Noncontact specular microscopy of human lens epithelium.

PURPOSE: To obtain in vivo specular images of human lens epithelial cells (LECs) from persons with or without age-related cataract (ARC); to identify features that describe individual aspects of these complex images; to develop feature scales to quantify the severity of each feature; and to study the association of these features with LEC count, age, Lens Opacity Classification System III (LOCS III) classifications and microscopic features of lens epithelium in ARC. METHODS: One hundred fifty-two individuals underwent ophthalmic examinations and LOCS III cataract classifications. Specular images of lenses were captured using a modified noncontact corneal specular microscope (SML-2; Konan, Hyogo, Japan). Enhanced images were graded in a masked fashion, and the presence or absence and severity of each of four features in the specular image ("columnar organization," "linear furrows," "puffy clouds," and "black holes") was graded on a four-step scale. The generalized linear model with intraclass correlation was used to ascertain the statistical significance of associations between age, sex, LOCS III grade, cell count, and feature grade. Capsulorrhexis specimens from 29 patients were studied with correlative light and electron microscopy. RESULTS: LEC density declined with age and was inversely correlated with the scalar grade for puffy clouds and for the size and number of black holes. The scalar grade for columnar organization was inversely associated with the severity of posterior subcapsular and nuclear cataracts, which was the only feature associated with the LOCS III grade of ARC. No statistically significant associations were found between average cell count and LOCS III grade. CONCLUSIONS: With the use of the corneal specular microscope excellent in vivo specular images of the LECs were obtained, the features in these images that correlated well with microscopic findings were classified, and cell density in vivo was estimated.

Correlation of nuclear color and opalescence with protein S-thiolation in human lenses.

Human lens nuclei were collected during routine cataract surgery and used to study the role of oxidation in cataract formation and brunescence. This study focused on the comparison of the intensities of nuclear opacity and pigmentation (brunescence) with the changes in free glutathione (GSH) and the three species of protein-thiol mixed disulfides: protein-S-S-glutathione (PSSG), protein-S S-cysteine (PSSC) and protein-S-S-gamma-glutamylcysteine (PSSGC). Eighty-one freshly excised human lens nuclei from a population with a mean age of 77 were used. The nuclear color was graded using the CCRG system, ranging from yellow to dark brown. The nuclear cataract opalescence of these lenses was also graded using the LOCS II system, ranging from LOCS II NO-1 to NO-4. Three normal human lenses (average age of 88 yr) were also included in the study as controls. The nuclear samples were each analyzed for free GSH and protein-thiol mixed disulfides, respectively. It was found that nuclear GSH decreased as the nuclear color increased from yellow to dark brown (from 0.73+/-0.13 to 0.13+/-0.03 micromole g wet wt-1) and as the nuclear opalescence increased from NO.1 to NO.4 (from 0. 80+/-0.19 to 0.20+/-0.01 micromole g wet wt-1). All these values were lower than that of GSH in normal controls (1.43+/-0.59 micromole g wet wt-1). Levels of both PSSG and PSSC progressively increased, however, as the nuclear color intensified. PSSG increased from 0.29+/-0.05 to 0.91+/-0.11 micromole g wet wt-1while PSSC increased from 0.13+/-0.04 to 0.41+/- 0.06 micromole g wet wt-1. PSSGC concentration progressively increased with increases in both nuclear pigmentation (from 0.05+/-0.01 to 0.23+/-0.05 micromole g wet wt-1) and nuclear opacity (from 0.02+/-0.00 to 0.20+/-0.02 micromole g wet wt-1). In comparison, normal controls had lower levels of all three mixed disulfide species: PSSG, 0.22+/-0.06; PSSC, 0.08+/-0.02; PSSGC, 0.02+/-0.06 micromole g wet wt-1, respectively. The correlation of lens nuclear color and opalescence intensity with nuclear protein S-thiolation indicates that protein-thiol mixed disulfides may play an important role in cataractogenesis and development of brunescence in human lenses.

Standardization and analysis of digitized photographic data in the longitudinal documentation of cataractous growth.

Age-related cataract formation in man can be documented with slit and retroillumination photographs. With digitization and image analysis of such photographs a cataract may be characterized by a frequency distribution of picture elements over a 255 step gray scale spectrum. Transition from a clear to a cataractous lens may be manifested as a change from a unimodal, Gaussian to a multimodal, non Gaussian frequency distribution respectively. How should one compare and contrast these two distributions, so to accurately describe the extent and significance of a change in lens opacification? The in vitro system of cold cataract formation in the rabbit lens was used as a model of the much slower process of age-related cataract formation in man. As in the human lens undergoing progressive opacification, the frequency distribution (number of pixels vs. intensity of gray) for a digitized image of a clear lens at 26 degrees C is unimodal and Gaussian; that of a fully developed cold cataract at 10 degrees C is multimodal and non-gaussian. In spite of the increasing multimodality of the frequency distribution as the temperature dropped and the cataract grew in density and size, the mean gray density proved to be a valid and useful measure to characterize the distribution and to compare different unaligned images. The Wilcoxon Rank Sum Test proved to be useless in comparing the frequency distributions from cataract images because it proved to be too sensitive to subtle changes in the degree of opacification. Anomalous behavior of the opacification process--i.e. clarification as well as opacification of the lens during cold cataract formation caused all pairs to appear statistically significantly different when in appearance there was no difference. The mean of the frequency distribution is less sensitive to this anomalous behavior and is useful as a comparative index. The method of calculating the threshold of significant change in the mean density of a cataract image is presented.

Lens hexokinase deactivation by near-UV irradiation.

Photodamage to lens hexokinase has been investigated by exposing the lenses of rat, rabbit and calf eyes to 300 nm irradiation. Hexokinase activity was diminished by 15.9% +/- 5.4 and 23.4% +/- 5.0 upon irradiation of the isolated rat lens for 1 and 2 hours respectively. Irradiation of the whole eye for 2 hours resulted in hexokinase deactivation of 13.6% +/- 5.8 and 19.2% +/- 6.2 for rat and rabbit lens homogenates and 55% +/- 7 for calf lens capsule plus epithelium. Enzyme deactivation was prevented when the isolated lens was irradiated with the vitreous attached. Glucose, catalase or ascorbate added to the medium prior to irradiation, each had a protective effect on hexokinase deactivation. The results are consistent with a mechanism in which photochemical generation of active species of oxygen, via the photosensitizing action of tryptophan photoproducts, plays a significant role in enzyme deactivation.

The protective effect of glucose on soluble rat lens hexokinase in the presence of oxidative stress.

An in vitro animal model was used to characterize the protective effect of glucose on lenses subjected to oxidative stress. Paired rat lenses were incubated in TC-199 medium for six hours in the presence of an oxidant (0.06 mM H2O2, superoxide produced from 5 mM purine, or hydroxyl radical) and 2 mM glucose (control) or no glucose (experimental). Soluble hexokinase (HK) specific activity and lactate production were measured. 0.06 mM H2O2 inactivates 48% of the hexokinase in the absence of glucose; with glucose present hexokinase activity is reduced only 26%. Control experiments without oxidants show a statistically insignificant difference between hexokinase activities in the 0 and 2 mM groups, suggesting that the changes observed are not simply due to the presence or absence of glucose. Hexosemonophosphate shunt activity increases nearly 2.5-fold in the presence of 0.06 mM H2O2 and 2.0, 4.0 or 5.5 mM glucose. This suggests that the loss of hexokinase (a -SH enzyme) in the presence of H2O2 and 0 mM glucose is due to NADPH production inadequate to offset the oxidative stress on enzyme -SH groups. FPLC analysis suggests that type II HK is more susceptible to oxidative inactivation than type I, and further studies have shown that this inactivation is localized to the capsule/epithelium. Lactate levels were measured and controls (without oxidants) were run, to obtain a baseline value for fresh lenses and assess the contribution of endogenous glucose to lactate production. H2O2 levels in superoxide and hydroxyl radical media were measured, and the protective effects of mannitol and catalase were also determined.

Inexpensive stereoscopic CCRG camera for lens/cataract photography in vitro.

In 1978 the American Cooperative Cataract Research Group (CCRG) adopted a stereoscopic camera system for photographing human cataracts in vitro based upon a Zeiss OPMI 1 operating microscope (Carl Zeiss, Inc.; Oberkochen, West Germany). Photographs obtained with this system were used to classify human cataractous change according to the standardized CCRG protocol. Classification data correlated with laboratory data furthered the attempt to define the biochemical or biophysical basis for specific types of cataractous change. Presently, the high cost of this camera (exceeding $20,000) precludes its use by many laboratories wishing to do human lens research. This study describes an inexpensive (less than $2,200) alternative camera for this type of photography. Adjacent frames on the film strip constitute stereo pairs which can be viewed in a modified stereo viewer. In the original CCRG camera both members of a stereo pair were included in the same frame. The quality of the stereo images obtained with this new system nearly equals that with the original Zeiss system. It is hoped that this inexpensive system will allow more scientists to participate in CCRG-related research and increase the supply of intracapsularly extracted cataracts available to all collaborating CCRG scientists.

Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus.

We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.

Classification of human senile cataractous change by the American Cooperative Cataract Research Group (CCRG) method: II. Staged simplification of cataract classification.

One thousand nine hundred and seventy-six cataracts extracted intracapsularly were classified according to the system adopted by the Cooperative Cataract Research Group (CCRG) Consortium. A nine-stage protocol for simplifying the classification data is presented. The method of simplifying the basic CCRG classification emphasizes anatomic similarities among lenses. Simplification is indicated when small numbers of cataracts in any class provide insufficient statistical power to allow detection of scientifically or clinically important differences in rates between comparison groups, if such differences exist; however, simplification unavoidably obscures the anatomic, biochemical, and biophysical features of individual cataracts. Age-specific characteristics of this population of 1976 cataracts are used to demonstrate the effects of the simplification process. The preponderance of mixed cataracts and the relative scarcity of pure cataracts are documented, and the implications of these numbers for cataract research are presented.

Classification of human senile cataractous changes by the American Cooperative Cataract Research Group (CCRG) method. I. Instrumentation and technique.

The American Cooperative Cataract Research Group (CCRG) has adopted a system of classifying human cataractous changes that is based on separate and independent photographic documentation of opacification and nuclear color. This system has been extremely useful to the laboratory scientist who wishes to know the significance of associations between laboratory data and the extent or type of cataractous change. It has been applied to the analysis of nearly 2500 cataracts since 1976. This study presents the details of the instrumentation and technique of this new system and the results of classifying 2231 intracapsularly-extracted cataracts.

Phase separation in rat lenses cultured in low glucose media.

Experimental evidence that indicates the nuclear opacity that develops in the hypoglycemic cataract in rat lenses is the result of a phase separation of the lens cytoplasm is presented. Phase diagrams of the cytoplasm of organ-cultured lenses were constructed for a series of incubation times in normal and hypoglycemic media. In normal media, the maximum phase separation temperature, Tmax, of the lens cytoplasm decreases with time. In hypoglycemic media, Tmax increases with time and exceeds 37 C after 24 hrs. The increase in Tmax can be stopped and reversed if the lens is returned to normal media within 8 hrs; in good agreement with an earlier biochemical analysis of the kinetics of hypoglycemic cataract formation.

Nonenzymatic glycosylation of bovine lens crystallins. Effect of aging.

We have investigated nonenzymatic glycosylation of crystallins from calf and mature bovine lenses (2-6 years old). The lens homogenates were treated with 200-fold molar excess of [3H]NaBH4 and the incorporation of radioactivity was determined. The extent of glycosylation was more precisely determined from the 6 N HCl hydrolysate of [3H]borohydride-treated proteins by analyzing the glucitol-lysine adduct on a high pressure cation exchange column. We found that the [3H]NaBH4 incorporation and the amount of glucitol-lysine detected increased with age, particularly in HM alpha crystallin, a high molecular weight aggregate which accumulates with aging. This age-related increase in nonenzymatic glycosylation was also demonstrated by a comparison of crystallins isolated from the cortex and nucleus of a single lens. Nonenzymatic glycosylation of lens crystallins exemplifies a new form of post-transitional modification of long-lived proteins in vivo.

The sorbitol pathway in the human lens: aldose reductase and polyol dehydrogenase.

The sorbitol pathway in human lenses is evaluated on the enzymic level. Adult lenses, normal and nondiabetic as well as diabetic cataracts, are found to contain limited levels of aldose reductase (AR) and high levels of polyol dehydrogenase (PD) relative to the animal lens. AR is confined primarily to the lens epithelium and is two to three times higher in juvenile lenses than in the adult lens. The level of AR in the epithelium of juvenile lenses is sufficient to cause significant osmotic stress. The Km of glucose of AR is roughly 200 mM, whereas the Km for NADPH is 0.06 mM. NADP inhibits human lens AR noncompetitively and has a Ki equivalent to the Km for NADPH. PD occurs in both the lens epithelium and cortex, remains persistently high with age, and decreases with increased cortical involvement. The Km of sorbitol for PD is 1.4 mM and for NAD is 0.06 mM. NADH (Ki 0.002 mM) competitively inhibits PD in the forward direction. PD purified 100-fold from diabetic and nondiabetic cataracts and normal lenses exhibit similar kinetic constants. PD has an extremely high Vmax in the fructose-to-sorbitol direction. The Km of fructose is 40 mM and for NADH is 0.02 mM. At high enough concentration, alrestatin also inhibits PD. The added activities of AR and PD in producing sorbitol and fructose in combination with decreased hexokinase with age may account for diabetic cataract formation in human lenses exposed to a high glucose stress. Nucleotide levels are reported for senile cataractous lenses.

Studies on human cataracts. I. Evaluation of techniques of human cataract preservation after extraction.

The effects of several media and temperatures on the features (i.e. classification) of human cataracts have been studied with the aim of defining those storage conditions that least alter the appearance of the cataract after extraction. It was found that distilled water, normal saline, TC 199 medium with extra bicarbonate and 5.5 mM glucose, and silicone oil at 4 degrees, 36 degrees, and 37 degrees C are all unsatisfactory for preserving cataracts for more than 1 hr. Normal saline and TC 199 medium with bicarbonate and glucose are satisfactory for period not exceeding 30 to 60 min at 4 degrees C. The best set of conditions, one that leads to no significant change in the appearance of the cataract, is storage in a small covered glass vial at 4 degrees C with no added fluid. In this moist chamber, no significant changes in cataract classification occurred during a 4 hr period. The effect of -20 degrees C for 15 min on the appearance of a nuclear cataract was negligible, and it was concluded that lenses so treated could be used for a correlation between the nuclear cataract's CCRG classification and its light scattering properties, as studied in Parts II and III.

Studies on human cataracts. II. Correlation between the clinical description and the light-scattering parameters of human cataracts.

Eight lenses with nuclear cataracts have been classified. The light-scattering properties of the eight lenses were obtained with thin sections cut perpendicularly to the posterior-anterior axis. Analysis of the light scattering envelop in the I and I+ models yielded eight structural parameters that describe the density and orientation fluctuations. The variation of these parameters within each less corresponded well to the clinical description obtained from the stereoscopic photos.

Differences in the susceptibility of various aldose reductases to inhibition. II.

The susceptibility of human lens aldose reductase (HLAR), human placental aldose reductase (HPAR), and rat lens aldose reductase (RLAR) to inhibition by 10 structurally diverse inhibitors is compared. Significant differences in the susceptibility of these enzymes was observed; however, no trends could be predicted. In general, HPAR appeared to be less susceptible to inhibition than either HLAR or RLAR, with the susceptibility of HLAR being more similar to RLAR than to HPAR. These findings indicate that the evaluation of aldose reductase inhibitors for potential clinical use may require the use of human aldose reductase from the appropriate target tissue.

Efficacy of Alrestatin, an aldose reductase inhibitor, in human diabetic and nondiabetic lenses.

Immediately after cataract extraction, lenses from diabetic and nondiabetic patients were collected, classified, and assayed or incubated in high-glucose medium. The distribution of cataract types within the diabetic and nondiabetic groups was almost identical. The aldose reductase (AR) inhibitor AY22,284 (Alrestatin) was as effective in blocking sorbitol formation in diabetic as in nondiabetic lenses. While there was no difference in the level of intralenticular glucose, the diabetic lens produced significantly more sorbitol than did the nondiabetic lens. Also, the activity of polyol dehydrogenase (PD) was much lower in the diabetic population. The diabetic lenses swelled slightly more (P <.2) than nondiabetic lenses in high glucose media, and AY22,284 was effective in reducing the swelling of diabetic lenses in 35.5 mM glucose medium. While these results are preliminary, they suggest that diabetes, in some way, may confer on the human lens an increased susceptibility to osmotic stress via the sorbitol pathway. It is also reassuring to note that an AR inhibitor is no less effective in blocking the more active AR in the diabetic than in the nondiabetic lens. The therapeutic implications of this are discussed.