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.

CYSTATIN RELATED EPIDIDYMAL SPERMATOGENIC PROTEIN RESIDES IN THE OUTER DENSE FIBRES AND LIKELY TRANSIENTLY ASSOCIATES WITH THE SURFACE OF EPIDIDYMAL MOUSE SPERMATOZOA

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm.

LEARNING IMPULSE CONTROL IN A NOVEL ANIMAL MODEL: SYNAPTIC, CELLULAR, AND PHARMACOLOGICAL SUBSTRATES

Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task. In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response. AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control. In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions. In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance. In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control.

IMPACT OF RNA VIRUSES ON THE REGULATION OF IL-23 IN MOUSE AND HUMAN MODELS OF INFECTION.

Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections.

Mouse Uterine Natural Killer Cell Functions During Early Pregnancy

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction.

Laboratory Investigation of Diluted Bitumen Trapping and Dissolution in Gravel

The Canadian economy is largely dependent on the distribution of large volumes of oil to domestic and international markets by a long network of pipelines. Unfortunately, accidents occur, and oil can leak or spill from these pipelines before it reaches its destination. Of particular concern are the long-term consequences of oil spills in freshwater, which include sinking of oil in water and the contamination of sensitive areas, such as where fish (e.g., salmon) deposit their eggs in gravel-dominated river sediments. There is a knowledge gap regarding the fate and behaviour of oil in river sediment. To this end, this study aimed at finding the potential for diluted bitumen (dilbit) oil to become trapped in gravel and to transfer hydrocarbons into water by dissolution, which are harmful to aquatic life. Two sets of laboratory experiments were conducted to simulate conditions of an oil spill on an exposed bank or in shallow water. In the first set, by conducting capillary pressure-saturation (Pc-Sw) experiments it was found that dilbit can enter gravel pores without much resistance and approximately 14% of the pore volume can be occupied by discontinuous single or multipore blobs of dilbit following imbibition of water. Air-water Pc-Sw experiments done in laboratory 1-D columns required gravity correction and could be successfully scaled to predict dilbit-water Pc-Sw curves, except for the trapped amount of dilbit. Trapped dilbit constituents can be dissolved into the water flowing through gravel pores (hyporheic flow) at different velocities. In the second set, dissolution experiments suggested that for the duration of the test, hydrocarbons that cause acute toxicity dissolve rapidly, likely resulting in a decrease in their effective solubility. However, dilbit saturation changed only <2% within that time. Chronically toxic PAH compounds were also detected in the effluent water. The total concentration of all detected PAHs and alkylPAHs exceeded the threshold literature value to protect early-life stage fish. Observations of decreased concentrations with increased aqueous velocities as well as less than equilibrium concentrations indicated that the mass transfer was rate-limited. A correlation was developed for the mass transfer rate coefficient to understand the mass transfer behaviour beyond the conditions used in the experiments, which had a Reynolds number exponent similar to the studies of NAPL dissolution in groundwater.