Intratracheal Inoculation and Ozone Exposure in Mice

Animals were anesthetized with an intramuscular injection of a mixture of Ketamine HCl (Ketaset, Fort Dodge Animal Health, IO) and Xylazine (XYLA-JECT, Phoenix Pharmacueticals Inc., St. Joseph, MO). The trachea was surgically exposed and ~450 CFU/mouse were inoculated intratracheally in 50 μl of PBS. Skin incision was closed with 7 mm wound clips. Deaths during the first 12 h post-infection period were considered to be due to surgical procedure rather than infection and those mice were excluded from study. Sixteen independent experiments (8 males and 8 females) were conducted. Each experiment consisted of 10 mice (5 exposed to FA or to O₃). The mice were monitored for survival for 14 days. The total number of mice used for Figure 1 was 147 mice (75 FA-exposed [39 males+36 females]), and 72 O₃-exposed [40 males + 32 females]). In cases (n = 14) where mice were moribund with no chance of recovery, these were euthanized to prevent unnecessary suffering according to Penn State University Institutional Animal Care and Use Committee recommendations and are included with the natural deaths. For the analysis shown in Figure 2, 130 mice were used out of the total 147 mice. Of these, 60 were males (6 independent experiments out 8) and 70 females (7 independent experiments out 8). Ratios from two experiments using males and from one with females were eliminated from analysis because the values measured were more than 2 standard deviations from the mean.