TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

EXAMINATION OF THE EFFECTS OF VALPROIC ACID EXPOSURE OR P300 PROTEIN INHIBITION ON THE BALANCE OF APOPTOSIS AND PROLIFERATION IN P19 EMBRYONAL CARCINOMA CELLS

Valproic acid (VPA), a commonly-used anticonvulsant drug, is associated with increased risk of fetal malformations, including neural tube defects (NTDs). Previous in vivo studies determined that VPA-exposed embryos with a NTD had altered expression of several proteins regulated by p300, a histone acetyltransferase (HAT) protein. p300 is capable of acetylating histones and non-histone proteins through its HAT activity, allowing it to transcriptionally regulate genes as well as modulate the stability and activity of specific proteins. NFκB, Stat3 and Egr1, all of which function as transcription factors, are regulated by p300 through its HAT activity. Together, these proteins all play an important role in maintaining the balance of apoptosis, proliferation and differentiation, the regulation of which is extremely important for proper embryonic development. The studies in this thesis utilized P19 embryonal carcinoma (EC) cells in order to determine the effects of VPA exposure on the expression of p300 and the aforementioned transcription factors, as well as apoptosis and proliferation, in vitro. P19 EC cells were exposed to C646, a selective p300 inhibitor, in order to assess whether the effects observed as a result of VPA exposure were due to p300 protein degradation. It was found that VPA exposure for 24 hours in P19 EC cells in vitro resulted in a significant decrease in p300 protein expression. VPA exposure also significantly decreased NFκB protein expression, while resulting in increased Stat3 protein expression. However, Stat3 acetylation and phosphorylation, which both contribute to Stat3 activation, were significantly decreased as a result of VPA exposure. p300 inhibition resulted in a significant decrease in NFκB, similar to what was observed as a result of VPA exposure, which suggests that VPA-mediated degradation of p300 may play a role in reduced NFκB protein expression following VPA exposure. Conversely, Stat3 protein expression, acetylation and phosphorylation were not significantly changed as a result of p300 inhibition, suggesting that p300 degradation does not play a role in VPA’s effects on Stat3 protein expression and activation. VPA exposure also resulted in a significant increase in apoptosis, while p300 inhibition did not significantly increase apoptosis. These data suggest that p300 degradation plays a role in VPA-mediated teratogenicity, and that VPA may target other cellular mechanisms in order to exert its teratogenic effects.