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House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis
对小鼠气道滴注尘螨提取物和细胞因子进行炎症分析   

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Abstract

Asthma is a complex disease of the airways primarily mediated by T helper 2 cells and innate lymphoid type 2 cells ( Licona et al.,2013). Mice do not develop spontaneous asthma and therefore models have been developed for the assessment of key processes that underlie human pathology (Nial et al.,2008). Exposure to House Dust Mite (HDM) extract induces many key features of acute airway inflammation including elevated IgE levels, eosinophilia, goblet cell metaplasia, epithelial hypertrophy and airway hyper responsiveness (AHR) in response to methacholine (Hammad et al., 2009; Dullaers et al., 2012; Coquet et al., 2015). The exact dose and duration of exposure to HDM can affect the type and extent of inflammation. In our case, we start with a low sensitizing dose that is increased on challenge, while others use differing schedules or a higher antigen concentration during sensitization of mice (Hondowicz et al., 2016; Trompette et al., 2014;  Zaiss et al., 2015). We believe that using a low sensitizing dose more accurately separates the primary and secondary immune responses and reduces the possibility that HDM given during sensitization continues to fuel the immune response during challenge (Coquet et al., 2015; Plantinga et al., 2013). Here, we outline in text, pictures and video how to administer HDM extracts or cytokines via the intranasal route and briefly touch upon the subsequent analysis of inflammation in the airways [covered otherwise in ( Han et al., 2013)].

Keywords: HDM(HDM), IL-33(IL-33), Asthma(哮喘), Lavage(灌洗), Airway Inflammation(气道炎症)

Materials and Reagents

  1. 1.5 ml Eppendorf tube (Sarstedt, catalog number: 72.269.001 )
  2. 100 μM Filter (BD, catalog number: 352360 )
    Note: Currently, it is “Corning, FalconTM, catalog number: 352360”.
  3. Sterile pipette tips (Corning, catalog number: 4804 )
  4. 23 Gauge (G) sterile needle (BD, catalog number: 300800 )
  5. Polyethylene tubing PE-10 (BD, IntramedicTM, catalog number: 427401 )
  6. 1 ml syringe (BD, catalog number: 309659 )
  7. 15 ml screw cap tube conical (Corning, FalconTM, catalog number: 352096 )
  8. 50 ml screw cap tube conical (Corning, FalconTM, catalog number: 352070 )
  9. Soft wood/styrofoam board
  10. Mice
    Note: We typically utilize C57BL/6 mice of at least 4 weeks of age. Either sex can be used although it is best not to mix genders in a particular experiment.
  11. Isoflurane (Baxter, catalog number: KDG9623 )
  12. House Dust Mite (HDM) extract (Greer Laboratories, catalog number: XPB70D3A2.5 )
  13. 1x Dulbecco’s phosphate buffered saline (DPBS) (Sigma-Aldrich, catalog number: D8537 )
  14. Ethanol
  15. Recombinant cytokines [e.g., rIL-21 (gifted by Zymogenetics); rIL-25 (Biolegend, catalog number: 587302 ) or IL-33 (Biolegend, catalog number: 580502 )]
  16. Fetal calf serum (FCS), heat inactivated (Sigma-Aldrich, catalog number: F4135 )
  17. Trypan blue (Sigma-Aldrich, catalog number: 93595 )
  18. Red blood cell lysis buffer (Sigma-Aldrich, catalog number: R7757 )

Equipment

  1. Laminar flow hood
  2. Rodent anesthesia system [e.g., Univentor 400 Anesthesia Unit or Univentor 410 Anesthesia unit (Univentor, catalog number: 8323101 )]
  3. Pipette p200 (Sigma-Aldrich, catalog number: Z678333 )
  4. Spray bottle with 70% ethanol (Sigma-Aldrich, catalog number: Z560847 )
  5. Surgical scissors (e.g., Sigma-Aldrich, catalog number: Z265969 )
  6. Surgical Thread 4-0 (0.17 mm), sterile (e.g., Agnthos, catalog number: 14757 )
  7. Stainless steel forceps (e.g., Sigma-Aldrich, catalog number: Z168785 )
  8. Hemocytometer
  9. Centrifuge [e.g., Hettich Rotina 420R (Sigma-Aldrich, catalog number: Z723630 )]


    Figure 1. Anesthesia setup. A. Mice should be anesthetized by isoflurane with the rate of breathing carefully observed to ensure a sufficient degree of anesthesia without being too light or too deep. B. Example of anesthesia system in this case is the Univentor 400 with air flow set to 388 ml/min and Isoflurane concentration of 3.8%. 

Procedure

Note: All experiments using mice should be approved by the relevant animal ethics committee in accordance with both national and international guidelines.
  1. Allergen sensitization (Day 0)
    1. Freshly prepare a solution of HDM extract in DPBS at a concentration of 1 μg per 40 μl of DPBS per mouse (Figure 2 shows the schedule of administration).
      Note: Sterile DPBS and good sterile technique in a tissue culture laminar flow hood are sufficient to reconstitute HDM extracts. No filtration of the solution is performed.


      Figure 2. Schematic of HDM induced airway inflammation

    2. Anesthetize the mouse taking care to ensure the breathing rate is normal, as breathing that is either too shallow or too deep will prevent efficient uptake of HDM solution (see Video 1). A good depth of breathing should be regular and smooth [approx. 1 breath per second (see Video 2)]. At our facility, we use a Univentor 400 with airflow at 380-400 ml/min and an isoflurane concentration of between 3.6-4.0% depending on the size of the mouse (Figure 1). Typically, only 1-2 mice are anesthetized simultaneously. When mice are not anesthetized enough, you will notice that the mice will wake up too quickly, be moving their legs and fail to inhale all 40 μl. When mice are too deeply anesthetized, you will notice that their breathing rate is slow, but that they take very deep breaths (gasps). Typically, this will result in bubbles being blown when trying to administer your volume.

      Video 1. Intranasal administration of HDM

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      Video 2. Correct depth of anesthesia

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    3. Hold the mouse gently on the backside of the neck (a very loose scruff) so that the head is up and the rear legs are facing down. Administer 40 μl of 1 μg HDM solution into the nostrils for each sensitized mouse (or DPBS alone for control mice) with care taken to ensure the uptake of the whole volume.
    4. After uptake, keep the mouse in an upright position for around 5 sec and check to ensure the mouse breathes normally.
    5. The mouse can be returned to its housing cage and left in a semi-upright position to recover from anesthesia.

  2. Allergen challenge (Days 7-11)
    1. Freshly prepare a solution of HDM extract in DPBS at a concentration of 10 μg per 40 μl per mouse.
    2. Anesthetize the mouse and administer HDM as described above using 10 μg HDM in a volume of 40 μl per mouse.
    3. Following this procedure, mice are challenged with HDM on Day 7, 8, 9, 10 and 11 post sensitization.

  3. Cytokine inhalation
    1. Freshly prepare the particular cytokine at the desired concentration in a volume of 40 μl DPBS per mouse. As is the case with HDM, sterile DPBS and good sterile technique are important at this step. The precise concentration will vary with each cytokine and the response wishing to be studied. Recombinant cytokines such as IL-25 and IL-33 seem to produce robust responses even at low doses (ng range), whereas IL-21 will elicit responses at a higher dose (μg range). Figure 3 shows the schedule for a previous cytokine inhalation protocol use in Coquet et al., 2015, although others exist that may better suit your own research. Other cytokine inhalation protocols are also common (Willart et al., 2012; Kayamuro et al., 2010; Hiromura et al., 2007).


      Figure 3. Schematic of cytokine inhalation

    2. Anesthetize the mouse taking care to ensure the breathing rate is normal, as breathing that is either too shallow or too deep will prevent efficient uptake of the cytokine solution.
    3. Administer into the nostrils of each mouse as described with HDM extract.
    4. This challenge can be repeated for 3 days with mice euthanized 24 h after the last challenge and tissues collected.

  4. Bronchoalveolar lavage (BAL) (D15 with HDM or D3 following cytokine inhalation)
    Note: A similar protocol by Han and Ziegler is also available although some details do vary to that described below (10). We provide a video of the lavage for instructional purposes (see Video 3).

    Video 3. Isolation of airway cells by bronchial alveolar lavage

    To play the video, you need to install a newer version of Adobe Flash Player.

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    Note: Mice should not be sacrificed by cervical dislocation as this method can damage the trachea resulting in poor lavage of airways. Moreover, it can also lead to bleeding around the trachea.
    1. Prepare the needle to be inserted into the trachea by attaching Polyethylene PE-10 tubing so that the tubing extends from the bevel point of the needle by approximately 3 mm. Ensure there are no sharp edges were the tubing has been cut (Figure 4A). Fill a syringe with 1 ml DPBS to be used for flushing out the lungs.
    2. Euthanize the mouse by CO2 asphyxiation or an approved anesthetic overdose protocol.
    3. Gently lay the mouse on its back, on a soft wooden/styrofoam surface, stretch the limbs and fix each paw with a pin (Figure 4B).


      Figure 4. Typical equipment required for isolation of cells by lavage. A. Example of equipment used to carry out BAL extraction. 23 G needle has PE-10 plastic tubing attached extending approximately 3 mm from the bevel point of the needle. B. The mouse should be pinned by each limb and fur wet with 70% ethanol.

    4. Wet the fur with 70% ethanol and make an incision with scissors at the base of the rib cage. Continue cutting the skin to the base of the jaw taking care not to rupture any of blood vessels found in the neck (Figure 5A).
    5. Separate the skin from the neck muscles and pin back to expose the neck.
    6. Using tweezers pull the surrounding salivary glands and muscles lining the trachea taking care not to damage the trachea itself (Figure 5B).


      Figure 5. Preparation of mouse prior to collection of lavage. A. Removal of the skin up to the jaw exposing the salivary glands and neck muscles, behind which lies the trachea. B. Neck muscles, both the sternocleidomastoids and those surrounding the trachea, are removed by pulling firmly with tweezers to reveal the trachea. Care should be taken not to damage the windpipe.

    7. Once the trachea is exposed, the surgical thread (min. 4 cm in length) can be passed underneath the trachea with tweezers. Using both tweezers, a loose knot can be made by looping the thread on one of the tweezers followed by passing the other end of the thread through the newly formed loop.
      Note: The knot also helps secure the needle in place throughout the lavage procedure.
    8. A small incision is made in the trachea roughly 2/3 from where it enters the rib cage. Avoid completely severing the trachea, as this will cause difficulties in subsequent steps (see Video 3).
    9. With care, the needle with plastic tubing attached can be inserted into the trachea until resistance is felt (approximately 10-15 mm in). Be careful not to push it through the lung tissue itself. Tighten the knot by pulling the ends of the thread in opposite directions. This will seal the lung and prevent the flow of PBS out of the opening during flushing.
    10. Attach a 1 ml syringe pre-filled with 1ml DPBS to the needle inserted in the trachea. With minimal movement of the needle, the plunger can be moved down to wash the lungs and airways with DPBS. It is critical that the needle is held in position with the non-injecting hand so as to avoid lung puncture. Expansion and contraction of the lungs or rib cage should be observed as DPBS is inserted and removed. Around 80-90% of the initial volume should be recovered. This can be transferred to a 15 ml tube and the lungs flushed with a further 1 ml of DPBS. The number of flushes should be consistent between mice to ensure the accuracy of subsequent cell counts. Video 2 depicts the mouse with an open chest cavity, but this was only for instructional purposes and for viewers to observe the inflation of the lungs. The chest cavity can remain closed during lavage.
    11. Top up each BAL sample in a 15 ml tube to 6 ml with DPBS and spin down for 5 min at 300 x g at 4 °C.
      Note: If cytokines or other soluble factors are to be detected by ELISA or Luminex based assays, it is best to separate the 1st and 2nd flushes of the airways. The 1st flush can be spun down for 5 min at 300 x g at 4 °C without the addition of any volume so as not to dilute the target proteins of interest. The supernatant can then be collected and stored at -20 °C and further analysis can be carried out on the cells.
    12. Discard the supernatant and resuspend the pellet in either media or FACs buffer (DPBS/2% FCS) depending on the downstream application.
      Note: Erythrocytes can be present in the BAL wash as a result of blood vessel rupture during the protocol or due to airway inflammation. Blood vessel rupture will lead to a dark red coloration of the BAL. In the case of overt airway inflammation leading to tissue damage, BAL can have a mild straw color that fades with subsequent lavages. In either case, erythrocytes should be eliminated using a hypotonic buffer prior to further steps. Hypotonic buffers can lead to degranulation in eosinophils so this should be considered when using such solutions. Consider exsanguination of mice through an incision at the inferior vena cava (IVC) prior to performing lavage to reduce erythrocyte contamination.
    13. Cell number can be determined by haemocytometer using trypan blue to exclude dead cells. Typical numbers range from 2.5 x 105-2.0 x 106 cells per sample in inflamed lungs (Coquet et al.,2015). In naïve or PBS-treated lungs, very few cells (1-5 x 104) should be observed (Cates et al., 2004). 

Representative data



Figure 6. Flow cytometry plots of cells isolated from BAL. BAL cells from C57Bl/6 mice were collected 4 days after the last HDM challenge. Representative FSC-A/SSC-A profiles from mice exposed to HDM or a naïve control are shown in A (HDM-exposed) and B (Naïve). The presence of eosinophils in the BAL can be defined as Siglec-F+CD11c- cells and alveolar macrophages as Siglec-F+CD11c+. The difference between inflamed BAL and naïve control is evident by the increased proportion of eosinophils (typically values range from 60-90%) in the former A and their almost complete absence in the latter B.

Notes

  1. The house dust mite model of asthma can give some variability, especially in terms of the numbers of eosinophils observed between mice, therefore it is wise to set up experiments with 5 or more mice per group, whenever possible. It is critical that the sensitization of mice on day 0 run smoothly, since mice that fail to be efficiently sensitized will not develop robust eosinophilic infiltration into the airways; our model relies on effective secondary adaptive immune responses. The number of flushes between animals must be kept constant to ensure comparable cell counts. Lysis of red blood cells in the case where blood is observed in the lavage fluid will help to minimize cell count errors.
  2. With regards to cytokine instillation, the type of immune response can be altered by the dose applied and more prolonged models of cytokine administration are also common.

Acknowledgements

The authors acknowledge the services of the MTC animal facility (Karolinska Institute, Sweden). This work was supported by a Swedish Research Council Young Investigator Grant and a grant from the Swedish Cancer Society. This protocol was adapted from Coquet et al., 2015.

References

  1. Cates, E. C., Fattouh, R., Wattie, J., Inman, M. D., Goncharova, S., Coyle, A. J., Gutierrez-Ramos, J. C. and Jordana, M. (2004). Intranasal exposure of mice to house dust mite elicits allergic airway inflammation via a GM-CSF-mediated mechanism. J Immunol 173(10): 6384-6392.
  2. Coquet, J. M., Schuijs, M. J., Smyth, M. J., Deswarte, K., Beyaert, R., Braun, H., Boon, L., Karlsson Hedestam, G. B., Nutt, S. L., Hammad, H. and Lambrecht, B. N. (2015). Interleukin-21-Producing CD4(+) T Cells Promote Type 2 Immunity to House Dust Mites. Immunity 43(2): 318-330.
  3. Dullaers, M., De, B. R., Ramadani, F., Gould, H.J., Gevaert, P., Lambrecht, B. N.(2012). The who, where, and when of IgE in allergic airway disease. Journal of Allergy and Clinical Immunology. 129(3):635-45.
  4. Hammad, H., Chieppa, M., Perros, F., Willart, M. A., Germain, R. N., Lambrecht, B. N.(2009). House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells. Nat Med. 15(4):410-6.
  5. Han, H., Ziegler, S. F., Bronchoalveolar lavage and lung tissue digestion.(2013). Bio-protocol 3(16):e859.
  6. Hiromura, Y., Kishida, T., Nakano, H., Hama, T., Imanishi, J., Hisa, Y., Mazda, O. (2007). IL-21 administration into the nostril alleviates murine allergic rhinitis. The Journal of Immunology 179(10):7157-65.
  7. Hondowicz, B. D., An, D., Schenkel, J. M., Kim, K. S., Steach, H. R., Krishnamurty, A. T., Keitany, G. J., Garza, E. N., Fraser, K. A., Moon, J. J., Altemeier, W. A., Masopust, D. and Pepper, M. (2016). Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma. Immunity 44(1): 155-166.
  8. Kayamuro, H., Yoshioka, Y., Abe, Y., Arita, S., Katayama, K., Nomura, T., Yoshikawa, T., Kubota-Koketsu, R., Ikuta, K., Okamoto, S., Mori, Y., Kunisawa, J., Kiyono, H., Itoh, N., Nagano, K., Kamada, H., Tsutsumi, Y., Tsunoda, S. (2010). Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus. Journal of Virology 84(24):12703-12.
  9. Licona-Limon, P., Kim, L. K., Palm, N. W. and Flavell, R. A. (2013). TH2, allergy and group 2 innate lymphoid cells. Nat Immunol 14(6): 536-542.
  10. Nials, A. T. and Uddin, S. (2008). Mouse models of allergic asthma: acute and chronic allergen challenge. Dis Model Mech 1(4-5): 213-220.
  11. Plantinga, M., Guilliams, M., Vanheerswynghels, M., Deswarte, K., Branco-Madeira, F., Toussaint, W., Vanhoutte, L., Neyt, K., Killeen, N., Malissen, B., Hammad, H. and Lambrecht, B. N. (2013). Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. Immunity 38(2): 322-335.
  12. Trompette, A., Gollwitzer, E. S., Yadava, K., Sichelstiel, A. K., Sprenger, N., Ngom-Bru, C., Blanchard, C., Junt, T., Nicod, L. P., Harris, N. L. and Marsland, B. J. (2014). Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 20(2): 159-166.
  13. Willart, M. A., Deswarte, K., Pouliot, P., Braun, H., Beyaert, R., Lambrecht, B. N. and Hammad, H. (2012). Interleukin-1alpha controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33. J Exp Med 209(8): 1505-1517.
  14. Zaiss, M. M., Rapin, A., Lebon, L., Dubey, L. K., Mosconi, I., Sarter, K., Piersigilli, A., Menin, L., Walker, A. W., Rougemont, J., Paerewijck, O., Geldhof, P., McCoy, K.D., Macpherson, A.J., Croese, J., Giacomin, P.R., Loukas, A., Junt, T., Marsland, B. J., Harris, N. L. (2015). The Intestinal microbiota contributes to the ability of helminths to modulate allergic inflammation. Immunity 43(5):998-1010.



简介

哮喘是主要由T辅助细胞2和先天淋巴2型细胞介导的气道的复杂疾病(Licona等人,2013)。小鼠不发展自发性哮喘,因此已经开发了用于评估作为人类病理学基础的关键过程的模型(Nial等人,2008)。暴露于房尘螨(HDM)提取物诱导急性气道炎症的许多关键特征,包括升高的IgE水平,嗜酸性粒细胞增多,杯状细胞化生,上皮肥大和响应乙酰甲胆碱的气道高反应性(AHR)(Hammad等人, ,2009; Dullaers等人,2012; Coquet等人,2015)。暴露于HDM的确切剂量和持续时间可以影响炎症的类型和程度。在我们的情况下,我们从在攻击时增加的低致敏剂量开始,而其他的在小鼠致敏期间使用不同的方案或更高的抗原浓度(Hondowicz等人,2016; Trompette < et al 。,2014;  Zaiss et al 。,2015)。我们认为使用低敏感剂量更准确地分离初级和次级免疫应答,并降低致敏过程中给予的HDM继续刺激攻击期间的免疫应答的可能性(Coquet等人,2015; Plantinga et al 。,2013)。在这里,我们在文本,图片和视频中概述了如何通过鼻内途径施用HDM提取物或细胞因子,并简要地谈谈随后在气道中的炎症的分析[在Han& 。,2013)]。

关键字:HDM, IL-33, 哮喘, 灌洗, 气道炎症

材料和试剂

  1. 1.5ml Eppendorf管(Sarstedt,目录号:72.269.001)
  2. 100μM过滤器(BD,目录号:352360)
    注意:目前,"Corning,Falcon TM ,目录号:352360"。
  3. 无菌移液管吸头(Corning,目录号:4804)
  4. 23号(G)无菌针(BD,目录号:300800)
  5. 聚乙烯管PE-10(BD,Intramedic TM ,目录号:427401)
  6. 1ml注射器(BD,目录号:309659)
  7. 15ml螺旋盖管锥形(Corning,Falcon ,目录号:352096)
  8. 50ml锥形螺旋盖管(Corning,Falcon ,目录号:352070)
  9. 软木/聚苯乙烯板
  10. 小鼠
    注意:我们通常使用至少4周龄的C57BL/6小鼠。可以使用性别,但最好不要在特定实验中混合性别。
  11. 异氟烷(Baxter,目录号:KDG9623)
  12. House Dust Mite(HDM)提取物(Greer Laboratories,目录号:XPB70D3A2.5)
  13. 1×Dulbecco磷酸盐缓冲盐水(DPBS)(Sigma-Aldrich,目录号:D8537)
  14. 乙醇
  15. 重组细胞因子[例如,rIL-21(由Zymogenetics赋予); rIL-25(Biolegend,目录号:587302)或IL-33(Biolegend,目录号:580502)]
  16. 胎牛血清(FCS),热灭活(Sigma-Aldrich,目录号:F4135)
  17. 台盼蓝(Sigma-Aldrich,目录号:93595)
  18. 红细胞裂解缓冲液(Sigma-Aldrich,目录号:R7757)

设备

  1. 层流罩
  2. 啮齿动物麻醉系统[例如,Univentor 400麻醉单位或Univentor 410麻醉单位(Univentor,目录号:8323101)]
  3. Pipette p200(Sigma-Aldrich,目录号:Z678333)
  4. 喷雾瓶用70%乙醇(Sigma-Aldrich,目录号:Z560847)
  5. 外科剪刀(例如,Sigma-Aldrich,目录号:Z265969)
  6. 外科线4-0(0.17mm),无菌(例如,Agnthos,目录号:14757)
  7. 不锈钢钳(,Sigma-Aldrich,目录号:Z168785)
  8. 血细胞计数器
  9. 离心机[例如,Hettich Rotina 420R(Sigma-Aldrich,目录号:Z723630)]


    图1.麻醉设置。A.小鼠应该用异氟烷麻醉,仔细观察呼吸速率,以确保足够程度的麻醉,而不会太浅或太深。 B.在这种情况下的麻醉系统的实例是Univentor 400,空气流量设置为388ml/min,异氟烷浓度为3.8%。

程序

注意:使用小鼠的所有实验都应该由相关动物伦理委员会根据国家和国际指南。
  1. 过敏原致敏(第0天)
    1. 新鲜制备HDM提取物在DPBS中的溶液,浓度为每40μlDPBS每只小鼠1μg(图2显示给药时间表)。
      注意:在组织培养层流罩中的无菌DPBS和良好的无菌技术足以重建HDM提取物。不进行溶液的过滤。


      图2. HDM诱导的气道炎症示意图

    2. 麻醉鼠标,注意确保呼吸频率正常,因为太浅或太深的呼吸都会阻碍HDM溶液的有效吸收(见视频1)。良好的呼吸深度应该有规律和顺利[约。每秒1次呼吸(见视频2)]。在我们的工厂,我们使用Univentor 400,气流速度为380-400 ml/min,异氟烷浓度在3.6-4.0%之间,具体取决于小鼠的尺寸(图1)。通常,只有1-2只小鼠同时麻醉。当小鼠没有足够麻醉,你会注意到,老鼠会醒得太快,移动他们的腿,未能吸入所有40微升。当老鼠被太深的麻醉,你会注意到他们的呼吸速率很慢,但他们采取非常深的呼吸(喘息)。通常,这会导致在尝试管理卷时出现气泡。

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      视频1. HDM的鼻内管理
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      视频2.正确的麻醉深度
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    3. 将鼠标轻轻地握在颈部的背面(非常宽松的颈部),使头部向上,后腿向下。管理40微升1微克HDM溶液到每个致敏小鼠的鼻孔(或DPBS单独作为对照小鼠),注意确保整个体积的摄取。
    4. 吸收后,将鼠标保持在垂直位置约5秒,并检查以确保鼠标正常呼吸。
    5. 鼠标可以返回其壳体笼,并保持在半直立的位置,以从麻醉中恢复
  2. 过敏原挑战(第7-11天)
    1. 新鲜制备HDM提取物在DPBS中的溶液,浓度为10μg/40μl/小鼠
    2. 麻醉小鼠和管理HDM如上所述使用10微克HDM体积为每只小鼠40微升。
    3. 按照该程序,在致敏后第7,8,9,10和11天用HDM攻击小鼠。

  3. 细胞因子吸入
    1. 以每只小鼠40μlDPBS的体积新鲜制备所需浓度的特定细胞因子。与HDM的情况一样,无菌DPBS和良好的无菌技术在这一步骤是重要的。精确的浓度将随着每种细胞因子和希望研究的反应而变化。重组细胞因子例如IL-25和IL-33似乎即使在低剂量(ng范围)下也产生鲁棒响应,而IL-21将在更高剂量(μg范围)引发响应。图3显示了在2015年Coquet等人之前使用的细胞因子吸入方案的时间表,尽管其他的存在可以更好地适应你自己的研究。其他细胞因子吸入方案也是常见的(Willart等人,2012; Kayamuro等人,2010; Hiromura等人,2007) 。


      图3.细胞因子吸入示意图

    2. 麻醉老鼠小心注意确保呼吸频率正常,因为太浅或太深的呼吸将阻止细胞因子溶液的有效摄取。
    3. 按照HDM提取物所述,给予每只小鼠的鼻孔。
    4. 该攻击可以重复3天,在最后一次攻击后24小时对小鼠实施安乐死,并收集组织
  4. 支气管肺泡灌洗(BAL)(D15与细胞因子吸入后的HDM或D3)
    注意:也可以使用Han和Ziegler的类似协议,但一些细节不同于下面描述的(10)。我们提供灌肠的视频用于教学目的(见视频3)。

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    视频3.通过支气管肺泡灌洗隔离气道细胞
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    注意:小鼠不应该被颈椎脱臼所牺牲,因为这种方法可能损伤气管,导致气道灌洗不良。此外,它还可能导致气管周围出血。
    1. 通过连接聚乙烯PE-10管准备插入气管的针,以使管从针的斜角点延伸大约3mm。确保没有锋利的边缘,管道已被切割(图4A)。填充注射器1毫升DPBS用于冲出肺。
    2. 通过CO 2窒息或批准的麻醉剂过量方案使小鼠安乐死。
    3. 轻轻地将鼠标放在背部,软木/聚苯乙烯泡沫塑料表面,伸展四肢,并用针固定每个爪子(图4B)。


      图4.通过灌洗分离细胞所需的典型设备。 A.用于进行BAL提取的设备示例。 23 G针具有附着的PE-10塑料管,从针的倾斜点延伸约3mm。 B.鼠标应该被每个肢体固定,并用70%乙醇湿毛。

    4. 用70%的乙醇湿毛,并用剪刀在胸腔底部做一个切口。继续将皮肤切到颌骨底部,注意不要破坏颈部发现的任何血管(图5A)。
    5. 将皮肤与颈部肌肉分开,然后向后张开以暴露颈部。
    6. 使用镊子拉动周围的唾液腺和衬在气管中的肌肉,注意不要损坏气管本身(图5B)。


      图5.在收集灌洗之前制备小鼠。A.去除皮肤直到下颚,暴露唾液腺和颈部肌肉,气管位于其后面。 B.通过用镊子牢固地拉动以去除气管,去除颈部肌肉,包括胸腔乳突肌和包围气管的肌肉。注意不要损坏气管。

    7. 一旦气管暴露,外科线(长度至少4cm)可以用镊子通过气管下面。使用两个镊子,可以通过将线缠绕在镊子中的一个上,然后使线的另一端穿过新形成的环来制造松结。
      注意:该结还有助于在灌洗过程中将针固定到位。
    8. 在气管大约2/3从其进入肋骨笼的小切口。避免完全切断气管,因为这会在后续步骤中造成困难(参见视频3)。
    9. 小心,连接有塑料管的针可以插入气管,直到感觉到阻力(大约10-15mm)。小心不要将其推过肺组织本身。通过沿相反方向拉动线的末端来拧紧结。这将密封肺并防止PBS在冲洗期间流出开口。
    10. 将预填充有1ml DPBS的1ml注射器连接到插入气管的针。随着针的最小移动,柱塞可以向下移动以用DPBS洗涤肺和气道。 关键是用非注射手保持针头的位置,以避免肺穿刺。在插入和取出DPBS时,应观察肺或肋骨的扩张和收缩。应该回收约80-90%的初始体积。这可以转移到15ml管中,并用另外1ml DPBS冲洗肺。冲洗次数应该在小鼠之间保持一致,以确保随后的细胞计数的准确性。视频2描绘了具有开放胸腔的小鼠,但这仅仅是为了教学目的,并且观察者观察肺的充气。胸腔可以在灌洗期间保持关闭
    11. 将每个BAL样品在15ml管中加入到具有DPBS的6ml中并在4℃下以300×g离心5分钟。
      注意:如果通过ELISA或基于Luminex的测定法检测细胞因子或其它可溶性因子,则最好分离第1次和第2次和第2次航空公司。第一次冲洗可以在300℃下在4℃下旋转5分钟,而不添加任何体积,以便不稀释感兴趣的目标蛋白质。然后可以收集上清液并在-20℃下储存,并且可以对细胞进行进一步分析。
    12. 弃去上清液,根据下游应用,在培养基或FACs缓冲液(DPBS/2%FCS)中重悬沉淀。
      注意:由于方案期间血管破裂或由于气道炎症,红细胞可存在于BAL洗涤液中。血管破裂将导致BAL的深红色。在明显的气道炎症导致组织损伤的情况下,BAL可以具有温和的稻草颜色,随着后续的灌溉而褪色。在任一情况下,在进一步的步骤之前,应使用低渗缓冲液消除红细胞。低渗缓冲液可导致嗜酸性粒细胞脱颗粒,因此在使用此类溶液时应考虑使用。在进行灌洗以减少红细胞污染之前,考虑通过在下腔静脉(IVC)切口对小鼠放血。
    13. 细胞数可以通过使用台盼蓝排除死细胞的血细胞计数器来测定。在发炎肺中,典型数量范围为每个样品2.5×10 5至2.0×10 6个细胞(Coquet等人,2015)。在初始或PBS处理的肺中,应当观察到非常少的细胞(1-5×10 4个)(Cates等人,2004)。

代表数据



图6.从BAL分离的细胞的流式细胞术图。在最后一次HDM攻击后4天收集来自C57Bl/6小鼠的BAL细胞。来自暴露于HDM或初始对照的小鼠的代表性FSC-A/SSC-A概况显示在A(暴露于HDM的)和B(NaIve)中。 BAL中嗜酸性粒细胞的存在可定义为Siglec-F sup + CD11c - 细胞和肺泡巨噬细胞,如Siglec-F sup + CD11c + 。发炎的BAL和初始对照之间的差异通过前A中嗜酸性粒细胞的比例增加(通常值在60-90%之间)以及在后者B中几乎完全不存在而明显。

笔记

  1. 哮喘的房尘螨模型可以给出一些变异性,特别是在小鼠之间观察到的嗜酸性粒细胞数目方面,因此,如果可能,建立每组5只或更多只小鼠的实验是明智的。至关重要的是,在第0天小鼠的致敏平稳地进行,因为未能有效致敏的小鼠不会发展强烈的嗜酸粒细胞浸润到气道中;我们的模型依赖于有效的次级适应性免疫应答。动物之间的冲洗次数必须保持恒定以确保相当的细胞计数。在灌洗液中观察到血液的情况下,红细胞的溶解有助于使细胞计数错误最小化。
  2. 关于细胞因子滴注,免疫应答的类型可以通过施用的剂量改变,并且更长时间的细胞因子施用模型也是常见的。

致谢

作者承认MTC动物设施(瑞典Karolinska研究所)的服务。这项工作得到了瑞典研究委员会青年研究员奖学金和瑞典癌症协会的资助。该协议改编自Coquet等人。 2015。

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引用:Tibbitt, C. and Coquet, J. M. (2016). House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis. Bio-protocol 6(14): e1875. DOI: 10.21769/BioProtoc.1875.
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