Este trabalho é parte de um projeto destinado a criar um equipamento médico, para ser aplicado com o protocolo de crosslinking de colágeno corneano, para o tratamento do ceratocone, do qual resultou o desenvolvimento de um sistema de irradiação UV-A de córneas para a aplicação clínica oftalmológica. O sistema desenvolvido neste trabalho consiste de um circuito de malha fechada, que estabiliza a potência luminosa emitida (com erro global, após a calibração, menor que 20% durante a emissão), possibilita seu ajuste até 5 mW (6,366 mW/\'CM POT. 2\') e permite selecionar a duração da emissão entre 10 s - 30 min. O sistema irradia com pico em 365 nm \'+ OU -\' 15 nm sendo que o design óptico proporciona spots homogêneos para três diâmetros selecionáveis: 10 mm; 8 mm; e 6 mm. O software desenvolvido tem a função de controlar todo o procedimento médico do crosslinking corneano, além de detectar falhas no sistema. Os resultados obtidos neste trabalho possibilitaram a criação de um equipamento robusto, flexível e estável, capaz de competir diretamente com os produtos internacionais comercializados mundialmente. Até a presente data tem-se conhecimento de apenas dois produtos, para mesma finalidade, comercializados em todo mundo, sendo ambos de empresas européias. Desta maneira, o sistema descrito nesta dissertação integra hoje o único aparelho de crosslinking totalmente projetado e produzido com tecnologia nacional. / This work is part of a project to create a medical device to be applied with the protocol for corneal collagen crosslinking for the treatment of keratoconus, which resulted in the development of an UV-A system for corneas irradiation for ophthalmic clinical application. The developed system consists of a closed loop circuit, which stabilizes the emitted light power (with global error, after calibration, less than 20% over the issue), allows its adjust up to 5 mW (6.366 mW/\'CM POT.2\') and allows to select the emission duration between 10 s - 30 min. The system irradiates with a peak wave length at 365 nm \'+ OU -\' 15 nm and the optical design provides homogeneous spots for three selectable diameter: 10 mm, 8 mm and 6 mm. The developed software has the function of controlling the medical procedure of corneal crosslinking, and detect system failures. The results of this work enabled the creation of a robust equipment, flexible, and stable that can compete directly with international products marketed worldwide. Until the present date, there has been aware of only two products for the same purpose, marketed worldwide, both from European companies. Thus, the system described in this dissertation integrates now the only unit of crosslinking completely designed and produced with national technology.

Orientador: Regina Paolucci El Dib / Coorientador: Amélia Kamegasawa / Banca: Eliane Jorge / Banca: Tais W. / Resumo: Introdução: A córnea é uma túnica transparente responsável por 60% do poder refrativo do olho. Segundo a Organização Mundial da Saúde (OMS), doenças que afetam a transparência da córnea são responsáveis por 5,1% do total de cerca de 45 milhões de cegos. O Brasil é o país que mais realiza transplantes de córnea na América Latina. A taxa de perda endotelial gira em torno de 0,6/ano em pessoas normais. Essa taxa é acelerada após cirurgias oculares ou glaucoma. Essa perda, após o transplante penetrante devido trauma inicial, reação imune, glaucoma secundário, interação celular entre o doador e receptor, seria de 7,8%/ano nos primeiros cinco anos e 4,2%/ano após 10 anos. Alguns estudos sugerem perda endotelial maior no Descemet's stripping automated endothelial keratoplasty/Ceratoplastia endothelial automatizada "stripping" de Descemet (DSAEK) (34% após seis meses) do que no transplante penetrante (11%) enquanto outros mostram que a perda endotelial após um ano é maior no transplante penetrante. A sobrevida do enxerto em ambas as técnicas foram semelhantes e o resultado óptico superior na técnica DSAEK. Há quase um século o transplante penetrante tem sido a técnica cirúrgica de escolha no manejo de alterações corneais. Apesar das vantagens de procedimentos lamelares, como menor risco de complicações intra-oculares e rejeição do enxerto, o transplante penetrante ainda mostra ser a técnica de preferência da maioria dos transplantadores mundiais. Entretanto, nos últimos anos, várias técnicas de ceratoplastia lamelar têm sido desenvolvidas, modificadas ou melhoradas, principalmente técnicas para substituição da porção posterior para correção da ceratopatia bolhosa. A escolha de ceratopatia bolhosa pós facectomia foi por esta ser a principal causa de transplantes relacionados a disfunções endoteliais. Objetivo: Avaliar a eficá cia e a seguranç a d a ceratoplastia endotelial quando... / Abstract: Introduction: The cornea is a transparent organ responsible for 60% of the refractive power of the eye. According to the World Health Organization (WHO), diseases affecting the transparency of the cornea are responsible for 5.1% of total of about 45 million blind. Brazil is the country that performs corneal transplants in Latin America. Endothelial loss rate is around 0.6 / year in normal people. This rate is accelerated after eye surgery or glaucoma. This loss after penetrating keratoplasty because initial trauma, immune reaction, secondary glaucoma, cell interaction between donor and recipient, it would be 7.8% / year in the first five years and 4.2% / year after 10 years. Some studies suggest a greater endothelial cell loss in DSAEK (34% after six months) than in penetrating keratoplasty (11%) while others show that endothelial cell loss after one year is higher in penetrating keratoplasty. Graft survival in both techniques were similar and higher optical result in DSAEK technique. For nearly a century the penetrating keratoplasty has been surgical technique of choice in the management of corneal changes. Despite the advantages of lamellar procedures such as intraocular lower risk of complications and graft rejection further shows penetrating keratoplasty is the preferred technique of most of the world transplant. However, in recent years, several lamellar keratoplasty techniques have been developed, modified or improved, especially techniques for replacement of the posterior portion for correction of bullous keratopathy. Objective: To evaluate the efficacy and safety in endothelial keratoplasty when compared to penetrating keratoplasty for the improvement of visual acuity in aphakic and pseudophakic bullous keratopathy. Methods: Systematic review of randomized controlled trials (RCTs) and / or quasirandomized studies that assessed endothelial keratoplasty versus penetrating keratoplasty in adults diagnosed with aphakic or ... / Mestre

INTRODUCTION: The cornea forms the anterior-most barrier of the eye, consisting of a non-keratinized pseudostratified squamous epithelium, a collagen-based stroma, and an endothelium. It is completely avascular, yet the most densely innervated structure in the human body. The sensory nerves project from the ophthalmic branch of the trigeminal cranial nerve into the limbal/stromal interface. From there, the nerves branch and ascend into Bowman’s membrane, a basal lamina delineating the epithelium from the stroma, and project into the epithelium as free nerve endings. Injury to the corneal epithelium can potentially lead to impaired vision if the wound healing process is not properly initiated. Immediately after injury, nucleotides such as ATP are released and bind to purinergic receptors known to be located in epithelial cell membranes, thereby initiating epithelial cell migration to close the wound. Malfunctions in the interactions between the corneal nerves and their epithelial counterparts during the wound healing process are thought to contribute to the attenuated wound healing characteristic of diabetes. However, the precise nature of these interactions, how they facilitate wound healing, and how they are impaired in diabetes, is not well understood. OBJECTIVES: Previously, our lab has shown that a member of purinergic family receptors (P2X7) is localized in the basal epithelial cells and becomes relocated to the leading edge of the wound after injury. When the relocation is inhibited, migration is attenuated. Additionally, it is known that diabetic mouse models display slower wound healing rates. The present study has three aims: (1) to replicate the characteristic sub-basal whorl organization of the corneal nerves in organ-cultured corneas; (2) to elucidate the connections between patterns of corneal innervation and purinergic receptor expression; and (3) to understand how these patterns interact to facilitate normal wound healing and how these interactions are disrupted in a diabetic model. METHODS: Our approach was to use immunohistochemistry of dissected mouse and to visualize the tissue using confocal microscopy. Sensory innervation profiles from diet induced obesity (DIO) mouse corneas and their wildtype C57Bl6 counterparts were compared in unwounded and wounded tissue. To image the nerves a methanol fixation protocol was optimized to examine the sub-basal plexus and the apical nerves. Corneas were dissected, stained with beta III-tubulin, which identifies nerves, and with an antibody to the P2X7 purinergic receptor, which is expressed in the epithelium and nerves. Trephine induced epithelial abrasion injuries were made on separate DIO and control models to compare re-epithelialization and re-innervation between the diseased and healthy states. Corneas were imaged using a Zeiss LSM 700 laser scanning confocal microscope and optical images were taken through the cornea over a distance averaging 115 microns. Corneas were imaged using a macro tiling plugin, stitching 3x3 optical z-stacks into composite images. The 3x3 tiles were created to image the central whorl, as well as the peripheral nerve fibers. Co-localization of P2X7 and betaIII tubulin were determined by thresholding using ImageJ/FIJI software. RESULTS: The elegant organization of the centralized sub-basal whorl of the control mouse was disrupted in the DIO mouse cornea, appearing fragmented and incomplete. Analysis of 7.5 and 15 wk corneas showed the whorl to be present at 7.5 wks. Average apical nerve fiber projection length was decreased in DIO cornea. Yet, analyses at each epithelial layer demonstrated overall increased apical nerve density in the DIO corneas as compared to control while sub-basal nerve density decreased dramatically. Stromal nerves remained equivalent. P2X7 did co-localize to the large stromal nerve fibers but it was difficult to show the localization along the sub-basal nerve plexus. However in cross-section images, P2X7 displayed an intracellular polarity, and was present along the apical surface of the columnar basal epithelial cells lining the basement membrane. This localization may suggest the presence of P2X7 expressing sensory nerves, which may be ideally poised for communication with the basal cells after injury. CONCLUSIONS: These data support the hypothesis that there is indeed a difference between diabetic and control corneal innervation. While wound healing differences due to the interaction between sensory nerves and the localization of P2X7 in epithelium at the leading edge remain to be fully elucidated, the novel finding of P2X7 expression in corneal nerves confirms a potential role of purinergic receptor and nerve coordination in conducting the wound healing response.

As junções GAP são canais intercelulares responsáveis pela comunicação de células vizinhas, por onde passam pequenas moléculas e íons que mantêm a homeostasia celular. A junção GAP é formada seis proteínas, as conexinas. Na célula endotelial encontram-se as conexinas 37, 40 e 43. Nesse estudo, estimulamos a angiogênese em córnea de camundongos, através da cauterização com cristal de nitrato de prata. Foram utilizados camundongos heterozigotos para o gene da conexina 43 (Cx43+/-) e camundongos selvagens (Cx43+/+). As córneas foram analisadas 2 e 6 dias após a cauterização atravéspor meio da morfologia vascular, detecção das Cx37, Cx40, Cx43, PCNA por meio de Western Blot e avaliação ultraestrutural das células endoteliais. Como resultado obtivemos uma menor área de preenchimento vascular nos animais Cx43+/- em 2 e 6 dias após a lesão corneal, porém, em relação a extensão dos vasos não foi observado diferenças entre os grupos. Uma menor proliferação celular foi verificada através da detecção do PCNA, nos animais heterozigotos, somente após 2 dias da lesão corneal. Não houve alteração da Cx37 e Cx40 entres os grupos. A Cx43 parece ser uma conexina importante para a célula endotelial durante o processo de angiogênese. / The GAP junctions are intercellular streams responsible for the communication between close cells, which allow small molecules and ions to pass through them maintaining the cellular homeostasis. The GAP junction is formed of six proteins, the connexin. In the endothelial cell, there are the connexin 37, 40 and 43. In this study, we stimulated the angiogenesis in the mice\'s cornea through its cauterization using silver\'s crystal glass. It was used heterozygote mice to the gene of connexin 43 (Cx43+/-) and wild mice (Cx43+/+). The corneas were analyzed 2 and 6 days after the cauterization through the vascular morphology, detection of Cx37, Cx40, Cx43, PCNA through Western Blot and ultrastructural evaluation of the endothelial cells. As a result, we obtained a smaller area of vascular fillness in the animals Cx43+/- with 2 and 6 days of corneal injury, however, in regard to the extensions of the vessels, it wasn\'t observed any changes between the groups. A smaller proliferation of cells was verified, through the detection of PCNA, in the heterozygote animals only 2 days after the corneal injury. There wasn\'t any modification of the Cx37 and Cx40 between groups. The Cx43 seems to be an important connexin to the endothelial cell during the process of angiogenesis.

Corneal endothelial cells have been shown to possess a uniform polygonal and complex multipolar shape at their apical and basolateral surface respectively. We established a morphological timetable to study how this complex shape arises postnatally. Corneas were collected from mice between postnatal day 8 to postnatal day 35 and labelled with antibodies specific for ZO-1 and NCAM at apical and basolateral region, respectively. Images were collected using wide-field fluorescence microscopy and morphometrically analyzed. Results showed that apical cell sizes increase linearly over the first 3 weeks, plateauing at 4-5 weeks postnatally with increased regularity. Basolateral membrane surfaces remained relatively smooth prior to eyelid opening and thereafter begins developing showing differences in development from periphery to the center until about 4 weeks postnatally when the wavy processes get vivid. Results indicate concurrent and independent development at both poles of the corneal endothelium, with more complexity seen at the basolateral surface.

Being the most abundant protein in the body, by mass, type I collagen provides the building blocks for tissues such as bone, extra-cellular matrix, tendons, cornea, etc[1-3]. The ability of a single protein to create structures with such various mechanical properties is not fully understood. Before one can engineer and assemble a complex tissue, such as cornea, the mechanisms underlying the formation and assembly, mechanical properties, and structure must be investigated and quantified. The work presented herein contains an extensive study of Type I collagen from the molecular to the tissue level. The engineering of collagenous tissues that mimic the mechanical and optical properties of native human cornea have been performed by a number of groups[4-7]. In all of these studies, the corneal-mimicking tissues have been created using a number of methods including repeated flow casting. To date, the ability to create self-assembled corneal tissue has not been achieved. Understanding the mechanisms of formation of native cornea will not only bring us closer to achieving self-assembled transplantable corneal tissue but will also aid in the engineering of all collagenous tissues and other structures comprised of filamentous units. Recently, the study of type I collagen has primarily focused on the tissue, fiber, and fibril scale[2, 8-21]. Grant, et al.[20] measured the elastic modulus of collagen fibrils in various solutions and found that by increasing ion concentration, in the solution around the fibril, the elastic modulus increased. The solution dependent behavior of the elastic modulus of collagen fibrils was measured but the cause of the dependence was unknown. Grant et al. state that due to the complex nature of the interactions between collagen fibrils and aqueous solutions, the exact cause of this effect is difficult to determine. Through work presented herein, not only do we show that this behavior is seen at the molecular level but also quantify the relationship between ionic concentration and molecular stiffness for a variety of ionic species. Studies of collagen mechanics, on the molecular level, are brief[22-26]. The most prominent of these studies in recent years was performed by Sun, et al.[27] wherein a persistence length of 14.5nm, for human type I procollagen, was measured. The persistence length of the molecule, which is a measure of flexibility, is a highly debated topic with quoted values of 14.5nm[27], 57nm[28], 130nm[29], 175nm[30], 308nm[31], and 544nm[32]. The broad range of values indicates that the flexibility of the collagen molecule is a complex question. It became apparent that the disagreement of the persistence length of molecular collagen in the literature may be due to the use of different ionic solutions. To address this, an initial atomic force microscope, AFM, study of the persistence length of molecular collagen diluted in DI water and two ionic solutions was conducted. This study showed that there is a strong solution dependence to the flexibility of the molecule. The ionic solutions presented molecules with a large persistence length, a straightened configuration, while the DI water dilution resulted in a persistence length that was a factor of 10 smaller. Because two different complex ionic solutions in the initial study showed different persistence lengths, an evaluation of the effect of each individual salt was performed. To elucidate the effects of individual ionic species on the conformations and persistence length of Type I collagen varying concentration of monovalent and divalent salts with different cations and anions were tested. It was found that increasing ionic concentration for all species types resulted in a higher persistence length but the rate of change in persistence length as a function of concentration is unique to each species. In 2002 Leikina, et at.[33] suggested that Type I molecular collagen is unstable at body temperature using differential scanning calorimetry. To examine these results, an AFM study was performed that imaged the collagen molecules after being held at body temperature for varying times. The density of molecules deposited onto mica, above a 200nm length cutoff, was calculated and it shows that the number of molecules above 200nm in length decreases with increasing incubation time. These environmental studies were performed with an aim to understanding the role of environment in creating a corneal mimicking tissue. Currently, the most promising method of collagen membrane fabrication for corneal replacement was developed by Tanaka, et al.[4]. This unique repeated flow casting method allows for the manufacturing of transparent collagen membranes with controllable thickness and fibrillar alignment. Using the repeated flow casting technique, orthogonally oriented collagen membranes were created and their optical properties were measured using the Generalized High Accuracy Universal Polarimeter, G-HAUP. When engineering a tissue for the eye, the optical properties of the tissue are of the utmost importance. Appropriately for corneal tissues, the measurements for linear birefringence and linear dichroism were negligible. It was clear, from the literature, that a fundamental understanding of molecular type I collagen was not available. In this work, the mechanical properties and environmentally sensitive behavior of bovine dermal type I molecular collagen is studied. The exploration into the unique behavior of these systems begins with documenting the rich ionic species and concentration dependent flexibility of molecular type I collagen and the temperature dependence on the stability of the molecule is tested. The study concludes with the construction of corneal mimicking tissues using the repeated flow casting method and measuring the complex optical properties of these tissues.

Orthokeratology (OK) is the reduction, modification or elimination of myopia through application of contact lenses. With the development of high Dk/t lens materials, overnight therapy has become the modality of choice for OK. Overnight OK lens wear has been previously investigated in terms of its efficacy to reduce myopia. However, the underlying effects of overnight OK lens wear on the human cornea have received less attention. As well as the clinical efficacy of overnight OK, this study investigated the effects of overnight OK on topographical corneal thickness and the overnight corneal edema response, and corneal tissue changes with overnight OK. Eighteen subjects participated as the OK lens-wearing group, wearing BE lenses (UltraVision, Brisbane, Queensland) in both eyes. A further ten subjects participated as control subjects, wearing conventional rigid lenses (J-Contour, UltraVision) in the right eye (RE) only. The left eye (LE) acted as a non-lens-wearing control. Both groups wore lenses overnight only, with no lens wear during the day. Measurements were conducted at baseline then on day 1, 4, 10, 30, 60, and 90 for the OK lens-wearing eyes; and up to day 30 for the control group, in the morning (after overnight lens wear) and in the evening (after 8-10 hours of lens removal). Variables measured included best vision sphere (BVS), unaided logMAR visual acuity (VA), refractive astigmatism, apical corneal power (ACP), simulated K readings (Medmont E300 corneal topographer), topographical corneal thickness (Holden-Payor optical pachometer), and keratocyte and endothelial cell densities (ConfoScan2 confocal microscope). Approximately 75% of myopia was corrected after the first night of OK lens wear and the changes in refractive error stabilised by day 10. By day 90, myopia reduction averaged 2.54 ?? 0.63 D. This was associated with significant improvement in unaided VA of about 82% after the first night of lens wear. There was no change in refractive astigmatism over the 3-month period. There was significant reduction in ACP in the OK lens-wearing eyes after the first night of lens wear, which accounted for more than 70% of the total ACP change over the 3-month period (RE: -2.16 ?? 0.53 D; LE: -2.11 ?? 0.86 D). There was significant central epithelial thinning (about 30%) and significant thickening (about 3%) in the mid-peripheral stroma in the OK lens-wearing eyes. Significant central epithelial thinning was found after the first night of lens wear while thickening in the mid-peripheral stroma reached statistical significance by day 4. Further analysis suggests that topographical corneal thickness changes account for the refractive error changes with overnight OK lens wear, rather than corneal bending. The central overnight stromal edema response was significantly reduced in the OK lens-wearing eyes (1.2 ?? 0.5%) to a level lower than in the conventional RGP (6.2 ?? 1.2%) and non-lens-wearing eyes (2.5 ?? 0.9%) in the control group. Mid-peripheral and peripheral stromal edema responses showed similar levels to those predicted based on lens Dk/t. A single overnight wear of BE and Paragon Corneal Refractive Therapy (CRT) lenses showed that the edema response to BE lens wear is significantly less than in the CRT lens-wearing eyes (BE: 2.5 ?? 0.7%; CRT 3.5 ?? 1.3%) immediately on eye opening. No significant changes were found in either central stromal keratocyte or endothelial cell densities in either OK or control groups over the study period. In conclusion, overnight OK lens wear induces significant reductions in myopia after the first night of lens wear associated with improvement in unaided VA. Overnight OK lens wear causes significant thinning in the central epithelium and significant mid-peripheral stromal thickening which results in flattening of the central cornea and steepening in the mid-periphery. Although there were no significant changes in central stromal keratocyte and endothelial cell densities, thinning of the central epithelial layer raises concerns regarding the safety of the procedure, especially with the alarming number of corneal infections reported recently in the literature.

Microbial contamination of contact lenses is a significant risk factor leading to adverse responses. Adhesion of microorganisms to a contact lens is the first step in a series of events that leads to contact lens-related infections or inflammation. Recently, some of the non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to have the ability to interfere with microbial biofilm formation. In this project, antibacterial properties of commonly used NSAIDs (salicylic acid, sodium diclofenac and ketorolac) were assessed and characterised using biological assays and molecular biological techniques. Salicylic acid, ketorolac and diclofenac reduced adhesion of a range of bacterial species isolated from corneal infection and inflammatory events to contact lenses in a dose-dependent manner. Salicylic acid also decreased the adhesion of Pseudomonas aeruginosa and Staphylococcus epidermidis to human corneal epithelial cells in a dose-dependent manner. Results further demonstrated that NSAIDs had a significant impact on the production of virulence factors such as Type IV pili mediated (twitching) motility, flagella mediated swimming, elastase, protease IV and alkaline protease and affected the production of acylated homoserine lactones of P. aeruginosa. Salicylic acid and ketorolac affect the expression of P. aeruginosa outer membrane proteins. In the presence of the salicylic acid and ketorolac more than 85% of all detectable outer membrane proteins changed and most were down-regulated. Moreover, in the presence of salicylic acid at least five gene products, including Na+ - translocating NADH (Nrq1), choline dehydrogenase (CHDH), a hypothetical protein of unknown function, a gene product with no similarity to any known sequence in the database and a sequence similar to 23S rRNA of P. aeruginosa, were down-regulated. The results of this study clearly demonstrated that NSAIDs have a significant impact on virulence factors and the expression of acylated homoserine lactones by P. aeruginosa. This thesis has illustrated the potential of NSAIDs for preventing bacterial contamination of contact lenses by ocular pathogens and highlights the potential for NSAIDs as antibacterial agents. Therefore, this class of compound should be investigated further for their therapeutic efficacy in vivo.

Purposes: To use optical coherence tomography (OCT) to image and quantify the effect of various corneal conditions, in terms of corneal, stromal and epithelial thickness, and light backscatter. To assess the changes caused by overnight orthokeratology (Corneal Refractive Therapy; CRT^TM) lens wear, keratoconus and laser in-situ keratomileusis (LASIK) refractive surgery, each of which may lead to topographical alterations in corneal thickness either by temporary moulding, degeneration, or permanent laser ablation, respectively. <br /><br /> Methods: Topographical thickness of the cornea was measured using OCT in all studies. The CRT^TM studies investigated myopic and hyperopic treatment, throughout the day. The myopic studies followed lens wear over a 4 week period, which was extended to 12 months, and investigated the thickness changes produced by two lenses of different oxygen transmissibility. CRT^TM for hyperopia (CRTH^TM) was evaluated after a single night of lens wear. <br /><br /> In the investigation of keratoconus, OCT corneal thickness values were compared to those obtained from Orbscan II (ORB) and ultrasound pachymetry (UP). A new fixation device was constructed to aid in the measurement of topographical corneal and epithelial thickness along 8 directions of gaze. Pachymetry maps were produced for the normal non-lens wearing cornea, and compared with the rigid gas permeable (RGP) lens wearing cornea and the keratoconic cornea. <br /><br /> Thickness changes prior to, and following LASIK were measured and monitored throughout six months. Myopic and hyperopic correction was investigated individually, as the laser ablation profiles differ for each type of procedure. The LASIK flap interface was also evaluated by using light backscatter data to monitor healing. <br /><br /> Results: Following immediate lens removal after myopic CRT^TM, the central cornea swelled less than the periphery, with corneal swelling recovering to baseline levels within 3 hours. The central epithelium decreased and mid-peripheral epithelium increased in thickness, with a more gradual recovery throughout the day. There also seemed to be an adaptation effect on the cornea and epithelium, showing a reduced amount of change by the end of the 4 week study period. The thickness changes did not alter dramatically during the 12 month extended study. In comparing the two lens materials used for myopic CRT^TM (Dk/t 91 vs. 47), there were differences in stromal swelling, but no differences in the central epithelial thinning caused by lens wear. There was a statistically insignificant asymmetry in mid-peripheral epithelial thickening between eyes, with the lens of lower Dk causing the greater amount of thickening. Hyperopic CRT^TM produced a greater increase in central stromal and central epithelial thickness than the mid-periphery. Once again, the stroma recovered faster than the epithelium, which remained significantly thicker centrally for at least six hours following lens removal. <br /><br /> Global pachymetry measurements of the normal cornea and epithelium found the periphery to be thicker than the centre. The superior cornea and epithelium was thicker than the inferior. In the measurement of the keratoconic cornea, OCT and ORB correlated well in corneal thickness values. UP measured greater values of corneal thickness. The keratoconic epithelium was thinner than normal, and more so over the apex of the cone than at the centre. The location of the cone was most commonly found in the inferior temporal region. Central epithelial thickness was thinner in keratoconics than in RGP lens wearers, which in turn was thinner than in non-lens wearers. <br /><br /> Following LASIK surgery for both myopia and hyperopia, the topographical OCT thickness profiles showed stromal thinning in the areas of ablation. The central myopic cornea showed slight regression at 6 months. During early recovery, epithelial thickness increased centrally in hyperopes and mid-peripherally in myopes. By the end of the 6 month study, mid-peripheral epithelial thickness was greater than the centre in both groups of subjects. The light backscatter profiles after LASIK showed a greater increase in backscatter on the anterior side of the flap interface (nearer the epithelium), than the posterior side (in the mid-stroma) during healing. The flap interface was difficult to locate in the OCT images at 6 months. <br /><br /> Conclusion: All the CRT^TM lenses used in this project produced more corneal swelling than that seen normally overnight without lens wear. In order for these lenses to be worn safely for long periods of time without affecting the health of the cornea, they need to be manufactured from the highest oxygen transmissible material available. The long-term effect of thinning on the epithelium's barrier properties needs to be monitored closely. <br /><br /> Global topographical thickness of the cornea and epithelium was measured using OCT in normal, RGP lens wearing and keratoconic eyes. Corneal and epithelial thickness was not symmetrical across meridians. The epithelium of RGP lens wearers was slightly thinner than normal, but not as thin as in keratoconics, suggesting that the epithelial change seen in keratoconus is mainly due to the condition. <br /><br /> Post-LASIK corneal and epithelial thickness profiles were not the same for myopic and hyperopic subjects, since the ablation patterns vary. Epithelial thickening in the mid-periphery had not recovered by six months in myopes or hyperopes, possibly indicating epithelial hyperplasia. Light backscatter profiles were used to monitor the recovery of the LASIK flap interface, showing the band of light backscatter around the flap interface to decrease as the cornea healed.

Abstract Both in the closed and open eye state the superior limbus is covered by the upper lid. This region is of physiological interest and clinical importance because in chronic hypoxia, neovascularization of the cornea commonly occurs here. The limbal region in general is additionally of importance as the stem cells which are the source of the new corneal cells are located in the epithelium of the limbus and these are vital for normal functioning and are affected under certain adverse conditions. Purpose: In this experiment I examined corneal morphology in the limbal area and in particular under the upper lid in order to primarily examine the variation in the corneal limbal epithelial and total thickness as well as epithelial and endothelial cell density. Methods: I measured 30 eyes OD/OS (chosen randomly) of thirty healthy subjects aged from 18 to 55 years in the first study and twelve participants in the second study, with refractive error ≤ ±4 D and astigmatism ≤ 2 D. The thickness and cell density of five positions: superior, inferior, temporal, nasal limbal and central cornea was determined with optical coherence tomography (OCT) and confocal microscopy. At least three scans of each position were taken in both studies with OCT. At least 40 of 100 adjacent sagittal scans of each image were measured using OCT software program. In the confocal study, image J software was used to determine cell densities. Results: The epithelial and corneal limbal thickness were significantly thicker than the epithelial and central corneal thickness (p<0.05). The limbal, inferior cornea is thinner than the three other positions and the temporal region of the cornea is the thickest both in epithelial and total cornea. Epithelial cell density was significantly lower in the superior cornea than the four other positions. There was no significant difference in the endothelial cell density. Conclusions: Using OCT with high resolution and cross-sectional imaging capability and confocal microscope with high magnification, I found that the limbal cornea is significantly thicker than the central cornea both in total and in epithelial thickness. In the limbus, one might expect the superior cornea (under the lid) to be thickest (because of the expected hypoxia) whereas I found the temporal cornea was thickest. The epithelial cell density was lower in the superior cornea but there was no significant difference in cell densities in the endothelium. Further morphological investigation is of interest.