Materials and Reagents

1.5 ml screw cap micro tube (SARSTEDT, catalog number: 72.703.600)
5 ml screw cap tube (SARSTEDT, catalog number: 60.9921.524)
25 ml screw cap tube (SARSTEDT, catalog number: 60.9922.243)
27 G x ½” 1 ml Insulin syringe (Terumo, catalog number: SS*10M2713A)
Aluminum foil
500 ml Bottle Top Filter, 0.2 µm pore size (Thermo Fisher Scientific, Nalgene^® Rapid-Flow^TM, catalog number: 595-4520)
50 ml serological pipette (SARSTEDT, catalog number: 86.1256.001)
21-day-old BALB/c mice (Animal Resource Centre, Murdoch, Australia)
Notes:
1. All mice were fed an OVA free diet.
2. Both male and female mice were used.
3. n ≥ 6 mice per experiment.
Ovalbumin lyophilized powder (Sigma-Aldrich, catalog number: A5503, store at 4 °C)
Aluminum hydroxide (SERVA, catalog number: 12261.02, store at room temperature in the dark)
Sterile Water for Irrigation (Baxter, catalog number: 2F7113, store at room temperature)
Sodium chloride (NaCl) (Rowe Scientific, catalog number: CS10363)
Potassium chloride (KCl) (Rowe Scientific, catalog number: CP2600)
Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Sigma-Aldrich, catalog number: RES20908-A704X)
Potassium phosphate monobasic anhydrous (KH₂PO₄) (Amresco, catalog number: 97062-350)
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331-500ML)
Sterile phosphate buffered saline (PBS) (see Recipes)
1 mg/ml ovalbumin stock solution (see Recipes)
10 mg/ml ovalbumin stock solution (see Recipes)
Ovalbumin-aluminum hydroxide (OVA-Alum) preparation for one mouse (see Recipes)

Equipment

Housing cages (Tecniplast, GM500)
500 ml measuring cylinder (Rowe Scientific, catalog number: GC1276)
100 ml Schott bottle (Sigma-Aldrich, Duran^®, catalog number: Z305170)
500 ml Schott bottle (Sigma-Aldrich, Duran^®, catalog number: Z305197)
5,000 ml Schott bottle (Sigma-Aldrich, Duran^®, catalog number: Z305227)
5,000 ml volumetric flask (Sigma-Aldrich, Duran^®, catalog number: 246787355)
S1 Pipet Filler (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 9521)
Rotary suspension mixer (Ratek, catalog number: RSM7DC)
Ultrasonic nebulizer (DeVilbiss Healthcare, UltraNeb, catalog number: U3000)
25 cm x 40 cm plexiglass aerosol chamber (AFM Plastics)
-20 °C freezer

Software

GraphPad Prism (GraphPad software, version 7.0a)

Procedure

Intraperitoneal sensitization of 21-day-old BALB/c mice (Figure 1)
1. Draw up 200 µl of the OVA-Alum preparation using a 27 G x ½” 1 ml Insulin syringe.
2. Scruff mouse and hold on a 45-degree downwards angle with the head away from you.
  Note: The holding technique used for intraperitoneal injections should adhere to independent in-house procedures.
3. Slowly inject the entire 200 µl OVA-Alum preparation intraperitoneally (i.p).
  Notes:
  1. Control mice received sham injection of 200 μl PBS.
  2. Always use a new sterile syringe for each intraperitoneal injection.
4. Monitor mice for 3 days post-sensitization or as per individual in-house ethics requirements.
  Note: OVA-sensitized mice are to be housed separately to control mice.
Intraperitoneal booster injection of 35-day-old mice (Figure 1)
1. Repeat Procedure A at 35 days of age.
2. Monitor mice for 3 days post-sensitization or as per individual in-house ethics requirements.
Ovalbumin aerosol challenge of 42-day-old sensitized mice (Figure 1)
1. Place sensitized mice in the plexiglass aerosol chamber connected to the ultrasonic nebulizer at a stock density of ≤ 10.
  Note: Do not overcrowd the plexiglass aerosol chamber. This ensures all mice receive an equivalent OVA aerosol dose.
2. Add 1x 25 ml aliquot of 10 mg/ml OVA solution to the ultrasonic nebulizer.
  Note: Control mice received PBS aerosol challenge.
3. Turn the ultrasonic nebulizer on with the following settings:
  1. Cloud setting: 4
  2. Fan setting: 3
  3. Run time: 30 min
4. Following the 30 min 10 mg/ml OVA aerosol challenge, return the mice to their housing cages.
5. Repeat OVA aerosol challenge on Days 43 and 44 of age using freshly defrosted 10 mg/ml OVA solution aliquots.
Figure 1. Timeline of intraperitoneal OVA-Alum injections and OVA aerosol challenges

Data analysis

As detailed in the original research article (Mincham et al., 2018), successful early life ovalbumin sensitization and aerosol challenge is clinically demonstrable by elevated levels of OVA-specific serum immunoglobulin E as determined by passive cutaneous anaphylaxis assay (Figure 1A in the original article), enhanced infiltration of inflammatory cell populations to the airways encompassing eosinophils, neutrophils, and lymphocytes (Figure 1B in the original article) and airways hyperresponsiveness following methacholine challenge (Figure 1C in the original article). Successful early life ovalbumin sensitization and aerosol challenge is also accompanied by elevated levels of Th2-associated cytokines interleukin (IL)-5 in the lung (Figure 2A) and IL-4 and IL-5 in the airway draining lymph nodes (Figure 2B).

Representative data:

Figure 2. Elevated Th2 cytokines following successful early life ovalbumin sensitization and aerosol challenge. A. Elevated levels of interleukin (IL)-5 in peripheral lung homogenates of early life OVA sensitized and aerosol challenged mice. B. Elevated levels of IL-4 and IL-5 in airway draining lymph node (ADLN) homogenates of early life OVA sensitized and aerosol challenged mice. Data shows mean ± SEM of n ≥ 4 independent experiments. Statistical significance was determined using Student’s t-test and presented as *P < 0.05, **P < 0.01, ****P < 0.0001.

Notes

Be gentle when adding OVA lyophilized power to sterile water for irrigation. Do not stir the solution to accelerate dissolving rate as this will cause the OVA to denature.
Gently pour the OVA solution when filtering. Rapid pouring will cause the OVA to denature.
All mice were autopsied 24 h after the final OVA aerosol challenge in the original research article (Mincham et al., 2018). However, this protocol could be used to assess allergic airway inflammation at any time point after the final OVA aerosol challenge.

Recipes

Sterile Phosphate buffered saline (to make 5 L)
1. Dissolve 43.9 g NaCl, 1 g KCl, 5.75 g Na₂HPO₄, 1 g KH₂PO₄ in 5,000 ml sterile water in a 5,000 ml volumetric flask and add 0.2 ml HCl
2. Once dissolved, filter PBS solution into a sterile 5,000 ml Schott bottle using a 500 ml bottle top filter
3. Aliquot the sterile PBS solution into 500 ml aliquots
1 mg/ml ovalbumin stock solution
1. Dissolve 0.1 g OVA lyophilized powder in 100 ml sterile water for irrigation by placing lyophilized OVA powder on the water surface and allow to slowly dissolve at room temperature. Do not stir the solution (Note 1)
2. Once dissolved, filter the 1 mg/ml OVA solution into a sterile 100 ml Schott bottle using a 500 ml bottle top filter (Note 2)
3. Aliquot the sterile 1 mg/ml OVA solution into 1 ml aliquots (1.5 ml screw cap micro tubes)
4. Store at -20 °C
10 mg/ml ovalbumin stock solution
1. Dissolve 5 g OVA lyophilized powder in 500 ml sterile water for irrigation by placing lyophilized OVA powder on the water surface and allow to slowly dissolve at room temperature. Do not stir the solution (Note 1)
2. Once dissolved, filter the 10 mg/ml OVA solution into a sterile 500 ml Schott bottle using a 500 ml bottle top filter (Note 2)
3. Aliquot the sterile 10 mg/ml OVA solution into 25 ml aliquots using a 50 ml serological pipette and S1 Pipet Filler (25 ml screw cap tubes)
4. Store at -20 °C
Ovalbumin-aluminum hydroxide (OVA-Alum) preparation for one mouse
1. For each mouse, emulsify 20 μl of 1 mg/ml OVA solution in 180 μl of aluminum hydroxide to give a final concentration of 20 μg OVA and 1.3 mg aluminum hydroxide in a total volume of 200 μl. Increase as necessary for the total number of mice to be sensitized
2. Wrap the tube (5 ml screw cap tube) containing the OVA-Alum preparation in aluminum foil and spin for 1 h at 20 rpm using a rotary suspension mixer

Acknowledgments

The authors would like to acknowledge the animal technicians at the Telethon Kids Institute Bioresources facility. The original research article (Mincham et al., 2018) utilizing this protocol was funded by the National Health and Medical Research Council of Australia.

Competing interests

The authors declare no financial or non-financial competing interests related to this work.

Ethics

All animal experiments relating to this protocol were formally approved by the Telethon Kids Institute Animal Ethics Committee, which operates under guidelines developed by the National Health and Medical Research Council of Australia for the care and use of animals in scientific research.

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