Exenteração e confecção de retalho de padrão axial da artéria temporal superficial para tratamento de carcinoma espinocelular periocular em um felino / Exenteration and axial pattern flap of the superficial temporal artery for the treatment of periocular squamous cell carcinoma in a feline

O carcinoma espinocelular é uma neoplasia maligna dos ceratinócitos que tem como principal causa a exposição a raios ultravioletas. Os felinos constituem a espécie doméstica mais susceptível para o aparecimento desta neoplasia, sendo a face a região mais comum de acometimento, incluindo áreas do plano nasal, pina e pálpebras. No presente trabalho relata-se um felino, sem raça definida, fêmea, 12 anos de idade, 3kg, castrada, de pelagem branca, que foi atendida apresentando lesão extensa de caráter ulcerativo em região periocular de globo ocular esquerdo, com evolução de aproximadamente três meses, envolvendo ambas as pálpebras, superior e inferior. Após realização de avaliação clínica, os achados obtidos por meio da anamnese juntamente com o exame citológico, sugeriram como principal diagnóstico o carcinoma espinocelular. Para tratamento foi preconizado remoção cirúrgica, na qual demonstrou boa efetividade para tratamento dessa afecção. Após, o material foi enviado para análise histopatológica, sendo confirmado o diagnóstico. O paciente apresentou boa recuperação e evolução clínica do quadro, não apresentando nenhuma complicação no decorrer do tratamento. O prognóstico para este caso foi considerado bom, uma vez que não havia indícios de metástase no momento da realização dos exames complementares, e as margens da neoplasia se encontravam livres de células neoplásicas.

Squamous cell carcinoma is a malignant neoplasm of keratinocytes whose main cause is exposure to ultraviolet rays. The original felines are the domestic species most susceptible to the onset of this neoplasm, the face being the most common region of involvement, including areas of the nasal plane, pinna and eyelids. In the present study, we report a feline, mixed breed, female, 12 years old, 3kg, neutered, with white fur, who was treated for an enlarged ulcerative lesion in the periocular region of the left eyeball, with an evolution of approximately three months. involving both the upper and lower eyelids. After conducting a clinical valuation, the findings obtained through the anamnesis together with the cytological examination, suggested squamous cell carcinoma as the main diagnosis. For treatment before surgical removal, in the qualification good effectiveness to treat this condition. Afterwards, the material sent for histopathological analysis, the diagnosis being confirmed. The patient shows good recovery and clinical evolution of the condition, there is no complication or complication during the treatment. The prognosis for this case was considered good, since there was no evidence of metastasis at the time of the complementary tests, and as the neoplasia margins were free of neoplastic cells.

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro / Avaliação do efeito da riboflavina e luz ultravioleta sobre os ceratócitos in vitro

ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.

RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.

A Case of Korean Patient with Macular Corneal Dystrophy Associated with Novel Mutation in the CHST6 Gene

To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.

Frecuencia de ulceras corneales de etiología fúngica en el Hospital Universitario de Caracas 2008-2012 / Frequency coneal ulcers fungal etiology University Hospital 2008-2012 Caracas

La queratitis micótica es una infección de curso subagudo que genera inflamación y ulceración de la córnea. La importancia de las queratitis micóticas en la práctica oftalmológica radica en su dificultad diagnóstica y terapéutica, la falla en el reconocimiento temprano del hongo y la instauración tardía del tratamiento adecuado incrementa la infiltración y ulceración del estroma corneal, lo que puede llegar a tener consecuencias graves en la integridad del globo ocular. Se propuso determinar la frecuencia de ulceras corneales de etiología fúngica en el Hospital Universitario de Caracas durante el periodo julio 2008-julio2012. Se diseñó un estudio retrospectivo, descriptivo, basado en la revisión de las 41 historias médicas de todos los pacientes con cultivo y/o examen directo positivo para hongos en muestras de úlceras corneales. El cultivo fue positivo en 97,56%, representando Fusarium spp el 37,5%, F. solani 20%, F. oxysporum 12,5%, Aspergillus spp 7,5%, y A. terreus, A. fumigatus, A. candidus, Penicillium spp, Candida spp, C. parapsilosis, P. boydii, Curvularia sp y Cladosporium sp representaron 2,5% cada uno. Al 58,54% se les realizo tratamiento quirúrgico. De 41 pacientes 73,17%, eran de sexo masculino, el grupo más afectado estuvo comprendido entre 20 y 59 años representando el 75,61%. El 58,54% procedía de zonas urbanas y el 41,56% de zona rural, y el 78,05% de la población atendida pertenecía al interior del país. Un 48,78% tuvo un evento traumático mientras que el 51,22% no y 87,80% pacientes ya habían recibido tratamiento previo. A pesar de los avances en diagnóstico y tratamiento para las queratomicosis siguen constituyendo un problema en los países subdesarrollados donde el diagnóstico tardío es directamente proporcional al grado de mejoría del paciente.

Fungal keratitis is a subacute infection that produces inflammation and ulceration of the cornea. The importance of fungal keratitis in ophthalmology practice lies in its diagnostic and therapeutic difficulties, failure of the fungus early recognition and appropriate treatment of late-onset increases infiltration and ulceration of the corneal stroma, which can have serious consequences in the integrity of the eyeball. Proposed to determine the frequency of fungal etiology corneal ulcers in Caracas University Hospital during the period July 2008- july 2012. We designed a retrospective descriptive study, based on a review of 41 medical records of all patients with culture and / or direct examination positive for fungal corneal ulcers samples. The culture was positive in 97.56%, representing 37.5% Fusarium spp, 20% F. solani, 12.5% F. oxysporum, Aspergillus spp 7.5%, and A. terreus, A. fumigatus, A. candidus, Penicillium spp, Candida spp, C. parapsilosis, P. boydii, Curvularia sp and Cladosporium sp accounted for 2.5% each. At 58.54% received surgical treatment. Of 41 patients 73.17% were males, the group most affected was between 20 and 59 years old representing 75.61%. , 58.54% were from urban areas and 41.56% in rural area, and 78.05% of the population served came from inside the country. A 48.78% had a traumatic event while 51.22% and 87.80% no patients had received previous treatment. Despite advances in diagnosis and treatment for keratomycosis remains a problem in developing countries where late diagnosis is directly proportional to the degree of patient improvement.

Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor β2 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transforming growth factor β (TGFβ) is one of the most important growth factors in the development of fibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFβ signal transduction. In recent years, effects of lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about the effects on cornea. This study aimed to determine the effects of lentiviral-mediated Smad7 gene expression on keratocyte proliferation and fibrosis induced by TGF β2 in vitro.</p><p><b>METHODS</b>Keratocytes were cultured from corneal tissue isolated from Sprague-Dawley (SD) rats and transfected with Smad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank). Following the exposure to TGFβ2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream event of TGFβ/Smad signaling. Expression of fibrotic markers α-smooth muscle actin (α-SMA), type III collagen (collagen III) were measured by Western blotting and quantitative real time polymerase chain reaction (PCR). Overall cell proliferation was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression of cell cycle-related marker Ki67 at both mRNA and protein levels.</p><p><b>RESULTS</b>The Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulating phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen III were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated.</p><p><b>CONCLUSION</b>Smad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.</p>

Effect of Mitomycin C, Dexamethasone, and Cyclosporine A 0.05% on the Proliferation of Human Corneal Keratocytes

PURPOSE: To investigate the biologic effect of mitomycin C, dexamethasone and cyclosporine A 0.05% on cultured human keratocytes in vitro. METHODS: Human corneal keratocytes were exposed to a concentration of mitomycin C (0.05%), dexamethasone (0.05%) and cyclosporine A (0.05%) for a period of 3, 5, and 10 minutes. MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and the concentration of type I procollagen COOH-terminal peptide (PIP) and laminin were measured. Cell damage was determined by using the lactate dehydrogenase (LDH) assay. Apoptotic response was evaluated utilizing flow cytometric analysis with Annexin V and propiodium iodide. RESULTS: The inhibitory effect of cellular proliferation and cytotoxicity in cultured human keratocytes showed a time-dependent response in all drugs. The production of PIP and laminin showed a time-dependent response in cultured cells. Apoptosis was observed in flow cytometry after being treated with mitomycin C, dexamethasone and cyclosporine A. Cyclosporin A resulted in less apoptosis of keratocytes than mitomycin C and dexamethasone. CONCLUSIONS: The apoptotic response of mitomycin C, dexamethasone and cyclosporine A is associated with the inhibitory effect of human corneal keratocyte proliferation. To decrease corneal opacity, mitomycin C and dexamethasone were more effective than cyclosporine A in the present study. Additionally, a high concentration of cyclosporine A greater than 0.05% is necessary to lower corneal opacity.

Effects of Gel Type Artificial Tears on Human Corneal Keratocytes and Conjunctival Epithelial Cells

PURPOSE: To evaluate the biological effects and cytotoxicity of gel-type artificial tears on human corneal keratocytes and conjunctival cells in vitro. METHODS: Human corneal keratocytes and conjunctival epithelial cells were exposed to Soothe(R) and Systane(R) at variable concentrations. Evaluations were conducted through an MTT-based calorimetric assay to measure the metabolic activity and through a lactate dehydrogenase (LDH) assay to assess cellular damage. Apoptotic response was examined using fluorescent microscopy and flow cytometric analysis, and cellular morphologic results were evaluated with a transmission electron microscope. RESULTS: The inhibitory effects of corneal keratocyte and conjunctival cell proliferations increased at higher concentrations and longer exposure times to Soothe(R) and Systane(R). The LDH titers increased after Soothe(R) exposure, but showed no significant difference after Systane(R) exposure. Soothe(R) and Systane(R) treatments both produced fluorescence, representing apoptotic cells. In flow cytometry, the maximal apoptotic response was observed for both types of artificial tears, although Systane(R) showed less edema, as well as reduced cytoplasmic and nuclear cell degeneration compared to those of Soothe(R). CONCLUSIONS: The apoptotic responses of Soothe(R) and Systane(R) are associated with inhibitory effects of human corneal keratocyte and conjunctival epithelial cell proliferations. To inhibit the cellular proliferation of human corneal keratocytes and conjunctival epithelial cells, Systane(R) may be less severe than Soothe(R) at higher concentrations and longer exposure times.

Effective Keratocyte Culture Using Amniotic Membrane Matrix and Differentiation of Mesenchymal Stem Cells

PURPOSE: To investigate the characteristics of cultured rabbit corneal keratocytes in vitro and evaluate the possibility of differentiation of mesenchymal stem cells to keratocytes using the keratocyte conditioned medium (KCM). METHODS: Isolated keratocytes were seeded on the stromal side of amniotic membranes (AM) or plastic dishes, and morphologic changes were evaluated. Rabbit mesenchymal stem cells were cultured on AM with alpha-MEM (minimum essential medium alpha) and KCM. The gene expression patterns of specific keratocyte markers (keratocan, lumican, and aldehyde dehydrogenase family, member A1 (ALDH1A1)) of cultured cells were evaluated by RT-PCR. RESULTS: Keratocytes on AM showed dendritic morphology with slow proliferation in contrast, cells on dishes were stellate in shape with fast proliferation. Cultured keratocytes on AM maintained the expression of keratocan, lumican and ALDH1A1 while keratocytes on plastic dishes steadily lost their keratocyte marker gene expression. Additionally, mesenchymal stem cells cultured with KCM on AM induced expression of keratocan and ALDH1A1. CONCLUSIONS: Keratocytes cultured on AM stromal matrix maintained their characteristic morphology and marker gene expression. Morphology changes and marker gene expressions of mesenchymal stem cells suggest an ability to differentiate into keratocytes when grown on AM with KCM.

Effects of Intrastromal Air Injection Compared to Hydro-injection on Keratocyte Apoptosis

PURPOSE: To evaluate the effects of intrastromal air injection and intrastromal balanced salt solution (BSS) injection on corneal keratocyte apoptosis. METHODS: Twelve right eyes of New Zealand White rabbits were divided into an air-injected group (n=6) and a hydro-injected group (n=6). Contralateral eyes served as a control. Air or Balanced salt solution (BSS(R), Alcon, USA) was injected into the deep corneal stroma at the paracentral area to propagate into nearly the entire cornea. To reduce the intraocular pressure, anterior chamber paracentesis was performed. The animals were sacrificed 4 hours (n=6) and 24 hours (n=6) after surgery. Central cornea buttons were retrieved to stain with Hematoxylin & Eosin and TUNEL (Apoptag(R), Chemicon). The mean number of apoptotic keratocytes was counted in 24.67+/-4.04 consecutive high power field (HPF). RESULTS: The mean number of TUNEL-positive cells at 4 hours was 12.85+/-7.25/HPF and 0.25+/-0.44/HPF in air-injected and hydro-injected eyes, respectively. It was reduced to 6.25+/-4.02/HPF and 0.15+/-0.37/HPF in air-injected and hydro-injected eyes after 24 hours. The air-injected group showed significantly more TUNEL-positive cells compared with the hydro-injected or control group until 24 hours (p=0.001, p=0.001, Mann-Whitney U test). CONCLUSIONS: Intrastromal air injection induces significant apoptosis of keratocytes suggesting some damages in the peripheral cornea when used in deep lamellar keratoplasty.

Effect of Amoebicidal Agents on the Human Corneal Keratocytes in Vitro

PURPOSE: To evaluate the effect of polyhexamethylene biguanide (PHMB) and chlorhexidine on Acanthamoeba cysts and cultured human keratocytes. METHODS: Each well of two-fold diluted PHMB and chlorhexidine were treated on the Acanthamoeba cyst suspension of 5 x 10(4) cysts/ml for 8, 24, and 48 hours to measure the minimal cysticidal concentration (MCC) of each disinfectant and was exposed to the human corneal keratocytes of 5 x 10(4) cells/ml for same hours to measure the survival rate of keratocytes. Inverted phase-contrast micrograph and electron microscopy for observing the morphologic changes were evaluated. RESULTS: MCC of PHMB was 9.42 microgram/ml, 5.62 microgram/ml, and 2.37 microgram/ml, and chlorhexidine was 24.32 microgram/ml, 10.02 microgram/ml, and 7.02 microgram/ml respecitvely in 8, 24, and 48 hours. The survival rate of keratocytes at MCC was 91.7%, 64.6%, and 49.7% in PHMB of which significant decreases were found at 24 and 48 hours, and 95.7%, 90.6%, and 78.1% in chlorhexidine of which significant decrease was only found at 48 hours. The higher the concentration of disinfectants, cysts and keratocytes demonstrated more damaged appearance. CONCLUSIONS: The amoebicidal efficacy of PHMB and chlorhexidine was similar. However, in consideration of toxic effect on keratocytes by disinfectants, chlorhexidine is suggested to be more clinically useful than PHMB.

Effects of Damaged Human Corneal Epithelial Cells on Differentiation of Human Mesenchymal Stem Cell

PURPOSE: To identify the effects of microenvironmental changes caused by human corneal epithelial damages to characteristics or differentiation of human mesenchymal stem cells (hMSCs). METHODS: Artificial corneal damage was induced onto a cultured monolayer of human corneal epithelial cells. hMSCs were then co-cultured with damaged human corneal epithelial cells (dIHCE). Morphological changes in the co-cultured hMSCs were observed. To elucidate the differentiation of hMSCs into corneal keratocytes or epithelial cells, the expressions of alpha-smooth muscle actin, keratin-3/-12, and E-cadherin were confirmed by immunofluorescence. RESULTS: hMSCs co-cultured with dIHCE showed enhanced adherence in the neighborhood of dIHCE and morphological change into dendritic shapes at 6 days post-seeding. Although the expression of alpha-smooth muscle actin, known as hMSCs marker, significantly decreased at the dIHCE-contacted site of hMSCs; there were no expressional changes on keratin-3/-12 and E-cadherin, the markers of corneal epithelial cells. Interestingly, positive expression of corneal epithelial marker keratin-3/-12 was observed in dIHCE co-cultured hMSCs. hMSCs co-cultured with normal human corneal epithelial cells (nIHCE) were unable to attach, and showed no change in the expression of alpha-smooth muscle actin. CONCLUSIONS: It is proposed that dIHCE causes a morphological change in hMSCs, and decreased expression of alpha-smooth muscle actin. These results suggest that dIHCE can affect a change in the characteristics and differentiation of hMSCs.

Gene Microarray Related with Apoptosis in Diabetic OLETF Keratocytes

PURPOSE: This study was to identified differentiated genes concerned with apoptosis that are up-regulated or down-regulated in OLETF corneal keratocytes stimulated with interleukin-1alpha. METHODS: Otsuka Long Evans Tokushima Fatty (OLETF) corneal keratocytes were primarily cultured and treated with 20 ng/ml of IL-1a for 6 hours. Hybridization was carried out using Oligonucleotide microarray. A sample of normal keratocytes was labeled with Cy 3, and a diabetic keratocytes sample was labeled with Cy 5. RESULTS Diabetes showed 20 down-regulated genes and 24 up-regulated genes compared with normal keratocytes. Also, IL-1alpha-treated diabetic keratocytes expressed 31 down-regulated and 33 up-regulated genes. Compared with diabetic keratocytes, the newly expressed genes in OLETF treated with IL-1alpha were Bcl, Lam, Timp, Mmp, Socs, Bmp, Ccl, Lcn, C7, etc. CONCLUSIONS: There were some genes related with apoptosis that expressed differently between normal and diabetic keratocytes of OLETF. Also, IL-1alpha may stimulate differing effects of apoptosis on diabetic corneal keratocytes. Studies analyzing the apoptotic significance of these differences identified in diabetic keratocytes are needed.

Gene Microarray Related with Apoptosis in Diabetic OLETF Keratocytes

PURPOSE: This study was to identified differentiated genes concerned with apoptosis that are up-regulated or down-regulated in OLETF corneal keratocytes stimulated with interleukin-1alpha. METHODS: Otsuka Long Evans Tokushima Fatty (OLETF) corneal keratocytes were primarily cultured and treated with 20 ng/ml of IL-1a for 6 hours. Hybridization was carried out using Oligonucleotide microarray. A sample of normal keratocytes was labeled with Cy 3, and a diabetic keratocytes sample was labeled with Cy 5. RESULTS Diabetes showed 20 down-regulated genes and 24 up-regulated genes compared with normal keratocytes. Also, IL-1alpha-treated diabetic keratocytes expressed 31 down-regulated and 33 up-regulated genes. Compared with diabetic keratocytes, the newly expressed genes in OLETF treated with IL-1alpha were Bcl, Lam, Timp, Mmp, Socs, Bmp, Ccl, Lcn, C7, etc. CONCLUSIONS: There were some genes related with apoptosis that expressed differently between normal and diabetic keratocytes of OLETF. Also, IL-1alpha may stimulate differing effects of apoptosis on diabetic corneal keratocytes. Studies analyzing the apoptotic significance of these differences identified in diabetic keratocytes are needed.

Effect of Tranilast on the Proliferation of Human Corneal Keratocytes in Vitro

PURPOSE: To evaluate the inhibitory effect of tranilast on proliferation of cultured human keratocytes, and to investigate the apoptotic response and the cellular morphologic changes associated with tranilast in vitro. METHODS: Human corneal keratocytes were exposed to tranilast at a concentration of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/ml for a period of 4, 24, and 48 hours. Evaluations were conducted with MTT-based-calorimetric assay for measuring the metabolic activity, flow cytometric analysis and fluorescent micrograph for assessing the apoptotic response, and inverted phase-contrast micrograph and electron microscopy for observing the morphologic changes. RESULTS: The inhibitory effect of human keratocyte proliferation was found to have a dose and time dependent pattern (p<0.05). In flow cytometry, the maximal apoptotic response developed at 0.8 mg/ml concentration after 4 and 24 hours of exposure time, and apoptotic cells were demonstrated in fluorescent micrograph. At higher concentration of Tratnilast, human corneal keratocytes were more swollen rather than having a spindle shape and being detached from the bottom of the dish. The damaged keratocytes had degenerative and apoptotic changes like the formation of phagolysosomal granule, marginal condensation in the nucleus, and bleb formation of the nuclear membrane. CONCLUSIONS: The apoptotic response of tranilast is concerned with the inhibitory effect of human corneal keratocyte proliferation. Therefore, tranilast shows promise in clinical use for the inhibition of postoperative excimer laser induced corneal opacity or haze with fewer side effects.

Effect of Dexamethasone and Diclofenac on the Proliferation of Human Corneal Keratocytes

PURPOSE: To evaluate the inhibitory effect of dexamethasone and diclofenac on the proliferation of cultured human keratocytes, and to investigate the apoptotic response and the cellular morphologic changes associated with dexamethasone and diclofenac in vitro. METHODS: Human corneal keratocytes were exposed to 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mM concentration of dexamethasone and diclofenac for 4, 24, and 48 hours. MTT based calorimetric assay, flow cytometric analysis, fluorescent micrograph, inverted phase-contrast micrograph, and electron microscopy were used to evaluate the results. RESULTS: The inhibitory effect of human keratocyte proliferation increased at higher concentrations and longer exposure times of dexamethasone and diclofenac (p<0.05). In flow cytometry, the maximal apoptotic response developed at 0.4 mM concentration of dexamethasone and 1.6 mM concentration of diclofenac after 4 hours. Apoptotic cells were demonstrated in fluorescent micrograph. Dexamethasone-treated cases showed a more damaged appearance, more swollen rather than spindle shaped, with greater detachment from the bottom of the dish and the chromatin of the nuclear remnant condensed along the nuclear periphery with cytoplasmic vesication and cytoplasmic blebs formation, and partial disruption of the nuclear membrane compared with diclofenac. CONCLUSIONS: The apoptotic response of dexamethasone and diclofenac is associated with the inhibitory effect of human corneal keratocyte proliferation. For inhibition of cellular proliferation of human corneal keratocytes, dexamethasone may be more effective at lower concentration and shorter exposure time than diclofenac.

Apoptosis of Keratocytes Induced by Mitomycin C

PURPOSE: The purpose of this study was to evaluate the effect of mitomycin C on rabbit keratocytes for their potential to modulate corneal stromal wound healing. We also investigated the pathway on which the modulation occurs. METHODS: Keratocytes were isolated from New Zealand White Rabbits and cultured. We used Hoechst stain and flowcytometric analysis with Annexin V to identify the kind of response that mitomycin C induced from the keratocytes. After cultured keratocytes were exposed to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C, we evaluated the response with LDH assay. Next, after exposing the keratocytes to 0.01% mitomycin C, we evaluated the responses with LDH assay at 6, 12, and 24 hours. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor (Z-IETD-FMK(R)), and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 0.01% mitomycin C. Twelve hours later, an LDH assay was performed. Cytochrome C immunostain was done after exposure to 0.01% mitomycin C. RESULTS: We observed shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation on Hoechst staining. In flowcytometric analysis, the cells showed apoptotic change. LDH activities increased significantly at a concentration of 0.005% and greater and were time-dependent until 24 hours. CPP32-like protease inhibitor decreased the LDH activity, but there was no statistical significance. Specific caspase-8 and -9 inhibitors significantly reduced the LDH activities that were induced by mitomycin C. The keratocytes which had been pretreated with mitomycin C were stained with cytochrome C antibody. CONCLUSIONS: Mitomycin C induces apoptosis, rather than necrosis, in cultured corneal keratocytes. This apoptosis occurs via the caspase pathway, and is especially related to the mitochondrial pathway, and caspases 8, and 9.

Ceramide-Induced Apoptosis in Cultured Keratocyte

PURPOSE: We investigated the effect of ceramide on keratocyte apoptosis and pathway of ceramide-induced keratocyte apoptosis. METHODS: Cultured Newzealand White Rabbit keratocytes were exposed to various concentrations of ceramide type II, VI and phytoceramide type II, VI. LDH level was measured for the evaluation of time and concentration related apoptosis. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK, diffuse caspase inhibitor), specific caspase-8 inhibitor (IETD-CHO) and specific caspase-9 inhibitor (Z-LEHD-FMK), then were exposed to 20 micro M of 4 types of ceramide. Cytochome C immune stainining was done after exposure of keratocyte to 4 types of ceramide. RESULTS: The lower effective dose of 4 types of ceramide was 20 micro M. Apoptosis of keratocytes was dependent on ceramide exposure time. Ceramide induced keratocyte apoptosis was inhibited by CPP32-like protease inhibitor, specific caspase-8 inhibitor and specific caspase-9 inhibitor. Apoptotic keratocytes induced by ceramide were immune-stained with cytochrome C antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal keratocytes. This apoptosis developed according caspase cascade, especially via mitochondria.

The Effect of Candida albicans and Dexamethasone on the Secretion of Tumor Necrosis Factor-alpha from Cultured Human Keratocytes

PURPOSE: To measure the secretion of TNF-alpha from cultured human keratocytes after inoculation of Candida albicans, and to evaluate the change of secretion of TNF-alpha following application of dexamethasone. METHODS: Human corneal keratocytes were cultured independently in vitro. The specimens were divided into 4 groups: Group I with only pure culture as control, Group II with C. albicans, Group III with C. albicans and dexamethasone, and Group IV with only dexamethasone. RESULTS: As a whole, Group II showed the highest secretion of TNF-alpha, followed by Group III, Group I, and Group IV respectively (P< 0.05). CONCLUSION: Keratocytes with the addition of both C. albicans and dexamethasone, secreted much lower level of TNF-alpha , but showed more proliferation in comparison to keratocytes with addition of C. albicans alone. Therefore, the author may conclude that in fungal keratitis, the early administration of dexamethasone may be beneficial in reducing inflammatory responses induced by increased TNF-alpha secretion. However, because steroids may stimulate the proliferation of fungus leading to tissue damages, more cautious use might be needed.

Gene Transfer into Corneal Keratocytes using a Hybrid EBV/retroviral Vector

PURPOSE: We tried to determine the feasibility and efficiency of foreign gene transfer into corneal keratocytes using a hybrid EBV/retroviral vector as an investigative trial for gene therapy in corneal diseases. METHODS: LZRSpBMN-Z, alac Z-transducing hybrid EBV/retroviral vector, was transfected into Phoenix(T M) amphotropic packaging cells based on a 293T cell line and then collected without/with puromycin selection (puro (-)/puro (+) vector respectively). Cultured human and rabbit keratocytes were transduced with lac-Z gene using the puro (-) or puro (+) vector solutions, then stained with 5-bromo-4-chloro-3-indolyl galactopyranoside (X-gal). FACS-Gal analysis of transduced corneal keratocytes was also performed for calculating gene transfer efficiency. In addition, as an in vivo trial, we tried to transduce rabbit keratocytes by topical application of the vector supernatants following PRK or lamellar dissection of rabbit corneas. RESULTS: In vitro, both cultred human and rabbit keratocytes were transduced successfully with lac - Z gene. Transduction efficiency was 22% and 16% for human and rabbit keratocytes respectively with puro (-) vector, and slightly increased to 24% and 22% with puro (+) vector. In vivo corneas, however, no keratocytes were stained with X-gal. CONCLUSIONS: A hybrid EBV/retroviral vector, LZRSpBMN-Z, successfully transduced corneal keratocytes in in vitro conditions but not in vivo corneas.

Inhibitory Effect of Mitomycin C (MMC) on the Proliferation of Human Corneal Keratocyte

PURPOSE: To evaluate the anti-proliferative effect of mitomycin C (MMC) on human corneal keratocyte, and to investigate the cellular morphology of keratocyte according to the concentration and exposure time in vitro. METHODS: Human corneal keratocytes using endothelium-free explant method were exposed to 0.005%, 0.01%, and 0.05% concentration of MMC for 3, 5, and 10 minutes. MTT based colorimetric assay was performed to assess the inhibition of cellular proliferation, and cellular morphology was evaluated by inverted phase-contrast light microscope and electron microscope. RESULTS: Use of higher concentration MMC and prolongation of exposure time resulted in greater inhibitory effect on cellular proliferation. When exposed to 0.005% MMC for 3, 5 and 10 minutes, the survival rate of keratocyte was 100%, 95.7% and 74.0% respectively. At 0.01% MMC, the survival rate was 98.6%, 92.9%, and 66.9%. At 0.05% MMC, it was 74.0%, 73.4%, and 38.8%. Exposure to the highest concentration (0.05%) among the 3 preparations for 3 or 5 minutes showed significant inhibition of keratocyte proliferation (p<0.05), and when exposed for 10 minutes, all 3 preparations showed significant inhibition of keratocyte proliferation (p<0.05). Inverted phase-contrast light microscopy showed that human corneal keratocytes lost their adherence to the bottom of the dish and assumed round and swollen shape rather than spindle shape when exposed to higher concentration of MMC for a prolonged time. The damaged keratocytes showed the degenerative changes like cellular membrane disruption, disappearance of microvilli, enlargement of rough surfaced endoplasmic reticulum and mitochondria, and vacuole formation by electronic microscope. CONCLUSIONS: When MMC is applied to inhibit the proliferation of keratocytes involved in corneal wound healing, it seems to be a valuable application at least 0.05% concentration for 3 minutes. Further studies should be followed for the biological effect of MMC including drug toxicity associated with human corneal tissue in vivo.