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Isolation of Nippostrongylus brasiliensis Larvae from Mouse Lungs
从小鼠肺部分离巴西日圆线虫幼虫   

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Abstract

The rodent parasite Nippostrongylus brasiliensis (N. brasiliensis) models the salient features of helminth infection including skin penetration, migration from tissues to lung, maturation and egg production in the gut. As a potent activator of systemic and mucosal Th2 immune responses, Nippostrongylus brasiliensis has been extensively used to study host protective immunity and in vivo regulation of Th2 immune response. Six to eight week old C57Bl/6J, Balb/c mice or any other strains are suitable, as all are susceptible to infection. Inocula of 150-650 L3 larvae can be administered by subcutaneous injection, but for greatest consistency a dose of 550 L3 larvae is routinely used for experimental purposes. We have optimized three different protocols for the isolation of larvae from the lungs of mice infected with the L3 stage of Nippostrongylus brasiliensis. Larvae can migrate to the lung between 18-60 h post inoculation from any site in the body. The numbers of larvae appearing in the lung peaks at 48 h after inoculation and it is recommended that isolation/harvesting be performed at 48 h for greatest consistency of each harvest method:

  1. Isolation by thermal induced migration-allows for the recovery of viable larvae but not dead or moribund larvae.
  2. Isolation by digestion of the lung-allows for the recovery of both dead and viable larvae.
  3. Isolation by bronchoalveolar lavage (BAL) -allows for the assessment of the number of molt 4 larvae on their way to the gut.
    These protocols can be used to follow the dynamics of worm migration during infection and the effect of the host immune system on worm viability and fecundity.

Part I. Isolation by migration

Materials and Reagents

  1. Cheesecloth cut into 8 x 8 cm squares (any fabric store)
  2. 8 x 8 cm plastic disposable weigh boats (Medi'Ray-Laboratory, Runlab Archives, catalog number: RL33102 )
  3. 50 ml centrifuge tubes (Thermo Fisher Scientific, FalconTM, catalog number: 352070 / 14-432-22 ) and corresponding racks
  4. Disposable Pasteur pipettes (Interab Supply, catalog number: KJ619-1 )
  5. 60 x 15 mm TC dish (Thermo Fisher Scientific, FalconTM, catalog number: 353002 / 08-772B )
  6. N. brasiliensis infected mice (Camberis et al., 2003)
  7. 70% Ethanol
  8. PBS (DPBS, no calcium, no magnesium) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )

Equipment

  1. Iris scissors (Medicon, catalog number: 202.20.11 )
  2. Forceps (Medicon, catalog number 07.55.20 )
  3. Water-bath set at 37 °C (Biolab, model: Grant SUB Aqua 18 ) with water set to a depth not exceeding 2 cm from the top of the 50 ml tubes
  4. Stereo-microscope (Olympus, model: SZX16 )

Procedure

  1. Prepare the Migration tube by adding 45 ml PBS to a 50 ml tube. Keep warm in 37 °C water bath until ready to use.
  2. Euthanize the infected mouse by cervical dislocation or any approved form of euthanasia.
  3. Place mouse on its back and wet with 70% ethanol.
  4. Expose the thoracic cavity; remove the lungs and place in a clean disposable weigh-boat.


    Figure 1. Day 2 lungs from N. brasiliensis infected mouse with visible petechiae (haemorraghic spots caused by the entry of larvae into the lung, indicated by the white arrows)

  5. Dice the lungs into fine tissue pieces of approximately 1-2 mm using the scissors.
  6. Place an 8 x 8 cm piece of cheesecloth into a prepared 50 ml tube with the edges of the cloth hanging over the side of the vessel; this should form an open pouch in the tube (Figure 2a).
  7. Transfer the diced lung into the pouch of the prepared 50 ml tube (Figure 2b).
  8. Take the exposed edges of the cheesecloth, and close the pouch in such a way that the exposed cheesecloth edge hangs over the edge of the 50 ml tube (Figure 2c). Seal the tube with the cap so as to trap the edge of the cloth under the cap (Figure 2d).
  9. Place in water bath and incubate for at least 3 h at 37 °C or up to overnight in water bath.
    Note: Ensure at least two-thirds of the tube is submerged in the water as the larvae migrate out of the tissue by following a thermal gradient and settle at the bottom of the tube.


    Figure 2. Tube set up for thermal gradient induced migration/isolation/enumeration of larvae from infected tissues

  10. Discard cheesecloth following incubation period.
    Note: Worms will have migrated through the tissue and collected at the bottom of the 50 ml tube.
  11. Without disrupting the pellet, discard all but 2 to 3 ml of the PBS by aspirating with a plastic disposable pipette.
  12. Transfer the contents of the tube into a 60 x 15 mm TC dish and visualize the larvae using a stereo microscope.
    Note: Volumes of more than 3 ml can make focusing on the larvae difficult.

Part II. Isolation by lung digest

Materials and Reagents

  1. 50 ml centrifuge tubes (Thermo Fisher Scientific, FalconTM, catalog number: 352070 / 14-432-22 ) and corresponding racks
  2. 6-well tissue culture dishes (Thermo Fisher Scientific, FalconTM, catalog number: 353046 / 08-772-1B )
  3. 100 μm Cell strainers (Thermo Fisher Scientific, FalconTM, catalog number: 352360 / 22-363-549 )
  4. 40 μm Cell strainers (Thermo Fisher Scientific, FalconTM, catalog number: 352340 / 22-363-547 )
  5. Disposable Pasteur pipettes (InterLab Supply, catalog number: KJ619-1 )
  6. N. brasiliensis infected mice (Camberis et al., 2003)
  7. 70% Ethanol
  8. PBS (DPBS, no calcium, no magnesium) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  9. LiberaseTM TL Research Grade (Sigma-Aldrich, catalog number: 5401020001 )
  10. DNase I (Sigma-Aldrich, catalog number: 10104159001 )
  11. IMDM, GlutaMAXTM Supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 31980-030 )
  12. Digestion mix (see Recipes)

Equipment

  1. Iris scissors (Medicon, catalog number: 202.20.11 )
  2. Forceps (Medicon, catalog number: 07.55.20 )
  3. Stereo-microscope (Olympus, model: SZX16 )
  4. Incubator Shaker/Rocker (Eppendorf, New Brunswick Scientific, model: innova 4200 ) set at 37 °C

Procedure

  1. Euthanize the mouse by cervical dislocation or any approved form of euthanasia.
  2. Place mouse on its back and wet with 70% ethanol.
  3. Expose the thoracic cavity; remove lungs and place into a well of the 6-well dish.
  4. Dice the lungs into fine tissue of approximately 1-2 mm pieces using the scissors.
  5. Add 5 ml Digestion mix to each well and gently mix by swirling the plate.
  6. Incubate for 45 min at 37 °C with slow agitation in a shaking incubator.
  7. Using a disposable pipette, transfer the lung digest to a 100 μm cell strainer attached to a 50 ml tube. Rinse the well with 5 ml IMDM and transfer to cell strainer
  8. Transfer the contents of the tube to a 40 μm cell strainer attached to another 50 ml tube, using a disposable pipette.
    Note: This separates the lung tissue from the larvae, the larvae staying trapped in on top of the filter.
  9. Transfer the 40 μm strainer to a 6 well TC dish and place the 40 μm strainer upside down.
  10. Rinse with 5 ml IMDM using a disposable pipette to dislodge larvae trapped on the cell strainer. Inspect the cell strainer for the presence of larvae using a stereo-microscope and rinse again if necessary.
  11. Visualize larvae using a stereo-microscope.

Part III. Isolation by bronchoalveolar lavage (BAL)

This protocol has been adapted from protocols used in measuring allergic airway inflammation (Harris et al., 1997; Erb et al., 1998).

Materials and Reagents

  1. 1 ml slip lip syringes (BD, catalog number: 302100 )
  2. 18 G Insyte catheter (BD, catalog number: 381247 )
  3. 15 ml Conical Centrifuge tubes (Thermo Fisher Scientific, FalconTM, catalog number: 352096 / 14-959-49B ) and corresponding racks
  4. 60 x 15 mm TC dish (Thermo Fisher Scientific, FalconTM, catalog number: 353002 / 08-772B )
  5. N. brasiliensis infected mice (Camberis et al., 2003)
  6. 70% Ethanol
  7. PBS (DPBS, no calcium, no magnesium) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )

Equipment

  1. Iris scissors (Medicon, catalog number: 202.20.11 )
  2. Forceps (Medicon, catalog number 07.55.20 )
  3. Stereo-microscope (Olympus, model: SZX16 )

Procedure

  1. Euthanize the mouse by cervical dislocation (or any approved form of euthanasia) but taking care to maintain the integrity of the trachea.
  2. Place mouse on its back and wet with 70% ethanol.
  3. Make a midline incision, pull back the skin and expose the trachea by carefully separating the muscle tissue above the trachea (Figure 3a).
  4. Make a small horizontal incision across the exposed trachea without going right through the trachea (Figure 3b and Figure 3c).
  5. Attach an 18 G Insyte catheter to a 1 ml syringe and fill with 1 ml PBS.
  6. Insert the catheter approximately 10 mm into the trachea and flush the contents of the syringe into the lungs (Figure 3d).
  7. Draw the fluid out (Figure 3e) and place contents of syringe into a 15 ml tube.
    The lavage fluid will be contaminated with blood due to the damage caused by the larvae entering the lung. To improve visualization of the larvae when lavage fluid is contaminated with blood, spin the BAL fluid down at 1,400 rpm for 2 min, remove the supernatant and lyse the red blood cells by the addition of 3ml of water to the pellet, then count larvae. Distinguishing features of the molt 4 larvae is the deposition of a dark-brown pigment that is clearly visible in the gut (Bouchery et al., 2015).
  8. Repeat twice more with fresh PBS for each lavage.
  9. Visualize the larvae by transferring the lavage into a 60 x 15 mm TC dish and inspecting the contents using a stereo microscope.


    Figure 3. Experimental procedure for performing bronchoalveolar lavage

Representative data


Figure 4. Comparison of larval recovery from lung using either thermal gradient induced migration, bronchoalveolar lavage (BAL) or lung digestion

Notes

For consistent results using the above protocols, it is important to always use larvae from 2-6 weeks old cultures for infecting mice. The infectivity of larvae is much reduced after 6 weeks culture (Camberis et al., 2003; Kassai, 1982).

Recipes

  1. Digestion mix
    0.1 mg/ml Liberase TL
    120 mg/ml DNase I
    Made up in IMDM
    Make up fresh, 5 ml per lung

Acknowledgments

This work has been adapted and modified from previous work carried out by Professor Graham Le Gros’s Allergy and Parasitic diseases laboratory, Malaghan Institute of Medical Research, New Zealand. This work was supported by The Health Research Council of New Zealand and the Marjorie Barclay Trust.

References

  1. Bouchery, T., Kyle, R., Camberis, M., Shepherd, A., Filbey, K., Smith, A., Harvie, M., Painter, G., Johnston, K., Ferguson, P., Jain, R., Roediger, B., Delahunt, B., Weninger, W., Forbes-Blom, E. and Le Gros, G. (2015). ILC2s and T cells cooperate to ensure maintenance of M2 macrophages for lung immunity against hookworms. Nat Commun 6: 6970.
  2. Camberis, M., Le Gros, G. and Urban, J., Jr. (2003). Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Curr Protoc Immunol Chapter 19: Unit 19 12.
  3. Erb, K. J., Holloway, J. W., Sobeck, A., Moll, H. and Le Gros, G. (1998). Infection of mice with Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) suppresses allergen-induced airway eosinophilia. J Exp Med 187(4): 561-569.   
  4. Harris, N., Campbell, C., Le Gros, G. and Ronchese, F. (1997). Blockade of CD28/B7 co-stimulation by mCTLA4-Hgamma1 inhibits antigen-induced lung eosinophilia but not Th2 cell development or recruitment in the lung. Eur J Immunol 27(1): 155-161.
  5. Harvie, M., Camberis, M., Tang, S. C., Delahunt, B., Paul, W. and Le Gros, G. (2010). The lung is an important site for priming CD4 T-cell-mediated protective immunity against gastrointestinal helminth parasites. Infect Immun 78(9): 3753-3762.
  6. Kassai, T. (1982). Handbook of Nippostrongylus brasiliensis, Akademia Kiado, Budapest, ISBN-10: 9630529769.

简介

啮齿动物寄生虫(Nippostrongylus brasiliensis)(<巴西> )模拟蠕虫感染的显着特征,包括皮肤渗透,从组织到肺的迁移,肠道中的成熟和产蛋。作为全身性和粘膜Th2免疫应答的有效激活剂,巴西尼罗巴戟天已广泛用于研究宿主保护性免疫和体内调节Th2免疫应答。 6至8周龄的C57Bl/6J,Balb/c小鼠或任何其它菌株是合适的,因为所有这些菌株都易于感染。 150-650LL幼虫的接种可以通过皮下注射给药,但是为了最大的一致性,通常将550LL幼虫的剂量用于实验目的。我们已经优化了三种不同的方案用于从感染了巴西尼罗巴戟甲L3阶段的小鼠的肺部分离幼虫。幼虫可以从体内任何部位接种后18-60小时迁移到肺部。在接种后48小时,出现在肺峰中的幼虫的数目,并且建议在48小时进行分离/收获以获得每种收获方法的最大一致性:

  1. 通过热诱导迁移的分离 - 允许回收有活力的幼虫,但不是死亡或垂死的幼虫。
  2. 通过消化肺分离 - 允许死亡和活的幼虫的恢复。
  3. 通过支气管肺泡灌洗(BAL)分离 - 允许用于评估在其到肠道的途中的蜕皮4幼虫的数量。
    这些协议可用于跟踪蠕虫在感染过程中的动态和宿主免疫系统对蠕虫生存力和繁殖力的影响。

第I部分:迁移隔离

材料和试剂

  1. 干酪布切成8 x 8厘米的方块(任何布料商店)
  2. 8×8cm塑料一次性称量舟(Medi'Ray-Laboratory,Runlab Archives,目录号:RL33102)
  3. 50ml离心管(Thermo Fisher Scientific,Falcon TM ,目录号:352070/14-432-22)和相应的架子
  4. 一次性巴斯德移液管(Interab Supply,目录号:KJ619-1)
  5. 60×15mm TC皿(Thermo Fisher Scientific,Falcon ,目录号:353002/08-772B)
  6. N。 brasiliensis 感染的小鼠(Camberis等人,2003)
  7. 70%乙醇
  8. PBS(DPBS,无钙,无镁)(Life Technologies,Gibco ,目录号:14190-144)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14190-144"

设备

  1. 虹膜剪刀(Medicon,目录号:202.20.11)
  2. 钳(Medicon,目录号07.55.20)
  3. 水浴设置在37℃(Biolab,型号:Grant SUB Aqua 18),水的深度距离50ml管的顶部不超过2cm
  4. 立体显微镜(奥林巴斯,型号:SZX16)

程序

  1. 通过加入45毫升PBS到50毫升管中准备迁移管。在37°C水浴中保温,直到准备使用。
  2. 安乐死受感染的小鼠颈椎脱位或任何批准形式的安乐死
  3. 将鼠标放在其背部,用70%乙醇湿润
  4. 暴露胸腔;取出肺并置于干净的一次性称量船中

    图1.来自N的第2天的肺。 (由白色箭头指示的幼虫进入肺部造成的血肿斑)感染的小鼠
  5. 使用剪刀将肺切成约1-2mm的细组织片
  6. 将8×8厘米的粗棉布放入准备好的50ml管中,使布的边缘悬挂在容器的侧面;这应该在管中形成开口袋(图2a)
  7. 将切成小块的肺转移到准备好的50ml管的小袋中(图2b)。
  8. 取出干酪布的暴露边缘,并以这样的方式关闭袋,暴露的干酪边缘悬挂在50ml管的边缘(图2c)。用盖子密封管子,以便将布的边缘卡在盖子下(图2d)。
  9. 置于水浴中,在37℃下孵育至少3小时或在水浴中孵育过夜 注意:确保至少三分之二的管浸没在水中,因为幼虫通过跟随热梯度从组织迁移出来并且沉积在管的底部。


    图2.用于热梯度诱导迁移/分离/计数受感染组织幼虫的管道

  10. 在孵育期后弃去干酪包布。
    注意:蠕虫将通过组织迁移并收集在50ml管的底部。
  11. 在不破坏沉淀的情况下,通过用塑料一次性移液管抽吸除去2至3ml的PBS而丢弃。
  12. 将管的内容物转移到60×15mm TC皿中,并使用立体显微镜观察幼虫。
    注意:超过3毫升的体积可能会使幼虫困难。

第二部分。肺消化隔离

材料和试剂

  1. 50ml离心管(Thermo Fisher Scientific,Falcon TM ,目录号:352070/14-432-22)和相应的架子
  2. 6孔组织培养皿(Thermo Fisher Scientific,Falcon ,目录号:353046/08-772-1B)
  3. 100μm细胞过滤器(Thermo Fisher Scientific,Falcon ,目录号:352360/22-363-549)
  4. 40μm细胞过滤器(Thermo Fisher Scientific,Falcon ,目录号:352340/22-363-547)
  5. 一次性巴斯德移液管(InterLab Supply,目录号:KJ619-1)
  6. N。 brasiliensis 感染的小鼠(Camberis等人,2003)
  7. 70%乙醇
  8. PBS(DPBS,无钙,无镁)(Life Technologies,Gibco ,目录号:14190-144)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14190-144"
  9. Liberase TM TL Research Grade(Sigma-Aldrich,目录号:5401020001)
  10. DNase I(Sigma-Aldrich,目录号:10104159001)
  11. IMDM,GlutaMAX TM Supplement(Life Technologies,Gibco TM ,目录号:31980-030)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:31980-030"
  12. 消解混合(参见配方)

设备

  1. 虹膜剪刀(Medicon,目录号:202.20.11)
  2. 钳(Medicon,目录号:07.55.20)
  3. 立体显微镜(奥林巴斯,型号:SZX16)
  4. 孵育器摇床/摇床(Eppendorf,New Brunswick Scientific,型号:innova 4200)设定为37℃

程序

  1. 安乐死小鼠颈椎脱位或任何批准形式的安乐死。
  2. 将鼠标放在其背部,用70%乙醇湿润
  3. 暴露胸腔;取出肺并放入6孔培养皿的孔中
  4. 使用剪刀将肺切成约1-2毫米的细小组织
  5. 向每个孔中加入5ml消解混合物,并通过旋转板轻轻混合
  6. 在37℃下在振荡培养箱中缓慢搅拌孵育45分钟
  7. 使用一次性移液管,将肺消化物转移到连接到50ml管的100μm细胞过滤器。用5ml IMDM冲洗孔并转移到细胞过滤器
  8. 使用一次性移液管将管的内容物转移到附接到另一个50ml管的40μm细胞过滤器。
    注意:这将肺组织与幼虫分离,幼虫停留在过滤器的顶部。
  9. 将40μm过滤器转移到6孔TC培养皿中,并将40μm过滤器上下颠倒
  10. 使用一次性移液管冲洗5ml IMDM,以移出捕获在细胞滤网上的幼虫。使用立体显微镜检查细胞过滤器是否存在幼虫,如有必要再次冲洗
  11. 使用立体显微镜可视化幼虫。

第三部分。支气管肺泡灌洗(BAL)隔离

该方案已经改变自用于测量过敏性气道炎症的方案(Harris等人,1997; Erb等人,1998)。

材料和试剂

  1. 1ml滑动唇形注射器(BD,目录号:302100)
  2. 18 G Insyte导管(BD,目录号:381247)
  3. 15ml锥形离心管(Thermo Fisher Scientific,Falcon TM,目录号:352096/14-959-49B)和相应的架子
  4. 60×15mm TC皿(Thermo Fisher Scientific,Falcon ,目录号:353002/08-772B)
  5. N。 brasiliensis 感染的小鼠(Camberis等人,2003)
  6. 70%乙醇
  7. PBS(DPBS,无钙,无镁)(Life Technologies,Gibco ,目录号:14190-144)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14190-144"

设备

  1. 虹膜剪刀(Medicon,目录号:202.20.11)
  2. 钳(Medicon,目录号07.55.20)
  3. 立体显微镜(奥林巴斯,型号:SZX16)

程序

  1. 安乐死的小鼠颈椎脱位(或任何批准形式的安乐死),但注意保持气管的完整性。
  2. 将鼠标放在其背部,用70%乙醇湿润
  3. 做一个中线切口,拉回皮肤,通过小心地分离气管上方的肌肉组织暴露气管(图3a)。
  4. 在暴露的气管上做一个小的水平切口,不要直接通过气管(图3b和图3c)。
  5. 将18 G Insyte导管连接到1 ml注射器,并加入1 ml PBS
  6. 将导管插入气管约10 mm,并将注射器的内容冲洗到肺部(图3d)。
  7. 抽出流体(图3e),将注射器的内容物放入15ml管中 由于幼虫进入肺部造成的损害,灌洗液将被血液污染。为了改善幼虫在灌洗液被血液污染时的可视化,将BAL液以1400rpm离心2分钟,除去上清液,通过向沉淀中加入3ml水溶解红细胞,然后计数幼虫。蜕皮4幼虫的区别特征是沉积在肠中清楚可见的深棕色色素(Bouchery等人,2015)。
  8. 每次灌洗时,用新鲜PBS重复两次。
  9. 通过将灌洗液转移到60×15mm TC皿中并使用立体显微镜检查内容来观察幼虫。


    图3.进行支气管肺泡灌洗的实验程序

代表数据


图4.使用热梯度诱导的迁移,支气管肺泡灌洗(BAL)或肺消化来自肺的幼虫恢复的比较。

笔记

对于使用上述方案的一致结果,重要的是总是使用来自2-6周龄培养物的幼虫感染小鼠。在培养6周后,幼虫的感染性大大降低(Camberis等人,2003; Kassai,1982)。

食谱

  1. 消化混合物
    0.1mg/ml Liberase TL
    120mg/ml DNase I在IMDM中制成
    补充新鲜,足够5毫升/肺

致谢

这项工作已经改编和修改,从前由Graham Le Gros的过敏和寄生虫疾病实验室,新西兰Malaghan医学研究所进行的工作。这项工作得到了新西兰健康研究委员会和Marjorie Barclay信托的支持。

参考文献

  1. Bouchery,T.,Kyle,R.,Camberis,M.,Shepherd,A.,Filbey,K.,Smith,A.,Harvie,M.,Painter,G.,Johnston,K.,Ferguson, Jain,R.,Roediger,B.,Delahunt,B.,Weninger,W.,Forbes-Blom,E.and Le Gros,G.(2015)。 ILC2和T细胞合作以确保M2巨噬细胞维持针对钩虫的肺免疫。 Nat Commun 6:6970.
  2. Camberis,M.,Le Gros,G。和Urban,J.,Jr。(2003)。 巴西尼日利亚动物模型和Heligmosomoides polygyrus。 < em> Curr Protoc Immunol 第19章:单位19 12。
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引用:Camberis, M., Bouchery, T. and Gros, G. L. (2016). Isolation of Nippostrongylus brasiliensis Larvae from Mouse Lungs. Bio-protocol 6(4): e1736. DOI: 10.21769/BioProtoc.1736.
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