A administração tópica de colírios é a maneira mais conveniente de se tratar doenças oculares. O grande desafio para a tecnologia farmacêutica é garantir que o fármaco administrado nessa forma farmacêutica chegue ao local de ação em concentrações adequadas, com efeitos adversos reduzidos, tempo de ação prolongado e dose única. Para tanto, o desenvolvimento de sistemas de liberação e de estratégias adequadas de administração tornam-se necessários. Assim, o objetivo deste trabalho foi avaliar a influência da iontoforese na penetração ocular de dendrímeros de poliamidoamina (PAMAM) de geração 4, com diferentes grupos superficiais (PAMAM G4, catiônico e PAMAM G3.5, aniônico), preparar complexos desses dendrímeros com um anti-inflamatório modelo, a dexametasona (Dexa), e avaliar a influência da associação de dendrímeros e iontoforese na penetração corneal da Dexa em modelos ex vivo e in vivo. Complexos Dexa-PAMAM foram obtidos e caracterizados por espectroscopia de infravermelho e ressonância magnética nuclear (H1-RMN, 13C-RMN e DOSY), espalhamento dinâmico de luz e espectroscopia UV/vis para avaliar a formação dos complexos, seu tamanho e potencial zeta, além de alterações na solubilidade da Dexa. A velocidade de liberação da Dexa dos complexos foi verificada por estudos de liberação in vitro utilizando membrana sintética. A penetração e distribuição dos PAMAMs na córnea e sua influência na penetração da Dexa foi avaliada ex vivo utilizando córnea de porco, microscopia confocal de varredura a laser (MCVL) e cromatografia de ultra performance aliada a um detector de massas para quantificação do fármaco permeado. A citotoxicidade dos PAMAMs foi avaliada em cultura de células epiteliais da retina e células epiteliais da córnea. Por fim, verificou-se in vivo a influência da iontoforese e dos PAMAMs sobre a quantidade de Dexa no humor aquoso de olhos de coelhos. Os estudos de caracterização indicaram que a Dexa foi incorporada aos PAMAMs e que esses complexos apresentaram cerca de 50 nm de tamanho médio pela técnica de NTA, com a presença de partículas pequenas e agregadas quando dispersos em meio fisiológico e potencial zeta de + 6,4 mV e -18,5 mV para Dexa-PAMAM G4 e Dexa-PAMAM G3.5, respectivamente. A solubilidade aparente da Dexa aumentou 3,9 e 10,3 vezes nos complexos com PAMAM G4 e PAMAM G3.5, respectivamente. O PAMAM G3.5 e PAMAM G4 diminuiram 82 e 1,7 vezes, respectivamente, o coeficiente de difusão da Dexa. Os estudos ex vivo indicaram que a iontoforese foi capaz de direcionar os dendrímeros para dentro da córnea, além de aumentar 2,9, 5,6 e 3,0 vezes a quantidade de Dexa permeada a partir das formulações que continham Dexa livre, Dexa-PAMAM G4 e Dexa-PAMAM G3.5, respectivamente. Aumentou também a quantidade de Dexa retida na córnea em aproximadamente 2 vezes para todas as formulações. Os experimentos de citotoxicidade evidenciaram a maior toxicidade do PAMAM G4 e sua dependência da concentração e tempo de incubação. Por fim, os experimentos in vivo mostraram que a iontoforese aumentou a concentração de Dexa no humor aquoso cerca de 2, 2,5 e 6,6 para a Dexa livre, Dexa-PAMAM G4 e Dexa-PAMAM G3.5, respectivamente. Portanto, a associação de dendrímeros PAMAM com a iontoforese representa uma estratégia promissora para a administração tópica direcionada e sustentada de fármacos na córnea. / Topical administration of eye drops is the most convenient way for treatment of eye diseases. The challenge for the pharmaceutical technology is to ensure that the drug administered in the eye drops reaches the site of action in appropriate concentrations with reduced side effects, prolonged effect and single dose. Therefore, the development of drug delivery systems and appropriate strategies become necessary. The objective of this work was to evaluate the influence of iontophoresis in the ocular penetration of generation 4 polyamidoamine dendrimers (PAMAM) with different surface groups (PAMAM G4, cationic, and PAMAM G3.5, anionic), prepare complexes of these dendrimers with an anti-inflammatory drug model, dexamethasone (Dexa), and evaluate the influence of dendrimers and iontophoresis association on Dexa cornea penetration using ex vivo and in vivo models. Dexa-PAMAM complexes were obtained and characterized by infrared spectroscopy, nuclear magnetic resonance (H1-NMR, 13C-NMR and DOSY), dynamic light scattering and UV/VIS spectroscopy to evaluate the formation of the complexes, their size and zeta potential, as well as changes in drug solubility. Dexa release rate from complexes was determined from the in vitro release studies using synthetic membrane. The penetration and distribution of PAMAMs into the cornea and their influence in the ex vivo Dexa penetration was assessed using pig\'s cornea, confocal scanning laser microscopy (CSLM) and ultra performance chromatography coupled to a mass spectrometer for quantification of the drug permeated. PAMAMs cytotoxicity was assessed in culture of retina epithelial cells and cornea epithelial cells. Finally, the influence of iontophoresis and PAMAMs on Dexa concentration in the aqueous humor of rabbit eyes was evaluated in vivo. The characterization results showed that Dexa was incorporated to PAMAMs and that these complexes had an average size of approximately 50 nm using the NTA technique, with the distribution of small particles and aggregates when dispersed in physiological medium. The zeta potential of Dexa-PAMAM G4 and Dexa PAMAM G3.5 complexes were +6.4 mV and -18.5 mV, respectively. PAMAM G4 and G3.5 PAMAM enhanced Dexa solubility by 3.9 and 10.3-fold, respectively. PAMAM G3.5 and PAMAM G4 decreased by 82 and 1.7-fold Dexa diffusion coefficient. The ex vivo studies indicated that iontophoresis directed dendrimers into the cornea, increasing the amount of Dexa permeated by 2.9, 5.6 and 3.0-fold for the formulations containing free Dexa, Dexa-PAMAM G4 and Dexa-PAMAM G3.5, respectively. Iontophoresis also increased approximately 2-fold the amount of drug retained into the cornea for all formulations. The cytotoxicity experiments revealed that PAMAM G4 toxicity was dependent on the concentration and incubation time. Finally, the in vivo experiments showed that iontophoresis increased Dexa concentration in the aqueous humor by 2, 2.5 and 6.6-fold for free Dexa, Dexa-PAMAM G4 and Dexa-PAMAM-G3.5, respectively. Therefore, the combination of iontophoresis with PAMAM dendrimers represents a promising strategy for targeted and sustained topical drug delivery to the cornea.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-20T17:53:34Z No. of bitstreams: 1 flaviabatistabarbosadesa.pdf: 1013858 bytes, checksum: 1e5c85e290deeb77a83c4367e5c61db1 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T11:27:01Z (GMT) No. of bitstreams: 1 flaviabatistabarbosadesa.pdf: 1013858 bytes, checksum: 1e5c85e290deeb77a83c4367e5c61db1 (MD5) / Made available in DSpace on 2016-07-02T11:27:01Z (GMT). No. of bitstreams: 1 flaviabatistabarbosadesa.pdf: 1013858 bytes, checksum: 1e5c85e290deeb77a83c4367e5c61db1 (MD5) Previous issue date: 2012-03-26 / Objetivo: Analisar os fatores que influenciam o processo de doação de córneas entre os potenciais doadores de um Hospital Público e de um Hospital Privado de Juiz de Fora. Métodos: estudo seccional do tipo descritivo. Foram rastreados os óbitos ocorridos em um Hospital Público e um Hospital Privado de Juiz de Fora no período de 01 janeiro de 2010 a 31 de dezembro de 2010, identificados os potenciais doadores de córneas, verificado se os óbitos foram notificados à Central de Notificação, Captação e Distribuição de Órgãos (CNCDO) da Zona da Mata de Juiz de Fora e se esta notificação resultou ou não em doação. Caso não tenha ocorrido doação, foi justificada a causa da não efetivação da mesma, e caso tenha ocorrido, foi analisado o destino da córnea captada (transplante ou perda). Os dados foram processados e discutidos utilizando-se a análise estatística feita através do Programa SPSS (Statistical Package for the Social Sciences), versão 13.0. Resultados: A população em estudo se caracteriza por serem em sua maioria pacientes do sexo masculino (53,1%), com idade entre 2 a 80 anos (69,1%), com causas mortis que não contraindicaram o transplante de córneas (59,1%). Dos 863 óbitos, 138 (16%) não foram notificados à CNCDO-Zona da Mata e destes, 54 eram potenciais doadores. Do total de óbitos, 210 (24,3%) foram classificados como potenciais doadores. O percentual de captação efetiva dos casos entrevistados foi de 34,9%. Foram realizadas 30 doações de córneas (19,2%). Observou-se que das 60 córneas captadas (30 doadores), 75,0% (45) foram descartadas. A não efetivação da doação foi justificada pela recusa familiar em 58 casos (7,0%), pelos problemas logísticos ou estruturais em 125 casos (15,0%) e pelas contraindicações médicas registradas na CNCDO-Zona da Mata em 650 casos (78,0%). Conclusão: Da população estudada, 24,3% eram potenciais doadores de córneas. A contraindicação médica foi a maior justificativa para a não efetivação da doação de córneas, correspondendo a 78,0% das causas. Considerando que apenas 25% das córneas captadas foram transplantadas, conclui-se que há uma grande desigualdade entre o número de doadores potenciais e o número de doadores reais para o transplante de córneas, pois muito se perde durante todo o processo de doação. / Objective: To analyze factors that influence the process of cornea donation among potential donors for a Public Hospital and a Private Hospital in Juiz de Fora. Methods: A descriptive cross sectional study. We traced the deaths occurred in a Public Hospital and a Private Hospital in Juiz de Fora in the period from January 1, 2010 to December 31, 2010, identified the potential donors of corneas, checked whether the deaths were reported to the Central Notification, Procurement and Distribution organs (CNCDO) from Zona of Mata in Juiz de Fora and whether or not this notification resulted in donation. If there were no donation, it was justified the cause of not being done, and if it has occurred, we analyzed the fate of the captured cornea (transplantation or loss). The data were analyzed and discussed using the statistical analysis performed using the SPSS (Statistical Package for Social Sciences) version 13.0. Results: The mortality profile of the institutions under study is characterized by being mostly male (53.1%), aged from 2 to 80 years (69.1%), with cause of death that did not prevent corneal transplantation to be done (59.1%). Of the 863 deaths, 138 (16%) were not notified to CNCDO-Zona of Mata and of these, 54 were potential donors. Of the total deaths, 210 (24.3%) were classified as potential donors. The percentage of effective capture of the cases surveyed was 34.9%. 30 donations of corneas were performed (19.2%). It was observed that from 60 corneal grafts (30 donors), 75.0% (45) were discarded. The effectiveness of the donation was not justified by family refusal in 58 cases (7.0%), by logistical or structural problems in 125 cases (15.0%) and recorded in the medical contraindications CNCDO-Zona da Mata in 650 cases (78.0 %). Conclusion: The study population, 24.3% were potential donors of corneas. The medical contraindication was the biggest reason for not effecting the donation of corneas, corresponding to 78.0% of the causes. Whereas only 25% of corneal grafts were transplanted, it is concluded that a considerable difference between the number of potential donors and the number of actual donors for transplantation of corneas, since much is lost during the donation process.

La cicatrisation cornéenne représente un processus de réparation complexe qui vise à restaurer l'intégrité, la structure et la transparence de la cornée. Cependant, dans un certain nombre de cas, ce processus peut évoluer de façon anormale et se stabiliser en entraînant la formation d'une opacité cornéenne installée. Les mécanismes impliqués dans la formation de ces cicatrices persistantes ne sont pas encore complètement élucidés, mais il est établi que la composition et l'organisation de la matrice extracellulaire du stroma jouent un rôle majeur dans la restauration de la transparence de la cornée. Ce projet s’est concentré sur la métalloprotéase extracellulaire BMP-1 (Bone Morphogenetic Protein 1), déjà connue pour son rôle dans l'assemblage de la matrice extracellulaire et l'activation du TGF-bêta. Afin d’identifier les processus contrôlés par BMP-1 dans la cornée, nous avons d’abord effectué une comparaison systématique des inhibiteurs de BMP-1 connus ou potentiels, de différentes origines, pour caractériser leurs propriétés à la fois in vitro et dans des cultures cellulaires. Ensuite, nous avons mené une étude approfondie du sécrétome des cellules stromales de la cornée humaine (kératocytes), et des conséquences de la différenciation de ces cellules en myofibroblastes. Enfin, nous avons analysé les événements protéolytiques médiés par BMP-1 dans le sécrétome des kératocytes en utilisant principalement une approche de protéomique quantitative basée sur le marquage iTRAQ des protéines entières (technique TAILS). La comparaison des inhibiteurs disponibles de BMP-1 a permis de mettre en évidence différents profils d’efficacité, de spécificité et de toxicité et a conduit à l’identification d’un inhibiteur hydroxamate et d’un inhibiteur protéique efficaces, peu toxiques et très spécifiques de BMP-1. Le sécrétome des kératocytes s’est avéré être un modèle adéquat pour l’étude des activités de BMP-1 dans le contexte cornéen. Plus de 2022 protéines ont été identifiées, dont la métalloprotéase BMP-1 et 16 de ses 33 substrats connus jusqu’à présent. Enfin, 76 protéines modifiées par l’activité de BMP-1 ont été identifiées dans le sécrétome des kératocytes. Ces résultats confirment les liens forts entre BMP-1, l'assemblage de la matrice extracellulaire et le TGF-bêta, mais suggèrent également de nouveaux rôles pour la protéase dans l'inflammation. Certains des substrats nouvellement identifiés (TGFBI, HSP47 et collagène VI) sont très pertinents dans le contexte de la cicatrisation de la cornée et ont été validés d’un point de vue biochimique. En conclusion, BMP-1 est confirmée comme une cible potentielle intéressante pour traiter ou prévenir la formation des opacités cornéennes et la caractérisation des inhibiteurs disponibles ouvre des perspectives importantes pour des études précliniques chez l’animal / When the cornea is injured, a complex multi-step healing process is triggered which aims at restoring corneal integrity, structure and transparency. However, in some cases, corneal healing results in the formation of a stable scar associated with a prolonged loss of corneal transparency and with functional blindness. The mechanisms involved in the formation of these persistent scars are still not fully understood but it is known that the composition and organization of the extracellular matrix significantly contributes to the maintenance of corneal transparency. This work focused on the extracellular metalloproteinase called BMP-1 (Bone Morphogenetic Protein 1), a major player in the control of extracellular matrix assembly and TGF-beta activation, which was previously shown to be up-regulated in corneal healing and scarring. In order to further probe BMP-1 functions in corneal healing, we first performed a systematic comparison of known or potential BMP-1 inhibitors from different origins to characterize their properties both in vitro and in cell cultures. We then carried out an in-depth study of the secretome of human corneal stromal cells (keratocytes) and of the consequences of the differentiation of these cells into myofibroblasts. Finally, we analyzed BMP-1-mediated proteolytic events in keratocyte secretomes, mainly using a quantitative proteomic approach based on iTRAQ labeling of proteins (TAILS technique). The comparison of BMP-1 available inhibitors revealed different profiles of efficacy, specificity and toxicity and led to the identification of one hydroxamate inhibitor and one protein inhibitor, which were very efficient, non-toxic and very specific of BMP-1. The keratocyte secretome was shown to be a suitable model for the study of BMP-1 activities in the corneal context. More than 2022 proteins were identified, including the BMP-1 metalloprotease and 16 of its 33 already known substrates. Finally, 76 proteins modified by BMP-1 activity were identified in the keratocyte secretome. These results confirm the strong links between BMP-1, extracellular matrix assembly and TGF-beta, but also suggest new roles for this protease in cell proliferation and inflammation. Some of the newly identified substrates (TGFBI, HSP47 and collagen VI) are highly relevant in the context of corneal healing and were validated at the biochemical standpoint. In conclusion, BMP-1 is confirmed as a potential target to treat or prevent the formation of corneal opacities and the characterization of available inhibitors opens up important perspectives for preclinical studies in animals

OBJETIVOS: 1) Avaliar os parâmetros de biomecânica ocular provenientes do Corvis ST (Oculus Corvis ST, Scheimpflug Technology; Wetzlar, Germany) obtidos de olhos saudáveis de uma população de pacientes brasileiros. 2) Correlacionar os parâmetros derivados do Corvis ST com a idade de pacientes jovens saudáveis. 3) Verificar se as técnicas de facoemulsificação (FC) e do laser de femtosegundo (LFS), empregadas na correção cirúrgica de catatara, influenciam os parâmetros de biomecânica ocular provenientes do Corvis ST. 4) Analisar as alterações da deformação da córnea observadas em um caso de ectasia pós LASIK com flap espesso. MÉTODOS: 1) Estudo clínico transversal conduzido em 90 pacientes (90 olhos saudáveis). Foram avaliados 11 parâmetros derivados do sistema de tonometria de não contato integrado com a câmera ultrarrápida de Scheimpflug (Oculus Corvis ST, Scheimpflug Technology; Wetzlar, Germany), a saber: deformidade de amplitude (DA); pressão intraocular; 1st A time; tempo de concavidade máxima; 2nd A time; 1st A Length (tempo da primeira aplanação); 2nd A Length (tempo da segunda aplanação); raio de curvatura de maior alcance; raio de curvatura normal; velocidade de entrada (Vin) e de saída (Vout). Estes parâmetros foram correlacionados com a espessura central corneana mensurada pela Tomografia de Córnea e Segmento Anterior (Pentacam® - Oculus, Wetzlar, Germany). 2) Estudo clínico observacional, retrospectivo, conduzido em 89 pacientes (89 olhos saudáveis). Os parâmetros derivados do Corvis ST foram correlacionados com a idade dos pacientes. 3) Estudo clínico prospectivo, envolvendo 151 olhos de 127 pacientes com diagnóstico de catarata nuclear. Setenta e cinco olhos de 65 pacientes foram submetidos à técnica do laser de femtosegundo (AlconLenSx, Aliso Viejo, USA) e 76 olhos de 62 pacientes à facoemulsificação convencional (Alcon Infinit, Fort Worth, USA). Foram avaliados os 11 parâmetros de biomecânica ocular derivados do Corvis ST antes (Pré) e após as cirurgias de catarata (D1, primeiro dia de pósoperatório). A densitometria do cristalino (scattering) foi realizada pelo PNS (Pentacam Nucleus Staging). 4) Avaliação com tomografia de coerência óptica de espessura dos flaps corneanos pós Lasik e análise dos parâmetros biomecânicos provenientes do Corvis ST em uma córnea com ectasia pós Lasik. RESULTADOS: 1) A média de idade dos pacientes foi de 35,80 ± 12,83 anos. A média do equivalente esférico foi de -3,29 ± 3,69 dioptrias. A média da espessura central corneana foi de 547,50 ± 32,00 ?m Os valores dos 11 parâmetros biomecânicos obtidos de olhos saudáveis, foram os seguintes: deformidade de amplitude 1,05 ± 0,08 mm; tempo de concavidade máxima 18,38 ± 0,93 ms; pressão intraocular 16,43 ± 2,15 mmHg; tempo da primeira aplanação (1st A time) 8,32 ± 0,33 ms; tempo da segunda aplanação 23,80 ± 0,44 ms; raio de curvatura de maior alcance 11,09 ± 2,06 mm; raio de curvatura normal 7,59 ± 0,67 mm; tempo da primeira aplanação (1st A Length) 2,07 ± 0,38 mm; tempo da segunda aplanação (2nd A Length) 2,37 ± 0,47 mm; velocidade de entrada (Vin) 0,21 ± 0,05 m/s e velocidade de saída (Vout) -0,33 ± 0,07 m/s. 2) A média de idade dos pacientes foi de 27,50 ± 6,30 anos. O tempo de concavidade máxima alcançada da córnea (HC-time) foi o único dos 11 parâmetros que apresentou correlação significativa com a idade (p=0,04, rs=0,18). 3) A média de idade dos pacientes dos grupos LFS (laser de femtosegundo) e FC (facoemulsificação convencional) foram, respectivamente, 67,6 ± 9,9 anos e 68,4 ± 11,8 anos. No grupo LFS, 9 dos 11 parâmetros foram estatisticamente significativos entre o Pré e D1; e no grupo FC, 7 dos 11 parâmetros foram estatisticamente significativos entre o Pré e D1. Entre os 11 parâmetros biomecânicos avaliados, somente o tempo de concavidade máxima da córnea (HC-time) foi significativamente diferente entre os dois grupos em D1 (p=0,0387). 4) Paciente do sexo feminino, 45 anos, submetida à Lasik em ambos os olhos. Com a utilização da tomografia de coerência óptica (OCT Rtvue, OptoVue, Fremont, CA,USA) foram identificados: um flap com espessura central de 392 ?m no OD e dois cortes, sendo um flap incompleto profundo e o outro mais fino superiormente, no OE. Os parâmetros derivados do Corvis ST como a deformidade de amplitude são diferentes em ambos os olhos. CONCLUSÕES: 1) Os valores de 8 dos 11 parâmetros derivados do Corvis ST foram influenciados pela espessura central da córnea, porém esta influência foi baixa. 2) Em olhos saudáveis de pacientes jovens foi obtida correlação significativa entre a idade e o tempo de concavidade máxima, que é o tempo do início de aplanação até a concavidade máxima alcançada da córnea. 3) O laser de femtosegundo para cirurgia de catarata e a técnica de facoemulsificação convencional induziram alterações nas propriedades biomecânicas da córnea no D1. Dos 11 parâmetros biomecânicos estudados apenas o tempo de concavidade máxima da córnea apresentou diferença significativa entre os grupos (LFS e FC) no D1. 4) A ectasia unilateral após LASIK pode ocorrer devido a flap espesso com falência biomecânica da córnea / PURPOSE: 1) To evaluate ocular biomechanical metrics given by the CorVis ST (Oculus, Inc., Berlin, Germany) in a population of healthy Brazilian patients. 2) To correlate parameters derived from corneal deformation resulting from non-contact tonometry integrated with an ultra-high-speed (UHS) Scheimpflug camera (Oculus Corvis ST, Scheimpflug Technology; Wetzlar, Germany) with age in normal eyes from young patients. 3) To evaluate the changes of corneal biomechanical after femtosecond laser - assisted cataract (FS) and to compare the parameters derived by Corvis ST between standard phacoemulsification (SP) and femtosecond laser - assisted in cataract surgery. 4) To report a case of post-LASIK corneal ectasia due to a thick flap, while the contralateral eye did not develop ectasia after an incomplete deep flap cut, followed by a thinner flap Lasik procedure. METHODS: 1) An observational and cross-sectional study involving 1 eye randomly selected from 90 healthy patients. Studied parameters (including deformation amplitude, first applanation time, highest concavity time, second applanation time, first applanation length, second applanation length, curvature radius highest concavity, curvature radius normal, velocity in, and velocity out) derived from the CorVis ST were correlated to central corneal thickness from the Pentacam (Oculus, Inc.). Differences between data on the basis of gender were evaluated. 2) Observational, retrospective study involving one eye randomly selected from study participants, totaling 89 healthy eyes. The Scheimpflug images were taken with an ultra-high-speed camera during each measurement by the Corvis ST. The deformation amplitude (DA) and other parameters (e.g., pachy apex, intraocular pressure, 1st A time, highest concavity-time, 2nd A time, 1st A Length, 2nd A Length, Wing-Dist, curvature radius highest concavity, curvature radius normal, Vin, Vout) measured by the corvis ST were correlated with age. 3) Prospective study: 151 eyes of 127 patients were underwent cataract surgery. 75 eyes of 65 patients were with femtosecond laser-assisted (FS)(Alcon Len Sx, Aliso Viejo,USA) and 76 eyes of 62 patients with standard phacoemulsification (SP) (Alcon Infinit, FortWorth, USA). 4) Case Report. RESULTS: 1) About the first study: Mean patient age was 35.80 ± 12.83 years (range: 21.07 to 78.84 years). Mean central corneal thickness was 547.50 ± 32.00 ?m (range: 490 to 647 ?m) and mean spherical equivalent refraction was -3.29 ± 3.69 diopters (range: -9.50 to +10.37 diopters). Mean deformation amplitude was 1.05 ± 0.08 mm (range: 0.91 to 1.26 mm). Highest concavity time was 18.38 ± 0.93 ms (range: 16.95 to 21.07 ms). Intraocular pressure was 16.43 ± 2.15 mm Hg (range: 11.50 to 21.0 mm Hg). First applanation time was 8.32 ± 0.33 ms (range: 7.53 to 9.12 ms) and second applanation time was 23.80 ± 0.44 ms (range: 22.76 to 24.95 ms). First applanation length (max) was 2.07 ± 0.38 mm (range: 1.20 to 3.10 mm) and second applanation length (max) was 2.37 ± 0.47 mm (range: 1.33 to 4.12 mm). Curvature radius highest concavity was 11.09 ± 2.06 mm (range: 7.58 to 15.98 mm) and curvature radius normal was 7.59 ± 0.67 mm (range: 6.82 to 11.02 mm). Velocity in was 0.21 ± 0.05 m/s (range: 0.16 to 0.72 m/s) and velocity out was -0.33 ± 0.07 m/s (range: - 0.72 to -0.20 m/s). Studied parameters were not associated with gender. 2) Mean patient age was 27.50 ± 6.30 years. The highest concavity-time was the only studied parameter statistically significantly correlated to age (i.e., p=0.04, rs=0.18). 3) In relation the surgery of cataract: In group of FS, 9 of 11 parameters derived from Corvis ST were statistically significant (ss). In group of SP, 7 of 11 parameters derived from Corvis ST were ss. Only the HC - time was statistically significant between two groups (FS;SP) with p = 0.0387. 4) Corneal OCT identified a deep meniscos-shaped Lasik flap, with a central thickness of a 392 ?m in the right eye, and a incomplete deep peripheral cut in the left eye with a thinner meniscos-shaped LASIK flap. CONCLUSIONS: 1) Eight of 11 ocular biomechanical metrics given by the CorVis ST were associated with central corneal thickness, but the influence of central corneal thickness on these measurements was low. 2) In healthy eyes, age and pressure or biomechanics as derived from the Corvis ST parameters were not associated with exception to highestconcavity-time, i.e., the time from starting until the highest concavity is reached. 3) The use of the femtosecond laser- assisted system for cataract surgery and standard phacoemulsification induzed changes of biomechanical properties of the cornea by Corvis ST. Only 1 of 11 parameters studied was different statistically in two groups. 4) Unilateral ectasia after LASIK may occur due to a thick flap which leads to biomechanical failure of the cornea

Zusammenfassung: Das Ziel dieser Arbeit ist die experimentelle und klinische Evaluierung eines neuartigen Messverfahrens zur Bestimmung biomechanischer Eigenschaften der Hornhaut. Das untersuchte Gerät (Corvis ST, Oculus, Wetzlar, Deutschland) ist ein Non-Kontakt Tonometer mit integrierter Scheimpflug-Technologie, welches zum Einsatz am menschlichen Auge weltweit zugelassen ist. Ein Luftimpuls wird dabei auf die Hornhaut appliziert, wodurch diese deformiert wird. Dieser Prozess hat zur Folge, dass die Hornhaut applaniert (erste Applanation), anschließend nach innen gedrückt (höchste Konkavität) und durch das Abschalten des externen Luftimpulses, vom Augeninnendruck (IOD) in ihre ursprüngliche, physiologische Form gedrückt wird, wobei sie die zweite Applanation durchläuft. Die klinisch relevanten Parameter des Corvis ST (DCR Parameter) sind der bIOP, ein biomechanisch korrigierter IOD; DAR2, das Verhältnis aus zentraler Deformation und peripherer Deformation (bei 2 mm) der Hornhaut; int. 1/R, Summe aus dem Kehrwert des Radius während der konkaven Phase der Hornhaut zwischen erster und zweiter Applanation; SP A1, Steifigkeitsparameter der ersten Applanation sowie CBI, eine Kombination aus verschiedenen DCR Parametern zur Trennung zwischen gesunden und Keratokonus Hornhäuten. Die Literaturrecherche hat ergeben, dass mechanische Kenngrößen nicht ohne Weiteres auf biologische Gewebe anwendbar sind, da insbesondere die Hornhaut anisotrope, nicht lineare und viskoelastische Eigenschaften aufweist. Aus diesem Grund kann für die Hornhaut beispielsweise kein einheitlicher E-Modul abgeleitet werden. Der E-Modul ist vielmehr eine Funktion der Dehnung. Für die Messung am Auge haben IOD und Hornhautdicke einen wesentlichen Einfluss auf das biomechanische Verhalten der Hornhaut. Diese und weitere Faktoren werden in dieser Arbeit untersucht und nach ihrem Effekt auf die Messparameter beurteilt. In experimentellen Untersuchungen wurde insbesondere der Einfluss des IOD betrachtet. Hierbei zeigt sich, je höher der IOD im Schweineauge induziert ist, desto weniger verformbar verhält sich die Hornhaut gegenüber dem eintreffenden Luftimpuls, wobei sich die Materialeigenschaften nicht verändern. Dies drückte sich unter anderem in geringeren Werten für DAR2 und int. 1/R sowie höheren Werte für SP A1 aus. Infolgedessen wird auf die IOD Konformität zwischen den zu untersuchenden Studienpopulationen geachtet, um die Messergebnisse richtig evaluieren zu können. In den klinischen Untersuchungen können zudem Hornhautdicke und Alter als weitere Einflussfaktoren auf die DCR Parameter bei Gesunden beobachtet werden. Eine dickere Hornhaut weist demnach einen höheren Widerstand (geringere Werte für DAR2, int. 1/R sowie höhere Werte für SP A1) gegenüber dem Luftimpuls auf als eine dünnere Hornhaut. Das Alter hat einen Versteifungseffekt auf die Hornhaut, was sich nachweislich durch eine positive Korrelation des Alters mit SP A1 zeigt. Eine weitere Voraussetzung für die Beurteilbarkeit der DCR Parameter ist die Bestimmung der Wiederholbarkeit und Reproduzierbarkeit. Sowohl am Schweineauge als auch bei Keratokonus-Patienten zeigt sich insgesamt eine hohe Genauigkeit der Wiederholbarkeit und Reproduzierbarkeit der Parameter, sodass von einer verlässlichen Messung ausgegangen werden kann. Diese Erkenntnis ist insbesondere für die Evaluierung von longitudinalen Fragestellungen enorm wichtig. Für die klinischen Untersuchungen wurde der Ocular Response Analyzer (ORA, Reichert Technologie, Buffalo, NY, USA), ein weiteres Non-Kontakt Tonometer zur Bestimmung biomechanischer Parameter der Hornhaut, als Vergleichsgerät herangezogen. Die Hauptparameter sind die korneale Hysterese (CH) und der korneale Widerstandsfaktor (CRF). Jedoch spiegeln diese Parameter nicht die Steifigkeit der Hornhaut wider, da sie viskoelastische Eigenschaften der Hornhaut beschreiben. Der Grund dafür ist die Integration der Druckwerte der ersten und zweiten Applanation während Ein- und Auswärtsbewegung der Hornhaut in die Berechnung von CH und CRF. Im Gegensatz dazu beschreiben die DCR Parameter des Corvis ST das Deformationsverhalten der Ein- und Auswärtsbewegung der Hornhaut. Es wird davon ausgegangen, dass die Einwärtsbewegung der Hornhaut vermehrt die elastische Komponente widerspiegelt, wodurch sich ein Zusammenhang zur Steifigkeit herleiten lässt. Die Evaluierung der DCR Parameter anhand von gesunden Probanden und Keratokonus Patienten ergibt, dass der CBI die beste Trennung mit hoher Sensitivität und Spezifität zwischen beiden Kohorten darstellt. Weiterhin zeigen sich DAR2, int. 1/R und SP A1 eine hohe Genauigkeit der Erkennung eines Keratokonus, sogar höher als CH und teilweise auch als CRF. Darüber hinaus können Unterschiede in bestimmten DCR Parameter zwischen verschiedenen Schweregraden des Keratokonus gefunden werden, so dass in der Folge eine weitere Analyse durchgeführt werden konnte, die anhand von Machine Learning Algorithmen den Schweregrad des Keratokonus vorhersagt. Das Modell erreicht eine gute Sensitivität und Spezifität zur Vorhersage von Gesunden, frühen und fortgeschrittenen Stadien, jedoch nicht für mäßige Stadien. Diese Arbeit weist weiterhin nach, dass die Hornhautvernetzung (CXL), ein Therapieverfahren bei progressiven Keratokonus, eine Änderung in den DCR Parametern erzeugt, die auf eine Zunahme der Hornhautfestigkeit hindeutet. Die experimentellen Versuche am Schweineauge leiten einen Zusammenhang zwischen Spannungs-Dehnungsmessung und Änderungen in den DCR Parameter her. Indizien für die Zunahme der Hornhautfestigkeit sind einerseits ein höher gemessener IOD trotz konstant induzierten IOD im Schweineauge und andererseits ein geringerer Wert für int. 1/R sowie ein höherer Wert für SP A1. Auch in den klinischen Untersuchungen wird diese Beobachtungen gemacht, auch wenn sich der Effekt weniger stark zeigt. Dennoch kann ebenfalls eine Zunahme des bIOP und eine Abnahme des int. 1/R beobachtet werden, wobei diese einen Monat postoperativ am stärksten ausgeprägt sind. Letztlich bietet das Corvis ST hilfreiche Informationen über die biomechanischen Eigenschaften der Hornhaut. Dies ist insbesondere im Vorfeld refraktiv-chirurgischer Eingriffe und zur Beurteilung des Keratokonus wichtig. In dieser Arbeit wird ein gewisser Einfluss des IOD und der Hornhautdicke auf die DCR Parameter nachgewiesen, so dass zukünftig computergestützte Verfahren verwendet werden sollten, um Materialeigenschaften der Hornhaut möglichst unabhängig von ihrer Dicke und dem vorliegenden IOD zu bestimmen. Dies ist auch für andere Augenerkrankungen, wie z. B. dem Glaukom, wichtig.:Vorwort IV Inhaltsverzeichnis VI Abbildungs- und Tabellenverzeichnis IX Abkürzungsverzeichnis XV 1 Einleitung 1 2 Wissenschaftlicher Hintergrund 2 2.1 Das Auge 2 2.2 Kornea – die Hornhaut des menschlichen Auges 3 2.2.1 Struktur und Eigenschaften des Hornhautstromas 8 2.2.2 Zelluläre Abnormitäten der Hornhaut bei Keratektasien 12 2.2.3 Strukturelle Veränderungen der Hornhaut bei okulären und systemischen Erkrankungen 14 2.3 Keratokonus 17 2.3.1 Inzidenz und Prävalenz 17 2.3.2 Risikofaktoren 18 2.3.3 Diagnose und klinische Zeichen 19 2.3.4 Behandlungsoptionen 25 2.4 Methoden zur Bestimmung von (bio-)mechanischer Eigenschaften 29 2.4.1 Übertragung mechanischer Kenngrößen auf Biomaterialien 29 2.4.2 Zusammenhang zwischen den chemischen, strukturellen Eigenschaften und der Biomechanik der Hornhaut 33 2.4.3 Bestimmung der Biomechanik der Hornhaut ex vivo 33 2.4.4 Bestimmung der Biomechanik der Hornhaut in vivo 37 2.4.5 Computer gestützte Modellierung von biomechanischen Modellen und Ektasien 43 2.5 Aktueller Stand der Forschung 46 2.5.1 Ocular Response Analyzer 46 2.5.2 Corvis ST 50 2.6 Fragestellungen und Hypothesen 53 3 Material und Methoden 54 3.1 Messgeräte 54 3.1.1 Ocular Response Analyzer 54 3.1.2 Corvis ST - Corneal Visualization Scheimpflug Technology 56 3.1.3 Scheimpflug-basierte Topografie und Tomografie 62 3.2 Experimentelle Untersuchungen 62 3.2.1 Beurteilung von Einflussfaktoren auf die DCR Parameter am Schweineauge 63 3.2.2 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking anhand von Schweineaugen 65 3.2.3 Statistische Auswertung der experimentellen Untersuchungen 69 3.3 Klinische Untersuchung 70 3.3.1 Normwerte von gesunden Probanden und Einflussfaktoren auf Messparameter des Corvis ST und ORA 70 3.3.2 Evaluierung biomechanischer Parameter anhand von gesunden Probanden und Keratokonus-Patienten – Eine Fall-Kontroll-Untersuchung 71 3.3.3 Klassifizierung der DCR Parameter anhand des Keratokonus-Schweregrades 73 3.3.4 Wiederholbarkeit und Reproduzierbarkeit der DCR Parameter anhand von Keratokonus-Patienten 75 3.3.5 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking 77 4 Ergebnisse 78 4.1 Experimentelle Untersuchungen 78 4.1.1 Beurteilung von Einflussfaktoren auf die DCR Parameter am Schweine-auge 78 4.1.2 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking anhand von Schweineaugen 83 4.2 Klinische Untersuchungen 88 4.2.1 Normwerte von gesunden Probanden und Einflussfaktoren auf Messparameter des Corvis ST und ORA 88 4.2.2 Evaluierung biomechanischer Parameter anhand von gesunden Probanden und Keratokonus-Patienten – Eine Fall-Kontroll-Untersuchung 101 4.2.3 Klassifizierung der DCR Parameter anhand des Keratokonus-Schweregrades 107 4.2.4 Wiederholbarkeit und Reproduzierbarkeit der DCR Parameter anhand von Keratokonus-Patienten 112 4.2.5 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking 118 5 Diskussion 121 5.1 Experimentelle Untersuchungen 121 5.1.1 Beurteilung von Einflussfaktoren auf die DCR Parameter am Schweine-auge 121 5.1.2 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking ex vivo und in vivo 123 5.2 Klinische Untersuchungen 129 5.2.1 Normwerte von gesunden Probanden und Einflussfaktoren auf Messparameter des Corvis ST und ORA 129 5.2.2 Evaluierung und Differenzierung gesunder Probanden und Keratokonus-Patienten hinsichtlich biomechanischer Parameter – Eine Fall-Kontroll-Untersuchung 134 5.2.3 Klassifizierung der DCR Parameter anhand des Keratokonus-Schweregrades 140 5.2.4 Wiederholbarkeit und Reproduzierbarkeit der DCR Parameter anhand von Keratokonus-Patienten 142 6 Zusammenfassung 145 7 Summary 148 Literaturverzeichnis 151 Stichwortverzeichnis 166 Anhang 168 Danksagung 187 Anlage 1 Erklärung zur Eröffnung des Promotionsverfahrens 188 Anlage 2 Erklärung zur Einhaltung aktueller gesetzlicher Vorgaben 189 Anlage 3 Angabe zu Bildrechten 190 Anlage 4 Kennzeichnung des Eigenanteils bereits veröffentlichter Publikationen 195 / Summary: The aim of this thesis is to investigate a novel method measuring biomechanical properties of the cornea in experimental and clinical conditions. The used device is a dynamic Scheimpflug Analyzer based non-contact tonometry (Corvis ST, Oculus, Wetzlar, Germany) and is approved for in vivo applications. An ultra-high speed Scheimpflug camera records the complete corneal deformation after applying an air-puff. Due to external air pressure, the cornea passes the 1st applanation, followed by a concave phase until highest concavity and afterwards 2nd applanation until it recovers to its initial physiological state. Several dynamic corneal response (DCR) parameters are derived from these measurements. Some of these DCR parameters show clinical relevance: The bIOP is a biomechanical corrected intraocular pressure (IOP) whose value is adjusted by age, corneal thickness and several DCR parameters. Further, the deformation amplitude ratio at 2 mm (DAR2) represents the ratio between central und peripheral deformations. The integrated inverse radius (int. 1/R) is the sum of the reciprocal curvature during the concave phase (between 1st and 2nd applanation). The overall corneal stiffness is represented by stiffness parameter at 1st applanation (SP A1). Finally, the Corvis Biomechanical Index (CBI) is a screening parameter that separates healthy from keratoconic eyes. Based on literature research, typical mechanical parameters from engineering or material sciences cannot be applied to the cornea easily, due to anisotropic, non-linear and visco-elastic properties of the cornea. Hence, it is not possible to determine a consistent value for Young’s modulus; instead, it can be seen as a function of strain. The measurement of biomechanical properties of the cornea are mainly influenced by IOP and corneal thickness. In this thesis, these and other factors were investigated to evaluate the impact on DCR parameters. During the experiment with porcine eyes, it has been found that the higher the induced IOP is, the less deformable the cornea behaves against the applied air puff, even though the material properties are not altered. While increasing the IOP, Corvis ST measurements were performed at each 5 mmHg steps. Among other findings, observations show decreased values for DAR2 and int. 1/R as well as increased values for SP A1. As a direct consequence, IOP conformity is taken into account for further investigations. In addition, clinical investigations also showed corneal thickness and age as influencing factors on the DCR parameter in healthy subjects. A higher corneal thickness is associated with a stiffer biomechanical behavior (lower DAR2 and int. 1/R, higher SP A1) than thinner corneas in healthy eyes. Furthermore, it could be found that age has a stiffening effect on the cornea (higher SP A1). Repeatability and reproducibility were investigated experimentally and clinically. Certain DCR parameters were repeatable in porcine eyes and keratoconic eyes. Therefore, it can be concluded that these measurements are reliable and can be used for longitudinal observations. The Ocular Response Analyzer (ORA, Reichert Technologies, Buffalo, NY, USA) is another clinical device to measure biomechanical properties of the cornea based on non-contact tonometry. Main parameters are corneal hysteresis (CH) and corneal resistance factor (CRF). However, CH and CRF are not associated with corneal stiffness because it reflects corneal visco-elastic properties due to the integration of pressure values of inward and outward movement in its calculations. In contrast, DCR parameters of Corvis ST describe corneal behavior of inward and outward movement separately. Parameters of inward movement are associated with the elastic component and thus to corneal stiffness. The investigations in healthy and keratoconic eyes have shown that CBI is the best parameter separating between these cohorts. Furthermore, DAR2, int. 1/R and SP A1 show higher values for sensitivity and specificity in differentiating healthy from keratoconus as CH and partly CRF. Regarding the severity of keratoconus, some DCR parameters are different between several stages. As a result, a classification model to predict the severity of keratoconus had been developed based on Machine learning algorithms. The prediction of healthy, early and advanced cases shows good sensitivity and specificity whereas mild cases show moderate accuracy. In this thesis, corneal biomechanical alterations after cross-linking (CXL) in progressive keratoconus are evaluated. Before the clinical study, porcine eyes were investigated ex vivo to evaluate the efficacy of CXL using common surgical protocols. These eyes were measured by Corvis ST and afterwards by stress-strain measurement (extensometry). In conclusion, a higher IOP, a higher SP A1 and a lower int. 1/R observed by Corvis ST after CXL can be associated with an increased corneal stiffness, measured by extensometry. In vivo, alterations in the same manner of bIOP and int. 1/R were observed one months after CXL and partly up to one year. However, it can be assumed that the biomechanical effect can be measured preferably in short-term follow-up. Hence, the Dynamic Scheimpflug Analyzer can be seen as a useful device to measure in vivo biomechanical properties of the cornea. Pre-operative examination in refractive surgery or early diagnosis of keratoconus can notably be improved by corneal biomechanical information. There is a certain relationship between DCR parameters and IOP as well as corneal thickness. In the future research, computer-aided data analysis of raw data from Corvis ST can help to determine advanced material properties of the cornea independently from its thickness and IOP. The investigation of e.g. glaucoma patients could be a further important application.:Vorwort IV Inhaltsverzeichnis VI Abbildungs- und Tabellenverzeichnis IX Abkürzungsverzeichnis XV 1 Einleitung 1 2 Wissenschaftlicher Hintergrund 2 2.1 Das Auge 2 2.2 Kornea – die Hornhaut des menschlichen Auges 3 2.2.1 Struktur und Eigenschaften des Hornhautstromas 8 2.2.2 Zelluläre Abnormitäten der Hornhaut bei Keratektasien 12 2.2.3 Strukturelle Veränderungen der Hornhaut bei okulären und systemischen Erkrankungen 14 2.3 Keratokonus 17 2.3.1 Inzidenz und Prävalenz 17 2.3.2 Risikofaktoren 18 2.3.3 Diagnose und klinische Zeichen 19 2.3.4 Behandlungsoptionen 25 2.4 Methoden zur Bestimmung von (bio-)mechanischer Eigenschaften 29 2.4.1 Übertragung mechanischer Kenngrößen auf Biomaterialien 29 2.4.2 Zusammenhang zwischen den chemischen, strukturellen Eigenschaften und der Biomechanik der Hornhaut 33 2.4.3 Bestimmung der Biomechanik der Hornhaut ex vivo 33 2.4.4 Bestimmung der Biomechanik der Hornhaut in vivo 37 2.4.5 Computer gestützte Modellierung von biomechanischen Modellen und Ektasien 43 2.5 Aktueller Stand der Forschung 46 2.5.1 Ocular Response Analyzer 46 2.5.2 Corvis ST 50 2.6 Fragestellungen und Hypothesen 53 3 Material und Methoden 54 3.1 Messgeräte 54 3.1.1 Ocular Response Analyzer 54 3.1.2 Corvis ST - Corneal Visualization Scheimpflug Technology 56 3.1.3 Scheimpflug-basierte Topografie und Tomografie 62 3.2 Experimentelle Untersuchungen 62 3.2.1 Beurteilung von Einflussfaktoren auf die DCR Parameter am Schweineauge 63 3.2.2 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking anhand von Schweineaugen 65 3.2.3 Statistische Auswertung der experimentellen Untersuchungen 69 3.3 Klinische Untersuchung 70 3.3.1 Normwerte von gesunden Probanden und Einflussfaktoren auf Messparameter des Corvis ST und ORA 70 3.3.2 Evaluierung biomechanischer Parameter anhand von gesunden Probanden und Keratokonus-Patienten – Eine Fall-Kontroll-Untersuchung 71 3.3.3 Klassifizierung der DCR Parameter anhand des Keratokonus-Schweregrades 73 3.3.4 Wiederholbarkeit und Reproduzierbarkeit der DCR Parameter anhand von Keratokonus-Patienten 75 3.3.5 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking 77 4 Ergebnisse 78 4.1 Experimentelle Untersuchungen 78 4.1.1 Beurteilung von Einflussfaktoren auf die DCR Parameter am Schweine-auge 78 4.1.2 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking anhand von Schweineaugen 83 4.2 Klinische Untersuchungen 88 4.2.1 Normwerte von gesunden Probanden und Einflussfaktoren auf Messparameter des Corvis ST und ORA 88 4.2.2 Evaluierung biomechanischer Parameter anhand von gesunden Probanden und Keratokonus-Patienten – Eine Fall-Kontroll-Untersuchung 101 4.2.3 Klassifizierung der DCR Parameter anhand des Keratokonus-Schweregrades 107 4.2.4 Wiederholbarkeit und Reproduzierbarkeit der DCR Parameter anhand von Keratokonus-Patienten 112 4.2.5 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking 118 5 Diskussion 121 5.1 Experimentelle Untersuchungen 121 5.1.1 Beurteilung von Einflussfaktoren auf die DCR Parameter am Schweine-auge 121 5.1.2 Beurteilung biomechanischer Änderungen nach kornealem Cross-Linking ex vivo und in vivo 123 5.2 Klinische Untersuchungen 129 5.2.1 Normwerte von gesunden Probanden und Einflussfaktoren auf Messparameter des Corvis ST und ORA 129 5.2.2 Evaluierung und Differenzierung gesunder Probanden und Keratokonus-Patienten hinsichtlich biomechanischer Parameter – Eine Fall-Kontroll-Untersuchung 134 5.2.3 Klassifizierung der DCR Parameter anhand des Keratokonus-Schweregrades 140 5.2.4 Wiederholbarkeit und Reproduzierbarkeit der DCR Parameter anhand von Keratokonus-Patienten 142 6 Zusammenfassung 145 7 Summary 148 Literaturverzeichnis 151 Stichwortverzeichnis 166 Anhang 168 Danksagung 187 Anlage 1 Erklärung zur Eröffnung des Promotionsverfahrens 188 Anlage 2 Erklärung zur Einhaltung aktueller gesetzlicher Vorgaben 189 Anlage 3 Angabe zu Bildrechten 190 Anlage 4 Kennzeichnung des Eigenanteils bereits veröffentlichter Publikationen 195

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

Les patients atteints de cécité cornéenne résultant d'une maladie ou d'une blessure dans de nombreux pays ne seront probablement pas transplantés avec des cornées de donneurs humains en raison d'une grave pénurie mondiale de tissus de donneurs. Cependant, même si des cornées de donneurs étaient disponibles, les patients présentant une inflammation ou une maladie grave ne seraient pas aidés car ils courent un risque élevé de rejet des cornées de donneurs car celles-ci contiennent des cellules allogéniques. Les implants cornéens sans cellules qui ne déclenchent pas de rejet ont été développés comme alternatives à la transplantation de donneurs humains par le laboratoire Griffith, et ont montré dans un premier essai clinique chez l'homme qu'ils régénèrent de manière stable le tissu et les nerfs cornéens. Ces implants comprenaient du collagène humain recombinant, la principale protéine structurelle trouvée dans la cornée humaine. Cependant, les collagènes de pleine longueur sont difficiles et coûteux à produire et ne peuvent pas être personnalisés. Une grande variété de peptides plus courts qui imitent le collagène et d'autres molécules de la matrice extracellulaire ont été développés et testés. Cela comprend les peptides hybrides combinant le collagène et la soie (VBsilk). Le but de ma thèse est de confirmer les simulations de VBsilk d'un peptide hybride collagène-soie produit au Griffith Lab. Un autre objectif est de déterminer les conditions de production et de purification pour montrer que le peptide simulé peut être converti en un peptide réel. En bref, l'ADN codant pour une séquence de VBsilk a été cloné dans ClearColi, une souche d'E. Coli à faible endotoxine. Les bactéries ont été cultivées dans des cultures à grand volume. Le VBsilk a été extrait et purifié par FPLC. SDS-PAGE a montré que des bandes de protéines de taille appropriée étaient obtenues. Par conséquent, il est possible de produire le peptide VBsilk. / Patients with cornea blindness resulting from disease or injury in many countries are unlikely to be transplanted with human donor corneas due a worldwide severe shortage of donor tissues. However, even if donor corneas were available, patients with inflammation or severe disease would not be helped as they are at a high risk of rejecting donor corneas as these contain allogeneic cells. Cell-free corneal implants that do not trigger rejection were developed as alternatives to human donor transplantation by the Griffith lab, and shown in a first-in-human clinical trial to stably regenerate corneal tissue and nerves. These implants comprised recombinant human collagen, the main structural protein found in the human cornea. However, full-length collagens are difficult and expensive to produce, and cannot be customized. A wide variety of shorter peptides that mimic collagen and other extracellular matrix molecules have been developed and tested. This includes hybrid peptides combining collagen and silk (VBsilk). The aim of my thesis is to is to confirm simulations of VBsilk, a hybrid collagen-silk peptide that was produced in the Griffith Lab. A further aim is to determine the conditions for the production and purification to show that simulated peptide can be converted into an actual peptide. Briefly, the DNA coding for a VBsilk sequence was cloned into ClearColi, a strain of E. coli with low endotoxin. The bacteria were grown up in large volume cultures. The VBsilk was extracted and purified by FPLC. SDS-PAGE showed that appropriate-sized bands of protein were obtained. Hence, it is possible to produce VBsilk peptide.

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...

Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...