Intact Human Lenses Research Articles

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens

Optical effects of exposing intact human lenses to ultraviolet radiation and visible light

BackgroundThe human lens is continuously exposed to high levels of light. Ultraviolet radiation is believed to play a causative role in the development of cataract. In vivo, however, the lens is mainly exposed to visible light and the ageing lens absorbs a great part of the short wavelength region of incoming visible light. The aim of the present study was to examine the optical effects on human lenses of short wavelength visible light and ultraviolet radiation.MethodsNaturally aged human donor lenses were irradiated with UVA (355 nm), violet (400 and 405 nm) and green (532 nm) lasers. The effect of irradiation was evaluated qualitatively by photography and quantitatively by measuring the direct transmission before and after irradiation. Furthermore, the effect of pulsed and continuous laser systems was compared as was the effect of short, intermediate and prolonged exposures.ResultsIrradiation with high intensity lasers caused scattering lesions in the human lenses. These effects were more likely to be seen when using pulsed lasers because of the high pulse intensity. Prolonged irradiation with UVA led to photodarkening whereas no detrimental effects were observed after irradiation with visible light.ConclusionsIrradiation with visible light does not seem to be harmful to the human lens except if the lens is exposed to laser irradiances that are high enough to warrant thermal protein denaturation that is more readily seen using pulsed laser systems.

Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling

Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 μm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.

α- and β-Crystallins Modulate the Head Group Order of Human Lens Membranes during Aging

To examine the physical properties of human lens cell membranes as a function of age. The environment of the phospholipid head groups in fiber cell membranes from human lenses, aged 22 to 83 years, was assessed with Laurdan and two-photon confocal microscopy. The effect of mild thermal stress on head group order was studied with lens pairs in which one intact lens was incubated at 50 °C. Dihydrosphingomyelin vesicles were preloaded with Laurdan, α-, β-, or γ-crystallin was added, and surface fluidity was determined. The membrane head group environment became more fluid with age as indicated by increased water penetration. Furthermore, these changes could be replicated simply by exposing intact human lenses to mild thermal stress; conditions which decreased the concentration of soluble α- and β-crystallins. Vesicle binding experiments showed that α- and β-, but not γ-, crystallins markedly affected head group order. The physical properties of cell membranes in the lens nucleus change substantially with age, and α- and β-crystallins may modulate this effect. β-Crystallins may therefore play a role in lens cells, and cells of other tissues, apart from being simple structural proteins. Age-dependent loss of these crystallins may affect membrane integrity and contribute to the dysfunction of lenses in older people.

Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment

To characterize age-related changes in the transmission of light through noncataractous human lenses. Department of Ophthalmology, Glostrup Hospital, Glostrup, Denmark. The spectral transmission of white light was measured along the visual axis in the most central part of the lens in vitro in intact human donor lenses over a wide range of ages. The study evaluated 28 intact human donor lenses of 15 donors aged 18 to 76 years. Increasing age was associated with gradually decreasing transmission at all visible wavelengths, most prominently at shorter wavelengths. Empirical formulas describing the age-related loss of transmission were created for each spectral color. At 480 nm, the absorption peak for melanopsin, transmission decreased by 72% from the age of 10 years to the age of 80 years. The age-related decrease in spectral transmission through the human lens could be modeled by a simple algorithm that may be useful in the design of intraocular lenses that mimic the characteristics of the human lens and in studies of color vision, psychophysics, and melanopsin activation.

Regional Differences in Store-Operated Ca2+Entry in the Epithelium of the Intact Human Lens

An elevated level of Ca(2+) is an important factor in cataract, yet precisely how Ca(2+) enters the lens is unknown. Lens epithelial cells contain a range of G-protein-coupled receptors and receptor tyrosine kinases that induce increases in intracellular Ca(2+). Receptor-associated Ca(2+) influx is, therefore, likely to be an important route for Ca(2+) influx to the lens. The authors investigated stimulated and passive Ca(2+) influx in in situ human lens epithelium. Ca(2+) changes in equatorial (E) and central anterior (CA) epithelial cells were monitored with the use of a Ca(2+) indicator (Fluo4) and confocal microscopy. Gene expression was monitored by RT-PCR and immunoblotting. Adenosine triphosphate (ATP) induced Ca(2+) responses that were smaller in CA than E. Ca(2+) store depletion, using ATP (100 microM) or thapsigargin (1 microM), revealed greater relative store capacity and Ca(2+) influx in E. Ca(2+) influx was blocked by La(3+) (0.5 microM) in both regions. Unstimulated Ca(2+) influx was greater in E than CA. Greater expression of Orai1 and STIM1 was detected in E than in CA. Greater Ca(2+) store capacity and Ca(2+) influx in E compared with CA reflects underlying differences in proliferation and differentiation between the regions. The relatively small resting Ca(2+) influx in CA epithelium suggests that store-operated Ca(2+) entry (SOCE) is the main route of Ca(2+) influx in these cells. Greater resting influx and SOCE in E cells suggests that these are a major route for Ca(2+) influx into the lens. Increased expression of Orai1 and STIM1 in E could account for the differences in Ca(2+) entry. Receptor activation will modulate Ca(2+) influx, and inappropriate activity may contribute to cortical cataract.

Spectroscopic and biochemical correlations during the course of human lens aging

BackgroundWith age, the human lens accumulates variety of substances that absorbs and fluorescence, which explains the color of yellow, brunescent and nigrescent cataract in terms of aging. The aim of this study was to assess lens fluorophores with properties comparable to those of advanced glycated end products (AGEs) in relation to age in human lenses. These fluorescent compounds are believed to be involved in the development of cataract.MethodsSpectroscopic (UV-Vis-NIR) and fluorescence photography (CCD-Digital based image analysis) studies were carried out in randomly selected intact human lenses (2–85 years). AGE-like fluorophores were also measured in water soluble and insoluble (alkali soluble) fractions of human lenses (20–80 years).ResultsOur experimental findings suggest that there was a progressive shift in the absorbance characteristic of intact lens in the range of λ210 nm-λ470 nm. A relative increase in the absorptivity at λ(511–520 nm), with age, was also observed. In addition, the ratio of absorptivity at λ(511–520 nm) versus the maximum absorbance recorded at blue-end cut-off (210–470 nm) was also found to increase, with age. The fluorescent intensity in the intact lens at both UV-B (λEx312 nm) and UV-A (λEx365 nm) were found to be positively correlated (r2 = 0.91 & 0.94, respectively; Confidence interval 95%) upto 50 years of age. In addition, a concomitant changes in AGE- like fluorophores were also observed in the processed lens samples (soluble and insoluble fractions) along the age. A significant increase in the concentration of AGE- like fluorophores, both in intact and processed lens was observed during the period of 40 – 50 years.ConclusionBased on the present investigation, it was concluded that significant changes do occur in the AGE-like fluorophores of human lenses during the period of 40–50 years.

Impact of UVR‐A on whole human lenses, supernatants of buffered human lens homogenates, and purified argpyrimidine and 3‐OH‐kynurenine

Yellow chromophores and fluorescent compounds accumulate in the lens with age. Some of these compounds are photochemically active. The present study aimed to examine the photochemical effect of ultraviolet radiation-A (UVR-A) on the human lens. Intact human lenses and supernatants of buffered lens homogenates were exposed to UVR-A. The effect of UVR-A was evaluated by time-resolved and steady-state fluorescence spectroscopy, visual evaluation of colour and protein gel electrophoresis. Intact lenses exposed to UVR-A showed no changes in time-resolved or steady-state fluorescence properties but the yellow coloration was visibly attenuated. The supernatants of buffered lens homogenates exposed to UVR-A demonstrated a reduction in time-resolved and steady-state fluorescent properties and protein cross-linking. Exposure of the intact lens to UVR-A causes chromophore bleaching without affecting fluorescence, indicating that non-fluorescent chromophores have been destroyed. After homogenization, both chromophores and fluorophores from the lens suffer damage and proteins aggregate. This indicates that powerful mechanisms of protection against UVR-A found in the intact lens are disturbed by homogenization of the lens, suggesting that isolated lens proteins cannot be used as a model system for studying cataractogenesis. Hypothetically, the protective mechanism could be related to the rigidly packed three-dimensional structure of the lens proteins or to the abundance of antioxidative and free radical scavenging defence systems.

Central surface curvatures of postmortem- extracted intact human crystalline lenses*1Implications for understanding the mechanism of accommodation

Central surface curvatures of postmortem- extracted intact human crystalline lenses*1Implications for understanding the mechanism of accommodation

Central surface curvatures of postmortem- extracted intact human crystalline lenses: Implications for understanding the mechanism of accommodation

Purpose To measure the radii of curvature of postmortem, whole, encapsulated human crystalline lenses, free of all zonular attachments, and to calculate their corresponding optical powers. Design Experimental study. Participants Thirty human crystalline lenses from donors with a mean age of 33.6±14.4 years. Methods Intact clear human crystalline lenses were obtained within an average of 21 hours of death. The lenses were removed from the eye by the contributing eye bank and shipped in Optisol-GS, a physiologic preservative storage medium. These lenses, with intact capsules, were freed of all zonular attachments. The lenses were stored at 7° C and were maintained in the same storage medium during the period that they were held for evaluation. Using a portable Keratron Scout corneal topographer (Eyequip, Ponte Vedra Beach, FL) fixed to an optical bench, the radii of curvatures of the anterior and posterior surfaces of the crystalline lens were measured daily for 10 days after receipt of the tissue. The capsules of the crystalline lenses remained intact, and the lenses were clear throughout the study. Measurements were made at room temperature after removing the lens from storage. Eight repetitions of the topography were made from each surface on each day to determine the accuracy and stability of the measurement. Profile photographs were taken daily to establish the central crystalline lens thickness. The corresponding optical power of each physiologically maintained crystalline lens was calculated. Main outcome measures The main outcome measures were the central anterior radius of curvature, the central posterior radius of curvature, the central thickness of each crystalline lens, and the amount of change in these parameters over 10 days. Results The means ± standard deviations of the central anterior and posterior radii of curvatures of the 30 adult lenses were 10.5±0.6 mm and 7.1±1.0 mm, respectively. The mean ± standard deviation of the central thickness, as measured from profile photographs, was 3.9±0.5 mm. These mean measurements remained stable for up to 5 days after receipt of the tissue. The mean and standard deviation of the calculated optical power of these postmortem, physiologically maintained, intact human crystalline lenses from donors with a mean age of 33.6 years was 19.8±1.7 diopters. Conclusions Lenses free of zonular tension are at an optical power that would be associated with optimal distance acuity.

Qualitative effect of zonular tension on freshly extracted intact human crystalline lenses: implications for the mechanism of accommodation.

To determine the topographic effects of zonular tension on the anterior surface of the human crystalline lens. Real-time topography of the anterior surface of seven fully relaxed, freshly extracted intact, clear, human crystalline lenses aged 3, 17, 45, 54, 54, 56, and 56 years was qualitatively obtained before, during, and after the application of zonular traction. Zonular traction was applied manually either by grasping a group of zonules 180 degrees apart with tying forceps (three lenses), or with micrometers by clamping four portions of the ciliary body that were 90 degrees apart (four lenses). Zonular tension began with the lenses in the fully relaxed, baseline state. As zonular tension was increased across one meridian of all seven lenses, the center of the anterior surface steepened while the periphery of the anterior surface flattened across that meridian of traction. When the tension was reduced across that meridian of traction, the center of the lens flattened while the periphery steepened in that meridian. Four-point zonular traction applied 90 degrees apart produced symmetrical central steepening (four lenses). Reduction of zonular tension across both orthogonal meridians caused symmetrical central flattening. These observations reveal that when zonular tension is applied to the fully relaxed lens, the center steepens and its periphery flattens in the meridian (or meridians) in which zonular tension is applied. The reverse of this process demonstrates that as tension is reduced, the center of the lens flattens while the periphery steepens either in the meridian of relaxation or symmetrically when zonular tension is released from two orthogonal meridians. These results are opposite to what would have been predicted on the basis of Helmholtz's theory of accommodation.

Calcium activates SK channels in the intact human lens.

Apamin-sensitive, calcium-activated SK potassium channels have been implicated in schizophrenia and myotonic dystrophy (MD), and both conditions carry an increased risk of cataract. The presence and functional activity of SK channels were therefore investigated in the human lens. The expression of all three members of the SK channel family was quantified by PCR. Their functional activity was investigated by using electrophysiological and calcium-imaging techniques. Lens voltage was monitored by inserting a single electrode into the intact human lens, and changes in intracellular calcium were recorded simultaneously after fura-2 incorporation. Expression of all three SK family members was detected in both anterior and equatorial lens epithelial cells. Application of either G-protein (e.g., adenosine triphosphate [ATP]) or tyrosine kinase (EGF) receptor agonists induced a hyperpolarization of lens voltage that was accompanied by an increase in intracellular calcium. The calcium ionophore ionomycin also induced a rapid hyperpolarization. The hyperpolarizing responses were abolished by apamin and trifluoperazine and were accentuated by the SK channel activator 1-ethyl-2-benzimidazolinone (1-EBIO). SK channels are an integral part of the G-protein and tyrosine kinase calcium signaling mechanisms in the human lens, and their activation is inhibited by certain anti-psychotic drugs. These findings help explain why a change in channel activity, whether by abnormal gene expression or by drug intervention, can lead to cataract.

Vitamin C Metabolomic Mapping in the Lens with 6-Deoxy-6-fluoro-ascorbic Acid and High-Resolution19F-NMR Spectroscopy

Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C. Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy. F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA. These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.

Radiolysis‐Induced Oxidation of Bovine α‐Crystallin

Radiolysis of water by ionizing radiation results in the production of pure hydroxyl radicals. This technique, combined with analysis by tandem mass spectrometry (MS/MS), has been used to study the effect of hydroxyl radicals on the intact bovine alpha-crystallin protein. After exposure to gamma-irradiation, the oxidized alpha-crystallin was digested with trypsin and the resulting peptides were fractionated by reverse-phase HPLC. The isolated fractions were analyzed by matrix-assisted laser desorption ionization and by MS/MS to determine the locations and identities of the modifications. Structural analysis revealed that methionine 1 of alpha A- and alpha B-crystallin and methionine 68 of alpha B-crystallin were oxidized to methionine sulfoxide. Hydroxytryptophan was formed from each tryptophan residue in alpha-crystallin, although only tryptophan 9 of alpha A-crystallin was converted into N-formylkynurenine. This study has, for the first time, identified the sites of modification and the structures produced in the intact alpha-crystallin protein by exposure to hydroxyl radicals. By determining the consequences of in vitro exposure of alpha-crystallin to pure hydroxyl radicals, the in vivo contribution of this reactive oxygen species to the overall oxidative stress of the lens will be achieved from the identification of the modifications to alpha-crystallin purified from intact human lenses.

Age-related changes in the human lens as monitored by detection of porphyrin excited states.

Previous studies have shown that the triplet state lifetimes of various porphyrins are increased by several orders of magnitude when they are bound to lens protein. Flash photolysis studies of mesotetra (p-sulfonatophenyl)porphyrin (TPPS) on intact bovine lenses indicated a biexponential decay of the triplet state with lifetimes of 160 microsecond and 1.6 ms. Here we extend those measurements to TPPS associated with intact human lenses. Steady-state fluorescence measurements indicate that TPPS binds to both young and old human lenses. In an intact young human lens, the TPPS triplet state is observed to decay biexponentially with lifetimes of 50 and 680 microsecond. As the age of the lens increases, the lifetime of the shorter-lived component lengthens while that of the longer-lived component decreases slightly. In order human lenses, the two lifetimes coalesce and the triplet decay exhibits purely monoexponential behavior. These photophysical characteristics apparently are due to age-related modification(s) of the protein in the human lens resulting in an increasingly more homogeneous environment around the porphyrin.

Combined Elastic and Raman Light Scattering of Human Eye Lenses

The distribution of the scattering coefficient (as defined in the appendix) at a wavelength of 647·1 nm along the visual axis of human eye lenses was investigated using a specially designed set-up for spatially resolved measurements of the intensity of the scattered light. For the same lenses, the distribution of the protein content was measured using confocal Raman microspectroscopy. Data collected by both methods were processed in terms of a recently developed theory of short-range, liquid-like order of crystallin proteins that accounts for eye lens transparency. Seven fresh intact human lenses of varying age have been investigated. In addition, elastic and Raman scattering measurements have been performed on fixed lenses. The main results and conclusions are: (1) Fixation significantly affects the light scattering properties of the eye lens. The average level of scattering increases and a change in the distribution of scattered light intensity along the visual axis occurs. Protein content and average distribution were not altered by fixation. (2) There are significant differences between the distribution of the scattering coefficient for lenses of different ages. For young lenses (18 and 20 years) regions with a low protein content (anterior and posterior cortex) show a higher level of elastic light scattering, while for older lenses (42-78 years old) there is no obvious correlation between the scattering level and protein content. (3) Changes in the level of light scattering along the visual axis of the lens cannot be explained by protein concentration effects. Therefore, these changes must be caused by changes in the supramolecular organization of lens proteins in the fibre cytoplasm. (4) The observed changes in light scattering may be related to the zones of discontinuity as observed in slitlamp and Scheimpflug photography of human lenses.

Photolysis of intact young human, baboon and rhesus monkey lenses.

Intact young human, baboon and rhesus monkey lenses were subjected to near-UV irradiation under identical conditions and fluorophore buildup was continuously monitored for several hours. The compositional changes occurring in the lenses were monitored by analyzing the ethanol extracts of the irradiated and control lenses using high-performance liquid chromatography and thin-layer chromatography (TLC). The chromatograms of the supernatant detected at 365 nm as well as the TLC scans showed the presence of 3-hydroxykynurenine glucoside (3-HKG) and two other kynurenine-type compounds. The 3-HKG and one of the compounds were found in all three species, while the remaining one was structurally different in the lower primates. A loss of 3-HKG as a result of irradiation was apparent in all the lenses and correlated with the buildup of the blue fluorophore, suggesting that the latter may be a photoproduct(s) of 3-HKG. The kinetic analysis of baboon and human lenses showed a slowdown in the fluorophore buildup as irradiation times increased. This was probably due to the competitive absorptions of 3-HKG and other chromophores present. Rhesus monkey lenses did not exhibit this slowdown.

Transformation of IR spectra of the human lens capsule due to traumatic lens subluxation and correlation with the excised and dried lens capsule under different treatments.

Transformation of the secondary conformational structure of the human lens capsule after traumatic lens subluxation in a patient was investigated by Fourier transform infrared (FT-IR) microscopic spectrometry. The result was compared with the IR spectra of type IV collagen in crystalline state, solid film and aqueous solution and those of the excised and dried lens capsule under compression or treated with ethyl alcohol. The results indicate that the IR spectra of the intact human lens capsule after traumatic lens subluxation were the same as those of the rabbit lens capsule, except in the 1,100-1,000 cm-1 proteoglycan region, but were different from those of type IV collagen aqueous solution at the amide I and II bands and the proteoglycan region, although type IV collagen is a predominant component of lens capsule. Two new peaks at 1,054 and 1,023 cm-1 appeared on the IR spectra of the intact human lens capsule and on those of type IV collagen in crystalline state and solid film after compression but not in normal rabbit lens capsule and native type IV collagen. It was also found that all the IR spectra of the excised and dried lens capsule were similar to those of native type IV collagen in crystalline state and solid film.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent structural changes in intact human lenses detected by synchrotron radiation X-ray scattering. Correlation with Maillard reaction protein fluorescence.

To understand alterations in the spatial organization of the crystallins, the major determinant of lens transparency, the x-ray scattering by intact normal human lenses from individuals 6-82 years of age was measured using synchrotron radiation. The angular dependence of the integrated scattering intensity is consistent with short-range order within the crystallin assemblies. A significant change in the scattering patterns of the lenses occurs after 55 years of age, in parallel with an increase of the fluorescence of the urea-insoluble crystallin fraction. This correlation suggests a gradual derangement of the short-range order as a result of cross-linking of the crystallin subunits by advanced Maillard reaction products that are generated by the continuous reaction of sugars, such as glucose or fructose, with proteins.

Age-related cysteine uptake as rate-limiting in glutathione synthesis and glutathione half-life in the cultured human lens

The study included human lenses of ages ranging from newborn to 92 years. Protein-free reduced glutathione decreased 14-fold, whereas protein-free oxidized glutathione increased 2·6-fold with increasing age. l-Cyst(e)ine uptake g −1 lens of very old cultured lenses decreased 70% from that exhibited in newborn lenses, demonstrating a marked decline of l-cyst(e)ine uptake as a function of age. In these same lenses the synthesis of reduced glutathione (μmol g −1 lens) decreased 73% with age. It was concluded that the glutathione decrease observed in the aging human lens was associated with decreased uptake of l-cyst(e)ine, decreased glutathione synthesis and possibly an increase in protein-free oxidized glutathione. The high correlation of l-cyst(e)ine uptake and glutathione synthesis supports the hypothesis that l-cyst(e)ine uptake is a rate-limiting factor of glutathione synthesis in the intact human lens. By the use of buthionine sulfoximine, the half-life of glutathione was estimated to be 90 hr in the cultured human lens.