The overall goal of this experiment is to induce chronic obstructive pulmonary disease or C-O-P-D via ozone exposure, O-E in mice. This new OE model can happen in the characterization of the underlying mechanism of COPD and it can be used for the screening of new drugs. The main adavantages of this OE model are that it recapitulates the pathological processes of COPD patients.
It can be generated in only six to eight weeks. Demonstrating the procedure with me will be Weijaian Sang, a research fellow in our group. To generate ozone, use an electric generator and a small air blower to blow the air out sealing Perspex box through an air vent pipe connected on the inside and outside of the box using an ozone probe to monitor the concentration of ozone in the box.
When the ozone concentration reaches 2.5 parts per million place the mice into the box for 3 hours. At the end of week 7, image the ozone treated animals by micro-computed tomography according to standard protocols. To test the ozone induced changes and functional capacities such as lung fatigue, first warm up the mice at a speed of 10 meters per minute on a treadmill.
After 5 minutes, increase the speed to 15 meters per minute for a period of 10 minutes. Continue to increase the speed by five meters per minute for every 30 minutes until the mice can no longer run. Recording the total running distance and running time for each animal as fatigue distance and fatigue time, respectively.
To measure the pulmonary function of the ozone exposed animals, place an anesthetized mouse in a body plethysmograph connected to a computer controlled ventilator. Use pressure controlled ventilation to impose an average breathing frequency of a 150 breaths per minute to the anesthetized animal until a regular breathing pattern and a complete expiration at each breathing cycle is obtained. Then record the forced expiratory volume in the first 25, 50, and 75 milliseconds of exhalation rejecting any suboptimal maneuvers.
Following terminal anesthesia, use a one millimeter diameter endotracheal tube to lavage the animals two times with one milliliter of PBS per lavage. Pool the retrieved lavage aloquats from each animal for centrifugation and collect the supernatants on liquid nitrogen. Re-suspend the pellets in fresh PPS for counting and equip two holders per mouse with a microscope slide, filter card, and funnel.
Add approximately 100 to 125 microliters of the re-suspended cells to each funnel. Load the holders on to a cyto centrifuge and spin the cells on to the slides. Then apply right staining solution to the cells according to the manufacturer's instructions and count 200 cells per mouse under a 400 times magnification.
For cardiac blood sampling, collect approximately 500 microliters of blood from the ozone exposed animals via cardiac puncture. And add the blood to 1.5 milliliter tubes on ice. After 30 minutes on ice, centrifuge the blood samples and transfer the serum supernatants into new tubes for storage on liquid nitrogen.
The samples can later be thawed for the assessment of serum cytokine expression levels by ELISA according to standard protocols. For lung morphometric analysis, place the first mouse in the supine position after cardiac sampling. Then open up the thoracic cavity and dissect away the platysma and the anterior tracheal muscles to access the tracheal rings.
Harvest the lungs and the trachea without separating the heart from the lungs. Next use a PE90 polyethylene tube to connect an endotracheal catheter to a syringe containing 4%power formaldehyde and completely inflate the lungs with approximately 200 microliters of paraformaldehyde. Then place the lungs in a 15 milliliter tube containing 10 milliliters of fresh four percent power formaldehyde for at least four hours.
As assessed by microcomputed tomography, ozone exposed mice exhibit a significantly larger total lung volume and percentage of low attenuation area than ambient air exposed control animals. The emphysema phenotype is also evidence by the enlarged lung alveoli and increased mean linear intercepts. Lung function assessment by plethysmograph demonstrates that all the parameters that decrease in ozone exposed mice are consistent with the typical lung functional defects observed in chronic obstructive pulmonary disease patients.
Further, ozone exposure significantly decreases the fatigue time and fatigue distance in an exercise tolerance tests. Ozone treated animals also display significant increase in inflammatory cells including macrophages and neutrophils as well as a significant decrease in IL-10 and an increase in IL-1 beta and TNF alpha demonstrating that the ozone exposure model recapitulates human chronic obstructive pulmonary disease like symptoms. Following this procedure on other strains of mice like the C57 BR6 mice can be used to generate this OE model After its development, this technique paves the way for researchers in the field of pulmonary disease to explore the underlying mechanisms of COPD.
Don't forget that working with the paraformaldehyde can be hazardous and that precautions such as wearing gloves, safety glasses, and using the solution inside the fume hood should always be taken while working with this reagent.
