouse CD16/CD32 (Mouse BD FC Block) | BD Bioscience | 553142 | |
| Recombinant Murine IFN-γ | Peprotech | 315-05 | |
| Recombinant Murine TNF-α | Peprotech | 315-01A |
References
- Olson, T. S., et al. The primary defect in experimental ileitis originates from a nonhematopoietic source. Journal of Experimental Medicine. 203 (3), 541-552 (2006).
- Jump, R. L., Levine, A. D. Mechanisms of natural tolerance in the intestine: implications for inflammatory bowel disease. Inflammatory Bowel Diseases. 10 (4), 462-478 (2004).
- Peeters, M., et al. Clustering of increased small intestinal permeability in families with Crohn’s disease. Gastroenterology. 113 (3), 802-807 (1997).
- Michielan, A., D’Inca, R. Intestinal permeability in inflammatory bowel disease: Pathogenesis, clinical evaluation, and therapy of leaky gut. Mediators of Inflammation. 2015, 628157 (2015).
- Chin, A. C., Parkos, C. A. Neutrophil transepithelial migration and epithelial barrier function in IBD: potential targets for inhibiting neutrophil trafficking. Annals of the New York Academy of Sciences. 1072, 276-287 (2006).
- Baumgart, D. C., Sandborn, W. J. Crohn’s disease. Lancet. 380 (9853), 1590-1605 (2012).
- Ordás, I., Eckmann, L., Talamini, M., Baumgart, D. C., Sandborn, W. J. Ulcerative colitis. Lancet. 380 (9853), 1606-1619 (2012).
- Muthas, D., et al. Neutrophils in ulcerative colitis: A review of selected biomarkers and their potential therapeutic implications. Scandanavian Journal of Gastroenterology. 52 (2), 125-135 (2017).
- Pai, R. K., et al. The emerging role of histologic disease activity assessment in ulcerative colitis. Gastrointestinal Endoscopy. 88 (6), 887-898 (2018).
- Parkos, C. A., Delp, C., Arnaout, M. A., Madara, J. L. Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction. The Journal of Clinical Investigation. 88 (5), 1605-1612 (1991).
- Brazil, J. C., Parkos, C. A. Pathobiology of neutrophil-epithelial interactions. Immunological Reviews. 273 (1), 94-111 (2016).
- Thomson, A., et al. The Ussing chamber system for measuring intestinal permeability in health and disease. BMC Gastroenterology. 19 (1), 98 (2019).
- Li, B. R., et al. In vitro and in vivo approaches to determine intestinal epithelial cell permeability. Journal of Visualized Experiments. (140), e57032 (2018).
- Srinivasan, B., et al. TEER measurement techniques for in vitro barrier model systems. Journal of Laboratory Automation. 20 (2), 107-126 (2015).
- Fan, S., et al. Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation. Molecular Biology of the Cell. 30 (5), 566-578 (2019).
- Parkos, C. A. Neutrophil-epithelial interactions: A double-edged sword. American Journal of Pathology. 186 (6), 1404-1416 (2016).
- Volynets, V., et al. Assessment of the intestinal barrier with five different permeability tests in healthy C57BL/6J and BALB/cJ mice. Digital Diseases and Sciences. 61 (3), 737-746 (2016).
- Wick, M. J., Harral, J. W., Loomis, Z. L., Dempsey, E. C. An optimized evans blue protocol to assess vascular leak in the mouse. Journal of Visualized Experiments. (139), e57037 (2018).
- Tateishi, H., Mitsuyama, K., Toyonaga, A., Tomoyose, M., Tanikawa, K. Role of cytokines in experimental colitis: relation to intestinal permeability. Digestion. 58 (3), 271-281 (1997).
- Mei, Q., Diao, L., Xu, J. M., Liu, X. C., Jin, J. A protective effect of melatonin on intestinal permeability is induced by diclofenac via regulation of mitochondrial function in mice. Acta Pharmacologica Sinica. 32 (4), 495-502 (2011).
- Vargas Robles, H., et al. Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis. Journal of Visualized Experiments. (119), e55095 (2017).
- Arques, J. L., et al. Salmonella induces flagellin- and MyD88-dependent migration of bacteria-capturing dendritic cells into the gut lumen. Gastroenterology. 137 (2), 579-587 (2009).
- Coombes, B. K., et al. Analysis of the contribution of Salmonella pathogenicity islands 1 and 2 to enteric disease progression using a novel bovine ileal loop model and a murine model of infectious enterocolitis. Infection and Immunity. 73 (11), 7161-7169 (2005).
- Everest, P., et al. Evaluation of Salmonella typhimurium mutants in a model of experimental gastroenteritis. Infection and Immunity. 67 (6), 2815-2821 (1999).
- Pron, B., et al. Comprehensive study of the intestinal stage of listeriosis in a rat ligated ileal loop system. Infection and Immunity. 66 (2), 747-755 (1998).
- Clayburgh, D. R., et al. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. The Journal of Clinical Investigation. 115 (10), 2702-2715 (2005).
- Palmblad, J., et al. Leukotriene B4 is a potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood. 58 (3), 658-661 (1981).
- Mandell, K. J., Babbin, B. A., Nusrat, A., Parkos, C. A. Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity. The Journal of Biological Chemistry. 280 (12), 11665-11674 (2005).
- Laukoetter, M. G., et al. JAM-A regulates permeability and inflammation in the intestine in vivo. Journal of Experimental Medicine. 204 (13), 3067-3076 (2007).
- Flemming, S., Luissint, A. C., Nusrat, A., Parkos, C. A. Analysis of leukocyte transepithelial migration using an in vivo murine colonic loop model. Journal of Clinical Investigation Insight. 3 (20), (2018).
- Luissint, A. C., Nusrat, A., Parkos, C. A. JAM-related proteins in mucosal homeostasis and inflammation. Seminars in Immunopathology. 36 (2), 211-226 (2014).
- Cesarovic, N., et al. Isoflurane and sevoflurane provide equally effective anaesthesia in laboratory mice. Lab Animal. 44 (4), 329-336 (2010).
- JoVE Science Education Database. Introduction to the Microplate Reader. Journal of Visualized Experiments. , e5024 (2020).
- Kelm, M., et al. Targeting epithelium-expressed sialyl Lewis glycans improves colonic mucosal wound healing and protects against colitis. Journal of Clinical Investigation Insight. 5 (12), (2020).
- Azcutia, V., et al. Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo. Mucosal Immunology. , (2020).
- Yu, Y. R., et al. A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues. PloS One. 11 (3), 0150606 (2016).
- Bradfield, P. F., Nourshargh, S., Aurrand-Lions, M., Imhof, B. A. JAM family and related proteins in leukocyte migration (Vestweber series). Arteriosclerosis, Thrombosis, and Vascular Biology. 27 (10), 2104-2112 (2007).
- Ebnet, K. Junctional Adhesion Molecules (JAMs): Cell adhesion receptors with pleiotropic functions in cell physiology and development. Physiological Reviews. 97 (4), 1529-1554 (2017).
- Sorribas, M., et al. FXR modulates the gut-vascular barrier by regulating the entry sites for bacterial translocation in experimental cirrhosis. Journal of Hepatology. 71 (6), 1126-1140 (2019).
- Mazzucco, M. R., Vartanian, T., Linden, J. R. In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin. Bio-Protocol. 10 (15), 3709 (2020).
- Kelly, J. R., et al. Breaking down the barriers: the gut microbiome, intestinal permeability and stress-related psychiatric disorders. Frontiers in Cellular Neuroscience. 9, 392 (2015).
- Fiorentino, M., et al. Blood-brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders. Molecular Autism. 7 (1), 49 (2016).
- Kelm, M., et al. Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18. FASEB Journal : An Official Publication of the Federation of American Societies for Experimental Biology. 34 (2), 2326-2343 (2020).
Cite This Article
Boerner, K., Luissint, A., Parkos, C. A. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model. J. Vis. Exp. (168), e62093, doi:10.3791/62093 (2021).