口腔插管和通气肺缺血再灌注手术小鼠模型
1Department of Anesthesia and Perioperative Care,University of California San Francisco and San Francisco General Hospital, 2Department of Physiology, School of Medicine,Tehran University of Medical Sciences, 3Department of Anesthesiology and Critical Care Medicine,Johns Hopkins University School of Medicine
Summary
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。
Abstract
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%。
Introduction
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。
Protocol
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 麻醉和插管准备<l…
Representative Results
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…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Equipment | |||
| Fiber Optic Light Pipe | Cole-Parmer | UX-41720-65 | Fiberoptic light pipe |
| Fiber Optic Light Source | AmScope | SKU: CL-HL250-B | Light source for fiberoptic lights |
| Germinator 500 | Cell Point Scientific, Inc. | No.5-1450 | Bead Sterilizer |
| Heating Pad | AIMS | 14-370-223 | Alternative option |
| Lithium.Ion Grooming Kits(hair clipper) | WAHL home products | SKU 09854-600B | To remove mouse hair on surgical site |
| Microscope | Nikon | SMZ-10 | Other newer options available at the company website |
| MiniVent Ventilator | Havard Apparatus | Model 845 | Mouse ventilator |
| Ultrasonic Cleaner | Cole-Parmer | UX-08895-05 | Clean tools that been used in operation |
| Warming Pad | Kent Scientific | RT-0501 | To keep mouse warm while recovering from surgery |
| Weighing Scale | Cole-Parmer | UX-11003-41 | Weighing scale |
| Surgery Tools | |||
| 4-0 Silk Suture | Ethicon | 683G | For closing muscle layer |
| 7-0 Prolene Suture | Ethicon Industry | EP8734H | Using for making a slip knot of left pulmonary artery |
| Bard-Parker (11) Scalpel (Rib-Back Carbon Steel Surgical Blade, sterile, single use) | Aspen Surgical | 372611 | For entering thoracic cavity (option 1) |
| Bard-Parker (12) Scalpel | Aspen Surgical | 372612 | For entering thoracic cavity (option 2) |
| Extra Fine Graefe Forceps | FST | 11150-10 | Muscle/rib holding forceps |
| Magnetic Fixator Retraction System | FST | 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) |
Small Animal Retraction System |
| Monoject Standard Hypodermic Needle | COVIDIEN | 05-561-20 | For medication delivery IP |
| Narrow Pattern Forceps | FST | 11002-12 | Skin level forceps |
| Needle holder/Needle driver | FST | 12565-14 | for holding needles |
| Needles | BD | 305110 | 26 gauge needle for externalizing slipknot (24 or 26 gauge needle okay too) |
| PA/Vessel Dilating forceps | FST | 00125-11 | To hold PA; non-damaging gripper |
| Scissors | FST | 14060-09 | Used for incision and cutting into the muscular layer durging surgery |
| Ultra Fine Dumont micro forceps | FST | 11295-10 (Dumont #5 forceps, Biology tip, tip dimension:0.05*0.02mm,11cm) | For passing through the space between the left pulmonary artery and bronchus |
| Reagents | |||
| 0.25% Bupivacaine | Hospira, Inc. | 0409-1159-02 | Topical analgesic used during surgical wound closure |
| Avertin (2,2,2-Tribromoethanol) | Sigma-Aldrich | T48402-25G | Anesthetic, using for anesthetize the mouse for IR surgery, the concentration used in IR surgery is 250-400 mg/kg. |
| Buprenorphine | Covetrus North America | 59122 | Analgesic: concentration used for surgery is 0.05-0.1 mg/kg |
| Eye Lubricant | BAUSCH+LOMB | Soothe Lubricant Eye Ointment | Relieves dryness of the eye |
| Povidone-Iodine 10% Solution | MEDLINE INDUSTRIES INC | SKU MDS093944H (2 FL OZ, topical antiseptic) | Topical liquid applied for an effective first aid antiseptic at beginning of surgery |
| Materials | |||
| Alcohol Swab | BD brand | BD 326895 | for sterilzing area of injection and surgery |
| Plastic film | KIRKLAND | Stretch-Tite premium | Alternative for covering the sterilized surgical field (more cost effective) |
| Rodent Surgical Drapes | Stoelting | 50981 | Sterile field or drape for surgical field |
| Sterile Cotton Tipped Application | Pwi-Wnaps | 703033 | used for applying eye lubricant |
| Top Sponges | Dukal Corporaton | Reorder # 5360 | Stopping bleeding from skin/muscle |
Riferimenti
- Shen, H., Kreisel, D., Goldstein, D. R. Processes of sterile inflammation. Journal of Immunology. 191 (6), 2857-2863 (2013).
- Fiser, S. M., et al. Lung transplant reperfusion injury involves pulmonary macrophages and circulating leukocytes in a biphasic response. The Journal of Thoracic and Cardiovascular Surgery. 121 (6), 1069-1075 (2001).
- Lama, V. N., et al. Models of lung transplant research: A consensus statement from the National Heart, Lung, and Blood Institute workshop. JCI Insight. 2 (9), 93121 (2017).
- Miao, R., Liu, J., Wang, J. Overview of mouse pulmonary embolism models. Drug Discovery Today: Disease Models. 7 (3-4), 77-82 (2010).
- Mira, J. C., et al. Mouse injury model of polytrauma and shock. Methods in Molecular Biology. 1717, 1-15 (2018).
- Krupnick, A. S., et al. Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology. Nature Protocols. 4 (1), 86-93 (2009).
- Gielis, J. F., et al. A murine model of lung ischemia and reperfusion injury: Tricks of the trade. The Journal of Surgical Research. 194 (2), 659-666 (2015).
- Nelson, K., et al. Animal models of ex vivo lung perfusion as a platform for transplantation research. World Journal of Experimental Medicine. 4 (2), 7-15 (2014).
- Dodd-o, J. M., Hristopoulos, M. L., Faraday, N., Pearse, D. B. Effect of ischemia and reperfusion without airway occlusion on vascular barrier function in the in vivo mouse lung. Journal of Applied Physiology. 95 (5), 1971-1978 (2003).
- Lawrenz, M. B., Fodah, R. A., Gutierrez, M. G., Warawa, J. Intubation-mediated intratracheal (IMIT) instillation: a noninvasive, lung-specific delivery system. Journal of Visualized Experiments. (93), e52261 (2014).
- Rayamajhi, M., et al. Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry. Journal of Visualized Experiments. (51), e2702 (2011).
- Dodd-o, J. M., Hristopoulos, M. L., Welsh-Servinsky, L. E., Tankersley, C. G., Pearse, D. B. Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice. Journal of Applied Physiology. 100 (5), 1590-1595 (2006).
- Prakash, A., et al. Lung ischemia reperfusion (IR) is a sterile inflammatory process influenced by commensal microbiota in mice. Shock. 44 (3), 272-279 (2015).
- Prakash, A., et al. Alveolar macrophages and toll-like receptor 4 mediate ventilated lung ischemia reperfusion injury in mice. Anesthesiology. 117 (4), 822-835 (2012).
- Dodd-o, J. M., et al. The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse. American Journal of Physiology. Lung Cellular and Molecular Physiology. 294 (4), 714-723 (2008).
- Prakash, A., Kianian, F., Tian, X., Maruyama, D. Ferroptosis mediates inflammation in lung ischemia-reperfusion (IR) sterile injury in mice. American Journal of Respiratory and Critical Care Medicine. 201, (2020).
- Tian, X., et al. NLRP3 inflammasome mediates dormant neutrophil recruitment following sterile lung injury and protects against subsequent bacterial pneumonia in mice. Frontiers in Immunology. 8, 1337 (2017).
- Tian, X., Hellman, J., Prakash, A. Elevated gut microbiome-derived propionate levels are associated with reduced sterile lung inflammation and bacterial immunity in mice. Frontiers in Microbiology. 10, 159 (2019).
- Liu, Q., Tian, X., Maruyama, D., Arjomandi, M., Prakash, A. Lung immune tone via gut-lung axis: Gut-derived LPS and short-chain fatty acids’ immunometabolic regulation of lung IL-1β, FFAR2, and FFAR3 expression. American Journal of Physiology. Lung Cellular and Molecular Physiology. 321 (1), 65-78 (2021).
- Dodd-o, J. M., et al. Interactive effects of mechanical ventilation and kidney health on lung function in an in vivo mouse model. American Journal of Physiology. Lung Cellular and Molecular Physiology. 296 (1), 3-11 (2009).
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Citazione di questo articolo
Liao, W., Maruyama, D., Kianian, F., Tat, C., Tian, X., Hellman, J., Dodd-o, J. M., Prakash, A. A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery. J. Vis. Exp. (187), e64383, doi:10.3791/64383 (2022).