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.

Unusual early recurrence of granular dystrophy after deep anterior lamellar keratoplasty: case report / Recorrência atípica e precoce de distrofia granular após transplante lamelar profundo: relato de caso

We report an atypical case of granular corneal dystrophy recurrence after deep anterior lamellar keratoplasty. We describe clinical features, histopathological analysis of the lamellar graft specimen and DNA analysis results. The slit-lamp examination and histopathological findings from the graft specimen indicated the confinement of the typical deposits of granular corneal dystrophy deep in the graft interface area. This localization is atypical, since in most cases recurrences in grafts tend to be initially superficial and situated in the epithelial or subepithelial corneal layers. Molecular genetic analysis revealed an already described mutation and a new intronic variant. The unusual localization and timing of this recurrence of granular corneal dystrophy after deep anterior lamellar keratoplasty suggests that corneal stromal keratocytes may play a role in the formation of granular deposits.

É relatado um caso atípico de recorrência de distrofia corneana granular após transplante lamelar anterior profundo. São descritas as características clínicas, a análise histopatológica do espécime do enxerto lamelar e os resultados de análises de DNA. O exame com lâmpada de fenda e a análise histopatológica do espécime do enxerto demonstram o confinamento dos depósitos típicos da distrofia corneana granular profundamente, na área de interface do enxerto. Esta localização é atípica, uma vez que, na maioria dos casos de recidivas em enxertos, estes tendem a ser no início localizados superficialmente, nas camadas epiteliais ou subepitelial da córnea. A análise genética molecular revelou uma mutação já descrita e uma nova variante intrónica. A localização incomum e o tempo de aparecimento da presente recorrência da distrofia corneana granular após transplante lamelar anterior profundo sugere que ceratócitos do estroma corneano possam desempenhar algum papel na formação dos depósitos granulares.

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.

Influence on cultured human keratocytes by liposome / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To observe the effects on human keratocytes by cationic liposome LipofectamineTM 2000(LF2000).to investigate the efficiency and safe range applied in human keratocytes,and establish basis for gene therapy of human keratocytes.METHODS: Human keratocytes cultured in vivo within 3 to 5 passages were used in experiment after being identified.The effects on proliferation of cultured human keratocytes by LF2000 with different concentrations and time were evaluated By MTT:the effects of LF2000 on the survival rate and its relation with 5,10,20,40.80mg/L concentration and time were detected by trypan blue staining.related with concentration and time.The cellular proliferation and survival rate declined when concentration of LF2000 was above certain level,and this effect increased as time became longer.LF2000 had no effect with concentration under 40mg/L for 24 hours. CONCLUSION:LF2000 did ont cause cytotoxicity during a concentration range"tested",and it is hoped to play an important role in gene therapy of human keratocytes.

Toxic effect of exotoxin A of Pseudomonas aeruginosa on keratocytes / 吉林大学学报(医学版)

Objective To study the toxic effect of exotoxin A of Pseudomonas aeruginosa on keratocytes.Methods Three-dimensional gels of type I collagen containing rabbit keratocytes were incubated in the presence of different concentrations of exotoxin A(0.1,1.0,10 mg?L-1),cultivated for 24 h at 37℃,the change of keratocytes in morphology was observed under the light microscope,and the amount of lactate dehydrogenase(LDH) was measured by enzyme linked immunosorbent assay(ELISA).Results The LDH contents in different concentrations(0.1,1.0,10 mg?L-1)of exotoxin A groups were higher than that in the group without exotoxin A(P

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.

The Effects of Tranilast gamma on the Cellular Proliferation and alpha-Smooth Muscle Actin Expression in the Cultured Keratocytes of Rabbit

PURPOSE: To evaluate the effects of Tranilast gamma on the cellular proliferation and expression of alpha-smooth muscle actin on the cultured keratocytes of the rabbit that influence the formation of corneal scar and haze in vitro. METHODS: After the keratocytes of the rabbit were cultured, they were exposed to Tranilast gamma (N-(3, 4-dimethoxycinnamoyl) anthranilic acid) 0.05, 0.1, 0.2, 0.4, 0.8, 1.0, 1.6 mg/ml with DMEM used as a control. MTT was used to measure the metabolic activity, and the Western blot assay was performed to confirm the alpha-smooth muscle actin expression, which was expressed with time by Tranilast gamma treatment. RESULTS: Higher the concentration and longer the exposure time of Tranilast gamma, the more the inhibition of the cellular proliferation. LD50 concentration was about 0.4 mg/ml in the exposure time of 24 hours and 0.2 mg/ml in the exposure time of over 48 hours(P<0.05). There was a decrease in response of alpha-smooth muscle actin expression with increasing concentrations of Tranilast gamma relative to the control. CONCLUSIONS: Tranilast gamma trends to have an inhibitory effect on the cellular proliferation and induction of alpha-smooth muscle actin expression. Tranilast gamma may inhibit excessive haze of the cornea and inhibit scar formation after the corneal wound healing and photorefractive surgery in the cornea.

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.

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.

Secretion of Interleukin-8 from Human Keratocyte Stimulated by Aspergillus fumigatus and Effect of Amphotericin B and Dexamethasone on The Secretion

PURPOSE: To measure the secretion of IL-8 from cultured human keratocytes after inoculation of conidia of A. fumigatus, and to compare the change of secretion of IL-8 following application of amphotericin B and dexamethasone. METHODS: Human corneal keraoctytes were cultured independently in vitro. The specimens were divided into 4 groups : Group I with only pure culture as control, Group II with conidia of A. fumigatus, Group III with conidia of A. fumigatus and amphotericin B, and Group IV with conidia of A. fumigatus and dexamethasone. The supernatants were aspirated from each group at different time intervals, and then were assayed for IL-8. RESULTS: Group II showed increased secretion of IL-8, at all selected time intervals except 12-h, in comparison with other three groups. Group III secreted IL-8 significantly less than the other groups(p<0.01). Group IV secreted IL-8 less than Group I and II at all selected time points(p<0.01), but more than Group III at the other time points except for 72-h(p<0.01). CONCLUSIONS: The secretion of IL-8 increased in the early stage of fungal keratitis but decreased in case of the administration of amphotericin B or dexamethasone. Amphotericin B was more potent than dexamethasone in decreasing the secretion of IL-8.

The Ultra structure of Rabbit Kerato cyte Infected by Herpes Simplex Virus

To evaluate the ultrastructure of cornea keratocyte infected by Herpes simplex virus, the cornea was excised from enucleated rabbit eyes. Cornea epithelium and endothelium of these was removed and cultivated in culture media. After 7 days, the Kos strain of Herpes simplex virus was inoculated into the cultured cornea keratocytes and co-cultivated until the cytopathic effect occurred. Cornea keratocytes was fixed in the 3%glutaraldehyde and examined with electron microscope. The infected cells were active cells and slightly more round appearance than normal cells. They had microvillies composed of cytoplasmic process protru-sions into collagen fibers. Virus particles was transmitted into neighboring cells easily since there was no barrier function of collagen fibers that was destroyed in the condition of culture. The nuclear changes of infected cells were marked :nuclear chromatin was degraded, condensated and displaced toward the nuclear membrane. There were many virus particles in the nucleus. There were also many degenerations in the cytoplasm, destructed numerous cytoplasmic organelles and many virus particles seen in the cytoplasm. The ultrastructural findings of keratocytes co-cultivated with Herpes simplex virus were identified from our result.

Effect of Poly Ethylene GlycolPEGGraft Polymerization onto Polymethy lmethacrylatePMMAon Cultured Keratocyte Adhesion

In this study, the effect of surface modification of polymethylmethacry-late[PMMA]by grafting of poly[ethylene glycol][PEG]on cell adhesion was investigated. PMMA surface was treated with ozone and then PEG-acry-late[PEGA]was graft-polymerized. Ozone treatment of the surface was car-ried out at room temperature by applying constant flow of oxygen[4.5liter/min]and 1 bar pressure. After ozone treatment, PMMA was immersed immediately in 20 wt%aq. PEG-acrylate solutions in glass ampoules. After degassing, the ampoule was sealed and kept at 60 degrees C for 24 hours to complete the graft polymerization. PMMA surface grafted with PEG revealed the enhanced oxygen content at ESCA analysis and the decreased dynamic receding contact angles. The adhesion of keratocytes onto modified PMMA was investigated. Keratocytes[4 x105cells/milliliter ]were layered on each PMMA discs which were glued to the bottom of 24-well culture plates, and cultured in a CO2 incubator for 24 hours. The adherent cells onto the surfaces were harvested by trypsinization and counted. The mean numbers of keratocytes on untreated PMMA, PEG-grafted PMMA with 1hour ozone treatment and PEG-grafted PMMA with 2 hour were 72.5 x104 and 6.5 x104 and 7.6 x104cells respectively, and there was a significant statistical difference [p=0.002], irrespective of ozone treatment period. This result suggests that surface modification of PMMA using PEG grafting may reduce etroprosthetic membrane formation of artificial cornea.

The Effects of Artificial Tear Formulations and Anti-inflammatory Agents on the Cultured Keratocytes of Rabbit

We investigated the biological effects and cytotoxicity of the artificial tear formulations and anti-inflammatory agents on the cultured keratocytes of rabbit in vitro by using lactate dehydrogenase (LDH) leakage assay and transmission electron microscopy. In the artificial tear fomualations, the LDH titer of the Tears naturale and Protagent were more increased with time, but the Tears naturale II and Tears naturale free was slowly progressed or has narrow range of LDH increasing. In the anti-inflammatory agent, the LDH titer of the all eye solutions was increased with time, especially Maxidex and Fluorometholone. Non-steroid, Decrol was less than other anti-inflammatory agents in Na+, K+, Cl- electrolyte compositon. Especially, the LDH titer of the artificial tear formulations and anti-inflammatory agents containing the benzalkonium chloride (BAC) or low Na+, K+, Cl- composition was more increased with time. The typical rabbit keratocytes were elongated and had not prominent nuclei. But the higher the titer of LDH, the keratocytes were visible round and swollen, and were injured with the disruption of plasma membrane, vacuole formation in cytoplasm, and damage of nuclei in transmission electron microscopy. As the use of topical drug containing low Na+, K+, Cl- or Benzalkonium chloride should be induced a particulary toxic effect on keratocyte, we should be carefully use these topical eye solutions, especially when the epithelium is denuded.

The Effect of Ascorbic Acid and its Derivative on Cultured Rabbit Keratocytes

The rabbit keratocytes were cultured to evaluate the effects of L-ascorbic acid(AA), ascorbic acid 2-sulfate(AA-2S), and ascorbic acid 2-phosphate(AA-2P) by measuring cell numbers and 3H-thymidine incorporation. AA at a concentration of 0.05mM enhanced the proliferation of the cells progressively. Addition of 0.1, and 0.5mM AA stimulated the proliferation of cells in the 3rd and 7th day after culture and inhibited in the 15th and 20th day in a dose-dependent manner. AA-2S showed a similar pattern to those of the AA effect, although the inhibitory effect was milder than AA. All concentrations of AA-2P enhanced the proliferation of the cells from the early phase. The effect was prominent in the late phase in a dose-dependent manner. These results suggest that AA-2P is the most stable and the least cytotoxic in the aqueous solution state or culture media and, 0.1-0.5mM concentration of it is best in the promotion of the proliferation of the cultured keratocytes.