The Scheimpflug principle was recommended as allowing distortion-free imaging; however, a detailed analysis reveals geometrical errors as well as distortions arising from absorption of light along the optical pathway. Correction formulas and factors will be presented and applied to the biometry of the eye. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Aberrations affect image quality of the eye away from the line of sight as well as along it. High amounts of lower order aberrations are found in the peripheral visual field and higher order aberrations change away from the centre of the visual field. Peripheral resolution is poorer than that in central vision, but peripheral vision is important for movement and detection tasks (for example driving) which are adversely affected by poor peripheral image quality. Any physiological process or intervention that affects axial image quality will affect peripheral image quality as well. The aim of this study was to investigate the effects of accommodation, myopia, age, and refractive interventions of orthokeratology, laser in situ keratomileusis and intraocular lens implantation on the peripheral aberrations of the eye. This is the first systematic investigation of peripheral aberrations in a variety of subject groups. Peripheral aberrations can be measured either by rotating a measuring instrument relative to the eye or rotating the eye relative to the instrument. I used the latter as it is much easier to do. To rule out effects of eye rotation on peripheral aberrations, I investigated the effects of eye rotation on axial and peripheral cycloplegic refraction using an open field autorefractor. For axial refraction, the subjects fixated at a target straight ahead, while their heads were rotated by ±30º with a compensatory eye rotation to view the target. For peripheral refraction, the subjects rotated their eyes to fixate on targets out to ±34° along the horizontal visual field, followed by measurements in which they rotated their heads such that the eyes stayed in the primary position relative to the head while fixating at the peripheral targets. Oblique viewing did not affect axial or peripheral refraction. Therefore it is not critical, within the range of viewing angles studied, if axial and peripheral refractions are measured with rotation of the eye relative to the instrument or rotation of the instrument relative to the eye. Peripheral aberrations were measured using a commercial Hartmann-Shack aberrometer. A number of hardware and software changes were made. The 1.4 mm range limiting aperture was replaced by a larger aperture (2.5 mm) to ensure all the light from peripheral parts of the pupil reached the instrument detector even when aberrations were high such as those occur in peripheral vision. The power of the super luminescent diode source was increased to improve detection of spots passing through the peripheral pupil. A beam splitter was placed between the subjects and the aberrometer, through which they viewed an array of targets on a wall or projected on a screen in a 6 row x 7 column matrix of points covering a visual field of 42 x 32. In peripheral vision, the pupil of the eye appears elliptical rather than circular; data were analysed off-line using custom software to determine peripheral aberrations. All analyses in the study were conducted for 5.0 mm pupils. Influence of accommodation on peripheral aberrations was investigated in young emmetropic subjects by presenting fixation targets at 25 cm and 3 m (4.0 D and 0.3 D accommodative demands, respectively). Increase in accommodation did not affect the patterns of any aberrations across the field, but there was overall negative shift in spherical aberration across the visual field of 0.10 ± 0.01m. Subsequent studies were conducted with the targets at a 1.2 m distance. Young emmetropes, young myopes and older emmetropes exhibited similar patterns of astigmatism and coma across the visual field. However, the rate of change of coma across the field was higher in young myopes than young emmetropes and was highest in older emmetropes amongst the three groups. Spherical aberration showed an overall decrease in myopes and increase in older emmetropes across the field, as compared to young emmetropes. Orthokeratology, spherical IOL implantation and LASIK altered peripheral higher order aberrations considerably, especially spherical aberration. Spherical IOL implantation resulted in an overall increase in spherical aberration across the field. Orthokeratology and LASIK reversed the direction of change in coma across the field. Orthokeratology corrected peripheral relative hypermetropia through correcting myopia in the central visual field. Theoretical ray tracing demonstrated that changes in aberrations due to orthokeratology and LASIK can be explained by the induced changes in radius of curvature and asphericity of the cornea. This investigation has shown that peripheral aberrations can be measured with reasonable accuracy with eye rotation relative to the instrument. Peripheral aberrations are affected by accommodation, myopia, age, orthokeratology, spherical intraocular lens implantation and laser in situ keratomileusis. These factors affect the magnitudes and patterns of most aberrations considerably (especially coma and spherical aberration) across the studied visual field. The changes in aberrations across the field may influence peripheral detection and motion perception. However, further research is required to investigate how the changes in aberrations influence peripheral detection and motion perception and consequently peripheral vision task performance.

Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.

Orientador: José Luiz Laus / Banca: Aline Adriana Bolzan / Banca: Cristiane dos Santos Honsho / Banca: Alexandre Lima de Andrade / Banca: Paola Castro Moraes / Resumo: Estudaram-se os efeitos da morfina 1% sobre a reparação corneal, avaliando-se a expressão das metaloproteinases-1, -2, -9, do colágeno tipo IV e da interleucina 10 (IL-10), em coelhos submetidos à ceratectomia lamelar. Dois experimentos foram concebidos, empregando-se 56 animais. No primeiro, 6 coelhos foram tratados com 50μl de morfina 1% (GM), a cada 4 horas, totalizando 4 aplicações diárias. Outros 6 coelhos receberam solução fisiológica (GC) nas mesmas condições adotadas para o GM. Após as ceratectomias, as córneas foram avaliadas até sua epitelização. No segundo, 40 animais foram empregados e as córneas colhidas decorridos 1, 3, 6, 9 e 12 dias das ceratectomias para histologia, imunoistoquímica (MMP-1, MMP-9 e colágeno tipo IV), zimografia (MMP-2 e MMP-9) e ELISA (IL-10). Mais 4 coelhos saudáveis foram utilizados para como controle negativo.O tempo médio de reetitelização não diferiu entre os grupos. Após a ceratectomia, observou-se elevação significativa quanto ao limiar de sensibilidade corneal da 6ª até 96a hora da avaliação. Não se observaram alterações histológicas e quanto ao índice de imunomarcação da MMP-1, -9 e do colágeno do tipo IV entre os grupos estudados . À zimografia, níveis mais elevados de MMP-2 e de MMP-9 foram observados no GM, ao 6º e ao 9º dias após as ceratectomias. Observou-se decréssimo das formas latente e ativa da MMP-9 ao 12º dia. Observou-se, ao 1º dia de avaliação, redução nos níveis de IL-10, comparativamente às córneas saudáveis, mas sem significância estatística entre os grupos, em qualquer dos períodos estudados. O uso local de morfina 1% promoveu analgesia corneal de 4 dias em coelhos submetidos à ceratectomia lamelar e não retardou a epitelização. Elevação do quantitativo da metaloproteinase-1, -2 e -9, em córneas tratadas com morfina 1%, não contraindica seu uso... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed evaluate the effects of continual topical administration of 1% morphine on corneal analgesia in rabbits subjected to lamellar keratectomy until completion of the corneal wound healing and to assess the expression of matrix metalloproteinases (MMPs)-1, -2 and -9, type IV collagen and interleukin-10 (IL-10) during the treatment. Fifty-six animals were used. In the first experiment, 12 rabbits were divided into two groups, one (n=6) received 50 μl of topical 1% morphine (MG) every 4 hours while the other group (n=6) received 0.9% NaCl instead (CG). After keratectomies, corneas were evaluated until healing. In the second experiment, 40 animals were divided, 4 rabbits of each group were euthanized 1, 3, 6, 9, and 12 days after keratectomy and corneal samples were processed for histology, immunohistochemistry (MMP- 1, -9 and IV collagen), zymography (MMP-2 and -9) and ELISA (IL-10). Other 4 healthy rabbits were used as negative control. In healthy corneas, CTT did not differ significantly before and after morphine instillation. Mean corneal reepithelization rate did not differ between groups. Following keratectomy, CTT increased from the 6h to 96h time points. Scores of leukocyte infiltration, MMP-1, MMP-9 and type IV collagen expression did not differ between groups at any time point. Zymography indicated that levels of the active forms of MMP-2 and MMP-9 increased on days 6 and 9 in the MG. The levels of both latent and active forms of MMP-9, as well as of the latent form of MMP-2 decreased to values close to those of healthy corneas on day 12. IL-10 levels measured on days 1 to 6 were reduced as compared to those of healthy corneal tissue and returned to levels close to those of healthy corneas. Topical application of 1% morphine promoted corneal analgesia for up to 4 days and did not delay corneal reepithelization... (Complete abstract click electronic access below) / Doutor

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.

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.

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.

INTRODUÇÃO: O endotélio corneal exerce papel primordial para a fisiologia da córnea. Seus dados morfológicos gerados pelo microscópio especular (MEC) como densidade endotelial (DE), área celular média (ACM), coeficiente de variação (CV) e porcentagem de células hexagonais (HEX) são importantes para avaliar sua vitalidade. Para interpretar estes dados de forma padronizada e reprodutível, foi utilizado um programa estatístico de análise amostral, Cells Analyzer PAT. REQ.(CA). OBJETIVO: Demonstrar valores de referência para DE, ACM, CV e HEX. Demonstrar o percentual de células endoteliais marcadas e desconsideradas no exame ao marcar-se 40, 100 e 150 células em uma única imagem do mosaico endotelial e o perfil do intervalo de confiança (IC) das variáveis estudadas ao se considerar 40, 100, 150 e tantas células quantas indicadas pelo CA. Demonstrar o erro amostral de cada grupo estudado. MÉTODOS: Estudo transversal. Os exames de MEC foram realizados com o aparelho Konan NONCON ROBO® SP-8000, nos 122 olhos de 61 portadores de catarata (63,97 ± 8,15 anos de idade). As imagens endoteliais caracterizaram se pelo número de células marcadas e consideradas para cálculo dos seguintes dados: DE, ACM, CV e HEX. Os grupos foram formados de 40, 100, 150 células marcadas numa única imagem endotelial e Grupo CA em que foram marcadas tantas células quanto necessárias em diferentes imagens, para obter o erro relativo calculado inferior ao planejado (0,05), conforme orientação do programa CA. Estudou-se o efeito do número de células sobre IC para as variáveis endoteliais utilizadas. RESULTADOS: A média dos valores de referência encontrados para DE foi 2395,37 ± 294,34 cel/mm2; ACM 423,64 ± 51,09 m2; CV 0,40 ± 0,04 e HEX 54,77 ± 4,19%. O percentual de células endoteliais desconsideradas no Grupo 40 foi 51,20%; no Grupo 100, 35,07% e no Grupo 150, 29,83%. O número médio de células calculado inicialmente pelo CA foi 247,48 ± 51,61 e o número médio de células efetivamente incluídas no final do processo amostral foi 425,25 ± 102,24. O erro amostral dos exames no Grupo 40 foi 0,157 ± 0,031; Grupo 100, 0,093 ± 0,024; Grupo 150, 0,075 ± 0,010 e Grupo CA, 0,037 ± 0,005. O aumento do número de células diminuiu a amplitude do IC nos olhos direito e esquerdo para a DE em 75,79% e 77,39%; ACM em 75,95% e 77,37%; CV em 72,72% e 76,92%; HEX em 75,93% e 76,71%. CONCLUSÃO: Os valores de referência da DE foi 2395,37 ± 294,34 cel/mm2; ACM foi 423,64 ± 51,09 m2; CV foi 0,40 ± 0,04 e HEX foi 54,77 ± 4,19%. O percentual de células endoteliais desconsideradas no Grupo 40 foi 51,20%; no Grupo 100 foi 35,07% e no Grupo 150 foi 29,83%. O programa CA considerou correto os exames nos quais 425,25 ± 102,24 células foram marcadas entre duas e cinco imagens (erro relativo calculado de 0,037 ± 0,005). O aumento do número de células diminuiu a amplitude do IC para todas as variáveis endoteliais avaliadas pela MEC / INTRODUCTION: Corneal endothelium plays an important role in physiology of the cornea. Morphological data generated from specular microscope such as endothelial cell density (CD), average cell area (ACA), coefficient of variance (CV) and percentage of hexagonal cells (HEX) are important to analyze corneal status. For a standard and reproducible analysis of the morphological data, a sampling statistical software called Cells Analyzer PAT. REC (CA) was used. PURPOSE: To determine normal reference values of CD, ACA, CV and HEX. To analyze the percentage of marked and excluded cells when the examiner counted 40, 100, 150 cells in one endothelial image. To analyze the percentage of marked and excluded cells according to the statistical software. To determine the confidence interval of these morphological data. METHODS: Transversal study of 122 endothelial specular microscope image (Konan, non-contact NONCON ROBO® SP- 8000 Specular Microscope) of 61 human individuals with cataract (63.97 ± 8.15 years old) was analyzed statistically using CA. Each image was submitted to standard cell counting. 40 cells were counted in study Group 40; 100 cells were counted in study Group 100; and 150 cells were counted in study Group 150. In study group CA, the number of counted cells was determined by the statistical analysis software in order to achieve the most reliable clinical information (relative error < 0,05). Relative error of the morphological data generated by the specular microscope were then analyzed by statistical analysis using CA software. For Group CA, relative planned error was set as 0.05. RESULTS: The average normal reference value of CD was 2395.37 ± 294.34 cells/mm2, ACA was 423.64 ± 51.09 m2, CV was 0.40 ± 0.04 and HEX was 54.77 ± 4.19%. The percentage of cells excluded for analysis was 51.20% in Group 40; 35.07% in Group 100; and 29.83% in Group 150. The average number of cells calculated initially by the statistical software was 247.48 ± 51.61 cells and the average number of cells included in the final sampling process was 425.25 ± 102.24 cells. The average relative error was 0.157 ± 0.031 for Group 40; 0.093 ± 0.024 for Group 100; 0.075 ± 0.010 for Group 150 and 0.037 ± 0.005 for Group CA. The increase of the marked cells decreases the amplitude of confidence interval (right and left eyes respectively) in 75.79% and 77.39% for CD; 75.95% and 77.37% for ACA; 72.72% and 76.92% for CV; 75.93% and 76.71% for HEX. CONCLUSION: The average normal reference value of CD was 2395.37 ± 294.34 cells/mm2, ACA was 423.64 ± 51.09 m2, CV was 0.40 ± 0.04 and HEX was 54.77 ± 4.19%. The percentage of excluded cells for analysis was 51.20% in Group 40; 35.07% in Group 100 and 29.83% in Group 150. CA software has considered reliable data when 425.25 ± 102.24 cells were marked by the examiner in two to five specular images (calculated relative error of 0.037 ± 0.005). The increase of the marked cells decreases the amplitude of confidence interval for all morphological data generated by the specular microscope