The heterozygous mutation, B6;C3-Opa1Q285STOP, leads to a 50% reduction in Opa1 transcript and protein in the mouse retina and neural tissues and models autosomal dominant optic atrophy and presents with visual dysfunction and structural changes in the retina and optic nerve. This thesis explores the intimate relationship between retinal ganglion cell dendritic architecture, health and synaptic connectivity as influenced by the mitochondrial fusion protein Opa1. Using a range experimental paradigms it is reported here retinal ganglion cell dendritic atrophy which is exacerbated with age and localised exclusively to sublamina b of the inner plexiform layer. There is a marked reduction in the number of glutamatergic synaptic sites and PSD95 levels on ON-centre retinal ganglion cells. In addition, there is a significant increase in synaptic vesicle number and density in both ON and OFF bipolar cells. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina and the changes shown here serve as an exciting biomarker for disease and rescue and recovery therapeutics.

The overall objective this work is to contribute to the understanding of how the precise structure of the corneal stroma is achieved during development, and to apply this knowledge to the latest attempts at engineering effective stromal constructs for use in transplantation. The cornea is the major refractive element of the human eye, accounting for two-thirds of total focusing power. Representing around 85% of corneal thickness, the stroma possesses the mechanical strength needed to protect intraocular tissues, whilst still achieving the high level of transparency necessary for light transmission. This is chiefly due to the small, uniform diameter collagen fibrils arranged into a precisely ordered series of orthogonal lamellae. Proteoglycans in the stroma are thought to regulate the arrangement and diameter of the collagen fibrils, although the mechanism by which this occurs is not fully understood. The deceptively complex organisation of the stroma may be responsible for the relatively little progress that has been made in engineering constructs that can reproduce the structural and functional characteristics of the cornea. Further study into the embryonic development of the cornea may aid attempts to recapitulate in vivo mechanisms for corneal construction. Of particular relevance would be the method of collagen organisation and deposition in the developing avian corneal stroma and the interactions that occur within the collagen fibril bundles as development progresses. Initially, en face sections were used to study the organisation and arrangement of collagen fibrils in the developing stroma. It is hypothesized that in tendon, the formation of parallel arrays of collagen fibrils occurs via fibroblast surface recesses and invaginations. It was evident through transmission electron microscopy that this process also occurs in the developing corneal stroma via surface recesses on stromal keratocytes. Analysis of the interactions between the collagen and proteoglycans within fibril bundles demonstrated that the developing cornea is less well structured than often considered and is possibly a much more fluid and dynamic system than originally thought. Proteoglycan size and orientation show a degree of variety and disorder and appear to follow no set organisation or positioning. The data suggests that proteoglycans were seen forming aggregates that were capable of bridging the gap between more distant neighbouring fibrils. Following the study of the developing corneal stroma, collagen gel based constructs were engineered and their structural and functional characteristics were analysed to assess their potential as stromal equivalents for use in tissue engineering. Manipulating the assembly of collagen fibrils by varying the pH and cross-linker concentration had a dramatic effect on the structure and functionality of the final gel construct. A range of collagen gels were then implanted into intra-stromal pockets to determine their biocompatibility and in vivo properties.

Understanding the biomechanical properties of the cornea is important in order to develop and improve new reliable standard procedures which can be used effectively to assess corneal behaviour in any disease condition, or before/after any ocular surgery. We believe that the Ocular Response Analyzer (ORA) is the only device that can measures the biomechanical properties of the cornea in vivo. However, it has been used for the first time both in vivo and in vitro. This thesis presents a clinical and laboratory investigation of the biomechanical properties of the cornea before/after LASIK and corneal cross-linking to improve our understanding of the knowledge required in both the laboratory and the clinic. Different machines were used in this project, including an ORA, an Oculus Pentacam, a spectrophotometer and a UV-X Illumination system. Laser in situ keratomileusis (LASIK) is, at present, one of the most well-known operations used to correct refractive errors; however, ocular problems arising from corneal thinning have been reported in some previous studies. Therefore, I looked at the effects of surgery on the central/peripheral thickness and the anterior/posterior curvature, and determined to what extent they affect the biomechanical properties of the cornea. During the past decade, much research has focused on improving and developing a new operation called corneal collagen cross-linking with riboflavin and UVA, which is used to stop the progression of keratectasia in the cornea (which occurs in keratoconus and sometimes follows refractive surgery). In the next phase, a range of experiments were conducted on cross-linking to determine to what extent this operation affects the molecular structure and biomechanical properties of the cornea. This thesis has shown for the first time that it is possible to obtain ORA signals in vitro and this opened up the possibility of examining whole eyes as well as excised corneas. It is also confirmed that the values of CH do not represent only a corneal biomechanical property, but rather depend on the presence of the rest of the eye. These in vitro studies have opened up a number of possibilities the future corneal biomechanical studies.

Autosomal dominant optic atrophy (ADOA) is a mitochondrial disorder caused by a nuclear DNA mutation. The mutation is within the OPA1 gene, which encodes a 120kDa protein product. The protein, OPA1, is a dynamin like GTPase, which is responsible for fusion of mitochondria. It is located in the inner mitochondrial membrane and functions in conjunction with outer membrane fission proteins to maintain mitochondrial integrity. The OPA1 protein is expressed ubiquitously and heterozygous mutations cause atrophy of the optic nerve. The variability in human ADOA phenotype may suggest some level of susceptibility to vision loss whereas other individuals appear symptom free. The Opa1 Q285STOP mouse model of ADOA provides a unique opportunity to examine the disease pathology with respect to mitochondrial haploinsufficiency. The tissue specificity of the disease suggests local control over the OXPHOS environment. The bioenergetic activity of Opa1Q285STOP mouse is currently unestablished. The status of antioxidant activity supporting bioenergetic function may be vital in maintaining stability within fusion deficient mitochondria. Mitochondrial stability could influence the extent of reactive species, and ultimately, ATP production. This thesis combined the study of mitochondrial haploinsufficiency with analysis of OXPHOS and antioxidant levels in Opa1Q285STOP mouse where a bioenergetic deficit was identified in all mitochondria tested. The addition of a plant derived antioxidant and its potential effects within the retinal environment and surrounding central nervous system of Opa1 Q285STOP mouse did not appear to influence the extended phenotypic profile observed in this mouse model. The administration of resveratrol to the wild type population conferred disadvantages both In Vivo and In Vitro.

In the diagnosis of dry eye, an evaluation of tear fluid volume is an important parameter. The tear menisci hold approximately 75-90% of the overall tear fluid volume and serve as reservoirs, supplying tears to the pre-corneal tear film. The measurement of the anterior curvature radius of the tear meniscus (TMR) is an indicator of tear film volume and when it is performed non-invasively has been found to have good dry eye diagnostic accuracies. Optical coherence tomography and meniscometry are existing techniques that can measure TMR non-invasively. These techniques have not found wide application among clinicians, either because they are not commercially available or they are too expensive. This PhD describes a series of experiments that investigated the development, evaluation and application of a new instrument for non-invasive tear meniscus measurement. From the results of these studies, it can be concluded: A Portable Digital Meniscometer (PDM) was developed. This consists of an application tool for the iPod-touch, a slit-lamp holder for the iPod-touch and an image analysis software for TMR calculation. A simple iPod-touch or an iPhone mounted on a commercially available digital slit-lamp can be used to project a grid of black and white lines on the tear meniscus. Using the principal of reflective meniscometry, the radius of the lower tear meniscus can be non-invasively measured. This newly developed instrument is a simple, mobile and useful device for measuring tear meniscus radius, and therefore tear volume, and is suitable for use by clinicians. The newly developed PDM was evaluated in vitro and in vivo. It produced accurate and reliable measurements and provided similar values for the tear meniscus radius, in human studies, to the existing video-meniscometer. PDM and OCT measurements of the TMR were significantly correlated. Since with the PDM no image calibration is needed, it seems to be a quick and non-invasive technique for evaluation of tear fluid quantity. The PDM appears to measure the radius of the central section of the tear meniscus The PDM was able to non-invasively measure alterations in TMR and TMH along the lower lid. The flatter TMR and higher TMH at the nasal and temporal locations may be caused by the LIPCOF degree of the underlying conjunctiva. To avoid any interference by LIPCOF, it is recommended that TMR and TMH be measured along the lower lid margin below the pupil centre. Furthermore, the PDM was able to usefully detect changes in TMR following the instillation of artificial tears. The difference in residence time is likely to reflect the different viscosity and Newtonian properties of these drops. An overload with a large drop may result in initial increased blink rate. Blink rate at baseline is significantly related to dry eye symptoms.

This thesis presents the results and conclusions of experiments designed to extend the current models for the origin of corneal transparency. The cornea is the transparent window at the front of the eye, which is responsible not only for the majority of refraction of light that enters, but also the protection against damage, infection and mechanical stress. The property of transparency is only realised by corneal and lens tissue in the human body. In the cornea, it has long been suspected to be caused by the precise arrangement of the fibrils of collagen that are contained within the central layer, the stroma, regulated by the sulphated proteoglycans (PG) that keep fibril spacing within acceptable boundaries. These models are consistent and give a complete description of the reasons for the transparency of a stroma that is entirely acellular. However, it is well known that the stroma is not acellular, and that the short-range order that is critical for transparency would necessarily be disturbed by the cells of the stroma, the keratocytes, which are at least an order of magnitude thicker than the maximum allowed range. Originally, an acellular stroma was considered to be a reasonable approximation due to the perceived sparsity of the cells, but more recent measurements have cast doubt upon this, and explanations have begun to focus on the properties of the cells themselves. One such property would be their refractive index (RI). If the cells could match their own RI to that of their surroundings then they would not scatter and hence would not cause a loss in transparency. This research attempts to measure that RI and by comparison with previously calculated values for the RI of the extra-cellular matrix, attempts to quantify the scale of the scattering that any mismatch would cause, using theoretical models based on both Mie scattering and finite-difference time-domain methods. In addition to models of healthy corneas, this thesis also provides results and conclusions drawn from studies of pathological corneas and discussions of how the pathology, and the treatments, can cause initial losses in transparency. The first such study concerned a cornea afflicted with keratoconus, a disorder of as yet unknown origin that causes the weakening of the corneal tissue, leading to a characteristic cone-shaped cornea, which had been treated with a full penetrating keratoplasty (PK) transplant before being donated. The study was conducted using the techniques of electron microscopy and x-ray diffraction, to both qualitatively and quantitatively analyse the properties of the fibrils and their spacing. This was done on both the original sections of diseased tissue and the donated sections, in order to investigate the idea of keratoconus recurring in previously healthy donated tissue. Any such discovery could provide evidence that keratoconus is not an entirely inherited disorder. The structural properties of the observed scar, that was present as a direct result of the PK procedure that was carried out decades before, were also investigated using the same methods. This investigation was designed to provide insights into the priorities of wound healing in the cornea, and whether any appreciable change in fibril spacing could account for the observed loss of transparency. The final study presented here is a novel tomographic reconstruction of a feature of the disease macular corneal dystrophy (MCD). MCD is a genetic disorder that affects the sulphation of the PG keratan sulphate. MCD gives rise to a multitude of abnormalities but one that has not been fully investigated is the apparent presence of areas within the stroma entirely populated by PGs, with no collagen present. This study attempts to reconstruct three-dimensional views of these lakes, as well as stromal lamellae, in order to investigate the interactions between free PGs and between PGs and collagen in MCD.

Purpose: To i) investigate the stimulus characteristics of the Belmonte OPM pneumatic aesthesiometer in vitro and in vivo; ii) explore the relationship between corneal sensitivity threshold (CST), spontaneous eye blink rate (SEBR), ocular surface temperature (OST) and tear film quality, using the non-contact aesthesiometer (NCCA), and iii) investigate the relationship between heating and cooling of the ocular surface on CST (with the NCCA). Methods: Stimulus flow rate (airflow meter), air stimulus volume (air capture) and force (weight scales) were recorded in vitro. Stimulus temperature was measured in vitro (using thermocouples) and in vivo (via a thermal infrared camera). SEBR, CST, OST, NIBUT and lipid pattern of the tear film were recorded. CST (using NCCA), OST and tear film quality were obtained at baseline, and after increasing and decreasing OST, using heated goggles or cooling gel mask, respectively, until OST baseline level was reached again. OST was recorded during stimulus presentation at CST, immediately after heating and cooling of the ocular surface. Conclusions: Stimulus characteristics measured in vitro showed little variability and may therefore be considered as reliable. Stimulus temperature recorded in vivo showed that a change in stimulus flow rate produced a small, but statistically significant effect on OST. Consequently, this air stimulus may not be exclusively ‘mechanical’, and may provoke an additional response from temperature sensitive C fibres. A moderate correlation between corneal sensitivity and blink frequency was established, and a strong correlation between tear film quality, SEBR and OST was obtained, emphasising that ocular surface condition represents one important trigger for the initiation of a blink. After both an increase and a decrease of OST, a greater localised OST decrease was required at CST, indicating that heating or cooling of the ocular surface may affect the nervous activity / sensitivity of cold thermoreceptors in the superficial cornea.

Children with delayed reading skills and/or poor academic achievement are increasingly being referred to Eye Care Professionals with suspected eye movement/“tracking” difficulties. However, current clinical techniques are highly subjective, poorly controlled, and relatively imprecise. It is therefore reasonable to suggest that Eye Care Professionals face challenges in recognising and diagnosing genuine eye movement disorders, and consequently, fail to support and/or manage these children. The principal aim of the studies described in this thesis was to characterise eye movements in children with learning related difficulties who are frequently considered to be at risk of eye movement disorders. Using a novel child-friendly method, we have shown that, in general, eye movement characteristics in children with reading/learning related difficulties are not different from those in typically developing age-matched children when compared as a group. The findings also showed that when eye movement characteristics in children with reading/learning related difficulties were compared on an individual basis, some of these children had eye movement parameters outside their age-matched norms. Further, our results suggested that children whose eye movements were outside their age-matched norms, generally corresponded to those who had specific, more complex and global difficulties (e.g. dyspraxia, general developmental delay). In conclusion, the studies presented in this thesis suggest that there is an association between specific learning difficulties and eye movement disorders, but challenge the view that eye movement disorders can be found in isolation in children with delayed reading/academic performance. Finally, based on the sum of results obtained, simple actionable guidelines are proposed to improve the examination of eye movements in clinical practice in order to recognise genuine eye movement disorders.

This thesis explored some aspects of the relationship between structural progression of the glaucomatous optic nerve head (ONH) and functional progression of the visual field. Sixty-one individuals with a longitudinal series of ONH images were manually identified from a database of approximately 2800 individuals attending a hospital glaucoma clinic. The ONH images obtained from the various photographic sources were equalized, for each individual, in terms of ONH size. Custom-software was designed to enable the viewing of consecutive and chronologically different ONH image-pairs under monoscopic and stereoscopic conditions, with and without sequential flicker. The efficacy, for the identification of progressive glaucomatous loss, amongst the 61 individuals, of the four viewing techniques was qualitatively evaluated by two ophthalmologists. Stereo-flicker identified the largest number of cases of progression, although little agreement was present between the two ophthalmologists. The digital characteristics of the ONH images from 27 of the 61 individuals enabled quantitative digital stereo-planimetry. A weak positive curvilinear association was present, at baseline, between the reduction in the neuroretinal rim area and the outcomes of perimetry, including residual retinal ganglion cell (RGC) count. However, little agreement was again present between the two ophthalmologists. Little association was present with either ophthalmologist between progressive structural damage and functional damage. A separate manual search of 1000 individuals with glaucoma archived in ‘Open eyes’ identified 112 individuals with a minimum of 5 visual field examinations over a minimum of 5 years. The outcomes at each stimulus location of the differential light sensitivity, expressed in decibels (dB), and of the residual RGC count, against time to follow-up, were compared using univariate linear regression analysis. In general, residual RGC count identified progression, in terms of a greater statistical significance and/ or of more stimulus locations, at an earlier stage of the disease than sensitivity expressed in dB.

Cross-linked Actin Networks (CLANs) were first identified in bovine trabecular meshwork (BTM) cells in response to steroid treatment. Since then, work within our group has identified CLANs in human TM (HTM) cells and human tissue ex vivo. An alteration in the cytoskeleton of TM cells has been associated with changes in outflow facility and is a promising therapeutic target in glaucoma research. CLANs which are believed to make TM cells more rigid, were found in increase numbers in tissue from glaucomatous donors, but were also present in tissue from non-glaucomatous donors. This would indicate that CLAN formation is not merely a steroid response but may be triggered by changes within the outflow system. The current work set out to identify CLAN inducing agents present within the normal outflow system and questioned whether age-related changes within the anterior chamber could influence CLAN number in a similar manner to glaucoma. Investigating the influence of aqueous humor (AH) and some of its constituent growth factors on CLAN formation in both BTM and HTM cells revealed that AH itself was capable of inducing CLANs and that the main CLAN inducing agent present was TGF-β2. Conversely mitogenic growth factors (FGF and HGF) did not increase CLAN incidence and implicated that CLAN incidence was linked to cell shape change linked and therefore to function. In vitro analysis of CLANs in reference to donor age revealed that TGF-β2 could induce a higher percentage of CLANs in HTM cells from older donors compared to HTM cells derived from younger donors. Ex vivo analysis run in parallel demonstrated that CLAN incidence increased with increasing donor age. In order to identify how age-related stress within the TM environment influenced CLAN incidence cells were treated with hydrogen peroxide. In BTM cells pre-treatment for 1 hour was sufficient to significantly increase CLAN incidence. In further experiments it would seem that CLANs are not associated with apoptotic cells nor with senescent cells. The findings of this study indicate that CLANs can form when no glaucomatous pathology is detected and that changes in the extracellular environment can influence their formation. This is especial evident in older tissue.