Mitochondrial oxygen metabolism in primary human lens epithelial cells: Association with age, diabetes and glaucoma.

PURPOSE: The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. METHODS: Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis. RESULTS: Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p<0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p<0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p<0.05), diabetic patients (p<0.01), and glaucoma patients (p<0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR. CONCLUSIONS: The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs.

Changes in adhesion complexes define stages in the differentiation of lens fiber cells.

PURPOSE: During their differentiation, lens fiber cells elongate, detach from the lens capsule, associate at the sutures, and degrade all cytoplasmic membrane-bound organelles. Changes in the expression or organization of cell adhesion and cytoskeleton-associated proteins were correlated with these events during fiber cell differentiation in chicken embryos. METHODS: Fixed or living lenses were sliced with a tissue slicer, permeabilized or extracted with detergents, stained with antibodies or fluorescent-labeled phalloidin, and viewed with a confocal microscope. The distribution of N-cadherin in elongating and mature fiber cells was determined by Western blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the distribution of vinculin and paxillin transcripts. RESULTS: Staining for N-cadherin and band 4.1 protein decreased soon after fiber cells detached from the capsule. Detergent extraction of lens sections and Western blots of dissected lens regions showed that much of this decrease in staining was due to epitope masking. Vinculin immunoreactivity was barely detectable on the lateral membranes of elongating fiber cells but increased markedly once these cells reached their maximum length and formed the sutures. Staining for paxillin was also low in elongating fiber cells but increased late in fiber cell differentiation, just before the cells destroyed their membrane-bound organelles. Spectrin and ankyrin immunoreactivity did not change when fiber cells reached the sutures. Staining for F-actin increased transiently in cells that had just reached the sutures. Messenger RNAs for vinculin and paxillin were more abundant in maturing than in elongating fiber cells. CONCLUSIONS: The adhesion complexes of lens fiber cells change in organization and composition soon after these cells finish elongating and detach from the capsule. Increased staining for vinculin and paxillin defines distinct stages of fiber cell differentiation that are intermediate between the completion of cell elongation and the time when lens fiber cells degrade their membrane-bound organelles. Remodeling adhesion complexes during fiber cell maturation may assure the stability of fiber-fiber associations, once these cells are no longer transcriptionally active.

Morphological observation on cell death and phagocytosis induced by ultraviolet irradiation in a cultured human lens epithelial cell line.

The purpose of this study is to observe dynamic morphological changes induced by ultraviolet (UV) irradiation in a cultured human lens epithelial cell line using electron microscopy, cell viability staining, time-lapsed videography and immunohistochemistry. Human lens epithelial cell line SRA 01-04 was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20% fetal bovine serum. Subconfluent cells were irradiated under a bank of UV lamps, which emitted 275-400 nm radiation with a maximum at 310 nm. The UV intensity was 20 microW cm(-2)at dosages from 0 to 10 mJ cm(-2). Alterations in the morphology of the living cells were monitored and recorded with phase-contrast microscopy and time-lapsed videography. At different times, the cells were fixed and examined by transmission electron microscopy (TEM), diamidinophenolindole (DAPI) staining, and in situ immunohistochemistry using TdT-mediated dUTP-biotin nick end labeling (TUNEL). Cell viability was also assessed with crystal violet staining. At low doses of UV exposure (2-5 mJ cm(-2)), time-lapsed videography revealed definitive cell death that appeared to be primarily apoptotic. The dead cell debris was engulfed and phagocytosed by neighboring living cells. Phase-contrast microscopy and TEM demonstrated that, at UV 10 mJ cm(-2), the cells not only showed typical apoptosis such as nuclear membrane shrinkage, chromatin condensation, and fragmentation into apoptotic bodies, but also necrosis such as swelling of the nucleus and cell body, and disruption of the plasma membrane. In support, DNA staining and in situ immunohistochemical reactions in the UV irradiated cells were both positive. The phagocytotic process was also seen with TEM. UV irradiation thus appears to cause both apoptosis and necrosis in the cultured human lens epithelial cell line. Active migration and phagocytosis of the cells appear to be stimulated by UV-induced damage. These findings may also aid in the understanding of UV injury and repair mechanisms of lens epithelial cells in vivo.

Location and severity of UVB irradiation damage in the rat lens.

We investigated the location and severity of lens opacities and epithelial alterations following ultraviolet-B (UVB) irradiation in vivo, using Brown Norway rats. A group of 9 rats received 65 mJ/cm2 UVB irradiation from overhead lamps every 6 days. Lens changes were documented and evaluated by an anterior eye segment analysis system. Lens epithelial cells were examined postmortem in flat preparations. After 8 weeks of the irradiation schedule (total dose: 0.6 J/cm2), an anterior polar opacity was apparent; at 16 weeks, the opacities had progressed more deeply into the cortex. At postmortem examination, cells in the central region displayed disorganization, clumping, some pyknotic nuclei and mitosis. There were deeper opacities and cell damage was more severe above the central horizontal plane than below it. This present study demonstrated that UVB damage differed in the superior and inferior parts divided by a horizontal plane through the lens anterior pole, when the UVB source was above and there was no reflection from below or laterally. The lens epithelial cells, and associated lens fibers, are the first target of UVB irradiation.

Experimentally induced steroid cataract in the rat: a scanning electron microscopic study.

To clarify the histopathological changes of experimentally induced steroid cataract in Brown-Norway rat eyes, a scanning electron microscopic study was performed. The biomicroscopic appearance of the cataracts, which was quite similar to that of human eyes, was induced by daily application, either topically or systemically, of prednisolone acetate for 12 months. A single dose of 2 Gy X ray was given to the right eyes of all the animals 2 weeks before drug administration. Twenty-seven rats were divided into three groups: a control group (CTL), a group receiving topically administered prednisolone (TOP), and a group receiving systemically administered prednisolone (SYS). In vivo observation was performed through a slit-lamp microscope, and the lens findings were documented and objectively analyzed by an anterior eye segment analysis system over a period of 12 months. At the end of the 12-month period, X-ray-irradiated right eyes in the CTL group showed some minor lens changes on biomicroscopy, and non-X-ray-irradiated left eyes were almost normal. In the TOP and SYS groups, either with or without X-ray irradiation, lenses showed anterior and posterior subcapsular opacification; however, the grade and increase of lens opacification were higher and faster in the eyes with X-ray irradiation. Scanning electron microscopic findings of the three groups at the 12th month were as follows. In the CTL group, the X-ray-irradiated right eyes showed minor changes. In the TOP group, the X-ray-irradiated right eyes showed marked damage in the lens fibers of the anterior and posterior cortices, while the nonirradiated left eyes showed minor changes. In the SYS group, the X-ray-irradiated right eyes showed prominent cataractous disorganization of lens fibers in the shallow anterior and posterior cortices, and the non-X-ray-irradiated lenses were almost the same as those in the TOP group. These findings might suggest that even a minimum invasion of a low dose of X-ray irradiation plays a cocataractogenic or syncataractogenic role during the formation of steroid cataracts.

Inhibition of steroid-induced cataract in rat eyes by administration of vitamin-E ophthalmic solution.

The efficacy of a vitamin-E (VE) ophthalmic solution was evaluated on a newly developed rat steroid-induced cataract model. Brown Norway rats irradiated with 2 Gy X-ray, right eyes only, were divided into 5 groups: the control group; 2 steroid (1 mg/kg/day)-treated groups with topic (Top) and systemic (Sys) administration, and 2 VE-treated groups, 1 with the same treatment as the Top group with the addition of 5% VE twice a day (Top + VE) and 1 with the same treatment as the Sys group with 5% VE twice a day (SYS + VE). The lens changes were documented with a Scheimpflug camera and changes in light scattering were evaluated quantitatively. The VE-treated groups (Top + VE and Sys + VE) showed a significant inhibition of the increase in the opaque area compared with each of the non-VE-treated groups. The VE ophthalmic solution was strong enough to prevent steroid-induced cataract in rats.

[1585 cases of retinal detachment surgery with homologous skin as buckling material]. / Verwendung von homologer Kutis als Plombenmaterial in 1585 Fällen von Ablatiooperation.

From January 1963 to December 1989, 1585 consecutive cases of retinal detachment were operated with homologous skin as buckling material, the rate of operative success being 91.6%. Human skin is easy to obtain, sterilize and preserve; because of its appropriate thickness and firm but elastic consistency it satisfactorily meets the need to produce sufficient height and to maintain necessary duration of the scleral buckling; in addition to a high rate of reattachment, homologous skin implantation was well tolerated, very rarely rejected (0.06%) and infected (0.25%), and no late complications occurred. Therefore, the authors prefer using human skin as buckling agent rather than conventional synthetic material, e.g., silicon sponge etc.