Name: a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation
Uploaded: 2017-07-26T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

作者要向北京国家生物科学研究所致谢。这项工作得到了北京市自然科学基金（Z17110200040000）的支持。

本议定书中使用的所有程序均按照北京国家生物科学研究所"实验动物护理和使用指南"中的建议进行。将8周龄的CD-1雄性小鼠饲养在特定的无病原体（SPF）设施中，直至进行实验。使用完全麻醉的小鼠进行手术（ 即 ，没有任何趾夹紧反应）。手术后，将小鼠保持在温暖，潮湿的房间内，食物和淡水充足。使用过量腹膜内注射的麻醉剂处死小鼠。 小鼠PNX手术<ol><li>通过腹膜内（IP）注射，用苯巴比妥钠（120mg / kg体重）和丁丙诺啡（0.1mg / kg体重）完全麻醉小鼠。当小鼠不再对脚趾捏住反应时，进行手术。 </li><li>用化学物质除去老鼠左胸部的头发肛门治疗（〜3×3厘米2 ）。 </li><li>将每只鼠标固定在插管平台上，其腹侧朝向操作者（ 图1A ）。 </li><li>拉出鼠标舌并用含有用于引导导管18 （ 图1A ）的切口的小型动物喉镜照亮声带。 </li><li>通过在呼吸过程中观察声带的运动来区分声带。轻轻地插入一个20 G静脉插管插管气管在前角19 。 </li><li>将小鼠放置在右侧卧位，并将插管连接到机械呼吸机（ 例如，压力控制;参见材料表 ）。通过观察鼠标胸部的呼吸动作，检查插管插入气管（ 图1B ）。 </li><li>设置吸气压力o将呼吸机调至12厘米H 2 O，并将呼吸频率设置为每分钟120次呼吸（ 图1B ）。 </li><li>用betadine和70％乙醇去除外科手术区域的皮肤。 </li><li>使2 -在第 5 次肋间空间，通过皮肤和肌肉与诺伊斯弹簧剪刀剪3厘米后外侧开胸切口（切削刃:14毫米;尖端直径:0.275毫米）（ 图2B，C）。用于开胸手术的手术器械在使用前进行灭菌。 </li><li>使在第 5肋间空间1.5厘米的切口，以暴露左肺（ 图2D，E）。在手术中，使用高温烧灼器止血。 </li><li>用钝头镊子从胸部抬起左肺叶三分之一（ 图2F ），然后用棉签拉出整个左侧肺（ 图2G ）。 </li><li>确定左肺叶肺动脉和支气管（ 图2G ）。 </li><li>用丝线外科缝合线将支气管和门口的血管紧密连接，并从连接处切开4〜4mm的左肺叶（ 图2H ，I ）。 注意:小心不要切断左门槛上的缝合线，这可能导致气胸（ 即胸腔腔内的空气或气体） 。 </li><li>用1条缝合物关闭胸壁，然后依次缝合肌肉层和皮肤层，使用5 - 6个中断的缝合线。在每个缝合线之间留出3 - 4 mm的间隙（ 图2M ，2N ）。 注意:将手术缝合针远离心脏;无心的心脏穿刺会导致立即死亡。 </li><li>用聚维酮碘消毒手术区域。 </li><li> AFTE在外科手术中，将鼠标放在38°C的热垫上，并将鼠标连接到呼吸机，直到自发呼吸运动开始（ 图2O ）。 </li></ol>假体植入<ol><li>执行PNX程序的步骤1.1 - 1.13（即，直到鼠标的左肺叶被移除的时候）。 </li><li>使用钝钳钳住硅胶假体的中心（客户制造，长度为12mm，厚度为3mm，宽度为7mm，椭圆形为0.2g）（ 图2J ）。插入硅胶假体前消毒。 </li><li>用镊子用一只手握住肋骨露出胸腔，然后用另一只手将假体插入左侧空胸腔。 注意:在假体的正面和胸部表面之间的插入角度约为45度（ 图2K ，L）。插入假体时要温柔。过度的力会导致胸膜破裂。 </li><li>用钝钳调整假体的方向，以确保假体占据左空胸腔。 </li><li>执行小鼠PNX程序的步骤1.14 - 1.16。 </li></ol> 3.测量ILV <ol><li>准备一个定制设备（"充气管"），该设备由从一次性血清移液管（10 mL）中取出的柱塞，带有针头适配器的40 cm长柔性管，流量控制阀和18 G针组成。组装后，将吸管固定在带有磁带的电路板上（ 图3A ）。移液器顶部和实验台之间的距离必须至少为30厘米。 </li><li>通过在55℃水浴中将20g PFA溶解在500mL预加热的1x磷酸盐缓冲盐水（PBS）中来准备新鲜的4％多聚甲醛（PFA）固定溶液，手动摇动每10分钟一次，直到溶液澄清。冷却至室温后，用0.45μm过滤器过滤溶液。 小心:处理PFA时，穿戴适当的个人防护装备（PPE）。 </li><li>用过量注射麻醉剂（0.8％苯巴比妥钠，1000U / mL肝素）服用牺牲小鼠。 </li><li>将每只小鼠固定在聚苯乙烯夹层板上，并用70％酒精喷雾。 </li><li>小心打开小鼠胸部，用剪刀切开胸骨，彻底暴露肺叶。 </li><li>使用剪刀去除多余的组织以暴露气管。确保将气管与食道分开。 </li><li>切开腹主动脉，并将25号针插入心脏右心室;在插入前将针连接到20 mL注射器。慢慢地将1x PBS推入心脏去除血细胞，直到肺变白。通常需要5-10毫升的PBS来清除肺血管。 </li><li>填充e定制的膨胀管，4％新鲜PFA，并从充气管中除去所有的气泡。 </li><li>将充气管的18号针插入气管，并用气管夹夹住气管，以避免流体泄漏。 </li><li> 在25厘米的恒定跨肺压/ H 2 O 2，20膨胀用4％PFA肺部。在室温下孵育肺2小时以达到充分扩张的肺。这种"预先修复"步骤对于保持肺形态至关重要。 <ol><li>通过监测通胀管，记录初始4％PFA量的值，并记录最终量。内部肺容积等于最初的4％PFA体积减去最终4％的PFA体积。 </li></ol></li><li>用气管和剪刀，轻轻地从周围的结缔组织中清除肺部（保持完整的肺）。非常温柔，以免损伤肺部。 </li><li> INCU使用4％PFA填充的50mL锥形管中的肺将其在4℃下轻轻晃动振荡器（50rpm）。继续进行组织加工和染色（见第4部分）。 </li></ol> 4.组织包埋，切片和苏木精与曙红（H＆E）染色<ol><li>固定后，使用Noyes弹簧剪刀剪除心脏和肺部过多的结缔组织。通过切断将肺叶连接到气管的支气管轻轻分开各肺叶。 </li><li>在50 ml 1x PBS（30 min /次）中，在定位摇床上（50 rpm）将肺叶大面积清洗3-4次。 </li><li>最终洗涤后，通过在4℃将它们浸入30％蔗糖溶液（1×PBS）中冷冻保护肺叶，直到组织沉入50mL锥形管的底部（约12小时）。 </li><li>在对组织进行嵌入和冷冻切片之前，用镊子从管中取出肺叶样品，保留一下用于组织学分析的裂片，从辅助叶片样品的表面吸取剩余的蔗糖溶液，然后将样品彻底浸入含有最佳切割温度（OCT）化合物的培养皿中约30分钟。 </li><li>使用低温枪将液氮中的OCT嵌入式附件叶片样品冷冻。将凸起的最大表面积与模具的底部平行。 </li><li>在冷冻切片过程中，为每个样品准备三个10μm厚的切片，用于组织学分析。弃去前1毫米的组织，收集一个10微米厚的部分，丢弃0.5毫米的组织，收集另一部分，丢弃0.5毫米的组织，并收集第三（最后）部分。 </li><li>在进行H＆E染色之前，将切片空气干燥1 h。 </li><li> 进行H＆E染色 <ol><li>洗涤3-4份自来水的切片，然后在新鲜的苏木精中染色切片2分钟;冲洗自来水下的部分;将该部分浸入1％HCl-70％乙醇溶液中以除去多余的苏木精。 </li><li>在新鲜曙红中染色3分钟;在95％乙醇中连续30秒洗涤两次，并用100％乙醇洗涤两次30s洗涤部分。清除二甲苯中的切片30秒，在新鲜的二甲苯中重复清除步骤一次;使用玻璃盖玻片安装带有安装介质的滑块。 </li></ol></li></ol> 5.定量MLI <ol><li>使用亮场显微镜获取H＆E染色的辅助叶片部分的数字图像（20倍放大）。 </li><li>为了量化MLI，从3个部分的合适区域（无动脉和静脉，主要气道和肺泡管）中随机选择总共15个非重叠视图（1,000μmx 1,000μm）。 </li><li>放置一个具有10个均匀分布的垂直线和10个均匀分布的水平线的网格使用标尺工具在所选择的视野上的gth（1,000μm）;因此每条线间隔100μm（ 图4B ）。 </li><li>将一个截距的值定义为两个相邻肺泡上皮之间的线性长度。测量每个1000 m长度线上所有截距的值。 </li><li>对于每个网格，量化10个水平1,000μm长度线和10个垂直1,000μm长度线中的所有截距的值。 注意:MLI是从为每个辅助波瓣准备的3个部分中分析的总共15个网格的截距长度的平均值。 </li></ol> 6.计算ISA <ol><li>使用公式1计算ISA（见导言 ）。有关ILV的测量，请参阅第3节，并参考第5节MLI的量化。 </li></ol>

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作者没有什么可以披露的。

在本协议中，我们提供关于肺小鼠左肺PNX和假体植入后肺参数测量的详细描述。 ISA现在被认为是评估许多肺部疾病和损伤引起的肺泡再生中呼吸功能的关键指标。然而，虽然肺部研究界对ISA的实用性有一致意见，但迄今为止，几乎没有考虑用于计算ISA的两个参数ILV和MLI的测量标准化。显然，与任何测量一样，重要的是尝试获得无偏见的数据。本研究工作的核心目标是建立一个标准化的方案供小鼠肺部研究界使用。 当我们开发这个协议时，我们试图以多种方式减少测量偏差的来源。我们发现ILV测量的变化可能是红色的通过确保插入到气管中的针的尺寸尺寸匹配来防止流体泄漏（我们发现18号针紧密匹配小鼠气管）。我们还发现，在PFA充气之前，需要彻底清除小鼠胸壁，因为这最小化胸壁对ILV测量的潜在影响。 MLI值在很大程度上取决于肺泡形态。因此，除了使用年龄匹配和性别匹配的小鼠的重要性之外，在肺固定程序期间适当维持肺泡形态至关重要。我们在这里使用广泛采用的固定方法:我们用新鲜制备的4％PFA，以25cm H 2 O的经肺压力充气肺，以充分扩张肺组织。根据我们的经验，较低的膨胀压力可导致组织收缩，异常肺泡形态，最终导致较低的MLI值。 观察我们以前的研究表明，在PNX再生后，剩余肺的辅助叶显示出与右肺其他三个叶相比最大的体积膨胀;剩余肺的附件叶也表现出形态学参数的值的最大增加（ 例如，MLI）21，22，23，24。因此，我们仅分析了附属瓣的部分，以避免不同肺叶的变化。为了减少在MLI定量中使用的各个部分之间的差异，我们在嵌入过程中控制了叶片的方向:我们将叶片的最大表面平行于低温温室底部放置。我们还仔细控制样品切片的厚度，切片顺序和切片位置。除了样品加工外，另外还有一种标准化的一个方面是，所有的动脉和静脉，胸膜，主要气道和肺泡管都需要从在MLI量化过程中评估的组织区域中排除。动脉，静脉和肺泡管均大于肺泡（4〜10倍），因此排除这些大结构对获得可靠的截距测量非常重要。对于每组，5只小鼠足以进行定量。对于每只小鼠，从辅助叶的3个部分的适当区域（无动脉和静脉，主要气道和肺泡管）随机选择总共15个非重叠视图（1,000μm×1,000μm）。样品包埋​​方法和MLI测量也可应用于石蜡处理的肺组织。 而其他人将MLI定义为总线长度除以具有交叉肺泡壁25的截距数，我们在此使用线性截距作为线性长度在MLI计算中两个相邻的肺泡上皮壁之间，不考虑来自MLI数据的间质的厚度。因此，我们使用ILV计算ISA，但不计算肺总体积。 PNX和假体植入手术中，PNX小鼠的存活率为85％〜90％。接受假体植入的小鼠的存活率约为80％。在所有手术中，应采取几个步骤来改善小鼠的生存。 1）导管在气管中的正确插入是PNX手术成功的先决条件。在喉镜的指导下，可以轻松观察小鼠的气管，以方便安全有效的气管插管。 2）手术过程中不要刺穿肺或心脏。确保第五左肋间空间的胸部被广泛打开，并且在叶切除术之前清楚地识别出左叶的门。执行左肺叶时通过使用钝钳确保左叶完整，以避免肺出血和/或肺叶破裂。在伤口闭合期间，将手术针的尖端远离心脏和肺。 3）将假体插入空胸腔时要温和，因为过度的力会导致胸膜破裂。 4）小鼠应放置在38°C的热垫上，并进行监测，直到感觉恢复，因为已知术后低温会增加小鼠的发病率。 去除左肺叶后，肺呼吸机和内肺气交换区均明显减少。手术后14天，PNX治疗组小鼠的剩余肺组织中ILV，MLI和ISA显着大于假体植入组，强烈表明假体插入阻断了PNX诱导的肺泡再生。因此，PNX和假体植入鼠标模式ls可用作研究在机械力引起的再肺泡中发生的细胞和分子事件的强大工具。此外， 邑 AT2空肺的ISA值在后PNX14天1较对照肺显著较小，这表明我们的协议也适用于检测遗传突变体小鼠的受损的再生。本研究中提出的严格和标准化的定量方法可用于测量发育研究中的肺参数和ISA，以及多种疾病的遗传修饰动物模型，包括慢性阻塞性肺疾病肺气肿，肺损伤后肺泡再生和肺发育缺陷。

肺的基本功能是血管和大气之间的氧气和二氧化碳的交换。肺部疾病，如支气管肺发育不良（BPD），慢性阻塞性肺病（COPD）和急性呼吸道感染，导致降低的ISA 2。研究人员在研究肺病已经开发出多种定量方法来评估肺形态变化，包括美林，ILV，3的气体交换单元的数量，ISA，和肺组织顺应性2。 Weibel 等人的开创性研究4和Duguid 等人 5一起确定了ISA可以作为人肺肺气交换能力的直接指标，可作为确定肺气肿严重程度的标准。过去五年发表的一些研究已经使用肺形态学参数（ 例如， </e米&gt; ISA和MLI）对发展6期间和从损伤PNX 1,7恢复期间评估在小鼠的肺中的形态和功能的变化。 ISA使用等式1 8,9计算: <img alt="方程" src="/files/ftp_upload/56114/56114eq1.jpg" /> ，其中ILV是内部肺容积，MLI是表示肺外周空间大小10的中间参数。 PNX，手术切除的一个或多个肺叶，已被广泛报道，以诱导在许多物种中肺泡再生，包括人类11，小鼠1，狗12，大鼠13，和兔14,15。一个螺柱PNX后14天的小鼠肺显示，既存在肺泡的扩张和肺泡从头形成有助于恢复其余肺组织中的ISA，ILV和肺泡数量1 。我们和其他人已经表明，在PNX（ 即 ，假体植入）后，将诸如海绵，蜡或定制假体的材料插入空胸腔会损害肺泡再生。现在已牢固地确立机械力功能的发起肺泡再生1，16，17最重要的因素之一。这些研究强调了使用PNX处理和假体植入肺的ISA值作为定量评估肺泡再生的标准的有效性。 已知观察者偏倚显着影响测量值肺部形态学参数（ 例如 ，MIL和ILV）。可以使用标准化协议来消除在确定ILV和MLI中的这种偏差，这是在ISA的计算中使用的两个参数。在这里，我们提供了高度详细的标准化方案来测量这些肺参数。重要的是，准确量化ISA的能力有望提高损伤诱导的肺泡再生模型中肺功能研究的可靠性和可重复性，并且有助于多种肺部疾病的机械发现。

<table><tbody><tr><td>Low cost cautery kit</td><td>Fine Science Tools</td><td>18010-00</td><td></td></tr><tr><td>Noyes scissors</td><td>Fine Science Tools</td><td>15012-12</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Castroviejo Micro Needle Holders</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>Vessel clips</td><td>Fine Science Tools</td><td>18374-44</td><td></td></tr><tr><td>I. V. Cannula-20 gauge</td><td>Jinhuan Medical Product Co., LTD.</td><td>29P0601</td><td></td></tr><tr><td>Surgical suture</td><td>Jinhuan Medical Product Co., LTD.</td><td>F602</td><td></td></tr><tr><td>Mouse intubation platform</td><td>Penn-Century, Inc</td><td>Model MIP</td><td></td></tr><tr><td>Small Animal Laryngoscope</td><td>Penn-Century, Inc</td><td>Model LS-2-M</td><td></td></tr><tr><td>TOPO Small Animal Ventilator</td><td>Kent Scientific</td><td>RSP1006-05L</td><td></td></tr><tr><td>Thermal pad</td><td>Stuart equipment</td><td>SBH130D</td><td></td></tr><tr><td>Pentobarbital sodium salt</td><td>Sigma</td><td>P3761</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Hematoxylin Solution</td><td>Sigma</td><td>GHS132</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma</td><td>HT110116</td><td></td></tr><tr><td>10 mL Pipette</td><td>Thermo Scientific</td><td>170356</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>O.C.T Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Disposable Base Molds</td><td>Fisher HealthCare</td><td>22-363-553</td><td></td></tr><tr><td>18 gauge needle</td><td>Becton Dickinson</td><td>305199</td><td></td></tr><tr><td>Povidone iodine</td><td>Fisher Scientific</td><td>19-027132</td><td></td></tr><tr><td>70% ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td></td></tr><tr><td>Infusion sets for single use</td><td>Weigao</td><td>SFDA 2012 3661704</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Tapes</td><td>3M Scotch</td><td>8915</td><td></td></tr><tr><td>Cotton pad</td><td>Vinda</td><td>Dr.P</td><td></td></tr><tr><td>Silicone prosthesis</td><td>Custom made</td><td></td><td></td></tr><tr><td>Brightfield microscope</td><td>Olympus</td><td>VS120</td><td></td></tr><tr><td>Ruler tool</td><td>Adobe Photoshop</td><td></td><td></td></tr></tbody></table>

a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

<html>我们在这里进行了PNX治疗组和假体植入（假体植入）组的实验。这些分组与我们研究组14之前发表的研究中使用的分组相同。 小鼠PNX和假体植入程序如图2所示。 8周龄的CD-1雄性小鼠用于手术和量化。在PNX治疗组和假体植入组中，左肺叶切除（ 图2A - 2I ）。在假体植入组中，在左肺叶被移除后，将模拟左肺叶大小和形状的假体插入胸部（ 图2J - 2L ）。 _content"fo:keep-together.within-page ="1"&gt;手术后14天，使用定制的充气管来确定剩余右肺的ILV（ 图3A ），剩余右侧的平均ILV 5只经PNX处理的小鼠的肺大约为1.4mL，明显高于5个假体植入小鼠右肺的1.05mL ILV值（ 图3B ，表1 ）。 对于MLI测量，从每只小鼠准备的3个部分中总共分析了15个视图。 图4A示出了来自辅助叶片部分的合并图像以及用于定量MLI的所选区域（ 例如，视图1-3）或非选择区域（ 例如，视图4-5）的形态学标准。在这里，以PNX治疗组的附属肺叶部分的视图3为例MLI的测量（ 图4B ）。还示出了放大的视图3，用于说明（ 图5A ）。第3行作为示例说明:由双头箭头线表示的被截留的肺泡空间的长度。我们的分析显示，PNX处理的小鼠的剩余右肺中的MLI值显着大于假体植入的小鼠的剩余右肺的MLI值（ 图5B ，表1 ）。所有数据表示为平均值±SEM （ 图5B ）。 使用等式1计算ISA。 表1显示所有肺的ILV值，MLI值和ISA。假体植入的小鼠的ISA显着小于PNX处理的小鼠的ISA，表明插入n假体受损PNX诱导再生。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56114/56114fig1.jpg" /> 图1:小鼠气管插管和机械通气。 （ A ）通过喉镜检查使用20 G静脉插管插管进行气管插管。 （ B ）将手术完全麻醉的鼠标连接到压力控制的机械呼吸机上。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56114/56114fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56114/56114fig2.jpg" /> 图2:小鼠肺切除术（PNX）和假体植入术。 （ A - C ）切割皮肤和肌肉层;停止b在外科手术过程中用高温烧灼器治疗。 （D - E）向位于第 5肋间1.5厘米的切口。 （ F - H ）用钝镊子拉出左肺叶，识别肺动脉和支气管，在门口连接。箭头代表左肺叶的肺动脉和支气管。 （ 一 ）左侧肺叶切除术后3〜4 mm。 （ J - L ）将假体插入左胸腔。 （ M，N ）缝合胸部，肌肉层和皮肤层。 （ O ）监测小鼠，直到自发呼吸运动开始。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56114/56114fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图3:剩余右肺的内部肺体积（ILV）的测量。 （ A ）用于测量内部肺容积的定制装置（"充气管"）。 （ B ）在PNX后14天测量PNX处理组和假体植入组剩余右肺的ILV（平均值±SEM）。 **， p &lt;0.01，学生t检验。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56114/56114fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56114/56114fig4.jpg" /> 图4:剩余右肺中附件的平均线性截距（MLI）的量化。 （ A ）合并的显示附件叶片的年龄。用于MLI量化的所选区域（ 例如，视图1-3）和非选择区域（ 例如，视图4-5）的示例。 （ B ）将10个均匀间隔的垂直线和10个均匀间隔的水平线（1,000μm）设置在选定的区域上。比例尺= 1 mm。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56114/56114fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/56114/56114fig5.jpg" /> 图5:PNX处理的肺和假体植入的肺的MLI的定量。 （ A ）示出了图4B中的视图3的放大图 。带有双箭头的红色线代表一个线性截距的长度。 （ B ）MLI值（平均值±SE在PNX后14天测量PNX处理的小鼠和假体植入的小鼠的附属裂片。 *， p &lt;0.05，学生t检验。刻度棒:100μm。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56114/56114fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 术后14天 </td><td> 内肺容积（ml） </td><td> 平均线性截距（mm） </td><td> 内肺表面积= 4ILV / MLI（cm 2 ） </td></tr><tr><td rowspan="5"> PNX处理 </td><td> 1.5 </td><td> 57.8 </td><td> 1038.06 </td></tr><tr><td> 1.35 </td><td> 49.6 </td><td> 1088.71 </td></tr><tr><td> 1.42 </td><td> 48.5 </td><td> 1171.13 </td></tr><tr><td> 1.4 </td><td> 51.5 </td&gt; <td> 1087.38 </td></tr><tr><td> 1.4 </td><td> 54.6 </td><td> 1025.64 </td></tr><tr><td rowspan="5"> 假体植入 </td><td> 1.1 </td><td> 49.6 </td><td> 887.10 </td></tr><tr><td> 1 </td><td> 50.5 </td><td> 792.08 </td></tr><tr><td> 1.1 </td><td> 47.3 </td><td> 930.23 </td></tr><tr><td> 1.15 </td><td> 44.8 </td><td> 1026.79 </td></tr><tr><td> 0.86 </td><td> 46.3 </td><td> 742.98 </td></tr></tbody></table> 表1:PNX处理和假体植入的小鼠的ISA值的计算。 PNX后14天附件叶的ILV，MLI和ISA的值。

Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

内肺表面积（ISA）是评估肺部疾病和损伤诱导肺泡再生的肺形态和生理学的关键标准。我们在这里描述了一种标准化方法，可以最大限度地减少肺肺切除术和假体植入小鼠模型中ISA的测量偏差。

通过小鼠肺切除术和假体植入测量内部肺表面积的标准方法

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

a-standardized-method-for-measuring-internal-lung-surface-area-via

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.1K 次观看.  Peking University. 内肺表面积（ISA）是评估肺部疾病和损伤诱导肺泡再生的肺形态和生理学的关键标准。我们在这里描述了一种标准化方法，可以最大限度地减少肺肺切除术和假体植入小鼠模型中ISA的测量偏差。 

视频: A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

科研

医学

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

研究癌症转移至肺部的永久窗口

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

癌症研究

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

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&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;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.

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.

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

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 ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

补偿性增长龙鼠标肺切除型号

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

在小鼠肺压力容积曲线的测量

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

灌输和固定方法有用的小鼠肺癌研究

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

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

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

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

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

免疫与感染

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

<meta charset="utf8"></head><body>小鼠肺移植中细胞动力学的实时监测

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>作者聚焦：通过活体成像推进肺移植免疫学

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植小鼠肺的活体双光子显微镜检查

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...