O endotélio corneano é uma monocamada de células poligonais localizadas na face posterior da córnea e é essencial para a manutenção da transparência corneana. Não foram encontradas referências na literatura a respeito dos parâmetros morfológicos e morfométricos do endotélio da córnea de galinhas, apesar destes animais serem amplamente utilizados como modelo experimental em estudos oftálmicos, devido à similitude com a córnea de humanos. O objetivo deste estudo foi o de avaliar os parâmetros morfométricos e o pleomorfismo da região central do endotélio da córnea de galinhas (Gallus gallus domesticus) de diferentes faixas etárias, utilizando a microscopia especular de contato. Avaliaram-se a densidade, a área celular média e o pleomorfismo das células do endotélio da córnea de 60 olhos de 30 galinhas da raça Leghorn branca. Os animais foram divididos em três grupos composto por 10 animais cada: G1 (animais com 30 dias de idade), G2 (animais com 45 dias de idade) e G3 (animais com 60 dias de idade). O presente estudo revelou que o endotélio da córnea de galinhas é composto por células poligonais de padrão regular, com predomínio de formato hexagonal. O endotélio corneano de galinhas sofreu alterações decorrentes da idade no que tange a morfometria, mas no que diz respeito ao pleomorfismo, não ocorreram alterações em resposta ao envelhecimento. / The corneal endothelium is a monolayer of polygonal cells located on the posterior face of the cornea and it is essential for the maintenance of corneal transparency. We found no references in the literature concerning the morphological and morphometric parameters of the corneal endothelium of chickens, although these animals are widely used as experimental model in ophthalmic studies due to the similarity with the human cornea. The objective of this study was to evaluate the morphometric parameters and the pleomorphism of central corneal endothelium of chickens (Gallus gallus domesticus) of different ages using the contact specular microscopy. The density, the average cell area and the pleomorphism of the corneal endothelial cells were evaluated on 60 eyes of 30 white Leghorn chickens. The animals were divided into three groups of 10 animals each: G1 (animals with 30 days of age), G2 (animals with 45 days of age) and G3 (animals with 60 days of age). The present study revealed that the corneal endothelium of chickens is composed of regular polygonal cells, with predominance of hexagonal shape. The corneal endothelium of chickens has changed due to age in respect to morphometry, but in regard to pleomorphism, no changes occurred in response to aging.

Diversos corantes vitais vêm sendo estudados e utilizados para a facilitação da capsulotomia curvilínea contínua (CCC) nas cirurgias de catarata no homem e nos animais. Além de corar adequadamente a cápsula anterior da lente e favorecer um melhor desempenho do cirurgião durante a realização da CCC, os corantes vitais devem ser seguros quanto aos seus efeitos sobre as estruturas oculares, em especial ao endotélio corneano, quando empregados com esta finalidade. O azul brilhante é um corante vital já empregado em cirurgias oculares do segmento posterior para coloração da retina e atualmente estudado sobre seu potencial de utilização em cirurgias de catarata para coloração da cápsula anterior da lente. Com o objetivo de avaliar os efeitos do uso intracameral do azul brilhante 0,05% na ultra-estrutura do endotélio corneano de suínos, vinte córneas de suínos foram avaliadas divididas em dois grupos: córneas dos bulbos oculares direitos (grupo controle) e esquerdos (grupo experimental). Todos os bulbos oculares foram previamente avaliados por microscopia especular. No grupo experimental foi realizada injeção intracameral de 0,2ml do corante azul brilhante 0,05% (OPTH-blue®) que permaneceu por um minuto antes de ser removido pela aplicação de solução salina balanceada. As córneas de ambos os grupos foram excisadas e avaliadas por microscopia eletrônica de varredura. Não houve diferença entre as imagens endoteliais obtidas em ambos os grupos. O uso intracameral do azul brilhante 0,05% não causou efeitos deletérios ao endotélio corneano dos suínos e pode, portanto, ser considerado uma escolha segura para a coloração da cápsula anterior da lente para cirurgias de catarata. / Several vital dyes have been studied and used to help the continuous curvilinear capsulotomy (CCC) in cataract surgery in men and animals. Besides staining the anterior capsule of the lens properly and providing the surgeons a better performance in the CCC, the vital dyes must be safe for their effects on ocular structures, particularly the corneal endothelium when used for this purpose. The brilliant blue is a vital dye already employed in the posterior segment eye surgeries for retinal staining and currently studied about its potential use in cataract surgeries to stain the anterior capsule of the lens. In order to evaluate the effects of the use of 0.05% intra-cameral brilliant blue in the ultra-structure of the corneal endothelium of pigs, twenty swine corneas were evaluated in two groups: right eye bulb corneas (control group) and left eye bulb corneas (experimental group). All eye bulbs were previously evaluated by specular microscopy. In the experimental group, a 0.2 ml intra-cameral injection of 0.05% brilliant blue dye (blue-OPTH ®) was given, which remained for a minute before being removed by the application of balanced salt solution. The corneas of both groups were excised and evaluated by scanning electron microscopy. There was no difference between the endothelial images obtained in both groups. The use of 0.05% intra-cameral brilliant blue caused no detrimental effects to the corneal endothelium of pigs and can therefore be considered a safe choice for staining the anterior capsule of the lens for cataract surgery.

O conhecimento da morfologia endotelial nas diferentes regiões da córnea é de suma importância para avaliação de endotélios corneanos saudáveis e doentes e de suas respostas ao uso de medicações. O objetivo do presente estudo foi descrever a morfologia do endotélio nas regiões central e periférica superior da córnea saudável de equinos, medir a área média das células pentagonais, hexagonais e heptagonais presentes nas regiões avaliadas, calcular o polimegatismo de cada tipo celular e correlacionar estes parâmetros entre os diferentes formatos celulares. Foram estudados dez equinos, machos ou fêmeas, de diferentes idades, provenientes de um abatedouro licenciado. Imagens da superfície posterior do endotélio da córnea foram obtidas com uso de microscopia eletrônica de varredura. A morfologia do endotélio de diferentes regiões da córnea foi avaliada. Além disso, foi correlacionada a variabilidade do tamanho celular médio com a morfologia endotelial. A análise estatística foi conduzida usando o teste de análise de variância (ANOVA) seguido do teste de Tukey (Post-Hoc), com nível de significância de 5%. As amostras avaliadas foram compostas em sua maioria por células hexagonais (60,5%), pentagonais (21,4%) e heptagonais (16,9%), células com quatro, oito ou nove lados compuseram 1,3%. A área celular média das células pentagonais foi 203,58 μm², das hexagonais foi 223,84 μm² e nas heptagonais foi 270,54 μm². O polimegatismo encontrado foi de 16% nas células pentagonais e hexagonais e de 20% nas heptagonais. A morfologia das células endoteliais de equinos saudáveis não diferiu entre as regiões central e periférica superior da córnea, sugerindo que a região central é representativa da região periférica. As células com sete lados apresentaram polimegatismo maior em relação às células de seis e de cinco lados. / The knowledge of the endothelial morphology in the different regions of the cornea is important for the evaluation of healthy and sick corneal endothelium and its response to drugs. In order to describe the endothelial morphology in the central and superior peripheral regions of the equines’ cornea, evaluate area from pentagonal, hexagonal and heptagonal cells present in the evaluated regions, calculate the polimegathism and correlate these parameters, two healthy corneas were collected of ten equine, male or female, of different ages. Images of the posterior surface of the corneal endothelium were taken with scanning electron microscope. The endothelial morphology was studied in the different regions of the equines’ cornea. In addition, the polimegathism and morphology was also correlated. A statistical analysis was conducted using the Analysis of Variance (ANOVA) followed by Tukey's Test (Post-Hoc), with a 5% level of significance. In the central region, the endothelium consisted of 58.8% hexagonal cells, 22.6% pentagon, 17.1% heptagonal and 1.4% cells with either four, eight or nine sides. In the superior peripheral region, 62.1% of the cells were hexagonal, 20.2% pentagon, 16.6% heptagonal and 1.1% cells with four or eight sides. The average cell area of the pentagonal cells was 210.77 ± 31.53 μm² in the central region and 196.39 ± 34.35 μm² in the superior peripheral region; in hexagonal cells, the average cell area found in the central region was 216.12 ± 37.09 μm² and 231.56 ± 34.95 μm² in the superior peripheral region; and in cells with seven sides, the average cell area was 261.76 ± 55.29 μm² in the central region and in the superior peripheral region was found 279.32 ± 52.37μm². When not considering the corneal regions, the polymegathism found was 16% in the pentagonal and hexagonal cells and 20% in the heptagonal cells. The morphology results obtained did not differ between the central and peripheral regions of the cornea, suggesting that the central region is representative of the peripheral region. The highest coefficient of variation was seen in cells with seven sides.

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

As técnicas de remoção de catarata evoluíram nas últimas décadas. Na tentativa de oclusão da córnea após incisão para remoção da catarata, diversas técnicas têm sido propostas. Objetivou-se avaliar experimentalmente a viabilidade do emprego do adesivo de fibrina na oclusão da incisão de córnea em coelhos. Além disso, comparar os efeitos do adesivo de fibrina e do fio de sutura na oclusão da incisão de córnea em coelhos, utilizando-se os aspectos clínicos, a microscopia eletrônica de varredura e a morfometria. Dezesseis coelhos (Oryctolagus cuniculus) da raça Nova Zelândia foram submetidos à incisão de córnea bilateral. Para a oclusão da incisão utilizou-se aleatoriamente em um bulbo do olho, adesivo de fibrina e no seu contralateral, fio de sutura. Os períodos de avaliação foram de 7 e 15 dias. As repercussões dos procedimentos foram avaliadas utilizando-se exame oftálmico. Ao final dos períodos determinados, procedeu-se à avaliação da área perincisional desprovida de células endoteliais por meio da microscopia eletrônica de varredura da morfometria e a análise estatística inferencial foi obtida pelo teste t de Student para amostrar pareadas. Clinicamente, observaram-se melhores resultados nas amostras ocluídas com fio de sutura. No que se refere à área perincisional desprovida de células endoteliais, comparando-se os dois tipos de oclusão, a área das amostras seladas com fibrina apresentou-se maior que a área ocluída com fio de sutura. Neste estudo, ambas as técnicas foram eficazes na oclusão da córnea de coelhos. Porém, a avaliação valendo-se da microscopia eletrônica de varredura e da morfometria das eletromicrografias das áreas perincisionais do endotélio da córnea desprovidas de células ocluídas com fio de sutura demonstrou maior nível de significância quando comparada ao adesivo de fibrina. / The techniques for cataract removal had developed in the last decades. As an attempt to repair the cornea after incision, different techniques are proposed for corneal sealing. The objective of this study was to evaluate experimentally the viability of the use of fibrin adhesive in occlusion of the incision of the cornea in rabbits. Also compare clinically and by scanning electron microscopy and morphometry the fibrin adhesive and the suture on the sealing the cornea incision in rabbits. In this study, 16 rabbits (Oryctolagus cuniculus) New Zealand breed were used. It was performed bilateral corneal incision. In one eye the incision was sealed with suture, in the other eye, with fibrin adhesive randomly. The periods of evaluation varied from 7 to 15 days. The repercussions at the eye were studied using the ophthalmic exam. At the end of the determinate period, it was performed the evaluation of the perincisional area without endothelial cell by means of scanning electron microscopy, morphometry and the inferential statistical analysis was made by Student t test for paired samples. Comparing the two types of sealing, the perincisional average area without endothelial cells was higher in the fibrin tissue than wired in both groups. In this study, both techniques had corneal sealing, however, the evaluation by scanning electron microscopy,and electromicrographs morphometry of the perinsional areas of the corneal endothelium without devoid cells occluded with suture wire has higher level significance when compared with fibrin adhesive.

As técnicas de remoção de catarata evoluíram nas últimas décadas. Na tentativa de oclusão da córnea após incisão para remoção da catarata, diversas técnicas têm sido propostas. Objetivou-se avaliar experimentalmente a viabilidade do emprego do adesivo de fibrina na oclusão da incisão de córnea em coelhos. Além disso, comparar os efeitos do adesivo de fibrina e do fio de sutura na oclusão da incisão de córnea em coelhos, utilizando-se os aspectos clínicos, a microscopia eletrônica de varredura e a morfometria. Dezesseis coelhos (Oryctolagus cuniculus) da raça Nova Zelândia foram submetidos à incisão de córnea bilateral. Para a oclusão da incisão utilizou-se aleatoriamente em um bulbo do olho, adesivo de fibrina e no seu contralateral, fio de sutura. Os períodos de avaliação foram de 7 e 15 dias. As repercussões dos procedimentos foram avaliadas utilizando-se exame oftálmico. Ao final dos períodos determinados, procedeu-se à avaliação da área perincisional desprovida de células endoteliais por meio da microscopia eletrônica de varredura da morfometria e a análise estatística inferencial foi obtida pelo teste t de Student para amostrar pareadas. Clinicamente, observaram-se melhores resultados nas amostras ocluídas com fio de sutura. No que se refere à área perincisional desprovida de células endoteliais, comparando-se os dois tipos de oclusão, a área das amostras seladas com fibrina apresentou-se maior que a área ocluída com fio de sutura. Neste estudo, ambas as técnicas foram eficazes na oclusão da córnea de coelhos. Porém, a avaliação valendo-se da microscopia eletrônica de varredura e da morfometria das eletromicrografias das áreas perincisionais do endotélio da córnea desprovidas de células ocluídas com fio de sutura demonstrou maior nível de significância quando comparada ao adesivo de fibrina. / The techniques for cataract removal had developed in the last decades. As an attempt to repair the cornea after incision, different techniques are proposed for corneal sealing. The objective of this study was to evaluate experimentally the viability of the use of fibrin adhesive in occlusion of the incision of the cornea in rabbits. Also compare clinically and by scanning electron microscopy and morphometry the fibrin adhesive and the suture on the sealing the cornea incision in rabbits. In this study, 16 rabbits (Oryctolagus cuniculus) New Zealand breed were used. It was performed bilateral corneal incision. In one eye the incision was sealed with suture, in the other eye, with fibrin adhesive randomly. The periods of evaluation varied from 7 to 15 days. The repercussions at the eye were studied using the ophthalmic exam. At the end of the determinate period, it was performed the evaluation of the perincisional area without endothelial cell by means of scanning electron microscopy, morphometry and the inferential statistical analysis was made by Student t test for paired samples. Comparing the two types of sealing, the perincisional average area without endothelial cells was higher in the fibrin tissue than wired in both groups. In this study, both techniques had corneal sealing, however, the evaluation by scanning electron microscopy,and electromicrographs morphometry of the perinsional areas of the corneal endothelium without devoid cells occluded with suture wire has higher level significance when compared with fibrin adhesive.

As técnicas de remoção de catarata evoluíram nas últimas décadas. Na tentativa de oclusão da córnea após incisão para remoção da catarata, diversas técnicas têm sido propostas. Objetivou-se avaliar experimentalmente a viabilidade do emprego do adesivo de fibrina na oclusão da incisão de córnea em coelhos. Além disso, comparar os efeitos do adesivo de fibrina e do fio de sutura na oclusão da incisão de córnea em coelhos, utilizando-se os aspectos clínicos, a microscopia eletrônica de varredura e a morfometria. Dezesseis coelhos (Oryctolagus cuniculus) da raça Nova Zelândia foram submetidos à incisão de córnea bilateral. Para a oclusão da incisão utilizou-se aleatoriamente em um bulbo do olho, adesivo de fibrina e no seu contralateral, fio de sutura. Os períodos de avaliação foram de 7 e 15 dias. As repercussões dos procedimentos foram avaliadas utilizando-se exame oftálmico. Ao final dos períodos determinados, procedeu-se à avaliação da área perincisional desprovida de células endoteliais por meio da microscopia eletrônica de varredura da morfometria e a análise estatística inferencial foi obtida pelo teste t de Student para amostrar pareadas. Clinicamente, observaram-se melhores resultados nas amostras ocluídas com fio de sutura. No que se refere à área perincisional desprovida de células endoteliais, comparando-se os dois tipos de oclusão, a área das amostras seladas com fibrina apresentou-se maior que a área ocluída com fio de sutura. Neste estudo, ambas as técnicas foram eficazes na oclusão da córnea de coelhos. Porém, a avaliação valendo-se da microscopia eletrônica de varredura e da morfometria das eletromicrografias das áreas perincisionais do endotélio da córnea desprovidas de células ocluídas com fio de sutura demonstrou maior nível de significância quando comparada ao adesivo de fibrina. / The techniques for cataract removal had developed in the last decades. As an attempt to repair the cornea after incision, different techniques are proposed for corneal sealing. The objective of this study was to evaluate experimentally the viability of the use of fibrin adhesive in occlusion of the incision of the cornea in rabbits. Also compare clinically and by scanning electron microscopy and morphometry the fibrin adhesive and the suture on the sealing the cornea incision in rabbits. In this study, 16 rabbits (Oryctolagus cuniculus) New Zealand breed were used. It was performed bilateral corneal incision. In one eye the incision was sealed with suture, in the other eye, with fibrin adhesive randomly. The periods of evaluation varied from 7 to 15 days. The repercussions at the eye were studied using the ophthalmic exam. At the end of the determinate period, it was performed the evaluation of the perincisional area without endothelial cell by means of scanning electron microscopy, morphometry and the inferential statistical analysis was made by Student t test for paired samples. Comparing the two types of sealing, the perincisional average area without endothelial cells was higher in the fibrin tissue than wired in both groups. In this study, both techniques had corneal sealing, however, the evaluation by scanning electron microscopy,and electromicrographs morphometry of the perinsional areas of the corneal endothelium without devoid cells occluded with suture wire has higher level significance when compared with fibrin adhesive.

Le contrôle qualité réalisé sur les cornées en organoculture dans les banques de cornées est primordial pour l'évaluation de la qualité des greffons. Dans une première partie, nous avons montré, sur 505 cornées en routine, que les paramètres mesurés avec notre analyseur d'images Sambacornea (CV de taille et hexagonalité cellulaire endothéliale) étaient tout à fait cohérents avec ceux mesurés en microscopie spéculaire in vivo chez les patients. Notre dispositif fournit une méthode fiable, validée pour la mesure de la densité cellulaire endothéliale DCE et de la morphométrie cellulaire. Dans une deuxième partie, nous avons analysé les erreurs inhérentes à la méthode de comptage à cadre fixe (grille calibrée ou réticule) lors de la mesure de la DCE par les banques de cornées. Nous avons mesuré sur 3000 zones, la DCE de 20 mosaïques cornéennes gravées, en utilisant des grilles dont la maille variait de 50 à 300 µm. La méthode était répétable avec des grilles de 200 µm à condition de considérer la moyenne sur 10 comptages et présentait une variabilité résiduelle de 5 % vs la DCE réelle. Les grilles classiques de 100 µm utilisées en comptage manuel devraient être abandonnées et remplacées par des grilles de 200 µm. L'analyse d'images numériques demeure cependant la meilleure solution. Dans une troisième partie, le développement d'un dispositif de mesure de la transparence et des diamètres utiles en greffe de cornée (cornée claire et diamètre total) est décrit. La transparence est appréciée via la mesure de la fonction de modulation de transfert à travers la cornée sur une mire originale dessinée dans notre laboratoire. Les mesures étaient reproductibles. D’après l’analyse de 358 cornées en routine, la transparence n'était pas corrélée à l’âge du donneur, ni à la DCE, et baissait au fil de la conservation. Ce dispositif simple et objectif permet de standardiser la «transparence» ; il devra être redéployé sur notre bioréacteur, et pourrait mener à la définition d'un seuil de transparence / The corneal quality control (QC) in eye banks is essential for the assessment of the quality of grafts. In the first part, we showed on 505 consecutive corneas, that the parameters measured by our image analyzer Sambacornea (size CV and hexagonality of endothelial cells) were entirely consistent with those measured by specular microscopy in vivo in patients. Our device provides a reliable validated method for the measurement of the Endothelial Cell Density ECD and cell morphometry. In a second part, we analyzed the inherent errors of the fixed-frame counting method (calibrated grid or graticule) for corneal ECD in eye banks. We measured in 3000 areas, the 20 corneal mosaic ECD using grids ranged from 50 to 300 µm. The method was repeatable with 200 µm long grids if the average of 10 counts were used: it showed a residual variability of 5 % vs the real ECD. Conventional 100 µm long grids used in manual counting should be abandoned and replaced by 200 µm grids. The digital image analysis, however, is the best solution. In the third part, the development of a device for measuring the transparency and effective diameters in corneal transplantation (clear cornea and total diameter) is described. Transparency is assessed by measuring the modulation transfer function through the cornea on an original pattern designed in our laboratory. The measurements were reproducible. Based on the analysis of 358 corneas routine, transparency was not correlated with donor age, or the ECD, and decreased with duration of stockage. This simple and objective device standardizes the QC "transparency" and it should be used with our bioreactor, and could lead to the definition of a level for transparency

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Bei der Hornhauttransplantation (Keratoplastik) handelt es sich um die häufigste Transplantation humanen Gewebes. Das Hornhautendothel, eine empfindliche Zellschicht auf der Innenseite der Kornea, verfügt nicht über die Fähigkeit, Zellverluste durch Proliferation auszugleichen. Zwei grundsätzliche Probleme ergeben sich aus dieser Eigenschaft des kornealen Endothels für die Keratoplastik: ein Endothelzellverlust tritt zum einen während der Hornhautkonservierung vor Keratoplastik auf und führt zu einem Ausschluss vieler Transplantate, zum anderen ist er oftmals im Zuge von immunvermittelten Abstoßungsreaktionen oder in Folge chronischer Vorgänge nach Keratoplastik zu beobachten. Aus diesem Grund ist eine möglichst hohe Endothelzelldichte auf kornealen Transplantaten eine Voraussetzung für den Erfolg einer Keratoplastik. Das erste Ziel dieser Arbeit war deshalb die Übertragung des Gens für den aziden FGF (aFGF) in korneale Endothelzellen mit Hilfe des nicht-viralen Gentransfers. Dazu wurden verschiedene Lipid-Formulationen für den Gentransfer in humane korneale Endothelzellen in vitro optimiert. Der Einsatz von DAC-30 und Lipofectin für den aFGF-Gentransfer führte zu einer deutlichen Proliferationssteigerung (um ca. 50 %) der Zellen, womit der Einsatz dieses Wachstumsfaktors eine gute Möglichkeit darzustellen scheint, die prä-operative Ausgangssituation kornealer Transplantate zu verbessern. Der zweite Teil dieser Arbeit befasste sich mit Untersuchungen zur Immunmodulation nach Keratoplastik. Eine Hauptrolle bei der Abstoßung kornealer Transplantate spielen CD4+ T- Lymphozyten. In anderen Transplantationsmodellen konnte durch die lokale Überexpression immunmodulatorischer Zytokine die Entstehung und Aktivierung dieser Zellen gehemmt und eine verlängerte Transplantatüberlebenszeit erzielt werden. Mit Hilfe gentherapeutischer Vektoren (Adenoviren, Liposomen) wurden die immunmodulatorischen Zytokine vIL-10 und rIL-4 ex vivo in korneale Transplantate eingebracht. Nach erfolgreichen in vitro- Untersuchungen zur Genexpression wurden die transduzierten/transfizierten Hornhäute in einem starken Abstoßungsmodell der Ratte transplantiert, was jedoch zu keiner signifikanten Verlängerung der Transplantatüberlebenszeit führte. Diese Arbeit liefert Hinweise darauf, dass der lokale Gentransfer von vIL-10 in der Keratoplastik nicht für eine Immunmodulation geeignet ist, und dass sowohl die Zytokindosis, als auch der Zeitpunkt und der Ort der Vektorapplikation eine entscheidende Rolle für den Transplantationserfolg spielen. / Keratoplasty is the most common transplantation of human tissue. The corneal endothelium constitutes a damagable cell layer on the inner surface of the cornea, unable to proliferate. From this, two general problems arise for the outcome of keratoplasty: loss of corneal endothelial cells occurs on the one hand during corneal long time storage before keratoplasty and enforces the lack of donor tissue, on the other hand it is often correlated with immune mediated rejections as well as with chronic processes after keratoplasty. Therefore an endothelial cell number as high as possible on corneal grafts displays a requirement for successful keratoplasties. The first aim of this study was non-viral gene transfer of the aFGF (acidic FGF) gene in corneal endothelial cells. Different lipid formulations were optimized for gene transfer in human corneal endothelial cells in vitro. Application of DAC-30 and Lipofectin for aFGF gene transfer clearly showed a stimulating effect on cell proliferation (approximately 50 %). Thus, the use of aFGF seems to be a good possibility for improving the pre-operative situation of corneal allografts. The second part of this study deals with the immune modulation after keratoplasty. CD4+ T- lymphocytes play a key role in rejection processes after keratoplasty. Local over expression of immunomodulatory cytokines in different transplantation models could inhibit the development and activation of these cells and was able to prolong the allograft survival time. Using different gene therapeutic vectors (adenoviruses, liposomes) the immunomodulatory cytokines vIL-10 and rIL-4 were transferred ex vivo in corneal allografts. After successfully determined gene expression in vitro, the transduced/transfected corneal allografts were transplanted in a strong rejection model of the rat. However, this was not sufficient in prolonging the graft survival time significantly. This study provides indications, that local gene transfer of vIL-10 in keratoplasty is not suitable for an immune modulation, and that both cytokine dose as well as time point and site of vector application play an important role for successful transplantation.