O objetivo deste trabalho e desenvolver um instrumento computadorizado para análise da superfície anterior da córnea humana, gerando para isto, um mapa topográfico bidimensional em código de cores. Utilizando a córnea como um espelho esférico convexo, projeta-se anéis luminosos sobre sua superfície e aquisiciona-se com uma câmera a imagem por ela refletida. Esta imagem e digitalizada e armazenada em um microcomputador para posterior processamento. Através de técnicas de computação gráfica e processamento de imagens digitais, extrai-se da imagem as informações necessárias para construção da topografia desejada. Por fim, a topografia e apresentada em forma de mapas coloridos, sendo cada cor associada a uma determinada dioptria, transmitindo ao médico oftalmologista uma noção exata da superfície da córnea do paciente em análise. / The main goal of this work is to develop a computerized instrument for the analysis of the anterior portion of the human cornea, which displays its result in topographic color maps. Approximating the cornea to a spherical convex mirror, and by projecting a known pattern over it, the reflected image is captured and stored. By means of computer graphics technology and image processing, the necessary information for mathematical calculations is extracted. The resulting maps are color-coded in accordance to the degree of power of each corneal region, that is, to the diopter value. The ophthalmologist can then make important diagnostics and surgery tactics from the analysis of these topographic maps.

A membrana amniótica tem se consolidado no tratamento das afecções da superfície ocular. Assim, o objetivo deste estudo foi avaliar a viabilidade e a eficácia do implante de MA eqüina, preservada em glicerina a 98%, na reparação de ceratoplastias lamelares em cães, por meio do estudo da avaliação clínica pós-operatória dos animais, do tempo de cicatrização, da reconstrução da arquitetura da córnea, da resposta inflamatória, e da composição colágena do estroma corneal no local do implante. Foram selecionados 12 cães, sem raça definida, machos ou fêmeas, divididos em quatro grupos de três, que tiveram tempos de observação distintos: 2, 7, 21 e 40 dias. Foi realizada ceratoplastia lamelar de 5 mm de diâmetro em um dos olhos de cada animal, seguida da aplicação do implante de membrana amniótica eqüina de 6 mm. Durante o período de observação, exame clínico oftalmológico foi realizado nos cães, com intervalos de 48 horas e ao final deste período, foram submetidos á eutanásia. Os olhos em estudo foram enucleados e fixados para posterior análise. Foram utilizados três métodos de coloração para o estudo histológico do tecido implantado: hematoxilina-eosina (HE), ácido periódico de Schiff (PAS) e picrossirius. Além disso, procedeu-se a imunomarcação para colágenos tipo I, III, e V, com uso de pepsina para digestão das fibras colágenas heterotípicas exposição dos epítopos. Clinicamente os implantes foram completamente epitelizados em aproximadamente 10 dias, os neovasos apresentaram involução progressiva a partir dos 20 dias de pós-operatório, estando ausentes ao final dos 40 dias de observação, restando apenas uma nébula no local da lesão. À microscopia óptica, observou-se resposta inflamatória moderada, presença de epitélio pavimentoso estratificado aos sete dias e epitelização completa aos 21 dias. Aos 40 dias a membrana basal do epitélio apresentou-se reconstituída. O colágeno tipo I teve sua expressão no estroma intensificada aos 21 dias de pós- operatório. O colágeno tipo III está presente na membrana amniótica, sua a ausência no local do implante, aos 21 dias, mostrou remodelamento do tecido implantado. O colágeno tipo V, presente no estroma da córnea, teve sua expressão aumentada aos 7 e 21 dias, retornando à distribuição normal aos 40 dias de pós-operatório. Assim concluí-se que: a membrana amniótica eqüina é viável como implante em córnea de cão, sendo incorporada ao estroma, resultando em restabelecimento parcial da transparência no local de implante; o colágeno do tecido implantado é remodelado e substituído já aos 21 dias de pós-operatório; a pepsina foi eficiente na digestão das fibras e exposição dos epítopos dos colágenos nas fibras heterotípicas / The amniotic membrane has consecrated itself in the treatment of ocular surface diseases. The objective of this study was to evaluate the efficiency and viability of the equine amniotic membrane graft, preserved in glycerin at 98%, in the lamellar keroplasty recovery in dogs. Evaluation was based on clinical post-surgical exam, healing time, corneal architectural reconstruction, inflammatory response and collagen composition of the corneal stroma at the graft site. Twelve mixed-breed, male and female dogs were divided into four groups of three dogs. Each group was submitted to different observation periods of 2, 7, 21 and 40 days. Each dog was submitted to a 5 mm lamellar keratoplasty in one eye, followed by a 6 mm equine amniotic membrane graft. Each animal was submitted to clinical ophthalmologic exam every 48 hours. At the end of the evaluation period, the animal was euthanized and the grafted eye was removed and fixated for posterior analysis. For the histological study of the tissue graft, three methods of coloration were used: hematoxylin eosin (HE), periodic acid of Schiff (PAS) and picrosirius. Immunolocalization for the collagen types I, III and V using pepsin for fiber digestion of heterotypic fibrils and epitope exposure, was made. Clinically, the grafts were completely epithelized in approximately 10 days and neovascularization regressed progressively 20 days after surgery, being completely absent after 40 days, when only a nebula remained at the graft site. Optic microscopy revealed mild inflammatory response and presence of stratified pavement epithelium after 7 days and complete epithelization 21 days after surgery. At the end of 40 days the basal membrane was reconstituted. Type I collagen had its expression in the stroma intensified 21 days after the surgery. By day 21 the absence of collagen III in the corneal stroma showed graft remodeling, since this was formerly present in the amniotic membrane. The expression of type V fiber in the corneal stroma showed a mildly intensified expression at 7 and 21 days of observation, but returned to its normal distribution 40 days after surgery. Conclusion was that the equine amniotic membrane is a viable graft for the dog\'s cornea as it is incorporated to the stroma, resulting in partial transparency at the site of the graft. Twenty-one days after surgery, collagen from the graft is already remodeled and substituted. Pepsin is efficient for fiber digestion and collagen epitope exposure in heterotypical fibers.

Two corneal dystrophies, posterior polymorphous corneal dystrophy (PPCD) and keratoconus, have been associated with missense mutations found in the transcription factor-encoding gene Visual System Homeobox 1 (VSX1). Despite this association, the pathogenic link between VSX1 and these diseases remains controversial. To address this issue, I utilized a variety of in vitro approaches to study how seven VSX1 missense mutations found in disease populations that span two highly conserved domains, the homeodomain (HD) and CVC domain affect VSX1 transcriptional activity, protein expression levels and subcellular localization. I also carried out an in vivo investigation by generating a mouse line carrying a mutation in Vsx1: P254R. Corneal morphology was examined through histology and ex vivo whole eye confocal imaging which was used to assess corneal thickness. Quantification of immunocytochemistry was used to characterize terminal marker expression in the inner retina compared to previously described phenotypes in Vsx1-null mice. My in vitro results showed that mutations found in both the HD and CVC domain alter the normal transcriptional repression activity in Vsx1. These changes were not due to changes to protein expression or subcellular localization. Characterization of corneal and retinal phenotypes in vivo revealed no significant differences in Vsx1 P254R mice when compared to wild-type and Vsx1-null controls. In conclusion, my work shows that Vsx1 P254R is not pathogenic for corneal dystrophies in a mouse model. However, my in vitro studies show that Vsx1 mutations have the ability to alter transcriptional activity and therefore still have the potential to be pathogenic in humans. Further investigation is needed to determine whether VSX1 mutations found in disease populations are, in fact, causative for corneal dystrophies. / Graduate / 2019-04-25

Brittle cornea syndrome (BCS) is an autosomal recessive, multisystemic connective tissue disorder characterised by extreme corneal thinning and fragility. Mutations in transcription factors ZNF469 and PRDM5 cause BCS types 1 and 2, respectively. Both genes are believed to regulate the transcription of extracellular matrix (ECM) components, particularly fibrillar collagens, and are suggested to act on a common pathway. Molecular diagnosis is available for affected patients, and those at risk of being heterozygous carriers. Chapter 3 presents the identification of mutations in ZNF469 in 14 families with BCS, and evidence for the downregulation of ECM-associated transcripts in skin fibroblasts from patients with ZNF469-associated disease by Q-PCR.Chapters 4 and 5 focus on PRDM5-associated disease. Chapter 4 highlights previously undescribed and potentially phenotype-related aspects of PRDM5- associated BCS. In chapter 4, a potential role for PRDM5 in development of Bruch’s membrane is suggested, by the observation of significantly reduced expression of major components of Bruch’s membrane, including collagens types I, III, and IV in patients with PRDM5-associated disease using immunohistochemistry. A first description of PRDM5 expression in the human eye is also presented in chapter 4. In chapter 5, a potential role for PRDM5 in retinal vasculogenesis is suggested. PRDM5-related disease also offers an in vivo opportunity to observe a subset of epigenetic regulatory mechanisms in an inherited eye disease, providing mechanistic insights, presented in chapter 5. Examination of PRDM5 interaction partners by pull-down and mass spectrometry reveals the diminished interaction of a PRDM5 construct carrying a BCS-associated mutation with repressive complexes, and, through studies on fibroblasts and retinal tissue from patients, we suggest a role for dysregulation of the repressive histone mark H3K9 di- methylation in vivo. These findings suggest a role for a molecular network surrounding dysregulated H3K9 di-methylation in PRDM5-associated disease. Finally, chapter 6 expands the study of a rare disease into more common diseases investigating the role of genetic variations in ZNF469 and PRDM5 in keratoconus, an ocular disorder resulting in progressive corneal thinning. I identified enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients, highlighting ZNF469 as the most significant genetic factor responsible for keratoconus identified to date. In conclusion, this study of a rare disease, BCS, has provided translational research insights (chapter 3), functional insights (chapter 4) mechanistic insights (chapter 5) and has expanded into other, less rare, diseases (chapter 6).

The cornea is the transparent, avascular and highly innervated outer anterior layer of the eye. The cornea is a very delicate structure and any traumatic insult may lead to damage and limbal stem cell deficiency (LSCD), leading to chronic discomfort, visual impairment and ultimately blindness. The resultant issues can have a significant effect on patients and reduce their quality of life. Whilst conservative and therapeutic management of these problems play a part in the treatment of corneal injuries often surgery is indicated. However, surgical repair of damaged corneas may be limited by the availability of suitable donor tissue and donor site morbidity. Corneal grafts or penetrating keratoplasty (PK) or donor limbal grafts may lead to surgical complications such as corneal scarring, infection and graft rejection. First described in 1908 by A. Maximow, stem cells offer the opportunity to produce functional cell specific tissues from undifferentiated “primordial” cells. By using stem cells from human adult or deciduous tooth pulp, repair and regeneration of the cornea may be possible. Furthermore, it may lead to development of new and innovative treatments of other corneal disorders and injuries. The aim of the investigations detailed in this thesis was to characterize dental pulp stem cells (DPSC), help establish their use in regenerative medicine and help enhance the repair and regeneration of damaged corneal epithelium. Using various laboratory techniques including PCR, western blot and immunostaining it was determined that DPSC possess adequate potency and plasticity to be differentiated into a number of cell-lines. Co-culture of DPSC with human cornea demonstrated that stem cells were attracted to the tissue and migrate towards it and attach to the surface of the limbal explant. Additionally, using soft contact lenses it has been shown that DPSC can be successfully transferred from culture to human cornea in vitro. Expression of terminally differentiated corneal epithelium markers such as cytokeratin 3 & 12 further supports the concept that DPSC were transdifferentiated into epithelial progenitor cells. Once transferred onto the corneal surface, DPSC supported corneal epithelium regeneration, allowed corneal epithelial like cells to grow and avert conjunctivalisation and thus maintained cornea transparency. Further studies are needed to provide a better understanding of the DPSC’s role in corneal regeneration, but it is clear that DPSC are promising candidates for this novel and non-invasive method of corneal epithelium regeneration.

M.Phil. / Cataracts have been identified as one of the leading causes of blindness, especially in the developing world. The only presently known effective treatment for this growing problem is surgical removal of the opaque lens followed by replacement with an artificial intra ocular lens. Newer methods have brought greater success, and greater costs. For people in the developing world, these newer methods are not always an option. Together with the increased cost, there is a growing demand because of this worldwide problem. This increased need for surgery has lead to the development of waiting lists in the state funded hospitals. To qualify for a cataract extraction in most state funded hospitals, a best visual acuity of 6/60 is required, compared to the 6/12 to 6/24 levels required in the industrial countries and private practices. With these levels of visual impairment in the developing world, many patients are left functionally blind for long periods of time until cataract extraction can be performed. Older methods such as extra-capsular cataract extraction are still being used in the developing world. This is mostly due to the increased density of the cataracts at the time when the extraction can be performed because of the long waiting time leading to further maturation of the cataract. This method requires a large corneal incision, which is normally closed with nylon sutures. With this method of surgery meticulous wound closure is very important, and in many cases surgically induced astigmatism is one of the unwanted consequences. It was therefore decided, for the purpose of this study, to use autokeratometric data to explore the refractive effects of two different methods of corneal wound closure following planned extra-capsular cataract extraction (ECCE). Astigmatism is a major problem associated with extra capsular cataract extraction, especially when the wound is closed by means of sutures. Studies by Minassian et al. (2001), Jacobi (2003) and Dowler et al. (2000) all show that newer methods of cataract extraction making use of smaller incisions and therefore fewer sutures show faster recovery and less astigmatism. These methods are however mostly restricted to private practice, and therefore potentially unsuited for use in developing countries. The type of material used for wound closure is another very important factor. Depending on the method of suturing wound gape and wound compression can cause increased amounts of astigmatism. Using a method of wound closure that would cause less traction on the cornea could therefore cause less of a problem postoperatively. Tissue adhesives such as Tisseel® fibrin glue could be such an alternative. Studies by Henrick et al. (1987), Kim and Kharod (2007) and Bhatia (2006) show that fibrin glue forms a watertight, non irritating wound while promoting the healing process by the cross linking of collagen fibres.

The cornea forms the anterior surface of the eye and is responsible for most of the eye’s refractive power. Injury to the outermost layer of the cornea, a non-keratinized stratified squamous epithelium, triggers a transient rise in intracellular calcium concentration that propagates radially from the wound. This calcium mobilization is initiated by the binding of nucleotides such as adenosine triphosphate (ATP), which are released from cells ruptured by the injury, to purinergic receptors (purinoreceptors) on undamaged cells near the wound. Downstream effects of this injury-induced "calcium wave" are generally thought to include the activation of signaling pathways that promote wound healing. However, the specific contributions of individual purinergic receptors to the overall wound response have in most cases not been well characterized. Purinoreceptors are classified into two broad categories: the P2Y class of G protein-coupled receptors, which act through second messengers to release calcium into the cytosol from the endoplasmic reticulum, and the P2X class of ligand-gated ionotropic receptors, which release calcium into the cytosol from the extracellular environment. Previously, our lab established the importance of the P2Y2 receptor to corneal epithelial wound healing by showing that P2Y2 activation makes a substantial contribution to the overall wound-induced calcium response, particularly in cells back from the leading edge, and promotes cell migration after injury. P2Y2 activation was also found to promote the phosphorylation of proteins involved in focal adhesions, which are multi-protein complexes that facilitate cell migration by transmitting the forces generated by the actin cytoskeleton to the extracellular environment. More recently, our lab has begun to demonstrate that P2X7 may play an equally important, yet distinct and perhaps complementary role in corneal epithelial wound healing. For instance, P2X7 was found to strongly influence the intensity of the injury-induced calcium response in cells immediately adjacent to the wound, and treatment with the P2X7 inhibitor oxidized ATP (oxATP) was shown to impair migration after injury both in vitro and in ex vivo rat corneas. Additionally, immunofluorescence of cells fixed eight hours after injury revealed an altered actin cytoskeletal architecture and localization of the focal adhesion proteins talin and vinculin in oxATP-treated cells compared to control cells. The goal of the present study was to further characterize P2X7’s role in the overall response to injury by using live cell imaging to examine actin cytoskeletal rearrangements and focal adhesion dynamics after injury under both control conditions and conditions of P2X7 inhibition. Human corneal limbal epithelial (HCLE) cells were transduced to express either actin or talin tagged with green fluorescent protein (GFP), grown into confluent monolayers, and scratch wounded in the presence or absence of oxATP. Cells at the leading edge of the wound were imaged using confocal microscopy every 10 minutes for 4 hours beginning 0.5 hours after injury. Analysis of the resulting actin-GFP movies revealed trends toward delayed extension of filopodia in oxATP-treated cells relative to control cells, as well as complex changes in the number of filopodia per cell over time. Additionally, while both groups formed lamella containing thick actin bundles that were oriented perpendicularly to the direction of migration, in oxATP-treated cells the formation of these structures was delayed. Furthermore, in oxATP-treated cells these actin bundles tended to persist once formed. This was in contrast to control cells, in which they tended to turn over to be replaced by thinner and shorter actin bundles that were oriented more obliquely relative to the direction of migration. Finally, analysis of talin-GFP movies demonstrated that focal adhesion lifespan was extended in oxATP-treated cells compared to control cells. Focal adhesions in oxATP-treated cells also exhibited a greater propensity to merge together or split apart, further suggesting impaired focal adhesion turnover. Overall, these findings suggest that P2X7 plays a critical role in promoting migration after corneal epithelial injury by coordinating rapid rearrangements of the actin cytoskeleton and turnover of focal adhesions at the leading edge.

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. An excellent model tissue is the corneal epithelium, which is an avascular stratified squamous tissue that responds to growth factors and nucleotides when the epithelial barrier is damaged. One signal that has a ubiquitous response in epithelial wound healing is the cellular release of the nucleotide ATP, which may occur because of mechanics forces and/or change in cell shape. Within milliseconds to seconds after injury, extracellular ATP binds to purinoreceptors and triggers a transient Ca2+ wave, which is used by cells to transduce mechanical signals into chemical signals and alter signaling pathways. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. In this study we developed a novel method to identify and characterize the degree of cell-cell communication that occurs through sustained Ca2+ mobilizations after injury, which are concentrated along the epithelial wound edge and reduced in cells distal to the injury. Using MATLAB analyses, we generated profiles of the sustained Ca2+ mobilizations, and demonstrated that the Ca2+ response was replicated in ex vivo organ culture models. The sustained Ca2+ mobilizations were present also after stimulation with either BzATP or UTP, which are agonists of P2X7 and P2Y2 respectively. The probability that cells would communicate was greater in response to BzATP compared to UTP. The specificity of these ligands was demonstrated using competitive inhibitors of P2Y2 and P2X7 receptors, AR-C 118925XX and A438079, respectively. An inhibitor of pannexin-1, 10Panx, attenuated both wound closure and BzATP agonist-initiated response. These sustained mobilizations are correlated with changes in cellular morphology and motility, which were prominent in cells at the leading edge of the wound during cell migration. Together, our results demonstrate that the sustained Ca2+ mobilizations mediated by purinoreceptors and pannexins are a vital component in regulating the long-term response to injury, as studied in organ culture.

Postoperative corneal complications from pars plana vitrectomy surgery on the retina have been studied extensively in the literature. Researchers are aware of possible issues with oil tamponades, laser techniques, and other methods used. There could be clear markers to focus on a pattern of retina diagnoses of the patients that seem more prone to these problems in the front of the eye. Studies have noted refractive issues and increased cataract or posterior capsule opacification (PCO) progression but it is inconclusive in many studies on a corneal safety standpoint. Furthermore, a 23 or 27gauge pars plana vitrectomy has varying protocols depending on the diagnosis of the retina patient including endolaser, tamponade exchange, or even endoscopic cyclophotocoagulation. This study was conducted to research data looking at varying combinations of surgical type, diagnosis, and other patient characteristics to gain statistical evidence or relative frequency to better understand what type of retinal demographics cause corneal complications. The list of corneal complications in this study include: visual distortion involving anisometropia or photophobia, increased intraocular pressure including Uveitis-Glaucoma Hyphema syndrome, allergic conjunctivitis, a lens subluxation, herpes virus (zoster or simplex), a corneal scar or lesion, neurotrophic cornea, bullous keratopathy and corneal neovascularization. This was a retrospective case study evaulating 57 patients and 58 eyes that underwent a retinal surgery, with corneal complications at Beth Israel Deaconess Medical Center between October 2013 to December 2016. Number of patients, systemic demographics including frequency of hypertension and diabetes, and frequency of retina surgery per eye was analyzed. Moreover, we looked at different retina diagnoses to view which groups have a higher occurrence of complications after surgery. We used silicone oil as a way to divide the corneal complication patients to determine if the use of oil had an effect on a higher rate of issues after surgery. Eyes were divided into treatment with silicone oil group (n=23) and a non-silicone oil group (n=34), and we found that the silicone group had a significantly higher frequency of retinal surgeries (p<0.001). Moreover, there was no significant evidence between certain systemic factors (p<0.05), that allowed us to include the silicone oil and non-silicone oil patients as a unified group. When looking at our retina diagnoses we saw some groups had a higher percentage of complications when we took total number of problematic postoperative outcomes and divided that by total number of surgeries. Over one quarter of the surgeries per category leading to corneal complications occurred in the categories of subluxed lens, endophthalmitis, trauma, and uveitis-glaucoma hyphema Syndrome or neovascular glaucoma. Vitreomacular traction similarly had a high percentage of patients with corneal complications. Retinal detachment and epiretinal membrane were largest quantities of a specific retinal problem with low percentages of fewer than 15% with complications postoperatively. The study found that in our patient demographic silicone oil did not seem to be a factor in causing more corneal complications but it did cause more retinal surgeries. Moreover, certain retina diagnoses seem more prone to cause challenging outcomes, which leaves room for further studies distinguishing certain factors that could cause such specific issues.

Yes / A confocal microscope provides a sequence of images of the corneal layers and structures at different depths from which medical clinicians can extract clinical information on the state of health of the patient’s cornea. Hybrid model based on snake and particle swarm optimisation (S-PSO) is proposed in this paper to analyse the confocal endothelium images. The proposed system is able to pre-process (quality enhancement, noise reduction), detect the cells, measure the cell density and identify abnormalities in the analysed data sets. Three normal corneal data sets acquired using confocal microscope, and two abnormal endothelium images associated with diseases have been investigated in the proposed system. Promising results are achieved and the performance of this system are compared with the performance of two morphological based approaches. The developed system can be deployed as clinical tool to underpin the expertise of ophthalmologists in analysing confocal corneal images.