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Jan 2020
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Murine Acute Pneumonia Model of Pseudomonas aeruginosa Lung Infection
铜绿假单胞菌肺部感染的小鼠急性肺炎模型   

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Abstract

Animal infection models play significant roles in studying bacterial pathogenic mechanisms, host pathogen interaction as well as evaluating drug and vaccine efficacies. We have been utilizing an acute pneumonia model to study bacterial colonization in lungs and assess virulence to the host by determination of bacterial loads and survival assays, as well as examine the bacterial gene expression in vivo. Additionally, the host's immune response to the pathogen can be explored through this infection model.

Background

Acute pneumonia is defined as an acute infection of the lungs by microbial pathogens. Hospital-acquired pneumonia, in particular, is often caused by multi-drug resistant pathogens and is difficult to treat. Common bacteria that cause pneumonia are Pseudomonas aeruginosa, Streptococcus pneumoniae, group A Streptococcus, Acinetobacter baumannii, Klebsiella pneumoniae,Staphylococcus aureus and Mycoplasma pneumonia (Ravi Kumar et al., 2018 ). To develop effective prevention and treatment strategies, it is critical to understand how bacteria sense and adapt to host in vivo environment and counteract host immune clearance. Currently, there are three types of pneumonia models: One hit acute pneumonia model, Ventilator-associated pneumonia model, and Agar-bead pneumonia model (Bielen et al., 2017). The model mentioned in this article belongs to the one hit acute pneumonia model. There are two methods for the infection, one is to inject bacterial suspension directly into the trachea or lungs. Here, we reported the other method in which bacteria are inoculated through nostrils, which is relatively easy to perform.


P. aeruginosa is a Gram negative opportunistic pathogenic bacterium that causes a variety of acute and chronic infections in human. It is one of the major pathogens that cause lung infections in patients with cystic fibrosis, chronic obstructive pulmonary disease and compromised immunity (Talwalkar and Murray, 2016). To study the virulence factors and gene expression of P. aeruginosa in host, we utilized gene expression (Wu et al., 2012). Additionally, this model is suitable for examining the immune response of host (Tian et al., 2019). We have also applied this model to study the infections by K. pneumoniae and A. baumannii.

Materials and Reagents

  1. Plastic EP tubes (1.5 ml, 2 ml, 4 ml, G SERVICEBIO, catalog numbers: EP-150-M , EP-200-M , EP-400-M )

  2. Syringe (1 ml, Anhui JINHUAN Co., Ltd, catalog number: 14001 )

  3. Pipette tips (20, 200, and 1,000 μl clear tips, Tianjin HUAXIN Co., Ltd, catalog number: HY130 )

  4. Six-week-old female BALB/c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd)

  5. Bacterial culture (P. aeruginosa wild type strain PA14 [ Liberati et al., 2006 ])

  6. 75% ethanol (Tianjin Chemicals Co., Ltd, catalog number: A1022 ). Diluent: double distilled water.

  7. 7.5% chloral hydrate (Tianjin Chemicals Co., Ltd, catalog number: 2223 ). Diluent: double distilled water

  8. 0.05 M Ethylenediaminetetraacetic acid (EDTA) disodium salt solution, pH 8.0 (Beijing Dingguo Biotech Co., Ltd, catalog number: LE568-1KG ). Diluent: double distilled water

  9. Carbon dioxide (Tianjin Baisida Co., Ltd, catalog number: N14 )

  10. 1% sterilized peptone (Oxiod Co., Ltd, catalog number: LP0037B ). Diluent: double distilled water.

  11. Trizol (BioFroxx Co., Ltd, catalog number: 15596026 )

  12. Isopropanol (Tianjin Chemicals Co., Ltd, catalog number: A1079 )

  13. Chloroform (Tianjin Chemicals Co., Ltd, catalog number: A1008 )

  14. Phosphate-buffered saline (PBS), pH 7.2 (see Recipes)

    NaCl (Invitrogen, catalog number: 24740011 )

    KCl (Aladdin, catalog number: P112134-12×500g)

    Na2HPO4·7H2O (Tianjin Chemicals catalog number: W2007)

    KH2PO4 (Macklin, catalog number: M822553-100g)

  15. LB broth (see Recipes)

    NaCl (Invitrogen, catalog number: 24740011 )

    Tryptone (Abbexa, catalog number: abx082523-500g )

    Yeast extract (Abbexa, catalog number: abx082370-500g )

Equipment

  1. Pipette (GILSON, catalog number: F167350 )

  2. Incubator (Shanghai Yihen Co., Ltd, catalog number: 2005A )

  3. -80 °C freezer

  4. Centrifuge (Eppendorf, model: 5810 )

  5. Vortex (BioExpress, GeneMate, catalog number: S-3200-1 )

  6. Spectrophotometer (BIORAD, model: Smart Spec plus )

  7. Surgical scissors

  8. Fine Forceps

  9. I.V. Catheter (BD, model: Angiocath 1.3 x 48 mm )

  10. Electronic homogenizer (Shanghai JIANLI Co., Ltd, catalog number: e76 )

Software

  1. GraphPad

Procedure

Time line of the procedures (Figure 1):



Figure 1. Time line of the procedures

  1. Infection

    1. Specific pathogen free (SPF) six-week-old female BALB/c mice are purchased from Beijing Vital River. After arrival, the mice are kept in animal facility for three to five days for acclimatization ( Wu et al., 2012 ).

    2. Streak the P. aeruginosa reference stain PA14 stored at -80 °C on a LB plate. Incubate the plate at 37 °C overnight.

    3. Inoculate a single colony on the plate into LB liquid medium and culture the bacteria at 37 °C for 16 h with shaking at 200 rpm.

    4. Dilute the bacterial culture 1:100 into 3 ml fresh LB broth.

    5. Culture the bacteria at 37 °C with shaking at 200 rpm for about 3 h until the OD600 reached 1.0.

    6. Collect 1 ml of the bacteria by centrifugation at 10,000 x g for 1 min at room temperature in a 1.5 ml EP tube. Discard the supernatant and resuspend the bacteria with 1 ml PBS at room temperature. Centrifuge the bacteria at 10,000 x g for 1 min at room temperature. Resuspend the bacteria with 1 ml PBS.

    7. Dilute the bacteria to the desired concentration in PBS (e.g., 1 x 109 CFU per ml of PA14).

    8. Anesthetize the mouse with an intraperitoneal injection of 7.5% chloral hydrate (100 µl per 20 g mouse).

    9. Flip the mouse onto its back and place it in the palm of your hand.

    10. Press the toe lightly to make sure the mouse has been anesthetized. Anesthetized mouse does not respond to the pressing.

    11. Hold the mouse’s body at a 45-degree angle.

    12. Place thumb lightly on the mouse's jaw to force the mouse to breathe through nose (Figure 2A).

    13. Take 10 μl of the bacteria suspension or PBS as control with a 20 μl pipette. Put the tip close to one of the nostrils of the mouse. Slowly pipette a drop of bacteria suspension onto the nostril. After the liquid is inhaled into the nostril, pipette the next drop until all the 10 μl is delivered. Repeat the process with another 10 μl of the bacteria suspension on the other nostril (Figure 2B, Video 1). During the process, pauses might be needed for restoring the normal breathing of the mouse.

    14. Lay the mouse on its back in the cage.



      Figure 2. Key steps of inoculation. A. Mouse on its back with jaw being pressed lightly. B. Pipette bacteria suspension onto the nostril.



      Video 1. An illustration of the process of the murine acute pneumonia model of P. aeruginosa


    15. Determination of bacterial load

      1. At desired time point post infection (e.g., 12 h), fill the closed container with carbon dioxide to sacrifice the mice by asphyxia.

      2. Spread the forelimbs of the mouse and affix to a working surface with syringe needles.

      3. Using fine scissors, cut the skin from the intersection of the abdomen and the chest to the leading edge of chest to open the chest cavity. Then cut the ribs on each side to expose the lung.

      4. Isolate the lung with a scissor and immerse the lung in 1 ml 1% sterilized peptone in a 4 ml EP tube (Steps B2-B4 are shown in Video 2).


        Video 2. Lung isolating for determination of the colonization of P. aeruginosa in the murine acute pneumonia model


      5. Grind the lung with an electronic homogenizer until it is completely homogenized (no particles larger than 1 mm in diameter). For our homogenizer and mice, the homogenization parameters of 65 HZ homogenize 20 s for 10 times with 10-second intervals work. If the readers uses a different instrument type or mouse type/weight, we recommend that new parameters should be tested to achieve completed homogenization with the shortest homogenization time.

      6. Centrifuge the homogenate at 10,000 x g for 1 min at room temperature and transfer the supernatant to a new EP tube.

      7. Add 50 μl of the supernatant to 200 μl LB in a 1.5 ml EP tube and mix well by vortex for 10 s. Transfer 50 μl of the acquired diluent to another 200 μl LB, repeat the dilution four to five times.

      8. Spot 15 μl of the last 3 diluent onto square LB plates and immediately tilt the plate to a 45 degree angle until the fluid flows about 2-3 cm long. Incubate the plates at 37 °C for 12 h before colony counting.

      9. After incubate for 12 h, plates should look like the Figure 3 shown. All colonies in each column are counted to calculate the bacteria load in an individual lung. For example, colonies in red box are counted as 85. If the sample is diluted 4 times, the colonization in this lung is 46 x 54 x (1,000/15) =1916667 CFU/lung.

      10. Analyze the results with the GraphPad software (Figure 4A). Every group should contain at least 6 mice.



        Figure 3. Illustration of plate for counting

      11. Survival assays

        1. After inoculation of the bacteria, check the mice at least twice a day, preferably in the morning and evening, record the survival number.

        2. Monitor the mice for 5 days post infection, make a survival curve by the GraphPad software (Figure 4B). Every group should contain at least 8 mice.



          Figure 4. Data presentation of (A) Bacterial load determination and (B) Survival assays. GraphPad is used for data analyses. Bars represent medians, and error bars represent standard errors (SEM). P value < 0.05 compared to wild type PA14 by the Kruskal-Wallis with Dunn’s multiple comparison test. PA14: wild type P. aeruginosa, PA14ΔpopB: PA14 with the popB gene deleted, PBS: phosphate buffer saline buffer as a negative control, ND: not detected.


        3. RNA isolation from bacteria in bronchoalveolar lavage fluid (BALF)

          1. Collection of the BALF

            1. At desired time point (6 h for PA14), sacrifice the mouse with CO2. Fix the mouse on a working surface as described in Procedure B.

            2. Using fine scissors, cut from the chest to the base of the intersection of the chin and neck. Open the chest to expose the lung and the trachea.

            3. Carefully clear the tissue around the trachea by using a fine forceps until the trachea is exposed, be careful not to cut any blood vessel.

            4. With the mouse head toward you, carefully insert an I.V. catheter into trachea from the top down (Figure 5A, Video 3).

            5. Once the I.V. Catheter is half an inch into the trachea, carefully pull out the metal part of the I.V. Catheter.

            6. Connect the syringe with 1 ml of 0.05 mM EDTA in PBS to the end of the I.V. Catheter.

            7. Carefully and slowly inject the solution into the lungs. The lungs will expand as a result, then wait 1 min prior to drawing back (Figure 5B, Video 3).

            8. Collect the liquid to the 2 ml tube.

            9. Repeat Steps D1e-D1g once with another 1 ml of 0.05 mM EDTA in PBS solution.

            10. Keep the BALF on ice until all the mice have been finished.



              Figure 5.Key steps of collection of bronchoalveolar lavage fluid (BALF). A. Insertion of an I.V. Catheter into trachea. B. Injection of the solution into lung through the trachea.


              Video 3. Lung lavage for mRNA isolation in the the murine acute pneumonia model


          2. RNA extraction

            1. Take 20 μl of the BALF for bacteria counting by serial dilution and plating (refer to Procedure B).

            2. Collect the remaining bacteria in the BALF by centrifugation at 10,000 x g for 1 min at 4 °C in a 1.5 ml EP tube.

            3. Immediately resuspend the bacteria with 1 ml Trizol for RNA isolation.

            4. Incubate the homogenized sample for 5 min at room temperature to permit complete dissociation of the nucleoprotein complex.

            5. Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent used for homogenization. Cap the tube securely.

            6. Shake tube vigorously by hand for 15 s.

            7. Incubate for 2-3 min at room temperature.

            8. Centrifuge the sample at 12,000 x g for 15 min at 4 °C.

              Note: The solution are separated into a lower red phenol-chloroform phase (protein), an interphase (DNA), and a colorless upper aqueous phase (RNA). RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume.

            9. Take the aqueous phase of the sample avoid drawing any of the interphase or organic phase into the pipette.

            10. Place the aqueous phase into a new tube.

            11. Add 0.5 ml of 100% isopropanol to the aqueous phase, per 1 ml of TRIzol Reagent used for homogenization. Mix vigorously.

            12. Incubate at room temperature for 10 min.

            13. Centrifuge at 12,000 x g for 10 min at 4 °C.

            14. Carefully remove the supernatant.

            15. Wash the precipitate twice with 0.5 ml 75% ethanol.

            16. Put the tubes at room-temperature for 5-10 min uncovering the lid.

            17. Add 25-50 μl H2O to the precipitate and incubate at room temperature to dissolve the RNA.

            18. The purified RNA can be used for q-RT PCR or RNA sequencing.

        Recipes

        1. Phosphate-buffered saline (PBS), pH 7.2

          137 mM NaCl

          2.7 mM KCl

          4.3 mM Na2HPO4·7H2O

          1.4 mM KH2PO4

        2. LB broth

          NaCl 5 g/L

          Tryptone 5 g/L

          Yeast extract 10 g/L

        Acknowledgments

        We thank members of the laboratory for improving on this technique over the years. This work was supported by the National Science Foundation of China (31900115).

        Competing interests

        We declare no conflict of interest or competing interests.

        All animal studies have been approved by the institutional animal care and use committee of the College of Life Sciences of Nankai University (permit number NK-04-2012).

        Ethics

        All animal studies have been approved by the institutional animal care and use committee of the College of Life Sciences of Nankai University (permit number NK-04-2012).

        References

        1. Bielen, K., Jongers, B., Malhotra-Kumar, S., Jorens, P. G., Goossens, H. and Kumar-Singh, S. (2017). Animal models of hospital-acquired pneumonia: current practices and future perspectives. Ann Transl Med 5(6): 132.
        2. Liberati, N. T., Urbach, J. M., Miyata, S., Lee, D. G., Drenkard, E., Wu, G., Villanueva, J., Wei, T. and Ausubel, F. M. (2006). An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103(8): 2833-2838.
        3. Ravi Kumar, S., Paudel, S., Ghimire, L., Bergeron, S., Cai, S., Zemans, R. L., Downey, G. P. and Jeyaseelan, S. (2018). Emerging Roles of Inflammasomes in Acute Pneumonia. Am J Respir Crit Care Med 197(2): 160-171.
        4. Talwalkar, J. S. and Murray, T. S. (2016). The Approach to Pseudomonas aeruginosa in Cystic Fibrosis. Clin Chest Med 37(1): 69-81.
        5. Tian, Z., Cheng, S., Xia, B., Jin, Y., Bai, F., Cheng, Z., Jin, S., Liu, X. and Wu, W. (2019). Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages. Infect Immun 87(12): e00695-19.
        6. Wu, W., Huang, J., Duan, B., Traficante, D. C., Hong, H., Risech, M., Lory, S. and Priebe, G. P. (2012). Th17-stimulating protein vaccines confer protection against Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med 186(5): 420-427.

简介

[摘要]动物感染模型在研究细菌致病机理,宿主病原体相互作用以及评估药物和疫苗效力方面起着重要作用。我们一直在利用急性肺炎模型来研究肺中的细菌定植并通过确定细菌载量和存活率分析来评估对宿主的毒力,以及检查体内细菌基因的表达。另外,可以通过这种感染模型探索宿主对病原体的免疫反应。


[背景]急性肺炎是指微生物病原体对肺部的急性感染。特别是医院获得性肺炎,通常是由耐多药病原体引起的,难以治疗。引起肺炎的常见细菌是铜绿假单胞菌,肺炎链球菌,A组链球菌,鲍曼不动杆菌,肺炎克雷伯菌,金黄色葡萄球菌和肺炎支原体(Ravi Kumar等,2018)。要制定有效的预防和治疗策略,了解细菌如何感测和适应宿主体内环境并抵消宿主免疫清除至关重要。目前,Ť这里有3吨肺炎模型YPES :一重击急性肺炎模型,呼吸机相关性肺炎模型和琼脂珠肺炎模型(比伦等人。,2017)。本文提到的模型属于单发性急性肺炎模型。有两种感染方法,一种是将细菌悬浮液直接注入气管或肺部。在这里,我们报道了另一种通过鼻孔接种细菌的方法,该方法相对容易实施。

铜绿假单胞菌是革兰氏阴性机会病原性细菌,可引起人类各种急性和慢性感染。它是引起囊性纤维化,慢性阻塞性肺疾病和免疫力下降的患者肺部感染的主要病原体之一(Talwalkar和Murray,2016)。为了研究宿主中铜绿假单胞菌的毒力因子和基因表达,我们利用基因表达(Wu等人,2012)。此外,该模型适用于检查宿主的免疫反应(Tian等人2019)。我们也应用这个模型来研究由感染肺炎克雷伯氏菌和A.杆菌。

材料和试剂


 1 P拉斯蒂克EP管(1.5毫升,加入2ml,4毫升,ģSERVICEBIO ,产品目录号:EP-150-M,EP-200-M,EP-400-M)
2 注射器(1 ml ,安徽金环股份有限公司,目录号:14001)
3 移液管头(20,200,和1000个微升清楚提示,天津HUAXIN有限公司,目录号:HY130)
4 六周大的BALB / c雌性小鼠(北京生命河实验动物技术有限公司)
5 细菌培养(铜绿假单胞菌野生型菌株PA14 [ Liberati等,2006 ] )
6 75%的乙醇(天津化学小号有限公司,目录号:A1022) 。稀释剂:双蒸馏水。
7 7.5%水合氯醛(天津化工有限公司,目录号:2223)。稀释剂:双蒸馏水
8 0.05 M乙二胺四乙酸(EDTA)二钠盐溶液,pH 8.0 (北京鼎国生物技术有限公司,北京目录号: LE568-1KG)。稀释剂:双蒸馏水
9 二氧化碳(天津百事达有限公司,目录号:N14)
10 1%灭菌的蛋白ept (Oxiod Co.,Ltd,目录号:LP0037B)。稀释剂:双蒸馏水。
11 Trizol (BioFroxx Co.,Ltd,目录号:15596026)
12 Isopropanol (天津化工小号有限公司,目录号:A1079)
13 Ç hloroform (天津化工小号有限公司,目录号:A1008)
14 磷酸盐缓冲液(PBS),pH 7.2(请参阅食谱)
NaCl(Invitrogen,货号:24740011)


 氯化钾(阿拉丁,目录号:P112134-12×500g)


Na 2 HPO 4 ·7H 2 O(天津化工目录号:W2007)


KH 2 PO 4 (Macklin,目录号:M822553-100g)


15 LB汤(请参阅食谱)
NaCl(Invitrogen,货号:24740011)


胰蛋白((Abbexa ,目录号:abx082523-500g)


酵母提取物(Abbexa ,目录号:abx082370-500g)


 


设备


1 移液器(GILSON,目录号:F 167350)
2 孵化器(上海亿亨有限公司,目录号:2005A)
3 -80 °C冷冻室
4 C离心机(Eppendorf,型号:5810)
5 涡流(BioExpress ,GeneMate ,目录号:S-3200-1)
6 小号pectrophotometer (BIORAD,型号:智能规格加)
7 手术剪刀
8 精细钳
9 静脉导管(BD ,型号:Angiocath 1.3 x 48 mm )
10 电子均质机(上海健力有限公司,目录号:e76)
 


软件


 1 绘图板
 


程序


 


过程s的时间线(图1):


 






图1. Ti me行的程序


 


A 感染
1 无特异性病原体(SPF)的六周龄雌性BALB / c小鼠购自北京生命河。到达后,将小鼠在动物设施中饲养三至五天以使其适应环境(Wu等人,2012)。
2 条纹的P.一个eruginosa基准污损PA14储存在-80 LB板上的℃。将板在37 °C下孵育过夜。
3 将平板上的单个菌落接种到LB液体培养基中,并在200 rpm摇动下于37 °C培养细菌16 h。
4 将细菌培养液1:100稀释到3 ml新鲜的LB肉汤中。
5 在37 °C下以2 00 rpm摇动培养细菌约3 h,直到OD 600达到1.0。
6 通过在1.5 ml EP管中于室温以10,000 xg离心1分钟收集1 ml细菌。弃去上清液,并在室温下用1 ml PBS重悬细菌。在室温下以10,000 xg离心细菌1分钟。用1 ml PBS重悬细菌。
7 在PBS中将细菌稀释至所需浓度(例如,每毫升PA14 1 x 10 9 CFU)。             
8 用7.5%的水合氯醛腹腔注射麻醉小鼠(每20 g小鼠100 µl )。
9 将鼠标翻转到其背面,然后将其放在手掌中。
10 轻轻按下脚趾以确保鼠标已被麻醉。一个nesthetized鼠标不能压制回应。
11 以45度角握住鼠标的身体。
12 将拇指轻轻放在鼠标的下巴上,以迫使鼠标通过鼻子呼吸(图2A)。
13 取10微升的菌悬液的或PBS作为对照用20微升移液管。将笔尖靠近鼠标的鼻孔之一。慢慢将一滴细菌悬浮液吸到鼻孔上。将液体吸入鼻孔后,用移液管吸取下一个液滴,直到所有10μl都输送完为止。用另一个鼻孔上的另一个10μl细菌悬液重复该过程(图2B ,视频1 )。在此过程中,可能需要暂停一下以恢复鼠标的正常呼吸。
14 将鼠标放在笼子里。
 






图2.接种的关键步骤。A.轻轻按下颚,将鼠标放在其背面。B.将细菌悬浮液吸到鼻孔上。


 






视频1.铜绿假单胞菌小鼠急性肺炎模型的过程图解


B 细菌含量的测定
1 在感染后的理想时间点(例如12小时),用二氧化碳填充密闭容器,以窒息杀死小鼠。
2 展开鼠标的前肢,并用注射器针头固定在工作表面上。
3 用小剪刀从腹部和胸部的交点到胸部的前缘切开皮肤,以打开胸腔。然后切开两侧的肋骨以露出肺部。
4 用剪刀隔离肺部, 将肺部浸入4 ml EP管中的1 ml 1%无菌蛋白p中(视频2中显示的步骤B2-B4 )。
 






视频2.肺隔离测定鼠急性肺炎模型中铜绿假单胞菌的定殖


 


5 用电子均质机研磨肺部,直至其完全均质(没有直径大于1毫米的颗粒)。对于我们的均质器和小鼠,65 HZ的均质参数以10秒的间隔均质20 s连续10次。如果读者使用不同的仪器类型或鼠标类型/重量,我们建议应测试新参数,以在最短的均质化时间内实现完全的d均质化。
6 离心机Ë的匀浆以10,000 ×g离心1分钟,在室温下和吨转让(BOT)的上清液到新的EP管。
7 加入50微升上清液至200微升LB在1.5ml EP管中,并通过涡旋10秒拌匀。转移50微升所获取的稀释剂的另一个200微升LB,重复稀释四个至五个次。
8 斑点1 5微升的最后的3稀释剂到正方形的LB平板,并立即倾斜板成45度角,直到约2-3厘米长的流体流动。孵育所述板在37 ℃下为12菌落计数之前小时。
9 后孵育12小时,将板应该像示出的图3。计算每列中的所有菌落,以计算单个肺中的细菌载量。例如,红色框中的菌落计数为85。如果将样品稀释4倍,则该肺中的菌落为46 x 5 4 x (1,000 / 15)= 1916667 CFU /肺。
10 使用GraphPad软件分析结果(图4 A)。每组至少应包含6只小鼠。
 






图3 。我llustration的平板计数


 


C 生存分析
1 接种细菌后,每天至少两次检查小鼠,最好在早晨和晚上,记录存活数。
2 莫尼TOR小鼠5天感染后,使存活曲线由的GraphPad软件(图4 B) 。每组至少应包含8只小鼠。
 






FIGUR ê 4 。(A)细菌负荷测定和(B)生存分析的数据表示。GraphPad用于数据分析。条形表示中位数,误差条表示标准误差(SEM)。通过Dunn多重比较检验,Kruskal-Wallis与野生型PA14相比,P值<0.05。PA14:野生型铜绿假单胞菌,PA14Δ POPB:PA14与POPB基因缺失,PBS:磷酸盐缓冲盐水缓冲液作为阴性对照,ND:未检测到。






D从支气管肺泡灌洗液(BALF)中的细菌中提取RNA
1 BALF的集合
a 在所需的时间点(对于PA14为6小时),用CO 2处死小鼠。按照步骤B所述,将鼠标固定在工作表面上。
b 用细剪刀从胸部切到下巴与颈部交点的底部。打开胸部,露出肺和气管。
c 仔细清除周围的组织 气管用一个细镊子,直到气管暴露,要小心,不要削减任何血管。
d 将鼠标头朝您的方向小心插入n从上向下将IV导管插入气管(图5 A ,视频3)。
e 将静脉导管插入气管半英寸后,小心地拉出静脉导管的金属部分。
f 将注射器与1 ml溶于PBS的0.05 mM EDTA连接到静脉导管的末端。
g 小心缓慢地将溶液注入肺部。结果,肺部将扩张,然后等待1分钟再抽回(图5 B ,视频3 )。
h 将液体收集到2 ml管中。
i 用另外1 ml PBS溶液中的0.05 mM EDTA重复步骤D1e-D1g 。
j 将BALF放在冰上,直到所有老鼠都吃完为止。
 






Figu重新5 。收集支气管肺泡灌洗液(BALF)的关键步骤。A.将静脉导管插入气管。B.通过气管将溶液注入肺。


 






视频3.在鼠急性肺炎模型中进行肺灌洗以分离mRNA


 


2 RNA提取
a 取20微升的BALF的细菌通过连续稀释和电镀计数(参见步骤B) 。
b COLLEC吨在通过离心BALF以10,000rpm的剩余的细菌XG 1分钟,在4 ℃下在1.5ml的EP管。
c 立即用1 ml Trizol重悬细菌以进行RNA分离。
d 在室温下将均质样品孵育5分钟,以使核蛋白复合物完全解离。
e 每1 ml用于均质化的TRIzol试剂添加0.2 ml氯仿。将管盖紧。
f 用手剧烈摇动试管15 s。
j 在室温下孵育2-3分钟。
h 在4 °C下以12,000 xg离心样品15分钟。
注意:溶液被分为较低的红色酚氯仿相(蛋白质),中间相(DNA)和无色的较高水相(RNA)。RNA仅保留在水相中。上层水相占总体积的〜50%。


i 取样品的水相,避免将任何中间相或有机相吸入移液管。
j 将水相放入新管中。
k 每1 ml用于均质化的TRIzol试剂,向水相中添加0.5 ml的100%异丙醇。剧烈混合。
l 在室温下孵育10分钟。
m 在4 °C下以12,000 xg离心10分钟。
n 小心除去上清液。
o 用0.5 ml 75%乙醇洗涤沉淀两次。
p 将试管在室温下放置5-10分钟,露出盖子。
q 25-50添加微升ħ 2 O到沉淀物,并在室温下孵育以溶解RNA。
r 纯化的RNA可用于q-RT PCR或RNA测序。
菜谱


1 磷酸盐缓冲液(PBS),pH 7.2
137毫米氯化钠


2.7毫米氯化钾


4.3 mM Na 2 HPO 4 ·7H 2 O


1.4毫米KH 2 PO 4


2 LB肉汤
氯化钠5克/升


胰蛋白5 5克/升


酵母提取物10克/升


 


致谢


 


我们感谢实验室成员多年来对这种技术的改进。这项工作得到了中国国家科学基金(31900115)的支持。


 


利益争夺


 


我们声明没有利益冲突或利益冲突。


 


伦理


 


所有动物研究均已获得南开大学生命科学学院动物保护与使用委员会的批准(许可号NK-04-2012)。


 


参考文献


 1 Bielen ,K.,Jongers ,B.,Malhotra-Kumar,S.,Jorens ,PG,Goossens ,H.和Kumar-Singh,S.(2017)。医院获得性肺炎的动物模型:当前的实践和未来的观点。Ann Transl Med 5(6):132。
2 新罕布什尔州的Liberati ,Urbach ,JM,Miyata,S.,Lee,DG,Drenkard ,E.,Wu G.,Villanueva,J.,Wei.T 。和Ausubel ,FM(2006)。铜绿假单胞菌菌株PA14转座子插入突变体的有序非冗余文库。PROC国家科科学院科学USA 103(8):2833至2838年。
3 Ravi Kumar,S.,Paudel ,S.,Ghimire,L.,Bergeron,S.,Cai,S.,Zemans ,RL,Downey,GP和Jeyaseelan ,S.(2018)。炎症小体在急性肺炎中的新兴作用。Am J呼吸急症护理医学197(2):160-171。
4 Talwalkar,JS和Murray,TS(2016)。囊性纤维化中铜绿假单胞菌的治疗方法。临床胸部医学杂志37(1):69-81。
5 田Z.,程S.,夏B.,金Y.,白F.,程Z.,金S.,刘X. 铜绿假单胞菌ExsA调节金属蛋白酶ImpA,抑制巨噬细胞的吞噬作用。 感染免疫87(12):e00695-19。
6 Wu,W.,Huang,J.,Duan B.,Traficante ,DC,Hong,H.,Risech ,M.,Lory,S. and Priebe,GP(2012)。Th17刺激性蛋白疫苗可预防铜绿假单胞菌肺炎。Am J呼吸急症护理医学186(5):420-427。
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引用:Pan, X. and Wu, W. (2020). Murine Acute Pneumonia Model of Pseudomonas aeruginosa Lung Infection. Bio-protocol 10(21): e3805. DOI: 10.21769/BioProtoc.3805.
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