Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis

PURPOSE: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro METHODS: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens. RESULTS: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking. CONCLUSIONS: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.     

Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter

PURPOSE: To measure lipid compositional and structural changes in lenses as a result of hyperbaric oxygen (HBO) treatment in vivo. HBO treatment in vivo has been shown to produce increased lens nuclear light scattering. METHODS: Guinea pigs, approximately 650 days old at death, were given 30 and 50 HBO treatments over 10- and 17-week periods, respectively, and the lenses were sectioned into equatorial, cortical, and nuclear regions. Lipid oxidation, composition, and structure were measured using infrared spectroscopy. Phospholipid composition was measured using (31)P-NMR spectroscopy. Data were compared with those obtained from lenses of 29- and 644-day-old untreated guinea pigs. RESULTS: The percentage of sphingolipid approximately doubled with increasing age (29-544 days old). Concomitant with an increase in sphingolipid was an increase in hydrocarbon chain saturation. The extent of normal lens lipid hydrocarbon chain order increased with age from the equatorial and cortical regions to the nucleus. These order data support the hypothesis that the degree of lipid hydrocarbon order is determined by the amount of lipid saturation, as regulated by the content of saturated sphingolipid. Products of lipid oxidation (including lipid hydroxyl, hydroperoxyl, and aldehydes) and lipid disorder increased only in the nuclear region of lenses after 30 HBO treatments, compared with control lenses. Enhanced oxidation correlated with the observed loss of transparency in the central region. HBO treatment in vivo appeared to accelerate age-related changes in lens lipid oxidation, particularly in the nucleus, which possesses less antioxidant capability. CONCLUSIONS: Oxidation could account for the lipid compositional changes that are observed to occur in the lens with age and cataract. Increased lipid oxidation and hydrocarbon chain disorder correlate with increased lens nuclear opacity in the in vivo HBO model.     

The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper

Oxidative effects on lens proteins have been linked with the formation of human age-related cataract, particularly nuclear cataract. This study investigated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on nuclear and cortical alpha-, beta- and gamma-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses were treated with 50 atm of either 100% N(2)(control) or 100% O(2)(experimental) for 3, 6, 16 and 48 hr. The levels of reduced glutathione (GSH) and water-soluble (WS) protein decreased more rapidly in the nucleus of the O(2)-treated lens than in the cortex. The first significant loss of WS protein in each of the two regions occurred when levels of GSH had decreased by at least 90% in either the nucleus (at 6 hr) or the cortex (at 16 hr). HPLC analysis of the nuclear WS proteins indicated that beta-crystallins were the first proteins affected by the oxidative stress. Soon after HBO-treatment was initiated (at 6 hr) and prior to insolubilization of protein, nuclear beta- and gamma-crystallins moved to the higher molecular weight alpha-crystallin fraction; 2-D gel electrophoresis and Western blotting indicated the presence of disulfide-crosslinked and non-crosslinked beta- and gamma-crystallins in this fraction. Significantly different HBO-induced effects were observed on lens cortical crystallins compared to those for the nucleus. For example, gamma-crystallins in the cortex shifted very soon after HBO-treatment (at 3 hr) to slightly higher molecular weights, possibly the result of protein/glutathione mixed disulfide formation; however, this phenomenon was not observed in the nucleus. Cortical beta- and gamma-crystallins remained in solution longer than nuclear proteins following HBO-treatment of the lenses, presumably the result of protection from the four-fold higher level of GSH (22 vs 6 m M) present in the lens periphery. Surprisingly, there was no movement of beta- and gamma-crystallins to alpha(H)- and alpha-crystallin fractions in the cortex of the O(2)-treated lens, in contrast to that observed for the nucleus. Cortical crystallins appeared to go directly from being soluble to being insoluble with no high molecular weight intermediate stage. The data suggested a possible chaperone-like function for alpha-crystallin in the nucleus of the stressed lenses, but not in the cortex. HBO-induced effects on lens nuclear supernatants, which mimicked those observed for intact lenses, could be nearly completely prevented by the copper-chelator bathocuproine, but not by the iron-chelator deferoxamine. Overall, the results provide additional evidence demonstrating an increased susceptibility of the lens nucleus to oxidative stress; the greater protective ability of the cortex may be linked to a higher capacity for beta- and gamma-crystallin/glutathione mixed disulfide formation, inhibiting disulfide-crosslinked insolubilization. The data also implicate copper as a catalyst for the autoxidation of -SH groups in the lens, and suggest that alpha-crystallin chaperone-like activity may play a greater role in the lens nucleus than in the cortex in preventing oxidative insolubilization of crystallins.     

Exposure of rabbit lens to hyperbaric oxygen in vitro: regional effects on GSH level

Previous studies have indicated that in vivo exposure to hyperbaric O2 may be associated with the development of nuclear cataract. In the present work, in vitro effects of hyperbaric O2 on rabbit lenses were investigated following culture of the lenses in an atmosphere of 99% O2 at pressures ranging between 1 and 100 atm. Treatment with O2 resulted in a significant decrease in the level of reduced glutathione (GSH) in the lenses even at the lower pressures studied (less than 8 atm). At 100 atm O2 the loss of GSH was 85% after a 3 hr exposure. At 8 atm O2 a significant drop in GSH concentration was shown to occur in the lens nucleus prior to loss of the tripeptide in the superficial cortex. O2-treated lenses became hazy in appearance, especially at the higher pressures, but did not become densely opaque. Pressures of N2 up to 100 atm had no effect on either lens transparency or on the concentration of GSH. Although oxidized glutathione (GSSG) was detected in the whole lens at pressures of O2 as low as 4 atm, no change in GSH level or evidence for GSSG accumulation was observed in the capsule-epithelium of the lens at pressures as high as 50 atm O2. Ninety percent of the GSSG present in lenses after exposure to 100 atm O2 could be reconverted to GSH by subsequent culture of the lenses under normal conditions. Exposure of lenses to 50 atm O2 produced a three-fold stimulation of hexose monophosphate shunt activity, equal to that which has been reported for treatment of lenses with 0.06 mM H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural characterization and Fourier analysis of fiber cell cytoplasm in the hyperbaric oxygen treated guinea pig lens opacification model

The structural characteristics of differentiated fiber cells in control and hyperbaric oxygen (HBO)-treated guinea pig lenses were examined by transmission electron microscopy (TEM). Emphasis was placed on cell damage, membrane integrity, and cytoplasmic texture. Given the faint gross opacities observed in HBO-treated lenses in previous studies, it was hypothesized that subtle but significant morphological differences due to oxidative damage exist between control and treated animals. Experimental animals received either 70 or 85 treatments with HBO (2.5 atm of 100% O(2) for 2.5 hr, 3 times per week for 5-7 months). All specimens were obtained within 24 hr of death. Freshly cut Vibratome lens sections were fixed and processed for low and high-magnification thin-section TEM analysis. Cytoplasmic texture was analyzed using Fourier and autocorrelation image processing techniques. Low-magnification analysis revealed relatively insignificant differences in general appearance between the fiber cells of the inner fetal and embryonic nuclei in control and HBO-treated guinea pigs. Both groups demonstrated cells of similar morphology with equivalent membrane complexity and homogeneous cytoplasmic texture. Evidence of any major cellular damage or extracellular space debris was not obvious. High-magnification analysis of the cytoplasm of the treated lenses exhibited a mild, yet detectable increase in texture compared with controls and was confirmed by Fourier analysis. Cytoplasmic texture increased in complexity with additional treatments. The absence of major cellular damage in the lenses of HBO-treated animals suggests a less conspicuous source of light scattering. The small changes in cytoplasmic organization observed between treated and control animals may entirely account for the increase in nuclear light scattering observed by slit lamp. The results obtained with this guinea pig/HBO model parallel many of the morphological data associated with human nuclear cataracts. The high-angle scattering observed in the lens of the HBO-treated guinea pig may represent the type of cytoplasmic reorganization that occurs with mild oxidation, effectively making it a valuable model for human lens aging.     

Glutathione: a vital lens antioxidant.

The reducing compound glutathione (GSH) exists in an unusually high concentration in the lens where it functions as an essential antioxidant vital for maintenance of the tissue's transparency. In conjunction with an active glutathione redox cycle located in the lens epithelium and superficial cortex, GSH detoxifies potentially damaging oxidants such as H2O2 and dehydroascorbic acid. Recent studies have indicated an important hydroxyl radical-scavenging function for GSH in lens epithelial cells, independent of the cells' ability to detoxify H2O2. Depletion of GSH or inhibition of the redox cycle allows low levels of oxidant to damage lens epithelial targets such as Na/K-ATPase, certain cytoskeletal proteins and proteins associated with normal membrane permeability. The level of GSH in the nucleus of the lens is relatively low, particularly in the aging lens, and exactly how the compound travels from the epithelium to the central region of the organ is not known. Recently, a cortical/nuclear barrier to GSH migration in older human lenses was demonstrated by Sweeney et al. The relatively low ratio of GSH to protein -SH in the nucleus of the lens, combined with low activity of the glutathione redox cycle in this region, makes the nucleus especially vulnerable to oxidative stress, as has been demonstrated with use of in vivo experimental animal models such as hyperbaric oxygen, UVA light and the glutathione peroxidase knockout mouse. Effects observed in these models, which are currently being utilized to investigate the mechanism of formation of human senile nuclear cataract, include an increase in lens nuclear disulfide, damage to nuclear membranes and an increase in nuclear light scattering. A need exists for development of therapeutic agents to slow age-related loss of antioxidant activity in the nucleus of the human lens to delay the onset of cataract.     

FTRaman和FTIR光谱对老年性白内障晶体的研究

目的探讨晶体混浊过程中晶体蛋白成分及其结构变化。方法首次用ＦＴＲａｍａｎ和ＦＴＩＲ（ＦｏｕｒｉｅｒｔｒａｎｓｆｏｒｍＲａｍａｎａｎｄｉｎｆｒａｒｅｄ）光谱及利用相关峰积分的半定量方法对老年性白内障不同混浊程度的晶体构像变化进行研究。结果按Ｃｈｙｌａｃｋ等的ＬＯＣＳ方法划分为Ⅲ级晶体的两种光谱特性介于０级和Ⅳ或Ⅴ级晶体之间；Ⅳ和Ⅴ级晶体的两种光谱构像非常相似，它们的一些谱峰与０级晶体的有明显区别。两种光谱对它们的检测既有一致性，也有互补性。结论晶体混浊发展不仅与老年性白内障晶体的三种晶体蛋白含量有关，也与其晶体蛋白中的ＳＨ、ｔｒｐ、ｐｈｅ和－ＣＨ２等受到各种损伤有关。     

Raman study of the lenses of spontaneously-occurring and streptozotocin-induced diabetic rats

The WBN/Kob rat is an animal model of spontaneously occurring diabetes only in males at the age of around one year. The diabetic cataract develops about a half year after the onset of diabetes. Using laser Raman spectroscopy we monitored WBN/Kob rat lenses which had chronic hyperglycemic stress and streptozotocin-induced diabetic rat lenses which showed relatively acute cataract formation. WBN/Kob rat lenses revealed a marked increase in the lens water content only in the cortical portion. On the other hand, streptozotocin-induced diabetic rat lenses showed a pronounced increase in the lens water content both in the cortical and the nuclear portion.     

Glutathione levels in human lens: regional distribution in different forms of cataract

The content of glutathione is high in the anterior lens cortex (plus epithelium) and the posterior lens cortex, whereas it is substantially lower in the lens nucleus. A decrease of glutathione in the lens cortex does not occur with lenses from adults up to the highest age. The glutathione content is highest in the cortex of clear fresh lenses and shows a decrease with deep supranuclear cataract, primary nuclear cataract (cataracta brunescens nigra) and clear lenses post-mortem (approximately 20 hr). The subcapsular cataract, especially with additional secondary nuclear cataract, with cataracta matura or intumescens, shows a rapid and pronounced progressive decrease in the glutathione content.     

Age-related nuclear cataract-oxidation is the key

Age is by far the biggest risk factor for cataract, and it is sometimes assumed that cataract is simply an amplification of this aging process. This appears not to be the case, since the lens changes associated with aging and cataract are distinct. Oxidation is the hallmark of age-related nuclear (ARN) cataract. Loss of protein sulfhydryl groups, and the oxidation of methionine residues, are progressive and increase as the cataract worsens until >90% of cysteine and half the methionine residues are oxidised in the most advanced form. By contrast, there may be no significant oxidation of proteins in the centre of the lens with advancing age, even past age 80. The key factor in preventing oxidation seems to be the concentration of nuclear glutathione (GSH). Provided that nuclear GSH levels can be maintained above 2 mm, it appears that significant protein oxidation and posttranslational modification by reactive small molecules, such as ascorbate or UV filter degradation products, is not observed. Adequate coupling of the metabolically-active cortex, the source of antioxidants such as GSH, to the quiescent nucleus, is crucial especially since it would appear that the cortex remains viable in old lenses, and even possibly in ARN cataract lenses. Therefore it is vital to understand the reason for the onset of the lens barrier. This barrier, which becomes apparent in middle age, acts to impede the flow of small molecules between the cortex and the nucleus. The barrier, rather than nuclear compaction (which is not observed in human lenses), may contribute to the lowered concentration of GSH in the lens nucleus after middle age. By extending the residence time within the lens centre, the barrier also facilitates the decomposition of intrinsically unstable metabolites and may exacerbate the formation of H(2)O(2) in the nucleus. This hypothesis, which is based on the generation of reactive oxygen species and reactive molecules within the nucleus itself, shifts the focus away from theories for cataract that postulated a primary role for oxidants generated outside of the lens. Unfortunately, due to marked variability in the lenses of different species, there appears at present to be no ideal animal model system for studying human ARN cataract.     

老年性白内障晶状体核硬度及相关因素研究

目的 检测老年性白内障晶状体核硬度并探讨其相关的因素。方法 ５０岁以上老年性白内障患者４７例（４７只眼）。术前散瞳,并按ＬＯＣＳⅡ分类,测量白内障囊外摘出晶状体核的重量、中心厚度和直径,采用测定穿透晶状体核力量的装置,测定晶状体核的硬度。多元回归分析晶状体核硬度与年龄、核颜色、病程等因素的关系。结果 切穿颜色为Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级核所需用的平均硬度与年龄、核颜色、病程等因素的关系。结果 切穿颜色     

UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious,oxidative effects

The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5 mWcm(-2), 340-410 nm wavelength, peak at 365 nm) for 4-5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70-90% of 340-400 nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30-35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein-thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, alpha- and beta- tubulin, vimentin and alpha-actinin (60-100%). The results indicate that a 4-5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O(2) present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.     

The presence of a human UV filter within the lens represents an oxidative stress

It has recently been demonstrated that, with age, UV filters such as 3-hydroxykynurenine glucoside, bind to proteins in the human lens. This covalent interaction leads to colouration of the normal lens, and results from the instability of the kynurenine side chain. Other primate UV filters, in addition to containing the same side chain, can also be readily oxidized. One such compound is 3-hydroxykynurenine (3OHKyn). It has been proposed that oxidation of bound and/or free UV filters, such as 3OHKyn may give rise to the lens colouration associated with age-related nuclear cataract. Therefore it has become important to understand the oxidation of 3OHKyn within the lens. In this study, intact bovine lenses (which lack UV filters) were incubated with 3OHKyn and various lens parameters monitored. The effect of exposure to hyperbaric oxygen (HBO) was also assessed, both alone, and in combination with prior 3OHKyn incubation. Glutathione (GSH), protein sulfhydryl and protein-bound sulfhydryl levels, as well as soluble protein content and gel filtration profiles, were obtained for cortical and nuclear regions after defined periods of incubation. The presence of the primate UV filter, 3OHKyn, at concentrations similar to those present in the human lens, was shown to produce considerable oxidative stress within the lens, as judged by its effect on GSH. This effect was noted under normobaric conditions, but was exacerbated by increased oxygen. Exposure of lenses to HBO caused a marked fall in GSH in cortical and nuclear regions. This effect was exaggerated in the presence of 3OHKyn. HBO treatment also lead to a fall in protein sulfhydryl content, however, this was only partial (approximately 1 mol SH per mol protein) and changed only slowly, even with extended periods of exposure to HBO, suggesting that most crystallin sulfhydryl groups may be buried. 3OHKyn did not appreciably affect this oxidation although it did cause an increase in the level of protein-bound sulfhydryl. HBO treatment produced a more than two-fold increase in protein-bound sulfhydryl content in the cortex. There was little influence of 3OHKyn alone on protein solubility, even with extended periods of incubation, however, incubation for 72 hr in the presence of HBO caused a significant increase in insoluble protein particularly in the nucleus. This insolubilization was further increased in the presence of 3OHKyn. FPLC profiles showed that the proportion of gamma and beta crystallins in the soluble fraction decreased following HBO, suggesting that these may be involved in disulfide bond formation. This study demonstrates that a readily oxidized compound, such as the primate UV filter 3OHKyn, represents an oxidative stress within the lens and that such oxidative processes can be exacerbated if the concentration of oxygen within the lens is increased. We speculate that this factor may account for the evolution of unusually high levels of glutathione reductase in human lenses.     

Hyperbaric oxygen in vivo accelerates the loss of cytoskeletal proteins and MIP26 in guinea pig lens nucleus

Previous studies have shown that treatment of guinea pigs with hyperbaric oxygen (HBO) produces certain changes in the lens nuclei of the animals which are typical of those occurring during aging. These include an increase in nuclear light scattering (NLS), elevation in levels of oxidized thiols, loss of water-soluble protein and damage to nuclear membranes. The present study investigated the effect of HBO-treatment in vivo on lens cytoskeletal proteins and MIP26 which are also known to undergo alteration with age. Young (2-month-old) and old (18-month-old) guinea pigs were treated 15 and 30 times with HBO (3 times per week with 2.5 atmospheres of 100% oxygen for 2.5 hr periods). SDS-PAGE and Western blotting showed that HBO-treatment of the older animals accelerated the age-related loss of five nuclear cytoskeletal proteins including actin, vimentin, ankyrin, alpha-actinin and tubulin, compared to levels present in age-matched controls (effects on spectrin and the beaded filaments were not investigated in this study). Treatment of the young animals with HBO produced losses which were primarily associated with concentrations of the nuclear alpha- and beta-tubulins; these cytoskeletal proteins were observed to be most sensitive to the induced oxidative stress, and were affected earliest in the study. Disulfide-crosslinking, rather than proteolysis, appeared to be the main cause of the HBO-induced cytoskeletal protein loss (elevated levels of calcium, which might have induced proteolysis, were not found in the experimental nuclei). Loss of MIP26 was observed only in the older guinea pigs treated 30 times with HBO; both disulfide-crosslinking and degradation to MIP22 were associated with the disappearance. Thus, nuclear MIP26 was susceptible to oxidative stress, but less so than the cytoskeletal proteins, particularly the tubulins. No cortical effects on either MIP26 or the cytoskeletal proteins were observed under any of the treatment protocols. No direct link was observed between an HBO-induced increase in NLS (observed in both the young and old animals using slit-lamp biomicroscopy) and losses of either MIP26 or the cytoskeletal proteins. The appearance of HBO-induced nuclear opacity without any change in the levels of nuclear sodium, potassium or calcium is similar to that observed previously for human senile pure nuclear cataracts. The results provide additional evidence that molecular oxygen can enter the nucleus of the lens and promote age-related events. The observed effects on MIP26 and the cytoskeletal proteins are indicative of an increased level of lens nuclear oxidative stress in the HBO model, possibly a precursor to nuclear cataract.     

Biochemical changes in selenite cataract model measured by high-resolution MAS H NMR spectroscopy

PURPOSE: To correlate certain levels of lens opacification with high-resolution magic-angle spinning proton nuclear magnetic resonance (HR-MAS (1)H NMR) spectroscopy analysis of the biochemical changes in rat lenses in a selenite cataract model. METHODS: Selenite cataract was induced by injecting 13-day-old Sprague-Dawley rat pups with a single subcutaneous dose of sodium selenite (3.28 mg/kg in 0.9% sodium chloride solution). Lens opacification was observed using a photographic slit-lamp microscope at selected time-points 3, 6 and 9 days after selenite injection and was then graded (levels 0, 1 and 2). The animals were killed after the slit-lamp microscopy, lenses were removed and HR-MAS (1)H NMR spectra from intact lenses were obtained. Relative changes in metabolite concentrations were determined after comparison with matched lenses from untreated animals. RESULTS: Photographic slit-lamp microscopy revealed different stages of cataract in all animals treated with selenite. In the high quality HR-MAS (1)H NMR spectra of the lenses, more than 30 different metabolites were identified in each lens. With the exception of taurine, the concentrations of all amino acids showed a significant increase (p < 0.05) in the second level of cataract. By contrast, glutathione (GSH), succinate and phosphocholine concentrations were significantly reduced. CONCLUSIONS: For the first time, this study demonstrates the potential to correlate the level of lens opacification with the biochemical changes obtained with HR-MAS (1)H NMR spectroscopy analysis in a selenite cataract model.     

大鼠半乳糖性白内障晶状体上皮细胞的病理变化

目的　探讨半乳糖性白内障大鼠晶状体上皮细胞的改变。方法　２４只ＳＤ雌性大鼠,分为正常对照组和白内障组,每组１２只。白内障组大鼠用半乳糖饲料喂养,正常对照组大鼠用普通颗粒饲料喂养。裂隙灯显微镜下观察大鼠晶状体混浊程度变化,观察至３０ｄ处死大鼠后取晶状体,在光镜和电镜下观察晶状体病理组织和超微结构改变。结果　观察至３０ｄ时,正常对照组大鼠晶状体保持透明,白内障组大鼠９眼（３７．５％）出现均一的皮质性混浊,１５眼（６２．５％）出现核混浊。白内障组光镜下可见晶状体皮质和核部大量纤维细胞水肿、崩解,前囊膜下及后囊膜下出现纤维细胞样的有核细胞堆积;透射电镜下可见晶状体上皮细胞变性、增生并突破晶状体上皮层向浅层皮质移行。结论　半乳糖性白内障不仅有晶状体纤维细胞水肿及结构破坏,还存在晶状体上皮细胞的异常增生、分化和移行。     

Variation of the concentration of sulfhydryl along the visual axis of aging lenses by laser Raman optical dissection technique

Laser Raman spectroscopy is a powerful, non-invasive analytical technique for studies of constituents in the intact lens. It was employed here to measure the variation of sulfhydryl levels (protein -SH plus glutathione -SH) along the visual axis (VA) of the lenses of different ages. The VA length of each rat and mouse lens was measured using a translation stage micrometer and the laser scattering. The rat VA length varied from 2·85 mm (26 days) to 4·32 mm (16 months). Spectra were obtained for 20 increments along the VA. The results are presented as VA profiles. The salient features in the series of curves are two maxima in the cortex (one anterior, one posterior) and a central minimum. The youngest rat lens (maintained at 37°C to avoid cold cataract) showed a bell-shaped curve. All curves were nearly symmetric for the rat, the minima slightly off center for the mouse. The two maxima of the second youngest rat lens were separated by a distance of 1·55 mm which increased to 2·95 mm in the oldest lens. In a $\text{7}\text{1}\text{2}$ month lens a 0·78 mm segment of the VA center contained too little sulfhydryl to be detected by this technique. This segment increased to 1·44 mm in the oldest lens. The apparent rate of decrease in SH, being quite pronounced in the nucleus, is different at other points along the VA. A plot of sulfhydryl level vs. age for several points at distance r from the center (VA midpoint) along the VA indicates a steady decrease in SH levels with age for r < 1·2 mm. For larger r, there is actually an increase in SH. These results are interpreted in terms of 2SH→S-S conversion, changing rates of synthesis of the different crystallins and of glutathione synthesis along the VA.     

Lens autofluorescence and light scatter in relation to the lens opacities classification system, LOCS III.

PURPOSE: To compare values of the human lens autofluorescence and back light scatter measurements with the improved Lens Opacities Classification System, LOCS III. METHODS: We measured autofluorescence and back light scatter of the lens from 122 smoking males aged 57 to 76 years who participated in a cancer prevention study. The retroillumination and slit-lamp photographs of the lenses were graded according to LOCS III by the Center for Ophthalmic Research in Boston. Lens fluorometry was carried out with a previously described technique using blue-green (495 nm/520 nm) autofluorescence range. Interzeag Lens Opacity Meter 701 was used for light scatter measurements. RESULTS: LOCS III nuclear opalescence and color grades were statistically significantly correlated with lens autofluorescence as well as with light scatter values. The lens transmission index of autofluorescence measurements showed the highest correlation with the nuclear color (r = -0.71; p < 0.0001) and the light scatter value with nuclear opalescence (r = 0.64; p < 0.0001). There was no correlation between autofluorescence measurements and LOCS III grades of cortical or posterior subcapsular cataract. A weak relation could be found between the grades of cortical cataract and light scatter values. CONCLUSIONS: The lens fluorometry provides a practical clinical technique to evaluate the yellow coloration and opalescence of the human lens nucleus. It may be a useful additional tool together with a subjective grading system in the follow-up of optical changes occurring in the nuclear region of the lens.     

Deferoxamine effect on selenite-induced cataract formation in rats.

A single subcutaneous dose of 30 nmol of sodium selenite per gram of body weight in 13-day-old rats resulted in posterior subcapsular cataract (PSC) after 24 hr and bilateral nuclear cataracts at 72-96 hr. Within 24 hr of treatment, a 60% decrease in lens glutathione was seen. A loss of calcium homeostasis observed by 48 hr resulted in increased lens calcium (4 mumol/g dry weight), which accompanied nuclear opacification. The iron chelator, deferoxamine (DF), was evaluated as a potential protective agent against these selenite-induced changes. Three doses each consisting of 1.1 mumol DF/g body weight were administered during the initial 24 hr of selenite exposure. Within 96 hr, all lenses from animals treated only with DF remained transparent, but 50% of these lenses showed cortical cataract at 3 wk postinjection. Concurrent administration of DF and selenite protected 80% of rats against PSC after 48 hr and 25% against nuclear cataract after 96 hr. No elevation in lens calcium occurred in the protected lenses. An additional 20% of animals were not protected fully but showed substantially less nuclear opacity than with selenite alone. They had a significant but moderate increase in lens calcium. After 3 wk (animal age, 35-40 d), cataract appeared in these "protected" lenses involving both the nucleus and cortex and loss of ion homeostasis. The glutathione content remained lower in lenses from animals treated with both selenite and DF compared with those from selenite-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of hydroxyl radicals in the human lens is related to the severity of nuclear cataract

Recent studies have identified specific hydroxylated amino acid oxidation products which strongly suggest the presence of hydroxyl radical (HO.)-damaged proteins in human cataractous lenses. In the present study, the ability of early stage (type II) and advanced (type IV) nuclear cataractous lens homogenates to catalyse HO. production in the presence of H(2)O(2)was investigated using electron paramagnetic resonance (EPR) spectroscopy with the free radical trap, 5,5-dimethyl-1-pyrroline- N -oxide (DMPO). Cataractous lens homogenates incubated with 1 m m H(2)O(2)generated a distinct HO. signal, which was significantly more intense in the nuclear region of the type IV compared to the type II lenses. The ability of individual lens nuclei and cortices to stimulate HO. production was positively correlated. The DMPO-HO. signal was competitively inhibited by ethanol, confirming that the DMPO-HO. signal was due to HO. formation and not DMPO-OOH degradation. The metal ion chelator, diethylenetriaminepentaacetic acid, also inhibited HO. formation, indicating that lenticular metal ions play a key role in HO. formation. Cataractous lens homogenates also stimulated ascorbyl radical production, further suggesting the presence of redox-active metal ions in the tissue. Analysis of lenses for total Fe and Cu (using atomic absorption spectrometry) showed that the more advanced type IV lenses tended to have higher Fe, but similar Cu, levels compared to the type II lenses. The levels of both metals were lower in non-cataractous lenses. These data support the hypothesis that transition metal-mediated HO. production may play a role in the aetiology of age-related nuclear cataract.