Heightened maternal inflammation is linked to placental oxidative and nitrosative stress associated with fetal growth restriction in the rat

Deficient trophoblast invasion and spiral artery remodeling are associated with pregnancy complications such as pre-eclampsia (PE) and fetal growth restriction (FGR). Using a model in which pregnant Wistar rats are given daily, low-dose, injections of bacterial lipopolysaccharide (LPS; 10 – 40 µg/kg) on gestational days (GD) 13.5 – 16.5, our group has shown that abnormal maternal inflammation is causally linked to shallow trophoblast invasion, deficient spiral artery remodeling, and altered utero-placental hemodynamics leading to FGR/PE; these alterations were shown to be mediated by TNF-a. The present research evaluated certain consequences of decreased placental perfusion; this was accomplished by examining placental alterations indicative of decreased placental perfusion. Additionally, the role of glyceryl trinitrate (GTN) was determined as a potential therapeutic to prevent the consequences of decreased placental perfusion. Results indicated that dams experiencing heightened maternal inflammation showed significantly greater expression of hypoxia-inducible factor-1a (HIF-1a) and nitrotyrosine, both of which are markers of decreased perfusion and oxidative/nitrosative stress. Contrary to expectations, inflammation did not appear to affect nitric oxide (NO) bioavailability, as revealed by a lack of change in placental or plasma levels of cyclic guanosine monophosphate (cGMP). However, continuous transdermal administration of GTN (25 µg/hr) on GD 12.5 – 16.5 prevented the accumulation of HIF-1a and nitrotyrosine in placentas from LPS-treated rats. These results support the concept that maternal inflammation contributes to placental hypoxia and oxidative/nitrosative stress. Additionally, they indicate that GTN has potential applications in the treatment and/or prevention of pregnancy complications associated with abnormal maternal inflammation.

Analysis of parameters of haemostasis in pre-eclamptic, normotensive pregnant and non-pregnant women.

Pregnancy is characterized by a state of heightened coagulation, which is exacerbated in pathological conditions such as pre-eclampsia (PET). PET is further associated with abnormal maternal inflammation and increased circulating microparticles (MP); however, a mechanistic link between these pathological features has never been established. It is proposed in this thesis that abnormal maternal inflammation is causally linked to pro-coagulant trophoblast MP shedding via a mechanism mediated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF), thereby contributing to maternal coagulopathies associated with PET. Using thromboelastography (TEG) and standard laboratory tests, haemostatic function was evaluated in PET and normotensive subjects at delivery and post-partum. Furthermore, the effects of the menstrual cycle and oral contraceptive (OC) use on haemostatic function were assessed in non-pregnant subjects in order to understand their influence on post-partum haemostasis. Plasma TNF and pro-coagulant MP levels were evaluated in the pregnant subjects. Using chorionic villi explants from human term placentas, MPs were quantified after TNF administration. The pro-coagulant potential of placental MPs was evaluated by TEG by spiking whole-blood with medium containing MPs from chorionic villi. TEG identified increased whole-blood coagulability in PET subjects at delivery, demonstrating its increased sensitivity over standard laboratory tests at identifying haemostatic alterations associated with PET. Haemostatic alterations were normalized by six weeks post-partum. TEG also identified cyclic haemostatic variations associated with OC use. Chorionic villi treated with TNF (1 ng/ml) shed significantly more MPs than untreated placentas. MPs from chorionic villi increased the coagulability of whole-blood. Together, results provide evidence supporting the concept that abnormal maternal inflammation is causally linked to the development of maternal coagulopathies in pregnancy complications. Moreover, TEG may be superior to standard laboratory tests in evaluating haemostasis in pregnant and non-pregnant subjects.

Identification and Development of Enzymes Associated With the Sperm Inner Acrosomal Membrane and Their Function during Fertilization

In order for mammalian fertilization to transpire, spermatozoa must transit through the female reproductive tract and penetrate the outer investments of the oocyte: the cumulus oophorus and the zona pellucida. In order to penetrate the oocyte, spermatozoa must undergo the acrosome reaction. The acrosome reaction results in the exposure of the inner acrosomal membrane (IAM) and proteins that coat it to the extracellular environment. After the acrosome reaction, the IAM becomes the leading edge of spermatozoa undergoing progressive movement. Thus the enzymes which effect lysis of the oocyte investments ought to be located on the IAM. An objective of this study was to identify and characterize enzymatic activity detected on the IAM and provide evidence that they play a role in fertilization. This study also describes procedures for fractionating spermatozoa and isolating the IAM and proteins on its intra- and extra-vesicular surfaces, and describes their development during male gametogenesis. Since the IAM is exposed to the extracellular environment and oviductal milieu after the acrosome reaction, this study also sought to characterize interactions and relationships between factors in the oviductal environment and the enzymes identified on the IAM. The data presented provide evidence that MMP2 and acrosin are co-localized on the IAM, originate from the Golgi apparatus in gametogenesis, and suggest they cooperate in their function. Their localization and results of in vitro fertilization suggests they have a function in zona pellucida penetration. The data also provide evidence that plasminogen, originating from the oviductal epithelium and/or cumulus-oocyte complex, is present in the immediate environment of sperm-egg initial contact and penetration. Additionally, plasminogen interacts with MMP2 and enhances its enzymatic action on the IAM. The data also provide evidence that MMP2 has an important function in penetration of the cumulus oophorus. Holistically, this thesis provides evidence that enzymes on the IAM, originating from the Golgi apparatus in development, have an important function in penetration of the outer investments of the oocyte, and are aided in penetration by plasminogen in the female reproductive tract.

The Function of Intestinal Afferent Neurons in Regulating Satiety During Health and Obesity

Background and aim: Within the gastrointestinal tract, vagal afferents regulate satiety and food intake via chemical and mechanical mechanisms. Cysteinyl Leukotrienes (CysLTs) are lipid mediators that are believed to regulate food intake and body weight. However, the involvement of vagal afferents in this effect remains to be established. Conversely, Glucagon like peptide-1 (GLP-1) is a satiety and incretin peptide hormone. The effect of obesity on GLP-1 mediated gut-brain signaling has yet to be investigated. Since intestinal vagal afferents’ activity is reduced during obesity, it is intriguing to investigate their responses to GLP-1 in such conditions. Methods: Extracellular recordings were performed on intestinal afferents from normal C57Bl6, low fat fed (LFF), and high fat fed (HFF) mice. To examine the effect on neuronal calcium signaling, calcium-imaging experiments were performed on isolated nodose ganglion neurons. Food intake experiments were conducted using LFF and HFF mice. Oral glucose tolerance tests (OGTT) were carried out. Whole cell patch clamp recordings were performed on nodose ganglion neurons from A) normal C57Bl mice to test the effect of CysLTs on membrane excitability, B) LFF and HFF mice to examine GLP-1 effect on membrane excitability during obesity. c-Fos immunohistochemical techniques were performed to measure the level of neuronal activation in the brainstem of both LFF and HFF mice in response to Ex-4. Results: CysLTs increased intestinal afferent firing rate and mechanosensitivity. In single nodose neuron experiments, CysLTs increased excitability. The GLP-1 agonist Ex-4 significantly decreased food intake in LFF but not HFF mice. However, Ex-4 markedly attenuated the rise in blood glucose in both LFF and HFF mice. The observed increase in nerve firing and mechanosensitivity following the application of GLP-1 and Ex-4 was abolished in HFF mice. Cell membrane excitability was significantly increased by Ex-4 in nodose from LFF but not HFF mice. Ex-4 significantly increased the number of activated neurons in the NTS area of LFF mice but not in their HFF counterparts. Conclusion: The previous observations indicate that the role CysLTs play in regulating satiety is likely to be vagally mediated. Also that satiety, but not incretin, effects of GLP-1 are impaired during obesity.