The cultured cells were derived from rabbit corneal stroma by explant technique following microdissection and serial passage. The ultrastructural features of fourteenth-passage keratocytes were examined with both scanning and transmission electron microscope. The cells can be divided into activated, intermediate and old cell according to the differences in electron density and surface microvillous pattern. The morphologic characteristics of the cultured keratocytes partially resemble those shown in corneal keratocytes in vivo.
Cells, Cultured ; Corneal Keratocytes* ; Corneal Stroma ; Microdissection ; Serial Passage

The authors performed an experiment to determine if human corneal keratocytes release IL-8 after stimulation with lipopolysaccharide (LPS). Human corneal keratocytes were isolated from human corneal buttoms and grown independently in vitro. Cultured keratocytes were treated with various concentrations of LPS (0.01, 0.1 1, 10 microgram/ml). At 6 hours, 12 hours, 24 hours, and 48 hours after the stimulation with LPS, culture supernatants were aspirated and frozen. Supernatants were assayed by enzyme-linked immunosorbent assay for IL-8 content. Exposure of corneal keratocytes to LPS induced IL-8 production. Initially, the secretion of IL-8 was detected at 6 hours and increased upto 48 hours. Between 12 and 24 hours, the IL-8 was increased rapidly. At 6 and 12 hours, keratocytes exposed to 10 microgram / ml LPS produced more IL-8 than those exposed to other concentrations of LPS. In this study, the ability of corneal keratocytes to produce IL-8 upto 48 hours suggests that these cells can play important roles in the induction of the inflammatory response in cornea.
Cornea ; Corneal Keratocytes ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Interleukin-8*

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.
Acanthamoeba ; Chlorhexidine ; Corneal Keratocytes* ; Disinfectants ; Humans* ; Microscopy, Electron ; Survival Rate

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.
Cell Line ; Cornea ; Corneal Diseases ; Corneal Keratocytes* ; Galactose ; Genetic Therapy ; Humans ; Product Packaging ; Puromycin

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.
Apoptosis ; Blister ; Corneal Keratocytes* ; Corneal Opacity ; Flow Cytometry ; Humans* ; Lasers, Excimer ; Microscopy, Electron ; Nuclear Envelope

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.
Candida albicans* ; Candida* ; Corneal Keratocytes ; Dexamethasone* ; Fungi ; Humans* ; Keratitis ; Steroids ; Tumor Necrosis Factor-alpha*

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.
Animals ; Apoptosis* ; Corneal Keratocytes ; Diabetes Mellitus ; Interleukin-1alpha ; Oligonucleotide Array Sequence Analysis ; Rats

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.
Animals ; Apoptosis* ; Corneal Keratocytes ; Diabetes Mellitus ; Interleukin-1alpha ; Oligonucleotide Array Sequence Analysis ; Rats

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.
Apoptosis* ; Caspase 8 ; Caspase 9 ; Corneal Keratocytes ; Cytochromes c ; Mitochondria ; Protease Inhibitors

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.
Animals ; Anterior Chamber ; Apoptosis* ; Cornea ; Corneal Keratocytes ; Corneal Stroma ; Corneal Transplantation ; Eosine Yellowish-(YS) ; Hematoxylin ; In Situ Nick-End Labeling ; Intraocular Pressure ; Paracentesis ; Rabbits