PURPOSE: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells. METHODS: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea. RESULTS: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species. CONCLUSIONS: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.
Adult ; Animal ; Cytokines/pharmacology ; Endothelium, Corneal/ultrastructure ; Endothelium, Corneal/metabolism* ; Endothelium, Corneal/cytology ; Human ; Microscopy, Electron ; Mucins/ultrastructure ; Mucins/metabolism* ; Owls ; Rabbits ; Staining and Labeling

OBJECTIVETo evaluate the feasibility of using cultured corneal endothelial cell (CECs) transplantation for cornea endothelium cell destruction with Gelatin membrane as the carrier in rabbits.

METHODSThe cultured CECs were labeled by Brdu and subcultured in vitro on glutaraldehyde-fixed Gelatin membranes and then the membranes were glued by alpha-cyanoacrylate alkyl to 7.00 mm autologous rabbit corneal bottons whose endothelium were mechanically removed previously. The buttons were sutured in place. With this method the right eyes of 21 rabbits were transplanted with CECs, and the right eyes of another 17 rabbits were transplanted with non-cells carrier as controls. The rabbits were bred and observed by slit microscopy and confocal microscopy at 1, 2, 4, 8, and 12 weeks after surgeries. Also, introcular pressure and corneal thickness were measured by Perkin's tonometer and ultrasonic pachymeter. After 12 weeks, all the animals were sacrificed and the grafts were examined by light microscopy and electronic microscopy.

RESULTSCECs grew well on the gelatine memberane, and formed confluent monolayers in 3-5 days; the cell density reached as high as 2700 cells/mm2. After 2 weeks of operation, all corneal buttons were edema and began to be opaque. The control eyes remained opaque throughout the observation period. In eyes with CECs transplanted, the grafts began to be clear and thin 4 weeks after operation. The cell density of grafts decreased along with time, and the mean cell density of CECs transplantation buttons was (2023.3 +/- 330.3) cells/mm2 12 weeks after operation. The transplanted cells were stained with the anti-Brdu monoclonal antibody.

CONCLUSIONIt is feasible to culture and translate CECs with the Gelatin membrane.

Animals ; Cells, Cultured ; Corneal Endothelial Cell Loss ; pathology ; therapy ; Endothelial Cells ; transplantation ; Endothelium, Corneal ; cytology ; Rabbits

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

BACKGROUNDThough there have been various methods for harvesting and preserving descemet membrane (DM) and intact endothelium, there is no literature about the morphological evaluation of endothelium after graft preparation for descemet membrane endothelial keratoplasty (DMEK). The aim of this study was to establish and improve a simple method for preparing, preserving, and morphologically evaluating the donor graft for DMEK.

METHODSTo obtain a donor graft, an air bubble was formed by injecting a 29 G needle with 1 ml sterile air into a small edge created outside the Schwalbe line. Another needle was inserted into the bubble through the stroma to aspirate the air or replace half the air with organ culture medium. Trypan blue was used to mark the location for small incision to improve the success rate. Frozen sections were stained with hematoxylin and eosin (HE). Based on the air bubble, DM grafts were divided into four groups: group A (normal control), graft without any operative technique; group B, graft with zero-pressure air bubble; group C, graft with full-pressure air bubble; group D, graft with half-pressure air bubble. The four groups of grafts were preserved for 24 hours to observe the effect of bubbles on cells. The gross and ultrastructure morphologies were evaluated using alizarin red and scanning electron microscopy (SEM), respectively.

RESULTSDonor grafts were harvested via the air bubble technique, facilitated by prior trypan blue staining. HE-stained sections revealed a pure graft without stroma. There were no significant changes under light microscope. In group A, SEM revealed a confluent layer of polygonal endothelium with distributed microvilli exhibiting characteristics of interdigitating junctions. In group B, intercellular borders became thinner. In group C, interdigitations were almost flat and microvilli were observed less frequently. In group D, other than less microvilli, there were minimal changes.

CONCLUSIONSThe donor graft preparation method appears to be effective and convenient. Properly decreasing the air pressure could protect and preserve the endothelium.

Animals ; Descemet Membrane ; cytology ; Descemet Stripping Endothelial Keratoplasty ; methods ; Endothelium, Corneal ; cytology ; Rabbits ; Tissue Donors

Twenty-six soft intraocular lenses were implanted in twenty-six senile cataract patients from luly 16, 1987, to April 15, 1988 at Kangnam St. Mary's Hospital, Seoul, Korea. The average age of the patients was seventy-six with a range from fifty-five to eight nine years old. Of the twenty-six patients at the 12 months follow up 87.5% have arhieved visual acuity of 20/40 or better. The average central corneal endothelial cell loss at postoperative 7 days and 3 months were 9.8% and 12.3% respectively. Complications were observed in six patients. Two patients had preexisting retinal and optic diseases and one had a fibrinous membrane. Three patients had transient pigmentary precipitates on IOL surface.
Aged ; Endothelium, Corneal/cytology ; Female ; Follow-Up Studies ; Humans ; *Lenses, Intraocular ; Male ; Middle Aged

OBJECTIVETo demonstrate the molecular expression of carbonic anhydrase IV (CA IV) in rabbit corneal endothelium.

METHODSReverse transcriptase polymerase chain reaction (RT-PCR) was performed using cultured and fresh rabbit corneal endothelial total RNA and specific primers for CA IV. The RT-PCR product was subcloned and sequenced. Immunoblotting and indirect immunofluorescence staining were performed to detect protein expression and distribution of CA IV using fresh and cultured rabbit corneal endothelium and rat anti-CA IV polyclonal antibody.

RESULTSRT-PCR screening gave positive bands at the predicted size for CA IV from fresh and cultured rabbit corneal endothelium. Sequencing further confirmed the identity of CA IV in corneal endothelium. Immunoblotting analysis showed a single band at 52 kDa for freshly isolated and cultured endothelial cells. Indirect immunofluorescence staining revealed an apparent positive staining in cultured endothelial cells.

CONCLUSIONCarbonic anhydrase IV is expressed in rabbit corneal endothelium, which could contribute to the transendothelial HCO(3)(-) flux that is necessary to maintain corneal hydration and transparency.

Animals ; Base Sequence ; Bicarbonates ; metabolism ; Carbonic Anhydrase IV ; analysis ; genetics ; Cells, Cultured ; Endothelium, Corneal ; cytology ; enzymology ; Fluorescent Antibody Technique, Indirect ; Molecular Sequence Data ; RNA, Messenger ; analysis ; Rabbits

A heterologous corneal endothelial transplantation was attempted using human endothelial cells and a Lewis rat penetrating keratoplasty model. Cultured human endothelial cells were seeded to a Lewis rat cornea, which was denuded of its endothelium. When grafted into the syngeneic Lewis rat, the graft remained clear for at least five days, and then became opaque and edematous because of immune rejection reaction. In contrast, corneas denuded of their endothelium became opaque and edematous immediately after transplantation. These results demonstrate that transplanted endothelial cells have enough antigens to induce rejection reaction even though they have the functional capacity to deturge the cornea.
Animals ; Endothelium, Corneal/cytology/immunology/*transplantation ; Female ; Graft Rejection/*immunology ; Humans ; Major Histocompatibility Complex/immunology ; Rats ; Rats, Inbred Lew ; Transplantation, Heterologous

Three hundred and forty-eight eyes (246 patients, both eye radial keratototmy in 102 patients) which could be followed-up for at least one year or more were included in this study. Postoperative uncorrected visul acuity which was 20/40 or more could be obtained in 79% of the lower myopic eyes (-1.75--2.75 D), in 73% of the moderate myopic eyes (-3.00--5.75 D) and in 34% of high myopic eyes (-6.00 D-). The postoperative refractive correction (spherical equivalent) ranged from plano to -9.0 D with a mean decrease of -3.86 D with a mean reduction of keratometry in moderate myopia of 3.33 D with a range from 0.5 D to 6.75 D. But its mean reduction in high myopia did not parallel its myopic degree. Glare and fluctuation of vision were the most frequent complaints following surgery. Some patients had continuous constant vision improvement for a long period while some patients (or other eyes) had episodes of decreased vision because of the recurrence of myopia or hyperopia shift. The mean central corneal endothelial cell loss determined 6-l2 months later was 5.31-5.61%. Microperforation in 11 eyes (3%) occurred during the early part of the study and improved naturally without any problem. An over-correction of more than +1.0 D (2%) and an under-correction of more than -1.0 D (1%) with having induced or residual astigmatism (17%) were observed. Radial keratotomy is suceessful in carefully selected patients with mild and moderate myopia, and also in anisometropic high myopia.
Adolescent ; Adult ; Endothelium, Corneal/cytology ; Female ; Follow-Up Studies ; Humans ; Keratotomy, Radial/*statistics & numerical data ; Korea ; Male ; Myopia/*surgery ; Prospective Studies ; *Visual Acuity

Primary and secondary corneal endothelial decompensation leads to stromal edema, corneal opacity and loss of visual acuity. The pathogenesis of corneal endothelial decompensation is that adult corneal endothelium in vivo lacks of a robust proliferative response to injury, does not divide sufficiently to replace the lost cells. Previous studies indicate that cell-cell contact inhibition and transforming growth factor-beta2 (TGF-β2) in aqueous humor may be responsible for maintaining human endothelial cells in a non-replicative state in vivo. The results of the experimental investigation by using immunofluorescent staining of the cell cycle-associated proteins and cell proliferation marker Ki67 in corneal endothelium indicate that human corneal endothelial cells in vivo are arrested in the G1-phase and have not exited from the cell cycle. Successful outgrowth in culture of human corneal endothelial cells in vitro and the establishment of the immortalized human endothelial cell line, provide strong evidence that corneal endothelial cells retain proliferative capacity. Experiments with cell culture ex vivo demonstrate that corneal endothelial cells cultured from young donors grow more robustly than those from older donors, and cells cultured from peripheral area of corneas show greater cell density than central regions. Studies have demonstrated that in vitro human corneal endothelia undergo mitotic changes in response to stimulation of growth promoting agents, such as growth factors, EDTA and extracellular matrix. Identification of corneal endothelial stem cells and isolation and culture of human endothelial precursor cells in vitro will be beneficial for further investigation regarding the mechanism of corneal endothelial regeneration as well as corneal endothelial cells in vitro culture.
Aqueous Humor ; metabolism ; Cell Count ; Cell Culture Techniques ; Cell Cycle ; Cell Proliferation ; Cells, Cultured ; Contact Inhibition ; Endothelium, Corneal ; cytology ; Humans ; Stem Cells ; Transforming Growth Factor beta2 ; metabolism

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