Ho Yi-Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-131). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.ix / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Corticosteroids in ophthalmology --- p.1 / Chapter 1.1.1 --- History of the clinical use of corticosteroids --- p.1 / Chapter 1.1.2 --- Administration --- p.1 / Chapter 1.1.3 --- General biological effects of corticosteroids --- p.4 / Chapter 1.1.4 --- Application of corticosteroids in ocular diseases --- p.5 / Chapter 1.1.5 --- Side effects of ocular corticosteroid treatment --- p.6 / Chapter 1.1.6 --- General introduction to commonly used corticosteroids in ophthalmology --- p.6 / Chapter 1.1.6.1 --- Hydrocortisone --- p.6 / Chapter 1.1.6.2 --- Dexamethasone --- p.7 / Chapter 1.1.6.3 --- Triamcinolone --- p.7 / Chapter 1.1.6.4. --- Chemical structures and relative anti-inflammatory potencies --- p.8 / Chapter 1.1.7 --- Cytotoxicity of triamcinolone --- p.12 / Chapter 1.2 --- Retinal ganglion cells --- p.13 / Chapter 1.2.1 --- Basic structures of the eye --- p.13 / Chapter 1.2.2 --- Anatomical structure of retina --- p.13 / Chapter 1.2.3 --- Functions of retinal ganglion cells --- p.18 / Chapter 1.2.4 --- Culture models to study RGCs --- p.20 / Chapter 1.3 --- Aim of study --- p.25 / Chapter Chapter 2 --- Methodology --- p.26 / Chapter 2.1 --- Intravitreal injection of TA (IVTA) --- p.26 / Chapter 2.1.1 --- Materials --- p.26 / Chapter 2.1.1.1 --- Animals --- p.26 / Chapter 2.1.1.2 --- Chemicals --- p.26 / Chapter 2.1.1.3 --- Instruments --- p.26 / Chapter 2.1.2 --- Procedures --- p.26 / Chapter 2.2 --- Peripheral Nerve - Optic Nerve Grafting (PN-ON) Procedure --- p.27 / Chapter 2.3 --- Retrograde Labeling of regenerating RGCs --- p.27 / Chapter 2.3.1 --- Materials --- p.27 / Chapter 2.3.2 --- Procedures --- p.27 / Chapter 2.3.3 --- Statistical analysis --- p.28 / Chapter 2.4 --- Immunohistochemistry --- p.28 / Chapter 2.4.1 --- Materials --- p.28 / Chapter 2.4.2 --- Procedures --- p.29 / Chapter 2.4.3 --- Statistical analysis --- p.29 / Chapter 2.5 --- Histology --- p.29 / Chapter 2.5.1 --- Materials --- p.29 / Chapter 2.5.2 --- Procedures --- p.29 / Chapter 2.6 --- Primary rat retinal ganglion cell culture --- p.30 / Chapter 2.6.1 --- Materials --- p.30 / Chapter 2.6.1.1 --- Animals --- p.30 / Chapter 2.6.1.2 --- Chemicals --- p.30 / Chapter 2.6.1.3 --- Solutions and buffers --- p.30 / Chapter 2.6.1.4 --- Instruments --- p.31 / Chapter 2.6.2 --- Preparations --- p.31 / Chapter 2.6.2.1 --- Working media --- p.31 / Chapter 2.6.2.2 --- Plate coating --- p.32 / Chapter 2.6.3 --- Cell culture process --- p.32 / Chapter 2.6.3.1 --- Dissection of retinal tissues --- p.32 / Chapter 2.6.3.2 --- Purification of RGCs --- p.33 / Chapter 2.6.3.3 --- Culture condition and cell seeding --- p.34 / Chapter 2.7 --- Corticosteroid treatment --- p.34 / Chapter 2.7.1 --- Materials --- p.34 / Chapter 2.7.2 --- Preparations --- p.34 / Chapter 2.7.3 --- Treatment --- p.35 / Chapter 2.8 --- Cell viability assay and morphometric study --- p.36 / Chapter 2.8.1 --- Materials --- p.36 / Chapter 2.8.2 --- Calcein-AM staining --- p.36 / Chapter 2.8.3 --- Cell viability --- p.37 / Chapter 2.8.4 --- Morphometry study --- p.37 / Chapter 2.9 --- TUNEL Assay --- p.38 / Chapter 2.9.1 --- Materials --- p.38 / Chapter 2.9.2 --- Procedure --- p.38 / Chapter 2.9.3 --- Statistical analysis --- p.39 / Chapter 2.10 --- Quantitative Reverse transcription - Polymerase Chain Reaction (qRT-PCR) --- p.39 / Chapter 2.10.1 --- Materials --- p.39 / Chapter 2.10.1.1 --- "Chemicals, reagents, and kits" --- p.39 / Chapter 2.10.1.2 --- Primers --- p.40 / Chapter 2.10.1.3 --- Equipment --- p.41 / Chapter 2.10.1.4 --- Software --- p.41 / Chapter 2.10.2 --- Procedures --- p.41 / Chapter 2.10.2.1 --- Cell collection and RNA isolation --- p.41 / Chapter 2.10.2.2 --- Reverse Transcription --- p.42 / Chapter 2.10.2.3 --- Real-time PCR --- p.43 / Chapter 2.10.3 --- Statistical analysis --- p.43 / Chapter 2.11 --- Western blotting --- p.44 / Chapter 2.11.1 --- Sample preparation --- p.44 / Chapter 2.11.1.1 --- Materials --- p.44 / Chapter 2.11.1.1.1 --- Chemicals and materials --- p.44 / Chapter 2.11.1.1.2 --- Equipment --- p.44 / Chapter 2.11.1.2 --- Procedures --- p.44 / Chapter 2.11.2 --- Protein assay --- p.45 / Chapter 2.11.2.1 --- Materials --- p.45 / Chapter 2.11.2.1.1 --- Chemicals and materials --- p.45 / Chapter 2.11.2.1.2 --- Equipment and software --- p.46 / Chapter 2.11.2.2 --- Procedures --- p.46 / Chapter 2.11.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.11.3.1 --- Materials --- p.46 / Chapter 2.11.3.1.1 --- Chemicals and reagents --- p.46 / Chapter 2.11.3.1.2 --- Equipment --- p.46 / Chapter 2.11.3.1.3 --- Solutions and buffers --- p.47 / Chapter 2.11.3.2 --- Gel preparation --- p.48 / Chapter 2.11.3.3 --- Electrophoresis --- p.49 / Chapter 2.11.3.4 --- Transblotting (semi-dry transfer) --- p.49 / Chapter 2.11.3.5 --- Band visualization --- p.49 / Chapter 2.11.4 --- Immunostaining --- p.50 / Chapter 2.11.4.1 --- Materials --- p.50 / Chapter 2.11.4.1.1 --- Antibodies --- p.50 / Chapter 2.11.4.1.2 --- Chemicals and reagents --- p.50 / Chapter 2.11.4.1.3 --- Equipment --- p.50 / Chapter 2.11.4.2 --- Procedures --- p.50 / Chapter 2.12 --- Gas-chromatography-electron-capture negative-ion mass spectrometry (GC-NCI-MS) --- p.51 / Chapter 2.12.1 --- Standard and reagents --- p.51 / Chapter 2.12.2 --- Chromatography and mass spectrometry --- p.52 / Chapter 2.12.3 --- Sample collection --- p.52 / Chapter 2.12.3 --- Standard and sample preparation --- p.53 / Chapter 2.12.4 --- Validation --- p.54 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effect of triamcinolone on RGCs in vivo --- p.55 / Chapter 3.2 --- Cell viability of RGCs after IVTA plus PN-ON grafting --- p.55 / Chapter 3.3 --- Abnormal retinal morphology under different IVTA conditions --- p.59 / Chapter 3.4 --- Cell viability assay --- p.66 / Chapter 3.4.1 --- Effects of triamcinolone on RGC viability --- p.66 / Chapter 3.4.2 --- Effects of dexamethasone on RGC viability --- p.68 / Chapter 3.4.3 --- Effects of hydrocortisone on RGC viability --- p.70 / Chapter 3.4.4 --- Effects of filtered fraction of triamcinolone on RGC viability --- p.72 / Chapter 3.5 --- Morphometric study analysis of RGCs --- p.74 / Chapter 3.5.1 --- Percentage of RGCs showing neurite outgrowth --- p.74 / Chapter 3.5.2 --- Average neurite length --- p.77 / Chapter 3.5.3 --- Neurite spanning area --- p.77 / Chapter 3.5.4 --- Neurite count --- p.80 / Chapter 3.5.5 --- Neurite branching --- p.83 / Chapter 3.6 --- Determination of concentration of TA in culture media by GC-NCI-MS --- p.85 / Chapter 3.7 --- TUNEL assay --- p.90 / Chapter 3.8 --- Real-time quantitative Reverse transcription - Polymerase Chain Reaction --- p.93 / Chapter 3.9 --- Western blot --- p.99 / Chapter Chapter 4 --- Discussion --- p.102 / Chapter Chapter 5 --- References --- p.120

INTRODUÇÃO: O tratamento padrão estabelecido pelo Early Treatment Diabetic Retinopathy Study (ETDRS) para retinopatia diabética proliferativa (RDP), com ou sem edema macular clinicamente significativo (EMCS), é a panfotocoagulação com laser. Essa forma de tratamento reduz o risco de perda visual, mas não proporciona ganho de visão. O objetivo deste estudo é avaliar os efeitos do acetonido de triancinolona associado à panfotocoagulação na RDP. MÉTODOS: Realizou-se um ensaio clínico randomizado, prospectivo e aberto com portadores de RDP bilateral e simétrica, submetidos à panfotocoagulação com laser em ambos os olhos. Portadores de EMCS foram tratados com fotocoagulação focal na região macular no primeiro episódio da panfotocoagulação. A injeção intravítrea de acetonido de triancinolona (4 mg/0,1 ml) foi administrada aleatoriamente em um dos olhos (grupo estudo), após último episódio da laserterapia, e o contralateral foi adotado para comparação (grupo controle), sendo seguidos por 6 meses. Os parâmetros adotados para avaliar os efeitos terapêuticos foram: acuidade visual com melhor correção óptica (tabela do ETDRS), medidas de espessura central e de volume macular por meio da tomografia de coerência óptica e taxa de sangramento (hemorragia pré-retiniana ou vítrea). As potenciais complicações da droga foram avaliadas durante o seguimento (pressão intra-ocular, catarata, endoftalmite e pseudo-endoftalmite). RESULTADOS: Foram incluídos 28 (vinte e oito) indivíduos com diabetes melito tipo 2. Quanto ao EMCS, 22 olhos apresentaram no grupo estudo e 23, no grupo controle (p= 0,317). A média de idade foi de 61,36 ± 5,77 anos, com predominância do sexo ferminino (57,1%). A maioria era portadora de hipertensão arterial sistêmica (82,1%) e usuária de insulina (75,0%). Não houve diferença significante nas médias de número de disparos da panfotocoagulação e da fotocoagulação na região macular entre os grupos. Quanto à acuidade visual, o grupo estudo apresentou pior acuidade antes do tratamento em relação ao grupo controle (p= 0,040), não havendo diferença significativa na primeira semana do tratamento. Durante o seguimento, o grupo estudo evoluiu com melhora na acuidade visual no primeiro (p< 0,001), no terceiro (p< 0,001) e no sexto meses (p< 0,001) em relação ao controle. Em relação às medidas de espessura central e de volume macular, os grupos não apresentaram diferença significativa antes do tratamento, no entanto, o grupo estudo apresentou medidas significativamente menores na primeira semana, no primeiro, no terceiro e no sexto meses em relação ao controle. Quanto à taxa de sangramento, 9 olhos (32,1%) do grupo controle evoluíram com sangramento e nenhum do grupo estudo (p< 0,001). Os grupos apresentaram diferença na pressão intra-ocular apenas na primeira semana do tratamento, quando as medidas do grupo estudo foram maiores que as do controle (p< 0,05). Nenhum dos olhos apresentou catarata com indicação cirúrgica, endoftalmite ou pseudo-endoftalmite. CONCLUSÃO: Os resultados deste estudo sugerem que a injeção intravítrea de triancinolona é um procedimento seguro e pode melhorar o prognóstico funcional e estrutural da mácula em olhos com RDP submetidos a panfotocoagulação com laser. / INTRODUCTION: The gold standard treatment for proliferative diabetic retinopahty (PDR) with and without clinically significant macular edema (CSME), as stablished by the Early Treatment Diabetic Retinopathy Study (ETDRS), is panretinal photocoagulation (PRP). This treatment lowers the rate of severe vision loss, but does not increase vision. The aim of this study is to evaluate the efficacy and safety of triamcinolone acetonide associated to PRP for the management of PDR. METHOD: This is a prospective, randomized clinical trial for patients with bilateral and symmetrical PDR who had undergone PRP in both eyes. Patients who had CSME were treated with macular focal photocoagulation on the first episode of the PRP. Intravitreal injection of triamcinolone acetonide (4 mg/0.1 ml) was given to the study eye after the last episode of PRP and the fellow eye was used as control. Follow up was 6 months long. Best-corrected visual acuity (BCVA) using ETDRS charts, central macular thickness and macular volume as measured by the optical coherence tomography software, and the amount of bleeding (both preretinal and vitreous) were the parameters chosen to analyse outcome. Side effects of triamcinolone acetonide such as intraocular pressure, cataracts and severe inflammation, were also followed during the study. RESULTS: Twenty eight diabetes type 2 patients were included. Twenty two study eyes and 23 fellow eyes (controls) presented with CSME (p= 0.317). Mean age was 61.36 ± 5.77 years, with 57.1% females. Many patients had hypertension (82.1%) and used insulin (75.0%). There was no significant difference on the number of spots used for PRP or macular photocoagulation in between the groups. The study eyes had lower BCVA on baseline than the control eyes (p= 0.040). One week after the treatment, there was no difference on BCVA between the study and control eyes. During the follow up, the study group increased their BCVA on the first (p< 0.001), third (p< 0.001) and sixth month (p< 0.001) compared to control. Even thought there was no significant difference on central macular thickness and macular volume between groups on baseline, the study eyes had significant lower measurements on the first week and first, third and sixth months in comparison to controls. Nine control eyes (32.1%) had hemorrhages and none study eyes (p< 0.001). Injected eyes had higher intraocular pressure than controls on the first week of treatment (p< 0.05). None of the eyes developed cataracts that needed surgery, endophthalmits or severe inflammation. CONCLUSION: This study suggests intravitreal injection of triamcinolone is a safe procedure that increases funcional and anatomical prognosis of the macula in PDR eyes that underwent PRP.

INTRODUÇÃO: O tratamento padrão estabelecido pelo Early Treatment Diabetic Retinopathy Study (ETDRS) para retinopatia diabética proliferativa (RDP), com ou sem edema macular clinicamente significativo (EMCS), é a panfotocoagulação com laser. Essa forma de tratamento reduz o risco de perda visual, mas não proporciona ganho de visão. O objetivo deste estudo é avaliar os efeitos do acetonido de triancinolona associado à panfotocoagulação na RDP. MÉTODOS: Realizou-se um ensaio clínico randomizado, prospectivo e aberto com portadores de RDP bilateral e simétrica, submetidos à panfotocoagulação com laser em ambos os olhos. Portadores de EMCS foram tratados com fotocoagulação focal na região macular no primeiro episódio da panfotocoagulação. A injeção intravítrea de acetonido de triancinolona (4 mg/0,1 ml) foi administrada aleatoriamente em um dos olhos (grupo estudo), após último episódio da laserterapia, e o contralateral foi adotado para comparação (grupo controle), sendo seguidos por 6 meses. Os parâmetros adotados para avaliar os efeitos terapêuticos foram: acuidade visual com melhor correção óptica (tabela do ETDRS), medidas de espessura central e de volume macular por meio da tomografia de coerência óptica e taxa de sangramento (hemorragia pré-retiniana ou vítrea). As potenciais complicações da droga foram avaliadas durante o seguimento (pressão intra-ocular, catarata, endoftalmite e pseudo-endoftalmite). RESULTADOS: Foram incluídos 28 (vinte e oito) indivíduos com diabetes melito tipo 2. Quanto ao EMCS, 22 olhos apresentaram no grupo estudo e 23, no grupo controle (p= 0,317). A média de idade foi de 61,36 ± 5,77 anos, com predominância do sexo ferminino (57,1%). A maioria era portadora de hipertensão arterial sistêmica (82,1%) e usuária de insulina (75,0%). Não houve diferença significante nas médias de número de disparos da panfotocoagulação e da fotocoagulação na região macular entre os grupos. Quanto à acuidade visual, o grupo estudo apresentou pior acuidade antes do tratamento em relação ao grupo controle (p= 0,040), não havendo diferença significativa na primeira semana do tratamento. Durante o seguimento, o grupo estudo evoluiu com melhora na acuidade visual no primeiro (p< 0,001), no terceiro (p< 0,001) e no sexto meses (p< 0,001) em relação ao controle. Em relação às medidas de espessura central e de volume macular, os grupos não apresentaram diferença significativa antes do tratamento, no entanto, o grupo estudo apresentou medidas significativamente menores na primeira semana, no primeiro, no terceiro e no sexto meses em relação ao controle. Quanto à taxa de sangramento, 9 olhos (32,1%) do grupo controle evoluíram com sangramento e nenhum do grupo estudo (p< 0,001). Os grupos apresentaram diferença na pressão intra-ocular apenas na primeira semana do tratamento, quando as medidas do grupo estudo foram maiores que as do controle (p< 0,05). Nenhum dos olhos apresentou catarata com indicação cirúrgica, endoftalmite ou pseudo-endoftalmite. CONCLUSÃO: Os resultados deste estudo sugerem que a injeção intravítrea de triancinolona é um procedimento seguro e pode melhorar o prognóstico funcional e estrutural da mácula em olhos com RDP submetidos a panfotocoagulação com laser. / INTRODUCTION: The gold standard treatment for proliferative diabetic retinopahty (PDR) with and without clinically significant macular edema (CSME), as stablished by the Early Treatment Diabetic Retinopathy Study (ETDRS), is panretinal photocoagulation (PRP). This treatment lowers the rate of severe vision loss, but does not increase vision. The aim of this study is to evaluate the efficacy and safety of triamcinolone acetonide associated to PRP for the management of PDR. METHOD: This is a prospective, randomized clinical trial for patients with bilateral and symmetrical PDR who had undergone PRP in both eyes. Patients who had CSME were treated with macular focal photocoagulation on the first episode of the PRP. Intravitreal injection of triamcinolone acetonide (4 mg/0.1 ml) was given to the study eye after the last episode of PRP and the fellow eye was used as control. Follow up was 6 months long. Best-corrected visual acuity (BCVA) using ETDRS charts, central macular thickness and macular volume as measured by the optical coherence tomography software, and the amount of bleeding (both preretinal and vitreous) were the parameters chosen to analyse outcome. Side effects of triamcinolone acetonide such as intraocular pressure, cataracts and severe inflammation, were also followed during the study. RESULTS: Twenty eight diabetes type 2 patients were included. Twenty two study eyes and 23 fellow eyes (controls) presented with CSME (p= 0.317). Mean age was 61.36 ± 5.77 years, with 57.1% females. Many patients had hypertension (82.1%) and used insulin (75.0%). There was no significant difference on the number of spots used for PRP or macular photocoagulation in between the groups. The study eyes had lower BCVA on baseline than the control eyes (p= 0.040). One week after the treatment, there was no difference on BCVA between the study and control eyes. During the follow up, the study group increased their BCVA on the first (p< 0.001), third (p< 0.001) and sixth month (p< 0.001) compared to control. Even thought there was no significant difference on central macular thickness and macular volume between groups on baseline, the study eyes had significant lower measurements on the first week and first, third and sixth months in comparison to controls. Nine control eyes (32.1%) had hemorrhages and none study eyes (p< 0.001). Injected eyes had higher intraocular pressure than controls on the first week of treatment (p< 0.05). None of the eyes developed cataracts that needed surgery, endophthalmits or severe inflammation. CONCLUSION: This study suggests intravitreal injection of triamcinolone is a safe procedure that increases funcional and anatomical prognosis of the macula in PDR eyes that underwent PRP.