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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23793号 / 医博第4839号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森 孝一, 教授 花川 隆, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Our lab has shown that conditionally disrupting the tcfap2beta gene, responsible for the activating protein-2beta (AP-2beta) transcription factor, exclusively in the craniofacial neural crest cells, leads to anterior segment dysgenesis. Subsequent loss of the corneal endothelium results in the adherence of the iris to the corneal stroma, causing closure of the iridocorneal angle. The activating protein-2beta neural crest cell knockout (AP-2beta NCC KO) model involves a complete blockage of the both the conventional (through the trabecular meshwork) and non-conventional (uveoscleral) pathways for aqueous humor drainage, and therefore it could be used as a powerful experimental model for glaucoma. As shown by our previous work, elevated intraocular pressure (IOP) and a 35% decrease in the number of cells in the retinal ganglion cell (RGC) layer was observed in AP-2beta NCC KO mice by 2 months; 6 to 11 months sooner than other reported mouse models of glaucoma. These observations suggested that the AP-2beta NCC KO mouse could be a novel and cost-effective experimental model for glaucoma if the RGC loss occurred progressively rather than due to a congenital defect. The purpose of this research project was to investigate how the retinal ganglion cell layer and macroglial activity changes with respect to age in the AP-2beta NCC KO mutant through immunofluorescence. Specifically, it was investigated whether the loss of RGCs was progressive and due to the increased IOP caused by the blockage of the uveoscleral drainage pathway. A significant decrease in the number of RGCs was observed between P4 and P10 in the retinal periphery of both WT and AP-2beta NCC KO mice (p<0.05), which is indicative of the programmed cell death that occurs due to retinal pruning during development. No statistical difference between WT and AP-2beta NCC KO mice phenotypes was observed at postnatal day 4 (P4), suggesting that no developmental defect resulted in the significant loss of RGCs at 2 months. In all other time points investigated, while no statistical difference was found between WT and the AP-2 NCC KO mutant, a clear downward trend was present in the AP-2 NCC KO mutant retinal ganglion cell layer from P10 to P40. There was also an expression of glial fibrillary acidic protein (GFAP) by Müller cells, indicating the presence of neuroinflammation at P35 and P40. This substantiates the potential P42 starting point of neurodegeneration our lab previously observed. This was further corroborated with Müller cell-associated expression of GFAP at P35 and P40 exclusively in the AP-2beta NCC KO mouse. Overall, we have shown that the retinal damage observed in our AP-2beta NCC KO mouse is not due to a developmental defect, but rather occurs over time. Thus, this mouse model, which appears to block both the conventional and unconventional uveoscleral pathways, has a profound effect on aqueous humor drainage. As a result, the model requires relatively little time to observe an increase in IOP and subsequent RGC loss. Our findings suggest that the AP-2beta NCC KO mouse can be a novel, powerful, and extremely cost-effective experimental model for glaucoma. / Thesis / Master of Science (MSc)

No / PURPOSE. The Activities of Daily Vision Scale (ADVS) has been extensively validated by traditional methodology. In the current study, Rasch analysis was used to explore further the validity of the ADVS and to determine whether improvements could be made. METHODS. Forty-three patients with cataract underwent visual acuity (VA) and contrast sensitivity (CS) testing and completed the ADVS. The data were Rasch analyzed and the value of response scale and item reduction explored. A shortened version and the original ADVS were tested for criterion validity by determining correlations with VA and CS. RESULTS. The ADVS data contained nonnormally distributed items and items with ceiling effects and empty response categories. Therefore, items benefited from shortening the response scale, the optimum length being three responses. There was poor targeting of item difficulty to patient ability, because many patients with cataract were sufficiently able that they had no difficulty with many activities. Items were eliminated if the task was too easy or did not fit with the overall concept of visual disability determined by the Rasch model. A reduced ADVS version was established that had adequate precision, equivalent criterion validity, and improved targeting of item difficulty to patient ability, but this version was still not ideal. CONCLUSIONS. Despite careful traditional validation, the ADVS data contained inadequacies exposed by Rasch analysis. Through Rasch scaling, particularly with response scale reduction, the ADVS can be improved, but additional questions seem to be needed to suit the more able, including patients undergoing second eye cataract surgery. There remains a need to develop Rasch-scaled measures of visual disability for use in ophthalmic outcomes research.

Introdução: Entre os possíveis Eventos Adversos (EA) mais importantes após cirurgias de catarata estão a endoftalmite, termo que define a infecção intraocular e a Síndrome Tóxica do Segmento Anterior (TASS), que consiste na reação inflamatória aguda pós-cirúrgica. Contudo, um sistema de vigilância epidemiológica (VE) da ocorrência destes EA não é uma realidade no Brasil, o que dificulta o monitoramento da incidência, detecção precoce de surtos e as medidas de prevenção. Devido as especificidades no campo da oftalmologia, nem sempre o enfermeiro está suficientemente instrumentalizado para contribuir na detecção e monitoramento de casos. A identificação de marcadores destes EA factíveis de serem acompanhados por este profissional irá favorecer o desenvolvimento de um sistema de VE. Objetivo: Identificar os marcadores mais adequados para o diagnóstico epidemiológico de EA após cirurgias de catarata visando à instituição de um sistema de VE específico. Métodos: Trata-se de um estudo longitudinal de série de casos de pacientes submetidos a cirurgias de catarata, realizado em duas etapas. Etapa I: abordagem retrospectiva por meio de revisão de prontuários dos pacientes com diagnóstico de EA (21 casos) no período de abril/2010 a fevereiro/2013. Etapa II: abordagem prospectiva por meio de revisão de prontuários dos pacientes submetidos à cirurgia de catarata sem EA (309 controles) nos meses de maio e junho/ 2013. A amostra baseou-se em um teste de proporções entre duas amostras assumindo que a amostra de controles seria 15 vezes maior que a de casos para detectar uma diferença de pelo menos 35 pontos percentuais na incidência da apresentação de características clínicas entre os dois grupos com erro tipo I de 5% e poder do teste de 90%. As variáveis pesquisadas foram os sinais e sintomas característicos do pós-operatório de cirurgias de catarata bem como informações demográficas e clínicas destes pacientes. Foi realizada estatística descritiva por meio de frequências relativas e absolutas. Resultados: Pacientes com EA apresentaram o diagnóstico de endoftalmite e TASS, respectivamente, em 19 (90,5%) e dois casos (9,5%). Dentre os casos, os sinais mais observados foram: córnea nebulosa, reação de câmara anterior (RCA), edema de córnea (>70%) e hipópio, hiperemia conjuntival, turvação vítrea e dobras na membrana Descemet (>40%). Dor ocular foi o sintoma relatado por 14 (66,7%) dos pacientes. Dentre os controles, no primeiro dia após a cirurgia os sinais mais apresentados (>50%) foram: RCA, edema de córnea, hiperemia conjuntival e dobras na Descemet. Córnea nebulosa e edema palpebral em mais que 30% dos pacientes. Os sinais e sintomas que apresentaram diferença maior que 35% entre os casos e controles foram: presença de hipópio, vítreo turvo, córnea nebulosa. RCA, hiperemia conjuntival, edema de córnea e dor ocular. A incidência de aplicação de antibiótico intra-vítreo e número de retornos foram maiores entre os casos do que nos controles. Uma ferramenta para auxiliar no sistema de VE foi sugerida a partir dos resultados obtidos no presente estudo. Conclusões: Os marcadores clínicos e epidemiológicos considerados mais adequados para compor uma ferramenta para VE foram: dor, edema de córnea, hiperemia conjuntival, hipópio, RCA, vítreo turvo, aplicação de antibiótico intra-vítreo e número de retornos. A ferramenta proposta por este estudo tem potencialidade para subsidiar a atuação do enfermeiro no sistema de VE de EA para cirurgias de catarata. / Introduction: Amongst the most important possible Adverse Events (AE) after cataract surgeries, are the endophthalmitis, a term which defines the intraocular infection and the Toxic Anterior Segment Syndrome (TASS), which is the acute inflammatory post-surgical reaction. However, an epidemiological surveillance (ES) system of these AE isnt a reality in Brazil, which makes difficult the incidence monitoring and early detection of outbreaks and preventive measures. Due to specifics in ophthalmology, nurses are not always enable to detect and monitoring the cases. The identification of markers of these AE achievable to be monitored by nurses will facilitate the development of an epidemiological ES system. Objective: Identifying most suitable markers for epidemiological diagnosis of AE after cataract surgeries aiming at the establishment of a specific ES system. Methods: This is a longitudinal case series study of patients who underwent cataract surgery performed in two phases. Phase I: a retrospective approach through reviews of medical records of patients that were diagnosed with AE (21 cases) from April/2010 to February/2013. Phase II: prospective approach through reviews medical records of patients without AE (309 controls) in the months of May and June of 2013. The sample size was based on a test of proportion between 2 samples assuming that control sample would be 15 times greater than of the cases to detect a difference of at least 35 percentage points in the incidence of clinical characteristics between the two groups, with type I error of 5% and power of testing of 90%. The variables studied were signs and symptoms of the post-operative presentation of cataract surgeries as well as demographic information and clinics of these patients. A descriptive statistics was carried out by using relative and absolute frequencies. Results: Patients with EA were diagnosed with endophthalmitis and TASS, respectively, in 19 (90,5%) and two cases (9,5%). Among cases, the signs most frequently observed were: corneal nebulosity, anterior chamber reaction (ACR), corneal edema (>70%), and hypopyon, conjunctival hyperemia, vitreous haze e Descemet\'s membrane folds (DMF) (>40%). Ocular pain was reported by 14 (66,7%) of patients. Amongst controls, on the first day after surgery the most frequent signals (>50%) were: ACR, corneal edema, conjunctival hyperemia and DMF. Corneal nebulosity and eyelid edema in more than (30,0%) patients. The signs that showed a difference greater than 35% between the cases and controls were: hypopyon, vitreous haze, corneal nebulosity, ACR, conjunctival hyperemia, corneal edema, and eye pain. The incidence of intravitreal antibiotic injection and the number of medical consultation returns were bigger amongst the cases than in the controls. A tool to assist in the ES system was suggested from the results obtained in the present study. Conclusions: The clinical and epidemiological markers considered the most adequate to form a tool for ES is: ocular pain, corneal edema, conjunctival hyperemia, hypopyon, ACR, vitreous haze, intravitreal antibiotic injection and the number of returns. The tool suggested by this study has the potential to support the nurses roles related to ES system for monitoring EA in cataract surgeries.

Cataract is one of the leading causes of vision loss worldwide. The rate of cataract surgery has been steadily increasing. Toxic Anterior Segment Syndrome (TASS) is a sterile inflammatory response in the anterior segment of the eye that may occur following cataract surgery. When left untreated, it can lead to permanent vision loss. Corneal endothelial cells are the cells most affected by TASS. These cells are unable to reproduce in vivo and consequently once the density of these cells drops below a certain level, vision is reduced and cannot be reversed. The damage is thought to be mediated by cytokines and endotoxins, primarily through the NF-κΒ pathway. It is hypothesized that anti-inflammatory drug delivery intraocular lenses may help reduce the occurrence of TASS and consequent vision loss. In this research thesis project, an in vitro model was developed as a tool to select drug and delivery material to be used in an anti-TASS ophthalmic biomaterial. In an attempt to find a novel and more effective approach to TASS prevention, dexamethasone, a potent anti-inflammatory steroid drug, was compared to triptolide, a cytokine inhibitor; aprotinin, a general protease inhibitor; and PPM-18, a NF-κΒ inhibitor. To assess the efficacy of these drugs, an in vitro assay using human umbilical vein endothelial cells (HUVEC) and lipopolysaccharide as a stimulant was developed. Cell response to dexamethasone (10 nM), triptolide (3 nM), aprotinin (20 μM) and PPM-18 (10 μM) with or without LPS was characterized by cell viability and flow cytometry analysis of cell activation. Activation was characterized using markers for cell adhesion and activation ICAM-1, PECAM-1, VCAM-1, β1-integrin, CD44 and E-selectin. Following preliminarily testing, the efficacy of dexamethasone (10 nM) and PPM-18 (10 μM) loaded polymer (PDMS) and copolymer (PDMS/pNIPAAm) interpenetrating polymer networks were evaluated over a 4 day release period. The results from soluble drug and LPS (100 ng/mL) testing indicated no decrease in cell viability after 24 h. Dexamethasone, triptolide, aprotinin, and PPM-18 did not reduce the significant ICAM-1 upregulation seen in HUVECs after exposure to LPS for 4 days. PPM-18 in combination with LPS significantly upregulated E-selectin iv and CD44 from unstimulated HUVEC cells. The polymer materials without drug loading did not influence the cell phenotype. However, PPM-18 delivering polymer and copolymer materials significantly upregulated VCAM-1, CD44 when compared to all other treatments. Propidium iodide uptake in HUVEC exposed to PPM-18 drug delivering polymer and copolymer treatments indicated that these treatments caused cell necrosis. None of the drugs, or the drug delivering materials were shown to counteract the upregulation seen from LPS stimulation of HUVEC cells. Future work should focus on validating the in vitro model to more closely replicate the in vivo environment of the anterior segment with the use of primary bovine corneal endothelial cells.

Cataract is one of the leading causes of vision loss worldwide. The rate of cataract surgery has been steadily increasing. Toxic Anterior Segment Syndrome (TASS) is a sterile inflammatory response in the anterior segment of the eye that may occur following cataract surgery. When left untreated, it can lead to permanent vision loss. Corneal endothelial cells are the cells most affected by TASS. These cells are unable to reproduce in vivo and consequently once the density of these cells drops below a certain level, vision is reduced and cannot be reversed. The damage is thought to be mediated by cytokines and endotoxins, primarily through the NF-κΒ pathway. It is hypothesized that anti-inflammatory drug delivery intraocular lenses may help reduce the occurrence of TASS and consequent vision loss. In this research thesis project, an in vitro model was developed as a tool to select drug and delivery material to be used in an anti-TASS ophthalmic biomaterial. In an attempt to find a novel and more effective approach to TASS prevention, dexamethasone, a potent anti-inflammatory steroid drug, was compared to triptolide, a cytokine inhibitor; aprotinin, a general protease inhibitor; and PPM-18, a NF-κΒ inhibitor. To assess the efficacy of these drugs, an in vitro assay using human umbilical vein endothelial cells (HUVEC) and lipopolysaccharide as a stimulant was developed. Cell response to dexamethasone (10 nM), triptolide (3 nM), aprotinin (20 μM) and PPM-18 (10 μM) with or without LPS was characterized by cell viability and flow cytometry analysis of cell activation. Activation was characterized using markers for cell adhesion and activation ICAM-1, PECAM-1, VCAM-1, β1-integrin, CD44 and E-selectin. Following preliminarily testing, the efficacy of dexamethasone (10 nM) and PPM-18 (10 μM) loaded polymer (PDMS) and copolymer (PDMS/pNIPAAm) interpenetrating polymer networks were evaluated over a 4 day release period. The results from soluble drug and LPS (100 ng/mL) testing indicated no decrease in cell viability after 24 h. Dexamethasone, triptolide, aprotinin, and PPM-18 did not reduce the significant ICAM-1 upregulation seen in HUVECs after exposure to LPS for 4 days. PPM-18 in combination with LPS significantly upregulated E-selectin iv and CD44 from unstimulated HUVEC cells. The polymer materials without drug loading did not influence the cell phenotype. However, PPM-18 delivering polymer and copolymer materials significantly upregulated VCAM-1, CD44 when compared to all other treatments. Propidium iodide uptake in HUVEC exposed to PPM-18 drug delivering polymer and copolymer treatments indicated that these treatments caused cell necrosis. None of the drugs, or the drug delivering materials were shown to counteract the upregulation seen from LPS stimulation of HUVEC cells. Future work should focus on validating the in vitro model to more closely replicate the in vivo environment of the anterior segment with the use of primary bovine corneal endothelial cells.

Introdução: Entre os possíveis Eventos Adversos (EA) mais importantes após cirurgias de catarata estão a endoftalmite, termo que define a infecção intraocular e a Síndrome Tóxica do Segmento Anterior (TASS), que consiste na reação inflamatória aguda pós-cirúrgica. Contudo, um sistema de vigilância epidemiológica (VE) da ocorrência destes EA não é uma realidade no Brasil, o que dificulta o monitoramento da incidência, detecção precoce de surtos e as medidas de prevenção. Devido as especificidades no campo da oftalmologia, nem sempre o enfermeiro está suficientemente instrumentalizado para contribuir na detecção e monitoramento de casos. A identificação de marcadores destes EA factíveis de serem acompanhados por este profissional irá favorecer o desenvolvimento de um sistema de VE. Objetivo: Identificar os marcadores mais adequados para o diagnóstico epidemiológico de EA após cirurgias de catarata visando à instituição de um sistema de VE específico. Métodos: Trata-se de um estudo longitudinal de série de casos de pacientes submetidos a cirurgias de catarata, realizado em duas etapas. Etapa I: abordagem retrospectiva por meio de revisão de prontuários dos pacientes com diagnóstico de EA (21 casos) no período de abril/2010 a fevereiro/2013. Etapa II: abordagem prospectiva por meio de revisão de prontuários dos pacientes submetidos à cirurgia de catarata sem EA (309 controles) nos meses de maio e junho/ 2013. A amostra baseou-se em um teste de proporções entre duas amostras assumindo que a amostra de controles seria 15 vezes maior que a de casos para detectar uma diferença de pelo menos 35 pontos percentuais na incidência da apresentação de características clínicas entre os dois grupos com erro tipo I de 5% e poder do teste de 90%. As variáveis pesquisadas foram os sinais e sintomas característicos do pós-operatório de cirurgias de catarata bem como informações demográficas e clínicas destes pacientes. Foi realizada estatística descritiva por meio de frequências relativas e absolutas. Resultados: Pacientes com EA apresentaram o diagnóstico de endoftalmite e TASS, respectivamente, em 19 (90,5%) e dois casos (9,5%). Dentre os casos, os sinais mais observados foram: córnea nebulosa, reação de câmara anterior (RCA), edema de córnea (>70%) e hipópio, hiperemia conjuntival, turvação vítrea e dobras na membrana Descemet (>40%). Dor ocular foi o sintoma relatado por 14 (66,7%) dos pacientes. Dentre os controles, no primeiro dia após a cirurgia os sinais mais apresentados (>50%) foram: RCA, edema de córnea, hiperemia conjuntival e dobras na Descemet. Córnea nebulosa e edema palpebral em mais que 30% dos pacientes. Os sinais e sintomas que apresentaram diferença maior que 35% entre os casos e controles foram: presença de hipópio, vítreo turvo, córnea nebulosa. RCA, hiperemia conjuntival, edema de córnea e dor ocular. A incidência de aplicação de antibiótico intra-vítreo e número de retornos foram maiores entre os casos do que nos controles. Uma ferramenta para auxiliar no sistema de VE foi sugerida a partir dos resultados obtidos no presente estudo. Conclusões: Os marcadores clínicos e epidemiológicos considerados mais adequados para compor uma ferramenta para VE foram: dor, edema de córnea, hiperemia conjuntival, hipópio, RCA, vítreo turvo, aplicação de antibiótico intra-vítreo e número de retornos. A ferramenta proposta por este estudo tem potencialidade para subsidiar a atuação do enfermeiro no sistema de VE de EA para cirurgias de catarata. / Introduction: Amongst the most important possible Adverse Events (AE) after cataract surgeries, are the endophthalmitis, a term which defines the intraocular infection and the Toxic Anterior Segment Syndrome (TASS), which is the acute inflammatory post-surgical reaction. However, an epidemiological surveillance (ES) system of these AE isnt a reality in Brazil, which makes difficult the incidence monitoring and early detection of outbreaks and preventive measures. Due to specifics in ophthalmology, nurses are not always enable to detect and monitoring the cases. The identification of markers of these AE achievable to be monitored by nurses will facilitate the development of an epidemiological ES system. Objective: Identifying most suitable markers for epidemiological diagnosis of AE after cataract surgeries aiming at the establishment of a specific ES system. Methods: This is a longitudinal case series study of patients who underwent cataract surgery performed in two phases. Phase I: a retrospective approach through reviews of medical records of patients that were diagnosed with AE (21 cases) from April/2010 to February/2013. Phase II: prospective approach through reviews medical records of patients without AE (309 controls) in the months of May and June of 2013. The sample size was based on a test of proportion between 2 samples assuming that control sample would be 15 times greater than of the cases to detect a difference of at least 35 percentage points in the incidence of clinical characteristics between the two groups, with type I error of 5% and power of testing of 90%. The variables studied were signs and symptoms of the post-operative presentation of cataract surgeries as well as demographic information and clinics of these patients. A descriptive statistics was carried out by using relative and absolute frequencies. Results: Patients with EA were diagnosed with endophthalmitis and TASS, respectively, in 19 (90,5%) and two cases (9,5%). Among cases, the signs most frequently observed were: corneal nebulosity, anterior chamber reaction (ACR), corneal edema (>70%), and hypopyon, conjunctival hyperemia, vitreous haze e Descemet\'s membrane folds (DMF) (>40%). Ocular pain was reported by 14 (66,7%) of patients. Amongst controls, on the first day after surgery the most frequent signals (>50%) were: ACR, corneal edema, conjunctival hyperemia and DMF. Corneal nebulosity and eyelid edema in more than (30,0%) patients. The signs that showed a difference greater than 35% between the cases and controls were: hypopyon, vitreous haze, corneal nebulosity, ACR, conjunctival hyperemia, corneal edema, and eye pain. The incidence of intravitreal antibiotic injection and the number of medical consultation returns were bigger amongst the cases than in the controls. A tool to assist in the ES system was suggested from the results obtained in the present study. Conclusions: The clinical and epidemiological markers considered the most adequate to form a tool for ES is: ocular pain, corneal edema, conjunctival hyperemia, hypopyon, ACR, vitreous haze, intravitreal antibiotic injection and the number of returns. The tool suggested by this study has the potential to support the nurses roles related to ES system for monitoring EA in cataract surgeries.

Glaucoma is a heterogeneous group of diseases that is currently considered to be the leading cause of irreversible blindness worldwide. Of the identified risk factors, elevated intraocular pressure remains the only modifiable risk factor that can be targeted clinically. Ocular hypertension is often a result of dysregulation of aqueous humour fluid dynamics in the anterior eye segment. Aqueous humour drainage is regulated by structures located in the anterior chamber of the eye. In some circumstances dysregulation occurs due to developmental abnormalities of these structures. The malformation of structures in the anterior segment is thought to be due to a defect in the differentiation and/or migration of the periocular mesenchyme during development. Unique to vertebrates, the neural crest cell (NCC) population contributes to the periocular mesenchyme and is instrumental to the proper development of structures in the anterior segment. For many years our laboratory has examined the role of the Activating Protein-2 (AP-2) transcription factors that are expressed in the neural crest and vital during the development of the eye. The purpose of this research project is to investigate the role of AP-2β in the NCC population during the development of the anterior segment of the eye. Conditional deletion of AP-2β expression in the NCC population demonstrated that mutants have dysgenesis of structures in the anterior segment including defects of the corneal endothelium, corneal stroma, ciliary body and a closed iridocorneal angle. Loss of retinal ganglion cells and their axons was also observed, likely due to the disruption of aqueous outflow, suggesting the development of glaucoma. The data generated from this research project will be critical in elucidating the role of AP-2β in the genetic cascade dictating the development of the anterior eye segment in addition to providing scientific research with a novel model of glaucomatous optic neuropathy. / Thesis / Master of Science (MSc)