Evidence of the oxidation of unsaturated fatty acids in cataracts

Gas chromatography analysis with the use of an electron captured detector including preparation of the halogen-substituted derivatives of fatty acids is a useful tool for the detection of lipid peroxidation products both in vitro and in vivo. This technique was applied to determine the content of fatty acid oxy-derivatives in lipid samples of transparent and completely opaque human lenses. At the stage of mature cataract a significantly increased level of oxyproducts was observed in the lens lipid fraction. It was concluded that accumulation of polar oxygroups in the lipid bilayer of plasma membranes of lens fibres is a plausible cause of their damage in cataracts.     

Short-term culturing of teleost crystalline lenses combined with high-resolution optical measurements

Culturing whole lenses is a frequently used method for studying regulatory events on the lens in controlled environments. The evaluation methods used often fall under two categories, molecular or optical. The main benefit from optical measurements is that they directly detect changes in the lens’ main function, i.e. refracting light. However, these measurements often have rather low resolution or yield results open for subjective interpretation. Here we present a short-term crystalline lens culturing technique combined with a high-resolution optical measuring method. There are two main advantages of using teleost lenses compared to mammalian lenses. Teleost tissue generally has a higher tolerance than mammalian tissue with regard to temperature and nutrient fluctuations. Teleost lenses are structurally more robust and can be excised from the eye without disturbing form or function. The technique is developed for short-term culturing (3 h), however, the lenses appear viable for at least 24 h and longer culturing may be possible. The technique is resistant to small variations in osmolarity and yields quantitative datasets for further analyses and statistical treatment.     

Delay of uv-induced eye lens protein damage in guinea pigs by dietary ascorbate

Large accumulations of postsynthetically oxidized proteins are observed in the aged and cataractous eye lens. Ascorbate has previously been used to delay photooxidative damage in vitro. The goals of this study were (1) to confirm that dietary ascorbate can be used to enhance lens ascorbate levels and (2) to determine if lenses with enhanced ascorbate can better withstand photooxidative stress in the form of ultraviolet (UV) light exposure. Guinea pigs were placed on high dietary ascorbate (HDA), 50 mg/day, and low dietary ascorbate (LDA), 2 mg/day, for 21 weeks. Lenses from HDA animals were found to contain 3.3 times more ascorbate than LDA animals. Prior to irradiation, SDS-PAGE protein profiles and exopeptidase activity in HDA and LDA lens soluble proteins were indistinguishable. However upon exposure to UV light, more protein damage (e.g., high-molecular-weight aggregates and enhanced loss of exopeptidase activity) was seen in lens preparations from LDA as compared to HDA animals. These results suggest that ascorbate protects lens components against cataract-like and agerelated postsynthetic changes in vivo. As in previous tests on lens preparations, attenuated exopeptidase activity was observed before protein aggregation.     

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.     

A passive pressure sensor for continuously measuring the intraocular pressure in glaucomatous patients

ObjectiveThe main risk factor for the development of glaucoma, a retinal disease leading to blindness, is an increase in the intraocular pressure (IOP). Reducing this IOP can be obtained by eye drops but unfortunately the disease can still progress because IOP increases are painless, can fluctuate and, thus remain undetected during a visit to an ophthalmologist. The “MATEO” ANR project aims to develop sensors embedded in a contact lens for continuously IOP monitoring.Materials and methodsPressure sensors were produced by MEMS technology and tested with pig eyes obtained at a local slaughterhouse. Solution was injected by 50 μL steps in the eye with a Hamilton syringe while IOP was monitored in parallel with a TonoVET system and an industrial pressure transducer inserted in the injection tubing system.ResultsOur first pressure sensor prototypes were generated and inserted in a lens compatible with eye application. A wireless system was developed to excite the sensor. At same time, it was recorded the data in components inserted into spectacles and a pocket recorder. In parallel, we showed that injecting a solution in the eye anterior chamber triggered an IOP increase smaller and more stable than injections in the posterior chamber. Finally, a direct correlation was observed between IOP measured on the corneal surface with the TonoVET and the pressure transducer placed close to eye injection point.DiscussionOur results indicate that our in vitro model on pig eyes is adequate to test our new lens sensor. Finally, the pressure sensor was successfully inserted in contact lens opening the way for their in vitro and in vivo preclinical validation.     

Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.     

Optical function and mitochondrial metabolic properties in damage and recovery of bovine lens after in vitro carbonyl cyanide m-chlorophenylhydrazone treatment

In order to elucidate the correlation between lens optical function and metabolic function, in vitro bovine lens optical quality and mitochondrial integrity was measured following treatment with carbonyl cyanide m-chlorophenylhydrazone (the mitochondrial depolarizing agent, CCCP). The results indicate that in vitro exposure to CCCP resulted in concentration and time-dependent loss of sharp focus. The concentrations tested included 65.0, 32.5, 16.25 and 8.125 microm CCCP. Lenses treated with two lower concentrations show recovery from damage at the 24-h scan point. In lenses treated with 65 microM CCCP, mitochondria in lens epithelial and superficial cortical fibre cells appeared short and swollen. The results of this study indicate that lens optical function and mitochondrial integrity are closely correlated.     

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 inPlasmodium cells and the mode of interaction of antimalarial drugs of the chloroquine class therein. We have also attempted to doin 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.     

Age-associated oxidative damage leads to absence of gamma-cystathionase in over 50% of rat lenses: relevance in cataractogenesis

Oxidative damage to lens proteins and glutathione depletion play a major role in the development of senile cataract. We previously found that a deficiency in gamma-cystathionase activity may be responsible for glutathione depletion in old lenses. The aims of this study were: (1) to investigate the mechanism that causes the age-related deficiency in gamma-cystathionase activity in the eye lens, and (2) to determine the role of gamma-cystathionase deficiency in cataractogenesis. Two populations of old rats were found, one (56%) whose lenses lacked gamma-cystathionase activity and the rest that exhibited detectable enzyme activity. gamma-Cystathionase protein was absent in lenses from old rats without gamma-cystathionase activity. Oxidative stress targeted gamma-cystathionase in the eye lens upon aging, since the enzyme contained more carbonyl groups in old lenses than in young ones. gamma-Cystathionase mRNA was also markedly reduced in old lenses, thus contributing to the age-associated deficiency in gamma-cystathionase. Inhibition of gamma-cystathionase activity caused glutathione depletion in lenses and led to cataractogenesis in vitro. In conclusion, the lack of gamma-cystathionase activity in over 50% of old lenses is due to decreased gene expression and proteolytic degradation of the oxidized enzyme. This results in a high risk for the development of senile cataract.     

Lens aging: Effects of crystallins

The primary function of the eye lens is to focus light on the retina. The major proteins in the lens—α, β, and γ-crystallins—are constantly subjected to age-related changes such as oxidation, deamidation, truncation, glycation, and methylation. Such age-related modifications are cumulative and affect crystallin structure and function. With time, the modified crystallins aggregate, causing the lens to increasingly scatter light on the retina instead of focusing light on it and causing the lens to lose its transparency gradually and become opaque. Age-related lens opacity, or cataract, is the major cause of blindness worldwide. We review deamidation, and glycation that occur in the lenses during aging keeping in mind the structural and functional changes that these modifications bring about in the proteins. In addition, we review proteolysis and discuss recent observations on how crystallin fragments generated in vivo, through their anti-chaperone activity may cause crystallin aggregation in aging lenses. We also review hyperbaric oxygen treatment induced guinea pig and ‘humanized’ ascorbate transporting mouse models as suitable options for studies on age-related changes in lens proteins.     

Oxidative modification of lens crystallins by H2O2 and chelated iron

Crystallins are the soluble structural proteins that constitute approximately 90% of the dry mass of the eye lens. The present study attempts to elucidate possible mechanisms whereby the H2O2 present in the eye could contribute to the oxidative modification of lens crystallins. The data indicate that exposure of solutions of crystallins to H2O2 and EDTA-chelated iron leads to covalent crosslinking of polypeptides, loss of intrinsic protein fluorescence, and the generation of a novel fluorophor emitting in the 420 nm range. These changes closely mimic oxidative modifications that occur in lens proteins in vivo. Exposure of the proteins to H2O2 in the absence of chelated iron failed to generate detectable levels of these modifications. These findings are contrasted with earlier studies of lenses in organ culture where H2O2 alone produced marked damage while the further addition of chelated iron protected the lenses from oxidation.     

Protective effects of selenium, vitamin C and vitamin E against oxidative stress of cigarette smoke in rats

Cataractous lenses have been found to have an altered distribution of the intracellular ionic environment: the concentrations of potassium and magnesium being decreased and the concentrations of sodium and calcium increased. These changes arise as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. In this study flame atomic absorption spectroscopy (AAS) was used for calcium, magnesium, iron and zinc determination, and flame atomic emission spectroscopy (AES) was used for sodium and potassium contents in normal and cigarette smoke-exposed rat lenses. The methods are sensitive enough to detect quantitatively all six cations in a single rat lenses. In this work, six elements, including Ca2+, K+, Na+, Zn2+, Fe2+ and Mg2+ in experimental rat eye lenses and normal transparent lenses were determined. It was found that the concentrations of Ca2+, Na+, Zn2+, and Fe2+ were increased dramatically while K+ and Mg2+ decreased in smoke-exposed rat lenses when compared to the control rat lenses. There were no significant changes between 'smoked' rats supplied with vitamin C and control groups. A positive correlation was found also in the other two groups of 'cigarette smoked' animals supplemented with selenium plus vitamin E and selenium when compared with 'cigarette smoked' without any supplements. These data provide support for the hypothesis that cigarette smoking increases the risk of cataract formation. We investigated whether vitamin C is the most important antioxidant in the body. The roles of diet with optimum amounts of antioxidant vitamins C and vitamin E and the antioxidant mineral selenium are discussed.     

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography.     

19F NMR quantitation of lens aldose reductase activity using 3-deoxy-3-fluoro-D-glucose

In the present study we have determined the kinetics of 3-deoxy-3-fluoro-D-glucose (3-FG) as a substrate for the aldose reductase reaction in vitro. In addition, we compared the 3-deoxy-3-fluoro-sorbitol (3-FS) production rates from 3-FG in the intact lens using 19F NMR with conventional aldose reductase determinations in extracts from the same lenses. The affinity of in vitro aldose reductase for 3-FG was approximately 20 times greater (9.3 mM) than that for glucose (188 mM). An excellent correlation between the rate of 3-FS production in the intact canine lens, determined with 19F NMR, and extracted aldose reductase activity was observed. The relatively high affinity of aldose reductase for 3-FG and the correlation of 3-FS production with enzyme activity in the intact lens suggests that 3-FS production from 3-FG detected by 19F NMR could provide an accurate noninvasive determination of aldose reductase activity in the eye lens.     

Limits of the confocal laser-scanning technique in measurements of time-resolved autofluorescence of the ocular fundus]

GOAL: The 2-dimensional measurement of the time-dependent autofluorescence has in combination of confocal laser scanner technique and time correlated single photon counting the potential for the investigation of the metabolism state at the eye ground. It was to be examined, to what extent these measurements are influenced through the auto-fluorescence of the crystalline lens. MATERIAL AND METHOD: The time-dependent auto-fluorescence was measured on eyes of 21 patients with age-related macular degeneration (AMD) and of 26 healthy subjects in 40 degrees fundus images. The experimental set-up used, was developed at the eye clinic of the University of Jena. In this scanning laser ophthalmoscope, the fundus was excited at 446 nm by pulses of 100 ps full width at half maximum and 40 MHz repetition rate. The auto-fluorescence was detected with a time resolution of 25 ps for wavelengths > 500 nm. The dynamic auto-fluorescence of each pixel was approximated by a tri-exponential model function. In order to determine the influence of the fluorescence of the crystalline lens, healthy eyes and eyes were compared with implanted artificial intra-ocular lenses. For comparison the frequency distributions were used, which the decay times t1, t2 and t3 in the fundus images had been calculated with. RESULTS: Clear differences were shown in the frequency distributions of the fluorescence lifetimes between AMD-patients and healthy subjects, It resulted, however, from a crucial analysis that the fluorescence of the crystalline lens is not suppressed by the confocal laser scanning principle sufficiently. In particular the long lifetime t3 is overlapped by the fluorescence of the crystalline lens of about 4.6 ns. To the complete suppression of the influence of the lens fluorescence in measurements of the time-resolved fundus auto-fluorescence it is suggested to combine the confocal laser scanning technique with the principle of the aperture diaphragm division. CONCLUSION: In order to be able to determine the capability of measurements of the time-dependent fundus auto-fluorescence as diagnostic tool exactly, the influence of the crystalline lens is to reduce through the combination of confocal laser scanning ophthalmoscopy with the principle of the aperture diaphragm division diversely.     

The eye lens weight and age in the common carp, Cyprinus carpio L.

The validity of the use of the weight of dried eye lenses as an indicator of age in the common carp, Cyprinus carpio L., is tested. There is a significant difference between males and females for fish of two and three years old. Otherwise no significant difference is noted between sexes. The linear relationship between the dry weight of eye lenses and age + 1 are: In Y + 1= 0.5202 + 16.4928 X ( r = 0.93) for female and ln Y + 1=0.4276 + 16.7364 X ( r = 0.94) for male.
The method for determining age is valuable for fish until three years old. However for older fish, in spite of a correlation coefficient between lens weight and age and even with a significant difference between annual groups, the age determination may be misleading due to the overlap in lens weights. In conclusion therefore this method may not be useful for back calculation of growth, for production estimates or studies in which accurate estimates of age are needed.     

Long-term in vivo and in vitro AAV-2-mediated RNA interference in rat retinal ganglion cells and cultured primary neurons

Viral vector-based expression of small interfering RNAs is a promising tool for gene regulation, both in cultured cells and in animal models. In this study, we analysed the ability of adeno-associated virus-2 to function as an RNAi vector in cultured primary hippocampal neurons in vitro and in retinal ganglion cells in vivo. We demonstrate a long-lasting, highly efficient, and specific down-regulation of gene expression in vivo and in vitro by the use of bicistronic vectors. This is the first evidence of a cell type-specific long-term (more than three-month-long) RNAi in the eye. Furthermore, our results constitute the prerequisite for the use of this technique in models of neurodegeneration and neuroregeneration in vivo and in vitro.     

A biochemical characterization of the photophore lenses of the midshipman fish,Porichthys notatus Girard

The present study is a biochemical characterization of the photophore lenses of the midshipman fish, Porichthys notatus, a species that bears 800 photophores distributed over the body surface. The biochemical properties of the photophore lenses were compared with those of the eye lens with which they share a similar developmental origin and analogous function. To achieve a high refractive index, the vertebrate eye lens has a relatively high concentration of structural proteins (20–50%, depending on species) and a simple protein composition, that is, relatively few proteins are synthesized in comparison to other tissues. Similarly, the photophore lenses of P. notatus had a relatively high protein concentration (average = 29%, n = 5) and approximately 60% of the total soluble protein was represented by two subunit species of 33 kD and 35 kD on denaturing polyacrylamide gels. The structural proteins of the eye lens are of two principle types: 1) and polypeptides which belong to vertebrate lens-specific crystallin families, and, 2) enzymes recruited into the lens which take on the function of structural proteins. Here, we report that the two major photophore lens subunits of 33 kD and 35 kD are biochemically similar to each other, but are clearly distinct from any of the previously characterized crystallins. Therefore, we propose that photophore lenses appear to recruit a novel protein.     

A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens

γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a two-fold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in 'shepherding' filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.     

Comparative x-ray diffraction study of the crystalline lens in a number of vertebrates including man

X-ray diffraction method has been applied for comparative investigation of native structure of eye lens proteins (crystallins). X-ray diffraction patterns of the whole lenses and/or their nuclear parts were obtained for man and vertebrate animals. Crystalline lenses of the fishes Acerina cernua and Pelmatochromis kribensis, frog Rana temporaria, bull and man contain crystallins with a very similar secondary and tertiary structure, whereas lenses of chicks and the tortoise Testudo horsfieldi contain mainly crystallins with other structure. The results obtained reveal evolutionary conservatism of crystallin structure in fishes, amphibians and mammals. It was also concluded that there is no correlation between crystallin structure of the lens, elasticity of the latter and accommodation mechanism.