Influence of gestational window on offspring vascular health in rodents with in utero exposure to electronic cigarettes

Abstract

Studies have shown cerebrovascular dysfunction in offspring with full-gestational electronic cigarette (Ecig) exposure, but little is known about how individual trimester exposure impacts offspring health. This study aimed to determine if there is a critical window during gestation that contributes to vascular and anxiety-like behavioural changes seen with full-term exposure. To test this, rats were time-mated, and the pregnant dams were randomly assigned to Ecig exposure during first trimester (gestational day, GD2-7), second trimester (GD8-14), third trimester (GD15-21) or full-term gestation (GD2-21). We also assessed the effect of maternal preconception exposure. Both male and female offspring from all maternal exposure conditions were compared to offspring from dams under ambient air (control) conditions. Ecig exposure consisted of 60-puffs/day (5 days/week) using either 5 or 30 watts for each respective exposure group. We found that maternal exposure to Ecig in the second and third trimesters resulted in a decrease (23-38%) in vascular reactivity of the middle cerebral artery (MCA) reactivity in 3- and 6-month-old offspring compared to Air offspring. Further, the severity of impairment was comparable to the full-term exposure (31-46%). Offspring also displayed changes in body composition, body mass, anxiety-like behaviour and locomotor activity, indicating that Ecigs influence neurodevelopment and metabolism. Maternal preconception exposure showed no impact on offspring body mass, anxiety-like behaviour, or vascular function. Thus, the critical exposure window where Ecig affects vascular development in offspring occurs during mid- to late-gestation in pregnancy, and both 5 W and 30 W exposure produce significant vascular dysfunction compared to Air. KEY POINTS: Exposure to electronic cigarettes (Ecigs) is known to increase risk factors for cardiovascular disease in both animals and humans. Maternal Ecig use during pregnancy in rodents is found to impair the vascular health of adolescent and adult offspring, but the critical gestation window for Ecig-induced vascular impairment is not known. This study demonstrates Ecig exposure during mid- and late-gestation (i.e. second or third trimester) results in impaired endothelial cell-mediated dilatation (i.e. middle cerebral artery reactivity) and alters anxiety-like behaviour in offspring. Maternal exposure prior to conception did not impact offspring's vascular or anxiety-like behavioural outcomes. Rodent models have been a reliable and useful predictor of inhalation-induced harm to humans. These data indicate maternal use of Ecigs during pregnancy should not be considered safe, and begin to inform clinicians and women about potential long-term harm to their offspring.

Keywords: brain; middle cerebral artery; myography; pregnancy; trimester; vaping.

Figure 1:. Maternal and Offspring Health

Maternal and offspring outcomes with in utero exposure to electronic cigarettes were evaluated by one-way ANOVA with Tukey post hoc testing. Weight gain of dams starting at their pre-pregnancy weight is defined as the weight before time-mating, until gestation day (GD)20 (panel A). The percentage of offspring loss due to reabsorption during pregnancy is determined by the difference in offspring litter size and number of implantation sites observed on the uterus (panel B, * p<0.05 compared to Air). Postpartum deaths in offspring occurring during weaning (i.e. from birth until postnatal day (PD)21) (panel C, * p<0.05 for offspring loss defined as the difference between # pups born vs # pups still alive at weaning compared to Air) (n=5-11/group).

Figure 2:. Weights of Offspring/Trimester

Offspring body mass at 3-months (panel A) and 6-months (panel B) of age corresponding to Ecig exposure at 5(w)atts or 30w evaluated by one-way ANOVA with Tukey post hoc testing (statistical comparisons are only shown relative to Air group). Groups are maternal Ecig exposed at 1^st, 2^nd, or 3^rd trimester, Full-term exposure, or exposure prior to conception (Pre). N=5-11/group. Mean ± SD.

Figure 3:. Offspring MCA

Pressure myography assessment of maximal middle cerebral artery (MCA) vasodilatory reactivity to acetylcholine (ACh at 10⁻⁴ M, panel A & B), sodium nitroprusside (SNP at 10⁻⁴ M, panel C & D), and vasoconstriction response to serotonin (5-HT at 10⁻⁴ M, panel E & F) in offspring at 3- and 6-months of age. (n=5-6/group). Mean ± SD.

Figure 3:. Offspring MCA

Pressure myography assessment of maximal middle cerebral artery (MCA) vasodilatory reactivity to acetylcholine (ACh at 10⁻⁴ M, panel A & B), sodium nitroprusside (SNP at 10⁻⁴ M, panel C & D), and vasoconstriction response to serotonin (5-HT at 10⁻⁴ M, panel E & F) in offspring at 3- and 6-months of age. (n=5-6/group). Mean ± SD.

Figure 4:. Offspring Tempol, L-NAME, Febuxostat

Pressure myography assessment of maximal middle cerebral artery (MCA) vasodilatory reactivity to acetylcholine (ACh) in offspring at 3- and 6-months with full-term maternal exposure to electronic cigarettes either at 5- or 30-(w)atts. Graphs depict the response of the MCA after incubation of a superoxide dismutase mimetic (TEMPOL, panel A & B), nitric oxide inhibitor (L-NAME, panel C & D), and a xanthine oxidase inhibitor (Febuxostat, panel E& F) (n=4-6/group). Mean ± SD.

Figure 4:. Offspring Tempol, L-NAME, Febuxostat

Pressure myography assessment of maximal middle cerebral artery (MCA) vasodilatory reactivity to acetylcholine (ACh) in offspring at 3- and 6-months with full-term maternal exposure to electronic cigarettes either at 5- or 30-(w)atts. Graphs depict the response of the MCA after incubation of a superoxide dismutase mimetic (TEMPOL, panel A & B), nitric oxide inhibitor (L-NAME, panel C & D), and a xanthine oxidase inhibitor (Febuxostat, panel E& F) (n=4-6/group). Mean ± SD.

Figure 5.. Open Field

Offspring anxiety-like behavior was assessed with open field and central tendency (panels A & B) and total activity (panels C & D) at 3- and 6-months of age. N=5-11/group. Mean ± SD.