A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Summary

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Abstract

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible. Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples. Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated. Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated. AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined. Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed. These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge. This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.

Protocol

Allergen challenge:

1. C57BL/6 mice, 4-8 weeks of age, are anesthetized in an airtight plexiglass chamber purged with a 3.2% isoflurane in oxygen vapor mixture for 10 minutes to achieve deep general anesthesia.
2. Intranasal allergen challenges (45μL OVA (22.5 μg) and 7μL A. Oryzae (7 μg), in PBS) are administered, every Tuesday, Thursday and Sunday, for a total of seven consecutive applications.

Anesthesia:

3. Prior to each allergen challenge, and following the 7th challenge, mice are administered an intraperitoneal injection of 48 mg/kg etomidate (2 mg/ml), prior to placement in a light-exc....

Discussion

The study of asthma, and various other airway obstructive diseases, constitutes an active and expanding field of biomedical research. An important component of asthma-related experimental research is the capacity to measure changes in airway size under varying conditions. Excessive airway narrowing in response to provocative challenge, a canonical feature of asthma and related lung diseases and a property of the airway termed airway hyperresponsiveness, is a major component of clinically significant attacks leading to sh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Airway physiology measurement software (Rescomp)	Millenium Premier Group; 415-519-4371		Custom prepared
PC workstation running Windows XP equipped with a Pentium III CPU	Intel		Data analysis
17-pin analog to digital signal converter	National Instruments	PC-LPM16)
0.5mm external diameter fiber-optic thread, connected to light source	Cole-Parmer	41722 series
Ventilator	Harvard Apparatus	687
10 mm, 27ga needle	BD Biosciences	309602
Heat lamp
1 ml syringe	BD Biosciences	305109
4 spring clamps	Pony	3200
0.5 mm external wire for intubation guide
Hemacytometer
Superfrost/plus microscope slides	Fisher Scientific	12-550-15
Shandon Filter Cards	Thermo Fisher Scientific, Inc.	5991022
Differential cell slide stain	Fisher Scientific	122911
Light microscope	Leica Microsystems
Cytospin 3	Shandon, Inc.
20 ga, 1.25 inch ProtectIV intravenous catheters	Smiths Medical
0.5 mm polymer optical fiber	Edmund Scientific	NT02-532
Small animal airway physiology workstation, Custom assembled by Millenium Premier Group using:
Commercially available pressure transducers	Buxco Research Systems	TRD5700
Commercially available pressure transducers	Buxco Research Systems	TRD4510
Preamp modules	Buxco Research Systems	MAX2270
Chassis	Buxco Research Systems	MAX1320
Customized small animal plethysmograph