PURPOSE: To evaluate and compare the toxic effects of eyedrops containing a fixed combination of 2.0% dorzolamide and 0.5% maleate timolol with or without preservatives on rabbit corneal endothelium. METHODS: This study was performed with 22 eyes of New Zealand white rabbits. Dorzolamide/timolol eyedrops with preservative (Cosopt group) or without preservative (Cosopt-S group) were diluted with a balanced salt solution at a 1 : 1 ratio. We injected 0.1 mL of diluted Cosopt into the anterior chamber of left eyes and an equal volume of diluted Cosopt-S into the anterior chamber of right eyes. Corneal thickness, corneal haze, and conjunctival injection were measured before and 24 hours after treatment. Endothelial damage was compared between both eyes by vital staining (alizarin red/trypan blue staining), live/dead cell assay, TUNEL assay, and scanning electron microscopy. RESULTS: Corneal endothelial damage was severe in the Cosopt group. Cosopt-treated eyes exhibited remarkable corneal edema and prominent apoptosis of endothelial cells. In addition, the live/dead cell assay revealed many dead cells in the endothelium, and scanning electron microscopy analysis showed that corneal endothelial cells exhibited a partial loss of microvilli on the surface as well as extensive destruction of intercellular junctions. However, in the Cosopt-S group, corneal edema was mild and the damage to the corneal endothelium was minimal. CONCLUSIONS: The main cause of corneal endothelial toxicity was due to the preservative in the dorzolamide/timolol fixed combination eyedrops, and not the active ingredient. Thus, it appears to be safer to use preservative-free eyedrops during the early postoperative period.
Animals ; Anterior Chamber/drug effects ; Apoptosis ; Corneal Edema/chemically induced/*pathology ; Disease Models, Animal ; Drug Combinations ; Endothelium, Corneal/drug effects/*pathology ; In Situ Nick-End Labeling ; Ophthalmic Solutions ; Rabbits ; Sulfonamides/administration & dosage/*toxicity ; Thiophenes/administration & dosage/*toxicity ; Timolol/administration & dosage/*toxicity

BACKGROUNDTrabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow in the eye. This study aimed to evaluate the role of HepII domain peptides V on corneal permeability, corneal endothelial cells, intraocular pressure (IOP) and morphology of trabecular meshwork in rats.

METHODSThe IOP of rat eyes was measured before and 3, 5, 7 and 8 hours after topical delivery of HepII domain peptides V through intracameral injections. The peptide's concentration in aqueous humor was assessed by high performance liquid chromatography (HPLC). The shape and density of endothelial cells were observed by laser confocal microscopy 8 hours, 3 and 14 days after intracameral injections of HepII domain peptides V. The morphological changes in TM of rat eyes were assessed by transmission electron microscopy (TEM).

RESULTSIntracameral injection of HepII domain peptides V significantly (P < 0.001) decreased IOP by (5.71 ± 2.10) mmHg in rats at 5 hours after injection. There were no obvious changes of the shape and the density of corneal endothelial cells. In addition, morphological changes in the TM of rats were observed including the expansion of intercellular spaces in the juxtacanalicular meshwork, removal of extracellular material, cellular relaxation, and cytoskeleton reorganization.

CONCLUSIONSHepII domain peptides V could not penetrate cornea and was safe to corneal endothelial cells. HepII domain peptides V could significantly decrease IOP in rat probably by disorganizing actin cytoskeleton and cell-junction in the TM.

Animals ; Chromatography, High Pressure Liquid ; Cornea ; cytology ; drug effects ; ultrastructure ; Endothelium, Corneal ; drug effects ; ultrastructure ; Female ; Fibronectins ; chemistry ; pharmacology ; Intraocular Pressure ; drug effects ; Male ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Rats ; Rats, Sprague-Dawley ; Trabecular Meshwork ; drug effects ; ultrastructure

OBJECTIVETo investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.

METHODSTotal RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively.

RESULTSPDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells.

CONCLUSIONSThe construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

Animals ; Cats ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Corneal ; cytology ; drug effects ; Humans ; Immunohistochemistry ; Phosphopyruvate Hydratase ; analysis ; Protein Folding ; Proto-Oncogene Proteins c-sis ; chemistry ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Wound Healing ; drug effects

PURPOSE: To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). METHODS: Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. RESULTS: Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15+/-10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. CONCLUSIONS: IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.
Transfection ; Reverse Transcriptase Polymerase Chain Reaction ; Repressor Proteins/genetics/*pharmacology ; RNA, Messenger/genetics ; Protein-Tyrosine Kinases ; Oncogene Proteins, Viral/genetics/*pharmacology ; Immunohistochemistry ; Humans ; Gene Expression Regulation, Viral ; Freeze Drying ; Endothelium, Corneal/*cytology/drug effects/metabolism ; Cell Line, Transformed ; Cell Count ; Amnion

In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.
Cell Proliferation/*drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelium, Corneal/*cytology ; Epithelium, Corneal/*cytology ; Nerve Growth Factor/*pharmacology

The goal of this study was to evaluate the function of hyaluronic acid (HA) on the active oxygen release from polymorphonuclear leukocytes (PMNs) and the protective effect of bovine corneal endothelial cells (BCEC) from activated PMNs. We used HA with three different molecular weights (MW 700, 000, 2, 000, 000, and 4, 000, 000) and five different concentrations (0, 0.1, 1, 2, and 3 mg/ml). We evaluated the amount of released superoxide from activated PMNs by using dismutase-inhibitable ferricytochrome C reduction. To compare the property and protective effect of HA with those of other viscoelastic substances, we used the same concentration of methylcellulose. HA suppressed superoxide release from PMNs and protected BCEC from activated PMNs in a dose-dependent, rather than a molecular weight-dependent, manner. The effect of HA reached almost a plateau at concentration above 2 mg/ml. However, methylcellulose, another viscoelastic substance, showed a similar effect. Therefore, it seems that the suppression of superoxide released from PMNs is not a property that is unique to HA, but is a general property of viscoelastic substances. Our results indicate that the action mechanism of HA proceeds not only through cell surface HA-receptor. We think that HA also acts as a physical barrier and/or a scavenger of superoxide.
Animals ; Cattle ; Cell Survival ; Cells, Cultured ; Comparative Study ; Cytochromes c/metabolism ; Cytoprotection ; Dose-Response Relationship, Drug ; Endothelium, Corneal/cytology/*drug effects ; Humans ; Hyaluronic Acid/*pharmacology ; Methylcellulose ; Molecular Weight ; *Neutrophil Activation ; Neutrophils/*drug effects/metabolism ; Superoxides/*metabolism

Acetates ; pharmacology ; Animals ; Bicarbonates ; pharmacology ; Drug Combinations ; Endothelium, Corneal ; drug effects ; pathology ; Female ; Glutathione ; pharmacology ; Hydrogen-Ion Concentration ; Minerals ; pharmacology ; Ophthalmic Solutions ; pharmacology ; Rabbits ; Retina ; drug effects ; pathology ; Sodium Chloride ; pharmacology ; Vitreous Body ; drug effects

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-beta 1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-beta 1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-beta 1 expression product TGF-beta 1 in the endothelia was detected. Specific TGF-beta 1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-beta 1 participating in local induction of corneal immune tolerance.
Animals ; Anterior Chamber ; Corneal Transplantation ; Endothelium, Corneal ; drug effects ; pathology ; Gene Transfer Techniques ; Immune Tolerance ; Rabbits ; Transforming Growth Factor beta ; administration & dosage ; biosynthesis ; genetics

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-beta 1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-beta 1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-beta 1 expression product TGF-beta 1 in the endothelia was detected. Specific TGF-beta 1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-beta 1 participating in local induction of corneal immune tolerance.
Anterior Chamber ; Corneal Transplantation ; Endothelium, Corneal/*drug effects ; Endothelium, Corneal/pathology ; Gene Transfer Techniques ; Immune Tolerance ; Transforming Growth Factor beta/administration &

The effect of topically applied 1% sodium hyaluronate (Na-HA) on the healing of a standardized corneal alkali wound was studied. The healing of the epithelium, stroma, and endothelium was evaluated separately, using quantitative methods. Central corneal alkali wound was produced in one eye of the rabbits by applying a 5.5-mm round filter paper, soaked in 1 N NaOH, for 60 seconds. 1% Na-HA in the treatment group and phosphate buffered saline (PBS) in the control group were given topically 4 times per day for 2 days, 1- and 3-weeks. Epithelial and endothelial healing was assessed morphometrically from standardized photographs and micrographs, respectively. Stromal healing was determined by counting polymorphonuclear leukocytes (PMN) and keratocytes in the central and marginal wound areas. A positive healing influence was observed in the epithelium. In stromal healing, 1% Na-HA treated corneas showed less PMNs and more keratocytes than the control group, suggesting that topically applied 1% Na-HA may suppress the stromal PMN infiltration and enhance the keratocyte repopulation during corneal alkali wound healing. However, no significant difference was found in morphometric evaluation of endothelial healing between the two groups.
Administration, Topical ; Animals ; Burns, Chemical/*drug therapy/etiology/pathology ; Cell Count ; Cornea/*drug effects/pathology ; Corneal Stroma/drug effects/pathology ; Endothelium, Corneal/drug effects/pathology ; Epithelium/drug effects/pathology ; Eye Burns/chemically induced/*drug therapy/pathology ; Hyaluronic Acid/administration & dosage/*pharmacology ; Ophthalmic Solutions ; Rabbits ; Sodium Hydroxide/toxicity ; Wound Healing/*drug effects