The placenta is a highly vascularized, temporary organ developed in pregnancy that is composed of both maternal and fetal cells. It plays a pivotal role in gestational health by facilitating embryo implantation and fostering nutrient exchange between mother and fetus. Placental malformation and the diffusion of harmful exogenous substances through the placental barrier can cause pregnancy complications and, in more severe cases, death of the mother or the fetus. Further, the placenta undergoes profound morphological and functional changes throughout pregnancy. Establishing models to mimic these phenomena at different stages of pregnancy informs prescription drug safety and expedites the development of placental disease treatments. Mouse models are often used to simulate human fetal development despite major interspecies differences. These limitations drive researchers to developing in vitro models consisting of human-derived cells. This thesis presents three 3D vascularized placental models utilizing human placental stem cells (PSCs) and human umbilical vein endothelial cells (HUVECs) which can model multiple placental phenomena across early- and late-stage pregnancy. The first model features a 3D fibrin hydrogel network with self-assembled vasculature and a monolayer of syncytialized human trophoblastic stem cells (STs) serving as a platform for barrier studies at the maternal-fetal interface. By tuning trophoblast differentiation and vascularization of this model to mimic the early- and late-stage placenta, it was revealed that placental barrier permeability was dependent on placental maturity and that the vascular barrier is also a critical determinant of what molecules can be passed from the mother to the fetus. The design and manufacturing of this model were then streamlined to meet the demands of large-scale drug studies in the second placental barrier model. Placental invasion into the maternal decidua is carefully orchestrated by multiple cell types to prevent over- and under-invasion, both of which can be dangerous to the mother and fetus. Understanding the biochemical and environmental cues that permit this healthy invasion can allow for improved diagnostics and treatments of placental diseases, such as preeclampsia and placenta accreta. Thus, the third model presented herein is a placental invasion model with chorionic villus-like structures seeded with invasive extravillous cytotrophoblasts (EVTs) and a perfusable vascular channel. Collectively, these models facilitate the exploration of placental morphogenesis and function throughout various stages of pregnancy. They offer a valuable tool for probing placental dysfunctions and assessing drug safety, ultimately contributing to advancements in fetal-maternal health. / Thesis / Doctor of Philosophy (PhD) / The placenta is an essential organ in pregnancy and is responsible for a variety of phenomena that assure the survival of the fetus. However, many women suffer from negative pregnancy outcomes due to placental disorders, such as preeclampsia, or due to the crossing of unsafe compounds through the placenta to the fetus. Trophoblasts are the most notable placental cell type originating from the fetus and they have the capacity to mature into more specialized subtypes that are responsible for placental barrier function and placental development via invasion into the maternal tissue. In this work, we have designed three systems that either model placental barrier function or trophoblast invasion by culturing primary endothelial cells with differentiated trophoblast cells on a gel-based device. Using the barrier models, it is possible to assess the rate of transport of different compounds that may be present in the mother’s blood to the fetus, to assess their safety. Whereas the invasion model has the capacity to model the genesis of the placenta and therefore may be used to shed light on the causes for placental dysfunctions at the early stage of pregnancy.

Doctor of Philosophy (Medicine) / Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Doctor of Philosophy (Medicine) / Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Cerebral small vessel pathology is now known to be associated with the development of cognitive impairment and mild motor impairments such as gait disturbance in a variety of neurodegenerative diseases. This dissertation explores the hypothesis that blood brain barrier dysfunction is an early event in cerebral ischemia and contributes to the development of cerebral small vessel disease (CSVD). A common rodent model of CSVD is permanent bilateral common carotid artery occlusion in the rat. This model was used to study several aspects of the progression of CSVD including the timecourse of blood brain barrier permeability changes following the onset of ischemia, gait disturbance, the expression of tight junction proteins and cytokine expression. It was determined that BBB permeability was elevated for 2 weeks following BCCAO and ischemic rats displayed lower gait velocity. There was no change in expression of TJ proteins. However, ischemic rats had higher levels of some proinflammatory cytokines and chemokines in brain tissue with no obvious changes in plasma levels. The mechanisms underlying the increase in BBB permeability were studied in vitro using artificial barriers made of confluent rat brain microvascular endothelial cells. Cerebral ischemia has been reported to cause an increase in plasma toxicity, likely by elevating the numbers of circulating microparticles (MPs). MPs isolated from the plasma of ischemic rats were applied to artificial barriers where it was found that they act mainly as vectors of TNF-α signaling. MPs induce activation of caspase-3 and the Rho/Rho kinase pathways. It is concluded that most of the increase in barrier permeability is due to apoptosis and disassembly of actin cytoskeleton and disruption of adherens junctions IV and not an increase in transcellular transport. The effects of treatment with the type III phosphodiesterase inhibitor cilostazol on dye extravasation in the brain, glial activation, white matter damage and motor performance were evaluated. It was determined that cilostazol could improve the increased BBB permeability and gait disturbance and microglial activation in optic tract following BCCAO. Also, the effects of treatment with cilostazol on plasma toxicity in vivo (24h and 14d following BCCAO) and artificial barriers (in vitro) were assessed. It was found that cilostazol could reduce plasma toxicity at 24h and improve increased endothelial barrier permeability that is induced by MP treatment respectively. In summary BBB dysfunction occurs in the rat model of chronic cerebral hypoperfusion with no differences in expression of TJ proteins. There is a mild motor disturbance in the form of lower gait velocity following BCCAO. Cytokines released in brain tissue may be associated with pathological consequences following BCCAO while there is no significant difference in plasma levels and circulating MPs may play a role in BBB dysfunction.

This thesis comprised patients with chronic rheumatic diseases. The studies aimed to elucidate food sensitivity by measuring mucosal inflammatory reactivity and thereby a possible link between the gut and joints. In all the studies, the mucosal path technique was used to evaluate the rectal mucosal response to rectal challenge with gluten and/or cow’s milk protein (CM). In some patients with primary Sjögren’s syndrome (pSS) and the genetic susceptibility genes HLA DQ2, mucosal reactivity measured with nitric oxide (NO) was found after rectal gluten challenge without detectable serum antibodies to gluten or transglutaminase. This gluten sensitivity was not linked to coeliac disease. After rectal CM challenge, a rectal mucosal inflammatory response measured with NO and myeloperoxidase (MPO) was detected in 38% of pSS patients, all of whom fulfilled the criteria for irritable bowel syndrome. In a questionnaire study of self-experienced adverse reactions to food, 27% of patients with rheumatoid arthritis (RA) reported intolerance to various foods and CM in particular. After rectal CM challenge performed in RA patients (n=27), strong mucosal reactivity to CM was observed in a few patients and a moderate increase in 23%. After gluten challenge, a moderate increase in mucosal reactivity was found in 35% of patients. No correlation to self-perceived intolerance and mucosal reactivity measured with NO and MPO was seen. Inflammation of the gut is a prominent feature of spondyloarthropathies (SpA). After rectal challenges with CM protein and gluten, an increase in rectal NO production was seen in 26% and 19% respectively (p<0.001). An increase in the mucosal release of MPO as a sign of neutrophil activation was seen in the CM- and gluten-sensitive patients. NO production in SpA patients was more enhanced compared with RA and pSS patients and could contribute to the increased barrier permeability described in SpA patients.

No description available.

<p>The gut and brain are involved in a bi-directional communication system, referred to as the gut-brain axis. While it has been established that antimicrobials induce dysbiosis in the gut, which further disrupts immune and metabolic homeostasis, research on brain and behaviour development is becoming a topic of interest. We propose that alterations via antibiotics at the level of the gut microbiota impacts the gut-brain axis. The primary interest of this thesis is to understand the effects that antibiotics have on brain and behaviour development in conjunction with changes in the immune system and metabolism using the antibiotic mouse model. Mice treated with antibiotics revealed behavioural differences in the open field apparatus and three-chamber social behaviour apparatus, but not in the elevated plus maze and auditory fear conditionings enclosures. Evaluation of intestinal permeability revealed that female Balb/C mice administered a combination of bacitracin, neomycin and primaricin and another group administered a combination of ampicillin, neomycin and primaricin showed reduced intestinal permeability. Furthermore, the immune system condition was evaluated using flow cytometric analysis of spleens, which revealed no effect of treatment on immune cell profiles in CD1 mice treated with ampicillin. Evaluation of serum cytokine levels showed minimal differences in Balb/C and C57Bl/6 mice treated with antibiotics. Body weight and water and food consumption were evaluated in mice administered antibiotics. Weight loss differences were observed in two groups of female Balb/C mice, with the first group administered bacitracin, neomycin and primaricin and the second group administered ampicillin , neomycin and primaricin. Antibiotic treatment dependent differences in water and food consumption were observed. Serum insulin and leptin level investigation revealed that female Balb/C mice administered ampicillin, neomycin and primaricin had reduced serum insulin levels compared to strain matched controls. These findings indicate that antibiotic treatment impact metabolic function. This pilot study using antibiotic treated mouse models provides insight on the microbiota’s effects on the gut-brain axis, which can help to potentially identify methods of preventing gut microbiota mediated pathology in humans.</p> / Master of Science (MSc)