Environmental Conditioning and Aerosol Infection of Mice

Materials and Reagents

1. 15 ml conical tube (BD Falcon, catalog number: 352096 )
2. Pipette tips (Fisherbrand, catalog numbers: 02-681-165 , 02-681-147 or equivalent)
3. C57BL/6 mice carrying a functional Mx1 allele (Horisberger et al., 1983)
   Note: Most laboratory mouse strains are highly susceptible to influenza infection due to a defective Mx1 gene which is an important interferon stimulated gene to combat influenza (Iwasaki, 2016). Therefore, we prefer to use Mx1 congenic B6 mice to study host responses to influenza viruses.
4. Highly virulent A/PR/8/34 (H1N1; hvPR8) (Grimm et al., 2007)
   Previously, the hvPR8 strain was generated by serial lung passages in Mx1 mice (Grimm et al., 2007).
5. 1x PBS (Sigma, catalog number: D8537 )
   

Equipment

1. Environmental chamber (Caron, model: 7000-10)
2. Nebulizer (Allied Healthcare Products, Schuco, model: S5000 )
3. Mouse pie cage (Braintree Scientific, model: MPC-3 AERO )
4. Weight scaler (OHAUS, Scout Pro, model: SP202 )
5. Pipettes (DENVILLE, P1000, P200)
   

Software

1. GraphPad Prism 7.0 (GraphPad Software; https://www.graphpad.com/)

Procedure

1. Pre-conditioning of mice in environmental chambers
   1. Transfer the mice to biosafety level 2 (BSL2) adaptive feeding for 3-7 days. Divide the mice randomly for housing under different humidity conditions at 20 °C, 5 mice for each group.
   2. House the mice in cages placed in chambers set to either 20% or 50% RH for 5-7 days.
      
      
      
2. Influenza infection via aerosol exposure (to be carried out under BSL2 condition)
   1. Dilute hvPR8 2 x 10⁵ pfu/ml in PBS and prepare 4 ml of diluted virus solution.
      Note: This virus concentration is the lethal dose 50 (LD₅₀) for this study. If you change influenza virus or mouse strain, you should determine LD₅₀ for your conditions before the experiment following this protocol. General protocol of measurement of LD₅₀ is reported in Zhao et al. (2018).
   2. Put mice (up to 12 mice simultaneously) in the pie cage.
   3. Set nebulizer and pour 4 ml of diluted virus solution (3 ml is exact volume that would be will be used during 15 min of aerosol delivery).
   4. Turn on the switch for the nebulizer and expose for 15 min (Figure 1 and Video 1).
      
      
      Figure 1. Representation of how to infect influenza virus in mice using a nebulizer. A. Mice are diagonally put into the pie cage if the number of mice is less than 12. B. Image of connected pie cage and nebulizer.
      
      
      
      Video 1. Infection via aerosol administration. (All procedures used in this study complied with federal and institutional policies of the Yale Animal Care and Use Committee (protocol #10365).)
      
      
   5. Put mice back in the cage and house them at respective environmental conditions.
   6. Monitor the weight and death of the mice for 2 weeks (Figure 2 and Video 2). Euthanize the animals according to the guidelines of the institutional animal care and use committee.
      (All procedures used in this study complied with federal and institutional policies of the Yale Animal Care and Use Committee (protocol #10365).)
      
      
      Figure 2. Measuring of body weight Mouse put on the weight scaler to measure the body weight
      
      
      
      Video 2. Measure the body weight

Data analysis

Survival curve and weight loss are analyzed using GraphPad Prism (Figure 3).


Figure 3. Low relative humidity leads to more severe disease. (A) Weight and (B) survival were monitored for 11 days. Data are representative of five experiments and means ± SEM *P < 0.05; one-way ANOVA; log-rank (Mantel-Cox) (Adapted from Kudo et al., 2019).

Acknowledgments

This work was supported in part by the Howard Hughes Medical Institute (A.I.), a gift from the Condair Group, the Naito Foundation (E.K.). An abbreviated version of this protocol was originally published in “Low ambient humidity impairs barrier function and innate resistance against influenza infection” (Kudo et al., 2019).

Competing interests

The authors declare no competing interests.

Ethics

All procedures used in this study complied with federal and institutional policies of the Yale Animal Care and Use Committee (protocol #10365).

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