使用全身吸入系统在小鼠中暴露香烟烟雾
1Program in Immunology,Baylor College of Medicine, 2Department of Pediatrics, Section of Infectious Diseases,Baylor College of Medicine, 3Department of Medicine – Immunology Allergy and Rheumatology,Baylor College of Medicine, 4Department of Medicine, Pulmonary, Critical Care, Sleep Medicine,Baylor College of Medicine, 5Department of Pathology and Immunology,Baylor College of Medicine, 6Department of Pediatrics, Research and Department of Epigenetics and Molecular Carcinogenesis,The University of Texas MD Anderson Cancer Center, 7Division of Pulmonary and Critical Care Medicine,The First Affiliated Hospital of Sun Yat-sen University, 8Center for Translational Research on Inflammatory Diseases (CTRID),Michael E. DeBakey VA Medical Center
Summary
该协议展示了对香烟烟雾（CS）的病理生理影响的研究，内部内建立了全身吸入（WBI）暴露系统（WBIS）。该系统可以在受控的可重复条件下使动物接触CS，用于研究CS介于肺气肿和血细胞瘤的影响。
Abstract
据报道，2018年美国有近14%的成年人吸烟。香烟烟雾（CS）对肺部和心血管疾病的影响已得到广泛研究，然而，CS对血液和骨髓等其他组织和器官的影响仍然未完全确定。找到适当的系统来研究CS对啮齿动物的影响可能过于昂贵，需要购买市售系统。因此，我们着手构建一个经济实惠、可靠且多功能的系统，以研究CS在小鼠中的病理影响。这种全身吸入暴露系统 （WBIS） 设置通过交替接触 CS 和清洁空气来模拟香烟的呼吸和吹气。在这里，我们表明，这种自己动手（DIY）系统在接触了4个月的香烟烟雾后，在小鼠身上诱发气道炎症和肺气肿。使用这种仪器，CS的全身吸入（WBI）对造血干细胞和骨髓中的祖细胞（HSPCs）的影响也显示出来。
Introduction
吸烟仍然是美国可预防疾病的主要原因之一，尽管在过去50-60年中吸烟的成年人数量持续下降。众所周知，吸烟与肺和血液的多种疾病有关，包括慢性阻塞性肺病（COPD），这是一组疾病，包括肺气肿和慢性支气管炎2、3、4。根据美国疾病控制中心（CDC）的数据，2014年，慢性阻塞性肺病是美国第三大死因，超过1500万美国人患有这种疾病。
CS最近也与患克隆造血症（CH）6、7的较高风险有关，在这种情况下，单个造血干细胞不成比例地产生一个人的外周血液的很大一部分。这一发现表明吸烟和骨髓功能之间有潜在的联系。鉴于CS对健康的广泛和非常重要的影响，鉴于穆林疾病模型是生物医学研究进步的基石，开发高效且经济实惠的系统来模拟小鼠的CS是有益的。
在这里，我们提供一个分步指南，建立一个负担得起的系统，用于治疗和研究CS对肺肺气肿和骨髓平衡的体内影响。此设备的组装不需要用户具有专业知识，因此允许 DIY 组装。
Protocol
参与实验和开发这项技术的所有动物都根据我们的动物使用协议，由机构动物护理和使用委员会 （IACUC） 和贝勒医学院和 MD 安德森机构批准，这些机构已获得实验室动物护理评估和认证协会 （AAALAC） 的认证。 1. 建造设备 用阀门系统组装空气压缩机。 使用 1 / 8 英寸螺纹男性适配器配件将流计（两个 15 L / min 带 Y 条和 2 个电源起飞）连接到微型压…
Representative Results
CS 暴露的主要特征之一是肺气肿，其特征是肺部气囊 （肺囊） 的损伤和破坏。因此，最初的实验侧重于DIY系统在反复接触CS后引起雌性小鼠肺部的肺部变化的能力。CS配药方案是根据我们先前的出版物选择的，我们利用此处描述的DIY系统用CS治疗小鼠，并研究肺气肿10、11、12、13、14、15、16的分子病理生理学。<sup class="…
Discussion
在这里，我们提供构建小鼠WBIS到CS的仪器所需的信息。系统安装后，调查人员根据动物中尼古丁或可替宁的交付剂量校准系统至关重要。该仪器包含一个定时器和压力计，可用于调节香烟泡芙量、泡芙频率、合并烟雾暴露期以及动物在每支香烟之间获得的休息间隔。此外，每天服用的香烟的实际数量可能因焦油和尼古丁含量而异。最后，必须定期清洁接触香烟烟雾的任何成分，以确保动物的烟雾?…
Declarações
The authors have nothing to disclose.
Acknowledgements
AR、XH和PE得到了NIH赠款R01HL140398和吉尔森·朗根博基金会赠款的支持。DEMM 和 KK 得到了 NIH 赠款 R01HL136333 和 R01HL134880 （KYK） 以及赫利斯医学研究基金会的赠款的支持。DEMM还得到霍华德·休斯医学研究所（HHMI）吉利亚姆高级研究奖学金的支持。PE 还得到精密环境卫生科学培训 NIEHS T32 ES027801 研究金计划的支持。JC 和 MF 由表观遗传学和分子致癌学系的烟草研究基金以及 MD Anderson 的表观遗传学中心（学者奖 MF）提供支持。FK 和 YZ 由 NIH 赠款 R01 ES029442-01 和 R01 AI135803-01 以及 VA 功绩赠款 CX000104 提供支持。该项目得到了贝勒医学院细胞测量和细胞分类核心的支持，并得到了CPRIT核心设施支持奖（CPRIT-RP180672）、NIH（CA125123和RR024574）和乔尔·塞德斯特伦的资助。
Materials
| 1 in fastener | Lowes | 756990 | |
| 1/4 in Barbed Y connector | VWR | 89093-282 | |
| 1/4 in straight tubing connector | VWR | 62866-378 | |
| 1/8 hex nipple | Lowes | 877221 | |
| 1/8 in threaded coupling fitting | Lowes | 877208 | |
| 1/8 in threaded male adapter nipple fitting | Lowes | 877243 | |
| 10/32 (M) threaded straight connector | Bimba | EB60 | |
| 3/4 in 90-degree elbow CPVC fitting | Lowes | 22643 | |
| 3/4 in chlorinated polyvinyl chloride (CPVC) pipe | Lowes | 23814 | |
| 3/4 in CPVC cap | Lowes | 23773 | |
| 3/4 in CPVC Drip irrigation female adapter | Lowes | 194629 | |
| 3/4 in diameter CPVC male adapter | Lowes | 23766 | |
| 8.5 L airtight container with lid (11.25in x 7.75in x 6 in) | Komax | N/A | Listed as "Komax Biokips Large Bread Box | (280-oz) Large Storage Container" |
| Glass drain tube (1.75 in diameter x 8 in length) | KIMAX | 6500 | |
| Isonic Solenoid Valves | Bimba | V2A02-AW1 | |
| Marlboro Red 100's | Marlboro | N/A | |
| Oxygen swivel barbed connector | Global Medical Solutions | RES002 | |
| Panasonic Timer LT4H-W | Panasonic | LT4HW | Item was built-in the valve controller by Shepherd Controls & Associates |
| Pressure regulator | Allied Electronics and Automation | 70600552 | Also listed as "Norgren R07-100-RGKA" |
| Rubber stopper # 1 (one hole) | VWR | 59581-163 | |
| Rubber stopper # 8.5 (one hole) | VWR | 59581-389 | |
| Scireq inExpose system | Scireq and Emka Technologies | N/A | Commercial system used for comparison with our DIY WBIS |
| Straight barbed fitting (8mm opening) | VWR | 10028-872 | |
| Thread Sealant tape | Lowes | 1184243 | |
| Threaded port adaptor | Bimba | P1SA1 | |
| Timeter Aridyne 2000 Medical Air Compressor | MFI Medical | AHC-TE20 | |
| Timeter flowmeter | Allied Healthcare Products | 15006-03YP2 | Also listed as "Puritan Air Meter" |
| Valve Control system | Shepherd Controls and Associates | N/A | Company custom designed the valve control system for this model. |
| Vinyl pipes | Vitality Medical | RES3007 |
Referências
- Current Cigarette Smoking Among Adults in the United States. Center for Disease Control and Prevention Available from: https://www.cdc.gov/tobacco/data_statistics/fact_sheets/adult_data/cig_smoking/index.htm (2018)
- Salvi, S. Tobacco smoking and evironmental risk factors for chronic obstructive pulmonary disease. Clinics in Chest Medicine. 35, 17-27 (2014).
- Sunyer, J., et al. Longitudinal relation between smoking and white blood cells. American Journal of Epidemiology. 144, 734-741 (1996).
- Freedman, D. S., Flanders, D., Barboriak, J. J., Malarcher, A. M., Gates, L. Cigarette smoking and leukocyte subpopulations in men. Annals of Epidemiology. 6, 299-306 (1996).
- Chronic Obstructive Pulmonary Disease (COPD). Center for Disease Control and Prevention Available from: https://www.cdc.gov/copd/basics-about.html (2019)
- Genovese, G., et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. New England Journal of Medicine. , (2014).
- Steensma, D. P. Clinical implications of clonal hematopoiesis. Mayo Clinic Proceedings. 93, 1122-1130 (2018).
- Tobacco. Federal Trade Comission Available from: https://www.ftc.gov/tips-advice/business-center/selected-industries/tobacco (2020)
- 3R4F Cigarettes. University of Kentucky – College of Agriculture Food and Environment Available from: https://ctrp.uky.edu/products/gallery/Reference (2020)
- Shan, M., et al. Cigarette smoke induction of osteopontin (SPP1) mediates T H 17 inflammation in human and experimental emphysema. Science Translational Medicine. 4, 1-10 (2012).
- Yuan, X., et al. Activation of C3a receptor is required in cigarette smoke-mediated emphysema. Nature Mucosal Immunology. 8, 874-885 (2014).
- Yuan, X., et al. Cigarette smoke – induced reduction of C1q promotes emphysema. JCI Insight. 4, 1-17 (2019).
- Shan, M., et al. Agonistic induction of PPAR g reverses cigarette smoke – induced emphysema Find the latest version: Agonistic induction of PPAR γ reverses cigarette smoke – induced emphysema. Journal of Clinical Investigation. 124, 1371-1381 (2014).
- Hong, M. J., et al. Protective role of gd T cells in cigarette smoke and influenza infection. Nature Mucosal Immunology. 11, 834-908 (2018).
- Kim, M., et al. Cigarette smoke induces intestinal inflammation via a Th17 cell-neutrophil axis. Frontiers in Immunology. 10, 1-11 (2019).
- Lu, W., et al. The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T H 17 cell – dependent emphysema. Nature Immunology. 16, 1185-1194 (2015).
- Hendrix, A. Y., Kheradmand, F. . The Role of Matrix Metalloproteinases in Development, Repair, and Destruction of the Lungs. Progress in Molecular Biology and Translational Science. 148, (2017).
- Siggins, R. W., Hossain, F., Rehman, T., Melvan, J. N., Welsh, D. A. Cigarette smoke alters the hematopoietic stem cell niche. Med Sci. 2, 37-50 (2014).
- Kheradmand, F., You, R., Gu, B. H., Corry, D. B. Cigarette smoke and DNA cleavage promote lung inflammation and emphysema. Transactions of the American Clinical and Climatological Association. 128, 222-233 (2017).
- Ha, M. A., et al. Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice. PLoS ONE. , 1-16 (2015).
- Moreno-Gonzalez, I., Estrada, L. D., Sanchez-Mejias, E., Soto, C. Smoking exacerbates amyloid pathology in a mouse model of Alzheimer’s disease. Nature Communications. 4, 1-10 (2013).
- Madison, M. C., et al. Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine. Journal of Clinical Investigation. 129, 4290-4304 (2019).
Citar este artigo
Morales-Mantilla, D. E., Huang, X., Erice, P., Porter, P., Zhang, Y., Figueroa, M., Chandra, J., King, K. Y., Kheradmand, F., Rodríguez, A. Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System. J. Vis. Exp. (164), e61793, doi:10.3791/61793 (2020).