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Improved corneal epithelial cell growth over artificial cornea materials is required to improve device retention within the eye. In this work, varying concentrations of epidermal growth factor (EGF), a potent mitogen for epithelial cells, were immobilized to polydimethylsiloxane (PDMS) substrates, and the cellular response was analyzed. Three methods were developed to bind EGF to PDMS via polyethylene glycol (PEG) tethers. 1) Plasma Modification: EGF was first reacted with homobifunctional NHS2PEG and then bound to allylamine plasma-modified PDMS. 2) Hydrosilylation: PDMS was modified with heterobifunctional allyl-PEG-NBS and then EGF was attached to the surface-bound PEG. 3) Thiol Modification: EGF was first reacted with heterobifunctional NHS-PEG-maleimide and then bound to thiol-modified PDMS. Using Method 1 (Plasma Modification), 40 to 90 ng/cm2 of EGF was bound, however 70% of this was adsorbed even under optimized EGF-PEG reaction conditions. Cells rapidly grew to confluence on these surfaces, and cell counts increased significantly compared to control surfaces. Extracellular matrix protein production was also increased on the EGF-modified surfaces, corresponding to significantly higher levels of cell adhesion observed under a detachment force. Modification by Method 2 (Hydrosilylation) resulted in 10 to 300 ng/cm2 of bound EGF, of which 20% was adsorbed. However, despite increased EGF binding homogeneity, the cell growth was slower on these surfaces than on those prepared by Method 1, and coverage was non-uniform at all EGF concentrations. This is likely due to a higher underlying PEG density, and binding of the PEG and EGF in clusters on the surface. Simultaneous tethering of the cell adhesion peptide YIGSR had no further effect on cell coverage. Using Method 3 (Thiol Modification), 24 to 65 ng/cm2 of EGF was bound, of which 22% was adsorbed. This method enables more homogeneous EGF surface binding than Method 1, with a lower PEG density than Method 2. However, free thiol groups were inhibitory to corneal epithelial cell growth, even in the presence of bound EGF. Defunctionalization of free thiols by reaction with 3-maleimidopropionic acid restored cell growth and morphology on the PDMS, and may hence allow for retention of the proliferative effect of the EGF. These results indicate that while tethering of EGF to PDMS can improve the coverage by corneal epithelial cells, and presents a promising strategy for modification of polymeric artificial cornea materials, the effects are highly dependent on the underlying surface chemistry. / Thesis / Doctor of Philosophy (PhD)

The purpose of this study was to determine the effects of three medical treatments, topical oxytetracycline, topical prednisolone acetate, and systemic flunixin meglumine, on the activity of two proteinases, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), in equine tear film. The study design consisted of twelve ophthalmically normal horses separated into three groups of four in a cross-over study design. Each group was treated for 5 days with flunixin meglumine (500mg IM bid), topical 1% prednisolone acetate (0.2ml tid), or topical oxytetracycline (0.2ml tid), followed by a 9-day washout period. All topical medications were applied to the left eye and the right eye was treated with a placebo. Tears were collected before the first treatment on day one and the morning following the last treatment on day 5. Tear film proteinase activity was measured using gelatin zymography and measurements of optical density. Statistical analysis of the difference between the treated and untreated eyes and the eyes before and after treatment was performed using mixed effects model for ANOVA. When eyes were compared after treatment, there was no significant difference between treated and placebo eyes for MMP-2 or MMP-9 for any of the treatments. When post-treated eyes were compared to pre-treated eyes, there was a significant decrease in MMP-2 activity in the left eye of horses treated with flunixin meglumine (P=0.0259). There were no differences in MMP-2 and MMP-9 activity for the other treatments. In conclusion, topical 1% prednisolone acetate and topical oxytetracycline did not significantly change MMP-2 or MMP-9 activity in normal equine tear film. Systemic flunixin meglumine had an inhibitory, but questionable, effect on MMP-2 activity in normal equine tear film. This project was funded by Patricia Bonsall Stuart Award for Equine Research. / Master of Science

Yes / Microbial keratitis can arise from penetrating injuries to the cornea. Corneal trauma promotes bacterial attachment and biofilm growth, which decrease the effectiveness of antimicrobials against microbial keratitis. Improved therapeutic efficacy can be achieved by reducing microbial burden prior to antimicrobial therapy. This paper assesses a highly-branched poly(N-isopropyl acrylamide) with vancomycin end groups (HB-PNIPAM-van), for reducing bacterial attachment and biofilm formation. The polymer lacked antimicrobial activity against Staphylococcus aureus, but significantly inhibited biofilm formation (p = 0.0008) on plastic. Furthermore, pre-incubation of S. aureus cells with HB-PNIPAM-van reduced cell attachment by 50% and application of HB-PNIPAM-van to infected ex vivo rabbit corneas caused a 1-log reduction in bacterial recovery, compared to controls (p = 0.002). In conclusion, HB-PNIPAM-van may be a useful adjunct to antimicrobial therapy in the treatment of corneal infections. / Medical Research Council and the Department of Biotechnology, India under grant number, MR/N50188/2.

Corneal sensitivity and adverse events following subconjunctival administration of three local anesthetics in twelve mares were evaluated. The subconjunctival space of the treated eye was injected with 0.2 mL of mepivacaine (2%), ropivacaine (1.3%) and liposomal bupivacaine (1.3%). All horses received each medication once and the contralateral eye received saline. The corneal touch threshold (CTT) was measured using a esthesiometer before sedation, after sedation, and at specified intervals until it returned to baseline. The mean total time of anesthesia (TTA) was 168.3 minutes for ropivacaine, 169.2 minutes for liposomal bupivacaine, 103.3 minutes for mepivacaine and 30.7 minutes for control. The TTA for liposomal bupivacaine (P<0.001) and ropivacaine (P=0.001) was longer than the control. The TTA for mepivacaine was not different from the control (P = 0.138), liposomal bupivacaine (P = 0.075) or ropivacaine (P = 0.150). Injection site hemorrhage reduced TTA regardless of treatments (P=0.047). No adverse effects were noted.

M.Phil.(Optometry) / The concept of contact lenses was conceived over 500 years ago and has now evolved into a fundamental component of optometric practice. Soft contact lenses have become a convenient, aesthetically pleasing and comfortable alternative to spectacles that are becoming increasingly popular. The use of rigid contact lenses is imperative in the management of conditions such as keratoconus due to spectacles being insufficient in providing adequate vision. Placing a contact lens onto the cornea is an invasive procedure. The contact lens is a foreign body to the eye hence it is expected that the eye would react to that foreign body. Literature has revealed that the general reactions of the eye to contact lens wear are initial tearing, alteration of the tear layer and oedema due to reduced oxygen transmission but these are just a few of the known consequences amongst the multitude of the unknown consequences. What exactly goes on under a contact lens remains an enigma which contact lens researchers have strived to uncover over the past century. The consequence of contact lens wear is a vast area of research and can best be investigated by focusing on one aspect at a time. The aim of this study was to use dioptric power matrices and multivariate statistics to explore the effects of both gas permeable and gas non-permeable rigid contact lenses on corneal curvature. This study involves auto-keratometric measurements of the corneal curvature before and after lens wear to establish if there are any curvature changes induced by the contact lens. Keratometric data was collected with an automated keratometer (Nidek ARK-700) and was analysed correctly and completely using multivariate statistics. This thesis presents the findings of a study done in an effort to establish the short-term effects of rigid contact lens wear on keratometric behaviour by using complete methods of multivariate statistical analysis. Twenty four subjects were equally divided into three groups. One group wore polymethyl methacrylate (PMMA) rigid lenses, another group wore rigid gas permeable (RGP) contact lenses and the third group served as the control. The control group was included in the study to establish a reference for normal diurnal changes in keratometric behaviour. Fifty autoii keratometric measurements were taken before and immediately after three hours of rigid contact lens wear for the experimental groups and 50 auto-keratometric measurements were taken before and immediately after three hours of no lens wear for the control group. Data collected was analysed using multivariate statistical methods that in the past have been used infrequently in this area of research.

Orientador: Alexandre Lima de Andrade / Resumo: Avaliaram-se os sinais clínicos oculares da ceratoplastia lamelar, utilizando-se a membrana de biocelulose (MB) embebida em ciprofloxacina em duas espessuras, no tratamento de úlceras de córneas profundas e/ou perfuradas, em cães e gatos, com ceratite ulcerativa com sequestro de córnea. A celulose possui aplicações importantes na engenharia de tecidos, como a reparação e cicatrização de feridas. Os principais exemplos são as aplicações que incluem engenharia de vasos sanguíneos, tecido neural, osso, cartilagem, fígado e tecido adiposo, reconstrução da uretra e dura-máter, reparação de tecido conjuntivo e defeitos cardíacos congênitos, construção de barreiras protetoras e aplicações oftalmológicas, principalmente construção de contato lentes. Os animais foram divididos igualmente em dois grupos, onde um (G-HE) recebeu a membrana hidratada espessa e outro recebeu (G-HD) hidratada delgada. Previamente ao emprego da MB, as mesmas foram recortadas em discos de 3-4mm e adaptadas ao leito das úlceras, utilizando-se sutura não perfurante total, em pontos simples isolados. Instituiram-se, por terapia pós-operatória, colírios à base de ciprofloxacina, diclofenaco sódico, atropina, lubrificante ocular e colar do tipo elizabetano. As causas das úlceras foram, também, tratadas. Avaliaram-se os sinais clínicos oculares no pós-operatório aos 3, 7, 15, 30 e 60 dias. A MB e os pontos de suturas foram removidos ao trigésimo dia. Sinais clínicos como blefarospasmo, presença de secreção ocular, ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Clinical ocular signs of lamellar keratoplasty were evaluated using a two-thickness ciprofloxacin-embedded biocellulose membrane (MB) in the treatment of deep and / or perforated corneal ulcers in dogs and cats with sequestered ulcerative keratitis. of cornea. Cellulose has important applications in tissue engineering, such as wound repair and healing. Top examples are applications that include blood vessel engineering, neural tissue, bone, cartilage, liver and adipose tissue, urethral and dura mater reconstruction, connective tissue repair and congenital heart defects, protective barrier construction and ophthalmic applications, mainly construction of contact lenses. The animals were divided equally into two groups, where one (G-HE) received the thick hydrated membrane and the other received thin hydrated (G-HD). Prior to the use of MB, they were cut in 3-4mm discs and adapted to the ulcer bed, using total nonperforating suture, in isolated single stitches. Postoperative therapy was followed by eye drops based on ciprofloxacin, diclofenac sodium, atropine, eye lubricant and Elizabethan collar. The causes of ulcers were also treated. Postoperative ocular clinical signs were evaluated at 3, 7, 15, 30 and 60 days. MB and suture stitches were removed on day 30. Clinical signs such as blepharospasm, presence of ocular discharge, conjunctival hyperemia, and corneal vascularization were present in both groups until the 15th postoperative day, gradually decreasing over time. No sign... (Complete abstract click electronic access below) / Mestre

Ziel der vorliegenden Arbeit war die Untersuchung des Einflusses von ausgewählten Cyclodextrinen (alpha-, HP-alpha-, beta-, HP-beta, gamma- und HP-gamma-Cyclodextrin) als Hilfsstoffe in Ophthalmika auf die enzymatische Esterspaltung von Dipivefrin im Corneaepithel. Dazu waren neben der Betrachtung der enzymatischen Spaltung von Dipivefrin durch corneale Enzyme von Rinderaugen und dem Reinenzym Butyrylcholinesterase Untersuchungen zu den Wechselwirkungen von Dipivefrin mit den verwendeten Cyclodextrinen und dem cornealem Gewebe notwendig. Zur Charakterisierung der Dipivefrin/Cyclodextrin-Wechselwirkungen in wässriger Lösung wurden die Ultrafiltration, die Hochleistungs-Flüssigchromatographie und die UV- bzw. H-NMR-Spektroskopie eingesetzt. Die Ermittlung der Dipivefrin- bzw. Cyclodextrin-Wechselwirkung mit dem cornealen Gewebe erfolgte mittels Thermoanalytik und Elektronenmikroskopie. Der Einfluss ausgewählter Cyclodextrine auf die in-vitro-Permeation von Dipivefrin durch die Schweinecornea und auf die dabei im Corneaepithel stattfindende enzymatische Freisetzung von Epinephrin wurde untersucht. Mit Hilfe einer Druckpermeationszelle wurde der Einfluss von Dipivefrin bzw. in Kombination mit alpha- und beta-Cyclodextrin auf die forcierte Permeation von Wasser durch die Cornea von endothelial nach epithelial untersucht und die Modelldaten auf den physiologischen Druck des Auges umgerechnet. / The aim of the present work was to study the influence of different (cyclodextrins alpha-, HP-alpha-, beta-, HP-beta-, gamma- und HP-gamma-cyclodextrin) as components of eye drops on the enzymatic hydrolysis of dipivefrine in the corneal epithelium. Therefor, enzymatic degradation of dipivefrine by corneal enzymes of bovine eyes and by the purified enzyme butyryl cholinesterase was investigated. The interaction of dipivefrine with cyclodextrins was tested in aqueous solution by the use of ultrafiltration, high performance liquid chromatography, UV- and H-NMR-spectroscopy. The interaction of dipivefrine with corneal tissue was tested by the use of thermal analysis and electron microscopy. The influence of some cyclodextrins on the in-vitro permeation of dipivefrine through porcine cornea and the simultaneous enzymatic release of epinephrine in the corneal epithelium was investigated. The forced permeation of water through cornea from the endothelial to epithelial site was tested with help of a pressurized permeation device and the modell data were calculated to meet the physiological pressure in the eye.

[Truncated abstract] The cornea plays a major role in the refraction of light and thus the maintenance of its transparency is critical for optimal vision. Infection or trauma can initiate a host inflammatory response, which can cause edema of the collagenous stroma. This tissue edema compromises vision by disrupting the regular arrangement of the corneal stromal lamellae, whose organization is critical to its refractive properties. Until recently, it was the accepted dogma that the cornea was an immune privileged tissue owing in part to its avascular nature and paucity of resident macrophages and dendritic cells (DCs) in the central region of the cornea. However, recent studies have identified heterogenous populations of macrophages and DCs in both the corneal stroma and epithelium. Despite the recognition of the existence of these cells in the cornea, very little is known about their biological role. The overall purpose of the experiments described in this thesis is to characterise corneal macrophages and DCs in homeostatic conditions and investigate their role in the initiation of inflammatory responses to bacterial ligands that induce corneal inflammation and contribute to the severity and resolution of bacterial keratitis. Experiments described in this thesis utilized a range of transgenic, knock-out and bone marrow (BM) chimeric mice to address the immunological function and characterization of BM-derived cells in the mouse cornea. Of particular importance was the use of Cx3cr1 transgenic mice, which contain an enhanced green fluorescent protein (eGFP) encoding cassette knocked into the Cx3cr1 gene that disrupts its expression but facilitates GFP expression under the control of the Cx3cr1 promoter. ... This highlights a novel functional role for corneal BM-derived cells in the recognition and initiation of inflammatory responses to LPS. Finally, a novel observation of a potential mechanism by which DC in the cornea communicate with neighbouring DCs via fine membrane extensions was identified in both chimeric and wild-type mice. These membrane nanotubes, found exclusively on MHC class II+ cells in the corneal stroma, significantly increased in density in the central cornea under inflammatory conditions, suggesting a role for these cell protrusions in the immune response. These data represent the first ever description of nanotubes in vivo, the only previous evidence of their presence being in vitro studies. In summary, the data presented in this thesis supports a role for Cx3cr1 in the homing of DCs to the normal corneal epithelium and also suggests that Cx3cr1-deficiency may influence the ability of corneal macrophages and DCs to respond to bacteria. In addition, the thesis supports a role for resident corneal macrophages and DCs in the initiation of immune responses following challenge with LPS, which is possibly supported by a newly discovered system of membrane nanotubes. A greater understanding of the biology of the resident corneal immune cells could lead to the development of potential therapies aimed at targeting macrophages and DCs as a means of regulating potentially harmful inflammatory responses in the cornea.