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Allergen Sensitization and Challenge to Ovalbumin
利用卵清蛋白刺激构建致敏模型   

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Abstract

This protocol describes the sensitization and challenge of mice with ovalbumin for use as an acute murine model of asthma. This protocol induces reproducible airway inflammation and remodelling, and bronchial hyperresponsiveness to methacholine as measured by barometric plethysmography, as well as by the Flexivent® technique in Balb/c mice.

Keywords: Asthma(哮喘), Allergen sensitization(过敏原), Ovalbumin(卵清蛋白), Intranasal challenge(鼻内挑战), Mouse(鼠标)

Materials and Reagents

  1. Sterile saline (0.9% NaCl) (B. Braun)
  2. Chicken egg albumin (ovalbumin, grade V) (Sigma-Aldrich, catalog number: A-5503 )
  3. Aluminium hydroxide (Sigma-Aldrich, catalog number: 23918-6 )
  4. Ketamine (Imalgene®, Mérial)
  5. Xylazine (2%) (Rompun®, Bayer)
  6. Ovalbumin (see Recipes)
  7. Anaesthetics (see Recipes)

Equipment

  1. "1 ml" sterile syringes (Terumo, catalog number: SS+01T1 )
  2. "25 G" 0.5 mm sterile needles (Terumo, catalog number: NN-2516R )
  3. Sterile tips (Starlab, catalog number: S1111-0800 )
  4. Precision pipette (20 μl)
  5. End-over-end rotator (SB2, Stuart)
  6. 15-ml Falcon tube

Procedure

Summary:

  • Nine week-old male Balb/c mice are sensitized on days 0 and 7 by intraperitoneal (i.p.) injections of 50 μg ovalbumin adsorbed with 2 mg aluminium hydroxide (alum) in saline.
  • Mice are challenged on days 18, 19, 20 and 21 by intranasal (i.n.) instillations of 10 μg ovalbumin in saline; control animals receive i.n. instillations of saline alone; i.n. administrations must be carried out under anesthesia (ketamine and xylazine diluted in sterile saline, see Recipes).
  • Assessments are performed 18-24 h after the last i.n. challenge.
  1. Allergen sensitization
    1. Freshly prepare a suspension containing 0.5 mg/ml of ovalbumin (see Recipes) and 20 mg/ml of alum in sterile saline (0.9% NaCl): Weigh 80 mg alum in a 5 ml tube, add 1 ml from a 2 mg/ml ovalbumin aliquot, and 3 ml of sterile saline (0.9% NaCl).
    2. Gently homogenize the suspension in a rotator for 4 h at 4 °C so that ovalbumin may adsorb on alum. The mixture appears as a white suspension, susceptible to rapidly settle down to the bottom of the tube. It should be homogenized by rapid reversals (3-4) of the tube/syringe just before use.
    3. Before use, bring suspension to room temperature (18-25 °C) on the rotator.
    4. Hold the mouse in your hand by the dorsal skin so that its head is up and its rear legs are down. Maintain its tail with fingers.
    5. Use "1 ml" syringes and "25 G" needles to inject i.p. 100 μl suspension per mouse.
      Note: Gently homogenize the suspension by 3-4 repeated reversals of the tube/syringe between each use so that the suspension does not drop at the bottom of the tube/syringe).
  2. Allergen challenge
    1. Before use, bring solutions (anaesthetics, 0.4 mg/ml ovalbumin aliquot (1 aliquot per 24 mice), and saline) to room temperature (18-25 °C). Vortex for 4 sec at the highest speed to mix.
    2. Hold the mouse in your hand by the dorsal skin so that its head is up and its rear legs are down. Maintain its tail with fingers.
    3. Use "1 ml" syringes and "25 G" needles to inject i.p. 100 μl per mouse (25 g) of the anesthetic solution (at room temperature).
    4. Wait until the mouse is anesthetized; check that vibrissae do not move any more.
    5. Hold the mouse in your hand so that its head is up and its rear legs are down. Administer 12.5 μl of the 0.4 mg/ml ovalbumin solution in each nostril for sensitized mice, and saline alone for control mice, by use of sterile tips (note: Solution must be  administered drop by drop, slowly and very carefully).
    6. Keep the mouse in your hand in a vertical position at least for 1 min, and check that the mouse breathes normally.
      Note: If the mouse does not breathe normally, perform a thorax massage by pressing the rib cage several times, quickly but carefully.
    7. Place the mouse in decubitus on a heated blanket until complete recovery.

Recipes

  1. Ovalbumin
    Prepare a sterile 2 mg/ml solution of ovalbumin as follows: Weigh 80 mg ovalbumin and dissolve it in 40 ml cold sterile saline in a 50-ml Falcon tube. Vortex for 5 min at 2,000 rpm to mix and distribute 30 ml of the solution as 1.1 ml aliquots into 1.5 ml microtubes; this will result in ovalbumin aliquots for 24 mice. Immediately store frozen at -20 °C for up to 2 months.
    Take the remaining 10 ml of the 2 mg/ml ovalbumin solution, dilute it 1/5 in sterile saline resulting in a 0.4 mg/ml solution, and distribute as 1 ml aliquots into microtubes (1 aliquot per 24 mice). Immediately store aliquots frozen at -20 °C for up to 2 months.
  2. Anaesthetics
    Iin a 15 ml Falcon tube, add 1.5 ml of a 100 g/L commercial solution of ketamine (Imalgene® 1000), 0.5 ml of a 20 g/L commercial solution of xylasine (Rompun® 2%) and 10 ml of sterile saline (0.9% NaCl). The prepared solution contains 12.5 mg/ml ketamine base and 0.83 mg/ml xylasine base from hydrochloride. Inject 100 μl of the anaesthetic solution per mouse (25 g), i.e. a 4 ml/kg.
    The administrated dose is 50 mg/kg ketamine and 3.3 mg/kg xylazine. This preparation may be stored at 4 °C for 10 days.

Notes

Other protocols based on this one may be implemented by modification of either the mouse strain, the dose or sequence of administration of ovalbumin, or the allergen (house dust mite or cockroach extracts for instance), with a sensitization step followed by challenges to the allergen. However, using standardized procedures for allergen sensitization and challenge would allow better reproducibility and comparison between results in the literature when studying airway inflammation and remodelling, and generation of bronchial hyper responsiveness.

Acknowledgments

This protocol was adapted from a previously published paper: Reber et al. (2012). FD was supported by the “fond de dotation recherche en santé respiratoire”, call for tenders 2010.

References

  1. Reber, L. L., F. Daubeuf, M. Plantinga, L. De Cauwer, S. Gerlo, W. Waelput, S. Van Calenbergh, J. Tavernier, G. Haegeman, B. N. Lambrecht, N. Frossard and K. De Bosscher (2012). A dissociated glucocorticoid receptor modulator reduces airway  hyperresponsiveness and inflammation in a mouse model of asthma. J Immunol 188(7): 3478-3487.

简介

该协议描述了用卵清蛋白作为哮喘的急性鼠模型的小鼠的致敏和攻击。 该协议诱导可重复的气道炎症和重塑,以及通过气压体积描记术以及通过Flexb/c小鼠中的Flexivent 技术测量的对乙酰甲胆碱的支气管高反应性。

关键字:哮喘, 过敏原, 卵清蛋白, 鼻内挑战, 鼠标

材料和试剂

  1. 无菌盐水(0.9%NaCl)(B.Braun)
  2. 鸡卵白蛋白(卵白蛋白,V级)(Sigma-Aldrich,目录号:A-5503)
  3. 氢氧化铝(Sigma-Aldrich,目录号:23918-6)
  4. 氯胺酮(Imalgene Mérial)
  5. 甲苯噻嗪(2%)(Rompun ,Bayer)
  6. 卵清蛋白(见配方)
  7. 美学(见配方)

设备

  1. "1ml"无菌注射器(Terumo,目录号:SS + 01T1)
  2. "25G"0.5mm无菌针(Terumo,目录号:NN-2516R)
  3. 无菌提示(Starlab,目录号:S1111-0800)
  4. 精密移液管(20μl)
  5. 端到端旋转器(SB2,Stuart)
  6. 15-ml Falcon管

程序

概要:

  • 通过腹膜内(i.p.)注射吸附有2mg氢氧化铝(明矾)的50μg卵清蛋白在盐水中的溶液,在第0和7天使九周龄的雄性Balb/c小鼠致敏。
  • 在第18,19,20和21天通过鼻内(i.n.)滴注10μg卵清蛋白在盐水中的攻击小鼠; 对照动物接受i.n. 滴注单独的盐水; 在。 给药必须在麻醉下进行(氯胺酮和赛拉嗪在无菌盐水中稀释,参见Recipes)。
  • 在最后一次给药后18-24小时进行评估。 挑战。
  1. 过敏原致敏
    1. 新鲜制备含有0.5mg/ml卵白蛋白(参见Recipes)和20mg/ml明矾在无菌盐水(0.9%NaCl)中的悬浮液:称取80mg明矾在5ml管中,从2mg/ml卵白蛋白等分试样和3ml无菌盐水(0.9%NaCl)
    2. 轻轻匀浆在旋转器中的悬浮液在4°C下4小时,使卵清蛋白可吸附在明矾上。混合物表现为白色悬浮液,易于快速沉降到管的底部。在使用之前,应通过管/注射器的快速反转(3-4)使其均匀化
    3. 使用前,将悬浮液置于旋转器上室温(18-25°C)下
    4. 将鼠标握在手中的背部皮肤,使其头部向上,其后腿向下。用手指保持其尾巴。
    5. 使用"1ml"注射器和"25G"针注射i.p。每只小鼠100μl悬浮液。
      注意:通过每次使用之间管/注射器的3-4次重复反转轻轻均匀化悬浮液,使得悬浮液不会在管/注射器的底部下降)。
  2. 过敏原挑战
    1. 使用前,将溶液(麻醉剂,0.4 mg/ml卵白蛋白等分试样(每24只小鼠1等分)和盐水)至室温(18-25°C)。以最高速度旋转4秒混合
    2. 将鼠标握在手中的背部皮肤,使其头部向上,其后腿向下。用手指保持其尾巴。
    3. 使用"1ml"注射器和"25G"针注射i.p。每只小鼠100微升(25克)的麻醉溶液(在室温下)
    4. 等待鼠标麻醉;检查是否动物不再移动
    5. 将鼠标握在手中,使其头部向上,其后腿向下。在致敏小鼠的每个鼻孔中给予12.5μl的0.4mg/ml卵白蛋白溶液,对于对照小鼠,使用无菌提示物给予盐水( n ote:慢慢地,非常仔细地) 。
    6. 将鼠标在垂直位置保持至少1分钟,并检查鼠标是否正常呼吸。
      注意:如果鼠标没有正常呼吸,可以快速但小心地按几下肋骨,进行胸部按摩。
    7. 将鼠标放在褥疮的加热毯子上,直到完全恢复

食谱

  1. 卵清蛋白
    准备无菌的2毫克/毫升卵白蛋白溶液如下:称重80毫克卵清蛋白,并溶解在40毫升冷无菌盐水的50毫升Falcon管。在2,000rpm涡旋5分钟以混合和分配30ml的溶液为1.1ml等分试样到1.5ml微管中;这将导致24只小鼠的卵白蛋白等分试样。立即储存在-20°C冷冻保存2个月。
    取剩余的10ml 2mg/ml卵清蛋白溶液,在无菌盐水中稀释1/5,得到0.4mg/ml溶液,并以1ml等分试样分配到微管中(每24只小鼠1等份)。立即储存等分试样冻结在 -20°C长达2个月。
  2. 美学
    在15ml Falcon管中,加入1.5ml的100μg/L的氯胺酮商业溶液(Imalgene 1000),0.5ml的20g/L的市售的西拉西林溶液(Rompun 2%)和10ml无菌盐水(0.9%NaCl)。制备的溶液含有12.5mg/ml氯胺酮碱和0.83mg/ml来自盐酸盐的甲硅氨酸碱。每只小鼠注射100μl麻醉剂溶液(25g),即4ml/kg。
    给药剂量为50mg/kg氯胺酮和3.3mg/kg赛拉嗪。该制剂可以在4℃下贮存10天

笔记

基于这种方法的其它方案可以通过修饰小鼠品系,卵白蛋白或者变应原(例如房尘螨或蟑螂提取物)的剂量或施用顺序,通过敏化步骤,然后挑战变应原。然而,使用用于过敏原敏化和挑战的标准程序将允许在研究气道炎症和重塑以及产生支气管高反应性时更好的重现性和文献中的结果之间的比较。

致谢

该协议改编自以前发表的论文:Reber等人(2012)。 FD由"fond de dotation recherche ensantérespiratoire"支持,呼吁招标2010年。

参考文献

  1. Reber,L.L.,F. Daubeuf,M。Plantinga,L.Deauau,S.Gerlo,W.Waelput,S.Van Calenbergh,J. Tavernier,G. Haegeman,B. N. Lambrecht,N. Frossard和K. De Bosscher(2012)。 A    解离的糖皮质激素受体调节剂减少 气道 高反应性和哮喘小鼠模型中的炎症。 J Immunol 188(7):3478-3487。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Daubeuf, F., Reber, L. and Frossard, N. (2013). Allergen Sensitization and Challenge to Ovalbumin. Bio-protocol 3(7): e644. DOI: 10.21769/BioProtoc.644.
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