Name: a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide
Uploaded: 2014-12-15T13:00:00
Description: Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

我们要感谢克斯廷Thim（阿斯利康，瑞典隆德）贝尼塔·达尔伯格和安德斯·埃克伦德博士（Karolinska研究所，斯德哥尔摩，瑞典）以及马丁Stampfli博士（麦克马斯特大学，汉密尔顿，ON，加拿大）的熟练援助和专家的意见。

动物研究批准了北斯德哥尔摩动物福利伦理委员会。在实验过程中符合瑞典法律进行的。 1.生成一个LPS气溶胶<ol><li>溶解0.5克纯化P.假单胞菌内毒素在50ml无菌盐水中以温和搅拌并验证溶解。稀释1毫升9毫升无菌盐水溶解的LPS，以1毫克/毫升的最终浓度。防止光用铝箔并储存在-20℃。 </li><li>解冻溶解的LPS在黑暗中在室温下以及在使用之前立即混合。 </li><li>在通风II级生?...

<ol>
	<li>King, J. D., Kocincova, D., Westman, E. L., Lam, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Lipopolysaccharide+biosynthesis+in+Pseudomonas+aeruginosa.">Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.</a> Innate Immun. 15, 261-312 (2009).</li><li>Grommes, J., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neutrophils+to+acute+lung+injury.">Contribution of neutrophils to acute lung injury.</a> Mol Med. 17, 293-307 (2011).</li><li>Pesci, A., 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=Inflammatory+cells+and+mediators+in+bronchial+lavage+of+patients+with+chronic+obstructive+pulmonary+disease.">Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease.</a> Eur Respir J. 12, 380-386 (1998).</li><li>Hoogerwerf, J. 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Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.</a> Pulm Pharmacol Ther. 13, 61-69 (2000).</li><li>Brand, P., 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=Total+deposition+of+therapeutic+particles+during+spontaneous+and+controlled+inhalations.">Total deposition of therapeutic particles during spontaneous and controlled inhalations.</a> Journal of pharmaceutical sciences. 89, 724-731 (2000).</li><li>Liu, F., Li, W., Pauluhn, J., Trubel, H., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+acute+lung+injury+in+rats:+comparative+assessment+of+intratracheal+instillation+and+aerosol+inhalation.">Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation.</a> Toxicology. 304, 158-166 (2013).</li><li>Skerrett, S. 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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=Lung+epithelial-C/EBPbeta+contributes+to+LPS-induced+inflammation+and+its+suppression+by+formoterol.">Lung epithelial-C/EBPbeta contributes to LPS-induced inflammation and its suppression by formoterol.</a> Biochem Biophys Res Commun. 423, 134-139 (2012).</li><li>Didon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+epithelial+CCAAT/enhancer-binding+protein-beta+is+necessary+for+the+integrity+of+inflammatory+responses+to+cigarette+smoke.">Lung epithelial CCAAT/enhancer-binding protein-beta is necessary for the integrity of inflammatory responses to cigarette smoke.</a> Am J Respir Crit Care Med. 184, 233-242 (2011).</li><li>Silverpil, 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=Negative+feedback+on+IL-23+exerted+by+IL-17A+during+pulmonary+inflammation.">Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.</a> Innate Immunity. 19, 479-492 (2013).</li><li>Mercer, P. F., 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=Proteinase-Activated+Receptor-1,+CCL2+and+CCL7+Regulate+Acute+Neutrophilic+Lung+Inflammation.">Proteinase-Activated Receptor-1, CCL2 and CCL7 Regulate Acute Neutrophilic Lung Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. , (2013).</li><li>Korkmaz, B., Horwitz, M. S., Jenne, D. E., Gauthier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases. 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雾化LPS产生的气道炎症反应，在上皮粘膜下层特征在于嗜中性粒细胞，围绕传导气道，以及肺泡空格的空格。这是，与增加的总蛋白含量BALF中，表示血浆渗漏，有代表性的急性肺损伤的病理在一起。作为脂多糖诱导无菌炎症，反应是独立的适应性免疫应答的，并且有限制的相关性细菌感染。该技术可能，但是，被用于通过排除适应性免疫应答来剖析炎症机制。 虽然该方法是?...

脂多糖（LPS）是革兰氏阴性细菌1细胞壁成分。挑战LPS是急性肺损伤，特点是急性嗜中性粒细胞炎症和水肿2综合征的证据充分的模式。此外，肺中性白细胞增多也是慢性阻塞性肺疾病（COPD）3，和在人类中LPS刺激的一个标志已被用于模拟COPD恶化4。因此，LPS暴露的实验模型在临床上有关和有价值的工具来理解人类的病理。 的肺递送这里描述雾化脂多糖的目标是产生在导通和呼吸道，一个中性的炎症反应，无全身参与。的LPS攻击的几种技术已如前所述。静脉内注射LPS是最常用的给药途径。虽然这种技术是方便，T他主损伤是对内皮，与肺上皮以下中性粒细胞迁移至肺二次破坏。静脉内给药也诱导全身性炎症2，它可以在动物模型复杂化的临床图像。全身性炎症是相不与气管内给药观察到。这种技术，但是，是劳动密集型的，并且需要麻醉剂以及相当训练5,6。此外，肺部沉积通过施用这条路线是依赖于呼吸7。因此，肺部沉积由所需的帧内气管施用和可变沉积在气道可以观察到麻醉深度的影响。与此相反，肺递送用气雾脂多糖需要最少的培训，并且可以容易地实现对大量的动物几乎没有或个人5,8之间不存在差异。最近的研究证实，气雾剂递送优于对于沉积的气管内的路线，并有更多的相关剂量的LPS诱导中性粒细胞炎症与这种模式8。 以前的研究已经表明，挑战气溶胶假单胞菌假单胞菌 LPS产生的气道内腔和肺实质，包括肺泡腔9,10的显着的炎症反应。炎症的特征在于嗜中性粒细胞和肺水肿的存在的优势，因此可以用来解决急性肺损伤的发病机制和增益有助于疾病病理机制的进一步了解。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

急性肺损伤（ALI）是一种严重的疾病，特点是肺泡中性粒细胞，用有限的治疗方法，死亡率高。 ALI的实验模型是提高我们对疾病发病机制的认识键。脂多糖（LPS）的革兰氏阳性菌引起得出中性粒细胞炎症的气道和小鼠的肺实质。有效的肺输送诸如LPS化合物，然而，难以实现。在这里所描述的方法中，肺递送在小鼠中通过挑战气溶胶绿脓杆菌脂多糖实现。溶解的LPS气溶胶由连接到压缩空气喷雾器。小鼠暴露于LPS气溶胶在一个有机玻璃盒子的连续流动10分钟，随后2分钟调理气溶胶被中断之后。气管插管和随后的支气管肺泡灌洗，接着福尔马林灌注在下一步进行，这允许无菌的p表征ulmonary炎症。雾化脂多糖产生肺部炎症特征在于肺泡中性粒细胞，在支气管肺泡灌洗和组织学评估检测。这种技术可以被设置在一个小的费用少用具及需要最少的培训和专门知识。曝光系统因此可以常规进行在任何实验室，有潜力，以提高我们的肺病理学的理解。

挑战雾化P.假单胞菌内毒素通常会产生在气道管腔和肺泡空间，在早期和晚期时间点的特征在于，中性粒细胞为主的显着的炎症反应。 雾化LPS诱导肺中性粒细胞增多 C57BL / 6BY和BALB / c小鼠暴露于雾化P.铜绿假单胞菌 LPS或单独的车辆和中性粒细胞列举BALF。对C57BL暴露于与车辆产生的烟雾/ 6BY小鼠的BALF细胞总数仅是通常大约或低于200,000个细胞，并?...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

采用雾化脂多糖一种产生肺中性粒细胞增多

我们描述诱导中性粒细胞肺部炎症通过挑战脂多糖雾化通过雾化，建模急性肺损伤的方法。此外，基本的手术技术对于肺隔离，气管插管和支气管肺泡灌洗也有所说明。

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

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标签胚胎小鼠血管的方法

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用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

免疫沉淀法研究受体与酶的功能相互作用

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...