Schwartz2 project protocol

Survey of ovalbumin-induced lung inflammatory changes in males of 8 inbred strains of mice   (2001)

Schwartz DA, Berman KG, Whitehead GS, Walker JK
With: Walker JKL, Foster WM

Ovalbumin 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, the genetic and molecular mechanisms underlying the responses are poorly understood. The biological variability of the responses indicates that there are environmental influences. To better understand the factors underlying these responses, sensitized and unsensitized mice from different inbred strains were subjected to a classical evaluation of allergen-induced asthma.

For more information, see Whitehead et al, 2003.


Days 0 and 14: Eighteen male mice (6-8 weeks old) were sensitized with 100 µL intraperitoneal (i.p.) injections of ovalbumin (OVA;10 µg chicken egg, Grade V, Sigma, St. Louis MO) complexed with aluminum hydroxide (Alum; AlumInject, Pierce Chemical Company, Rockford IL). Eighteen males from each strain received i.p. injections of Alum alone at the same time points.

Day 20: All mice were subjected to a standard methacholine challenge (0, 5, 10, 20 mg/mL in saline), after which airway hyperreactivity was measured (see below). Three control and three OVA-exposed mice were euthanized via CO2; tissues were collected (see below).

Day 21: The remaining mice were exposed to aerosolized OVA (1% in saline) for one hour. The airway hyperreactivity of 30 mice from each strain (15 exposed, 15 controls) were measured at 24, 48 or 72 hr post-exposure. Mice were euthanized with CO2; tissues were collected (see below).


Lavage fluid cellularity (differentials) data available in Schwartz2 Project Data and Supplemental Data File
Lavage cell counts in whole-lung lavage fluid data available in Supplemental Data File
Airway hyperreactivity (enhanced pause (Penh)) data available in Supplemental Data File

Whole-lung lavage fluid preparation, cell counts, and cell differentials

Standard methods have been described (Deetz et al. 1997, Schwartz et al. 1997). Briefly, red blood cells were cleared from the lungs by infusing isotonic saline (at a pressure of 25 cm H2O) through a catheter inserted from the right ventricle to the pulmonary artery. The lungs were lavaged with 6 mL of saline, and the lavage fluid was collected and centrifuge for 10 min at 2500 rpm. The supernatant was divided into two equal portions and stored at -80°C for later cytokine assessment. The cell pellet was resuspended in lysis buffer to eliminate red blood cells and centrifuged 10 min at 2500 rpm. The cell pellet was resuspended in 1.0 mL Hank's balanced salt solution. Cells were counted with a hemacytometer and cellular differentiation was determined with a Cytospin 3 Centrifuge (Shandon Inc., Pittsburgh PA). Data for selected cell differentials are in the Schwartz2 Project Dataset (Eosinophils, Neutrophils, Lymphocytes). All other data are available in the Supplementary Data File (including total lavage cell count, macrophages, and other airway cells).

• Graphic showing cell differentials at all time points.

Whole Body Plethysmograph (Model PLY 3211 V2.1, Buxco Electronics, Sharon, CT USA).

The validity of Penh as a measure of bronchoconstriction has been examined and demonstrated (Hamelmann et al., 1997, Kline et al., 1998, Quinn et al. 2000). Mice were assessed in a whole body plethysmograph (Buxco Electronics, Inc., Sharon CT). Real time calculations of frequency and breath waveform (expiratory time (Tc), relaxation time (Tr), peak expiratory flow (PEF), peak inspiratory flow (PIF) were performed and recorded electronically. Estimates of airway responsiveness (Penh) were derived from the ventilation and flow-derived parameters as follows: Penh=((T3 ÷ 0.4(Tr-1) X (PEF ÷ PIF)) X 0.67 . The change in airway responsiveness was computed for each animal by subtracting pre-exposure levels from post-exposure levels. The full set of Penh data are available in the Supplementary Data File only.


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