A manutenção da morfologia normal do endotélio da córnea é um importante indicador da integridade funcional. A reparação endotelial frente a um trauma, em coelhos, ocorre por migração, hipertrofia e mitose celular. Objetivou-se comparar a morfologia das células do endotélio, da região periférica perincisional à região central, da córnea de coelhos (Oryctolagus cuniculus) em diferentes períodos pós-operatórios. Foram designados três grupos, com 5 animais cada, para avaliação pós-operatória, sendo G1 (7 dias); G2 (15 dias) e G3 (45 dias). Trinta bulbos dos olhos de coelhos, da raça Nova Zelândia, foram submetidos à incisão de córnea clara uniplanar com 3,2 mm. Ao fim dos períodos determinados, procedeu-se a avaliação da morfologia endotelial valendose da microscopia eletrônica de varredura. Realizaram-se seis eletromicrografias de varredura, de cada região da córnea, com aumento de 1000 vezes. Para análise do percentual do número de lados celular, foram analisadas 100 células endoteliais. Na região periférica perincisional, avaliada ao 7° dia de pós-operatório, foram encontradas células com 6 lados (47,8%), 5 lados (31,3%), 7 lados (13,9%), 3 lados (0,1%), 4 lados (4,9%), 8 lados (1,8%) e 9 lados (0,2%). Na avaliação ao 15° dia de pós-operatório, observaram-se células com 6 lados (45,6%), 5 lados (32,6%), 7 lados (17,4%), 4 lados (1,7%) e 8 lados (2,7%). No 45° dia de pós-operatório, verificou-se a presença de células com 6 lados (57%), 5 lados (24%), 7 lados (17,2%), 4 lados (0,1%), 8 lados (1,6%) e 9 lados (0,1%). Na área central, ao 7° dia de pós-operatório, detectaram-se células com 6 lados (75,6%), 5 lados (13,3%), 7 lados (10,8%) e 8 lados (0,3%). Na avaliação, ao 15° dia de pós-operatório, foi possível observar células com 6 lados (78,9%), 5 lados (11,5%) e 7 lados (9,6%). No 45° dia de pós-operatório identificaram-se células com 6 lados (74,8%), 5 lados (13,6%) e 7 lados (11,6%). Os resultados demonstraram que na região periférica perincisional ocorreu diminuição das células com seis lados e aumento do número de células com cinco e sete lados. Na região central manteve-se o padrão regular de hexagonalidade das células endoteliais nos diferentes períodos pós-operatórios. Conclui-se que houve alteração na morfologia das células endoteliais, da região periférica perincisional comparada à região central, da córnea de coelhos nos diferentes períodos pós-operatórios. / The maintenance of the normal corneal endothelium morphology is an important indicator of its functional integrity. In rabbits, endothelial repair in the event of traumas is made through cell migration, hypertrophy and mitosis. The purpose of this study was to compare the morphology of endothelial cells of the perincisional area with the central area of the cornea of rabbits (Oryctolagus cuniculus), in different post-operative periods. Three groups containing 5 animals each were designed for post-operative evaluation: G1 (7 days); G2 (15 days) and G3 (45 days). The clear cornea of thirty New Zealand rabbits was subjected to a single-planed incision of 3.2 mm. At the end of the established periods, a morphological evaluation of the endothelium was carried out using scanning electron microscopy. Six scanning electron micrographs of each corneal area were performed using a magnification of 1000 x. One hundred endothelial cells were analyzed to obtain the cell side count percentage. In the perincisional peripheral area, which was evaluated at the 7 post-operative day, 6-sided (47.8%), 5-sided (31.3%) and 7-sided (13.9%) cells were found, in addition to, 3-sided cells (0.1%), 4-sided cells (4.9%), 8-sided cells (1.8%) and 9-sided cells (0.2%). In the evaluation made on the 15th post-operative day, 6-sided (45.6%), 5-sided (32.6%) and 7-sided (17.4%) cells were observed, as well as 4-sided (1.7%) and 8-sided cells (2.7%). On the 45th postoperative day, the presence of 6-sided (57%), 5-sided (24%), 7-sided (17.2%), 4-sided (0,1%), 8-sided (1.6%) and 9-sided cells (0.1%) was verified. On the 7th post-operative day, 6-sided (75.6%), 5-sided (13.3%), 7-sided (10.8%) and 8-sided cells (0.3%) were observed in the central area .Upon evaluation made on the 15th post-operative day, it was possible to observe 6-sided (78.9%), 5-sided (11.5%) and 7-sided (9.6%) cells. Results have shown that there was a reduction of six-sided cells and an increase in the number of five and seven-sided cells in the perincisional peripheral area. The regular hexagonal standard of the endothelial cells was maintained in the central area in different post-operative periods. In comparison to the central area, there was a morphological alteration of the endothelial cells of the peripheral perincisional area in different post-operative periods of the cornea of rabbits.

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

No description available.

Limbal stem cells maintain the corneal epithelium through a process of clonal growth and ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6 weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to represent chords of clonally related, inwardly migrating cells. By measuring the number and width of stripes and correcting for the effects of different proportions of LacZ-positive cells, an estimate of the number of coherent stem cell clones maintaining the tissue can be derived. Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks. An automated method was developed using image analysis software to analyse these striping patterns. This method produced results that did not differ significantly from the above. The dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6 heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at 15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77 transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics, estimated clone number was similarly reduced to ~50 with no further decline. Mice heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an increase in coherent clones. When the corneal epithelium is injured the surrounding epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged using time-lapse confocal microscopy. This technique demonstrated that clones remain contiguous throughout their migration. Healing of peripheral wounds was observed to form de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away from and towards the limbal region.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in the fulfillment of the requirements for the degree of Master of Medicine in Ophthalmology. Johannesburg, February 2017 / Purpose: To evaluate if corneal collagen crosslinking carried out on patients with keratoconus, slows down or halts the progression of keratoconus. To determine which group of keratoconus patients benefited most from the procedure. Methods: A retrospective record review of 41 eyes of 29 patients. Visual acuity and keratometry measurements were recorded for the involved eye pre-crosslinking and at 3 months and 6 months post-crosslinking. A comparison of these variables pre-crosslinking and at 6 months post-crosslinking was made to determine if there was a flattening of corneal curvature (keratometry readings) and an improvement in visual acuity. Patients were further divided into 3 groups of keratoconus, based on their keratometry readings (measured in diopters): mild keratoconus (≤47 diopters), moderate keratoconus (48 – 54 diopters) and advanced keratoconus (≥55 diopters), to determine which group of keratoconus had the best keratometry reduction readings. Results: After crosslinking took place on 41 eyes, the UnVA of 16(39%) eyes showed an improvement at 6 months, 17(41%) eyes showed no change and 8(20%) eyes showed a decrease in UnVA at 6 months, compared to pre-CXL values. For BCVA, 12(29%) eyes showed an improvement at 6 months, 18(44%) eyes showed no change and 11(27%) eyes showed a decrease in BCVA at 6 months, compared to pre-CXL values. Keratometry readings however showed that 23(56%) eyes had an average flattening of corneal curvature readings of 0.7 D and the remaining 18(44%) eyes showed more steepening (worsening) of the corneal curvature readings of 0.9 D after 6 months post-CXL. 30(73%) eyes had mild keratoconus, 7(17%) had moderate keratoconus and 4(10%) had advanced keratoconus. 19 of the 30 eyes in the mild keratoconus group (73%) showed an average flattening of corneal curvature of 0.6 D. 4 of the 7 eyes in the moderate keratoconus group (17%) showed an average flattening of corneal curvature of 0.7 D. All 4 patients in the advanced group (10%) had steepening (worsening) of their corneal curvatures with an average of 1.2 D. Conclusion: Corneal collagen crosslinking performed on keratoconus patients at least halts the progress of keratoconus. 6 months after CXL most patients showed minimal change from pre-CXL to 6 months in both visual acuity and keratometry. However a longer follow up period and larger sample size is needed to determine if vision and keratometry readings can improve significantly. / MT2017

The validity of corneal confocal microscopy (CCM) in assessing peripheral neuropathy has been studied extensively in several studies with a large cohort of subjects with diabetes and in a handful of studies with small sample sizes in subjects with other systemic conditions. The non-invasive nature of this technique as well as its high reproducibility, moderate to high sensitivity and specificity, and ease of use make it an ideal biomarker for diagnosing onset, severity and progression of peripheral neuropathy. This thesis aims to further investigate the potential of CCM by evaluating abnormalities in the corneal sub-basal nerve plexus, Langerhans Cells (LCs) and epithelial cells in neuropathy related to diabetes and cancer. This thesis has established that evaluating the sub-basal nerve plexus in the centre and at the inferior whorl increases the diagnostic performance of CCM. In addition to diagnosing clinical and subclinical neuropathy in children and adults with diabetes CCM can also identify sub-clinical nerve damage in patients with upper gastrointestinal cancer and assess the effects of chemotherapy. CCM also identifies differences in small fibre pathology between diabetic patients with and without painful neuropathy. Although there was an increased prevalence and severity of dry eye and LCs' density, this was not related to an abnormality of corneal nerves in diabetic patients with no or mild neuropathy. Epithelial cell morphology was not associated with corneal nerve damage and did not alter in patients with Type 1 diabetes. In conclusion, CCM has been shown to be an ideal marker for quantifying early small fibre pathology and assessing peripheral neuropathies.

The corneal epithelium is a vital tissue which must retain its integrity to preserve vision and protect against harmful bacterial infections and other insults. Corneal disease represents the second most common cause of world blindness after cataract.1 Examination of this tissue is therefore important in any ophthalmic routine, and in particular in contact lens practice where an increased number of factors, such as lens material, lens fit, care solution and contamination may directly affect its integrity. The ocular surface cell collection apparatus (OSCCA) allows safe and efficacious collection of human corneal epithelial cells2 and may provide the ability to examine cytological changes to the human cornea during lens wear. The overall objective of this project was to demonstrate the efficacy and reliability of the OSCCA as a tool to collect human corneal epithelial cells and examine cytological changes to the human cornea. This was achieved by characterizing the phenotype and viability status of cells collected from the ocular surface using the OSCCA and by comparing the obtained results with samples collected using other non-invasive techniques. There was a high level of uncertainty whether or not the cells collected were in fact corneal or conjunctival epithelial cells. Chapter 2 and 3 showed the Hoechst and PI were not optimal stains to measure the viability status of cells collected with the OSCCA because there was an unanticipated overlap of the fluorescence from PI+ nucleated cells into the blue spectrum and the Hoechst stained both live and dead cells. Chapter 4 looked at other cytological stains and concluded that the LIVE⁄DEAD® Viability⁄Cytotoxicity Kit (calcein AM/ethidium homodimer-1) was the most appropriate stain to use with the OSCCA collected cells due to the lack of overlap between stains. Chapter 3 showed that cells that stained with sodium fluorescein stained with only Hoechst and not PI. Since Hoechst stains live and early apoptotic cells and PI stains cells that are late stage apoptotic, necrotic and dead cells, we can conclude that sodium fluorescein stains live and early apoptotic cells. Similarly in chapter 5 it was found that cells that stained with sodium fluorescein stained exclusively with calcein blue AM and not ethidium homodimer-1.

The corneal epithelium is a vital tissue which must retain its integrity to preserve vision and protect against harmful bacterial infections and other insults. Corneal disease represents the second most common cause of world blindness after cataract.1 Examination of this tissue is therefore important in any ophthalmic routine, and in particular in contact lens practice where an increased number of factors, such as lens material, lens fit, care solution and contamination may directly affect its integrity. The ocular surface cell collection apparatus (OSCCA) allows safe and efficacious collection of human corneal epithelial cells2 and may provide the ability to examine cytological changes to the human cornea during lens wear. The overall objective of this project was to demonstrate the efficacy and reliability of the OSCCA as a tool to collect human corneal epithelial cells and examine cytological changes to the human cornea. This was achieved by characterizing the phenotype and viability status of cells collected from the ocular surface using the OSCCA and by comparing the obtained results with samples collected using other non-invasive techniques. There was a high level of uncertainty whether or not the cells collected were in fact corneal or conjunctival epithelial cells. Chapter 2 and 3 showed the Hoechst and PI were not optimal stains to measure the viability status of cells collected with the OSCCA because there was an unanticipated overlap of the fluorescence from PI+ nucleated cells into the blue spectrum and the Hoechst stained both live and dead cells. Chapter 4 looked at other cytological stains and concluded that the LIVE⁄DEAD® Viability⁄Cytotoxicity Kit (calcein AM/ethidium homodimer-1) was the most appropriate stain to use with the OSCCA collected cells due to the lack of overlap between stains. Chapter 3 showed that cells that stained with sodium fluorescein stained with only Hoechst and not PI. Since Hoechst stains live and early apoptotic cells and PI stains cells that are late stage apoptotic, necrotic and dead cells, we can conclude that sodium fluorescein stains live and early apoptotic cells. Similarly in chapter 5 it was found that cells that stained with sodium fluorescein stained exclusively with calcein blue AM and not ethidium homodimer-1.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 4 uppsatser.

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-07-19T18:28:21Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) colacao - cecilia escariao.pdf: 1217403 bytes, checksum: 5bc515a017c6ca8024249a926f827192 (MD5) / Made available in DSpace on 2016-07-19T18:28:21Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) colacao - cecilia escariao.pdf: 1217403 bytes, checksum: 5bc515a017c6ca8024249a926f827192 (MD5) Previous issue date: 2012-09-03 / Objetivo: Avaliar o efeito do crosslinking (CXL) no tratamento de ceratite infecciosa, resistente ao tratamento clínico, e investigar a relação com o agente etiológico. Métodos: Foram incluídos 11 pacientes com diagnóstico de ceratite infecciosa de etiologia bacteriana (sete olhos) e fúngica (quatro olhos) na Fundação Altino Ventura (FAV) no período de outubro de 2011 a maio de 2012. Os pacientes incluídos estavam em uso de colírios há pelo menos sete dias e não apresentavam melhora da infecção. Estes foram avaliados antes da realização do CXL e no período pós-operatório, até cicatrização da úlcera. Para realização do CXL foram instilados gotas de riboflavina a 0,1% e dextrano a 20%, a cada cinco minutos em um período de 30 minutos antes do procedimento, e durante a aplicação da luz ultravioleta A (UVA). A córnea foi exposta a UVA com comprimento de onda de 370m ± 5m e uma irradiância de 3mW/cm². Resultados: Os pacientes com infecção bacteriana obtiveram cura do processo infeccioso após o CXL e nenhum paciente com ceratite fúngica apresentou cicatrização. Observou-se associação significante (p=0,003) entre o agente etiológico e a cicatrização. Conclusão: O CXL mostrou-se eficaz no tratamento de ceratite bacteriana resistente ao tratamento clínico, evitando a realização de transplante tectônico. Em relação a ceratite fúngica, o este procedimento não influenciou para a melhora do processo infeccioso. / Purpose: To evaluate the effect of corneal crosslinking (CXL) in the treatment of infectious keratitis resistant to medical treatment, and investigate the relation with the CXL outcome to the etiologic agent. Methods: The study included 11 patients who were diagnosed with bacterial (seven eyes) or fungal keratitis (four eyes) at Fundação Altino Ventura from October 2011 to May 2012. All patients were using antibiotic eye drops for at least 7 days and have had no infection improvement. Patients were evaluated prior to CXL and the postoperative period until healing of the keratitis. For CXL, eyes were first instilled with a solution containing 0.1% riboflavin and 20% dextran for 30 min at a 5-minutes interval. Riboflavin-soaked eyes were then irradiated with UVA light (370 ± 5m) at 3mW/cm² for 30 minutes. Results: Eyes with bacterial infection exhibited improvement of infectious symptoms after CXL whereas eyes with fungal keratitis showed no improvement. Thus, there was a statistically significant correlation (p = 0.003) between the etiologic agent and the effectiveness of healing. Conclusion: CXL was effective in the treatment of bacterial keratitis resistant to clinical treatment, eliminating the need for surgery. However, CXL was not effective in managing fungal keratitis.