小鼠肺气肿评价持续压力下的肺固定
Summary
这里介绍的是肺固定的有用方案，为肺气肿小鼠模型肺标本的组织学评估创造了一个稳定的条件。该模型的主要优点是，它可以修复许多肺具有相同的恒定压力，没有肺崩溃或通缩。
Abstract
肺气肿是慢性阻塞性肺病（COPD）的一个重要特征。涉及肺气肿小鼠模型的研究需要最佳的肺固定，以产生可靠的肺组织学标本。由于肺的结构组成的性质，主要由空气和组织组成，在固定过程中有它崩溃或放气的风险。各种肺固定方法存在，每种方法各有其优缺点。这里介绍的肺固定方法利用恒定压力，利用肺气肿小鼠肺模型为研究提供最佳的组织评估。主要优点是，它可以修复许多肺与相同的条件在同一时间。肺标本是从慢性香烟烟雾暴露小鼠获得的。肺固定使用专用设备进行，能够产生恒定的压力。这种恒定的压力使肺保持在合理的膨胀状态。因此，该方法产生一个肺组织标本，适合评估香烟烟雾引起的轻度肺气肿。
Introduction
慢性阻塞性肺病是全球主要死亡原因之一。香烟烟雾是COPD最重要的病因，但发病机制仍不完全确定。COPD表现出两个主要特征，包括气流的逐渐限制和肺的异常炎症反应。肺气肿紊乱经常发生在慢性阻塞性肺病患者2的肺部。肺气肿的病理发现以肺泡壁破坏为特征3。一些动物物种已经被用来生成在体内的COPD模型（即狗，豚鼠，猴子和啮齿动物）4。然而，鼠标已成为在COPD模型构造中最常用的。这有许多优点，包括成本低，具有转基因能力，广泛的基因组信息可用性，抗体的可用性，以及使用各种小鼠菌株的能力5。目前，没有小鼠模型可以模仿人类COPD的全部特征;因此，个别研究人员必须选择哪种模型最适合特定的慢性阻塞性肺病研究6。肺气小鼠模型是目前可用的众多 COPD 小鼠模型之一。其他模型包括加重小鼠模型、系统性共病模型和COPD易感性模型7。
肺气鼠模型可以由几种外源性物质产生，包括化学剂和香烟烟雾暴露4。化学品接触（例如，对乳化酶）会产生一种严重的肺气肿，而香烟烟雾则导致轻度肺气肿8，9。香烟烟雾被认为是COPD发病的主要原因;因此，选择香烟烟雾作为创建COPD小鼠模型的手段是合理的10。许多研究都利用香烟烟雾在老鼠体内制造肺气肿。例如，Nikula等人成功地从B6C3F1雌性小鼠身上培育出一种肺气肿小鼠模型，将它们暴露在香烟烟雾中7个月或13个月。我们还建立了肺气肿小鼠模型，通过衰老标记蛋白/SMP-30 KO小鼠12。执行肺固定方法至关重要，该方法可以通过香烟烟雾暴露来正确可视化这种温和的肺气肿模型。
各种肺固定方法已经建立13。然而，没有金标准肺组织固定方法用于评估肺气肿14。该实验室的几项研究表明，这里介绍的固定系统是有用的，为评估肺气肿12，15，16，17，18创建一个稳定的条件。目前系统的主要优点是，它可以修复许多肺与相同的条件在同一时间没有肺崩溃或通缩。目前的肺固定系统使用一些特殊设备，允许肺标本在给定时期内以适当的恒定压力膨胀。这种特殊设备由三个部分组成，包括下部容器、上部容器和泵。肺试样被放置在与加压固定剂相连的下容器中，导致上容器和下容器之间的代理水平存在25 cmH2O的压力差异19。
Protocol
以下方法已获得君登多大学医学院动物护理和使用委员会的批准。2006年6月1日，日本科学委员会遵循了《动物实验正确进行指南》。这种方法有三个主要步骤：1）小鼠解剖，2）肺外泄，和3）在专用设备辅助的肺组织的固定。通常，肺标本在48小时后被加工嵌入12，15，16，17，18。<sup class="xref"…
Representative Results
如前所述，产生扩展恒定压力的专用设备可分为三部分（图3A）。下部是插入肺样本的点（图4A）。肺通过导管（20 G）连接到形式素流动的尖端使用三向停止公鸡（图4B）。压力由下部和上容器之间的固定剂的不同表面水平产生（图5）。压力差为25 cmH2O;但是，使…
Discussion
这里介绍的啮齿动物肺的固定程序并不新颖;然而，该系统有几个优点。首先，它可以修复许多肺（最多20个）与相同的条件在同一时间。毒理学病理学学会指出，重力灌输的压力从22-25 cmH2O22不等。值得注意的是，一些研究在25 cmH2O13，19，23，24，<sup class="xref"…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了JSPS KAKENHI赠款号26461199（佐藤）和Juntendo大学医学院环境与性别特定医学研究所的支持，助学金编号为E2920（佐藤）。在设计现有方法和撰写手稿方面，受发人没有作用。
Materials
| 10% formalin (formalin neutral buffer solution) | Wako | 060-01667 | |
| Bent forceps | Hammacher | HSC187-11 | |
| Cannula, size 20G | Terumo | SR-FS2032 | |
| Cannula, size 22G | Terumo | SR-OT2225C | Cannula to exsanguinate lung |
| Forceps | Hammacher | HSC184-10 | |
| Kimtowel | Nippon Paper Crecia (Kimberly Clark) | 61000 | |
| Kimwipe | Nippon Paper Crecia (Kimberly Clark) | 62011 | |
| Lower container (acrylic glass material) | Tokyo Science | Custom-made | Pressure equipment component |
| Roller pump | Nissin Scientific Corp | NRP-75 | Pump machine to exsanguinate lung |
| Roller pump RP-2000 | Eyela (Tokyo Rikakikai Co. Ltd) | 160200 | Pressure equipment pump |
| Silicone tube Ø 9 mm | Sansyo | 94-0479 | Pressure equipment component |
| Somnopentyl (64.8 mg/mL) | Kyoritsu Seiyaku | SOM02-YA1312 | Pentobarbital Sodium |
| Surgical scissor | Hammacher | HSB014-11 | |
| Suture thread, size 0 | Nescosuture | GA01SW | |
| Syringe, 1 mL | Terumo | SS-01T | |
| Syringe, 1 ml with needle | Terumo | SS-01T2613S | |
| Syringe, 10 mL | Terumo | SS-10ESZ | |
| Three-way stopcock | Terumo | TS-TR1K01 | |
| Upper container (acrylic glass material) | Tokyo Science | Custom-made | Pressure equipment component |
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Cite This Article
Karasutani, K., Baskoro, H., Sato, T., Arano, N., Suzuki, Y., Mitsui, A., Shimada, N., Kodama, Y., Seyama, K., Fukuchi, Y., Takahashi, K. Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice. J. Vis. Exp. (151), e58197, doi:10.3791/58197 (2019).