Defense Date

2020

Document Type

Thesis

Degree Name

Master of Science

Department

Anatomy & Neurobiology

First Advisor

Andrew Ottens

Abstract

Ambient outdoor ozone, a common of component of photochemical smog and urban air pollution, is linked to various neurological and vascular pathologies. Its immediate reaction with lung surfactant after inhalation results in complete reactivity of the gas, with no active ozone passing into circulation. This indicates the presence of secondary and tertiary mediators in ozone-related systemic pathologies after pulmonary insult. In vasculature, ozone exposure is associated with an acute hypertensive phenotype apparent at least 24 hours after dose, such as experienced on a hot summer afternoon in a large metropolitan area like Los Angeles or Mexico City. However, the effects of ozone have been underexplored during gestation, when inducible teratogenesis during critical periods for development can result in catastrophic fetal outcomes, but also when more subtle impacts on placental supply can hamper sensitive developmental aspects such as in the brain. As the interface of maternal-fetal circulation, the placenta is a target organ of toxicological interest in hypertensive disorders of pregnancy such as preeclampsia. Moreover, the developing fetal blood-brain barrier (BBB) is underexplored, especially in relation to toxicological study. In the study at hand, proteomic analysis of amniotic fluid (AF) was assessed after exposure of pregnant Sprague-Dawley rats at two gestational timepoint representing critical periods during rat development. The AF proteome is an understudied window into the privileged amniotic environment and its response to toxicological stressors. Pregnant dams experienced a single 4-h exposure to ozone at GD10, a critical period of placental and BBB development, or at GD20, a period just prior to term and one of anatomical significance in brain development. AF samples were then collected at term for detailed proteomic analysis. Studies assessed: AF proteomic impacts between GD10 and GD20 time points; the differential response to 0.3 ppm or 1.0 ppm ozone dosage at GD20; and the overlap between AF proteomic and maternal serum proteomic responses to 0.3 ppm or 1.0 ppm GD20 ozone exposures. Subsequently, a plan was devised for targeted immunofluorescence microscopy assessments on the pathological effects within the placental and blood-brain barriers linked with the toxicological ozone response observed within the AF proteome.

Overall, we identified 231 significant AF proteomic responses across all time points and ozone doses within the amniotic fluid. To our knowledge, this is the most comprehensive assessment of the AF proteome. Overlap was low between the AF proteomic responses to gestational exposure time or ozone dose, while considerably more consistent for maternal serum responses. The highly dynamic AF proteomic response to ozone, however, consistently related to extracellular matrix and vascular remodeling processes, many proteins with well-defined roles in the placenta and developing conceptus. Results with maternal serum indicated activation of the maternal compliment cascade system and systemic inflammation. Additionally, limited overlap between maternal serum and matched AF proteomes indicated the placenta or fetus as the primary contributors to the AF proteomic response to ozone. With placentation and cerebral angiogenesis just beginning in rat at GD10, we hypothesized a dramatic effect on these structures to be assessed with the devised immunofluorescence analysis. In placenta, we expect to observe conditions consistent with a hypertensive preeclamptic-like state. Our AF proteomic results showed significant fold changes in proteins associated with this disease such as galectin-1, MMP-2, TIMP-2, and osteopontin. In follow-up targeted analysis of these proteins in placenta, we expect to observe classic signs of preeclampsia and general placental dysfunction—shallow invasion of the maternal endometrial decidualized tissue layer and inadequate remodeling of the maternal spiral arteries resulting in inadequate fetal circulation. AF proteomic data also showed shifts in proteins important for vascular development in the fetal brain such as TGFβ, PKM and neuropilin-1. Thus, we devised follow-up studies to test for ozone-induced BBB barrier dysfunction within the fetal brain to include augmented tight-junction proteins, albumin leakage and promoted astrocytic and microglial reactivity proximal to the cerebrovasculature. Overall, the outlined studies have potential to demonstrate stark effects of critical-period ambient ozone exposure on the placenta and developing fetus.

Rights

© Alexander I. Hamm 2020

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-2-2020

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