Name: lung fixation under constant pressure for evaluation of emphysema in mice
Uploaded: 2019-09-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice Video at JoVE.com

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。

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"...

<ol>
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W., Ghosh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Relevance+of+the+Mouse+Cigarette+Smoke+Model+of+COPD:+Peering+through+the+Smoke.">Understanding the Relevance of the Mouse Cigarette Smoke Model of COPD: Peering through the Smoke.</a> American Journal of Respiratory Cell and Molecular Biology. 57 (1), 3-4 (2017).</li><li>Wright, J. L., Cosio, M., Churg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+chronic+obstructive+pulmonary+disease.">Animal models of chronic obstructive pulmonary disease.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 295 (1), L1-L15 (2008).</li><li>Rennard, S. I., Togo, S., Holz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+inhibits+alveolar+repair:+a+mechanism+for+the+development+of+emphysema.">Cigarette smoke inhibits alveolar repair: a mechanism for the development of emphysema.</a> Proceedings of the American Thoracic Society. 3 (8), 703-708 (2006).</li><li>Nikula, K. 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D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 299 (6), L843-L851 (2010).</li><li>Hsia, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Kasagi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+juice+prevents+senescence-accelerated+mouse+P1+strain+from+developing+emphysema+induced+by+chronic+exposure+to+tobacco+smoke.">Tomato juice prevents senescence-accelerated mouse P1 strain from developing emphysema induced by chronic exposure to tobacco smoke.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 290 (2), L396-L404 (2006).</li><li>Koike, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+lack+of+vitamin+C+intake+generates+pulmonary+emphysema+in+senescence+marker+protein-30+knockout+mice.">Complete lack of vitamin C intake generates pulmonary emphysema in senescence marker protein-30 knockout mice.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 298 (6), L784-L792 (2010).</li><li>Koike, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+C+prevents+cigarette+smoke-induced+pulmonary+emphysema+in+mice+and+provides+pulmonary+restoration.">Vitamin C prevents cigarette smoke-induced pulmonary emphysema in mice and provides pulmonary restoration.</a> American Journal of Respiratory Cell and Molecular Biology. 50 (2), 347-357 (2014).</li><li>Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-rich+pure+water+prevents+cigarette+smoke-induced+pulmonary+emphysema+in+SMP30+knockout+mice.">Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice.</a> Biochemical and Biophysical Research Communications. 492 (1), 74-81 (2017).</li><li>Saad, M., Ruwanpura, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Processing+for+Stereological+Analyses+of+Lung+Structure+in+Chronic+Obstructive+Pulmonary+Disease.">Tissue Processing for Stereological Analyses of Lung Structure in Chronic Obstructive Pulmonary Disease.</a> Methods in Molecular Biology. 1725, 155-162 (2018).</li><li>Thurlbeck, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+surface+area+of+nonemphysematous+lungs.">The internal surface area of nonemphysematous lungs.</a> The American Review of Respiratory Disease. 95 (5), 765-773 (1967).</li><li>Saetta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Destructive+index:+a+measurement+of+lung+parenchymal+destruction+in+smokers.">Destructive index: a measurement of lung parenchymal destruction in smokers.</a> The American Review of Respiratory Disease. 131 (5), 764-769 (1985).</li><li>Renne, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Schneider, J. P., Ochs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+mouse+lung+tissue+dimensions+during+processing+for+morphometry:+a+comparison+of+methods.">Alterations of mouse lung tissue dimensions during processing for morphometry: a comparison of methods.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 306 (4), L341-L350 (2014).</li><li>Wright, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+pulmonary+arterial+pressure+and+airflow+obstruction+to+emphysema.">Relationship of pulmonary arterial pressure and airflow obstruction to emphysema.</a> Journal of Applied Physiology. 74 (3), 1320-1324 (1993).</li><li>Wright, J. L., Churg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+causes+physiologic+and+morphologic+changes+of+emphysema+in+the+guinea+pig.">Cigarette smoke causes physiologic and morphologic changes of emphysema in the guinea pig.</a> The American Review of Respiratory Disease. 142 (6 Pt 1), 1422-1428 (1990).</li><li>Thurlbeck, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+surface+area+and+other+measurements+in+emphysema.">Internal surface area and other measurements in emphysema.</a> Thorax. 22 (6), 483-496 (1967).</li><li>Wright, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+remodeling+in+the+smoke+exposed+guinea+pig+model.">Airway remodeling in the smoke exposed guinea pig model.</a> Inhalation Toxicology. 19 (11), 915-923 (2007).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Roos, A. B., Berg, T., Ahlgren, K. M., Grunewald, J., Nord, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+generating+pulmonary+neutrophilia+using+aerosolized+lipopolysaccharide.">A method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide.</a> Journal of Visualized Experiments. (94), (2014).</li><li>Laucho-Contreras, M. E., Taylor, K. L., Mahadeva, R., Boukedes, S. S., Owen, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+measurement+of+pulmonary+emphysema+and+small+airway+remodeling+in+cigarette+smoke-exposed+mice.">Automated measurement of pulmonary emphysema and small airway remodeling in cigarette smoke-exposed mice.</a> Journal of Visualized Experiments. (95), 52236 (2015).</li><li>Nakanishi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarithromycin+prevents+smoke-induced+emphysema+in+mice.">Clarithromycin prevents smoke-induced emphysema in mice.</a> American Journal of Respiratory and Critical Care Medicine. 179 (4), 271-278 (2009).</li><li>Maeno, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> Journal of Immunology. 178 (12), 8090-8096 (2007).</li><li>Sato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+fixation+for+total+preanalytic+phase+evaluation+in+pathology+laboratories:+a+comprehensive+study+including+immunohistochemistry,+DNA,+and+mRNA+assays.">Optimal fixation for total preanalytic phase evaluation in pathology laboratories: a comprehensive study including immunohistochemistry, DNA, and mRNA assays.</a> Pathology International. 64 (5), 209-216 (2014).</li></ol>

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"...

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。

<table>
	<tbody>
		<tr>
			<td>10% formalin (formalin neutral buffer solution)</td>
			<td>Wako</td>
			<td>060-01667</td>
			<td></td>
		</tr>
		<tr>
			<td>Bent forceps</td>
			<td>Hammacher</td>
			<td>HSC187-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula, size 20G</td>
			<td>Terumo</td>
			<td>SR-FS2032</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula, size 22G</td>
			<td>Terumo</td>
			<td>SR-OT2225C</td>
			<td>Cannula to exsanguinate lung</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Hammacher</td>
			<td>HSC184-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtowel</td>
			<td>Nippon Paper Crecia (Kimberly Clark)</td>
			<td>61000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Nippon Paper Crecia (Kimberly Clark)</td>
			<td>62011</td>
			<td></td>
		</tr>
		<tr>
			<td>Lower container (acrylic glass material)</td>
			<td>Tokyo Science</td>
			<td>Custom-made</td>
			<td>Pressure equipment component</td>
		</tr>
		<tr>
			<td>Roller pump</td>
			<td>Nissin Scientific Corp</td>
			<td>NRP-75</td>
			<td>Pump machine to exsanguinate lung</td>
		</tr>
		<tr>
			<td>Roller pump RP-2000</td>
			<td>Eyela (Tokyo Rikakikai Co. Ltd)</td>
			<td>160200</td>
			<td>Pressure equipment pump</td>
		</tr>
		<tr>
			<td>Silicone tube &#216; 9 mm</td>
			<td>Sansyo</td>
			<td>94-0479</td>
			<td>Pressure equipment component</td>
		</tr>
		<tr>
			<td>Somnopentyl (64.8 mg/mL)</td>
			<td>Kyoritsu Seiyaku</td>
			<td>SOM02-YA1312</td>
			<td>Pentobarbital Sodium</td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Hammacher</td>
			<td>HSB014-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture thread, size 0</td>
			<td>Nescosuture</td>
			<td>GA01SW</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 mL</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml with needle</td>
			<td>Terumo</td>
			<td>SS-01T2613S</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Terumo</td>
			<td>SS-10ESZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>Terumo</td>
			<td>TS-TR1K01</td>
			<td></td>
		</tr>
		<tr>
			<td>Upper container (acrylic glass material)</td>
			<td>Tokyo Science</td>
			<td>Custom-made</td>
			<td>Pressure equipment component</td>
		</tr>
	</tbody>
</table>

lung fixation under constant pressure for evaluation of emphysema in mice

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。

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...

Watch this Scientific Journal Video about Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice Video at JoVE.com

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice Video (Video) | JoVE

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。

小鼠肺气肿评价持续压力下的肺固定

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

固定装置内的恒定压力使肺部保持合理膨胀状态，产生一个组织学肺标本，适合评估轻度香烟烟雾诱发肺气肿。该模型的主要优点是，它可以修复许多肺在同一时间使用相同的恒定压力，没有肺崩溃或通缩。演示这个程序的将是 Naoko Arano， 一个研究生从我的实验室。在确认对反射运动缺乏反应后，沿着小鼠的中线在皮肤和肌肉组织进行切口，瞄准头型区域，并横向切割以创造更宽的工作空间。暴露膜片层后，用钳子刺穿组织并打开胸腔空间。切开胸骨区域，使肺部和心脏被可视化。切左中庭和右心室将连接到灌注泵的 24 量表管插入右心室区域，并将管管插入脑腔区域，直到到达肺动脉。然后，打开泵，用大约200毫升PBS将组织吸气一小时，直到所有的肺组织都变成白色。在灌注结束时切割气管、肺和心脏周围的结缔组织，并使用缝合线将右主支气管绑起来。接下来，收获右肺的所有个体叶，将左肺的心脏和叶放入含有适当固定剂的单个10毫升注射器中。缩回柱塞，在注射器中产生真空，使肺部膨胀，并从注射器中去除肺部。将 20 量表管插入气管，并使用缝合线将管管固定到位。将装满固定剂的一毫升注射器连接到管内，并给肺部加注额外的固定剂，以检查泄漏。确保插入位点已固定，以防止肺标本在固定过程中从肺板分离。然后将组织转移到肺固定压力设备，取出样品，并在固定期结束时用结绑掉气管。将肺样本插入的肺固定压力设备的下部组件通过 20 量表管连接到甲醛流尖端，并配有三向止动器。压力来自下部和上部隔间之间的固定器的不同表面水平。压力差为 25 厘米的水，但高度调节旋钮可用于将压力调节到 25 到 30 厘米的水。泵连接下部和上部容器，在固定表面高度中保持 25 厘米的差异，并指示代理的流动。经过48小时的固定后，SMP30敲除小鼠的肺部暴露在环境空气中，不会表现出明显的空域扩大。然而，在SMP30淘汰暴露于慢性香烟烟雾的小鼠中观察到大量空域扩大和肺肺壁破坏。此外，在烟雾中暴露的SMP30敲击小鼠中，肺标本的线性截取和破坏性指数明显高于暴露在空气中的动物。我们的实验室使用10%的甲醛，但研究人员可以根据他们的实验需要使用其他固定剂。正如我们所报告使用演示的固定系统肺样本可以从各种小鼠肺气肿模型中采集，用于其形态评估。由于固定剂是危险的，始终穿戴适当的人员防护设备，并在通风良好的房间工作。

Research

JoVE Journal

Medicine

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

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