Der Keratokonus ist eine Erkrankung, bei der sich die Hornhaut des Auges, auch als Cornea bezeichnet, fortschreitend ausdünnt. Dies führt zu einer Formveränderung und damit zu Einschränkungen des Sehvermögens. Bei den Betroffenen tritt häufig Kurzsichtigkeit auf, die aufgrund der unregelmäßigen Wölbung der Cornea nur schwer durch Sehhilfen ausgeglichen werden kann. In schweren Fällen ist eine Hornhauttransplantation erforderlich. Die Ursachen der Erkrankung sowie der Einfluss äußerer Faktoren auf den Krankheitsverlauf sind bislang nicht hinreichend erforscht, sodass eine Prognose schwierig ist. Quervernetzungen (Crosslinks) im Kollagen spielen eine entscheidende Rolle für die optischen und biomechanischen Eigenschaften der Cornea. Therapeutische Ansätze zielen darauf ab, zusätzliche Crosslinks zu erzeugen und damit das Gewebe zu stabilisieren. Die photochemische Vernetzung der Cornea (engl. Corneal Crosslinking, CXL) mit Riboflavin und UV-Licht wurde entwickelt, um die biomechanischen Eigenschaften der Cornea zu verbessern und dem Fortschreiten des Keratokonus entgegenzuwirken. Es wird angenommen, dass die UV-Bestrahlung Riboflavin aktiviert, welches daraufhin Sauerstoffmoleküle in der Cornea in hochreaktive Formen umwandelt. Diese reaktiven Sauerstoffspezies initiieren chemische Reaktionen, welche zur Bildung neuer Crosslinks zwischen den Kollagenfasern führen. Obgleich makroskopische Effekte wie eine erhöhte Gewebesteifigkeit, eine erhöhte Resistenz gegenüber enzymatischem Abbau sowie Veränderungen in der Fibrillenarchitektur dokumentiert sind, existieren nur wenige Studien, welche die CXL auf molekularer Ebene untersuchen. Im Rahmen dieser Arbeit werden die molekularen Mechanismen der CXL in der Cornea mit schwingungsspektroskopischen Methoden wie Infrarot- (IR) und Raman-Spektroskopie untersucht. Diese Methoden bieten eine hohe Sensitivität gegenüber kleinsten molekularen Veränderungen und ermöglichen die Untersuchung von Gewebe in nahezu nativem Zustand. Die Experimente wurden an vom Schwein stammenden Corneae durchgeführt, da diese über eine große Ähnlichkeit mit der humanen Cornea verfügen. Zum Nachweis der kovalenten Bindungen wurden IR-Imaging-Messungen an Dünnschnittpräparaten und ATR-spektroskopische Experimente (engl. attenuated total reflection) durchgeführt. Letztere ermöglichten die oberflächennahe Detektion molekularer Veränderungen in situ, d. h. zeitgleich mit der UV-Bestrahlung. Die Messungen zeigen gemeinsame spektrale Veränderungen, insbesondere im Amid-I- und Amid-II-Bereich, sowie eine Zunahme der C–O-Schwingungen. Aufgrund der im Rahmen dieser Arbeit gesammelten ATR-spektroskopischen Daten kann insgesamt davon ausgegangen werden, dass eine Kombination mehrerer Effekte zu einer Verstärkung der Biomechanik durch die CXL führt. Die ATR-Spektren liefern keine eindeutigen Hinweise für die Entstehung neuer kovalenter Bindungen, die durch einen einheitlichen Reaktionsmechanismus (z. B. Addition von Stickstoffbasen an Carbonylgruppen, Vernetzung via Histidinseitenketten) erklärt werden können. Im zweiten Teil der Arbeit wurde die Raman-Spektroskopie als ergänzende Methode zur Untersuchung der CXL eingesetzt. Durch den zusätzlichen Einsatz der SERS-Messtechnik (engl. surface-enhanced Raman spectroscopy) sollten gezielt oberflächennahe Reaktionsprodukte, die durch die CXL gebildet werden, hervorgehoben werden. Da SERS spezielle Strukturen für die Oberflächenverstärkung benötigt, wurden Cornea-Proben nach der CXL-Behandlung mit Silber bedampft und anschließend untersucht. Die systematische Auswertung der Messungen zeigte vor allem Veränderungen in den Bereichen, die mit Prolin und Hydroxyprolin sowie CH2/CH3-Gruppen assoziiert sind. Darüber hinaus zeigen die Untersuchungen, dass Vernetzungssignale bereits ohne Bestrahlung auftreten, die Vernetzungsbehandlung jedoch nicht bei jeder individuellen Behandlung bzw. Cornea zu einem nachweisbaren Effekt führt. Die Ergebnisse werfen ein neues Licht auf die Funktionsweise der CXL und erfordern eine Neubewertung bisheriger Annahmen über mögliche Reaktionsmechanismen. Aufgrund fehlender Hinweise können kovalente Bindungen als primäre Quelle für die Erhöhung der Stabilität der Cornea ausgeschlossen werden. Die Stabilisierung der Cornea durch die CXL lässt sich vermutlich auf eine Kombination von physikalischen, chemischen und strukturellen Veränderungen zurückführen. Es ist davon auszugehen, dass nicht-kovalente Interaktionen wie Dipolwechselwirkungen und Wasserstoffbrückenbindungen zwischen Kollagenmolekülen einen weitaus größeren Beitrag zur erhöhten biomechanischen Stabilität der Cornea leisten als bisher angenommen.:1. Einleitung 1.1 Hintergrund und Motivation 1.2 Zielsetzung 2. Cornea und Keratokonus 2.1 Aufbau und Funktion der Cornea 2.2 Zusammensetzung der Cornea 2.3 Molekularer Aufbau des Kollagens in der Cornea 2.4 Kollagen-Crosslinks 2.4.1 Enzymatische Crosslinks durch Lysyloxidase 2.4.2 Nicht-enzymatische Crosslinks durch Glykierung 2.5 Keratokonus 3. Therapie CXL 3.1 Die Funktion von Riboflavin, UV-Licht und Sauerstoff 3.2 Methoden zur Untersuchung der CXL 3.3 Stand der Forschung zur Untersuchung der CXL 4. Material und Methoden 4.1 Präparation der untersuchten Proben 4.1.1 Präparation der Cornea-Flaps 4.1.2 Präparation der Dünnschnittpräparate 4.2 CXL nach dem Dresden-Protokoll 4.3 Vernetzungsbehandlung mit Glutaraldehyd 4.4 Chemikalien 4.5 Transmissions-IR-Mikrospektroskopie 4.6 ATR-Spektroskopie 4.7 Oberflächenverstärkte Raman-Spektroskopie (SERS) 4.8 Konfokale Raman-Messungen 4.9 Datenvorbehandlung 5. Ergebnisse und Diskussion 5.1 IR-mikroskopisches Imaging der Cornea 5.2 ATR-spektroskopische Untersuchung der CXL 5.2.1 Messungen der CXL unter in-situ-äquivalenten Bedingungen 5.2.2 Entwicklung eines Aufbaus zur ATR-Messung mit Sauerstoffzufuhr 5.2.3 Ergebnisse der ATR-Messungen unter erhöhter Sauerstoffzufuhr 5.2.4 Fazit 5.3 Raman-spektroskopische Untersuchung der CXL 5.3.1 Raman-Spektrum der Cornea 5.3.2 Raman-Messungen der CXL an Cornea-Flaps 5.3.3 Raman-Messungen der CXL von ganzen Augen 5.3.4 Oberflächenverstärkte Raman-Spektroskopie 5.3.5 Abscheidung der Silberschicht 5.3.6 Ergebnisse der SERS-Experimente 5.3.7 Raman-Messungen der Vernetzung mit Glutaraldehyd 5.3.8 Fazit 6. Zusammenfassung 7. Literaturverzeichnis 8. Anhang Veröffentlichungen Versicherung

Objetivo: Comparar as características inflamatórias da superfície ocular de pacientes com ceratocone (CE) às de pacientes com alergia ocular (AO). Métodos: Realizou-se um estudo transversal, envolvendo 134 participantes, divididos em três grupos: alergia ocular (AO)(n=55), ceratocone com ou sem alergia ocular associada (CE) (n=61) e controle (CO) (n=18). Os participantes do estudo foram recrutados e avaliados em clínica privada na cidade de Chapecó-SC, no período de polinização, em dezembro de 2013 e de outubro de 2014 a janeiro de 2015. Para análise dos grupos, todos os pacientes foram avaliados clinicamente por especialistas em alergia e oftalmologia e submetidos a exame de tomografia de córnea com tecnologia de Scheimpflug (Pentacam HRR), medida da osmolaridade da lágrima por impedanciometria (TearLabR) em ambos os olhos e preenchimento de questionários padronizados para alergia ocular (QA) e para doença de superfície ocular (OSDI). Em 107 de 134 pacientes (CE=50, AO=42, CO=15) foram dosadas as citocinas IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-15, IL-31, IL-33, IL-17 A, IL-23, TNF-alfa, TNF-beta e INF-gama na lágrima, por meio de tecnologia multiplex com beads magnéticas. A coleta das amostras de lágrimas (5 microlitros no mínimo) foi realizada por meio de aspiração no menisco lacrimal temporal inferior, com capilares de vidro em um único olho. As amostras foram congeladas imediatamente após coleta e mantidas a -80 oC, até o momento da dosagem e análise em agosto de 2016. Resultados: Constatou-se alta prevalência de alergia no grupo com ceratocone 68,85% (42/61), sendo que 42,62% (26/61) dos pacientes desse grupo apresentaram alergia ocular. A intensidade do prurido ocular foi maior nos pacientes AO e no grupo CE, do que no grupo controle (p < 0,001 e p=0,047) e maior no grupo AO do que no CE (p < 0,001). As citocinas INF-gama, IL-13, IL-15, IL-17A, IL-1beta, IL-2, IL-4, IL-5, IL-31, TNF-beta foram estatisticamente mais elevadas no grupo AO, quando comparadas ao grupo CE. No grupo controle, houve correlação direta da diferença absoluta da osmolaridade (delta osmol) com o nível encontrado para todas as citocinas dosadas. Observou-se, ainda, correlação direta entre níveis de IL-1beta, IL-6 e TNF-alfa com OSDI (p=0,044, p=0,010 e p=0,047) no grupo CE. A osmolaridade foi maior no grupo CE, quando comparado ao AO (p=0,043). Conclusões: Pacientes com ceratocone apresentaram alterações inflamatórias na superfície ocular, tais como: correlação direta do índice OSDI com IL-1beta, IL-6 e TNF-alfa na lágrima; intensidade de prurido mais elevada que no grupo controle e osmolaridade maior que no grupo com AO. No grupo AO, observou-se índices OSDI maiores que no grupo controle e aumento de várias citocinas dosadas, quando comparadas ao grupo CE. Nos pacientes controle, a instabilidade no filme lacrimal (delta osmol), correlacionou-se diretamente com a inflamação ocular (citocinas dosadas). Observou-se alta prevalência de formas leves de alergia ocular no grupo CE (40,98%) e apenas um caso de alergia ocular grave (ceratoconjuntivite atópica [1,3%]), esses dados estão em desacordo com a maior parte da literatura disponível sobre o assunto. Em conjunto, os achados do presente estudo podem estar relacionados a características específicas da região estudada / Purpose: To compare the inflammatory profile of the ocular surface of patients with keratoconus with that of patients with ocular allergy. Methods: A cross sectional study was carried out and involved 134 participants, divided into three groups: ocular allergy group (OA) (N=55), keratoconus with or without ocular allergy (KC) (N=61) and a control group (CO) (N=18). The study participants were recruited and evaluated in a private clinic in the city of Chapecó-SC, during the pollination period in 2013 (December) and from 2014 to 2015 (October to January). In order to analyze the three groups, all patients were evaluated clinically by allergy and ophthalmology specialists, submitted to a corneal tomography exam with Scheimpflug technology (Pentacam HRR), to impedanciociometry to assess tear osmolarity (Tear LabR) of both eyes and to standardized questionnaire rates for ocular allergy (AQ) and for ocular surface disease index (OSDI). Cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-15, IL-31, IL-33, IL-17 A, IL-23, TNF-alfa, TNF-beta and INF-gama) levels were assessed in 107 out of 134 patients using multiplex technology with magnetic beads. Tear collection of at least 5 microliters was carried out on a single eye by suction in the inferior temporal lacrimal meniscus with glass capillaries. The samples were frozen at -800C, immediately after collection to the time of dosing and analysis in August 2016. Results: The prevalence of allergy in the keratoconus group was 68.85% (42/61), within the same group 42.62% (26/61) of the patients had ocular allergy. Ocular pruritus intensity was higher in the OA group and in the KC group than in the control group (p < 0,001 e p=0,047), and higher in the OA group than in the KC group (p < 0,001). Cytokines INF-gama, IL-13, IL-15, IL-17A, IL-1beta, IL-2, IL-4, IL-5, IL-31, TNF-beta were statiscally higher in the OA group in comparision to the KC group. In the control group, there was a positive correlation of the difference measurement of osmolarity (osmol delta) between the eyes with all cytokines dosed. In the KC group, IL-1beta, IL-6 e TNF-alfa correlated positively with OSDI (p=0,044, p=0,010 e p=0,047). Tear osmolarity was higher in the KC group than in the OA group (p=0,043). Conclusions: Patients with keratoconus had inflammatory changes in the ocular surface, such as: direct correlation between OSDI and tear IL-1beta, IL-6 and TNF-alpha levels; higher pruritus intensity than the control group and higher osmolarity than the OA group. In the OA group, OSDI levels were higher than in the control group and some cytokines had higher levels than the KC group. In the control patients, tear film instability (delta osmol) was direct correlated to ocular inflammation (dosed cytokines). In the KC group, there was a high prevalence of mild forms of ocular allergy (40.98%) and only one case of severe ocular allergy (atopic keratoconjunctivitis [1.3%]). However, most studies avaiable have associated keratoconus with severe forms of ocular allergy. Together, these findings may be related to specific characteristics of the region of study

There has been widespread interest in corneal biomechanics over recent years, driven largely by the advancements in, and the popularity of refractive surgery techniques and subsequent concerns over their safety. Lately there has been interest into whether crosslinking, which is currently used for the treatment of keratoconus, could be developed as a minimally invasive technique to change the refractive power of the cornea by selectively changing the corneal biomechanics in specific regions to induce a shape change. Successful application of this technique requires a detailed understanding of corneal biomechanics and so far, little is known about the biomechanics of this complex tissue. The current lack of understanding can be mostly attributed to the absence of a suitable measurement technique capable of examining the dynamic behaviour of the cornea under physiological loading conditions. This thesis describes the development of a novel full-field, ex vivo, measurement method incorporating speckle interferometric techniques, to examine the biomechanics of the cornea before and after crosslinking in response to hydrostatic pressure fluctuations representative of those that occur in vivo during the cardiac cycle. The eventual measurement system used for the experiments detailed in this thesis incorporated; an Electronic Speckle Pattern Interferometer (ESPI), a Lateral Shearing Interferometer (LSI) and a fringe projection shape measurement system. The combination of these systems enabled the 3-dimensional components of surface displacement and the 1st derivative of surface displacement to be determined in response to small pressure fluctuations up to 1 mmHg in magnitude. The use of both ESPI and LSI together also enabled the applicability of LSI for measurement of non-flat surfaces to be assessed, and limitations and error sources to be identified throughout this work. To enable the measurement of corneal biomechanics, part of this thesis was concerned with the design of a bespoke loading rig. A chamber was designed that could accommodate tissue of both porcine and human origin. This chamber was linked to a hydraulic loading rig, whereby the cornea could be held at a baseline pressure representative of normal intraocular pressure and small pressure variations could be introduced by the automated vertical movement of the reservoir supplying the chamber. Experiments were conducted on a range of non-biological samples with both flat and curved surface topography, and both uniform and non-uniform mechanical properties, to determine if the measurement configuration was giving the expected measurement data and the loading rig was stable and repeatable. Following experiments on non-biological samples, a range of experiments were conducted on porcine corneas to develop a suitable testing methodology and address some of the challenges associated with corneal measurement, including transparency and hydration instability. During these investigations, a suitable surface coating was identified to generate an adequate return signal from the corneal surface, while not interfering with the response. Alongside this, the natural variation in the response of the cornea was investigated over the total experimental time, and a range of data was presented on corneas before and after crosslinking, which confirmed the suitability of the measurement methods for the assessment of crosslinking. Ultimately, a small sample size of six human corneas were investigated before and after crosslinking in specific topographic locations. From the experiments on human and porcine corneas, full-field maps of surface deformation have been presented, and a compliant region incorporating the peripheral and limbal areas has been identified as being fundamental to the response of the cornea to small pressure fluctuations. In addition to this, the regional effects of crosslinking in four different topographic locations on corneal biomechanics have been evaluated. From this, it has been demonstrated that crosslinking in specific regions in isolation can influence the way the cornea deforms to physiological-scale fluctuations in hydrostatic pressure and this could have implications for refractive correction.

This study aimed to create computer models to aid the diagnosis of keratoconus using an edge segmentation algorithm, wavelet transforms and machine-learning techniques based on biomechanical parameters of the cornea derived from Corvis ST. 102 normal eyes, and 73 eyes with keratoconus grade I and II for training and validation of the models were included. Initially the 31 original equipment parameters were studied to assess its power in separating the control groups and research group. Then, it was used the Canny algorithm for edges segmentation of the selected images, so that 400 central points of these images could be processed with the wavelet transforms and later with machine learning techniques. The best result was achieved using the Haar wavelet transform and a Multilayer Perceptron Neural Network, reaching the sensitivity of 84.93%, a specificity of 89.22% and an Area Under the ROC Curve (AUC) of 0932. It concludes that the developed models can help make the diagnosis of early keratoconus. / O presente trabalho objetivou criar modelos computacionais de auxílio ao diagnóstico de ceratocone, utilizando um algoritmo de segmentação de borda, transformadas de wavelet e técnicas de aprendizagem de máquina, baseados em parâmetros biomecânicos da córnea derivados do CorVis ST. Foram incluídos 102 olhos normais, e 73 olhos com ceratocone grau I e II para o treinamento e validação dos modelos criados. Inicialmente foram estudados os 31 parâmetros originais do equipamento, para avaliar seu poder em separar os grupos de controle e pesquisa. Foi então utilizado o algoritmo Canny para segmentação das bordas das imagens selecionadas, para que 400 pontos centrais extraídos dessas imagens pudessem ser processados com as transformadas de wavelet e posteriormente com técnicas de aprendizado de máquina. O melhor resultado foi alcançado utilizando a transformada de Wavelet do tipo Haar e uma Rede Neural Multilayer Perceptron, alcançando a sensibilidade de 84.93%, uma especificidade de 89.22% e uma Area Under de ROC Curve (AUC) de 0.932. Conclui-se que os modelos desenvolvidos podem contribuir para tornar o diagnóstico de ceratocone mais precoce.

Objetivo: Comparar as características inflamatórias da superfície ocular de pacientes com ceratocone (CE) às de pacientes com alergia ocular (AO). Métodos: Realizou-se um estudo transversal, envolvendo 134 participantes, divididos em três grupos: alergia ocular (AO)(n=55), ceratocone com ou sem alergia ocular associada (CE) (n=61) e controle (CO) (n=18). Os participantes do estudo foram recrutados e avaliados em clínica privada na cidade de Chapecó-SC, no período de polinização, em dezembro de 2013 e de outubro de 2014 a janeiro de 2015. Para análise dos grupos, todos os pacientes foram avaliados clinicamente por especialistas em alergia e oftalmologia e submetidos a exame de tomografia de córnea com tecnologia de Scheimpflug (Pentacam HRR), medida da osmolaridade da lágrima por impedanciometria (TearLabR) em ambos os olhos e preenchimento de questionários padronizados para alergia ocular (QA) e para doença de superfície ocular (OSDI). Em 107 de 134 pacientes (CE=50, AO=42, CO=15) foram dosadas as citocinas IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-15, IL-31, IL-33, IL-17 A, IL-23, TNF-alfa, TNF-beta e INF-gama na lágrima, por meio de tecnologia multiplex com beads magnéticas. A coleta das amostras de lágrimas (5 microlitros no mínimo) foi realizada por meio de aspiração no menisco lacrimal temporal inferior, com capilares de vidro em um único olho. As amostras foram congeladas imediatamente após coleta e mantidas a -80 oC, até o momento da dosagem e análise em agosto de 2016. Resultados: Constatou-se alta prevalência de alergia no grupo com ceratocone 68,85% (42/61), sendo que 42,62% (26/61) dos pacientes desse grupo apresentaram alergia ocular. A intensidade do prurido ocular foi maior nos pacientes AO e no grupo CE, do que no grupo controle (p < 0,001 e p=0,047) e maior no grupo AO do que no CE (p < 0,001). As citocinas INF-gama, IL-13, IL-15, IL-17A, IL-1beta, IL-2, IL-4, IL-5, IL-31, TNF-beta foram estatisticamente mais elevadas no grupo AO, quando comparadas ao grupo CE. No grupo controle, houve correlação direta da diferença absoluta da osmolaridade (delta osmol) com o nível encontrado para todas as citocinas dosadas. Observou-se, ainda, correlação direta entre níveis de IL-1beta, IL-6 e TNF-alfa com OSDI (p=0,044, p=0,010 e p=0,047) no grupo CE. A osmolaridade foi maior no grupo CE, quando comparado ao AO (p=0,043). Conclusões: Pacientes com ceratocone apresentaram alterações inflamatórias na superfície ocular, tais como: correlação direta do índice OSDI com IL-1beta, IL-6 e TNF-alfa na lágrima; intensidade de prurido mais elevada que no grupo controle e osmolaridade maior que no grupo com AO. No grupo AO, observou-se índices OSDI maiores que no grupo controle e aumento de várias citocinas dosadas, quando comparadas ao grupo CE. Nos pacientes controle, a instabilidade no filme lacrimal (delta osmol), correlacionou-se diretamente com a inflamação ocular (citocinas dosadas). Observou-se alta prevalência de formas leves de alergia ocular no grupo CE (40,98%) e apenas um caso de alergia ocular grave (ceratoconjuntivite atópica [1,3%]), esses dados estão em desacordo com a maior parte da literatura disponível sobre o assunto. Em conjunto, os achados do presente estudo podem estar relacionados a características específicas da região estudada / Purpose: To compare the inflammatory profile of the ocular surface of patients with keratoconus with that of patients with ocular allergy. Methods: A cross sectional study was carried out and involved 134 participants, divided into three groups: ocular allergy group (OA) (N=55), keratoconus with or without ocular allergy (KC) (N=61) and a control group (CO) (N=18). The study participants were recruited and evaluated in a private clinic in the city of Chapecó-SC, during the pollination period in 2013 (December) and from 2014 to 2015 (October to January). In order to analyze the three groups, all patients were evaluated clinically by allergy and ophthalmology specialists, submitted to a corneal tomography exam with Scheimpflug technology (Pentacam HRR), to impedanciociometry to assess tear osmolarity (Tear LabR) of both eyes and to standardized questionnaire rates for ocular allergy (AQ) and for ocular surface disease index (OSDI). Cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-15, IL-31, IL-33, IL-17 A, IL-23, TNF-alfa, TNF-beta and INF-gama) levels were assessed in 107 out of 134 patients using multiplex technology with magnetic beads. Tear collection of at least 5 microliters was carried out on a single eye by suction in the inferior temporal lacrimal meniscus with glass capillaries. The samples were frozen at -800C, immediately after collection to the time of dosing and analysis in August 2016. Results: The prevalence of allergy in the keratoconus group was 68.85% (42/61), within the same group 42.62% (26/61) of the patients had ocular allergy. Ocular pruritus intensity was higher in the OA group and in the KC group than in the control group (p < 0,001 e p=0,047), and higher in the OA group than in the KC group (p < 0,001). Cytokines INF-gama, IL-13, IL-15, IL-17A, IL-1beta, IL-2, IL-4, IL-5, IL-31, TNF-beta were statiscally higher in the OA group in comparision to the KC group. In the control group, there was a positive correlation of the difference measurement of osmolarity (osmol delta) between the eyes with all cytokines dosed. In the KC group, IL-1beta, IL-6 e TNF-alfa correlated positively with OSDI (p=0,044, p=0,010 e p=0,047). Tear osmolarity was higher in the KC group than in the OA group (p=0,043). Conclusions: Patients with keratoconus had inflammatory changes in the ocular surface, such as: direct correlation between OSDI and tear IL-1beta, IL-6 and TNF-alpha levels; higher pruritus intensity than the control group and higher osmolarity than the OA group. In the OA group, OSDI levels were higher than in the control group and some cytokines had higher levels than the KC group. In the control patients, tear film instability (delta osmol) was direct correlated to ocular inflammation (dosed cytokines). In the KC group, there was a high prevalence of mild forms of ocular allergy (40.98%) and only one case of severe ocular allergy (atopic keratoconjunctivitis [1.3%]). However, most studies avaiable have associated keratoconus with severe forms of ocular allergy. Together, these findings may be related to specific characteristics of the region of study

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-06-06T12:33:44Z No. of bitstreams: 2 Tese - Leonardo Mariano Reis - 2016.pdf: 3509741 bytes, checksum: e1f5a9a506233e87a4136a0c0016ae8e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-06T15:32:50Z (GMT) No. of bitstreams: 2 Tese - Leonardo Mariano Reis - 2016.pdf: 3509741 bytes, checksum: e1f5a9a506233e87a4136a0c0016ae8e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-06T15:32:50Z (GMT). No. of bitstreams: 2 Tese - Leonardo Mariano Reis - 2016.pdf: 3509741 bytes, checksum: e1f5a9a506233e87a4136a0c0016ae8e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2016-02-23 / Keratoconus is a non-inflammatory ocular dysfunction characterized by thinning, protrusion and conical shape of the cornea. The progression of this dysfunction leads to significant decrease in visual acuity and occasionally, in more severe cases, to corneal transplantation. The etiology of keratoconus is complex and the genetic component is among the main factors associated with the development of this disease. The objective of this study was to evaluate the occurrence of mutation in candidate genetic loci and its relation with keratoconus in patients attended in Brazil compared to healthy volunteers, through analysis of single nucleotide polymorphism in the gene DOCK9. In this clinical study, 108 participants were evaluated: 46 keratoconus patients and 62 healthy volunteers (controls). DNA samples were extracted from collected blood from keratoconus patients and controls. The genotyping of the single nucleotide polymorphism rs7995432 in the gene DOCK9 was determined through realtime polymerase chain reaction (qPCR). Single nucleotide polymorphism mutations were observed in both patients and controls. There were no significant differences on the frequency and discrimination of the mutant and wild alleles between patients and controls. The frequency of the mutant allele (C) was 4.8% in patients and 7.6% in controls. For the wild allele (T), the frequencies were 95.2% in patients and 92.4% in controls. The heterozygous genotype was present in 9.5% of patients and 11% of controls, while the homozygous genotype for the wild allele (TT) was found in 90.5% and 87% for patients and controls, respectively. Thus, these results confirm no association of these mutations and the occurrence of keratoconus for this population. / O ceratocone é uma disfunção ocular não-inflamatória caracterizada por afinamento, protrusão e formato cônico da córnea. A progressão desta disfunção ocular induz a diminuição significativa da acuidade visual em pacientes e ocasionalmente, nos casos mais avançados, ao transplante de córnea. A etiologia do ceratocone é complexa e o componente genético está entre os principais fatores associados ao seu desenvolvimento. O objetivo deste estudo foi avaliar a ocorrência de mutação em loci gênicos candidatos e sua relação com ceratocone em pacientes atendidos no Brasil comparados a voluntários saudáveis, através da análise de polimorfismo de nucleotídeo único no gene DOCK9. Neste estudo clínico foram avaliados 108 indivíduos, sendo 46 pacientes com ceratocone e 62 voluntários saudáveis (controles). Amostras de DNA foram obtidas do sangue coletado de pacientes com ceratocone e controles para a realização de análise de genotipagem. O genótipo do polimorfismo de nucleotídeo único rs7995432 no gene DOCK9 foi determinado através de reação em cadeia da polimerase em tempo real (qPCR). As mutações de polimorfismos de nucleotídeo único foram observadas em pacientes e controles. Não foram observadas diferenças significativas na frequência e discriminação dos alelos mutante e selvagem entre os pacientes com ceratocone e os controles. A frequência do alelo mutante (C) foi de 4,8% para os pacientes e 7,6% para os controles. Para o alelo selvagem (T), as frequências foram de 95,2% para os pacientes e 92,4% para os controles. O genótipo heterozigótico esteve presente em 9,5% dos pacientes e 11% dos controles, enquanto o genótipo homozigótico para o alelo selvagem (TT) foi encontrado em 90,5% e 87% para os pacientes e controles, respectivamente. Assim, não foi possível fazer uma associação destas mutações com a ocorrência do ceratocone para esta população.

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...

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...

INTRODUÇÃO: O ceratocone é uma doença não inflamatória, sem etiologia definida, caracterizada pelo afilamento estromal e protrusão da córnea. Geralmente esta doença torna-se clinicamente evidente na adolescência. Apesar de possuir sinais clínicos bem conhecidos, a detecção do ceratocone em estádios iniciais pode representar uma tarefa de difícil execução, mesmo quando a videoceratografia computadorizada ou outros métodos são utilizados para avaliar a córnea. Anteriormente, diagnosticar o ceratocone apenas após a identificação de sinais clínicos inequívocos era uma conduta aceitável. Com o advento da cirurgia refrativa porém, a identificação precoce do ceratocone tornou-se um procedimento de vital importância para evitar complicações pós-operatórias. O objetivo principal deste estudo é avaliar o uso de máquinas de vetor de suporte e redes neurais artificiais como métodos auxiliares para identificação de ceratocone e suspeita de ceratocone em exames realizados com o Orbscan II. MÉTODOS: Foram avaliados retrospectivamente dados de 344 pacientes. Os exames selecionados foram classificados em 6 categorias: normal (n=172), astigmatismo (n=89), ceratocone (n=46), ceratocone forma frustra (n=10), suspeita de ceratocone (n=16) e cirurgia refrativa (n=11). Para cada paciente 10 atributos foram obtidos ou calculados a partir de dados fornecidos pelo Orbscan II. O método do holdout e da validação cruzada foram utilizados para encontrar a melhor configuração, treinar e testar os classificadores. Além da acurácia, sensibilidade e especificidade, curvas ROC foram obtidas para cada classificador, e as áreas sob as curvas ROC foram calculadas. RESULTADOS: Os dois classificadores selecionados alcançaram um bom desempenho, com áreas sob as curvas ROC de 0,99. Não houve diferença estatística entre as suas performances. O desempenho dos classificadores foi superior ao desempenho de todos os atributos individuais do Orbscan II. (p<0,05). CONCLUSÃO: Os resultados alcançados sugerem que xi x máquinas de vetor de suporte e redes neurais artificiais podem representar técnicas úteis para a detecção de ceratocone em exames realizados com o Orbscan II. / PURPOSE: Keratoconus is a bilateral and non-inflammatory condition characterized by progressive thinning, protrusion and scarring of the córnea. The disease usually becomes clinically evident at puberty, and its etiology remains unknown. Although it has well-described clinical signs, early forms of the disease may be undetected, even when computer-assisted videokeratography techniques or other methods are used to evaluate the cornea. Prior to the development of refractive surgery, it was considered sufficient to diagnose clinically evident keratoconus. However, given the spread of refractive surgery, a careful differentiation between normal and early keratoconus cases is essential to avoid postoperative complications. This study evaluated the performance of support vector machine and multilayer perceptron neural network, as auxiliary tools to identify keratoconus from Orbscan II maps. METHODS: A total of 344 maps were retrospectively selected and classified into six categories: normal (n=172), astigmatism (n=89), keratoconus (n=46), forme fruste keratoconus (n=10), keratoconus suspect (n=16), and photorefractive keratectomy (n=11). For each map 10 attributes were obtained or calculated from data provided by the Orbscan II. Holdout method and ten-fold cross-validation was used to train and test the classifiers. Besides accuracy, sensitivity and specificity, ROC curves for each classifier were generated and the areas under the curves were calculated. RESULTS: The two selected classifiers provided a good performance and there were no differences between their performances. The area under the ROC curve of the support vector machine and multi-layer perceptron were significantly larger than those for all individual Orbscan II attributes evaluated (p<0.05). CONCLUSION: Overall, our results suggest that support vector machine and multi-layer perceptron classifiers, trained on Orbscan II data, could represent useful techniques for keratoconus detection