Name: a mouse model of orotracheal intubation and ventilated lung ischemia reperfusion surgery
Uploaded: 2022-09-09T15:00:00
Description: Watch this Scientific Journal Video about A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery at JoVE.com

这项工作由加州大学旧金山分校和旧金山综合医院麻醉和围手术期护理系的部门支持以及NIH R01奖（AP）：1R01HL146753资助。

下面描述的所有程序和步骤均已获得加州大学旧金山分校机构动物护理和使用委员会（IACUC）的批准。可以使用任何小鼠品系，尽管与其他菌株相比，某些菌株具有更强烈的肺IR炎症反应12。大约12-15周龄（30-40g）或更大的小鼠比年轻小鼠更好地耐受和存活肺IR手术。雄性和雌性小鼠都可用于这些手术。
1. 小鼠插管方案
<ol><li>麻醉和插管准备<ol><l...

<ol>
	<li>Shen, H., Kreisel, D., Goldstein, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processes+of+sterile+inflammation.">Processes of sterile inflammation.</a> Journal of Immunology. 191 (6), 2857-2863 (2013).</li><li>Fiser, S. 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=Lung+transplant+reperfusion+injury+involves+pulmonary+macrophages+and+circulating+leukocytes+in+a+biphasic+response.">Lung transplant reperfusion injury involves pulmonary macrophages and circulating leukocytes in a biphasic response.</a> The Journal of Thoracic and Cardiovascular Surgery. 121 (6), 1069-1075 (2001).</li><li>Lama, V. N., 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=Models+of+lung+transplant+research:+A+consensus+statement+from+the+National+Heart,+Lung,+and+Blood+Institute+workshop.">Models of lung transplant research: A consensus statement from the National Heart, Lung, and Blood Institute workshop.</a> JCI Insight. 2 (9), 93121 (2017).</li><li>Miao, R., Liu, J., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+mouse+pulmonary+embolism+models.">Overview of mouse pulmonary embolism models.</a> Drug Discovery Today: Disease Models. 7 (3-4), 77-82 (2010).</li><li>Mira, J. 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=Mouse+injury+model+of+polytrauma+and+shock.">Mouse injury model of polytrauma and shock.</a> Methods in Molecular Biology. 1717, 1-15 (2018).</li><li>Krupnick, A. 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=Orthotopic+mouse+lung+transplantation+as+experimental+methodology+to+study+transplant+and+tumor+biology.">Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology.</a> Nature Protocols. 4 (1), 86-93 (2009).</li><li>Gielis, J. 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=A+murine+model+of+lung+ischemia+and+reperfusion+injury:+Tricks+of+the+trade.">A murine model of lung ischemia and reperfusion injury: Tricks of the trade.</a> The Journal of Surgical Research. 194 (2), 659-666 (2015).</li><li>Nelson, K., 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=Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research.">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World Journal of Experimental Medicine. 4 (2), 7-15 (2014).</li><li>Dodd-o, J. M., Hristopoulos, M. L., Faraday, N., Pearse, 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=Effect+of+ischemia+and+reperfusion+without+airway+occlusion+on+vascular+barrier+function+in+the+in+vivo+mouse+lung.">Effect of ischemia and reperfusion without airway occlusion on vascular barrier function in the in vivo mouse lung.</a> Journal of Applied Physiology. 95 (5), 1971-1978 (2003).</li><li>Lawrenz, M. B., Fodah, R. A., Gutierrez, M. G., Warawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intubation-mediated+intratracheal+(IMIT)+instillation:+a+noninvasive,+lung-specific+delivery+system.">Intubation-mediated intratracheal (IMIT) instillation: a noninvasive, lung-specific delivery system.</a> Journal of Visualized Experiments. (93), e52261 (2014).</li><li>Rayamajhi, 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=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> Journal of Visualized Experiments. (51), e2702 (2011).</li><li>Dodd-o, J. M., Hristopoulos, M. L., Welsh-Servinsky, L. E., Tankersley, C. G., Pearse, 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=Strain-specific+differences+in+sensitivity+to+ischemia-reperfusion+lung+injury+in+mice.">Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.</a> Journal of Applied Physiology. 100 (5), 1590-1595 (2006).</li><li>Prakash, 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=Lung+ischemia+reperfusion+(IR)+is+a+sterile+inflammatory+process+influenced+by+commensal+microbiota+in+mice.">Lung ischemia reperfusion (IR) is a sterile inflammatory process influenced by commensal microbiota in mice.</a> Shock. 44 (3), 272-279 (2015).</li><li>Prakash, 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=Alveolar+macrophages+and+toll-like+receptor+4+mediate+ventilated+lung+ischemia+reperfusion+injury+in+mice.">Alveolar macrophages and toll-like receptor 4 mediate ventilated lung ischemia reperfusion injury in mice.</a> Anesthesiology. 117 (4), 822-835 (2012).</li><li>Dodd-o, J. 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+role+of+natriuretic+peptide+receptor-A+signaling+in+unilateral+lung+ischemia-reperfusion+injury+in+the+intact+mouse.">The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 294 (4), 714-723 (2008).</li><li>Prakash, A., Kianian, F., Tian, X., Maruyama, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferroptosis+mediates+inflammation+in+lung+ischemia-reperfusion+(IR)+sterile+injury+in+mice.">Ferroptosis mediates inflammation in lung ischemia-reperfusion (IR) sterile injury in mice.</a> American Journal of Respiratory and Critical Care Medicine. 201, (2020).</li><li>Tian, 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=NLRP3+inflammasome+mediates+dormant+neutrophil+recruitment+following+sterile+lung+injury+and+protects+against+subsequent+bacterial+pneumonia+in+mice.">NLRP3 inflammasome mediates dormant neutrophil recruitment following sterile lung injury and protects against subsequent bacterial pneumonia in mice.</a> Frontiers in Immunology. 8, 1337 (2017).</li><li>Tian, X., Hellman, J., Prakash, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+gut+microbiome-derived+propionate+levels+are+associated+with+reduced+sterile+lung+inflammation+and+bacterial+immunity+in+mice.">Elevated gut microbiome-derived propionate levels are associated with reduced sterile lung inflammation and bacterial immunity in mice.</a> Frontiers in Microbiology. 10, 159 (2019).</li><li>Liu, Q., Tian, X., Maruyama, D., Arjomandi, M., Prakash, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+immune+tone+via+gut-lung+axis:+Gut-derived+LPS+and+short-chain+fatty+acids'+immunometabolic+regulation+of+lung+IL-1&#946;,+FFAR2,+and+FFAR3+expression.">Lung immune tone via gut-lung axis: Gut-derived LPS and short-chain fatty acids' immunometabolic regulation of lung IL-1&#946;, FFAR2, and FFAR3 expression.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 321 (1), 65-78 (2021).</li><li>Dodd-o, J. 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=Interactive+effects+of+mechanical+ventilation+and+kidney+health+on+lung+function+in+an+in+vivo+mouse+model.">Interactive effects of mechanical ventilation and kidney health on lung function in an in vivo mouse model.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 296 (1), 3-11 (2009).</li></ol>

这份手稿详细介绍了执行由 Dodd-o 等人开发的通气肺 IR 模型所涉及的步骤9。该模型有助于确定参与分离肺IR的炎症产生和解决的分子途径14，15，16，17，肺IR与共存感染的结合18，以及肺IR与肠 - 肺轴的关系以及肠道微生物组的贡献13，18，<s...

缺血再灌注 （IR） 损伤可能发生在中断一段时间后恢复到器官或组织床的血流。在肺中，IR 可单独发生或与其他损伤过程相关，例如感染、缺氧、肺不张、容积伤（机械通气期间潮气量增加）、气压伤（机械通气期间出现峰值或持续压力）或钝性（非穿透性）肺挫伤1，2，3.我们对LIRI的机制以及并发过程（例如感染）对LIRI结局的影响的认识仍然存在一些差距，而且LIRI的治疗选择也很有限。需要纯LIRI的体内模型来单独识别肺IR损伤的病理生理学，并研究其对任何多命中过程的贡献，其中肺损伤是其组成部分。
鼠肺 IR 模型可用于研究多个过程的肺特异性病理生理学，包括肺移植3、肺栓塞4 和复苏后出血性创伤后的肺损伤5。目前使用的模型包括外科肺移植6，肺门钳夹7， 离体 肺灌注8和通气肺IR9。在这里，我们为无菌性肺损伤的小鼠通气肺IR模型提供了详细的方案。这种方法有多种好处（图2），包括它诱导最小的缺氧和最小的肺不张，并且它是一种允许长期研究的生存手术模型。
选择这种LIRI模型而不是其他模型（如肺门钳夹和 离体 灌注模型）的原因如下：该模型最大限度地减少了肺不张，机械通气和缺氧的炎症贡献;它保留了周期性通风;它保持完整的 体内 循环免疫系统，可以对IR损伤做出反应;最后，作为一种生存程序，它允许对继发性损伤产生（2次命中模型）和损伤消退的机制进行长期分析。总体而言，我们相信这种通气肺IR模型提供了可以通过实验研究的“最纯净”形式的IR损伤。
其他出版物描述了使用小鼠的口腱插管进行IT注射或安装10，11，但不像该模型那样作为生存手术的起点。口腔导管的放置允许通过允许手术肺塌陷来进行肺部手术。它还允许在手术结束时重新充气肺，这对于气胸和小鼠在手术结束时恢复自主通气的能力至关重要。最后，移除固定的口腔导管是一个简单的程序，与侵入性气管切开术不同，它与生存手术兼容。这允许长期研究，重点是了解LIRI和相关疾病的进展和解决，以及创建慢性损伤模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Light Pipe</td>
			<td>Cole-Parmer</td>
			<td>UX-41720-65</td>
			<td>Fiberoptic light pipe</td>
		</tr>
		<tr>
			<td>Fiber Optic Light Source</td>
			<td>AmScope</td>
			<td>SKU: CL-HL250-B</td>
			<td>Light source for fiberoptic lights</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Cell Point Scientific, Inc.</td>
			<td>No.5-1450</td>
			<td>Bead Sterilizer</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>AIMS</td>
			<td>14-370-223</td>
			<td>Alternative option</td>
		</tr>
		<tr>
			<td>Lithium.Ion Grooming Kits(hair clipper)</td>
			<td>WAHL home products</td>
			<td>SKU 09854-600B</td>
			<td>To remove mouse hair on surgical site</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ-10</td>
			<td>Other newer options available at the company website</td>
		</tr>
		<tr>
			<td>MiniVent Ventilator</td>
			<td>Havard Apparatus</td>
			<td>Model 845</td>
			<td>Mouse ventilator</td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Cole-Parmer</td>
			<td>UX-08895-05</td>
			<td>Clean tools that been used in operation</td>
		</tr>
		<tr>
			<td>Warming Pad</td>
			<td>Kent Scientific</td>
			<td>RT-0501</td>
			<td>To keep mouse warm while recovering from surgery</td>
		</tr>
		<tr>
			<td>Weighing Scale</td>
			<td>Cole-Parmer</td>
			<td>UX-11003-41</td>
			<td>Weighing scale</td>
		</tr>
		<tr>
			<td>Surgery Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Silk Suture</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>For closing muscle layer</td>
		</tr>
		<tr>
			<td>7-0 Prolene Suture</td>
			<td>Ethicon Industry</td>
			<td>EP8734H</td>
			<td>Using for making a slip knot of left pulmonary artery</td>
		</tr>
		<tr>
			<td>Bard-Parker (11) Scalpel (Rib-Back Carbon Steel Surgical Blade, sterile, single use)</td>
			<td>Aspen Surgical</td>
			<td>372611</td>
			<td>For entering thoracic cavity (option 1)</td>
		</tr>
		<tr>
			<td>Bard-Parker (12) Scalpel</td>
			<td>Aspen Surgical</td>
			<td>372612</td>
			<td>For entering thoracic cavity (option 2)</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>FST</td>
			<td>11150-10</td>
			<td>Muscle/rib holding forceps</td>
		</tr>
		<tr>
			<td>Magnetic Fixator Retraction System</td>
			<td>FST</td>
			<td>1. Base Plate (Nos. 18200-03) 
			2. Fixators (Nos. 18200-01) 
			3. Retractors (Nos. 18200-05 through 18200-12) 
			4. Elastomer (Nos.18200-07) 5. Retractor(No.18200-08)</td>
			<td>Small Animal Retraction System</td>
		</tr>
		<tr>
			<td>Monoject Standard Hypodermic Needle</td>
			<td>COVIDIEN</td>
			<td>05-561-20</td>
			<td>For medication delivery IP</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>FST</td>
			<td>11002-12</td>
			<td>Skin level forceps</td>
		</tr>
		<tr>
			<td>Needle holder/Needle driver</td>
			<td>FST</td>
			<td>12565-14</td>
			<td>for holding needles</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>BD</td>
			<td>305110</td>
			<td>26 gauge needle for externalizing slipknot (24 or 26 gauge needle okay too)</td>
		</tr>
		<tr>
			<td>PA/Vessel Dilating forceps</td>
			<td>FST</td>
			<td>00125-11</td>
			<td>To hold PA; non-damaging gripper</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14060-09</td>
			<td>Used for incision and cutting into the muscular layer durging surgery</td>
		</tr>
		<tr>
			<td>Ultra Fine Dumont micro forceps</td>
			<td>FST</td>
			<td>11295-10 (Dumont #5 forceps, Biology tip, tip dimension:0.05*0.02mm,11cm)</td>
			<td>For passing through the space between the left pulmonary artery and bronchus</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Bupivacaine</td>
			<td>Hospira, Inc.</td>
			<td>0409-1159-02</td>
			<td>Topical analgesic used during surgical wound closure</td>
		</tr>
		<tr>
			<td>Avertin (2,2,2-Tribromoethanol)</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25G</td>
			<td>Anesthetic, using for anesthetize the mouse for IR surgery, the concentration used in IR surgery is 250-400 mg/kg.</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Covetrus North America</td>
			<td>59122</td>
			<td>Analgesic: concentration used for surgery is 0.05-0.1 mg/kg</td>
		</tr>
		<tr>
			<td>Eye Lubricant</td>
			<td>BAUSCH+LOMB</td>
			<td>Soothe Lubricant Eye Ointment</td>
			<td>Relieves dryness of the eye</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% Solution</td>
			<td>MEDLINE INDUSTRIES INC</td>
			<td>SKU MDS093944H (2 FL OZ, topical antiseptic)</td>
			<td>Topical liquid applied for an effective first aid antiseptic at beginning of surgery</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Swab</td>
			<td>BD brand</td>
			<td>&#160;BD 326895</td>
			<td>for sterilzing area of injection and surgery</td>
		</tr>
		<tr>
			<td>Plastic film</td>
			<td>KIRKLAND</td>
			<td>Stretch-Tite premium</td>
			<td>Alternative for covering the sterilized surgical field (more cost effective)</td>
		</tr>
		<tr>
			<td>Rodent Surgical Drapes</td>
			<td>Stoelting</td>
			<td>50981</td>
			<td>Sterile field or drape for surgical field</td>
		</tr>
		<tr>
			<td>Sterile Cotton Tipped Application</td>
			<td>Pwi-Wnaps</td>
			<td>703033</td>
			<td>used for applying eye lubricant</td>
		</tr>
		<tr>
			<td>Top Sponges</td>
			<td>Dukal Corporaton</td>
			<td>Reorder # 5360</td>
			<td>Stopping bleeding from skin/muscle</td>
		</tr>
	</tbody>
</table>

a mouse model of orotracheal intubation and ventilated lung ischemia reperfusion surgery

缺血再灌注 （IR） 损伤通常由涉及短暂的血流中断的过程引起。在肺中，孤立的IR允许通过持续的肺泡通气对这一特定过程进行实验研究，从而避免缺氧和肺不张的复合有害过程。在临床环境中，肺缺血再灌注损伤（也称为肺IRI或LIRI）由许多过程引起，包括但不限于肺栓塞，复苏性出血性创伤和肺移植。目前，LIRI的有效治疗选择有限。在这里，我们提出了肺IR的可逆手术模型，包括首先耳廓气插管，然后是单侧左肺缺血和再灌注，保留肺泡通气或气体交换。小鼠接受左开胸术，通过该切开术，左肺动脉暴露，可视化，隔离和使用可逆滑结压缩。然后在缺血期间关闭手术切口，唤醒动物并拔管。随着小鼠自主呼吸，通过释放肺动脉周围的滑结来建立再灌注。这种临床相关的生存模型允许评估肺IR损伤，消退阶段，对肺功能的下游影响，以及涉及实验性肺炎的两次命中模型。虽然技术上具有挑战性，但这种模式可以在几周到几个月内掌握，最终生存率或成功率为80%-90%。

单侧通气无菌肺缺血再灌注（IR）损伤引起的炎症：缺血1小时后，我们观察到ELISA和qRT-PCR在再灌注后1小时血清和肺组织中的细胞因子水平升高，在再灌注后12-24小时内迅速恢复到基线13。对于再灌注后3小时收集的样品，我们观察到左肺组织内的强烈中性粒细胞浸润，并注意到炎症的强度取决于所使用的小鼠菌株（图1）。值得注意的是，在没有共存或随后的...

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A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

一种小鼠手术模型，用于在保持通气和避免缺氧的同时产生左肺缺血再灌注 （IR） 损伤。

口腔插管和通气肺缺血再灌注手术小鼠模型

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

通气肺缺血再灌注手术可用于研究多个过程的肺部特异性病理生理学，包括肺移植、肺栓塞和复苏后出血性创伤后的肺损伤。该模型最大限度地减少了肺不张、机械通气和缺氧的炎症贡献。它保持完整的体内循环免疫系统，并允许长期研究。该模型可以深入了解无菌炎症如何在肺内得到控制和调节。显微外科技术首先需要数小时的练习。在开始之前，仔细的组织和计划很重要。此外，它可以由安乐死的小鼠最初练习，而不会分散心脏活动及其引起的运动。左侧PA的血瘀使其更容易可视化和操纵。首先，轻轻地将光纤柔性灯放在麻醉小鼠的气管上，略低于声带。调整照明水平，使在观察小鼠口腔咽部时只看到暗场，除了从声带下方发出的红光。对于插管，用惯用手握住镊子，用它们轻轻握住舌头并将舌头从口腔中拉出。使用非惯用手握住的镊子打开下颌，并将镊子推入喉部以抬起会厌。此时，从镊子中松开舌头。观察声带。它们应该根据每次呼吸打开和关闭。在预装导丝的情况下握住套管，将导线尖端插入声带。接下来，要非常小心，不要通过握住套管外但声带上方的一部分来移动电线，撤回套管，只留下电线，其远端在气管内。此时，对声带进行第二次可视化，以确认钢丝远端尖端仍然穿过发光的声带并进入气管，并且不在未点燃的食道中。接下来，将导线放在嘴外，左手的弯曲镊子稳定在坚硬的表面上，并小心地将带有胶带翅膀的 20 号导管推进到导线上。一旦导线的远端从导管或气管插管的后端出来，用弯曲的镊子握住该端，并将导管平稳地推进到气管中。接下来，用弯曲的镊子小心地从导管的远端取下导线，而不会移开导管的位置。接下来，在固定之前将导管短暂连接到呼吸机，以确认正确放置在气管而不是食道中。通过观察机械通气依赖性双侧胸壁运动和无胃充气来确认气管放置。插管后，将导管连接到呼吸机上，呼吸机的标题容积为 0.2 至 0.225 毫升，呼吸频率为 120 至 150 次/分钟，以确认口腔插管的气管位置是否正确。然后断开鼠标通过口腔管自主呼吸。剃掉左胸区到左肩胛骨的小鼠毛发。使用酒精棉签去除多余的剃光头发并对手术区域进行消毒。然后将鼠标放在左侧或四分之三转动位置的加热垫上。将气管插管连接到呼吸机。在使皮肤切口后，在肌肉层下方插入三个无菌牵开器。接下来，确定第二个肋间隙并用超细镊子握住第二根肋骨。然后，向上拉肋骨，使用无菌的12号弯曲手术刀刀片通过分离并切割第二至第三空间的肋间肌肉进入复数空间。考虑暂停通气以减少左肺尖损伤。沿肋骨方向使用最小、最窄的牵开器头，沿第三肋向左使用中等大小的牵开器，沿第二肋骨表面向右使用最大的牵开器。使用弹性牵开器绳索缓慢而渐进地回缩胸部。用无菌棉签或手术海绵移开左肺尖，暴露并识别左肺动脉或 PA。使用右手的微型镊子超细镊子和左手的PA或血管扩张钳轻轻暴露并创建左侧PA和支气管都可见的区域。使用PA钳，拿起左侧PA并轻轻但牢固地向上拉动，头部以可视化下面的透明支气管。将解剖显微镜上的放大倍数增加到四倍，同时将PA从支气管上收回时，小心地将封闭的超细镊子穿过左PA和支气管之间的空间。然后用这些镊子握住并拉动 7-0 或 8-0通过上方左肺动脉和下方支气管之间的空间缝合前匕首。接下来，通过打一个滑结来包围左侧 PA，以在 PA 中形成遮挡。此时血流中断很容易可视化，标志着缺血期的开始。使用 24 至 28 号针通过左前胸的不同入口点将结的自由端外部化，并用一小块胶带固定缝合线的末端，以便以后更容易识别。接下来，在啮齿动物呼吸机上使用正压和呼气压力阀或管道，重新充气肺以将尽可能多的空气排出胸腔。然后，用两条中断的 4-0 尼龙缝合线关闭胸腔，然后关闭肌肉和皮肤层，并应用局部麻醉。断开鼠标与呼吸机的连接，小心地将其放在加热垫上进行术后护理，以在恢复期间保持体温。确保在鼠标移动过程中清晰地控制和可视化外部化的滑结。在缺血期结束时轻轻拉动外部化的滑结。此处显示了品系C3H和C57黑六的野生型小鼠肺切片的组织学。经过1小时缺血和3小时再灌注后，两种菌株均观察到肺左组织内强烈的中性粒细胞浸润。然而，与C57黑六相比，C3H菌株显示出明显更高的炎症水平。在左PA和左支气管之间通过单丝期间的错误可能导致未挽救的手术，左PA灾难性出血或左支气管不可逆转的损伤。这是技术上最具挑战性的步骤，在第一次学习此过程时需要反复练习。再灌注后约1小时或更短时间内，小鼠从肺缺血再灌注手术中恢复后，可在3小时或24至48小时后进行气管内滴注活菌，以模拟缺血再灌注引起的无菌肺损伤后的感染。在缺血再灌注后，也可以以类似的方式在气管内安装其他可以调节无菌炎症的药物。该技术有助于发现和确认中性粒细胞的逐步运输和激活，以响应肺部的无菌损伤和感染。

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Lung Dendritic Cells

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Pseudomonas aeruginosa Induced Lung Injury Model

Pseudomonas aeruginosa Induced Lung Injury Model

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Influenza A Virus Studies in a Mouse Model of Infection

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

成年斑马鱼的口服插管: 一种评价生物活性化合物肠道吸收的模型

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...