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Instillation of Particulate Suspensions to the Lungs
将颗粒悬浮物滴注至肺部   

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Abstract

Inhaled fine particulates are thought to cause chronic pulmonary inflammation through the deposition of particulates into the lungs. To investigate the effect of fine particulates on the lungs, instillation of suspension of particulates into the lungs is required. This protocol describes direct injection of suspensions of fine particulates into the airway. We also show examples of typical lung immune responses after particulate administration.

Keywords: Particulates(颗粒), Lung inflammation(肺炎), Pulmonary aspiration(肺部异物吸入), Intranasal administration(鼻内给药)

Background

Recently, many studies have demonstrated that particulate pollutants such as diesel exhaust particles, sand dusts and particulate matter 2.5 (PM 2.5), are involved in chronic pulmonary inflammation leading to lung cancer or allergic asthma. Epidemiological analysis revealed that increased particulate air pollution is related to increased asthma hospitalization. In general, upon inhalation, fine particles, such as PM 2.5, are known to reach deep into the lungs. Instillation of suspensions of particulates into the lungs has been widely used for understanding pulmonary inflammation induced by deposited particulates (Morimoto et al., 2016).

Materials and Reagents

  1. Pipet tip for gel loading (Vertex-GL 200 μl gel-loading tip) (SSIbio, catalog number: 4837-S0S )
  2. Parafilm
  3. 1 ml sterile syringe (without needle) (TERUMO, catalog number: SS-01T )
  4. Mice (C57BL/6, BALB/c etc.)
    Note: For training, bigger mice (aged male mice) are better.
  5. Alhydrogel (InvivoGen, catalog number: vac-alu-250 ) as particulate for instillation
    Note: Alhydrogel (alum) is suspended in dH2O. For instillation of alum into the lungs, buffer exchange is required. Centrifuge a suspension of alum in a microtube at 2,000 x g for 2 min. Discard supernatant (H2O) and add an equal volume of saline or PBS. Mix well and centrifuge again. Repeat this procedure five times to exchange H2O to saline or PBS. Finally, adjust the concentration of alum to 2 mg/ml in saline and use for instillation.
  6. Anesthetic (ketamine/xylazine mixture)
    Note: 10 ml of Ketalar (Ketamine, 50 mg/ml, Daiichi Sankyo Co. Ltd., Tokyo, Japan) is mixed with 2.2 ml of Selactar (Xylazine, 20 mg/ml, Bayer HealthCare Ltd., Tokyo, Japan). Anesthetize mice with 50 to 75 μl of ketamine/xylazine mixture by s.c. injection into the back.

Equipment

  1. Ear pick earwax remover with light (Japan Smile Kids)
    Note: Before use, silicone rubber at the tip of ear pick will be removed (Figure 1).
  2. Stainless steel micro spatula (Figure 1) (ASONE, catalog number: 9-891-02 )
    Note: This is used as a tongue depressor, so small size is better.


    Figure 1. Ear puck with light and spatula

  3. Platform for instillation into the lungs. As shown in Figure 2, stretch a string across the wooden (cork) board (approximate size: 20 x 30 cm)


    Figure 2. Platform for instillation into the lungs

  4. Injector for instillation (Figure 3)
  5. FACS analyzer


    Figure 3. Injector for instillation. A. Fix a thin pipet tip to the ear pick by Parafilm. B. Attach a syringe to the pipet tip. C. If needed, cut the top of pipet tip to adjust for syringe insertion. D. Cut the edge of the pipet tip (arrow) for injection.

Procedure

  1. As shown in Figure 4A, stand up the platform for instillation on the bench.
  2. Anesthetize mice with 50 to 75 μl of ketamine/xylazine mixture by s.c. injection into the back.
  3. As shown in Figure 4B, hang anesthetized mice on the platform by their incisors.
  4. Take 50 μl of prepared alum suspension into an injector for instillation. In general, 50 μl of suspension (containing 50-100 μg of alum or 50-500 μg other particulates you want to inject, such as silica or nickel oxide) is the maximum volume for injection into the lungs.
  5. As shown in Figure 4C, use a spatula as a tongue depressor and insert injector into the throat, check the trachea (in order to find the trachea easily, put out mouse’s tongue using spatula and injector, first). It is difficult to check the trachea when young mice are used for the experiment. Because aged male mice are bigger, they are easier to find the trachea especially for training. Then, slowly drop suspensions of alum toward trachea. Dropped suspensions are aspirated into the lungs by breathing (aspiration method).


    Figure 4. Administration into the lungs

  6. Intranasal administration (i.n.) is also effective for administration into the lung. As shown in Figure 5, hold anesthetized mice and drop 30 μl (15 μl in each nostril) of suspensions of particulates into the nose. Dropped suspensions are also aspirated by breathing. In general, it is known that i.n. administration of 30 μl of solution reaches the lung.


    Figure 5. Intranasal administration

Data analysis

  1. As shown in Figure 6, we examined the efficacy of administration into the lungs between i.n. administration and the aspiration method, using black ink. Lungs were only partially stained by i.n. administration. On the other hand, lungs were more diffusely stained by the aspiration method, indicating that this method is more effective for administration into the lung.


    Figure 6. Comparison of efficacy of intranasal administration and aspiration method. Upper two samples are lungs from mice administered black ink by aspiration. Lower samples are those administrated by intranasal administration.

  2. A change in the number of alveolar macrophages 3 days after administration of alum into the lungs were observed. Alum is known to induce cell death by phagocytosis in macrophages. As shown in Figure 7, the percentage of alveolar macrophages (CD11c+ and siglec F+ cells) in bronchoalveolar-lavage fluid (BALF) were significantly decreased after the direct administration of alum into the lungs.


    Figure 7. FACS analysis of BAL cells from saline- or alum-administered mice. BAL cells were collected and stained by anti-CD11c and anti-Siglec F antibodies (Leukocytes in BAL cells were gated as CD45+ cells). A. BAL cells from saline-administered mice. B. BAL cells from alum-administered mice. This data is representative of at least two independent experiments (n = 4-5).

Notes

  1. Similar experiments can be performed for other particulates such as crystalline silica and nickel oxide nanoparticles (Kuroda et al., 2016).
  2. Suspensions more than 50 μl volumes sometimes choke mice to death. If mice often die by suffocation after administration of 50 μl of suspensions, administration of 30 μl of solution would be safer than administration of 50 μl. However we recommend 50 μl volumes for administration of solutions into the lungs, because of less variation among samples.

Acknowledgments

We thank Dr. Patrick Lelliotte and Burcu Temizoz for helpful discussion. Authors have no conflict of interest to declare.

References

  1. Kuroda, E., Ozasa, K., Temizoz, B., Ohata, K., Koo, C. X., Kanuma, T., Kusakabe, T., Kobari, S., Horie, M., Morimoto, Y., Nakajima, S., Kabashima, K., Ziegler, S. F., Iwakura, Y., Ise, W., Kurosaki, T., Nagatake, T., Kunisawa, J., Takemura, N., Uematsu, S., Hayashi, M., Aoshi, T., Kobiyama, K., Coban, C. and Ishii, K. J. (2016). Inhaled fine particles induce alveolar macrophage death and interleukin-1α release to promote inducible bronchus-associated lymphoid tissue formation. Immunity 45(6): 1299-1310.
  2. Morimoto, Y., Izumi, H., Yoshiura, Y., Tomonaga, T., Lee, B. W., Okada, T., Oyabu, T., Myojo, T., Kawai, K., Yatera, K., Shimada, M., Kubo, M., Yamamoto, K., Kitajima, S., Kuroda, E., Horie, M., Kawaguchi, K. and Sasaki, T. (2016). Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles. Nanotoxicology 10(5): 607-618.

简介

吸入的细微颗粒被认为是通过颗粒沉积到肺中引起慢性肺部炎症。 为了研究细颗粒对肺的影响,需要将颗粒悬浮液滴注到肺中。 这个协议描述了直接注入气道细粒悬浮物。 我们还展示了颗粒给药后典型的肺部免疫反应的例子。

【背景】近来,许多研究已经证明,诸如柴油机尾气颗粒物,沙尘和颗粒物质2.5(PM2.5)之类的颗粒污染物参与导致肺癌或过敏性哮喘的慢性肺部炎症。 流行病学分析显示,颗粒物空气污染增加与哮喘住院率增加有关。 通常,在吸入时,已知细颗粒如PM2.5深入肺部。 将颗粒悬浮液滴注到肺中已被广泛用于理解由沉积颗粒诱导的肺部炎症(Morimoto等人,2016)。

关键字:颗粒, 肺炎, 肺部异物吸入, 鼻内给药

材料和试剂

  1. 用于凝胶加样的移液头(Vertex-GL 200μl凝胶加样头)(SSIbio,目录号:4837-S0S)
  2. Parafilm
  3. 1毫升无菌注射器(无针)(TERUMO,目录号:SS-01T)
  4. 小鼠(C57BL / 6,BALB / c等等)
    注意:对于训练,更大的老鼠(老年雄性老鼠)更好。
  5. Alhydrogel(InvivoGen,目录号:vac-alu-250)作为滴注颗粒
    注意:Alhydrogel(明矾)被悬浮在dH <2:em> O中。为了将明矾注入肺中,需要进行缓冲液交换。离心的明矾悬浮液在2,000×g的微管2分钟。弃去上清液(H 2 O),加入等体积的生理盐水或PBS。充分混合并再次离心。重复此过程五次,将H / em> O O转换为盐水或PBS。最后,在盐水中将明矾的浓度调整到2mg / ml并用于滴注。
  6. 麻醉剂(氯胺酮/甲苯噻嗪混合物)
    注意:将10ml Ketalar(氯胺酮,50mg / ml,Daiichi Sankyo Co.Ltd。,Tokyo,Japan)与2.2ml Selactar(甲苯噻嗪,20mg / ml,Bayer HealthCare Ltd.,Tokyo,日本)。将50至75μl氯胺酮/赛拉嗪混合物麻醉小鼠麻醉注入后面。

设备

  1. 耳朵摘耳垢卸妆灯( Japan Smile Kids
    注意:在使用之前,耳塞尖端的硅橡胶将被去除(图1)。
  2. 不锈钢微铲(图1)(ASONE,产品目录号:9-891-02)
    注意:这是用作压舌板,所以小尺寸更好。


    图1.带有光和刮刀的耳柄

  3. 滴入肺部的平台。如图2所示,在木制软木板(大小约20×30厘米)上拉一根绳子。


    图2.滴入肺部的平台

  4. 注射器(图3)
  5. FACS分析仪


    图3.注射器的注射器。 :一种。用Parafilm将一个薄的移液器尖端固定在耳塞上。 B.将注射器连接到移液器尖端。 C.如果需要,切割移液器吸头的顶部以调整注射器插入。 D.切割移液枪头的边缘(箭头)注射。

程序

  1. 如图4A所示,站起来在凳子上灌输平台。
  2. 将50至75μl氯胺酮/赛拉嗪混合物麻醉小鼠麻醉注入后面。
  3. 如图4B所示,通过切牙将麻醉的小鼠悬挂在平台上。
  4. 将50μl制备好的明矾混悬液注入注射器中进行滴注。一般来说,50μl悬浮液(含有50-100微克明矾或50-500微克其他想要注射的微粒,如二氧化硅或氧化镍)是注射到肺部的最大体积。
  5. 如图4C所示,使用刮刀作为压舌板并将注射器插入喉部,检查气管(为了找到气管容易,先用刮铲和注射器拔出鼠标的舌头)。当年轻的小鼠用于实验时,很难检查气管。由于老年雄性小鼠较大,特别是对于训练更容易找到气管。然后,缓缓地将明矾悬浮液滴向气管。滴下的悬浮液通过呼吸(抽吸方法)被吸入肺部。


    图4.进入肺部

  6. 鼻内给药(i.n.)对肺内给药也是有效的。如图5所示,保持麻醉的小鼠,并将30μl(每个鼻孔中15μl)颗粒悬浮液滴入鼻中。落下的悬浮液也是通过呼吸吸入的。一般来说,我知道。
    注射30μl溶液到达肺部

    图5.鼻内管理

数据分析

  1. 如图6所示,我们研究了在美国之间肺部给药的疗效。使用黑色墨水的管理和吸入方法。肺只有部分沾染。行政。另一方面,通过吸入方法使肺更加弥漫性染色,表明这种方法对于肺内施用更有效。


    图6.鼻内给药和吸入方法的功效比较。上面的两个样品是来自吸入黑色墨水的小鼠的肺。较低的样品是鼻腔给药的样品。

  2. 观察到明矾注入肺部3天后肺泡巨噬细胞数量的变化。已知明矾在巨噬细胞中通过吞噬作用诱导细胞死亡。如图7所示,支气管肺泡灌洗液(BALF)中肺泡巨噬细胞(CD11c + sup和Fs + Fs +细胞)的百分比在直接施用
    明矾进入肺部

    图7.来自盐水或明矾给药小鼠的BAL细胞的FACS分析。收集BAL细胞并通过抗CD11c和抗Siglec F抗体(BAL细胞中的白细胞作为CD45 +细胞进行门控)染色。 A.来自盐水施用小鼠的BAL细胞。 B.来自明矾给药小鼠的BAL细胞。这个数据是至少两个独立实验的代表(n = 4-5)。

笔记

  1. 对于其他颗粒如结晶二氧化硅和氧化镍纳米颗粒可以进行类似的实验(Kuroda等人,2016)。
  2. 超过50μl的悬浮液有时会使小鼠死亡。如果在给予50μl悬浮液后小鼠经常因窒息而死亡,则给予30μl溶液比给予50μl更安全。然而,我们建议50μl体积的溶液注入肺部,因为样品间的差异较小。

致谢

我们感谢Patrick Lelliotte博士和Burcu Temizoz博士的有益讨论。作者没有利益冲突要申报。

参考

  1. Kuroda,E.,Ozasa,K.,Temizoz,B.,Ohata,K.,Koo,CX,Kanuma,T.,Kusakabe,T.,Kobari,S.,Horie,M.,Morimoto,Y.,Nakajima S.,Kabashima K.,Ziegler SF,Iwakura Y.,Ise W.,Kurosaki T.,Nagatake T.,Kunisawa J.,Takemura N.,Uematsu S.,Hayashi, M.,Aoshi,T.,Kobiyama,K.,Coban,C.和Ishii,KJ(2016)。 吸入细小颗粒诱导肺泡巨噬细胞死亡和白细胞介素-1α释放,促进可诱导的支气管相关淋巴组织形成。 免疫 45(6):1299-1310。
  2. Morimoto,Y.,Izumi,H.,Yoshiura,Y.,Tomonaga,T.,Lee,BW,Okada,T.,Oyabu,T.,Myojo,T.,Kawai,K.,Yatera,K.,Shimada M.,Kubo,M.,Yamamoto,K.,Kitajima,S.,Kuroda,E.,Horie,M.,Kawaguchi,K.和Sasaki,T。(2016)。 气管内灌注和吸入纳米颗粒后肺部炎症反应的比较纳米毒理学 10(5):607-618。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kuroda, E., Morimoto, Y. and Ishii, K. J. (2017). Instillation of Particulate Suspensions to the Lungs. Bio-protocol 7(22): e2618. DOI: 10.21769/BioProtoc.2618.
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