通过直接内切内脂多糖灌输诱导小鼠急性肺损伤
Summary
这里介绍的是一个逐步的过程,通过直接的内切内脂多糖灌输诱导小鼠急性肺损伤,并执行FACS分析血液样本,支气管洗浴液和肺组织。微创性、简单处理、良好的可重复性和疾病严重程度的滴定是这种方法的优点。
Abstract
脂多糖(LPS)气道管理是研究小动物模型中肺部炎症和急性肺损伤(ALI)的常用方法。已经描述了各种方法,例如吸入气溶胶LPS以及鼻腔或内气浸灌输。提出的方案描述了通过直接宫内LPS灌输诱导小鼠ALI的详细分步程序,并执行对血液样本、支气管性(BAL)液体和肺组织的FACS分析。腹管内镇化后,气管暴露,LPS通过22G静脉导管施用。根据使用的LPS剂量,在数到几天内诱导一种强健且可重复的炎症反应,其内为白细胞入侵、前列腺炎细胞因子的增调和白细胞屏障的中断。协议详细介绍了采集血液样本、BAL液体和肺采集以及FACS分析的处理。虽然无菌LPS的使用不适合研究传染病的药理干预,但所述方法提供最小的侵入性、简单的处理和良好的可重复性来回答机械免疫学问题。此外,剂量滴定以及使用替代LPS制剂或小鼠菌株允许调节临床效果,这可能表现出不同程度的ALI严重程度或早期与晚发病症状。
Introduction
实验动物模型在基础免疫研究中不可或缺。管理整个细菌或微生物成分已经常用于小动物模型,以诱发局部或全身炎症1。脂多糖(LPS,或细菌内毒素)是一种细胞壁成分和克阴性细菌的表面抗原(例如,肠杆菌、伪多糖,或军团菌)。热稳定和大分子(分子量1-4 x 106 kDa)由脂质莫伊蒂(脂质A)、核心区域(寡糖)和O多糖(或O抗原)组成。脂质A,其疏水性脂肪酸链,锚定分子到细菌膜和调解(在细菌降解)的免疫活性和毒性的LPS。继与LPS结合蛋白(LBP)结合后,LPS:LBP复合物可使位于许多细胞类型的表面的CD14/TLR4/MD2受体复合物结合,通过NF-βB核易位和随后的上升调节诱导强烈的前列腺炎反应细胞因子表达2。
急性肺损伤(ALI)被定义为急性低氧呼吸衰竭与双边肺水肿,在没有心力衰竭3。LPS的气道管理是诱发肺部炎症和ALI4,5,6,7的常见方法。虽然无菌物质不适合研究传染病的药理干预,但机械免疫学问题可以足够精确地回答。LPS 注入气管诱导强大的炎症反应与白细胞入侵, 前列腺炎细胞因子的调节, 和中断的白细胞 – 毛细血管屏障在几个小时到几天内, 取决于 LPS 剂量3, 6,7.
提出的协议描述了通过内切液LPS灌输诱导小鼠的ALI的详细分步程序。该模型已经通过评估细胞因子表达、中性粒细胞细胞入侵和肺泡内白蛋白泄漏进行了验证,如前所述8。
Protocol
此动物协议由当地动物护理委员会(德国莱克林豪森的LANUV;第84-02.04.2015号协议)批准,并根据国家卫生研究院使用活体动物的准则(NIH出版物)执行第85-23号,1996年修订)。 1. 阿里公司感应 在10-12周的年龄使用成年C57BL/6小鼠。将动物安置在单独通风的笼子里,免费获得水和标准啮齿动物。然而,有可能对年轻的动物和其他小鼠菌株执行这种方法。 在-20°C下以5mg/mL的浓度将…
Representative Results
LPS灌输后,通过评估细胞因子表达、中性粒细胞渗透和肺毛细血管屏障破坏24小时和72小时后,通过评估小鼠诱导ALI的方法得到了验证。PBS注射的动物充当控制。内切术LPS管理诱导一个强大的肺前列腺炎反应。与对照动物相比,肺组织中TNF-α的表达明显增强,达到持续和超过50倍的增幅[RQ(TNF-+/18s);24小时:53.7(SD = 11.6);72小时:55.0(SD = 20.6);p <0.05)](图3A)。白细胞侵入组织和藻?…
Discussion
微创性、简单处理和良好的可重复性是所呈现的方法的关键特征,用于在小型啮齿动物模型中诱导 ALI。在动物模型中使用LPS代替整个细菌具有优势。它是一种稳定和纯净的化合物,可以储存在冻干形式,直到使用。它是通过TLR4途径进行先天免疫反应的有力兴奋剂,其生物活性易于量化,促进疾病严重程度的滴定,具有良好的可重复性。此外,使用LPS已被证明作为安全模型,诱导急性支气管炎在人类健康志愿…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢扬·克莱纳和苏珊娜·舒尔茨提供技术支持。作者肯定了波恩大学医学院对流式细胞学核心设施的出色支持。提交人没有得到任何外部组织的任何资助。 结果部分给出如图3所示的部分数据已在上一份出版物8中显示。
Materials
| 1 ml syringes | BD, Franklin Lakes, NJ, USA | 300013 | |
| 10 ml syringes | BD, Franklin Lakes, NJ, USA | 309110 | |
| Anti-CD115 (c-fms) APC | Thermo Fisher, Waltham, MA, USA | 17-1152-80 | |
| Anti-CD11b (M1/70) – FITC | Thermo Fisher, Waltham, MA, USA | 11-0112-81 | |
| Anti-CD45 (30-F11) – eF450 | Thermo Fisher, Waltham, MA, USA | 48-0451-82 | |
| Anti-F4/80 (BM-8) – PE Cy7 | Thermo Fisher, Waltham, MA, USA | 25-4801-82 | |
| Anti-Gr1 (RB6-8C5) | BD Biosciences, Franklin Lakes, NJ, USA | 552093 | |
| Anti-Ly6C (HK1.4) PerCP-Cy5.5 | Thermo Fisher, Waltham, MA, USA | 45-5932-82 | |
| Anti-Ly6G (1A8) APC/Cy7 | Bio Legend, San Diego, CA | 127623 | |
| Buprenorphine hydrochloride | Indivior UK Limited, Berkshire, UK | ||
| C57BL/6 mice, female, 10 – 12 weeks old | Charles River, Wilmongton, MA, USA | ||
| CaliBRITE APC-beads (6µm) | BD Biosciences, Franklin Lakes, NJ, USA | 340487 | |
| Canula 23 gauge 1'' | BD, Franklin Lakes, NJ, USA | 300800 | |
| Canula 26 gauge 1/2'' | BD, Franklin Lakes, NJ, USA | 303800 | |
| Cell strainer 70 µm | BD Biosciences, Franklin Lakes, NJ, USA | 352350 | |
| Collagenase Type I | Sigma-Aldrich, St. Louis, MO, USA | 1148089 | |
| Deoxyribonuclease II | Sigma-Aldrich, St. Louis, MO, USA | D8764 | |
| Dulbecco's Phosphate Buffered Saline (PBS), sterile | Sigma-Aldrich, St. Louis, MO, USA | D8662 | |
| Dulbecco’s Phosphate Buffered Saline (PBS), without calcium chloride and magnesium chloride, sterile | Sigma-Aldrich, St. Louis, MO, USA | D8537 | |
| Ethylenediaminetetraacetic acid (EDTA) solution | Sigma-Aldrich, St. Louis, MO, USA | E7889 | |
| FACS tubes, 5 ml | Sarstedt, Nümbrecht, Germany | 551579 | |
| Fetal calf serum (FCS) | Sigma-Aldrich, St. Louis, MO, USA | F2442 | |
| Forceps | Fine Science Tools, Heidelberg, Germany | 11049-10 | |
| Isoflurane | Baxter, Unterschleißheim, Germany | ||
| Ketamine hydrochloride | Serumwerk Bernburg, Bernburg, Germany | ||
| Lipopolysaccharides (LPS) from Escherichia coli O111:B4 | Sigma-Aldrich, St. Louis, MO, USA | L2630 | |
| LIVE/DEAD Fixable Dead Cell Green Kit | Thermo Fisher, Waltham, MA, USA | L23101 | |
| Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™), Clone 2.4G2 | BD, Franklin Lakes, NJ, USA | 553141 | |
| Red blood cell lysis buffer | Thermo Fisher, Waltham, MA, USA | 00-4333-57 | |
| RPMI-1640, with L-glutamine and sodium bicarbonate | Sigma-Aldrich, St. Louis, MO, USA | R8758 | |
| Scissors | Fine Science Tools, Heidelberg, Germany | 14060-09 | |
| Sodium azide (NaN3) | Sigma-Aldrich, St. Louis, MO, USA | S2002 | |
| Spring scissors | Fine Science Tools, Heidelberg, Germany | 15018-10 | |
| Tissue forceps | Fine Science Tools, Heidelberg, Germany | 11021-12 | |
| Tubes | Eppendorf, Hamburg, Germany | 30125150 | |
| Venous catheter, 22 gauge | B.Braun, Melsungen, Germany | 4268091B | |
| Xylazine hydrochloride | Serumwerk Bernburg, Bernburg, Germany |
References
- Fink, M. P. Animal models of sepsis. Virulence. 5 (1), 143-153 (2014).
- Lu, Y. -. C., Yeh, W. -. C., Ohashi, P. S. LPS/TLR4 signal transduction pathway. Cytokine. 42 (2), 145-151 (2008).
- Matute-Bello, G., Frevert, C. W., Martin, T. R. Animal models of acute lung injury. American Journal of Physiology – Lung Cellular and Molecular Physiology. 295 (3), 379-399 (2008).
- Rabelo, M. A. E., et al. Acute Lung Injury in Response to Intratracheal Instillation of Lipopolysaccharide in an Animal Model of Emphysema Induced by Elastase. Inflammation. 41 (1), 174-182 (2018).
- Liu, F., Li, W., Pauluhn, J., Trübel, H., Wang, C. Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation. Toxicology. 304, 158-166 (2013).
- Rittirsch, D., et al. Acute Lung Injury Induced by Lipopolysaccharide Is Independent of Complement Activation. Journal of Immunology. 180 (11), 7664-7672 (2008).
- D’Alessio, F. R., et al. CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. The Journal of Clinical Investigation. 119 (10), 2898-2913 (2009).
- Ehrentraut, H., Weisheit, C., Scheck, M., Frede, S., Hilbert, T. Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages. Journal of Inflammation. 15, 12 (2018).
- Szarka, R. J., Wang, N., Gordon, L., Nation, P. N., Smith, R. H. A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. Journal of Immunological Methods. 202 (1), 49-57 (1997).
- Reutershan, J., Basit, A., Galkina, E. V., Ley, K. Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury. American Journal of Physiology. Lung Cellular and Molecular Physiology. 289 (5), 807-815 (2005).
- Hoegl, S., et al. Capturing the multifactorial nature of ARDS – approach to model murine acute lung injury. Physiological Reports. 6 (6), (2018).
- Weisheit, C., et al. Ly6Clow and Not Ly6Chigh Macrophages Accumulate First in the Heart in a Model of Murine Pressure-Overload. PLoS ONE. 9 (11), (2014).
- Grommes, J., Soehnlein, O. Contribution of Neutrophils to Acute Lung Injury. Molecular Medicine. 17 (3-4), 293-307 (2011).
- Müller-Redetzky, H. C., Suttorp, N., Witzenrath, M. Dynamics of pulmonary endothelial barrier function in acute inflammation: mechanisms and therapeutic perspectives. Cell and Tissue Research. 355 (3), 657-673 (2014).
- Fujita, M., et al. Endothelial cell apoptosis in lipopolysaccharide-induced lung injury in mice. International Archives of Allergy and Immunology. 117 (3), 202-208 (1998).
- Doyen, V., et al. Inflammation induced by inhaled lipopolysaccharide depends on particle size in healthy volunteers. British Journal of Clinical Pharmacology. 82 (5), 1371-1381 (2016).
- Stephens, R. S., Johnston, L., Servinsky, L., Kim, B. S., Damarla, M. The tyrosine kinase inhibitor imatinib prevents lung injury and death after intravenous LPS in mice. Physiological Reports. 3 (11), (2015).
- Yu, Y., Jing, L., Zhang, X., Gao, C. Simvastatin Attenuates Acute Lung Injury via Regulating CDC42-PAK4 and Endothelial Microparticles. Shock. 47 (3), 378-384 (2017).
Citer Cet Article
Ehrentraut, H., Weisheit, C. K., Frede, S., Hilbert, T. Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation. J. Vis. Exp. (149), e59999, doi:10.3791/59999 (2019).