肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量
Summary
The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.
Abstract
COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill’s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson’s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.
Introduction
利用动物模型，研究慢性阻塞性肺病是具有挑战性的，因为没有模式可以完美地复制人类疾病（2）的所有功能。大多数研究者用老鼠COPD模型，因为在他们的肺的生理，病理，遗传，代谢和小鼠和人类之间的相似之处。此外，老鼠是相对便宜的学习，和6个月的CS曝光（5,7-9）内均肺气肿和小气道重塑发展。
香烟烟雾诱导的慢性阻塞性肺病 ：有几种方法可诱导的COPD小鼠。大多数研究人员暴露小鼠的CS，这是人类慢性阻塞性肺病的主要的致病因素。 CS暴露6个月导致肺气肿和小气道重塑（SAR）的小鼠的发展，但根据不同的小鼠品系研究这种疾病的被诱导的严重程度而变化。例如，NZWLacZ小鼠抗CS诱发肺气肿的发展而AKR / J小鼠是extremelŸ敏感（10）。大多数研究者研究C57BL / 6小鼠品系在CS曝光模式尽可能多的基因打靶小鼠可在这株。 6个月后的CS曝光，肺气肿和小气道纤维化的发展中的野生型（WT）C57BL / 6小鼠，并且两个病变的严重程度（5,10）相对温和。研究人员使用两种类型的CS曝光：鼻式和全身暴露。鼻式暴露技术的主要缺点是：1）它是一种劳动密集型方法;和2）小鼠在小室能够诱导在动物（11）的应力响应和热疗受到抑制。的全身暴露量（本文中所述）的主要缺点是，动物可以摄取（以及吸气）的尼古丁和焦油的产品时，他们清洁他们的皮毛。暴露于全身的CS小鼠也具有较低的碳氧水平，当与动物暴露于鼻式的CS（12）相比，减少的体重减轻。
<p class ="“jove_content”"> 肺功能检查（肺功能）：肺顺应性和顺应性的措施通常是在C57BL / 6野生型相似（WT）由于相对温和肺气肿，开发时，这种暴露在空气中或CS 6个月小鼠应变被暴露到CS（10）。然而，当气肿性破坏更加严重，增加肺顺应性和左移位，在压力容积（PV）流可以检测环路。后者可以被观察到，例如，在小鼠菌株更容易受到CS的效果（10），在具有比C57BL / 6野生型小鼠更严重的肺气肿类型的CS-暴露的C57BL / 6株基因靶向的小鼠（13），或者在经受使得它们更容易受到CS的效果（14）的环境变化的CS-暴露的小鼠。该协议使用小动物呼吸机来测量减少肺的弹性回缩力（增加准静态肺顺应性[Cst中]和减少组织顺应性[H]），光伏流动回路，并改变在麻醉小鼠（15,16）的气道和组织电阻。肺气肿的措施：肺气肿发展的CS暴露C56BL / 6小鼠品系分析是具有挑战性的，因为它的分布空间异质性。几种不同的方法进行量化的小鼠空域放大图。所用的第一种方法是将平均线性截距（L M）（17）。然而，L M法是一种缓慢的，手动的过程，可能无法捕捉疾病（除非肺的所有部分是随机取样的）和它的用途，因此可能会引入偏见观察者到分析的异质性。破坏性指数[DI，（18）]也量化使用的透明片，用50均等地分布点放置在一个苏木精和伊红染色的肺部的印刷数字化图像空域放大。 PI法得分周围的每一点交流区盘带到何种程度上在此区域内的肺泡管和肺泡壁破坏。在DI法的主要缺点是，它是费时和不大于其他的方法（19,20）更加准确。
该协议的措施平均肺泡弦长和肺泡面积上沾满吉尔氏染色石蜡包埋的肺切片。形态软件转换的肺切片，以二值图像的图像（在该组织是白色的，空域是黑色），然后叠加水平和垂直线（和弦）的均匀网格，该软件然后量化内确定的区域的每个弦的长度软件领空。使用该方法，可以测量在标准化和相对自动化的方式（21）在肺的所有部分肺泡的大小。
小气道重塑（SAR）：ECM蛋白的沉积增多（尤其是interstitia升胶原蛋白）周围小气道发生在CS暴露的动物，并有助于气流阻塞。研究人员不学习特区COPD的动物模型那样频繁肺气肿发展（22）。量化特区中的CS-暴露的小鼠，这种协议使用图像分析软件来测量ECM蛋白的该沉积周围小气道（气道具有300到899微米之间的平均直径）在石蜡包埋肺切片的层的厚度沾上Masson三色染色。
Protocol
Representative Results
Discussion
Most researcher use mice to model the main chronic lung pathologies and abnormal lung physiology in COPD (airspace enlargement, SAR, and increases in lung compliance) present in the human disease. A comprehensive approach to assess the effect of molecules of interest on both emphysema development and SAR is needed in mice in order to comprehensively assess the activities of molecules of interest in these chronic disease processes.
There are several critical steps in this protocol. First, dur…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢弗朗西斯Polverino医学博士，研究员布莱根妇女医院为她对本文的贡献，同时也莫妮卡姚明，BS和凯特韦迪尔，BS他们用小鼠饲养和暴露的小鼠香烟烟雾的援助。
这项工作是由公共卫生服务，美国国家心脏，肺和血液研究所资助HL111835，HL105339，HL114501，乘务员医学研究所格兰特＃CIA123046中，布莱根妇女医院 – 洛夫莱斯呼吸研究所协会，以及剑桥NIHR生物医学支持研究中心。
Materials
| Whole-body smoke exposure device | Teague Enterprise | TE-10z | Chronic Smoke exposures to induce chronic lung disease in mice |
| Research Cigarette | University of Kentucky | 3R4F reference cigarettes | |
| Pallflex® Air Monitoring Filters, Emfab Filters TX40HI20WW, 25 mm | Pall Corporation | 7219 | For measurement of TPMs |
| 25 mm filter holder | Pall Corporation | ||
| Filter sampler | Intermatic | Metal T100 | |
| Gas meter | AEM | Gas meters G1.6; G2.5; G4 | |
| Tracheal Cannula for mouse 18 gauge | Labinvention | Analysis of pulmonary function | |
| Mechanical ventilator | Scireq | FlexiVent | |
| Gill's hematoxylin solution | Sigma-Aldrich | GSH316 | For Gill staining, work under a fume hood |
| Hematoxylin solution, Harris modified | Sigma-Aldrich | HHS16 | |
| Cytoseal-60 | Thermo Scientific | 8310-16 | |
| Micro-Slide-Field-Finder | Andwin Scientific INC | 50-949-582 | For analysis of emphysema |
| Scion Image Program | Scion Corporation | ||
| Mason's trichrome stain | Sigma-Aldrich | HT15 | For analysis of small airway fibrosis |
| MetaMorp Offline version 7.0 | Molecullar Devices LLC | 31032 |
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