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.

Characteristics of cataracts in the Chinese Singaporean.

PURPOSE: To conduct an epidemiological survey of cataracts and examine the characteristics of lens opacities in Chinese Singaporeans. The results are then compared with those from two similar surveys previously done in Japan in Noto Area, Honshu, and Amami, Kyushu, respectively. SUBJECTS AND METHODS: 468 subjects of > or = 50 years of age, who responded to the invitation to participate, were examined. Examination principally included photo-documentation of the anterior and posterior segments of both eyes. Evaluation and grading of lens opacities were done using graphical analysis of Scheimpflug and retro-illumination images. Inter-group comparisons were based on statistical analysis of cataract prevalence and distribution. RESULTS: The prevalence of clear lenses decreased with aging with no significant difference between males and females--a finding common to Singapore and the two Japanese study groups. The prevalence of cataracts (or lens opacities of Grade II and above) in 60-79 year-old Singapore subjects was significantly higher than Noto and Amami subjects in the same age group. Further, cortical opacity was the main type in Singapore subjects in their 50s and which was significantly higher than Noto subjects of the same age group. In all age groups, the distribution and prevalence of both nuclear and subcapsular types in the Singapore group were higher than the two Japanese study groups. CONCLUSIONS: Cataracts in Chinese Singaporeans are characterized by a high prevalence of nuclear opacities which was generally seen in tropics and sub-tropics. Our study also suggested the involvement of solar-UV in cortical cataracts as well as that of additional risk factors, such as environmental temperature and race, in nuclear and subcapsular cataract formation.

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.

Refractive errors in Singapore and Xiamen, China--a comparative study in school children aged 6 to 7 years.

PURPOSE: To compare and contrast the prevalence of myopia and other refractive errors in Xiamen city, Xiamen countryside (Southern China), and Singapore. METHODS: One hundred thirty-two schoolchildren aged 6 to 7 years from Xiamen city, 104 from Xiamen countryside, and 146 from Singapore city were recruited to join the study. Cycloplegic autorefraction, keratometry, and biometry measurements were performed on all children. RESULTS: The prevalence of myopia was 12.3% in Singapore city, 9.1% in Xiamen city, and 3.9% in Xiamen countryside. The prevalence of astigmatism was higher in Singapore compared with Xiamen. The rates of hyperopia and anisometropia were similar in all three locations. CONCLUSIONS: The myopia rate in Singapore city was higher than in Xiamen city; the lowest rates were found in Xiamen countryside. As the Chinese population from all three sites is of similar genetic stock (predominantly from Southern China), it is postulated that the differences in myopia rates in these three localities may be related to environmental factors.

Epidemiological studies on UV-related cataract in climatically different countries.

Cataract epidemiological surveys applying objective judgement through lens images in the climatically different places of Noto and Amami, Japan, Singapore and Reykjavik, Iceland yielded several significant results about the influence of solar UV. 1) The percentage of transparent and of lens opacification was significantly higher in the Reykjavik subjects than in the Singaporeans. 2) The percentages including early changes were higher in Amami and Singapore than in Noto and Reykjavik. 3) Progressed lens opacification was highest in Singapore. While the main type of lens opacification was cortical in Noto and Reykjavik, that of Singapore was nuclear. 4) A significant correlation between cortical opacification and the history of time spent outdoors was noticed. The UV risk for formation and/or progression of cortical opacification should be acceptable from the epidemiological standpoint.

UV damage to the eye lens: further results from animal model studies: a review.

UV irradiation has the potential to induce the development of lens opacities. This has been demonstrated since long with animal experiments. Unfortunately these animal cataracts did not explain or elucidate the epidemiological observation that the frequency of human cataracts--such as the so called senile cataract--is remarkably higher in regions with increased cosmic UV irradiation or in the population being in close professional contact with UV-irradiation. The main problem was that the type of UV induced animal cataracts differs remarkably with respect to onset, localization of the opacity, size and its timely progression from the cataract classes observed in human. The research of the last 10 years comes to the conclusion that beside the direct (acute) damage--as seen in animal studies due to high UV dosages--we have to realize a syn- or co-cataractogenic potential of UV irradiation even below the threshold dose which is able to accumulate in the lens and to initiate together with other risk factors (chronic damage) the opacification of the lens. The mechanism for the animal cataract and the human cataract (with an UV risk participation) are different. The epidemiological research about cataract frequency in different regions of the world have to take into account that UV irradiation--even below a threshold dose--is a possible risk among the multifactorial pathogenesis of human cataract.

In vitro UV-B effect on lens protein solutions.

To investigate the effects of UV-B exposure on the protein solutions of different lens parts, rabbit lenses were separated into the equator (Eq), anterior cortex (Ac), nucleus (Nu) and posterior cortex (Pc). After homogenization, the water-soluble protein from each part was irradiated with UV-B at 0 to 0.225 J/cm2. Alterations in the content of protein SH, carbonyl groups, light scattering intensity and SDS-PAGE pattern were measured to compare the effect of UV-B on the protein solutions of various lens parts with or without additional GSH to test its preventive effect. The results showed that after UV-B irradiation, the protein sulfhydryl groups are gradually reduced. The nonprotein thiol (GSH added to the protein solution) was lost more rapidly than the protein sulfhydryl. The high molecular bands on the SDS-PAGE pattern mainly aggregated with disulfide. UV-B damage also increased the content of carbonyl groups and light scattering, irrespective of the lens parts. Lens proteins from the equator suffered the least damage while those of the nucleus were most strongly affected by UV-B exposure. This study suggests that the lens proteins from various lens parts have different responses to UV-B exposure; the sensitivity was in the following order: Eq < Ac < or = Pc < Nu.

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.

A new steroid-induced cataract model in the rat: long-term prednisolone applications with a minimum of X-irradiation.

In order to induce experimental steroid cataracts in rat eyes similar morphologically to those seen in human eyes, prednisolone acetate was administered either topically or systemically for 12 months with a low dose of X-irradiation as a cocataractogenic factor. Twenty-seven Brown-Norway rats were randomly divided into a control group (group I) with no steroid administration; an eyedrop group (group II) with a daily 1% prednisolone acetate instillation of a total volume of 1.0 mg/kg in both eyes, and a systemic group (group III) with a daily intramuscular injection of 0.8-1.0 mg/kg prednisolone acetate. The right eyes of animals in each group were X-irradiated with a single dose of 2 Gy. Topical and systemic steroid administrations started 2 weeks after X-irradiation. Anterior segment changes were documented with a slitlamp microscope and an anterior eye segment analysis system once a month. Body weight and blood glucose levels were examined every week and every 2 weeks, respectively. The mortality rates in groups I, II and III were 0, 11 (1/9) and 25% (3/12), respectively. The both lenses in group I showed a gradual increase in light-scattering intensity in the nuclear and supranuclear regions over time. Initial lens changes in both steroid-treated groups were Y-suture dissociation and a slight increase in light-scattering intensity in the posterior supranuclear region 3 months after prednisolone administration. Opacification of the anterior shallow cortex and the posterior subcapsular layer was observed after 10 months. X-irradiated eyes showed more prominent lens opacification as compared with nonirradiated eyes after 10 months in both group II and group III. Either topical or systemic administrations of prednisolone acetate over a long term successfully induced morphological lenticular changes in the rat similar to those found in human steroid-induced cataracts. A low dose of X-irradiation effectively accelerated opacification as a cocataractogenic risk. This new model will allow future investigation of steroid cataracts.

In vivo morphological changes in rat lenses induced by the administration of prednisolone after subliminal X-irradiation. A preliminary report.

In order to induce steroid cataracts in rat lenses, prednisolone acetate was administered together with a single subliminal dose of X-irradiation, which was applied unilaterally before steroid application started. The rats were divided into a control group (without prednisolone acetate administration), a group with a topically administered daily dose of 1 mg/kg prednisolone acetate suspension and a group with a systemically applied daily dose of 0.8-1.0 mg/kg prednisolone acetate suspension. Changes in the lens were objectively evaluated with in vivo Scheimpflug slit images during a 30-week period. Although body weight increase was significantly affected in both groups administered prednisolone, animals survived until the end of the observation period. The initial changes in the lens were the dissociation of the Y-suture and a slight increase in scattering light intensity at the posterior embryonic nucleus after both topical and systemic steroid administration.

Topographic distribution of prednisolone in the lens after organ culture.

Topographic distribution of prednisolone in the lens after organ culture was investigated. Freshly enucleated pig lenses were divided into two groups; the complete vitreous was removed from the lenses in group A, and the adherent parts of the vitreous were left in group B. Groups A and B were each divided into two subgroups which were separated according to drug application: two groups underwent drug application by a divided chamber (Iwata, 1982, DC) and two groups by a closed chamber. The incubation medium was TCM-199 with 2 micrograms/ml prednisolone, and the incubation was performed for 8 h. In order to know the participation of the vitreous in drug penetration into the lens, prednisolone was applied by a divided chamber from the anterior or posterior side, with or without vitreous, and incubated for 4 h as a first incubation. A secondary incubation was performed for another 4 h with a medium that did not contain prednisolone. After the lens incubation, the lenses were separated into single lens layers, and the drug concentration of each lens layer was measured by high performance liquid chromatography. The highest prednisolone concentration in the lens was detected by a closed chamber in the equatorial part followed by the posterior cortex, anterior cortex and nucleus. There were no significant differences in drug concentration due to the direction of drug application or the absence or presence of vitreous. The prednisolone distribution pattern in the lens by DC depended on the direction from which the drug was applied. The highest drug concentrations were detected in the lens cortex of the drug-applied side followed by the equator. No drug was detected in the side of the lens opposite the drug-applied side or in the nucleus. When a drug was applied from the posterior lens side, the presence of the vitreous made a big difference in the amount of drug penetration. The comparison of drug concentration between the absence or presence of the vitreous after secondary incubation in a medium that did not contain prednisolone indicated that the vitreous participates in the drug kinetics of the lens. The experiments suggest that while prednisolone penetration into the posterior lens part is small compared to that of the anterior part, when the steroid penetrated into the posterior part it remained there for a long period.

[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.