Neuroinflammatory and Behavioral Outcomes Measured in Adult Offspring of Mice Exposed Prenatally to E-Cigarette Aerosols

Abstract

Background: In an effort to decrease the rates of smoking conventional tobacco cigarettes, electronic cigarettes (e-cigarettes) have been proposed as an effective smoking cessation tool. However, little is known about their toxicological impacts. This is concerning given that e-cigarette use is perceived as less harmful than conventional tobacco cigarettes during pregnancy for both the mother and fetus.

Objective: The goal of this study was to test the neurodevelopmental consequences of maternal e-cigarette use on adult offspring behavior and neuroimmune outcomes.

Methods: Pregnant female CD-1 mice were randomly assigned to one of three treatment groups ($n=8-10\text{per}\text{group}$) and exposed daily to either filtered air, propylene glycol and vegetable glycerol (50:50 PG/VG vehicle), or to PG/VG with $16\mathrm{mg}/\mathrm{mL}$ nicotine ($+\text{Nic}$). Whole-body exposures were carried out for 3 h/d, 7 d/week, from gestational day (GD)0.5 until GD17.5. Adult male and female offspring (8 weeks old) were assessed across a battery of behavioral assessments followed by region-specific quantification of brain cytokines using multiplex immunoassays.

Results: Adult offspring of both sexes exposed to $+\text{Nic}$ exhibited elevated locomotor activity in the elevated plus maze and altered stress-coping strategies in the forced swim task. Moreover, male and female offspring exposed to PG/VG with and without nicotine had a 5.2% lower object discrimination score in the novel object recognition task. In addition to differences in offspring behavior, maternal e-cigarette exposure with nicotine led to a reduction in interleukin (IL)-4 and interferon-gamma ($\text{IFN}\gamma$) in the diencephalon, as well as lower levels of hippocampal $\text{IFN}\gamma$ (females only). E-cigarette exposure without nicotine resulted in a 2-fold increase of IL-6 in the cerebellum.

Discussion: These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.

Figure 1.

Diagram of experimental procedures and sample size. The number of male and female mice in each treatment group is included for each behavioral task. Brains from a subset of mice were used for region-specific brain cytokine measures. Note: F, female; FA, filtered air; M, male; PG/VG, propylene glycol/vegetable glycerin; $+\text{Nic}$, propylene glycol/vegetable glycerin plus nicotine.

Figure 2.

Weights of pregnant dams and offspring exposed to filtered air (FA), e-cigarette aerosol (PG/VG), or e-cigarette aerosol with nicotine. (A) Pregnant dams were weighed daily throughout gestation ($+\text{Nic}$). Offspring were weighed on (B) PND21 and again in (C) adulthood at the completion of behavioral testing, and body mass (in grams) were compared across treatment groups. For maternal weight, plots are mean $\pm \text{SE}$ and trend lines represent estimated growth curves using linear mixed-effects modeling with treatment and day as fixed effects and dam as random effects. For offspring weights, *$p<0.05$, **$p<0.01$ as determined by linear mixed-effects modeling with treatment and sex as fixed effects and litters as random effects. Plots represent individual mice; bars represent marginal means $\pm \text{SE}$. FA (10 litters), males [$n=55$ (PND21) and $n=23$ (adulthood)], females [$n=41$ (PND21) and $n=23$ (adulthood)]; PG/VG (9 litters) [males $n=41$ (PND21) and $n=20$ (adulthood)], females [$n=45$ (PND21) and $n=23$ (adulthood)]; $+\text{Nic}$ (8 litters) males [$n=33$ (PND21) and $n=19$ (adulthood)], females [$n=45$ (PND21) and $n=18$ (adulthood)]. Note: PG/VG, propylene glycol/vegetable glycerin; PND, postnatal day; SE, standard error; $+\text{Nic}$, propylene glycol/vegetable glycerin plus nicotine.

Figure 3.

Stress-coping behaviors as determined by the elevated plus maze, open field, and forced swim tests, respectively, in male and female adult offspring of mice treated with filtered air (FA), E-cigarette aerosol (PG/VG), or E-cigarette aerosol with nicotine ($+\text{Nic}$). Pregnant female mice were exposed to either FA or e-cigarette aerosol (PG/VG) with or without nicotine, and offspring were tested using the (A,B) elevated plus maze, (C,D) open field, and (E,F) forced swim tasks. Reported is (A) the percentage of time offspring spent in the open arm of the elevated plus maze as a percentage of the total time spent in both open and closed arms; (B) total distance traveled during the 5-min elevated plus maze task; (C) time spent in the center of an open field arena during the 20-min open field task; (D) the total distance traveled in the open field task; and the total time spent (E) swimming and (F) immobile in the forced swim task. *$p<0.05$, **$p<0.01$ as determined by linear mixed-effects modeling with treatment and sex as fixed effects and litters as random effects. Plots represent individual mice; bars represent marginal means $\pm \text{SE}$. FA (10 litters) [males ($n=23–27$), females ($n=23–24$)]; PG/VG (9 litters) [males ($n=19–21$), females ($n=22–23$)]; $+\text{Nic}$ (8 litters) [males ($n=19$), females ($n=18$)]. Note: EPM, elevated plus maze; FST, forced swim task; OF, open field; PG/VG, propylene glycol/vegetable glycerin; SE, standard error; $+\text{Nic}$, propylene glycol/vegetable glycerin plus nicotine.

Figure 4.

Measure of short-term memory in offspring of dams exposed to filtered air (FA), e-cigarette aerosol (PG/VG), or e-cigarette aerosol with nicotine ($+\text{Nic}$). Offspring were given 10 min to explore two identical objects and then assessed 24 h later for memory performance by replacing one familiar object with a novel object. (A) Total time spent sniffing two objects during the initial learning phase; (B) percentage of novel object recognition determined as the time spent with the novel object over total object exploration time; and (C) time spent sniffing the familiar and novel object 24 h later during the testing phase. *$p<0.05$, **$p<0.01$ as determined by liner mixed-effects model with treatment and sex as fixed effects (A,B) or as separate models for each treatment group (C) and individual animals nested in litter as random effects. Plots represent individual mice; bars represent marginal means $\pm \text{SE}$. FA (10 litters) [males ($n=25$), females ($n=24$)]; PG/VG (9 litters) [males ($n=20$), females ($n=23$)]; $+\text{Nic}$ (8 litters) [males ($n=19$), females ($n=18$)]. Note: NOR, novel object recognition; PG/VG, propylene glycol/vegetable glycerin; SE, standard error; $+\text{Nic}$, propylene glycol/vegetable glycerin plus nicotine.

Figure 5.

Brain cytokine measurement in offspring of dams exposed to filtered air (FA), e-cigarette aerosol (PG/VG), or e-cigarette aerosol with nicotine ($+\text{Nic}$). Following behavioral assessments, offspring from e-cigarette-treated and control dams were sacrificed and brains processed for cytokine quantification using bead-based multiplex assays. Reported are (A) $\text{IFN}\mathrm{\gamma }$ levels in the hippocampus and (B) correlation with swimming behavior in the forced swim test, (C) $\text{IFN}\mathrm{\gamma }$ in the diencephalon and (D) correlation with swimming behavior, (E) IL-4 concentrations in the diencephalon, and (F) IL-6 concentrations in the cerebellum. *$p<0.05$, **$p<0.01$ as determined by two-way factorial ANOVA followed by simple main effects analysis and Tukey post hoc. Correlations were tested using Spearman’s rho. Plots represent individual mice; bars represent marginal means $\pm \text{SE}$; scatterplots include both male and female offspring. FA (8 litters) [males ($n=7–8$, females $n=7–8$)]; PG/VG (8 litters) [males ($n=7–8$), females ($n=7–8$)]; $+\text{Nic}$ (8 litters) [males ($n=7–8$, females $n=7–8$)]. Note: ANOVA, analysis of variance; IFN, interferon; IL, interleukin; PG/VG, propylene glycol/vegetable glycerin; SE, standard error; $+\text{Nic}$, propylene glycol/vegetable glycerin plus nicotine.