Representative examples of ciliated epithelial cells in the trachea of control and drug-treated mice.

<p>Tissue was evaluated at 100 X and at 40 X magnification for the control (A and B) and drug-treated mice (C and D), respectively. Black arrows point to cilia. Qualitatively, the presence of ciliated epithelial cells in the airways in each of the representive animals is similar.</p

iNOS staining in airways of vagotomized mice.

<p><b>A.</b> Increased iNOS staining (brown) in bronchial epithelium of adult mouse treated with immunosuppressive agents (Black arrow). iNOS staining was qualitatively increased in the bronchial epithelium of five of the five drug-treated mice. <b>B.</b> Minimal iNOS staining in airway of adult mouse that was not treated with immunosuppressive agents. Minimal iNOS staining was observed in five of the five mice not treated with immunosuppressive agents. <b>C.</b> No first antibody control.</p

PAS staining of seromucous cells in tracheal glands of vagotomized mice.

<p><b>A.</b> Trachea of mouse treated with immunosuppressive agents. <b>B.</b> Trachea of mouse not treated with immunosuppressive agents. Black arrows point to PAS staining in seromucous cells. Qualitatively, both the drug-treated animals and control mice appeared to have similar PAS staining in the seromucous cells of the tracheal glands.</p

Mean MCC (±SD) from the right lung between 1–1.5 hours and 6–6.5 hours in 7 C57BL/6 control mice (dark bar) and 8 drug-treated C57BL/6 mice (light bars).

<p>There was a trend toward slower clearance in the drug-treated mice, compared to controls, at 1–1.5 hours, but differences in MCC were not statistically significant. Mucociliary clearance was statistically significantly slower in the drug-treated mice, compared to controls, at 6–6.5 hours (p = 0.006).</p

Adult Behavior in Male Mice Exposed to E-Cigarette Nicotine Vapors during Late Prenatal and Early Postnatal Life

<div><p>Nicotine exposure has been associated with an increased likelihood of developing attention deficit hyperactivity disorder (ADHD) in offspring of mothers who smoked during pregnancy. The goal of this study was to determine if exposure to E-cigarette nicotine vapors during late prenatal and early postnatal life altered behavior in adult mice.</p><p>Methods</p><p>Timed-pregnant C57BL/6J mice were exposed to 2.4% nicotine in propylene glycol (PG) or 0% nicotine /PG once a day from gestational day 15 until delivery. After delivery, offspring and mothers were exposed to E-cigarette vapors for an additional 14 days from postnatal day 2 through 16. Following their last exposure serum cotinine levels were measured in female juvenile mice. Male mice underwent behavioral testing at 14 weeks of age to assess sensorimotor, affective, and cognitive functional domains.</p><p>Results</p><p>Adult male mice exposed to 2.4% nicotine/PG E-cigarette vapors had significantly more head dips in the zero maze test and higher levels of rearing activity in the open field test compared to 0% nicotine/PG exposed mice and untreated controls. In the water maze test after reversal training, the 2.4% nicotine/PG mice spent more than 25% of time in the new location whereas the other groups did not.</p><p>Conclusion</p><p>Adult male mice exhibited increased levels of activity in the zero maze and open field tests when exposed to E-cigarette vapor containing nicotine during late prenatal and early postnatal life. These findings indicate that nicotine exposure from E-cigarettes may cause persistent behavioral changes when exposure occurs during a period of rapid brain growth.</p></div

(A) Daily weights during 14 days of postnatal exposure to E-cigarette vapors starting at PN day 2. From 7–14 days of postnatal exposure, pups exposed to 0% nicotine/PG or 2.4% nicotine/PG E-cigarette vapors had significantly lower weights compared to untreated mice (RA) (* p< 0.02). Mice exposed to 0% Nic/PG E-cigarette vapors were significantly smaller than 2.4% Nic/PG and room air mice throughout the exposure (p<0.03, error bars represent standard error of the means) (n = 11–31). (B) Serum cotinine levels from female pups exposed to 14 days of 2.4% Nic/PG or 0% Nic/PG E-cigarette vapors or room air controls (n = 4–7, error bars represent standard deviations).

<p>(A) Daily weights during 14 days of postnatal exposure to E-cigarette vapors starting at PN day 2. From 7–14 days of postnatal exposure, pups exposed to 0% nicotine/PG or 2.4% nicotine/PG E-cigarette vapors had significantly lower weights compared to untreated mice (RA) (* p< 0.02). Mice exposed to 0% Nic/PG E-cigarette vapors were significantly smaller than 2.4% Nic/PG and room air mice throughout the exposure (p<0.03, error bars represent standard error of the means) (n = 11–31). (B) Serum cotinine levels from female pups exposed to 14 days of 2.4% Nic/PG or 0% Nic/PG E-cigarette vapors or room air controls (n = 4–7, error bars represent standard deviations).</p

Open Field Test.

<p>(A-B) There was a main effect of Day (F_1,25 = 4.994, p = 0.035). There was no effect of Treatment or Day x Treatment interaction. (C) <i>t</i>-tests were used to compare distance travelled on Day 1 between the 2.4% nicotine/PG exposed mice and each of the other two groups. Mice exposed to 2.4% nicotine/PG travelled significantly greater total distances than mice exposed to 0% nicotine/PG (t₁₉ = 2.205, p<0.04) and tended to travel a greater distance than mice exposed to room air (t₁₃ = 1.857, p<0.09). (D) Rearing behavior was significantly increased in mice exposed to 2.4% nicotine/PG compared to the other groups. This was confirmed in a significant main effect of Treatment (F_1,25 = 7.438, p = 0.003) in the absence of an effect of Day or Day x Treatment interaction. Analysis of each individual day revealed mice exposed to 2.4% nicotine/PG had a greater number of rears than mice exposed to 0% nicotine/PG on both days (t test p<0.0001, Day1; p<0.007, Day 2), and also a greater number of rears than mice exposed to room air on Day 1 (p<0.008). (n = 7–13)</p

Water Maze testing with reversal learning.

<p>(A) Latency to the hidden platform during the initial place training phase. Over the course of training all three groups learned equally well to navigate to the hidden platform. (B) Illustration of data from trials in which the platform was unavailable. The figure shows the percent of time each group spent in the quadrant where the platform is normally located (Quad 1- target platform) versus the quadrant opposite of that (Quad 3). All three groups spent more time in the Target quadrant (Quad 1) relative to Quad 3 (p<0.001), (white line represents chance levels (25%). (C) Latency to locate hidden platform at new location in reversal training trials. Similar to training during initial place learning, all three groups decreased the time to the escape platform in a similar manner (p<0.001). (D) The percent time in each quadrant during the final probe trial after all training has been completed. In this trial the 2.4% nicotine/PG exposed mice trended towards a spatial bias for the new platform location, however analysis yielded only a marginal effect of Quadrant (F_1,25 = 2.986, p < 0.096). The marginal effect prompted an analysis by one sample <i>t</i>-tests to compare each groups time in the new location to chance (25%), and only the 2.4% nicotine/PG exposed mice spent significantly more than 25% of time in the new location (t₇ = 2.632, p < 0.034, n = 7–13).</p

Elevated Zero Maze Test.

<p>Head dips were significantly greater in the 2.4% nicotine/PG mice compared to either the 0% nicotine/PG or the untreated mice (RA). (n = 7–13)</p

Rotarod Test.

<p>Mean latency to fall during the three training sessions. There was no difference between any of the treatment groups from day 1 through day 3. As training progressed all groups of mice demonstrated increased latencies before falling which was confirmed by ANOVA, which yielded a main effect of Day (p < 0.001). (n = 7–13)</p