No / To present a computational model mapping visual field (VF) locations to optic nerve head (ONH) sectors accounting for individual ocular anatomy, and to describe the effects of anatomical variability on maps produced. A previous model that related retinal locations to ONH sectors was adapted to model eyes with varying axial length, ONH position and ONH dimensions. Maps (n = 11,550) relating VF locations (24-2 pattern, n = 52 non–blind-spot locations) to 1° ONH sectors were generated for a range of clinically plausible anatomical parameters. Infrequently mapped ONH sectors (5%) were discarded for all locations. The influence of anatomical variables on the maps was explored by multiple linear regression. Across all anatomical variants, for individual VF locations (24-2), total number of mapped 1° ONH sectors ranged from 12 to 90. Forty-one locations varied more than 30°. In five nasal-step locations, mapped ONH sectors were bimodally distributed, mapping to vertically opposite ONH sectors depending on vertical ONH position. Mapped ONH sectors were significantly influenced (P < 0.0002) by axial length, ONH position, and ONH dimensions for 39, 52, and 30 VF locations, respectively. On average across all VF locations, vertical ONH position explained the most variance in mapped ONH sector, followed by horizontal ONH position, axial length, and ONH dimensions. Relations between ONH sectors and many VF locations are strongly anatomy-dependent. Our model may be used to produce customized maps from VF locations to the ONH in individual eyes where some simple biometric parameters are known. / ustralian Research Council Linkage Project LP100100250 (with Heidelberg Engineering GmbH, Germany); Australian Research Council Future Fellowship FT0990930 (AMM); Australian Research Council Future Fellowship FT0991326 (AT)

published_or_final_version / Anatomy / Master / Master of Philosophy

Purpose: Retinal ganglion cell (RGC) loss is a measure of the progression of many visual disorders. It is important to identify RGCs with good specificity, so RGC numbers can be reliably analyzed. The purpose of this study was to analyze the effectiveness of two current RGC markers: Brn3a immunohistochemistry and the expression of Thy1-CFP in the Thy1-CFP transgenic mouse. Methods: Rhodamine-?-isothiocyanate (RITC) retrograde labeling, immunohistochemistry, wholemount retinal imaging, western blot, cross sectional analysis and cell densities in uninjured control animals and 3, 5, 7 and 14 days post-optic nerve crush (ONC) or transection (ONT) were tabulated. Results: Brn3a positive (Brn3a+) cell density was significantly less than RITC positive (RITC+) cell density in control mice. After ON injury, Brn3a+ cell density did not decrease at the same rate as RITC+ cell density. The density of RGCs that express Brn3a was significantly less than the individual Brn3a+ and RITC+ cell density at all experimental time points. Thy1-CFP positive (Thy1-CFP+) cell density was significantly less than RITC+ in control mice and significantly more than RITC+ cell density 14 days after ON injury. Thy1-CFP co-localized with ChAT positive (ChAT+) cells 7 days after ONT. Conclusion: Brn3a and Thy1-CFP are not reliable markers of RGCs. Retrograde labeling remains one of the most reliable methods of labeling RGCs in mice.

Thesis (Ph.D.)--University of Western Australia, 2007.

Thesis: M.S., Massachusetts Institute of Technology, Department of Psychology, 1973 / Vita. / Bibliography: leaves 67-73. / M.S. / M.S. Massachusetts Institute of Technology, Department of Psychology

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Several research groups have applied elevated hydrostatic pressure to ONH astrocytes cultured on a rigid substrate as an in vitro model for glaucoma. These studies have shown significant biological effects and this hydrostatic pressure model is now becoming generally accepted in the ophthalmic community. However, since the applied pressures were modest the finding of significant biological effects due to pressure alone is surprising. We hypothesized that the application of hydrostatic pressure as described in these studies also altered gas tensions in the culture media. Our goal was to design equipment and carry out experiments to separate the biologic effects of pressure from those of hypoxia on cultured astrocytes. We designed equipment and carried out experiments to subject cultures of DITNC1 astrocytes to the four combinations of two levels of each parameter. We explored the morphology and migration rates of astrocytes, but observed no significant change in any of these properties.

Glaucoma is a common cause of blindness worldwide, yet the etiology of the disease is unclear. A leading hypothesis is that elevated intraocular pressure (IOP) affects the biomechanical environment within the tissues of the optic nerve head (ONH), and that the altered biomechanical environment contributes to optic nerve damage and consequent loss of vision. The biomechanical environment of the ONH is strongly dependent on the biomechanical properties of sclera, particularly scleral stiffness. However there is significant variability in reported stiffness data for human sclera. Therefore, our research goal was to measure the stiffness of human sclera and incorporate this information into finite element models of the human eye to characterize and quantify the biomechanical environment within and around the optic nerve head region at different IOP levels. Human sclera adjacent to the optic nerve head showed highly nonlinear, nearly isotropic and heterogeneous stiffness which was found to be substantially lower than that previously assumed, particularly at lower levels of IOP. The products c*c1 and c*c2, measures of stiffness in the latitudinal and longitudinal directions from the Fung constitutive model, were 2.9 ± 2.0 MPa and 2.8 ± 1.9 MPa, respectively, and were not significantly different (two-sided t-test; p = 0.795). Scleral stiffness was not statistically different between left and right eyes of an individual (p = 0.952) and amongst the quadrants of an eye (p = 0.412 and p = 0.456 in latitudinal and longitudinal directions, respectively). Three stress-strain relationships consistent with the 5th, 50th and 95th percentiles of the measured scleral stiffness distribution were selected as representatives of compliant, median and stiff scleral properties and were implemented in a generic finite element model of the eye using a hyperelastic five-parameter Mooney-Rivlin material model. Models were solved for IOPs of 15, 25 and 50 mmHg. The magnitudes of strains at the optic nerve head region were substantial at even the lowest applied IOP (15 mmHg) and increased at elevated IOPs (e.g. the third principal strain in the compliant model reached as much as 5.25% in the lamina cribrosa at 15mmHg and 8.84% in the lamina cribrosa at 50 mmHg). Scleras that are “weak”, but still within the physiologic range, are predicted to lead to appreciably increased optic nerve head strains and could represent a risk factor for glaucomatous optic neuropathy. As IOP increased from 15 to 50 mmHg, principal strains in the model with a compliant sclera increased at a lower rate than in the model with a stiff sclera. We quantified the biomechanical environment within and around the optic nerve head region using a range of experimentally measured mechanical properties of sclera and at different IOPs. We showed that IOP-related strains within optic nerve head tissues can reach potentially biologically significant levels (capable of inducing a range of effects in glial cells) even at average levels of IOP and for typical human scleral biomechanical properties.

Central nervous system (CNS) myelination is important for proper nervous system function in vertebrates. In demyelinating diseases such as multiple sclerosis, autoimmune-mediated myelin destruction results in neurological impairment; and although remyelination does occur spontaneously, it is poorly understood and insufficient in humans. Zebrafish (Danio rerio) are known to harbour tremendous regenerative capacity of various CNS tissues; however, there is presently only little knowledge of their myelin repair efficiency. An experimental model of myelin injury in zebrafish would permit study of the mechanisms involved in successful remyelination and could potentially guide the development of novel therapeutic agents for mammalian remyelination. This doctoral thesis describes the characterisation of the novel myelin protein Claudin k in zebrafish, demonstrates the establishment of adult zebrafish as an experimental model for CNS de- and remyelination and explores some mechanisms underlying myelin repair. A variety of myelin markers have previously been investigated in zebrafish, including myelin basic protein and myelin protein zero. However, the use of these is limited by either late developmental expression or presence in compact myelin only. Claudin k is a novel tight junction protein specific to zebrafish CNS and PNS, which can be observed early in development and throughout nervous system regeneration. Utilising specific antibodies and a novel transgenic zebrafish line, in which the claudin k promoter drives the expression of green fluorescent protein in myelinating cells, the studies herein characterise the expression of Claudin k, demonstrate the fidelity of the transgenic construct, and investigate the relationship of Claudin k with established myelin and CNS inflammation markers. Data demonstrate that Claudin k expression closely resembles expression patterns of the endogenous gene, and as such provides a key tool for examining CNS myelination in zebrafish. For the study of de- and remyelination in the zebrafish, the experiments herein describe the use of lysophosphatidylcholine (LPC), a detergent-like myelin toxin, which is used widely in rodent models to demyelinate axons. Its application to the adult zebrafish optic nerve induced focal demyelinating lesions, critically without detectable axonal injury, and permitted the study of time course and efficiency of remyelination. Myelin in the lesion area was reduced as detected by both immunohistochemistry and electron microscopy at 8 days post lesion (dpl), and return of the markers to control levels suggested regeneration by 28 dpl. In addition microglial activation was observed along the optic pathway, which also returned to levels compared to unlesioned control by 28 dpl. In young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres showed no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. However, in old fish (aged 18+ months), remyelinated fibres presented with thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. While the zebrafish as an experimental system has tremendous benefits, such as potential for drug screens using the transparent larvae, capacity for transgenesis and live imaging, experimental models in zebrafish potentially bear several limitations, in particular their distant relationship to humans. To determine whether zebrafish remyelination involves homologous signalling mechanisms to mammals, demyelinated zebrafish optic nerves were treated with human recombinant Semaphorin 3A, an axonal guidance molecule which is well known to inhibit oligodendrocyte precursor cell (OPC) recruitment and remyelination in mammals. Results demonstrated fewer oligodendroglial cells at 14 dpl and less myelinated fibres at 28 dpl in the optic nerve lesion area compared to control treated animals, supporting the hypothesis that zebrafish remyelination may indeed respond to human signalling molecules. Taken together, the findings in this doctoral thesis suggest that this new experimental zebrafish-based model of CNS remyelination can be added to the suite of current models to better understand the remyelination process and that some signalling mechanisms observed in mammals around myelination and OPC recruitment are likely conserved in the zebrafish. In addition, it could potentially be used to discover novel therapeutic targets that promote myelination in injury.