Purpose: Solution-induced corneal staining (SICS) has been the subject of much debate in the clinical literature. While it has been suggested that this form of staining indicates toxicity of the cornea, microscopy studies have suggested that cells treated with multi-purpose solutions (MPS) known to produce SICS in vivo are undamaged. There is further debate in the literature as to whether or not sodium fluorescein (‘fluorescein’) actually enters epithelial cells or not. The aim of this work was to investigate the cellular mechanisms involved in SICS by developing an in vitro cell culture model to mimic the clinical presentation of this phenomenon. Methods: An in vitro model of SICS was developed using cultured cells that were exposed overnight to ReNu MultiPlus® (Bausch + Lomb) MPS. After addition of fluorescein, cells were imaged using an automated fluorescence microscope. Hyperfluorescent cells were identified using predetermined threshold of intensity in fluorescence microscope, and confocal microscopy was used to investigate where fluorescein was situated within the cells. The extent of cell toxicity was assessed using propidium iodide and Annexin V. In order to examine the contribution of passive and active transport mechanisms in fluorescein uptake and release, levels of hyperfluorescent staining were measured at 37°C and 4°C. In all described experiments, fluorescein staining was expressed by the proportion of hyperfluorescent cells in the total cells. Results: All cultured cells readily took up fluorescein at room temperature, however a sub-population of cells stained more intensely with fluorescein. These cells were termed ‘hyperfluorescent’ cells. Exposure to ReNu MultiPlus® resulted in a significant increase in the proportion of hyperfluorescent cells compared with control cells. In addition, the staining profiles of individual cells showed no correlation between cell death and hyperfluorescence. The data also showed that hyperfluorescence did not occur extensively in deliberately lysed cells.Addition of fluorescein to the cells at 4°C resulted in very low levels of hyperfluorescence compared to high levels at 37°C. Fluorescein was rapidly released from cells at 37°C but not from those at 4°C. Conclusion: In this work, an effective in vitro model of SICS was developed in order to provide a better understanding of the mechanisms involved in fluorescein staining. This work suggests that corneal fluorescein staining may reflect a simple cellular uptake of fluorescein. Levels of staining in the cells appear to be unrelated to cellular toxicity or cell damage. Staining appears to occur in the cytoplasm and the nucleus of the cells. Finally, fluorescein uptake and release are likely to occur through active transports mechanisms.

Orientador: Cláudia Valéria Seullner Brandão / Resumo: O estudo objetivou descrever valores de referência para importantes testes diagnósticos oftalmológicos, como sensibilidade corneal, produção lacrimal, pressão intraocular, espessura corneal e microbiota conjuntival em saguis de tufo preto (Callithrix penicillata) saudáveis e sem alterações oculares. Foram utilizados 13 saguis de tufo preto, totalizando 26 olhos, provenientes do CEMPAS da Faculdade de Medicina Veterinária e Zootecnia - UNESP, Botucatu, São Paulo, Brasil. Os saguis foram submetidos a contenção química com anestesia dissociativa à base de cetamina e midazolam, para realização dos exames. Os resultados obtidos para sensibilidade corneal com estesiômetro Cochet-Bonnet foi de 5,8 ± 0,6cm; produção lacrimal com ponta de papel absorvente endodôntica estéril de 13 ± 3mm/min; pressão intraocular com tonômetro de rebote Tonovet Plus Icare® de 22,2 ± 2,9mmHg; espessura corneal 340,7 ± 25,5µm; e predomínio de bactérias Gram-positivas nas amostras de microbiota conjuntival. Não houve diferença significativa considerando lateralidade e sexo dos animais (P>0,05). Os resultados obtidos nos testes diagnósticos oftalmológicos poderão auxiliar médicos veterinários oftalmologistas a diagnosticar e tratar doenças oculares em saguis de tufo preto, além de contribuir para pesquisas na área de visão. / Abstract: The study aimed to describe reference values for important ophthalmic diagnostic tests, such as corneal sensitivity, tear production, intraocular pressure, corneal thickness and conjunctival microbiota in black tufted marmosets (Callithrix penicillata). Thirteen black tuft marmosets were used, totaling 26 eyes, followed by CEMPAS by the Faculty of Veterinary Medicine and Zootechnics - UNESP, Botucatu, São Paulo, Brazil. The procedures were used for chemical containment with dissociative anesthesia based on ketamine and midazolam, to perform the exams. The results obtained for corneal sensitivity with a Cochet-Bonnet esthesiometer were 5.8 ± 0.6 cm; tear production with sterile endodontic absorbent paper tip of 13 ± 3mm / min; intraocular pressure with Tonovet Plus Icare® rebound tonometer of 22.2 ± 2.9mmHg; corneal thickness 340.7 ± 25.5 µm; and predominance of Gram-positive bacteria in conjunctival microbiota. There was no significant difference considering the laterality and sex of the animals (P> 0.05). The results obtained in ophthalmological diagnostic tests can help veterinary ophthalmologists to diagnose and treat eye diseases in the black tufted marmosets, in addition to contributing to research in the field of vision. / Mestre

Purpose: The aim of the study was to evaluate the outcomes of corneal manual epithelial debridement (MED) for the treatment of painful bullous keratopathy (BK). Methods: In a prospective interventional case series, 15 eyes of 15 consecutive patients presenting with painful BK of varying aetiology underwent MED. Patients were followed up at 10 days, 1 month, 2 months, 3 months and 6 months post procedure. Outcome parameters evaluated include numeric rating pain score (NRS), visual acuity (VA), corneal transparency and size of corneal bullae. Results: The mean NRS was significantly decreased from its baseline value of 7.2 +- 1.7 at all follow-up visits (p < 0.02). Mean VA and corneal transparency remained stable for the duration of the study. In most patients the average size of corneal bullae was initially reduced, but returned to baseline by the end of the study. Conclusion: MED reduces mean pain scores and temporarily reduces the size of corneal bullae in BK. MED may be considered as a simple, low cost alternative for reducing pain in patients awaiting corneal transplant. Further studies are required to evaluate MED for the treatment of BK and compare outcomes against other palliative treatment options.

This body of work encompasses two studies: the collection of canine tears via a novel polyester<br />rod and the comparison of VEGF-A concentrations in tears from dogs with normal and<br />vascularized corneas. The first study used polyester rods for tear collection in dogs. Fluid volume and VEGF recovery characteristics, as well as potential binding of VEGF to the rod, were determined. Tears were harvested from normal dogs using rods and glass capillary tubes. Tears were assayed for tear film VEGF using a commercial canine VEGF sandwich ELISA kit. Dilutions of VEGF standard were wicked into the rods or drawn into capillary tubes, eluted, and assayed. Percent volume recovery is adequate for polyester rods as is percent VEGF recovery. VEGF is detectable in normal canine tears.The second study harvested tear samples from eyes of dogs with vascularizing corneal disease, as well as the contralateral unaffected eye of unilaterally diseased dogs, and normal dogs. Vascularization scores were assigned to diseased eyes and tear film VEGF concentration was assayed as above. Mean tear film VEGF concentration of diseased eyes did not differ from control eyes, and was not correlated with disease process, extent of vascularization, or other parameters. Tear film VEGF in unaffected eyes was significantly higher than control and vascularized eyes. Canine tear film VEGF exceeds biologically active concentrations, but does not correlate with state of corneal vascularization. VEGF-related control of corneal vascularization may be mediated by other proangiogenic factors. / Master of Science

Aim; This study looks at the effect of orthokeratology on a number of biometric parameters and refractive error in an adult population. Method; Forty three myopic subjects were recruited to a twelve month study into the effects of orthokeratology on ocular biometry and refractive error. Two different back surface lens designs were applied right eye) pentacurve and left eye) aspheric. The aspheric design was chosen to more closely mimic the cornea¿s natural shape. Anterior and posterior apical radii and p-values; corneal thickness and anterior chamber depth were measured using the Orbscan IIz; together with ocular biometry by IOL Master and a standard clinical refraction. All measurements were repeated at one night, one week, one, three, six and twelve months. Refractive changes were analysed against biometric changes. Results; Twenty seven participants completed one month of lens wear. Twelve subjects completed twelve months of lens wear. Subjects with myopia ¿ -4.00DS were successfully treated with orthokeratology. Both anterior and posterior apical radii and p values were altered by orthokeratology. Corneal thickness changes were in agreement with previously published studies. Axial length and anterior chamber depth were unaffected by the treatment. Conclusion; Orthokeratology should be available as an alternative to laser refractive surgery. It is best restricted to myopes of up to -4.00DS with low levels of with the rule corneal astigmatism. The use of an aspheric back design contact lens did not produce a significant benefit over that of a pentacurve.

Keratoconus is an eye disease that manifests as progressive thinning and steepening of the cornea. While there is no singular cause for keratoconus, both genetic and environmental factors have been proposed to influence the onset and progression. This review aims to explore the pathogenesis, identification, classification, and treatment of this corneal disease. Since early detection is essential in treatment success, various imaging methods have been developed to analyze multiple aspects of the corneal surface. Keratoconus can be identified with a reflection based system, elevation based system, as well as a combination of the two. Once diagnosed, the Belin ABCD classification can be used to monitor the stage of keratoconus and treat it accordingly. Current treatment options prioritize halting disease progression with corneal crosslinking before considering visual rehabilitation. Individuals with mild keratoconus can improve vision with non-surgical options such as spectacles and contact lenses. As the disease progresses, patients may need surgical intervention such as intrastromal corneal ring segments or photorefractive keratectomy (PRK). In advanced keratoconus cases, the cornea may need to be replaced with partial or full-thickness keratoplasty. The goal of this review is to evaluate the more current treatment options that have become available today.

BACKGROUND: The cornea is the outer portion of the eye and protects the eye from infection or debris. When the cornea becomes compromised due to age and disease (specifically Diabetes Mellitus), it becomes impaired and can have profound impacts on an individual’s quality of life by leading to vision loss or blindness. The different layers of the cornea all contain many proteins and collagen, and have varying degrees of thickness and biomechanical properties. Stiffness in the cornea has either been measured via the use of AFM (Atomic Force Microscopy) which involves removing a slice of the cornea and adhering to the surface, as a function of IOP (Intraocular Pressure), or tensile testing. Previous research has also used the nanoindenter to measure the stiffness of different layers in the intact globe (eyeball) within the mouse head or by adhering to PEG submerged in PBS. However, no studies to our knowledge have used the intact globe exposed to air and placed on a 3D printed model to measure different corneal layers via the use of nanoindentation. METHODS: 6 C57BL/6J mice were obtained between 8-12 and 27 weeks of age, had the eyes extracted, and half remained with intact epithelium while the other half had the epithelium abraded with a 1.5 mm trephine. The eyes were placed in keratinocyte solution (KCM) for preservation while they were transported to the site with a nanoindenter. The globes were then placed on a 3D printed holder, cornea facing up, and irrigated with KCM solution in between indentation measurements. The PIUMA Optics 11 Nanoindenter was used to measure the Effective Young’s Modulus of the epithelium, basement membrane, and stroma. The Oliver & Pharr modeling was used as opposed to the Hertzian Model due to the biomechanical and adhesion properties of the eye. RESULTS: A comparison of control mice at 9 weeks shows an average Effective Young’s Modulus of 30.73 kPa, and an average Effective Young’s Modulus for 15 week old mice of 62.50 kPa for the epithelium. The average Effective Young’s Modulus of the basement membrane for 9 week control mice was ~6.2 kPa and for older 27 week mice was ~6 kPa. The Effective Young’s modulus for the stroma of 9 week old mice was ~68.3 kPa and for 27 week old mice was ~ 222.7 kPa. CONCLUSION: It was observed that stiffer substrates (in this instance, stiffer layers) require stiffer probes. The opposite is true of softer substrates, which require softer probes. It is beneficial in either instance to use a larger tip radius as there will be more contact and surface area measurement, so the probe has less recoil due to the adhesion from the corneal layers. The values observed in this study correlated with the values seen in the study conducted by Xu et al. However, the basement membrane values were different and could be due to probe specifications or layer thinness. Additional studies are needed to observe changes in Young’s Modulus based on probe characteristics with diseases such as Diabetes Mellitus (DM).

El queratocono (QC) se trata de una enfermedad corneal caracterizada por un adelgazamiento progresivo de la córnea y ectasia, que conduce a un astigmatismo irregular y finalmente, a una pérdida de la agudeza visual. Aunque existen diferentes modalidades de tratamiento del QC, el único tratamiento que ha demostrado retrasar la progresión del QC es el crosslinking corneal (CXL), con el objetivo de detener la progresión del QC, fortaleciendo las propiedades biomecánicas de la córnea. A pesar de que el CXL estándar o convencional (CXL-S), sigue siendo el "gold standard", con las posibles complicaciones secundarias relacionadas con esta técnica, en la actual tesis se ha demostrado, que la capacidad de la técnica de CXL epi-on (epithelium-on, por sus siglas en inglés) de detener la progresión del QC es similar y comparable, a la técnica de CXL epi-off (epithelium off, por sus siglas en inglés). Además se ha observado que la mayoría de los artículos publicados, comparan diferenes técnicas de CXL en QC. Sin embargo, para optimizar el tratamiento de estos pacientes, es importante establecer una seria de factores predictivos preoperatorios de los cambios visuales tras el CXL, para así elegir la mejor técnica. De esta manera, en la actual tesis se ha comprobado los factores predictivos del cambio visual a tener en cuenta a priori en las córneas sometidas a CXL, epi-on y epi-off, y se ha comprobado que los factores difieren para ambas técnicas, sugiriendo que el mecanismo de acción es distinto entre ellas. Otra opción de tratamiento en pacientes con QC, son la implantación de los anillos corneales intraestromales (ACI), que reducen las aberraciones de alto orden, y secundariamente mejoran la calidad visual. Sin embargo, los estudios disponibles presentan una gran variabilidad, que podría ser explicada por diferentes nomogramas disponibles o los distintos criterios de implantación de los ACI. En la actual tesis se ha desarrollado un nomograma de implantación de ACI optimizado, para lograr mejores resultados y más predecibles. En los últimos años, el uso de las lentes de contacto esclerales (LCE) han adquirido gran importancia en el manejo del QC, para una adecuada rehabilitación visual. Sin embargo, existe una gran variabilidad entre pacientes por varios factores, como puede ser el vault de la LCE. En la actual tesis, se ha demostrado una buena eficacia con la LCE ICD 16.50, en lo que respecta a la rehabilitación visual y lo más importante es que ésta mejora en la AV, se ha podido predecir a partir de varios factores. En definitiva, la actual tesis ha sido clave para lograr una optimización del manejo del QC. Las técnicas de CXL epi-on y epi-off, presentan una capacidad similar para detener la progresión del QC; se han obtenido los factores predictivos del efecto del tratamiento del CXL y uso de LCE en el QC; y se ha desarrollado un nomograma optimizado para la implantación de los ACI.

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