脓毒症诱导免疫抑制的切卡结扎和穿刺和鼻内感染双重模型的设计
Summary
该协议描述了在免疫抑制条件下测量继发医院感染的传染性结果的技术,首先通过建立结扎/穿刺小鼠,然后挑战他们与鼻内感染创建一个临床相关的免疫抑制败血症模型。
Abstract
败血症是一种严重而复杂的危及生命的感染,其特征是多个器官的亲炎和抗炎反应之间不平衡。随着疗法的发展,大多数患者在高炎阶段存活,但进展到免疫抑制阶段,这增加了继发性感染的出现。因此,更好地了解脓毒症期间免疫抑制阶段继发性医院获得性感染的发病机制非常重要。这里报道的模型是通过在小鼠中制造双重感染来测试传染性结果。标准外科手术用于通过结扎和穿刺(CLP)诱导多微生物性囊中膜炎,然后对金黄色葡萄球菌进行鼻内感染,以模拟在免疫抑制中发生的肺炎,这是常见的在败血症患者。这种双重模型可以反映长期败血症患者的免疫抑制状态,以及对非典型肺炎继发性感染的易感性。因此,该模型为研究败血症引起的继发性细菌性肺炎的病理生理学提供了一种简单的实验方法,可用于发现脓毒症及其并发症的新疗法。
Introduction
败血症启动宿主亲炎和抗炎过程的复杂相互作用,其特点是高炎反应和随后的免疫功能障碍1,2。败血症是全球卫生优先事项,在重症监护病房(Ioc)中造成大量死亡。据估计,全世界每年脓毒症的发病率超过3000万例,尽管ICU管理取得了进步,但死亡率仍高达30%。2017年,世界卫生组织通过决议,改善这一致命疾病的预防、诊断和管理。然而,最近的研究表明,死亡不是由严重败血症患者的原发性感染引起的,而是由免疫抑制6,7引起的继发性感染(特别是肺炎).因此,迫切需要了解败血症患者为何发展为继发感染的机制,并发现更有效的治疗方法。本文描述了一种双模型,也称为双击模型,用于研究长期败血症患者的免疫抑制现象。
作为多微生物败血症、囊性结扎和穿刺(CLP)研究的黄金标准实验模型,是一种以囊性结扎和穿孔为特征的手术,有助于多微生物性囊炎8、9.病理生理学过程和细胞因子轮廓,以及动力学和量级,类似于临床败血症。结扎的位置、用于穿刺的针体尺寸以及尖刺次数是影响 CLP 后死亡率的主要因素。
肺炎是败血症危重病人死亡的主要原因。引起严重败血症的主要生物类型包括金黄色葡萄球菌(20.5%)、伪多米诺类(19.9%)、肠杆菌(主要是大肠杆菌,16.0%)和真菌(19%)。 同时,最近的研究表明,克阳性生物的发病率在增加,现在几乎和克阴性感染3一样常见。
该协议中描述的方法涉及CLP,作为”第一击”来诱导亚致死多微生物性包炎,表现为免疫抑制。该程序还涉及后续的鼻内灌输S.aureus作为”第二击”,以提供一个临床相关的研究平台。
Protocol
Representative Results
Discussion
作为败血症研究的黄金标准模型,中电具有三种侮辱的组合,包括腹腔切除术造成的组织创伤、因结扎而导致的坏死,以及微生物泄漏导致腹腔炎和易位性腹腔炎引起的感染。细菌进入血液8。因此,CLP 比许多其他模型更能模仿人类败血症的复杂性。然而,目前CLP模式的一个主要限制是不能反映在ICU3、4、5患者中看到的脓毒症的更长期阶段。<sup class="xr…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院支持 R01 AI138203-01、AI109317-04、AI101973-01 和 AI097532-01A1 到 M. W.北达科他大学核心设施由NIH赠款(INBRE P20GM103442和COBRE P20GM113123)提供支持。这项工作还得到了国家自然科学基金(81530063)国家重点项目(81530063)对江建新的支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有作用。我们感谢马文·莱尔(北达科他大学农村卫生中心)制作了这段视频。
Materials
| 21 G 1 ½ Needle | BD | BD305167 | |
| ACK lysing buffer | Gibco | A10492-01 | |
| Anti-mouse CD11b antibody | Biolegend | 101201 | |
| Anti-mouse Ly-6G/Ly-6C (Gr-1) antibody | Biolegend | 108401 | |
| C57BL/6 mice | Harlan (Indianapolis) | C57BL/6NHsd | |
| Desk light | General Supply | General Supply | |
| Disinfecting wipes | Clorox | B07NV5JMCS | |
| Electric razor | General Supply | General Supply | |
| ELISA kits (mouse IL-1β, IL-6 and TNFα) | Invitrogen | 88-7013, 88-7064, and 88-7324 | |
| Iodine | Dynarex | B003U463PY | PVP Iodine Wipes |
| Ketamine | FORT DODGE | NDC 0856-2013-01 | Amine hydrochloride injection |
| Laboratory scale | General Supply | General Supply | |
| LB Agar, Miller | Fisher Scientific | BP1425-500 | Molecular genetics, powder |
| Micropipette | ErgoOne | 7100-1100 | |
| Normal saline | General Supply | General Supply | |
| Polylined towel | CardinalHealth, Convertors | 3520 | Surgical drape, sterile, for single use only |
| Silk suture, 4-0 | DAVIS & GECK | 1123-31 | |
| Small animal needle holder | General Supply | General Supply | |
| Small animal surgery scissors | General Supply | General Supply | |
| Small animal surgical forceps | General Supply | General Supply | |
| Staphylococcus aureus | ATCC | 13301 | |
| Warm pad | General Supply | General Supply | |
| Xylazine | Alfa Aesar | 7361-61-7 |
References
- Singer, M., et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 315 (8), 801-810 (2016).
- Delano, M. J., Ward, P. A. The immune system’s role in sepsis progression, resolution, and long-term outcome. Immunological Reviews. 274 (1), 330-353 (2016).
- Mayr, F. B., Yende, S., Angus, D. C. Epidemiology of severe sepsis. Virulence. 5 (1), 4-11 (2014).
- Fleischmann, C., et al. Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations. American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).
- Reinhart, K., et al. Recognizing Sepsis as a Global Health Priority – A WHO Resolution. The New England Journal of Medicine. 377 (5), 414-417 (2017).
- Boomer, J. S., et al. Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA. 306 (23), 2594-2605 (2011).
- Hotchkiss, R. S., Monneret, G., Payen, D. Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nature Reviews Immunology. 13 (12), 862-874 (2013).
- Dejager, L., Pinheiro, I., Dejonckheere, E., Libert, C. Cecal ligation and puncture: the gold standard model for polymicrobial sepsis?. Trends in Microbiology. 19 (4), 198-208 (2011).
- Rittirsch, D., Huber-Lang, M. S., Flierl, M. A., Ward, P. A. Immunodesign of experimental sepsis by cecal ligation and puncture. Nature Protocols. 4 (1), 31-36 (2009).
- Hugunin, K. M. S., Fry, C., Shuster, K., Nemzek, J. A. Effects of tramadol and buprenorphine on select immunologic factors in a cecal ligation and puncture model. Shock. 34 (3), 250-260 (2010).
- He, S., et al. Annexin A2 Modulates ROS and Impacts Inflammatory Response via IL-17 Signaling in Polymicrobial Sepsis Mice. PLoS Pathogens. 12 (7), 23 (2016).
- Pu, Q. Q., et al. Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Pseudomonas Sepsis. Journal of Immunology. 198 (8), 3205-3213 (2017).
- Zanotti-Cavazzoni, S. L., et al. Fluid resuscitation influences cardiovascular performance and mortality in a murine model of sepsis. Intensive Care Medicine. 35 (4), 748-754 (2009).
- Chin, W., et al. A macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset. Nature Communications. 9 (1), 917 (2018).
- Nascimento, D. C., et al. IL-33 contributes to sepsis-induced long-term immunosuppression by expanding the regulatory T cell population. Nature Communications. 8, 14919 (2017).
- Deng, D., et al. Systematic investigation on the turning point of over-inflammation to immunosuppression in CLP mice model and their characteristics. International Immunopharmacology. 42, 49-58 (2017).
