Intact Lens Research Articles

Protective Effect of the Anterior Lens Capsule during Extracapsular Cataract Extraction: Part I. Experimental Animal Study

In an experimental study using albino Rex rabbits, the intercapsular cataract extraction (ICCE) technique was performed in 20 eyes with a small anterior capsulotomy. A large, can opener capsulotomy was performed in another 20 eyes, and 10 unoperated eyes served as controls. Endothelial cell loss was determined by vital staining with Trypan blue and Alizarin red S stains. Average endothelial cell loss with the ICCE technique was 1.2%; with the can opener technique, the average cell loss was 6.6%. This difference was statistically significant (P < 0.01). The percentage of endothelial cell loss in the control eyes was 0.5. A positive correlation between endothelial cell loss related to phacoemulsification time and/or the amount of irrigating fluid used existed for the can opener group only (P < 0.01). These results demonstrate that the presence of an almost intact anterior lens capsule during removal of lens substance is protective to the corneal endothelium.

Packing interactions in the eye-lens: Structural analysis, internal symmetry and lattice interactions of bovine γIVa-crystallin

γ-Crystallins are a family of low molecular weight proteins found in high concentration in the densely packed regions of high refractive index in vertebrate lenses. Certain members have the characteristic property of a high critical temperature ( t c) for phase separation. We report the three-dimensional structure determination of such a protein, bovine lens γIVa-crystallin, which has been refined to give an X-ray R-factor of 0.143. Its high t c contrasts with the low t c γII-crystallin, whose structure we have already published. The root mean square difference between the α-carbon atoms of these two proteins is 0.70 Å and γIVa has an internal symmetry even higher than that of γII. The presence of a protein that exhibits the phenomenon of phase separation at body temperature renders the lens very susceptible to a transformation from transparent to an opaque state due to irregularities in the refractive index. Protein interactions of γIVa-crystallin have implications for the mechanism of cataract formation. Modes of self-association behaviour of γIVa-crystallin have been inferred from an analysis of the lattice interactions in the crystalline state, where the protein packing density is similar to that of the intact lens. It appears that the point mutation at position 103 from a serine residue in γII to a valine in γIVa gives rise to a lattice contact formed by two four-stranded β-sheets in γIVa. A group-specific mutation at position 118 from leucine to phenylalanine induces subtle differences in core packing, leading to a reorganization around residue 103. However, the final phase separation determinant may be a complex combination of many side-chain functions.

Effect of inhibition of the glutathione redox cycle on the ultrastructure of peroxide-treated rabbit epithelial cells

Our previous studies have shown that exposure of cultured rabbit lenses to physiological levels of hydrogen peroxide, following inhibition of the glutathione redox cycle, leads to the formation of distinct vacuoles in the anterior region of the lens at the germinative zone between the epithelium and lens fibers. In the present study the ultrastructure of H2O2-induced membrane damage in the intact lens and in cultured lens epithelial cells was examined by scanning and transmission electron microscopy (SEM and TEM), following the inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Lenses treated with BCNU/H2O2 exhibited swollen epithelial cells which were observed only above the peroxide-induced vacuoles. The apical surface of the swollen cells had membrane blebs which protruded into the underlying vacuolar space. The appearance of the blebs coincided with a change in the organization of the layer of microfilaments which is normally associated with the apical surface of the cell. Cultured lens epithelial cells treated with BCNU/H2O2 showed membrane blebs which increased in size and number with the duration of exposure. Initially, the blebs were seen only on certain regions of the cell surface with other regions appearing normal. TEM revealed a disorganization of microfilaments in the BCNU/H2O2 treated cells. Neither BCNU nor H2O2 alone affected the morphology of intact lenses or of cultured lens epithelial cells. In culture, isolated lens epithelial cells exposed to BCNU/H2O2 were more susceptible to damage than contiguous cells. While the exact mechanism by which H2O2-induced damage leads to bleb formation on the cell surface is not known, the inability of the cells to detoxify H2O2 due to the inhibition of glutathione reductase results in the disturbance of membrane cytoskeleton and a focal weakening of the cell surface. These results indicate a correlation between the active glutathione redox cycle in lens epithelium and maintenance of normal cytoskeletal protein organization.

Raman spectroscopy of calf lens γ-II crystallin: Direct evidence for the formation of mixed disulfide bonds with 2-mercaptoethanol and glutathione

This study presents Raman spectra of calf lens gamma-II crystallin and its reaction products with reduced glutathione, 2-mercaptoethanol and p-hydroxymercuribenzoate. The absence of a disulfide vibration in gamma-III crystallin (both in aqueous solution and in lyophilized state) indicates that the seven thiol groups in this protein are resistant to air oxidation, and are capable of maintaining their reduced state in the absence of added reducing agents during isolation. However, treatment of the protein with low molecular weight thiols such as glutathione and 2-mercaptoethanol results in mixed disulfide bonds. We have detected, for the first time, the S--S bond stretching vibration from the mixed disulfides at 510 cm-1, which is very similar to the 508 cm-1 reported for the inter/intramolecular disulfide bonds in intact mouse lenses (Yu, N.-T., DeNagel, D.C., Pruett, P.L. and Kuck, J.F.R., Jr. (1985). Proc. Natl. Acad. Sci. U.S.A., 82, 7965-8). Upon titration with five equivalents of p-hydroxymercuribenzoate, a strong Raman line was detected at 345 cm-1, which is tentatively attributed to the Hg--S stretching vibration of the mercaptide complex. The S--H vibration region (2500-2700 cm-1) exhibits two resolved peaks at 2562 and 2580 cm-1 with an intensity ratio of 2:5. Both reactive surface thiol groups and buried cysteines give rise to the S--H vibration at 2580 cm-1.

Drug uptake and release by a hydrogel intraocular lens and the human crystalline lens

Implantation of a hydrogel (IOGEL) intraocular lens in humans has been reported. The polyhydroxyethyl methacrylate (poly HEMA) matrix of this hydrogel is permeable to water soluble drugs and may adsorb agents used intracamerally during cataract extraction or topically during the postoperative period. This study compared the in vitro uptake and release of chloramphenicol, dexamethasone, epinephrine, pilocarpine, and bovine serum albumin by polymethylmethacrylate and hydrogel intraocular lenses with that of the intact crystalline lens of humans and rabbits. An in vivo study compared the uptake and release of chloramphenicol and dexamethasone by hydrogel lenses implanted in the anterior chamber of rabbit eyes with that of the rabbit's crystalline lens. The in vitro uptake and washout of epinephrine and pilocarpine by the hydrogel lens was comparable to the human lens. Uptake of chloramphenicol and dexamethasone by the hydrogel lens exceeded that of the human lens and, following a two-hour washout period, the dexamethasone content of the hydrogel lens remained significantly greater than the human lens. The uptake and washout of bovine serum albumin by the hydrogel lens was half that of the human lens. In vivo, the hydrogel lens efficiently eluted both chloramphenicol and dexamethasone. These studies show that a hydrogel lens will not act as a significant depot for drugs in the eye.

Regional sensitivity to hypoglycemia within the rabbit lens epithelium.

Paired rabbit lenses were kept in organ culture; one of the lenses was subjected to hypoglycemia, and the other served as control. The lens changes were followed and examined by photomacrography, light microscopy of the intact lens, and light and electron microscopy at different time intervals. The possibility of reversing the morphological changes was evaluated at different times by replacing the hypoglycemic medium with a normal glycemic one. The central zone of the lens epithelium was affected first through the formation of large extracellular vacuoles. These changes were reversible. Subsequently, the central lens epithelial cells showed severe swelling and breakdown of the lens membranes. These changes were not reversible. With time the epithelial changes spread from the central zone towards the equator. Lens fiber swelling followed the severe epithelial damage. These findings show that there is a regional sensitivity to hypoglycemia within the lens epithelium. The results are discussed in relation to hexokinase activity.

The effect of an aldose reductase inhibitor on lens phosphorylcholine under hyperglycemic conditions: Biochemical and NMR studies

Phosphorylcholine (P-choline) is a precursor of the phospholipids in the lens membrane. A human lens normally contains approx. 1 m m P-choline but this is significantly lowered in some cataractous lenses. A normal rat lens contains a very high concentration (11 m m). We found that rat lens P-choline was depleted drastically when the lenses were exposed to hyperglycemic conditions either in culture, with galactose or xylose, or in vivo by streptozotocin-induced diabetes. The lens P-choline level was measured by fractionating the organic phosphates in the lens homogenate using an ion exchange column, or by quantitating the P-choline 31P NMR intensity in intact lenses. The results of both the chemical method and the noninvasive method agreed remarkably well. Besides the change in P-choline, the choline influx was also drastically reduced both in lenses from diabetic rats and in lenses incubated with 30 m m xylose. In addition, the ATP concentration was greatly diminished under similar conditions. The changes in P-choline, choline, and ATP could all be prevented in the presence of an aldose reductase inhibitor (ARI). It is thus concluded that these changes in phospholipid precursors may result from lenticular membrane defects caused by hyperglycemic stress. The effect of the lowered precursors on lipid biosynthesis was observed, and surprisingly showed a more rapid phospholipid-biosynthesis in the 2-week diabetic rat lens than in the 3-day diabetic rat lens.

Experimental parameters affecting the ultraviolet-induced tryptophan modification in the lens

Experimental parameters affecting photolysis of tryptophan in the lens were evaluated, including: (a) the use of previously-frozen vs. freshly excised lenses, (b) the use of lenses in aqueous buffered solutions vs. in air, (c) the species source of the lens, and (d) the effects of oxygen depletion. Lenses were subjected to monochromatic ultraviolet radiation followed by monitoring of the tryptophan fluorescence spectra while exciting at two or more wavelengths. Previously frozen lenses showed faster UV-induced degradation of tryptophan, particularly in aqueous media. A rapid leakage of fluorescent components (including amino acids and peptides) from intact lenses was observed. Evidence for the artifactual production of free radicals during photometric monitoring of samples in solution was obtained. Species dependent differences were also observed.

Human T-cell mediated response to homologous lens antigen

T-Cell response to a suspension of homologous whole lens antigen was investigated in three groups; (1) adults with normal eyes; (2) patients with mature and hypermature cataract with an intact lens capsule; and (3) patients, who had had extracapsular cataract surgery or lens injury, some of whom had clinical evidence of severe lens-induced inflammation. The results show that sensitized T-cells occur only in some patients in the third group. To our knowledge, this is the first direct evidence of a difference in the response to T-cells to lens antigen released from mature and hypermature lenses, and to lens antigen exposed by lens injury or extracapsular cataract surgery. The test may be useful to confirm clinical suspicion of delayed hypersensitivity to lens antigen in patients with severe sterile purulent inflammation or milder delayed sterile non-purulent inflammation following lens injury or extracapsular surgery, in the absence of histological proof.

In situ optical spectroscopy of some systems of biological interest.

Monitoring the optical absorption or emission spectrum of a condensed phase sample offers information about the supramolecular assembly, packing effects and other features characteristic of the phase that would be missed when one studies solution-state spectra. We have used the technique of photoacoustic spectroscopy to study intact biological specimens, such as algae, parasite cells and the eye lens. Such a study has offered information about the status of endogenous hemin in Plasmodium cells and the mode of interaction of antimalarial drugs of the chloroquine class therein. We have also attempted to do in situ fluorescence spectroscopy on isolated intact eye lenses, which has enabled us to follow the photochemistry and the status of the photoproduct of the oxidation of the trp residues of the crystallins of the lens.

Cellular pH and Na+-H+ exchange activity in lens epithelium of Bufo marinus toad.

Incubation of lenses with the acetoxymethyl ester of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) led to selective, prolonged entrapment of the deesterified, pH-sensitive, membrane-impermeable form of the dye (BCECF) in the lens epithelial cells allowing, thereby, for the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. Under anaerobic conditions in physiological solution, the epithelium maintained a pHi of 7.36 +/- 0.14 (mean +/- SD, n = 6 lenses). In the absence of CO2 and HCO3-, the pHi increased to 7.83 +/- 0.38 (n = 55). In this condition, either the removal of Na+ or the addition of amiloride to the medium resulted in a decrease of pHi. This acidification was increased in amiloride-inhibitable fashion when lenses were loaded with Na+ before extracellular Na+ removal. The extent of artificially generated cellular acidosis (induced by the "NH4+ loading" technique) was markedly decreased by the presence of extracellular Na+ or reverted on its addition when the ion was initially absent; the latter reversal was inhibited by amiloride. This inhibition was by-passed by monensin addition. The existence of a Na+-H+ antiport in the epithelial cells is concluded.

Intracellular protein degradation in cultured bovine lens epithelial cells.

Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life greater than 50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life less than 10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43.

Calibrating Multichannel Raman Spectrometers

Abstract Although multichannel Raman spectroscopy has a history of more than a decade [I], it was only recently that the real potential of this technique became fully appreciated. The use of advanced optical multichannel detectors (OMDs) is now bringing about a substantial change in Raman spectroscopy. For example, the application of this technique to short-lived transient species has established a new frontier of Raman spectroscopy known as transient and time-resolved Raman spectroscopy [2]. The technique has made it possible to measure the spectrum even from an extremely weak scatterer like a monolayer film deposited on a substrate [3, 41. A recent experiment on an intact mouse lens [5] demonstrated that only 1 mW laser power with 1 s exposure time is enough to obtain a good quality Raman spectrum of the lens protein, which is of clinical importance for the prevention of cataract formation. A number of stimulating reports are also notable in a variety of fields including chemistry, physics, biology, med...

Metabolism of D-glucose anomers in rat lens.

In lens homogenates, hexokinase displayed a greater affinity for alpha-D-glucose but a higher maximal velocity with beta-D-glucose. However, in intact lenses incubated at 10 degrees C in the presence of 4-7 mM alpha- or beta-D-glucose, no anomeric difference could be found in either the intracellular content in D-glucose and D-glucose 6-phosphate or the utilization of D-[5-3H]glucose and oxidation of D-[1-14C]glucose. Even the sorbitol content of the lens was not significantly different after 180 min perifusion at 37 degrees C in the presence of either alpha- or beta-D-glucose. It is proposed that the lack of anomeric specificity in D-glucose metabolism is attributable, at least in part, to the low turnover rate of glucose 6-phosphate in the lens, allowing for the interconversion of glucose 6-phosphate anomers.

Age and the transmittance of the human crystalline lens.

1. Conflicting data both on the transmission of the human crystalline lens in the ultra-violet part of the spectrum and on its variation with age necessitate a re-examination of the subject. 2. Twenty-four excised lenses in the age range of 0-85 years were studied with a Perkin-Elmer spectrophotometer between 327 and 700 nm. 3. Some lenses were homogenized and the homogenates were similarly examined. 4. A systematic increase in absorbance with age was observed both in the visible and the ultra-violet parts of the spectrum. 5. An exponential function describes the data, facilitating comparison with, and prediction of, other values. 6. The absorbance of homogenized material was found to be predictable from that of intact lenses, and does not support the notion that mechanical trauma may account for high values in earlier studies.

Na+-K+-ATPase pumping activity is not directly linked to myo-inositol levels after sorbinil treatment in lenses of diabetic rats.

Changes in tissue levels of sorbitol, myo-inositol, and Na+-K+-ATPase enzyme activity have been implicated in the development of diabetic complications in animal models of the disease and in humans. The ability of the aldose reductase inhibitor sorbinil to reverse the hyperglycemia-induced changes in these lenticular metabolite and enzyme-activity levels in the streptozocin-induced diabetic rat was examined to determine what, if any, relationship exists between these changes. Two weeks of untreated diabetes did not change ouabain-inhibitable ATPase enzyme activity assayed in lens homogenates but did result in a decrease in the Na+-K+-ATPase transport activity as measured by 86Rb uptake in the intact lens. This was accompanied by a 100-fold increase in the levels of sorbitol and significant decreases in the levels of myo-inositol, ATP, and glutathione in the lens. Whereas all of these changes could be reversed by sorbinil treatment, the dose required for restoration of the depleted myo-inositol level (ED50 greater than 20 mg.kg-1.day-1) was much higher than the dose required to reverse the other changes (ED50 range 2-5 mg.kg-1.day-1). These results suggest that the restoration of lenticular Na+ -K+ -ATPase activity is not secondary to a normalization of myo-inositol levels and may provide evidence that the two parameters are not strictly associated in diabetic tissues.

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging.

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.

Limited proteolysis of gap junction protein is intrinsic in mammlian lens fiber-cell plasma membranes

We demonstrate that the limited proteolysis of the lens fiber-cell gap junction protein, MP26, is intrinsic in mammalian lens fiber plasma membranes. Incubations of isolated intact bovine lens fiber plasma membranes in buffer alone did not elicit proteolysis of MP26. Incubations in the buffer with detergent, however, resulted in the limited proteolysis of MP26 which was totally inhibited by calcium chelators, thiol-alkylating agents, and protease inhibitors. As the limited proteolysis required the presence of detergent, it must depend on an enzymatic activity intrinsic in the lens fiber plasma membranes or in MP26 itself.

Multichannel Raman Spectroscopy of an Intact Lens: Raman Measurement with Laser Irradiation below the Threshold for Retinal Damage

While cataract formation is not a fatal condition, it still remains one of the diseases which people greatly fear. Prevention of cataract formation was a hopeless dream for mankind until quite recently. However, recent rapid progress in laser light scattering and Raman spectroscopy has given rise to the expectation that the dream of cataract prevention can become a reality.

The utilization of 13C and 31P nuclear magnetic resonance spectroscopy in the study of the sorbitol pathway and aldose reductase inhibition in intact rabbit lenses

Nuclear magnetic resonance (NMR) spectroscopy has been utilized in the study of the metabolism of intact, functioning rabbit lenses maintained in organ culture. The sorbitol pathway and aldose reductase inhibition have been studied using carbon-13 NMR spectroscopy. Incubation of lenses in high concentration [1-13C] glucose medium with and without added inhibitors allows the sorbitol pathway and glycolysis to be monitored. Various aldose reductase inhibitors have been studied and are ranked based on percentage of inhibition as follows: tolrestat greater than or equal to sorbinil greater than sulindac greater than sulindac sulfide much greater than indomethacin greater than acetylsalicylic acid greater than quercetin greater than tandearil greater than salicylic acid greater than 3,3-Tetramethyleneglutaric acid (TMG). It has been demonstrated that 13C NMR spectroscopy provides an effective method of screening potential inhibitors of aldose reductase. The aspirin substitutes ibuprofen and acetaminophen have been studied and are found to reduce sorbitol accumulation in intact rabbit lenses. The effects of myo-inositol and vitamin E on sorbitol accumulation have also been investigated. Results suggest that the various metabolic pathways within the lens are intricately connected. In a preliminary manner, the effect of diabetes on metabolism in intact lenses has been investigated using 13C NMR spectroscopy. Increased sorbitol production has been observed for diabetic lenses. 31P NMR spectroscopy has also been utilized in the study of lens metabolism and aldose reductase inhibitors. Inclusion of various inhibitors in the high concentration glucose medium results in maintenance of essentially normal phosphorus-containing metabolite levels in the lens. No clear relationship was observed between lens clarity and phosphorus metabolite levels as determined using NMR.