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 âbarrier characteristicsâ 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.
| Identifer | oai:union.ndltd.org:ADTP/283250 |
| Date | January 2005 |
| Creators | Tretiach, Marina Louise |
| Publisher | University of Sydney., Department of Clinical Ophthamology |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_AU |
| Detected Language | English |
| Rights | The author retains copyright of this thesis., http://www.library.usyd.edu.au/copyright.html |
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