The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient’s healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker ..- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP ... These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP .. in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

Thesis (Ph. D. in Biomedical Engineering)--Vanderbilt University, Dec. 2004. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2004. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed Feb. 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 142-169). Online version of the print edition.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 117-136.

Wound healing in the corneal epithelium is an essential process to maintain corneal clarity and organism health. The earliest events of cellular injury response include the release of nucleotides and the activation of P2 purinergic receptors. While the purinergic receptor P2X7 has been shown to promote cell migration, its role in corneal epithelial wound healing is still poorly understood. The goal of this work is to better understand the role of P2X7 in the injury response. We analyzed P2X7 expression after epithelial injury in rat corneal organ cultures and found that the receptor localizes to the leading edge of the corneal epithelium. However, overall mRNA and protein expression of P2X7 decreased after injury. Inhibition of P2X7 activation significantly delayed wound closure and prevented the leading edge-localization after injury. We found that P2X7 inhibition altered the wound-induced calcium wave in epithelial cells and altered the number and distribution of focal adhesions in the migrating cells. Live cell imaging of epithelial cells showed that P2X7 inhibition led to altered actin rearrangement, with thick actin bundles in the treated cells. In order to determine the importance of P2X7 in epithelial differentiation and stratified cell migration, we developed a stratified culture model. The cells in the stratified model expressed proliferative and differentiation markers similar to organ cultured corneas, as well as similar P2X7 expression and localization after injury. Together, these results show the importance of P2X7 in the overall purinergic response to injury, and provide tools to study P2X7 in stratified corneal cell migration. To determine if P2X7 may contribute to pathologic delayed wound healing in diseases such as type 2 diabetes, we analyzed P2X7 expression in diabetic human corneas and diabetic model rodent corneas. We showed that P2X7 expression is significantly elevated in unwounded diabetic corneas, and that wound healing is delayed in the rodent model. These data show that elevated P2X7 expression may contribute to the delayed healing in disease and may be a possible therapeutic target.

Orientador: José Luiz Laus / Coorientador: Marcela Aldrovani / Banca: Karin Werther / Banca: Paola Castro Moraes / Banca: Claudia Valéria Seullner Brandão / Banca: Gabriela Rodrigues Sampaio / Resumo: Cachorros-do-mato (Cerdocyon thous) figuram entre os animais selvagens mais assistidos em centros de reabilitação, tornando imperioso ampliarem-se conhecimentos sobre parâmetros morfofisiológicos próprios à espécie. Na presente pesquisa, avaliaram-se as condições oftálmicas de 16 cachorros do mato (n=32 olhos). No estudo clínico, avaliaram-se oito indivíduos, sob contenção química, à teste lacrimal de Schirmer I (TLS I), à tonometria digital de aplanação (PIO), à microscopia especular de não contato (espessura corneal, densidade, área e hexagonalidade celulares endoteliais corneais), à ultrassonografia em modos A e B (biometria, notadamente o diâmetro axial, a profundidade das câmaras anterior e vítrea, e a espessura da lente) e à eletrorretinografia (amplitudes e tempos implícitos de ondas a e b, nas fases escotóptica e fotóptica). Córneas, colhidas post mortem, de outros oito animais, foram avaliadas à histologia, quanto às espessuras da córnea e do epitélio, e aos parâmetros de birrefringência (que informam sobre agregação e orientação) de fibras lamelares colágenas estromais, empregando-se morfometria, luz polarizada e "software" de análise de imagens. Parâmetros de birrefringência foram quantificados como níveis de cinza médio (GA). Em relação ao TLS I e a PIO, verificaram-se valores médios de 13,37±0,94 mm e 10,43±0,96 mmHg, respectivamente. A espessura corneal média foi de 0,53±0,01mm. Relativamente às células endoteliais corneais, os valores médios de densidade e de área foram, respectivamente, de 2.850,38±137,28 cell/mm2 e de 389,00±0,17 μm2. O valor médio de hexagonalidade foi 60,06±4,66 %. Em relação à biometria, verificaram-se valores de 15,93±0,28 mm para diâmetro axial, de 3,29±0,14 mm para profundidade de câmara anterior, de 6,02±0,11 mm para espessura da lente e de 6,61±0,21 mm para profundidade da câmara vítrea. À eletrorretinografia, na fase... / Abstract: Crab-eating foxes (Cerdocyon thous) are one of the wild animals most attended in rehabilitation centers, making it imperative to extend current knowledge of morphophysiological parameters specific to the species. This study assessed the ophthalmic conditions of 16 crab-eating foxes (n=32 eyes). Eight individuals were clinically assessed, under chemical restraint, by the Schirmer tear test I (STT I), digital applanation tonometry (IOP), non-contact specular microscopy (corneal thickness, density, area and corneal endothelial cell hexagonality), ultrasound in A and B modes (biometrics, particularly axial diameter, anterior and vitreous chamber depths and lens thickness) and electroretinography (amplitudes and implicit times of a- and b-waves, in scotopic and photopic phases). Corneas, collected post mortem from another eight individuals, were evaluated histologically to determine cornea and epithelium thickness and the birefringence parameters (data regarding aggregation and orientation) of lamellar collagenous stromal fibers, using morphometry, polarized light and image analysis software. Birefringence parameters were quantified as gray average (GA) values. Regarding the STT I and IOP, mean values of 13.37±0.94 mm and 10.43±0.96 mmHg were respectively verified. The mean corneal thickness was 0.53±0.01 mm. The mean density and area values of corneal endothelial cells were respectively 2850.38±137.28 cell/mm2 and 389.00±0.17 μm2. The mean value for hexagonality was 60.06±4.66 %. Concerning biometrics, the following mean values were verified: axial diameter, 15.93±0.28 mm; anterior chamber depth, 3.29±0.14 mm; vitreous chamber depth, 6.61±0.21 mm; and lens thickness, 6.02±0.11 mm. Electroretinography in the low amplitude scotopic phase showed values for mean amplitude and b-wave implicit time (20 min adaptation) of 87.33±5.36 μV and 73.10±1.12 ms, respectively. In the high amplitude scotopic phase, the mean amplitude value ... / Doutor

La cornée est le hublot transparent de l’oeil. Bien que de nombreux modèles alternatifs utilisant des cornées animales ex vivo aient vu le jour durant ces 30 dernières années, les recherches préclinique (étude de nouvelles stratégies diagnostiques et thérapeutiques) et fondamentale sur la cornée ont toujours besoin de l’expérimentation animale in vivo. Elle fait aujourd’hui l’objet d’une réglementation stricte afin d’éviter tout abus et maltraitance. Les animaux les plus fréquemment utilisés en recherche cornéenne sont des mammifères (souris, rat, lapin, chat, chien, cochon, boeuf et primate non humain). Malgré leur proximité phylogénétique de l’Homme, ces animaux peuvent présenter des différences notables avec la cornée humaines qui doivent être connues pour ne pas induire de biais dans l’expérimentation. Les objectifs de cette thèse sont de mettre au point les modèles animaux et les méthodes alternatives nécessaires aux travaux du laboratoire BiiGC (EA 2521, Université de Saint-Étienne, France). Ils sont illustrés par 3 projets innovants : 1/une étude préclinique utilisant un modèle de kératoplastie transfixiante chez le lapin pour évaluer la prévention du rejet d’allogreffe de cornée par implant sous conjonctival de déxamethasone ; 2/Le développement d’un bioréacteur cornéen porcin pour l’analyse de la cicatrisation épithéliale ; 3/ l’utilisation d’un modèle lapin de lésion endothéliale pour l’étude de la régénération endothéliale. Ces 3 travaux innovant démontrent la diversité des modèles animaux nécessaires en recherche fondamentale et translationnelle. / The cornea is the clear window of the eye. Although many alternative models using ex vivo animal corneas have emerged during the last 30 years, preclinical research (study of new diagnostic and therapeutic strategies) and fundamental corneal research still need animal experiments in vivo. The most commonly used animals in corneal research are mammals (mouse, rat, rabbit, cat, dog, pig, beef and non-human primate). Despite their phylogenetic proximity to humans, these animals may exhibit notable differences with the human cornea, which must be known so as not to induce bias into the experiment. The aims of this thesis are to develop the animal models and the alternative models necessary for the work of the BiiGC laboratory (EA2521, University of Saint-Etienne, France). They illustrated by 3 innovative projects: 1/ a preclinical study using penetrating keratoplasty model in rabbits to evaluate the prevention of corneal allografts rejection by a conjunctival implant of dexamethasone; 2/ The development of a porcine corneal bioreactor for the analysis of epithelial wound healing; 3/ The use of rabbit endothelial lesion model for the study of endothelial regeneration. These 3 innovative works demonstrate the diversity of animal models needed in fundamental and translational research

O endotélio é uma monocamada de células achatadas, poligonais e interligadas que recobrem a superfície posterior da córnea, sendo fundamental na manutenção da transparência desta estrutura. Embora as chinchilas (Chinchilla lanigera) sejam utilizadas em pesquisas oftálmicas, não existem dados referentes ao endotélio da córnea nesta espécie. Portanto, objetivou-se avaliar a densidade celular, a paquimetria, a área celular média e a morfologia do endotélio da córnea de chinchilas em diferentes faixas etárias. Foram estudados 60 bulbos oculares de 30 chinchilas, machos ou fêmeas, de diferentes faixas etárias. As chinchilas foram subdivididas em três grupos com 10 animais cada, designados por: G I (animais com idade entre dois a quatro meses); G II (animais com 11 meses de idade) e G III (animais com quatro anos de idade). A densidade celular endotelial média nos animais dos grupos G I, G II e G III foi de respectivamente 3385,39 ± 507,53, 2565,51 ± 220,46 e 1812,82 ± 117,92 células/mm2. A área celular média foi de respectivamente 301,51 ± 43,94, 392,46 ± 33,34 e de 553,79 ± 35,70 μm2 para G I, G II e G III. Os resultados apontam decréscimo da densidade, do pleomorfismo, e aumento da área celular média com o aumento da idade. A espessura da córnea aumentou em animais mais velhos. Conclui-se que o endotélio da córnea de chinchilas sofre alterações com o avanço da idade. / The corneal endothelium is a monolayer of interconnected flattened polygonal cells that cover the back surface of the cornea, it is essential for the maintenance of its transparency. Due to the complete lack of data on the endothelial cells of chinchilla (Chinchilla lanigera) and its possible use as an animal model in ophthalmic research, the objective was to evaluate the cellular density, pachymetry, the average cell area and morphology of the corneal endothelium of chinchillas in different age groups. It was studied 60 eyes of 30 chinchillas, male or female, of different ages. Chinchillas were divided into three groups with 10 animals each, designated by: G I (animals aged two to four months), G II (animals with 11 months) and G III (animals with four years of age). The mean endothelial cell density in the animals of G I, G II and G III were respectively 3385,39 ± 507,53, 2565,51 ± 220,46 and 1812,82 ± 117,92 cells/mm2. The average cell area was respectively 301,51 ± 43,94, 392,46 ± 33,34 and 553,79 ± 35,70 μm2 for GI, G II and G III. The results indicate a decrease in density, pleomorphism, and increased in average cell area with increasing age. Corneal thickness increased in older animals. The corneal endothelium of chinchillas changes with advancing age.

Made available in DSpace on 2016-04-01T17:55:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-11-19. Added 1 bitstream(s) on 2016-04-01T18:00:54Z : No. of bitstreams: 1 000860264.pdf: 11975335 bytes, checksum: 933d99aa32827700877c6ba0ecab0dd4 (MD5) / Cachorros-do-mato (Cerdocyon thous) figuram entre os animais selvagens mais assistidos em centros de reabilitação, tornando imperioso ampliarem-se conhecimentos sobre parâmetros morfofisiológicos próprios à espécie. Na presente pesquisa, avaliaram-se as condições oftálmicas de 16 cachorros do mato (n=32 olhos). No estudo clínico, avaliaram-se oito indivíduos, sob contenção química, à teste lacrimal de Schirmer I (TLS I), à tonometria digital de aplanação (PIO), à microscopia especular de não contato (espessura corneal, densidade, área e hexagonalidade celulares endoteliais corneais), à ultrassonografia em modos A e B (biometria, notadamente o diâmetro axial, a profundidade das câmaras anterior e vítrea, e a espessura da lente) e à eletrorretinografia (amplitudes e tempos implícitos de ondas a e b, nas fases escotóptica e fotóptica). Córneas, colhidas post mortem, de outros oito animais, foram avaliadas à histologia, quanto às espessuras da córnea e do epitélio, e aos parâmetros de birrefringência (que informam sobre agregação e orientação) de fibras lamelares colágenas estromais, empregando-se morfometria, luz polarizada e software de análise de imagens. Parâmetros de birrefringência foram quantificados como níveis de cinza médio (GA). Em relação ao TLS I e a PIO, verificaram-se valores médios de 13,37±0,94 mm e 10,43±0,96 mmHg, respectivamente. A espessura corneal média foi de 0,53±0,01mm. Relativamente às células endoteliais corneais, os valores médios de densidade e de área foram, respectivamente, de 2.850,38±137,28 cell/mm2 e de 389,00±0,17 μm2. O valor médio de hexagonalidade foi 60,06±4,66 %. Em relação à biometria, verificaram-se valores de 15,93±0,28 mm para diâmetro axial, de 3,29±0,14 mm para profundidade de câmara anterior, de 6,02±0,11 mm para espessura da lente e de 6,61±0,21 mm para profundidade da câmara vítrea. À eletrorretinografia, na fase... / Crab-eating foxes (Cerdocyon thous) are one of the wild animals most attended in rehabilitation centers, making it imperative to extend current knowledge of morphophysiological parameters specific to the species. This study assessed the ophthalmic conditions of 16 crab-eating foxes (n=32 eyes). Eight individuals were clinically assessed, under chemical restraint, by the Schirmer tear test I (STT I), digital applanation tonometry (IOP), non-contact specular microscopy (corneal thickness, density, area and corneal endothelial cell hexagonality), ultrasound in A and B modes (biometrics, particularly axial diameter, anterior and vitreous chamber depths and lens thickness) and electroretinography (amplitudes and implicit times of a- and b-waves, in scotopic and photopic phases). Corneas, collected post mortem from another eight individuals, were evaluated histologically to determine cornea and epithelium thickness and the birefringence parameters (data regarding aggregation and orientation) of lamellar collagenous stromal fibers, using morphometry, polarized light and image analysis software. Birefringence parameters were quantified as gray average (GA) values. Regarding the STT I and IOP, mean values of 13.37±0.94 mm and 10.43±0.96 mmHg were respectively verified. The mean corneal thickness was 0.53±0.01 mm. The mean density and area values of corneal endothelial cells were respectively 2850.38±137.28 cell/mm2 and 389.00±0.17 μm2. The mean value for hexagonality was 60.06±4.66 %. Concerning biometrics, the following mean values were verified: axial diameter, 15.93±0.28 mm; anterior chamber depth, 3.29±0.14 mm; vitreous chamber depth, 6.61±0.21 mm; and lens thickness, 6.02±0.11 mm. Electroretinography in the low amplitude scotopic phase showed values for mean amplitude and b-wave implicit time (20 min adaptation) of 87.33±5.36 μV and 73.10±1.12 ms, respectively. In the high amplitude scotopic phase, the mean amplitude value ...

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Keratoconjunctivitis sicca (kCS) is a chronic inflammatory disease common in dogs, resulting from deficiency of the precorneal tear film. It can occur as a quantitative deficiency of the aqueous component of the tear, as well as a qualitative deficiency of the lipid or mucin components of the tear film, generating instability of that film. The ocular complications of this disease affect the ocular surface provoking vascularization, pigmentation and corneal edema. In more severe cases, it can also lead to loss of corneal epithelium, ocular pain and blindness. The treatment normally used for KCS is medicated and mainly includes tear, lacrimomimetics, mucolytics and topical antibacterials. Considering the importance of keratoconjunctivitis sicca, the objective of this study was to investigate and compare the treatment with different eye drops in healthy patients with KCS. For the study, a literature review on the KCS was carried out, addressing the main treatments (article 1), 13 dogs divided into two studies were used for the second study. The animals were divided into three groups of three animals each: in the CsA col group the treatment of the cornea was performed with cyclosporin A (0,05%) eye drops, Tacro group with tacrolimus eye drops (0,03%) and the CsA pom group with cyclosporin A ointment (0,05%). In the second study (article 3), the dogs were divided into two groups of two animals each, in the CsA col group collagen treatment was performed with cyclosporin A eye drops (0,05%) and the Tacro group with tacrolimus eye drops (0,03%). A drop of the medication was instilled every 12 hours in all animals and ophthalmic evaluations were performed on days 0, 15, 30, 45, 60, 75 and 90 days. Clinical signs of conjunctival hyperemia, type of present ocular secretion, corneal opacity, corneal vascularization and corneal pigmentation in healthy dogs and with KCS were evaluated through Schirmer's Lacrimal Test (TLS), Fluorescein Test, Rose Bengal Test. Based on the literature review on KCS, the best treatment described is tacrolimus eye drops. Among the surgical techniques, nasolacrimal duct occlusion was the best outcome. When analyzed the behavior of cyclosporin A in the formulation eye drops and ointment and tacrolimus in healthy animals, cyclosporin A in the ointment formulation was the one with the best lachrymal stimulation. In patients with CCS, when compared to treatments with CsA eye drops and tacrolimus, the dogs with tacrolimus were the most successful. / A ceratoconjuntivite seca (CCS) é uma doença inflamatória crônica comum em cães, resultante de deficiência do filme lacrimal pré-corneal. Pode ocorrer como uma deficiência quantitativa do componente aquoso da lágrima, bem como uma deficiência qualitativa dos componentes lipídico ou mucínico do filme lacrimal, gerando instabilidade desse filme. As complicações oculares dessa doença afetam a superfície ocular provocando vascularização, pigmentação e edema corneal. Em casos mais graves, pode ainda cursar com perda do epitélio da córnea, dor ocular e cegueira. O tratamento normalmente utilizado para CCS é medicamentoso e inclui principalmente lacrimogênicos, lacrimomiméticos, mucolíticos e antibacterianos tópicos. Considerando-se a importância da ceratoconjuntivite seca, o objetivo deste estudo foi investigar e comparar o tratamento com diferentes colírios em pacientes hígidos e com CCS. Para o estudo realizou-se inicialmente uma revisão bibliográfica sobre a CCS abordando os principais tratamentos (artigo 1), para a segunda pesquisa foram utilizados 13 cães divididos em dois trabalhos. O primeiro trabalho (artigo 2) os animais foram divididos em três grupos de três animais cada: no grupo CsA col o tratamento da córnea foi realizado com colírio de ciclosporina A (0,05%), o grupo Tacro com colírio tacrolimus (0,03%) e o grupo CsA pom com pomada de ciclosporina A (0,05%). No segundo trabalho (artigo 3), os cães foram divididos em dois grupos de dois animais cada, no grupo CsA col o tratamento da córnea foi realizado com colírio de ciclosporina A (0,05%) e o grupo Tacro com colírio tacrolimus (0,03%). Em todos os animais foi instilado uma gota da medicação a cada 12 horas e foram realizadas avaliações oftálmicas nos dias 0, 15, 30, 45, 60, 75 e 90 dias. As avaliações ocorreram através do Teste Lacrimal de Schirmer (TLS), Prova de Fluoresceína, Teste Rosa de Bengala, além dos sinais clínicos de hiperemia conjuntival, tipo de secreção ocular presente, opacidade corneal, vascularização corneal e pigmentação corneal em cães hígidos e com CCS. Com base na revisão bibliográfica sobre CCS, o melhor tratamento descrito é o colírio de tacrolimus, entre as técnicas cirúrgicas a que obteve melhor resultado foi a oclusão do ducto nasolacrimal. Quando analisado o comportamento da ciclosporina A na formulação colírio e pomada e o tacrolimus em animais hígidos, a que apresentou melhor estimulação lacrimal foi a ciclosporina A na formulação de pomada. Em paciente com CCS, quando comparados os tratamentos com CsA colírio e o tacrolimus, os que obtiveram melhor resultado foram os cães tratados com tacrolimus.