All authors disclose no financial or any other conflicts of interests.
GRANTS:
This study was supported by a grant from the Deutsche Forschungsgemeinschaft to P. Pickerodt and W. Boemke (Pi795/2-2).

实验在实验医学的Charité部进行 - Universitätsmedizin，柏林，德国（根据EN DIN ISO 9001 CERTI网络版:2000），并在柏林，德国前实验联邦当局对动物的研究获得批准。实验动物管理，这是在所有的实验中使用的原则，分别按照实验动物科学的欧洲和德国社会的准则。 1.动物福利和动物实验<ol><li>所有的实验都是在完全麻醉公猪进行（德国长×大）的3-4个月的年龄，体重30-60公斤。 </li></ol> 2.麻醉，插管，机械通气<ol><li>扣压食物前麻醉，避免了满肚子猪的12小时，但允许自由饮水，以减少压力。 </li><li>对于术前用药，注入一个合作二甲苯胺噻嗪的mbination（3毫克/千克），阿托品（0.03毫克/千克），氯胺酮（25毫克/千克）和甲苯噻嗪（3.5毫克/千克）到猪的颈部肌肉，而动物仍保存在盒子最大限度地减少压力。 <ol><li>动物放置到担架和一旦麻醉的适当水平达到与运输的布覆盖的眼睛。 </li><li>运输猪的手术剧院和确保在任何时候都充分的自主呼吸通过保持鼻子通畅。 </li><li>放置猪俯卧位和preoxygenate用适合使用氧的高流量（ 例如 10L /分钟）动物的鼻子的掩模。 </li></ol></li><li>开始监控的周氧饱和度（S P 2 O 2）通过夹紧显示器的各自的传感器到耳朵中的一个。增益放入耳缘静脉之一，酒精掉期擦拭过程后应用于临床外​​周静脉导管（通常是18或20 G）静脉通路。 <ol&gt; <li>用500毫升丸剂随后约4毫升/公斤/小时的连续输注平衡晶体溶液开始输注（在认为取决于实验临床需要），并确保了导管的正确位置为麻醉剂的后续输注。 注:较大生理盐水代替平衡晶体溶液可导致高氯酸中毒的体积的输液，而含有乳酸可导致增加的血清乳酸浓度，并因此与血液气体分析的解释或干扰的溶液注入随后的实验的结果。 </li></ol></li><li>充分预吸氧（期间外周静脉通路的完整的时间预吸氧，测定小号P 2 O 2的95％-100％）注入异丙酚（约5-10毫克/公斤体重后-确切剂量依赖于术前用药和不同的效果从使用外围静脉导管动物动物）。 </li><li>插管使用气管插管的临床应用（7.5-8.5 ID）和专为大型动物喉镜（约25厘米长的直叶片）俯卧位的猪。 <ol><li>通过动物（第一调查）的吻表带2绷带。向上拉一包扎头部移动到正确的位置，摆正口咽结构，拉动其他绷带向下打开吻。舌头拉向一侧（第二次调查）。 </li><li>按舌头打倒喉镜的叶片，推动刀片朝着会厌。请注意，在这个位置上，会厌常常楔入猪的软腭的后面。 </li><li>动员会厌与管，向下按用喉镜的叶片和可视化声带插管。 </li><li>通过声带推进管而向上转动管与如由Theisen 等人详细描述方框袖带5 </s了&gt;。 注:插管也可以是可能在仰卧位，这取决于研究者的训练以及在一定机构的标准程序。大管径支持由于更快的流入和灌洗液流出表面活性剂冲洗。 </li><li>检查使用二氧化碳分析和听诊管的正确位置。对于这一点，确保二氧化碳描记图是"正常的"形和听诊两肺为在诊所做等于呼吸音。 注:机械通风与胸外按压猪失败或延迟插管的情况。这需要从两侧肋笼的手动压缩，同时经由紧身面罩高流量供给氧气。 </li></ol></li><li>开始机械通气，设定吸入氧的分数（F I O 2）为1时，呼吸器频率15-20 /分，8-9毫升/公斤体重潮气量，灵感到期比例:（一E）1:1.5，并且施加5厘米水柱2 O的呼气末正压（PEEP）调整设置到目标二氧化碳的35-40毫米汞柱的端呼气分压（P 等 的 CO 2）和95％以上的一台S P 2 O 2。 <ol><li>保持用硫喷妥钠连续静脉输注（20毫克/千克/小时）和芬太尼（7微克/千克/小时）麻醉。 注:必需的剂量可能会有所不同，从动物到动物。不要让动物无人值守。保证实验动物福利和科学的原因，在任何时候都足够的麻醉。 </li><li>检查无角膜反射消失及仪器仪表在严密监控动物的应力/疼痛反应。仪器应在不如果麻醉足以给予肌肉松弛剂是可能的。辖泮库溴铵（0.15毫克/公斤体重静脉推注接着的0.15毫克/ kgBW /小时连续输注）如肌肉松弛是必要的实验（ 例如 </em&gt;肺顺应性测量）。 </li></ol></li></ol> 3.仪表技术<ol><li>将动物成仰卧位，用绷带拉伸上述计划切口部位皮肤缩回腿。使用手术前皮肤消毒等的醇/ 1％碘溶液消毒的操作区。 注:我们使用擦拭方法消毒的经营面积，但不使用完整aseptical技术，因为这是一个nonsurvival模式。手术无菌的程度取决于肺损伤的诱导下进行调查。 </li><li>使在连接下颌骨和用手术刀为中心静脉线和肺动脉导管的导引器鞘的放置通过皮肤切割胸骨（左侧或右侧）可在线路10厘米的切口。重新评估麻醉深度，并在必要增加剂量。 </li><li>分离皮下组织和板状SMA使用组织镊子和手术剪。一旦头臂和sternocephalic肌肉是可见执行钝削减方法，用类似镊子或手指文书可见分离的肌肉之间的筋膜直到外部颈静脉。 </li><li>导管插入与中央静脉导管和通过改性Seldinger技术的装置中的导引器鞘管颈外静脉。引进他们之前冲洗用生理盐水所有导管。 <ol><li>为此，推进从导引集合各自的针刺入静脉，直到静脉血（暗，没有脉动）可以被吸出。推进通过导管导丝进入静脉15厘米左右。取出针，推进导引鞘进入静脉。移除导丝。重复同样的步骤为中心静脉导管，如果必要的实验的放置。 </li></ol></li><li>通过诉心愿控制导管的正确位置源性血。与标准的缝合线紧密。 注意:不要用扩张血管扩张静脉，你会在经皮方法的情况下，因为这会撕裂静脉（ 图1）。一个超声引导方法也是可能的，如果研究者在猪超声引导cannulization技术培训。 </li><li>识别股薄和后腿的缝匠肌之间的折叠（左或右是可能的）的动脉导管的放置。这是股动脉的脉动能摸到折叠。 </li><li>请通过沿用手术刀在皮肤褶皱切5厘米的切口。 </li><li>分开使用组织镊子和手术剪皮下组织。使用钝削减过程的肌肉之间的筋膜分离股动脉的水平。请注意，避免因执行他们的砍伐过程颅切断隐船只。 </li><li>借助于导管插入股动脉在3.2中描述的改良Seldinger技术。连字可以围绕动脉被环和在穿刺部位出血的情况下关闭。此步骤，应避免，如果可能的话，因为它损害了血液流向后腿。与标准的缝合线紧密。 </li><li>连接动脉导管和中心静脉线到换能器系统和校准都对大气（零），或者200毫米汞柱（动脉线）或50毫米汞柱（中央静脉线）来开始监视。 </li><li>发生在右心房的高度所有的压力传感器（在猪约胸部高度的一半仰卧位）。 </li><li>执行小（4-5厘米）的切口用手术刀为膀胱插管通过气囊上方的皮肤切割。同样，使用钝器分离皮下组织。 </li><li>一旦它被显现在膀胱的壁进行荷包缝合（直径1-2厘米）。 注:缝合应该ñOT穿透膀胱壁的所有层，因为这将导致尿通过穿刺的损失。 </li><li>做一个小切口的缝合线的中间，介绍导尿管，方框用10ml水作空白的气球，拉导管背面直到光感觉到阻力，并​​紧密围绕所述导管的荷包缝合。关闭使用标准缝合皮肤。 </li></ol> 4.肺动脉导管简介<ol><li>注入0.5-1毫升空气进入肺动脉导管（取决于导管的大小）的球囊，并检查该气囊的可能的损害。再次放气气球。 </li><li>肺动脉导管连接到压力换能器系统和校准所述PAC上日气氛（零）和100毫米汞柱（ 图2和3）。 <ol><li>通过导引器鞘（瘪气球）10至1介绍的肺动脉导管5厘米，取决于护套长度。 </li><li>充气气球（气球具有已经离开此护套）和推进肺动脉导管进一步同时监测压力和血液动力学监视器上的典型波形。 </li><li>推进PAC而波形这是典型的右心房，右心室和肺动脉出现和停止时，肺毛细血管楔压（PCWP）曲线出现（ 图4）推进。放气气球。 注意:一旦气囊被放气的PCWP波形必须消失和肺动脉压的波形必须是可见的。否则该导管是最有可能的太多插入导致动脉（自动楔位置）的永久性闭塞肺动脉。在这种情况下，拉导管背面直至肺动脉压波形再次出现，以避免严重的并发症（ 例如血管破裂）6。 </li> &lt;li&gt;确保每当导管被拉回，以避免严重的并发症气球放气。 注:肺动脉导管经常通过偶然猪下腔静脉推进入肝静脉。因此，回拉导管和重新开始，如果经过约30厘米没有达到右心室。 </li></ol></li></ol> 5.肺动脉热稀释技术和血流动力学测量<ol><li>复制所有的血流动力学值，如心脏率，收缩压，舒张压，平均从血流动力学监测动脉压（MAP），肺动脉压力，中心静脉压（CVP）。 </li><li>测量PCWP及时。为此，膨胀肺动脉导管的囊，并确保一个正确PCWP曲线被显示（ 图4）。从监视器呼气末肺毛细血管楔压（PCWP）复制。立即放气气球之后（见4.2.4）。 DEFL吃了气球，拉导管回来，重新定位它，如果你无法看到在4.2.2所描述的正确PCWP曲线。 <ol><li>连接热敏电阻和适当的流过壳体的肺动脉导管的中央静脉管腔和显示器，用于测量心输出量（CO）。接下来，将显示器连接导管（红帽子）的远端温度端口。 </li><li>启动监视器，并选择"推有限公司"监视时间-温度曲线，从而测量心输出量（CO）与肺动脉热稀释技术7。 </li><li>按&#39;注入体积&#39;和选择冷却盐水的体积（5毫升这里介绍的实验）。回到前面的画面。按"导管"，并选择肺动脉导管，用于的大小。回到前面的画面。 </li><li>选择&#39;启动丸药&#39;，并使用作为快速注入溶于5ml 4℃的温度下的生理盐水的尽可能通过壳体的流动。等到测量完成和各自的时间 - 温度曲线出现在监视器上。二氧化碳值从监视器复制。 </li><li>如在5.3.4中描述的随机顺序在通气机呼吸周期进行快速连续5次测量。忽略的最高和最低值，并使用剩余的三个来计算心输出量的平均值。 注:此设置的监控是为爱德华兹警惕显示器，型号VGS1描述。该设置可以根据显示器上的不同。尽管如此，有必要选择盐水的正确的注射体积以及导管的大小。有些显示器需要计算常数的选择，守则盐水和导管尺寸所牵涉的金额。常数通常在导管的包装内的小册子中。保持在相同的温度的盐水在整个实验（&lt;5℃），以确保正确的测量电对此语句。用5％葡萄糖溶液代替生理盐水涉及电解质摄入和平衡的精确测量研究。 </li></ol></li><li>确保所有参数都被记录，并且动脉和混合静脉血样品被采取以使肺内从右到左分流的计算。 </li><li>记录所有需要的呼吸数据及时象峰和平台吸气压力从呼吸器，或执行类似的测量肺压在实验中的任何给定的时间点，完成数据的附加测量。 </li></ol> 6.肺灌洗诱导肺癌损伤<ol><li>确保动物通风与1.0 F I O 2和PEEP设置为2-4厘米H 2 O数灌洗手术。断开呼吸器动物。 </li><li>填充温热正常无菌盐水（37℃，50毫升/公斤体重）的肺部。为此，预填一个漏斗，并将其连接到气管内管与接头的弹性管。提高漏斗1米动物的上方和尽快倒入盐水进入肺部。静水压力将分配生理盐水所有肺段。 注:无菌生理盐水用于避免在病原体和动物的可能，感染性代偿肺洗涤。使用0.9％的盐水中是至关重要的，因为低渗流体将导致立即肺水肿，电解质紊乱和死亡的动物。不要重复使用生理盐水灌洗后，以最大限度地提高表面活性剂洗出。 </li><li>停车时MAP低于50毫米汞柱填充。 注意:只有血液动力学参数和S P 2 O 2可用于监测代偿动物，因为该动物从呼吸机中肺灌洗断开。 </li><li>手动放下漏斗地面平整，排水被动的灌洗液并重新连接动物呼吸机进行充氧。 </li><li>等待联合国直到动物补偿（在MAP和S P 2 O 2的增加），并尽快重复灌洗越好。对于重新灌洗的期限不得超过5分钟。 注:血流动力学失代偿的案例二灌洗液之间的动物的稳定供应，以避免使用升压药物来治疗全身性低血压，并进一步避免招聘演习遇到低氧血症。动物用的灌洗液中1.0 F I O 2和下面的实验中，尽管通风降低P至维持氧合 O 2 / F I O 2比值。灌洗期间在2-4 cm H2O的设置PEEP将促进肺不张的快速形成。但是，PEEP必须达到或超过5 cm H2O的肺损伤诱导后进行设置以满足ARDS柏林定义。在没有招聘演习或改变PEEP允许在实验中，以防止任何方面的调查引起的偏差s至肺损伤的严重性。 </li><li>取依赖于S中P 2 O 2中的血流动力学恶化和妥协的第二或第三灌洗后动脉血气体样品。 </li><li>直到在P 一 O 2 / F I O 2的比例（霍洛维茨指数）重复灌洗低于100毫米汞柱的持续测量至少60分钟，F I O 2 1.0和PEEP≥5 公分水柱。 </li><li>在灌洗期间调整呼吸速率，以保持动脉pH高于7.25，以防止血液动力学失代偿。 </li><li>启动基于表面活性剂冲洗模式一旦 O 2 / F I O 2的比例（霍洛维茨指数）低于100毫米汞柱的持续测量60分钟在P实验/治疗。 注:肺损伤所描述的诱导后，在肺功能的变化将保持数小时稳定，恶化，或者甚至取决于呼吸机设置改善。 注:该动物模型是基于表面活性剂冲洗和肺不张随之形成。因此，从指定的呼吸机设置，这可能会导致不张肺区域的招募任何偏差（增加PIP或PEEP），将部分扭转灌洗液的有害影响，阻碍该模型的标准化。 </li></ol> 7.结束实验和安乐死<ol><li>确保所有的测量被执行，并在实验结束前的数据被固定。 </li><li>注射0.5毫克芬太尼此外，对于连续麻醉，并等待至少5分钟。注入使用中心线的硫喷妥钠（至少1000毫克）过量紧接着至少60钾毫摩尔。 </li></ol>

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	<li>Rubenfeld, G. D., 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=Incidence+and+Outcomes+of+Acute+Lung+Injury.">Incidence and Outcomes of Acute Lung Injury.</a> N Engl J Med. 353 (16), 1685-1693 (2005).</li><li>Ballard-Croft, C., Wang, D., Sumpter, L. R., Zhou, X., Zwischenberger, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-animal+models+of+acute+respiratory+distress+syndrome.">Large-animal models of acute respiratory distress syndrome.</a> Ann Thorac Surg. 93 (4), 1331-1339 (2012).</li><li>Lachmann, B., Robertson, B., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+lung+lavage+as+an+experimental+model+of+the+respiratory+distress+syndrome.">In vivo lung lavage as an experimental model of the respiratory distress syndrome.</a> Acta Anaesthesiol Scand. 24 (3), 231-236 (1980).</li><li>Donaubauer, B., 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=Low-dose+inhalation+of+an+endothelin-A+receptor+antagonist+in+experimental+acute+lung+injury:+ET-1+plasma+concentration+and+pulmonary+inflammation.">Low-dose inhalation of an endothelin-A receptor antagonist in experimental acute lung injury: ET-1 plasma concentration and pulmonary inflammation.</a> Exp Biol Med (Maywood). 231 (6), 960-969 (2006).</li><li>Theisen, M. 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=Ventral+recumbency+is+crucial+for+fast+and+safe+orotracheal+intubation+in+laboratory+swine.">Ventral recumbency is crucial for fast and safe orotracheal intubation in laboratory swine.</a> Lab Anim. 43 (1), 96-101 (2009).</li><li>Kelly, C. R., Rabbani, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Videos+in+clinical+medicine.+Pulmonary-artery+catheterization.">Videos in clinical medicine. Pulmonary-artery catheterization.</a> N Engl J Med. 369 (25), 35 (2013).</li><li>Forrester, J. 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=Thermodilution+cardiac+output+determination+with+a+single+flow-directed+catheter.">Thermodilution cardiac output determination with a single flow-directed catheter.</a> Am Heart J. 83 (3), 306-311 (1972).</li><li>Deja, 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=The+inhaled+ET(A)+receptor+antagonist+LU-135252+acts+as+a+selective+pulmonary+vasodilator.">The inhaled ET(A) receptor antagonist LU-135252 acts as a selective pulmonary vasodilator.</a> Clin Sci (Lond). 103, 21-24 (2002).</li><li>Matute-Bello, G., Frevert, C. W., Martin, T. R. <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+acute+lung+injury.">Animal models of acute lung injury.</a> Am J Physiol Lung Cell Mol Physiol. 295 (3), 379-399 (2008).</li><li>Kloot, T. E., 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=Recruitment+maneuvers+in+three+experimental+models+of+acute+lung+injury.+Effect+on+lung+volume+and+gas+exchange.">Recruitment maneuvers in three experimental models of acute lung injury. Effect on lung volume and gas exchange.</a> Am J Respir Crit Care Med. 161 (5), 1485-1494 (2000).</li></ol>

We gratefully acknowledge the help of Prof. Burkhard Lachmann when contemplating the intricacies of PEEP settings during surfactant washout and the excellent technical assistance of Birgit Brandt and Sabine Molling.

本文介绍了一步步指令来诱导猪严重肺损伤，由于表面活性剂冲刷反复灌洗肺。这种特定的方法使肺功能和肺血管阻力可再现和可比较的恶化。当务之急是要灌洗，直到猪在P 一 O 2 / F I O 2比值下降低于100毫米汞柱，并保持低于100毫米汞柱为1小时。一次，这是取得的动物通常不会因为没有招募演习被进行4,8-从肺损伤至少4至8小时，只要恢复。遵守该协议有?...

尽管超过40年的深入研究人类急性呼吸窘迫综合征（ARDS）的死亡率仍然高，40和50％之间的1值。肺损伤动物模型研究中的复杂病机变化或新的治疗方法来降低死亡率和限制长期残疾发挥重大作用。 各种型号已经建立诱发肺损伤，模拟的或大（ 如猪）人ARDS或小动物（ 如啮齿动物）的各个方面。方法强烈不同，包括肺动脉灌注油酸的，静脉内（IV）的细菌的输注，和内毒素或盲肠结扎引起败血症引起的ARDS穿刺（CLP）的模型。此外，直接肺损伤由于大潮气量和高峰吸气压力（呼吸机相关性肺损伤; VILI），烟雾/烧伤或肺缺血/再灌注（I / R）模型经常使用2。CLP模型，以及与内毒素的工作模型的一个主要缺点，是阻碍所造成的单独肺损伤biotrauma的分析潜在的炎症。此外，可能需要几个小时到几天导致肺损伤，如在大型动物为VILI的情况。 肺损伤的诱导通过表面活性剂的洗脱反复肺灌洗液，因为它首先由拉赫曼等人所述。豚鼠3，是诱导的肺损伤与再现的功能和机械的妥协，以及肺血管阻力的变化的时间有效的方法。该模型适配到机械通气的约30-60公斤体重的猪支持与临床上使用的机械通风，导管和监视器的基础研究，而在气体交换和血流动力学的妥协是在同一时间4高度可重复的。此外，感应肺损伤通过灌洗不需要特定的设备，是不是在设计用于大型动物实验呼吸实验室常用的。在这篇文章中提出的模型是适用于设计用于人类，进而保证了肺功能的恶化产生一个高重复性的研究苛刻的设备（ 如呼吸机）。该模型的标准化，有助于确保实验之间的可比性并减少所需的动物数量。萎陷的肺区有故意或未知的招聘演习的潜力recruitability是这个特定模型的一个严重的限制。在下面的文章中，我们给出了肺损伤的诱导的灌洗模型的详细说明，并提供表示数据以表征在肺功能的妥协的稳定性。

<table><tbody><tr><td>Evita Infinity V500</td><td>Dr&amp;auml;ger</td><td></td><td>intensive care ventilator</td></tr><tr><td>Vigilance I&amp;nbsp;</td><td>Edwards</td><td></td><td>monitor</td></tr><tr><td>Vasofix Braun&amp;uuml;le 20G</td><td>B Braun</td><td>4268113B</td><td>peripheral vein catheter</td></tr><tr><td>Mallinckrodt Tracheal Tube Cuffed</td><td>Covidien</td><td>107-80</td><td>&amp;nbsp;8.0 mm ID</td></tr><tr><td>MultiCath3</td><td>Vygon</td><td>157,300</td><td>3 lumen central venous catheter, 20 cm length</td></tr><tr><td>Leader Cath Set</td><td>Vygon</td><td>115,805</td><td>arterial catheter</td></tr><tr><td>Percutaneus Sheath Introducer Set</td><td>Arrow</td><td>SI-09600</td><td>introducer sheath for pulmonary artery catheter of 4-6 Fr., 10 cm length</td></tr><tr><td>Swan-Ganz True Size Thermodilution Catheter</td><td>Edwards</td><td>132F5</td><td>pulmonary artery catheter, 75 cm length</td></tr><tr><td>Flow through chamber thermistor</td><td>Baxter</td><td>93-505</td><td>for measuring cardiac output</td></tr><tr><td>urinary catheter</td><td></td><td></td><td>no specific model requiered</td></tr></tbody></table>

lavage-induced surfactant depletion in pigs as a model of the acute respiratory distress syndrome (ards)

肺损伤的各种动物模型中存在研究人类急性呼吸窘迫综合征（ARDS）的复杂病理机制并评估未来的疗法。严重的肺损伤肺部气体交换和血流动力学的可重复的恶化可以麻醉用猪用温0.9％生理盐水反复灌洗肺（50毫升/公斤体重）诱导。包括标准呼吸和血液动力学监测与在此模型中临床应用的设备允许的新的治疗策略（药物，现代换气机，体外膜式人工氧合，ECMO）的评价，并且桥接长凳和床头之间的差距。此外，肺损伤诱导肺灌洗不需要对亲和抗炎细胞因子的测量病原体/内毒素影响的注入。该模型的缺点是不张的肺组织高recruitability。模型的标准化有助于避免陷阱，保证Ç实验之间omparability，并减少所需的动物数量。

P 上 O 2 / F I O期间肺灌洗2 -ratio下降，但单灌洗的确切影响很难预测。我们开始采取从第三灌洗动脉血气体样品开始检测P中减少一 O 2 / F I O 2 -ratio低于100毫米汞柱。一旦P中减少一 O 2 / F I O 2 -ratio低于100毫米汞柱实现时，我们要求这一比率仍低于100毫米汞柱一小时在PEEP≥5厘?...

Watch this Scientific Journal Video about Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS at JoVE.com

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

在麻醉的猪反复灌洗肺引起的肺损伤类似人类急性呼吸窘迫综合征（ARDS）的主要方面。为了这个目的，肺用0.9％盐水在37℃下反复灌洗。该协议的目标是在ARDS研究气体交换和血流动力学的可重复的缓解作用。

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

lavage-induced-surfactant-depletion-pigs-as-model-acute-respiratory

Research

JoVE Journal

Medicine

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

11.2K 次观看.  Charité - Universitätsmedizin, Berlin. 在麻醉的猪反复灌洗肺引起的肺损伤类似人类急性呼吸窘迫综合征（ARDS）的主要方面。为了这个目的，肺用0.9％盐水在37℃下反复灌洗。该协议的目标是在ARDS研究气体交换和血流动力学的可重复的缓解作用。 

视频: Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

科研

医学

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心肌梗死的猪模型主要成果评估

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome (ARDS)

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% in severe cases. Further research efforts are needed to better understand the complex pathophysiology of this disease. There are different well-established animal models to induce acute lung injury but none has been able to adequately mimic the complex pathomechanisms of ARDS. The most crucial factor for the development of this condition is the damage to the alveolar capillary unit. The combination of two well-established lung injury models allow us to mimic in more detail the underlying pathomechanism. Bronchoalveolar lavage (BAL) leads to surfactant depletion as well as alveolar collapse. The repeated instillation of fluid volumes causes subsequent hypoxemia. Surfactant depletion is a key factor of ARDS in humans. BAL is often combined with other lung injury approaches, but not with a second hit followed by oleic acid injection (OAI) yet. Oleic acid injection leads to severely impaired gas exchange, a deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The OAI mimics most of the expected effects of ARDS consisting of extended inflammation of lung tissue with an increase of alveolar leakage and gas exchange impairment. A disadvantage of the combination of different models is the difficulty to determine the influence to the lung injury caused by BAL alone, OAI alone or both together. The model presented in this report represents the combination of BAL and OAI as a new double-hit lung injury model. This new model is easy to implement and an alternative to study different therapeutic approaches in ARDS in the future.

<meta charset="utf8"></head><body>猪支气管肺泡灌洗和油酸注射作为急性呼吸窘迫综合征 ARDS 的双重打击模型

Different, complex animal models exist to study the pathophysiology of acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage and oleic acid injection induced lung injury is suitable as a new double-hit animal model for studying the acute respiratory distress syndrome.

猪支气管肺泡灌洗和油酸注射作为急性呼吸窘迫综合征 （ARDS） 的双重打击模型

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

重症监护病房型装置在猪模型急性呼吸窘迫综合征中的卤化剂递送

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial infusion of oleic acid, infusion of endotoxins or bacteria, cecal ligation and puncture, various pneumonia models, lung ischemia/reperfusion models and, of course, surfactant depletion models, among others. Surfactant depletion produces a rapid, reproducible deterioration of pulmonary gas exchange and hemodynamics and can be induced in anesthetized pigs using repeated lung lavages with 0.9% saline (35 mL/kg body weight, 37 &#176;C). The surfactant depletion model supports investigations with standard respiratory and hemodynamic monitoring with clinically applied devices. But the model suffers from a relatively high recruitability and ventilation with high airway pressures can immediately reduce the severity of the injury by reopening atelectatic lung areas. Thus, this model is not suitable for investigations of ventilator regimes that use high airway pressures. A combination of surfactant depletion and injurious ventilation with high tidal volume/low positive end-expiratory pressure (high Tv/low PEEP) to cause ventilator induced lung injury (VILI) will reduce the recruitability of the resulting lung injury. The advantages of a timely induction and the possibility to perform experimental research in a setting comparable to an intensive care unit are preserved.

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome ARDS

A combination of surfactant washout using 0.9% saline (35 mL/kg body weight, 37 &#176;C) and high tidal volume ventilation with low PEEP to cause moderate ventilator induced lung injury (VILI) results in experimental acute respiratory distress syndrome (ARDS). This method provides a model of lung injury with low/limited recruitability to study the effect of various ventilation strategies for extended periods.

表面活性剂耗竭与有害通风相结合导致急性呼吸窘迫综合症 （ARDS） 的可重复模型

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial ...

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

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