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Berndt1 project protocol

Methacholine-induced changes in airway resistance in 37 inbred strains of mice   (2010)

Berndt A, Leme AS, Paigen B, Shapiro SD




Berndt1_Protocol

Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure for measuring airway resistance
Definitions
Data
References

Workflow and sampling

Workflow

Step

Procedure performed
Methacholine

Age (wk)

Data collected
1
All mice raised and maintained in colonies at The Jackson Laboratory
0
<6-11
-
2
Mice strains shipped to the University of Pittsburgh, where they are maintained under the similar conditions as vendor
0
>6-11
-
3
 Mice are allowed to acclimate in their new facility for <3 wks
0
9-12
-
4a
Mice are weighed, anesthetized, and undergo pre-treatment testing
0 (pre-treatment)
9-12
pre-treatment airway resistance (R)
4b
While still under anesthesia, mice are challenged with saline and tested again to obtain baseline measurements
0 (saline)
9-12
baseline airway resistance (R)
4c
Following saline challenge, mice are challenged with 4 incremental and consecutive methacholine doses (mg/kg BW)
1, 3, 10, 30 mg/mL
9-12
airway resistance (R)

Equipment and supplies

  • Balance scale
  • Electric hair clippers such as the Oster Finisher Trimmer (Cat. # 76059-030, Oster Professional Products, McMinnville, TN)
  • 19G cannula
  • small rodent dissecting/surgical pack (forceps, scissors)
  • Computer-controlled ventilator (flexiVent, SCIREQ, Montreal, QC, Canada)

Reagents and solutions

  • 5-0 USP surgical suture
  • Methacholine (Sigma–Aldrich Inc., St. Louis, MO, USA)
  • Pentobarbital sodium (Lundbeck Inc./Ovation Pharmaceuticals Inc., DeerWeld, IL, USA)

Procedure for measuring airway resistance

Methacholine sensitivity
Airway hyper-responsiveness is one of the defining characteristics of allergen-induced asthma. Although it is well documented that asthmatic patients hyper-respond to a variety of bronchoconstrictor agonists (i.e. methacholine), the genetic and molecular mechanisms underlying variability in individual responses are poorly understood, suggesting that there are environmental factors involved. To better understand these factors, inbred strains of mice, both sensitized and unsensitized, are subjected to a classical evaluation of allergen-induced asthma.

a. Body weights are measured for the calculation of anesthetic dose.
b. Mice are anesthetized by intraperitoneal (i.p.) injection of 60 mg/kg pentobarbital sodium.
b. The mice are positioned ventro-dorsally (supine) for tracheotomy (see Figure A-D below).
d. A 19G cannula is gently inserted into the trachea and secured using 5-0 USP surgical suture.
e. The cannula is then attached to the ventilator.
f. For the initial readings, a deep lung inflation of 30 cm H2O pressure is applied to each naive untreated mouse.
g. Aerosolized saline is then administered through the tracheotomy for baseline measurements.
h. Using a computer-controlled ventilator, airway resistance in response to methacholine inhalation is measured.
i. Following baseline readings, increasing doses of methacholine concentrations (1, 3, 10, and 30 mL/kg) are administered via nebulizer for 10 s for the measurement of airway hyper-responsiveness (AHR).
j. The mice are sacrificed after measuring AHR.

Definitions and calculations

For the determination of airway resistance, a constant phase model is fitted to the data from multiple frequencies simultaneously applied at the airway opening. After each of the 5 challenges, airway resistance (Raw) in 12 cycles of 30–60 s is recorded. Changes in Raw as a function of the log-transformed methacholine concentration administered is then plotted. AHR is summarized as the slope of the curve Raw versus log-transformed methacholine concentration.

Resistance and Compliance are most commonly estimated using mathematical model-fitting techniques such as Multiple Linear Regression that take all data points into account.

Measurements in passive subjects
• passive anaesthetized subjects, the single compartment model is commonly fit to airway opening pressure and flow obtained from a body plethysmograph during mechanical ventilation with passive expiration.
• However, it is advantageous to fit the model to data obtained during a Single-frequency Forced Oscillation maneuver where amplitude and frequency content are precisely standardized. This is the technique used by the flexiVent.
• In both cases, measurements represent respiratory system mechanics that include both lung and chest wall components (unless the chest wall is surgically opened).

R = Resistance = Dynamic resistance quantitatively assesses the level of constriction in the lungs.

MPD calculation: percent airway resistance = ((airway resistance with methacholinesaline - airway resistance with saline) ÷ airway resistance with saline) X 100

Data collected by investigator

Airway resistance (R)