AR、XH和PE得到了NIH赠款R01HL140398和吉尔森·朗根博基金会赠款的支持。DEMM 和 KK 得到了 NIH 赠款 R01HL136333 和 R01HL134880 （KYK） 以及赫利斯医学研究基金会的赠款的支持。DEMM还得到霍华德·休斯医学研究所（HHMI）吉利亚姆高级研究奖学金的支持。PE 还得到精密环境卫生科学培训 NIEHS T32 ES027801 研究金计划的支持。JC 和 MF 由表观遗传学和分子致癌学系的烟草研究基金以及 MD Anderson 的表观遗传学中心（学者奖 MF）提供支持。FK 和 YZ 由 NIH 赠款 R01 ES029442-01 和 R01 AI135803-01 以及 VA 功绩赠款 CX000104 提供支持。该项目得到了贝勒医学院细胞测量和细胞分类核心的支持，并得到了CPRIT核心设施支持奖（CPRIT-RP180672）、NIH（CA125123和RR024574）和乔尔·塞德斯特伦的资助。

参与实验和开发这项技术的所有动物都根据我们的动物使用协议，由机构动物护理和使用委员会 （IACUC） 和贝勒医学院和 MD 安德森机构批准，这些机构已获得实验室动物护理评估和认证协会 （AAALAC） 的认证。
1. 建造设备
<ol><li>用阀门系统组装空气压缩机。
	<ol>
<li>使用 1 / 8 英寸螺纹男性适配器配件将流计（两个 15 L / min 带 Y 条和 2 个电源起飞）连接到微型压力调节器。请确保在所有螺纹端使用螺纹密封胶带。</li>
		<li>使用以下方式将组装的压力调节器与流计连接到医用空气压缩机仪器：压缩机气口上的 1 / 8 英寸六角、 1 / 8 英寸螺纹耦合配件以及连接到压力调节器的 1 / 8 英寸螺纹男性适配器配件。</li>
		<li>在每个 （4） 流表上安装氧气旋转带刺接头。</li>
		<li>在空气压缩机的顶部空气出口上安装男性适配器（部分包含在医用空气压缩机仪器中）。</li>
	</ol></li>
	<li>组装曝光室（制作4个单元）
	<ol>
<li>将一根 3/4 英寸氯化聚氯乙烯 （CPVC） 管道切成 8 个 4 英寸段。</li>
		<li>将每个段插入一个 3/4 英寸 90° 肘部 CPVC 配件，并将肘部的配件侧连接到直径为 3/4 英寸的 CPVC 男性适配器上。应有八个 CPVC 段，每个部分连接到一个 CPVC 肘部安装和一个 CPVC 男性适配器。</li>
		<li>在距离对方最远的 8.5 L 密封容器（11.25 x 7.75 x 6 英寸）上钻两个孔（直径 1 1/4 英寸），盖子（见图 1 曝光室）。孔的定位是从上到下和从左到右。</li>
		<li>将之前组装的 CPVC 男性适配器的螺纹侧插入容器中的每个孔中。</li>
		<li>从容器内部，在另一侧（室内烟雾输入）上安装一个 3/4 英寸 CPVC 盖，在一侧附加 3/4 英寸 CPVC 滴灌女性适配器（室烟输出）。</li>
		<li>在室内烟雾输出的 CPVC 盖顶部以昆昆克斯（淋浴头）模式钻出五个 3 毫米孔。这将使香烟烟雾以更高的速度进入房间，并确保它均匀地分布在室内的所有方向。</li>
	</ol></li>
	<li>组装卷烟室（最多4个穆林暴露单位）
	<ol>
<li>采取一个单孔橡胶塞（制造商大小8.5），并在较宽的一侧插入一个1/4英寸的带刺Y连接器和一个直刺配件（8毫米开口）在较窄的一侧。香烟将在吸烟过程中（香烟基座）放置在这里。</li>
		<li>将 12 英寸长医用级乙烯基管的一端连接到连接到橡胶塞的 Y 连接器上的一个带刺连接器，将另一端连接到 1/4 英寸的配件上，并将此配件的对面插入一个单孔橡胶塞子（制造商尺寸 1）。</li>
		<li>在另一个橡胶塞子（制造商尺寸 8.5），插入一个 1/4 英寸直管连接器在塞子的较宽的一侧，并将配件的外端连接到 7 英尺医用级乙烯基管。</li>
		<li>将之前组装的两个橡胶塞件结构（步骤 1.3.1–1.3.3）与实验室玻璃排水管中的 8 英寸 x 1.75 英寸玻璃缸连接起来。</li>
	</ol></li>
	<li>阀门控制系统
	<ol>
<li>该系统由有节奏的电磁阀打开和关闭控制，这些阀门模拟吸入（吹）香烟烟雾和清洁空气。控制电磁阀的系统是商业设计的（见 材料表）。</li>
	</ol></li>
	<li>将所有组件组装在一起（见 图 1）
	<ol>
<li>使用 1 英寸紧固件将四个电磁阀安装到阀门控制系统的两侧。</li>
		<li>按照制造商的指示将电磁阀连接到阀门控制系统。</li>
		<li>将 10+32 （M） 螺纹直接器连接到电磁阀上的排气 （"EXH"） 连接，以及同一电磁阀的"IN"和"OUT"连接上的螺纹端口适配器。</li>
		<li>使用 7 英尺医用级乙烯基管通过"OUT"连接将连接到压缩机的流计连接到电磁阀。</li>
		<li>将 7 英尺乙烯基管与第 1.3.3 步的橡胶塞连接起来。在电磁阀上的"IN"连接器上。</li>
		<li>在烟室烟雾输入上插入卷烟室的小橡胶塞。</li>
		<li>将电磁阀连接到香烟基座上按步骤 1.3.1 组装的带刺 Y 连接器的第二个连接。</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61793/61793fig01.jpg"> 图1：我们WBIS与CS接触的连接示意图。此图演示了所有组件的组装方式，以形成工作装置。该图仅显示机器能够操作的四个烟室中的一个组装吸烟室。 <a href="https://www.jove.com/files/ftp_upload/61793/61793fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
2. 香烟烟雾暴露
注意：避免二手和三手接触香烟烟雾。香烟和暴露室应在 II 类 B2 层流生物安全柜中使用。在进行烟雾暴露实验时应佩戴适当的PPE（即口罩、手套、发网、礼服）。
<ol><li>
设置压力和气流
	<ol>
<li>一旦所有部件都按照 图 1的显示进行组装，请打开空气压缩机，等待安全警报自行关闭。</li>
		<li>通过打开压力调节器的旋钮，将空气压缩机的压力调整到 40–50 psi。</li>
		<li>使用流量计将空气压缩机的气流调整到 5 升。</li>
		<li>打开阀门控制器。</li>
		<li>通过按下 SET/LOCK 键，同时按住计时器第一位数字的 UP 键，将阀门控制器上的数字定时器调整为 PULSE-C（显示屏上显示为"Pu-c"）操作模式。然后，按上键，直到达到 Pu-c 模式。按重置键将显示的操作模式（即 Pu-C）设置为工作模式。</li>
		<li>按下 SET/LOCK 更改定时器 1（显示屏上显示为"T1"）。</li>
		<li>按上或向下键设置 T1 到 20 s。</li>
		<li>按下 SET/LOCK 更改定时器 2（显示屏上显示为"T2"）。</li>
		<li>按上或向下键设置T2到3 s。 注：步骤 2.1.5 到 2.1.9 是定制用于特定定时器（参见 材料表）。有关本产品其他用途的进一步说明，请参阅其相应的用户手册。</li>
	</ol>
</li>
	<li>
香烟烟雾处理 注意：该系统允许同时使用1–4个穆林暴露室。<ol>
<li>打开空气压缩机，等待安全警报自行关闭。</li>
		<li>打开阀门控制器。</li>
		<li>将 5 只小鼠转移到四个暴露室中，每个暴露室配有气密可拆卸盖，体积为 8.5 L。 将四个暴露室与小鼠放在 II 类 B2 层层流生物安全柜内。</li>
		<li>在层流生物安全柜内，点亮香烟，将香烟插入卷烟室内。使用市售香烟，其中含有15毫克/烟焦油和1.1毫克/西格尼古丁8相比，肯塔基州3RF4研究香烟（9.5毫克/西格焦油和0.73毫克/西格尼古丁）9。</li>
		<li>打开与当前使用的机舱对应的阀门控制器上的阀门。暴露分为两个阶段：（T1）清洁空气被泵入暴露室20s和（T2）气流导致香烟燃烧，从烟室抽到暴露室的烟雾3s。让香烟完全烧坏，直到它到达过滤器。<ol>
<li>调整定时器设置，在~4分钟的时间内平均执行约10次泡芙/香烟。请注意，定时器和系统很容易根据调查人员的研究需要定制，用于增强或降低CS给注方案。</li>
		</ol>
</li>
		<li>取下香烟过滤器，将烟头放入玻璃烧杯中，用水灭火并抑制气味。</li>
		<li>确保烟室再次关闭，无需香烟。让机器泵清洁空气10分钟。保持对接触CS的脊椎动物的持续监测至关重要。这种暴露方案针对每个暴露室中9周以上的5只雌性小鼠进行了优化。</li>
		<li>重复步骤 2.3.4 到 2.3.7 三次，每天总共 4 支香烟。只要研究人员需要进行实验，这个程序每周重复5天。</li>
		<li>将小鼠从暴露室中取出，带回相应的笼子里。</li>
		<li>关闭阀门控制器和空气压缩机。</li>
		<li>取出暴露和烟室，用水和肥皂清洗，以去除焦油残留物。</li>
		<li>让房间完全干燥，然后再使用它们。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Current Cigarette Smoking Among Adults in the United States. Center for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/tobacco/data_statistics/fact_sheets/adult_data/cig_smoking/index.htm">https://www.cdc.gov/tobacco/data_statistics/fact_sheets/adult_data/cig_smoking/index.htm</a> (2018)</li><li>Salvi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoking+and+evironmental+risk+factors+for+chronic+obstructive+pulmonary+disease.">Tobacco smoking and evironmental risk factors for chronic obstructive pulmonary disease.</a> Clinics in Chest Medicine. 35, 17-27 (2014).</li><li>Sunyer, J., 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=Longitudinal+relation+between+smoking+and+white+blood+cells.">Longitudinal relation between smoking and white blood cells.</a> American Journal of Epidemiology. 144, 734-741 (1996).</li><li>Freedman, D. S., Flanders, D., Barboriak, J. J., Malarcher, A. M., Gates, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoking+and+leukocyte+subpopulations+in+men.">Cigarette smoking and leukocyte subpopulations in men.</a> Annals of Epidemiology. 6, 299-306 (1996).</li><li>Chronic Obstructive Pulmonary Disease (COPD). Center for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/copd/basics-about.html">https://www.cdc.gov/copd/basics-about.html</a> (2019)</li><li>Genovese, G., 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=Clonal+hematopoiesis+and+blood-cancer+risk+inferred+from+blood+DNA+sequence.">Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.</a> New England Journal of Medicine. , (2014).</li><li>Steensma, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+implications+of+clonal+hematopoiesis.">Clinical implications of clonal hematopoiesis.</a> Mayo Clinic Proceedings. 93, 1122-1130 (2018).</li><li>Tobacco. Federal Trade Comission Available from: <a target="_blank" href="https://www.ftc.gov/tips-advice/business-center/selected-industries/tobacco">https://www.ftc.gov/tips-advice/business-center/selected-industries/tobacco</a> (2020)</li><li>3R4F Cigarettes. University of Kentucky - College of Agriculture Food and Environment Available from: <a target="_blank" href="https://ctrp.uky.edu/products/gallery/Reference">https://ctrp.uky.edu/products/gallery/Reference</a> (2020)</li><li>Shan, 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=Cigarette+smoke+induction+of+osteopontin+(SPP1)+mediates+T+H+17+inflammation+in+human+and+experimental+emphysema.">Cigarette smoke induction of osteopontin (SPP1) mediates T H 17 inflammation in human and experimental emphysema.</a> Science Translational Medicine. 4, 1-10 (2012).</li><li>Yuan, X., 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=Activation+of+C3a+receptor+is+required+in+cigarette+smoke-mediated+emphysema.">Activation of C3a receptor is required in cigarette smoke-mediated emphysema.</a> Nature Mucosal Immunology. 8, 874-885 (2014).</li><li>Yuan, X., 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=Cigarette+smoke+-+induced+reduction+of+C1q+promotes+emphysema.">Cigarette smoke - induced reduction of C1q promotes emphysema.</a> JCI Insight. 4, 1-17 (2019).</li><li>Shan, 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=Agonistic+induction+of+PPAR+g+reverses+cigarette+smoke+-+induced+emphysema+Find+the+latest+version:+Agonistic+induction+of+PPAR+&#947;+reverses+cigarette+smoke+-+induced+emphysema.">Agonistic induction of PPAR g reverses cigarette smoke - induced emphysema Find the latest version: Agonistic induction of PPAR &#947; reverses cigarette smoke - induced emphysema.</a> Journal of Clinical Investigation. 124, 1371-1381 (2014).</li><li>Hong, M. J., 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=Protective+role+of+gd+T+cells+in+cigarette+smoke+and+influenza+infection.">Protective role of gd T cells in cigarette smoke and influenza infection.</a> Nature Mucosal Immunology. 11, 834-908 (2018).</li><li>Kim, 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=Cigarette+smoke+induces+intestinal+inflammation+via+a+Th17+cell-neutrophil+axis.">Cigarette smoke induces intestinal inflammation via a Th17 cell-neutrophil axis.</a> Frontiers in Immunology. 10, 1-11 (2019).</li><li>Lu, W., 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+microRNA+miR-22+inhibits+the+histone+deacetylase+HDAC4+to+promote+T+H+17+cell+-+dependent+emphysema.">The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T H 17 cell - dependent emphysema.</a> Nature Immunology. 16, 1185-1194 (2015).</li><li>Hendrix, A. Y., Kheradmand, 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> The Role of Matrix Metalloproteinases in Development, Repair, and Destruction of the Lungs. Progress in Molecular Biology and Translational Science. 148, (2017).</li><li>Siggins, R. W., Hossain, F., Rehman, T., Melvan, J. N., Welsh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+alters+the+hematopoietic+stem+cell+niche.">Cigarette smoke alters the hematopoietic stem cell niche.</a> Med Sci. 2, 37-50 (2014).</li><li>Kheradmand, F., You, R., Gu, B. H., Corry, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+and+DNA+cleavage+promote+lung+inflammation+and+emphysema.">Cigarette smoke and DNA cleavage promote lung inflammation and emphysema.</a> Transactions of the American Clinical and Climatological Association. 128, 222-233 (2017).</li><li>Ha, M. 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=Menthol+attenuates+respiratory+irritation+and+elevates+blood+cotinine+in+cigarette+smoke+exposed+mice.">Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.</a> PLoS ONE. , 1-16 (2015).</li><li>Moreno-Gonzalez, I., Estrada, L. D., Sanchez-Mejias, E., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smoking+exacerbates+amyloid+pathology+in+a+mouse+model+of+Alzheimer's+disease.">Smoking exacerbates amyloid pathology in a mouse model of Alzheimer's disease.</a> Nature Communications. 4, 1-10 (2013).</li><li>Madison, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+cigarettes+disrupt+lung+lipid+homeostasis+and+innate+immunity+independent+of+nicotine.">Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine.</a> Journal of Clinical Investigation. 129, 4290-4304 (2019).</li></ol>

作者没有什么可透露的。

在这里，我们提供构建小鼠WBIS到CS的仪器所需的信息。系统安装后，调查人员根据动物中尼古丁或可替宁的交付剂量校准系统至关重要。该仪器包含一个定时器和压力计，可用于调节香烟泡芙量、泡芙频率、合并烟雾暴露期以及动物在每支香烟之间获得的休息间隔。此外，每天服用的香烟的实际数量可能因焦油和尼古丁含量而异。最后，必须定期清洁接触香烟烟雾的任何成分，以确保动物的烟雾?...

吸烟仍然是美国可预防疾病的主要原因之一，尽管在过去50-60年中吸烟的成年人数量持续下降。众所周知，吸烟与肺和血液的多种疾病有关，包括慢性阻塞性肺病（COPD），这是一组疾病，包括肺气肿和慢性支气管炎2、3、4。根据美国疾病控制中心（CDC）的数据，2014年，慢性阻塞性肺病是美国第三大死因，超过1500万美国人患有这种疾病。
CS最近也与患克隆造血症（CH）6、7的较高风险有关，在这种情况下，单个造血干细胞不成比例地产生一个人的外周血液的很大一部分。这一发现表明吸烟和骨髓功能之间有潜在的联系。鉴于CS对健康的广泛和非常重要的影响，鉴于穆林疾病模型是生物医学研究进步的基石，开发高效且经济实惠的系统来模拟小鼠的CS是有益的。
在这里，我们提供一个分步指南，建立一个负担得起的系统，用于治疗和研究CS对肺肺气肿和骨髓平衡的体内影响。此设备的组装不需要用户具有专业知识，因此允许 DIY 组装。

<table>
	<tbody>
		<tr>
			<td>1 in fastener</td>
			<td>Lowes</td>
			<td>756990</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in Barbed Y connector</td>
			<td>VWR</td>
			<td>89093-282</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in straight tubing connector</td>
			<td>VWR</td>
			<td>62866-378</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 hex nipple</td>
			<td>Lowes</td>
			<td>877221</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded coupling fitting</td>
			<td>Lowes</td>
			<td>877208</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded male adapter nipple fitting</td>
			<td>Lowes</td>
			<td>877243</td>
			<td></td>
		</tr>
		<tr>
			<td>10/32 (M) threaded straight connector</td>
			<td>Bimba</td>
			<td>EB60</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in 90-degree elbow CPVC fitting</td>
			<td>Lowes</td>
			<td>22643</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in chlorinated polyvinyl chloride (CPVC) pipe</td>
			<td>Lowes</td>
			<td>23814</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC cap</td>
			<td>Lowes</td>
			<td>23773</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC Drip irrigation female adapter</td>
			<td>Lowes</td>
			<td>194629</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in diameter CPVC male adapter</td>
			<td>Lowes</td>
			<td>23766</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 L airtight container with lid (11.25in x 7.75in x 6 in)</td>
			<td>Komax</td>
			<td>N/A</td>
			<td>Listed as &#34;Komax Biokips Large Bread Box | (280-oz) Large Storage Container&#34;</td>
		</tr>
		<tr>
			<td>Glass drain tube (1.75 in diameter x 8 in length)</td>
			<td>KIMAX</td>
			<td>6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isonic Solenoid Valves</td>
			<td>Bimba</td>
			<td>V2A02-AW1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marlboro Red 100&#39;s</td>
			<td>Marlboro</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen swivel barbed connector</td>
			<td>Global Medical Solutions</td>
			<td>RES002</td>
			<td></td>
		</tr>
		<tr>
			<td>Panasonic Timer LT4H-W</td>
			<td>Panasonic</td>
			<td>LT4HW</td>
			<td>Item was built-in the valve controller by Shepherd Controls &#38; Associates</td>
		</tr>
		<tr>
			<td>Pressure regulator</td>
			<td>Allied Electronics and Automation</td>
			<td>70600552</td>
			<td>Also listed as &#34;Norgren R07-100-RGKA&#34;</td>
		</tr>
		<tr>
			<td>Rubber stopper # 1 (one hole)</td>
			<td>VWR</td>
			<td>59581-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber stopper # 8.5 (one hole)</td>
			<td>VWR</td>
			<td>59581-389</td>
			<td></td>
		</tr>
		<tr>
			<td>Scireq inExpose system</td>
			<td>Scireq and Emka Technologies</td>
			<td>N/A</td>
			<td>Commercial system used for comparison with our DIY WBIS</td>
		</tr>
		<tr>
			<td>Straight barbed fitting (8mm opening)</td>
			<td>VWR</td>
			<td>10028-872</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Sealant tape</td>
			<td>Lowes</td>
			<td>1184243</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded port adaptor</td>
			<td>Bimba</td>
			<td>P1SA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter Aridyne 2000 Medical Air Compressor</td>
			<td>MFI Medical</td>
			<td>AHC-TE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter flowmeter</td>
			<td>Allied Healthcare Products</td>
			<td>15006-03YP2</td>
			<td>Also listed as &#34;Puritan Air Meter&#34;</td>
		</tr>
		<tr>
			<td>Valve Control system</td>
			<td>Shepherd Controls and Associates</td>
			<td>N/A</td>
			<td>Company custom designed the valve control system for this model.</td>
		</tr>
		<tr>
			<td>Vinyl pipes</td>
			<td>Vitality Medical</td>
			<td>RES3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

cigarette smoke exposure in mice using a whole-body inhalation system

据报道，2018年美国有近14%的成年人吸烟。香烟烟雾（CS）对肺部和心血管疾病的影响已得到广泛研究，然而，CS对血液和骨髓等其他组织和器官的影响仍然未完全确定。找到适当的系统来研究CS对啮齿动物的影响可能过于昂贵，需要购买市售系统。因此，我们着手构建一个经济实惠、可靠且多功能的系统，以研究CS在小鼠中的病理影响。这种全身吸入暴露系统 （WBIS） 设置通过交替接触 CS 和清洁空气来模拟香烟的呼吸和吹气。在这里，我们表明，这种自己动手（DIY）系统在接触了4个月的香烟烟雾后，在小鼠身上诱发气道炎症和肺气肿。使用这种仪器，CS的全身吸入（WBI）对造血干细胞和骨髓中的祖细胞（HSPCs）的影响也显示出来。

CS 暴露的主要特征之一是肺气肿，其特征是肺部气囊 （肺囊） 的损伤和破坏。因此，最初的实验侧重于DIY系统在反复接触CS后引起雌性小鼠肺部的肺部变化的能力。CS配药方案是根据我们先前的出版物选择的，我们利用此处描述的DIY系统用CS治疗小鼠，并研究肺气肿10、11、12、13、14、15、16的分子病理生理学。<sup class="...

Watch this Scientific Journal Video about Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System at JoVE.com

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

该协议展示了对香烟烟雾（CS）的病理生理影响的研究，内部内建立了全身吸入（WBI）暴露系统（WBIS）。该系统可以在受控的可重复条件下使动物接触CS，用于研究CS介于肺气肿和血细胞瘤的影响。

使用全身吸入系统在小鼠中暴露香烟烟雾

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

cigarette-smoke-exposure-in-mice-using-a-whole-body-inhalation-system

Research

JoVE Journal

Immunology and Infection

6.1K 次观看.  Baylor College of Medicine. 该协议展示了对香烟烟雾（CS）的病理生理影响的研究，内部内建立了全身吸入（WBI）暴露系统（WBIS）。该系统可以在受控的可重复条件下使动物接触CS，用于研究CS介于肺气肿和血细胞瘤的影响。

视频: Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

用于阴道内艾滋病毒暴露和艾滋病毒感染的人性化 NOG 小鼠

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...