Mechanisms and Treatment of Halogen Inhalation-Induced Pulmonary and Systemic Injuries in Pregnant Mice

Abstract

Inhalation of oxidant gases has been implicated in adverse outcomes in pregnancy, but animal models to address mechanisms and studies to identify potential pregnancy-specific therapies are lacking. Herein, we show that inhalation of bromine at 600 parts per million for 30 minutes by pregnant mice on the 15th day of embryonic development results in significantly lower survival after 96 hours than an identical level of exposure in nonpregnant mice. On the 19th embryonic day, bromine-exposed pregnant mice have increased systemic blood pressure, abnormal placental development, severe fetal growth restriction, systemic inflammation, increased levels of circulating antiangiogenic short fms-like tyrosine kinase-1, and evidence of pulmonary and cardiac injury. Treatment with tadalafil, an inhibitor of type 5 phosphodiesterase, by oral gavage 1 hour post-exposure and then once daily thereafter, attenuated systemic blood pressures, decreased inflammation, ameliorated pulmonary and cardiac injury, and improved maternal survival (from 36% to 80%) and fetal growth. These pathological changes resemble those seen in preeclampsia. Nonpregnant mice did not exhibit any of these pathological changes and were not affected by tadalafil. These findings suggest that pregnant women exposed to bromine may require particular attention and monitoring for signs of preeclampsia-like symptoms.

Keywords: blood pressure; heart failure; hemodynamics; preeclampsia; pregnancy.

Figure 1. Pregnant mice exhibit increased body weight loss and mortality and fetal growth restriction

Non-pregnant (NP) and pregnant (P) (E14.5) mice were exposed to air or to Br₂ at 600 ppm for 30 min and returned to room air; they received tadalafil (TAD; 2 mg/kg BW in 0.1 ml of sterile saline) or vehicle via oral gavage at 1 h post-exposure and every 24 h thereafter. Body weights and survival times were recorded daily. A) Kaplan-Meyer curves of pregnant and non-pregnant mice with tadalafil or vehicle, post Br₂ exposure. Non-pregnant mice exposed to Br₂ and returned to room air lived longer than similarly exposed pregnant mice (* = P<0.05). Tadalafil improved survival times of pregnant mice post Br₂ (# = P<0.05) but not of non-pregnant mice; n=10–19; Log-Rank Test. B) Body weights normalized to weights of air-exposed mice. Pregnant mice exposed to Br₂ and returned to room air exhibit more severe weight loss compared to similarly exposed non-pregnant mice (* = P<0.05). Tadalafil administration mitigated weight loss in pregnant mice at four days post-exposure, but has no effect in non-pregnant mice; n=6–8; ANOVA. C) Representative ultrasound of a fetus showing how fetal length was measured. D) Summary data of fetal length measurements at E14.5, E16.5, & E18.5. Exposure to Br₂ resulted in decreased fetal lengths which were restored to their air control values at E18.5 in the tadalafil group; n= 6–10 pups (2 pups per litter) for each condition; ANOVA; p values as compared to the corresponding air controls for the indicated gestational age. E) Representative photograph of paraformaldehyde-fixed fetuses at E18.5 for the indicated conditions. Fetuses of Br₂-exposed pregnant mice exhibit severe fetal growth restriction, and tadalafil improves fetal growth. F) Fetal weights were recorded after extraction of fetuses at E18.5. Fetal weights of fetuses from Br₂-exposed pregnant mice weighed considerably less and were partially rescued by tadalafil (TAD); n=pups (11–25) (2 pups per litter); ANOVA. All data are means±S.E.M.

Figure 2. Exposure of pregnant mice to Br₂ damages their placentas

Pregnant (E14.5) mice were exposed to air or 600ppm Br₂ for 30 minutes, returned to room air and received tadalafil (TAD) or vehicle. A–B) Representative H&E stained (A) placenta sections at E18.5 with the junctional zone demarcated with yellow highlighting (A) as well as (B) PAS staining (left) and CDX2 staining (right) of Br₂-exposed pregnant mice revealed a reduced junctional zone (B: black bars) at E18.5; tadalafil administration restored junctional zones to normal size. C) Junctional zone areas at E18.5 for the indicated groups; n=9–20; ANOVA. D–E) TNFα mRNA (n=6–23) was reduced in Br₂-exposed pregnant mice treated with tadalafil. sFLT-1/FLT-1 mRNA (=12–18) was increased in placentas of Br₂-exposed pregnant mice at E18.5 (ANOVA), and was reduced to air control values by tadalafil. F) sFLT-1 in plasma at E18.5 increased in pregnant mice exposed to Br₂ and was reduced with tadalafil; ANOVA. Only one placenta per pregnant mother was used. All data are means±S.E.M.

Figure 3. Pregnant mice exhibit pulmonary injury 96 h post-exposure

Non-pregnant and pregnant (E14.5) mice were exposed to air or 600ppm Br₂ for 30 minutes, returned to room air, then administered tadalafil (TAD) or vehicle. A) Lung wet/dry weight ratios at E18.5 are increased in pregnant mice exposed to Br₂ and are reduced by tadalafil. B) Protein concentrations in the bronchoalveolar lavage fluid (BALF) of pregnant mice were increased at E18.5 and returned to the air control values following administration of tadalafil (TAD). C–F) P_aO₂ was unchanged, P_aCO₂ increased, [H⁺] increased and S_aO₂ was decreased in pregnant mice exposed to Br₂ at E18.5. Administration of Tadalafil returned [H⁺] and S_aO₂ to their air control values but did not improve P_aCO₂; All data n=6–14 mice; ANOVA; means±S.E.M.

Figure 4. Exposure to Br₂ increases the systemic blood pressure of pregnant mice and decreases cGMP in the lungs, placentas and aortas

Non-pregnant and pregnant (E14.5) mice were exposed to air or 600ppm Br₂ for 30 minutes, returned to room air, then administered tadalafil (TAD) or vehicle. At E18.5, a pressure-transducer catheter was inserted into either the aortic arch via the carotid or into the right ventricle through the jugular vein in anesthetized mice. A–B) Br₂ has no significant effect on systemic systolic or diastolic blood pressures in non-pregnant mice (NP). However, systemic diastolic pressures were increased in pregnant mice (P). Both systemic systolic and diastolic pressures were decreased to their corresponding air controls following tadalafil administration; n=6–11; ANOVA. C) Right ventricular (RV) systolic pressures remained unchanged in pregnant mice exposed to Br₂. D–F) Lung tissue, placenta, and aorta cGMP levels of Br₂-exposed pregnant mice decreased significantly. Lung and placenta cGMP levels were restored following tadalafil administration; n=6–7 per group; ANOVA. All data are means±S.E.M.

Figure 5. Pregnant mice exposed to Br₂ demonstrated systemic inflammation at E18.5

Non-pregnant and pregnant (E14.5) mice were exposed to air or 600ppm Br₂ for 30 minutes, returned to room air, then administered tadalafil (TAD) or vehicle. A–C) Plasma TNFα, IL-6, and KC/GRO increased in pregnant mice exposed to Br₂ but only IL-6 increased in non-pregnant mice post-Br₂. All three cytokines in pregnant mice exposed to Br₂ returned to air control values following tadalafil administration; n=6–12 per group; ANOVA. All data are means±S.E.M.

Figure 6. Br₂-exposed pregnant mice exhibit diminished cardiac function at E18.5

Non-pregnant and pregnant (E14.5) mice were exposed to air or 600ppm Br₂ for 30 minutes, returned to room air, then administered tadalafil (TAD) or vehicle. A) Representative echosonography LV and RV traces at E18.5 of pregnant mice exposed to air or Br₂ with demarcation of ventricular sizes. HR = Heart rate, A = Area, A;s = Area systole, A;d = Area diastole, FAC = Fractional area change. B) Cardiac output (LV) determined by echosonography was decreased in Br₂-exposed pregnant mice, which increased following administration of tadalafil; ANOVA: n=6–8. C) Ejection fraction (LV) was unchanged in all groups; ANOVA. D) LV end-systolic volume was similar in all groups. E) However, LV end-diastolic volume was diminished in Br₂-exposed pregnant mice; n=6–8; ANOVA. All data are means±S.E.M.