JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Please note that all translations are automatically generated. Click here for the English version.
Immunologie et infection

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

Published: March 15, 2018
doi:
10.3791/56685
, , , , , , ,
1Translational Experimental Pediatrics, Experimental Pulmonology, Department of Pediatrics and Adolescent Medicine,University of Cologne, 2Department of Pediatrics and Adolescent Medicine,University of Cologne, 3Division of Experimental Pediatrics and Metabolism, University Children’s Hospital,Heinrich Heine University Düsseldorf

Summary

肥胖的发病率在上升, 并增加了慢性肺病的风险。为了建立基本机制和预防战略, 需要有明确定义的动物模型。在这里, 我们提供三种方法 (葡萄糖耐受性测试, 身体 plethysmography 和肺固定), 以研究肥胖对肺结局的影响小鼠。

Abstract

肥胖和呼吸系统疾病是主要的健康问题。肥胖正在成为一个新兴的流行病, 预计到2030年世界各地有超过10亿的肥胖人口, 这就代表了社会经济负担的增加。同时, 肥胖相关的并发症, 包括糖尿病以及心脏和慢性肺病, 都在不断上升。虽然肥胖与哮喘加重的风险、呼吸道症状恶化和控制不力有关, 但肥胖和摄动代谢在慢性肺病发病中的功能作用往往被低估,而潜在的分子机制仍然难以捉摸。本文旨在探讨肥胖对代谢的影响以及肺结构和功能的评估方法。在这里, 我们描述了三技术的老鼠研究: (1) 评估腹腔葡萄糖耐受 (ipGTT), 以分析肥胖对葡萄糖代谢的影响;(2) 测量气道阻力 (Res) 和呼吸系统依从性 (Cdyn), 分析肥胖对肺功能的影响;(3) 肺的制备和固定, 供随后的定量组织学评估。肥胖相关的肺部疾病可能是多因素的, 其产生于全身炎症和代谢失调, 可能对肺功能和治疗反应产生不利影响。因此, 研究分子机制和新疗法效果的标准化方法是必不可少的。

Introduction

根据世界卫生组织 (卫生组织) 的数据, 在 2008年, 超过14亿个年龄在20岁以上的成年人超重, 身体质量指数 (BMI) 大于或等于 25;此外, 2亿多名男子和近3亿名妇女肥胖 (BMI≥30)1。肥胖和代谢综合征是多种疾病的主要危险因素。肥胖和随之增加的白脂肪组织质量与2型糖尿病有密切关系2,3, 心血管疾病包括冠心病 (CHD), 心力衰竭 (HF), 心房颤动4和骨关节炎5, 它们在呼吸道疾病发病机制中的功能作用仍然不甚了解。然而, 流行病学研究表明, 肥胖与慢性呼吸道疾病密切相关, 包括劳力呼吸困难、阻塞性睡眠呼吸暂停综合征 (OSAS)、肥胖低通气综合征 (职业职业治疗)、慢性阻塞性肺病 (COPD), 肺栓塞, 吸入肺炎和支气管哮喘6,7,8,9。将肥胖和摄动代谢、例如、胰岛素抵抗和 II 型糖尿病与慢性肺病发病机制联系起来的潜在机制, 不仅包括对通气的体重增加的机械和物理后果, 而且还诱发慢性亚急性炎症状态10,11。过去十年中肥胖和肺部疾病的增加, 加上缺乏有效的预防策略和治疗方法, 突显出需要调查分子机制, 以确定管理肥胖相关肺的新途径。疾病.

在这里, 我们描述了三标准测试, 这是研究肥胖及其对小鼠肺结构和功能的影响的重要基础知识: (1) 腹腔葡萄糖耐受 (ipGTT) (2) 气道阻力 (Res) 和呼吸的测量系统遵从性 (Cdyn);(3) 肺的制备和固定, 供随后的定量组织学评估。ipGTT 是一个强有力的筛选测试, 以测量葡萄糖摄取量, 从而影响肥胖对新陈代谢。这种方法的简单性使得标准化很好, 因此实验室之间的结果可比性。更复杂的方法, 如高血糖钳或研究孤立胰岛, 可用于详细分析代谢表型12。在这里, 我们评估葡萄糖耐受性, 以确定肥胖相关的系统性和代谢紊乱的状态, 作为进一步研究肺部结局的基础。为了评估肥胖和代谢紊乱对肺功能的影响, 我们测量了气道阻力 (Res) 和呼吸系统依从性 (Cdyn)。为描述肺部疾病, 有无节制的和克制的方法评估肺功能。无拘无束的 plethysmography 在自由运动的动物模仿自然状态, 反映呼吸模式;与此相反, 侵入性方法, 如输入阻抗测量的 cDyn 在深麻醉小鼠评估动态肺力学, 是更准确的13。由于慢性呼吸道疾病是由肺组织的组织学改变反映出来的, 因此适当的肺部固定术是迫在眉睫的。组织固定和制备方法的选择取决于肺的隔间, 例如传导气管或肺实质14。在这里, 我们描述了一种方法, 允许定性和定量评估传导气道, 以研究肥胖对哮喘发展的影响。

Protocol

所有动物程序都是按照地方政府当局批准的议定书进行的 (土地北威州, 亚利桑那州: 2012. A424), 并符合德国动物福利法和关于动物福利的条例, 用于试验或为其他科学目的。由于肺功能分析可能会影响肺结构, 因此随后的组织学分析, 应在不同的动物中进行 Cdyn 的测定和肺形态计量学的制备和固定。然而, ipGTT 后的 Cdyn 的测量是可能的。由于 ipGTT 期间的压力可能会干扰肺功能测试所需的麻醉, 因此建?…

Representative Results

腹腔葡萄糖耐受试验 (ipGTT) (图 4)、肺功能测试 (图 5) 的代表性结果, 以及说明苏木精和红红染色肺部的代表性图像 (图 6)。 ipGTT 7 周高脂饮食 (风) 后, 在肥胖小鼠 (蓝色) 中进行。标准膳食喂养的老鼠作为控制 (黑色)。肥胖小鼠在腹腔葡萄糖注射液后显示血糖水…

Discussion

本报告为三种不同方法分析肥胖对葡萄糖代谢和肺部结局的影响提供三项议定书。首先, 葡萄糖耐受性试验提供了分析细胞内葡萄糖吸收的机会, 并可以表明胰岛素抵抗。第二, 全身 plethysmography 是一种测量肺功能的技术, 从而有助于测试新疗法的疗效。第三, 标准化的固定协议是定量形态学分析的关键, 以评估肥胖对结构变化的影响。

饮食诱发肥胖在动物研究中的作用

<p class="…

Divulgations

The authors have nothing to disclose.

Acknowledgements

实验得到了 Marga 和基金会, Kerpen, 德国的支持;项目 210-02-16 (MAAA), 项目 210-03-15 (MAAA) 和由德国研究基金会 (DFG;AL1632-02;MAAA), 波恩, 德国;分子医学的中心科隆 (CMMC;大学医院科隆;职业发展计划;MAAA), 科隆财富 (医学学院, 科隆大学;KD)。

Materials

GlucoMen LX A.Menarini diagnostics, Firneze, Italy 38969 blood glucose meter
GlucoMen LX Sensor A.Menarini diagnostics, Firneze, Italy 39765 Test stripes
Glucose 20% B. Braun, Melsung, Germany 2356746
FinePointe Software DSI, MC s´Hertogenbosch, Netherlands 601-1831-002
FinePointe RC Single Site Mouse Table DSI, MC s´Hertogenbosch, Netherlands 601-1831-001
FPRC Controller DSI, MC s´Hertogenbosch, Netherlands 601-1075-001
FPRC Aerosol Block DSI, MC s´Hertogenbosch, Netherlands 601-1106-001
Aerogen neb head-5.2-4um DSI, MC s´Hertogenbosch, Netherlands 601-2306-001
Forceps FST, British Columbia, Canada 11065-07
Blunt scissors FST, British Columbia, Canada 14105-12
Micro scissors FST, British Columbia, Canada 15000-00
Perma-Hand 4-0 Ethicon, Puerto Rico, USA 736H Surgical suture
Roti-Histofix 4% Roth P087.1 4% Paraformaldehyd
Ketaset Zoetis, Berlin, Germany 10013389 Ketamine
Rompun 2% Bayer, Leverkusen, Germany 770081 Xylazine
DOWNLOAD MATERIALS LIST

References

  1. Kelly, T., Yang, W., Chen, C. S., Reynolds, K., He, J. Global burden of obesity in 2005 and projections to 2030. Int J Obes (Lond). 32, 1431-1437 (2008).
  2. Freemantle, N., Holmes, J., Hockey, A., Kumar, S. How strong is the association between abdominal obesity and the incidence of type 2 diabetes?. International journal of clinical practice. 62, 1391-1396 (2008).
  3. Wassink, A. M. J., et al. Waist circumference and metabolic risk factors have separate and additive effects on the risk of future Type 2 diabetes in patients with vascular diseases. A cohort study. Diabetic Medicine. 28, 932-940 (2011).
  4. Oktay, A. A., et al. The Interaction of Cardiorespiratory Fitness with Obesity and the Obesity Paradox in Cardiovascular Disease. Progress in cardiovascular diseases. , (2017).
  5. Azamar-Llamas, D., Hernandez-Molina, G., Ramos-Avalos, B., Furuzawa-Carballeda, J. Adipokine Contribution to the Pathogenesis of Osteoarthritis. Mediators Inflamm. 2017, 5468023 (2017).
  6. Koenig, S. M. Pulmonary complications of obesity. The American journal of the medical sciences. 321, 249-279 (2001).
  7. Stunkard, A. J. Current views on obesity. The American journal of medicine. 100, 230-236 (1996).
  8. Murugan, A. T., Sharma, G. Obesity and respiratory diseases. Chron Respir Dis. 5, 233-242 (2008).
  9. Zammit, C., Liddicoat, H., Moonsie, I., Makker, H. Obesity and respiratory diseases. International journal of general medicine. 3, 335-343 (2010).
  10. Ouchi, N., Parker, J. L., Lugus, J. J., Walsh, K. Adipokines in inflammation and metabolic disease. Nat Rev Immunol. 11, 85-97 (2011).
  11. McArdle, M. A., Finucane, O. M., Connaughton, R. M., McMorrow, A. M., Roche, H. M. Mechanisms of obesity-induced inflammation and insulin resistance: insights into the emerging role of nutritional strategies. Front Endocrinol (Lausanne). 4, 52 (2013).
  12. Ayala, J. E., et al. Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice. Disease models & mechanisms. 3, 525-534 (2010).
  13. Bates, J. H., Irvin, C. G. Measuring lung function in mice: the phenotyping uncertainty principle. J Appl Physiol. 94 (1985), 1297-1306 (2003).
  14. Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R. An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure. Am J Respir Crit Care Med. 181, 394-418 (2010).
  15. Hoogstraten-Miller, S. L., Brown, P. A. Techniques in aseptic rodent surgery. Curr Protoc Immunol. Chapter 1, (2008).
  16. Heydemann, A. An Overview of Murine High Fat Diet as a Model for Type 2 Diabetes Mellitus. Journal of diabetes research. 2016, 2902351 (2016).
  17. Asha, G. V., Raja Gopal Reddy, M., Mahesh, M., Vajreswari, A., Jeyakumar, S. M. Male mice are susceptible to high fat diet-induced hyperglycaemia and display increased circulatory retinol binding protein 4 (RBP4) levels and its expression in visceral adipose depots. Archives of physiology and biochemistry. 122, 19-26 (2016).
  18. Jovicic, N., et al. Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding. PLoS One. 10, e0134089 (2015).
  19. Fontaine, D. A., Davis, D. B. Attention to Background Strain Is Essential for Metabolic Research: C57BL/6 and the International Knockout Mouse Consortium. Diabetes. 65, 25-33 (2016).
  20. Muniyappa, R., Lee, S., Chen, H., Quon, M. J. Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. Am J Physiol Endocrinol Metab. 294, E15-E26 (2008).
  21. Heijboer, A. C., et al. Sixteen hours of fasting differentially affects hepatic and muscle insulin sensitivity in mice. Journal of lipid research. 46, 582-588 (2005).
  22. Heikkinen, S., Argmann, C. A., Champy, M. F., Auwerx, J. Evaluation of glucose homeostasis. Current protocols in molecular biology. Chapter 29, (2007).
  23. McGuinness, O. P., Ayala, J. E., Laughlin, M. R., Wasserman, D. H. NIH experiment in centralized mouse phenotyping: the Vanderbilt experience and recommendations for evaluating glucose homeostasis in the mouse. Am J Physiol Endocrinol Metab. 297, E849-E855 (2009).
  24. Ayala, J. E., Bracy, D. P., McGuinness, O. P., Wasserman, D. H. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes. 55, 390-397 (2006).
  25. Lodhi, I. J., Semenkovich, C. F. Why we should put clothes on mice. Cell Metab. 9, 111-112 (2009).
  26. Swoap, S. J., Gutilla, M. J., Liles, L. C., Smith, R. O., Weinshenker, D. The full expression of fasting-induced torpor requires beta 3-adrenergic receptor signaling. J Neurosci. 26, 241-245 (2006).
  27. Geiser, F. Metabolic rate and body temperature reduction during hibernation and daily torpor. Annu Rev Physiol. 66, 239-274 (2004).
  28. Mead, J. Mechanical properties of lungs. Physiological reviews. 41, 281-330 (1961).
  29. Lundblad, L. K., Irvin, C. G., Adler, A., Bates, J. H. A reevaluation of the validity of unrestrained plethysmography in mice. J Appl Physiol. 93, 1198-1207 (2002).
  30. Lundblad, L. K., et al. Penh is not a measure of airway resistance!. Eur Respir J. 30, 805 (2007).
  31. Adler, A., Cieslewicz, G., Irvin, C. G. Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice. J Appl Physiol. 97, 286-292 (2004).
  32. Fairchild, G. A. Measurement of respiratory volume for virus retention studies in mice. Applied microbiology. 24, 812-818 (1972).
  33. Brown, R. H., Wagner, E. M. Mechanisms of bronchoprotection by anesthetic induction agents: propofol versus ketamine. Anesthesiology. 90, 822-828 (1999).
  34. Goyal, S., Agrawal, A. Ketamine in status asthmaticus: A review. Indian journal of critical care medicine: peer-reviewed, official publication of Indian Society of Critical Care Medicine. 17, 154-161 (2013).
  35. Doi, M., Ikeda, K. Airway irritation produced by volatile anaesthetics during brief inhalation: comparison of halothane, enflurane, isoflurane and sevoflurane. Canadian journal of anaesthesia = Journal canadien d’anesthesie. 40, 122-126 (1993).
  36. Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. A comparison of fixation methods on lung morphology in a murine model of emphysema. Am J Physiol Lung Cell Mol Physiol. 299, L843-L851 (2010).
  37. Weibel, E. R., Limacher, W., Bachofen, H. Electron microscopy of rapidly frozen lungs: evaluation on the basis of standard criteria. Journal of applied physiology: respiratory, environmental and exercise physiology. 53, 516-527 (1982).
  38. Rolls, G. . Process of Fixation and the Nature of Fixatives. , (2017).
  39. Winsor, L., Woods, A., Ellis, R. Tissue processing. Laboratory histopathology. , 4.2-1-4.2-39 (1994).
  40. Pearse, A. . Histochemistry, theoretical and applied. , (1980).
  41. Weibel, E. R. Morphological basis of alveolar-capillary gas exchange. Physiological reviews. 53, 419-495 (1973).
  42. Bur, S., Bachofen, H., Gehr, P., Weibel, E. R. Lung fixation by airway instillation: effects on capillary hematocrit. Experimental lung research. 9, 57-66 (1985).
  43. Bachofen, H., Ammann, A., Wangensteen, D., Weibel, E. R. Perfusion fixation of lungs for structure-function analysis: credits and limitations. Journal of applied physiology: respiratory, environmental and exercise physiology. 53, 528-533 (1982).
  44. Balcombe, J. P., Barnard, N. D., Sandusky, C. Laboratory routines cause animal stress. Contemporary topics in laboratory animal science. 43, 42-51 (2004).

Tags

Intraperitoneal Glucose Tolerance TestLung Function MeasurementLung FixationObesityImpaired MetabolismPulmonary OutcomesRespiratory ResearchMetabolic DisordersLung StructureLung FunctionChronic Lung DiseasesTherapeutic ApproachesCOPDAsthmaFasted Blood GlucoseGlucose InjectionLung Function AssessmentAnesthesiaAnimal Models

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
Dinger, K., Mohr, J., Vohlen, C., Hirani, D., Hucklenbruch-Rother, E., Ensenauer, R., Dötsch, J., Alejandre Alcazar, M. A. Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes. J. Vis. Exp. (133), e56685, doi:10.3791/56685 (2018).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image