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About 99% of the global population resides in areas with air pollution surpassing World Health Organization standards. Air pollution is associated with adverse neonatal health outcomes such as low fetal birth weight and an increased risk for maternal pre-eclampsia. A particularly reactive air pollutant is ozone, which forms reactive oxygen species that induce cellular damage. Research exists on the dispersion of reactive oxygen species through the bloodstream leading to fetal vulnerability during pregnancy, specifically via the placenta. Yet, placental and fetal development is a temporal process with varied susceptibility to negative gestational outcomes.
To addressing this gap, our laboratory utilized non-targeted proteomic analysis of amniotic fluid collected at term after either gestational day (GD) 10 or GD20 ozone exposure. Results provided a comprehensive list of proteins that indicated distinct outcome phenotypes. The acute GD20 exposure resulted in a potent acute-phase increase in antioxidant factors while the subacute GD10 exposure had a greater influence of growth factors. In follow-up, selected markers of these phenotypes will be assessed within matched placentas. Relevant to the antioxidant GD20 response, we will assess superoxide dismutase 1 (SOD1) and catalase, which catalyze superoxide and hydrogen peroxide, respectively. Per the GD10 subacute response, connective tissue growth factor (CTGF) is produced by cells involved with structure and stabilization of the ECM and affects cellular growth, migration, adhesion, and vascularization. Together with CTGF, collagen T1A2 plays a vital role in the extracellular matrix (ECM) and has been linked to pregnancy complications such as miscarriage, gestational diabetes, and preeclampsia. To assess differential impacts on the placental vasculature, we will be investigating vascular endothelial cell adhesion molecule 1 (VCAM-1) and vascular endothelial cadherin (VE-Cadherin), which have both been identified as biomarkers of preeclampsia.
In our experiments, pregnant Sprague-Dawley rats were exposed once to 0.3 ppm of ozone (O3) or filtered air (FA) via whole-body inhalation at GD10 or GD20 while control animals received a sham filtered air exposure at both times. Placentas were collected and snap-frozen at GD21 followed by thin-sectioning using a frozen microtome and formaldehyde fixation. Primary antibodies to our protein targets are incubated overnight at 4C followed by secondary alexa-fluor conjugated antibodies to allow for multi-channel immunofluorescence detection. Images are generated on a Zeiss Axio Imager.M2 microscope at 200x magnification.
Ongoing experiments are set at optimizing primary antibody concentrations. The experimental design involves creating three wells of two sample placental tissues per slide that are prepared and marked with primary and then secondary antibodies specific to the protein of interest. Each well contains a different dilution of the antibody that yields different fluorescence. Densitometric analysis is used to determine the concentration with the greatest signal-to-noise ratio. Once optimized, antibodies will be co-imaged on placenta tissues across five replicate animal exposure per experimental group. Quantification of mean fluorescence intensity will then be tabulated across decidual, labyrinth and chorionic placental lamina. Results will be assessed using analysis of variance with post-hoc testing for group differences. Expected outcomes will demonstrate the relationship between prior amniotic fluid proteomic findings and effects within the placenta while differentiating placental vulnerability across windows of gestation. These findings will prove significant in understanding outcomes at term for both the mother and fetus when exposed to ozone pollution.
Anatomy and Neurobiology
placenta, ozone pollution, maternal pre-eclampsia, reactive oxygen species, fetal vulnerability during pregnancy, acute/subacute exposure, growth factors, superoxide dismutase 1 (SOD1), catalase, connective tissue growth factor (CTGF), collagen T1A2, vascular endothelial cell adhesion molecule 1 (VCAM-1), vascular endothelial cadherin (VE-Cadherin)
Biochemistry | Developmental Biology | Disorders of Environmental Origin | Female Urogenital Diseases and Pregnancy Complications
Current Academic Year
Dr. Andrew Ottens
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