这项工作是由美国国立卫生研究院资助HL093196（RCR）和亚特兰大研究和教育基金会（于AREF）支持。

一，过敏原致敏和挑战（ 见图1） 答:对于气管挑战<ol><li>初步敏，雄性或雌性C57BL / 6，BALB / c小鼠（6-8周龄）腹腔0天，第7天再次注入20微克的卵（卵，Sigma-Aldrich公司，圣路易斯，密苏里州）乳化0.2毫升无菌磷酸盐缓冲液（PBS）含有2毫克氢氧化铝（Sigma-Aldrich公司）或2毫克，氢氧化铝0.2毫升无菌作为对照PBS。 </li><li>适当的挑战与抗原（例如，14天，16，18，20）。挑战过程如下。 </li><li>麻醉小鼠腹腔内（IP）注射氯胺酮的混合物（90毫克/千克）和甲苯噻嗪（10毫克/千克）。确保鼠标完全是至少10分钟的麻醉。 </li><li>在45度或更大角度的操作表面。将鼠标保持腹面向上的表面，在头顶部。 </li><li>钩ţ根据前面的门牙hread举行的后脑勺。水平与另一个爪子，以确保气管是直和使用标签的磁带，抱腿。手术部位浸泡于70％酒精和棉签。 </li><li>切口部位的局部应用布比卡因（0.25％的溶液0.1〜0.2毫升）。 </li><li>聂皮肤上用钳子的喉咙，轻轻向外拉。请与垂直小切口手术剪。尽量减少切口的大小。 </li><li>准备用50μL的PBS或0.1％的卵在PBS 1 ml注射器和重复移液器（Tridak步进，托林顿，CT）的插入。一方面采取吸管和使用其他组织举行镊子，暴露气管。 </li><li>担任平行气管尽可能注射器，通过插入气管墙针和注入的解决方案。 </li><li>保持鼠标在垂直方向注射后让时间解决方案，以解决在肺部。 </li><li>轻轻地和无菌密切伤口面积镊子和密封缝合。 </li><li>地方鼠标胸骨下一个加热垫，并允许它恢复，直至完全卧床。恢复后，返回鼠标动物保健设施和日常监察积液，炎症或感染，伤口裂开的迹象，直到伤口完全愈合。 </li></ol> B.通过雾化的挑战<ol><li>敏感0天小鼠腹腔注射20微克的OVA（Sigma-Aldrich公司）在0.2毫升的无菌PBS含2毫克氢氧化铝（Sigma-Aldrich公司）或2毫克氢氧化铝作为控制在0.2毫升无菌PBS乳化。 </li><li>第14天，提高腹腔注射致敏，如上所述。 </li><li>在21 日，22日，23日，24 日和25 日的日子后，初步敏，挑战小鼠暴露30分钟雾化1％OVA或PBS仅通过超声波雾化器（Buxco研究系统，Wilmi的交付ngton，NC）。 </li><li>地方小鼠人造板主室体积描记法室内至少10分钟，他们逐渐适应。 </li><li>地点1毫升0.1％的卵在无菌PBS或无菌PBS，仅在下面的步骤4-6所述，通过喷雾器杯。 Nebulize为30分钟。 </li><li>删除雾化杯和丢弃任何剩余的解决方案。 </li><li>同时使用雾化室，雾化可以执行所有的老鼠。 </li></ol>二。乙酰甲胆碱气道高反应性的测定答:非侵入性测量全身体积描记法（人造板;研究系统Buxco，威尔明顿，北卡罗来纳州）的气道高反应性<ol><li>允许粉状乙酰胆碱瓶开幕前预热至室温（乙酰胆碱是非常吸湿性，如果允许吸收水分），将成为无用的团块。准备了200毫克/毫升无菌PBS原液，然后进行连续2倍稀释（例如，100，50，25，12.5％和60.25毫克/毫升）。保持解决方案的寒冷。 </li><li>设备设置如下:连接的偏见流入口空气泵，雾化器，气藏采用紧身橡胶管和人造板出口人造板的主要入口。压力传感器连接，以弥合人造板的主要和参考室网点。连接压力传感器提供的电缆与前置放大器，前置放大器连接到电脑上使用特定的数据采集卡。 </li><li>校准的前置放大器，使用该软件，根据制造商的建议。 </li><li>地方小鼠人造板主室体积描记法室内至少10分钟，然后记录基线读数为3分钟（Penhbase）适应环境。 </li><li>地方1毫升无菌PBS在喷雾器杯。 Nebulize 2分钟，然后在干燥阶段，额外的6分钟监测呼吸变量。删除雾化杯，并丢弃任何剩余的PBS。 </li><li>地方1毫升6.25毫克/毫升乙酰胆碱在雾化杯和rEPEAT雾化2分钟加6分的监测周期。 </li><li> 12.5，25，50和100毫克/毫升乙酰胆碱重复测量，使用相同的2分钟雾化期间和6分钟监测周期。 </li><li>从商会的小鼠，并返回他们自己的笼子。 </li><li>笔芯喷雾器1毫升无菌PBS杯和运行另一序列冲洗油管。 </li><li>关闭空气流动，拆解，并擦拭干净运行前的动物第二套商会。 </li></ol> B.创检测气道反应性的由电脑控制的换气扇（flexiVent SCIREQ公司，蒙特利尔，加拿大） <ol><li>权衡鼠标和（IP）腹腔注射60毫克每公斤体重的戊巴比妥钠麻醉。 </li><li>足够的麻醉，腹，背部为气管位置的小鼠。 </li><li>用70％乙醇消毒颈部皮肤。我申请布比卡因（0.1〜0.2毫升0.25％的溶液）局部ncision网站。切割和打开的颈部皮肤。分离颈部肌肉，暴露气管。 </li><li>使1 - 2毫米的细剪刀气管切口（一定不能割断气管）和气管导管插入谨慎。配合周围的气管缝合，以防止漏气。 </li><li>在身体体积描记室打下鼠标和插入气管插管连接呼吸机。 </li><li>启动机械通气。设置适当的呼吸频率和潮气/每搏输出量（150次/分和200μL，分别为20-G鼠标）。可以肯定，胸部呼吸机同步移动。如果鼠标"打架"与呼吸器（自主呼吸），注入更多的麻醉剂，并等待同步。 </li><li>基线测量，另外3分钟保持下基线通风小鼠，然后2 ND阻抗测量。设置基线测量这2 ND用于钙ulate平均基线值。 </li><li>窜从呼吸机的吸气流量通过超声波雾化器，提供PBS或乙酰甲胆碱（MCH）挑战（6.25，12.5，25，50和100毫克/毫升）。 </li><li>每个的MCH挑战（6.25，12.5，25，50和100毫克/毫升），提供10毫升/公斤的Vt，在120次/分返回活塞，并采取阻抗测量。 </li></ol>第三。进入空域测量细胞浸润 A.执行支气管肺泡灌洗（BAL） <ol><li> AHR的测量后，安乐死与CO 2的鼠标，每个鼠标定位在其背部手术垫。 </li><li>用70％乙醇浸泡的区域。 </li><li>从小腹开始，剖开腹腔，去除皮肤/上部肌肉，肋骨走向向上。 </li><li>一旦肋骨可见，用剪刀小心刺破隔膜。肺部应该远离膈肌折叠。尤其是CAreful尼克肺部或心脏。 </li><li>切去胸腔，充分暴露肺/心（避免削减任何重大的血管保持血液充盈网站）。 </li><li>使用27号针头（屋宇署注射器，富兰克林湖，新泽西州），穿刺心脏脑室，慢慢地小心地拉了回来收集血液的注射器1毫升注射器。照顾，以避免崩溃的心脏。 </li><li>从这个使用标准协议的血液收集的血清。贮存于-70°C，直到使用。 </li><li>切去皮肤和组织从喉咙直到气管透露。扫清足够的组织轻松的工作领域内的（再次，避免削减任何重大的血管）。 </li><li>使用弯剪，切下的气管，清除路径。 </li><li>通过气管下弯钳点，并抓住了一块缝合结束。画下气管的缝合线。 </li><li>配合有关气管宽松的半结，喉咙低。 </li><li> carefully切开一个缺口，在套管的大小是足够的，上面的缝合线。 </li><li>小心地插入孔和过去的缝合线的点下气管插管。轻轻地向前推进，直到套管只是出现在入口（太远肺部穿刺肺部;太短:崩溃气管试图恢复灌洗时）。 </li><li>收紧缝合线和完整的结密封解决气管插管。 </li><li>锁注射器套管（含1毫升PBS），并轻轻按压液体进入肺部。肺叶应单独慢慢地膨胀。不要过度填充。对于一个完整的生长鼠标0.9-1.0毫升，是绝对最大。 0.8毫升可能会更安全。锁定注射器松散插管，否则很可能造成损害时，试图以脱离。 </li><li>退出润肺生津。如果遇到阻力（组织吸入套管），按导管慢慢进入肺部的进一步恢复删除。也可以尝试旋转的导管到位。如果所有的ELSé失败，退出套管的一部分的方式;气管是在这种情况下更容易崩溃。 </li><li>脱离插管，注射器存款BAL液容器中，并重复2次，新鲜的PBS溶液。 </li><li> BAL解决方案，直到降速置于冰上。 </li><li>使用BAL液和血清在OVA特异性IgE使用市售小鼠IgE的ELISA试剂盒（MD生物制品，圣保罗，MN）来衡量。 </li></ol> B.计数细胞，并确定差异<ol><li>离心机〜600×G BAL液中5分钟，4°C。 </li><li>重悬细胞沉淀在PBS轻轻，并保持在冰上。 </li><li>加载标准纽鲍尔血球稀释细胞悬液，并计数细胞。 </li><li>取出等分2×10 4细胞，在10至40为cytospins微升量。如果有必要，稀释细胞。 </li><li>为cytospins，混合2×10 4细胞，130μLPBS和10μLFBS的。整个细胞混合DOUB细胞离心涂片乐漏斗和700转离心10分钟，采用双cytoslides重复样品。 </li><li>允许的幻灯片，在室温下干燥1小时前染色。 </li><li>染色使用区分快速染色（西门子，纽瓦克，DE）的幻灯片。 </li></ol>四。代表结果过度挑衅刺激呼吸道收缩是临床哮喘的一个突出特点。我们介绍两种方法测量在OVA致敏和挑战小鼠气道高反应性乙酰胆碱:全身体积描记法（ 图2）和强迫振荡使用的flexiVent的系统（ 图3）。这两种方法都表明，致敏和挑战，产生在小鼠气道高反应性。 丰富的嗜酸性粒细胞气道炎症是临床哮喘小鼠呼吸道过敏性疾病的另一个突出的特点。在图4所示</st荣&gt;，致敏和挑战大大增加灌洗可以从呼吸道恢复细胞的总数量。嗜酸性粒细胞，并在较小程度上的数字，特别是增加中性粒细胞。 有证据表明，呼吸道过敏性疾病导致生产过剩IgE抗体致敏抗原。宣传和使用我们所描述的协议卵的挑战，增加治疗的小鼠的血清和BAL液（ 图5）IgE水平。 <img alt="图1" src="/files/ftp_upload/3771/3771fig1.jpg" /> 图1。 OVA诱发过敏性哮喘的实验模式。小鼠致敏两次IP 2毫克氢氧化铝乳化卵在0.2毫升的无菌PBS，0.2毫升无菌PBS单独或2毫克氢氧化铝与20微克，在由它指定的时间点挑战OVA或0.1％，无菌PBS溶液或每日前<html>仅30分钟，以PBS或PBS雾化吸入1％卵posure交付通过超声波雾化器（Buxco）。二十四小时后，最后卵暴露，气道反应性测定。随后，BAL液，血液样本，肺细胞，组织收集作进一步分析。 <img alt="图2" src="/files/ftp_upload/3771/3771fig2.jpg" /> 图2。由过敏原引起的气道高反应性的一种非侵入性方法的评估。的小鼠（每组4/group）是致敏和与卵质疑。二十四小时后，最后的挑战，吸入乙酰甲胆碱气道反应性测定采用在协议中所述的整体体积描记法。金边决心和金边的比例（平均超过分为平均金边与乙酰胆碱的8分钟的时间间隔8分钟的时间间隔在用PBS金边）表示。 * P &lt;0.05与的PBS。 iles/ftp_upload/3771/3771fig3.jpg"/&gt; 图3。评估过敏原引起的气道高反应性，侵入性的方法（强迫振荡）。小鼠（每组4/group）以OVA致敏和挑战。二十四小时后，最后的挑战，气道高反应性增加吸入乙酰胆碱的浓度被强迫振荡（flexiVent）在协议中所述的方法确定。 ，A，B）气道阻力）肺顺应性。 * P &lt;0.05与的PBS。 <img alt="图4" src="/files/ftp_upload/3771/3771fig4.jpg" /> 图4。 BAL液细胞计数。小鼠（每组4/group）以OVA致敏和挑战。二十四小时后，最后的挑战，（ 前 ） BAL细胞和总细胞计数在协议中所述。 （ 底 ）细胞离心涂片幻灯片为Prepared和与变化的快速染色。 TOT =总细胞，Eos的嗜酸性粒细胞;东大=中性粒细胞，MAC =巨噬细胞; LYM =淋巴细胞。 * P &lt;0.05与的PBS。 <img alt="图5" src="/files/ftp_upload/3771/3771fig5.jpg" /> 图5。 OVA特异性IgE水平 。小鼠（N = 4/group）以OVA致敏和挑战。二十四小时后，最后的挑战，IGE在BAL液和血清心脏穿刺采集的血液，在协议中所述的测量。 * P &lt;0.05与的PBS。

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Care Med. 174, 1173-1178 (2006).</li><li>Rayamajhi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-surgical+Intratracheal+Instillation+of+Mice+with+Analysis+of+Lungs+and+Lung+Draining+Lymph+Nodes+by+Flow+Cytometry.">Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry.</a> J. Vis. Exp. (51), e2702 (2011).</li><li>Swedin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+aerosol+and+intranasal+challenge+in+a+mouse+model+of+allergic+airway+inflammation+and+hyperresponsiveness.">Comparison of aerosol and intranasal challenge in a mouse model of allergic airway inflammation and hyperresponsiveness.</a> Int. Arch. Allergy Immunol. 153, 249-258 (2010).</li><li>Gueders, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+asthma:+a+comparison+between+C57BL/6+and+BALB/c+strains+regarding+bronchial+responsiveness,+inflammation,+and+cytokine+production.">Mouse models of asthma: a comparison between C57BL/6 and BALB/c strains regarding bronchial responsiveness, inflammation, and cytokine production.</a> Inflamm. Res. 58, 845-854 (2009).</li><li>Zhu, W., Gilmour, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+allergic+lung+disease+in+three+mouse+strains+after+systemic+or+mucosal+sensitization+with+ovalbumin+antigen.">Comparison of allergic lung disease in three mouse strains after systemic or mucosal sensitization with ovalbumin antigen.</a> Immunogenetics. 61, 199-207 (2009).</li><li>Flandre, T. D., Leroy, P. L., Desmecht, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+somatic+growth,+strain,+and+sex+on+double-chamber+plethysmographic+respiratory+function+values+in+healthy+mice.">Effect of somatic growth, strain, and sex on double-chamber plethysmographic respiratory function values in healthy mice.</a> J. Appl. Physiol. 94, 1129-1136 (2003).</li><li>Hoymann, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+and+noninvasive+lung+function+measurements+in+rodents.">Invasive and noninvasive lung function measurements in rodents.</a> J. Pharmacol. Toxicol. Methods. 55, 16-26 (2007).</li><li>Chong, B. T., Agrawal, D. K., Romero, F. A., Townley, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+bronchoconstriction+using+whole-body+plethysmograph:+comparison+of+freely+moving+versus+restrained+guinea+pigs.">Measurement of bronchoconstriction using whole-body plethysmograph: comparison of freely moving versus restrained guinea pigs.</a> J. Pharmacol. Toxicol. Methods. 39, 163-168 (1998).</li></ol>

亚特兰大VAMC IACUC委员会提出的指导方针和＃V010-10根据协议规定，进行动物实验。

呼吸道过敏性疾病的动物模型，为临床哮喘的相关研究提供了重要的工具。许多不同的机型，采用不同的物种和抗原，已被开发出来。鼠标，一个有吸引力的和常用的实验室物种，也为呼吸道过敏性疾病模型提供了优势。9,10虽然这种模式不模仿在各方面的哮喘，11与慢性疾病方面是特别困难的重现， 12,13我们确认这里，许多主要功能是复制。我们还表明，在人类哮喘，这?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>卵清蛋白</td><td> Sigma-Aldrich公司 密苏里州圣路易斯</td><td> A5503 </td><td></td></tr><tr><td>氢氧化铝</td><td> Sigma-Aldrich公司</td><td> 239186 </td><td></td></tr><tr><td>乙酰-β-methylcholine氯化</td><td> Sigma-Aldrich公司</td><td> A2251 </td><td></td></tr><tr><td>戊巴比妥钠盐</td><td> Sigma-Aldrich公司</td><td> P3761 </td><td></td></tr><tr><td>全车身体积描记法 （人造板）系统</td><td> buxco研究系统 北卡罗莱纳州威尔明顿</td><td></td><td> <a href="http://www.buxco.com" target="_blank">http://www.buxco.com</a> </td></tr><tr><td> FlexiVent </td><td>SCIREQ，公司 加拿大蒙特利尔</td><td></td><td> <a href="http://www.scireq.com" target="_blank">http://www.scireq.com</a> </td></tr><tr><td>光学显微镜</td><td>莱卡微系统公司 水牛城树丛，白细胞介素</td><td></td><td></td></tr><tr><td>细胞离心涂片4 </td><td> Thermo Scientific的 北卡罗来纳州阿什维尔， </td><td></td><td></td></tr><tr><td>变化的快速染色</td><td>西门子 纽瓦克，DE </td><td> B4132-1A </td><td></td></tr><tr><td>重复移液器</td><td> tridak 托林顿，CT </td><td> STP4001-0025 </td><td></td></tr></tbody></table>

murine model of allergen induced asthma

哮喘是一种发病率和死亡率的重要原因，影响约300万世界各地的人们。1超过8％的美国人口有气喘病，患病率增加。与其他疾病，呼吸道过敏性疾病的动物模型，极大地方便2基本病理生理学的了解，有助于确定潜在的治疗靶点，并允许可能的新疗法的临床试验。在几种动物模型过敏性气道疾病已开发，但小鼠模型特别有吸引力，由于成本低，随时可用，和良好的特点，这些动物的免疫系统。3多种转基因株系的可用性进一步提高吸引力这些模型。在这里，我们描述了两种小鼠模型过敏性气道疾病，无论是用人卵清蛋白作为抗原。腹腔注射致敏的初始模型，一个交付ERS的雾化，气管内交付的其他抗原的挑战。这两种模式提供优势互补，相互模仿人类哮喘的主要特征。 哮喘急性发作的主要功能包括夸张的气道反应，如乙酰胆碱（气道高反应性，AHR）刺激和丰富的嗜酸性粒细胞气道炎症。这些也都是过敏原的挑战突出的影响，在我们的小鼠模型，5,6和我们描述了测量他们的技术，从而评价实验操作的影响。具体来说，我们描述都测量气道高反应性，以及评估到气管和肺的炎性细胞浸润的方法7侵入和非侵入性技术。收集支气管肺泡气道炎症细胞，而肺组织病理是用来评估整个器官的炎症标志物。这些技术提供强大的工具，学习的方式，将不会在人类可能哮喘。

Watch this Scientific Journal Video about Murine Model of Allergen Induced Asthma at JoVE.com

Murine Model of Allergen Induced Asthma

过敏性哮喘的实验小鼠模型为研究疾病的发病机制和开发新疗法提供了新的可能性。这些模型非常适合过敏的免疫反应，呼吸道炎症，肺的病理生理测量因素。

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Immunology and Infection

Unit 1

Lower Respiratory Disorders

SCIENTISTS IN ACTION

40.0K 次观看.  Emory University and Atlanta VA Medical Center. 过敏性哮喘的实验小鼠模型为研究疾病的发病机制和开发新疗法提供了新的可能性。这些模型非常适合过敏的免疫反应，呼吸道炎症，肺的病理生理测量因素。

视频: Murine Model of Allergen Induced Asthma

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

科研

生物学

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

免疫与感染

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

医学

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

接触超敏反应作为过敏性接触性皮炎的小鼠模型

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

哮喘鼠模型中的针灸

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

过敏性鼻炎大鼠模型中鼻组织的多色免疫荧光

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

作者聚焦:利用多色免疫荧光推进过敏性鼻炎研究

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

通过多色免疫测定法对过敏性鼻炎大鼠鼻粘膜组织切片进行免疫荧光标记

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

过敏原诱发的哮喘小鼠模型

摘要

探索更多视频

此视频中的章节

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

研究微生物群落

IL-8瞬时转化的小鼠模型

HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎

双室 Plethysmography 对清醒小鼠呼吸功能的评价

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

接触超敏反应作为过敏性接触性皮炎的小鼠模型

哮喘鼠模型中的针灸

通过多色免疫测定法对过敏性鼻炎大鼠鼻粘膜组织切片进行免疫荧光标记

通过多色免疫测定法对过敏性鼻炎大鼠鼻粘膜组织切片进行免疫荧光标记