Involvement of inducible nitric oxide synthase in cataract formation in Shumiya cataract rat (SCR)

PURPOSE: Our previous results showed that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly suppresses lens opacification in Shumiya cataract rat (SCR). Therefore, we examine whether iNOS is upregulated and involved in cataract formation in SCR. METHODS: The expressions of iNOS mRNA and iNOS protein in SCR lenses were examined by RT-PCR and Western blotting, respectively. Calpain-mediated alphaB-crystallin proteolysis was analyzed by Western blotting using antibody specific to the calpain-generated fragment of alphaB-crystallin. Lens opacification was analyzed using computerized image analysis software connected to the Anterior Eye Segment Analysis System (EAS-1000, Nidek). Calcium contents in lenses were measured by atomic absorption spectrophotometry. RESULTS: High levels of iNOS mRNA and iNOS protein are expressed in cataractous lenses compared with normal lenses. The increases in their expression are markedly suppressed by the oral administration of AG, which acts to prevent lens opacification. The induction of iNOS protein is observed before the elevation in calcium content and the acceleration of calpain-mediated proteolysis, both of which are closely related to the development of lens opacification. CONCLUSIONS: These findings strongly suggest that iNOS is involved in cataract formation in SCR. The induction of iNOS occurs prior to the elevation of calcium content and its induction is inhibited by AG-treatment. Considering our previous result that the elevation of calcium content is also prevented by AG-treatment, it is conceivable that upregulation of iNOS causes calcium influx into lens cells and the subsequent activation of calpain.     

Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice

PURPOSE: The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS: Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day-old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS: Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of alphaA-crystallin. CONCLUSIONS: Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.     

Lens reconstruction after mature cataract in SCR rat

PURPOSE: To conduct a long-term observation study of SCR rats that had developed a mature cataract at 11 weeks of age at 3-month intervals until the rats were 12 months old. METHODS: Lenses of 15 rats were examined with both light and electron microscopes. RESULTS: At 12 weeks, opacity was observed in the perinuclear zone and the cortical intermediate layer. Liquefaction of the posterior subcapsular area and regression of cortical superficial fibers were also observed at this stage. Epithelial cells at the anterior polar area were multilayered. At 12 months, the lens recovered as a result of the regenerated lens fibers in the intermediate layer and the cortical superficial layer, although the opacity remained in the perinuclear zone. The multilayered cellular structure in the center of the epithelium returned to its original monolayer form. However, the equatorial epithelial cells became vacuolated and swollen with age, showing regression from the bow region. CONCLUSIONS: These results suggest that the decrease of opacity in SCR rats is merely a temporary phenomenon that reflects the differentiating and metabolizing functions of the epithelial cells. With initiation of epithelial regression, the regeneration of the lens fibers ceased, suggesting that further decrease in opacity was no longer possible.     

珠子草对大鼠半乳糖性白内障的抑制作用

目的:本研究旨在评价珠子草在体外及体内抑制半乳糖性白内障的作用。方法:研究珠子草提取液在体内及体外抑制半乳糖性白内障的作用。300g/L半乳糖饮食诱导半乳糖性白内障大鼠,75, 150及300mg/kg3剂量水平口服珠子草。在体外小鼠晶体培养基中掺入半乳糖(30mmol/L)建立渗透性应激,研究珠子草(720及880μg/mL)对谷胱甘肽(GSH)及山梨糖醇水平的影响。结果:在体内,珠子草显著延缓的白内障的发生和发展。除了延缓各期白内障的发展,直至试验周期结束,低剂量组未发展至IV期。在体外,880μg/mL珠子草培养晶状体显示较高浓度的GSH和山梨糖醇浓度的降低。在体内,与对照组比较,75mg/kg珠子草显著延缓了白内障的发生和发展。结论:在试验模型中珠子草延缓了白内障的发展。然而,将其延伸运用在人类预防白内障需要进一步的研究。     

Anti cataract potential of phyllanthus niruri in galactose induced cataractogenesis of rat

AIM: To evaluate anti cataract effect of phyllanthus niruri (PN) both in vitro and in vino galactose induced cataract. METHODS: Aqueous extract of PN was evaluated against galactose-induced cataract both in vitro and in vivo. Galactosaemic cataract was induced in rats by feeding 300g/L galactose diet. PN was administered orally at three-dose levels 75, 150 and 300mg/kg of body weight. Rat lenses were subjected to osmotic stress in vitro by incorporating galactose (30mmol/L) in the culture medium. The effect of PN (720 and 880ug/mL) on the glutathione (GSH) and polyols levels was studied. RESULTS: PN significantly delayed the onset and progression of cataract in vivo. In addition to the delay in reaching various stages of development of cataract, stage IV did not develop with lower doses till the completion of experimental period. Lenses treated with PN 880ug/mL concentration showed higher levels of GSH and decreased levels of polyols in vitro.In vivo , 75mg/kg significantly delayed the onset and progression of cataract as compared to control. CONCLUSION: Phyllanthus niruri delays the process of cataracto-genesis in the experimental models. However, further study is required to extrapolate the use in human beings for the prevention of cataract.     

Dipeptidyl peptidase Ⅲ在先天性白内障大鼠晶状体中的免疫活性变化

目的 观察8、10、12、14周龄正常及先天性白内障大鼠晶状体中dipeptidyl peptidaseⅢ(DPPⅢ)的免疫活性变化，探讨其与白内障发病的关系。方法 分别于8、10、12、14周龄鼠，摘取正常对照组以及先天性白内障大鼠晶状体，应用抗生物素蛋白．生物素-过氧化物酶复合体法，观察不同时期大鼠晶状体中DPPⅢ的免疫活性变化。结果 各周龄先天性白内障大鼠晶状体纤维部DPPⅢ的免疫活性明显高于正常对照组，特别是12、14周白内障晶状体中DPPⅢ的免疫阳性产物扩展至晶状体核周部。结论 DPPⅢ在白内障大鼠晶状体中免疫活性增强，它有可能在白内障形成过程中参与晶状体蛋白的水解分化，从而促进白内障的形成。     

Biochemical and morphological changes during development of sugar cataract in Otsuka Long-Evans Tokushima fatty (OLETF) rat

The relationship between the polyol pathway and sugar cataracts has been studied extensively using streptozotocin-induced diabetic rats and galactose fed rats as animal models for insulin-dependent diabetes mellitus (IDDM). In these models, sugar cataracts progress quickly, leading to rapid lenticular polyol accumulation in the early stages of cataract formation. In 1992, a new animal model of non-insulin-dependent diabetes mellitus (NIDDM), the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, was established. In the present study, we examined both biochemical and morphological changes in the lenses of the OLETF rats to determine whether these changes reflect those associated with diabetic cataract formation and to clarify their relationship with the polyol pathway. For the biochemical analysis, we measured the enzyme activity of aldose reductase (AR) and sorbitol dehydrogenase (SDH) and the sorbitol levels using 20, 40 and 60 week old OLETF or control Long-Evans Tokushima Otsuka (LETO) rats. Enzyme activities of AR and SDH, which were lower in 20 week old OLETF rats than in LETO rats, were increased in 60 week old OLETF rats. The lenticular sorbitol level of the OLETF rats was similar to the control level at 20 weeks of age, but it was markedly increased at 40 weeks of age, and slightly decreased at 60 weeks of age compared with rats at 40 weeks but not compared with controls. Slight lens fiber swelling was observed in the anterior and/or posterior subcapsular regions of 40 week old OLETF rats, accompanying elevated sorbitol level and slightly increased SDH activity in the lens. Swelling and liquefaction of lens fibers were observed in the subcapsular and supranuclear region of 60 week old OLETF rats, as well as decreased lenticular sorbitol, and markedly increased SDH activity compared with rats at 40 weeks. AR activity was also increased causing the elevation of sorbitol in lenses of OLETF rats during the early stages of cataract formation. Despite differences in the etiology of diabetes mellitus, the strain of rat and the rate of disease progression in the OLETF rat model compared with other diabetic models, the present results support the theory that the polyol pathway via AR is a factor in the development of sugar cataracts.     

Modelling cortical cataractogenesis. IV. Induction by hygromycin B in vivo (swine) and in vitro (rat lens)

The cataractogenic effect of the anthelminthic antibiotic hygromycin B was examined. Histochemical staining and scanning electron microscopy of field samples of cataracts from pigs fed hygromycin B were examined in vitro in isolated rat lenses, in vivo in pigs maintained on several defined dietary regimens.In vivo, field samples showed globular degeneration similar to that which we have previously found for human senile posterior cortical subcapsular cataracts (PCSC), including intensified immunofluorescence for α-, β- and γ-crystallins and actin, and similar morphology by scanning electron microscopy. Four dietary regimens were tested: (1) 66 g/t, continuously, (2) 13·2 g/t, continuously, (3) 13·2 g/t intermittently (8 weeks on, 8 weeks off), the manufacturer's suggested regimen, and (4) control. All but one pig on regimens 1 and 2 had developed cataracts with associated globular degeneration when the experiment was terminated after 14 months, but no pigs on regimens 3 and 4 had cataracts.In vitro, in rat lenses incubated in M199 cortical lenticular opacities (cataracts) with associated globular degeneration were found at concentrations of hygromycin B as low as 10^?10m; vitamin E seemed to offer some protection, in that no cataracts were found at 10^?10m-hygromycin if vitamin E (2·4 μm) was also present.The possibility exists that this drug may provide another system for modelling human senile PCSC, as well as a potential cataractogenic agent for human population.     

Characterization of peptide inducing cataractogenesis in lens of hereditary cataractous rat (ICR/f RAT)

A material with inhibitory action to Na+/K+ ATPase was found in the lens of the ICR/f rat, a recessive hereditary cataractous rat. The material also induced lens opacification in vitro. From the results of amino acid analysis and by secondary ion mass spectroscopy, it was suggested that the material might contain approximately equimolar amounts of four amino acids, ie, aspartic acid, serine, glutamic acid and glycine, and that the molecular weight was 444. These facts suggested that this material with Na+/K+ ATPase inhibitory action might be a peptide. However, there is not yet any corroborating evidence to show whether this peptide is only a single material or not. The peptide significantly increased with aging in the lens of the ICR/f rat until approximately 90 days, when cataract became manifest, but its content decreased thereafter. This study suggests that one of the causes of cataractogenesis in the ICR/f rat might be this peptide, which is transformed in the lens with aging, and also that the peptide might accelerate lens opacification after cataractogenesis.     

Lp82 is the dominant form of calpain in young mouse lens

The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC.The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a unique N -terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.     

Age-related cataract in the hereditary cataract rat (ICR/1): development and classification.

Lens opacification of the Ihara hereditary cataract rat (ICR/1 rat) was followed up with slit lamp biomicroscopy and photography up to 1 year after birth. The development of lens opacities was first recognized in the anterior superficial cortex of the equatorial region between 30 and 44 days of age. It progressed to maturity around 93-107 days after birth. Cataract development was classified into six stages. The increase of lens weight indicated a continuous lens growth after birth which seemingly even continued after the occurrence of lens opacities. After around 60 days of age, the lens showed a continuous increase of water content, suggesting a participation of increased water content in lens opacification.     

Limited proteolysis of MP26 in lens fiber plasma membranes of the galactose-induced cataract in the rat

Lenses of rats maintained on a 50% galactose diet displayed the development of a progressive cataract which was cortical at 3-11 days, and progressively internalized (nuclear as well) and mature at 16-20 days of feeding. Lens fiber plasma membranes were isolated from female rats subjected to the galactose diet and from controls at 11, 19, and 31 days of feeding, and analyzed by SDS-PAGE. Examination of the fiber plasma membranes from whole lenses of galactose-fed rats demonstrated the limited proteolysis of MP26 into MP23-24, in both the cortical and mature stages of the resultant cataracts. The limited proteolysis of MP26 was first evident in the lens cortex at 11 days of galactose feeding, and was evident as well, and more severe in proportion, in the lens nucleus at 19 days of feeding. The greatest proportion in MP26 limited proteolysis was observed in whole lenses at 31 days of galactose feeding. The regional progression of MP26 limited proteolysis closely paralleled the morphological progression of the galactose-induced cataract in the rat. The proportion of lens MP26 which underwent limited proteolysis into MP23-24 increased the longer the animals were kept on the galactose diet.     

Comparative diets of a idiopathic senile cataract and normal population: dietary risk factors in cataractogenesis

A survey was carried out contrasting the diets of population with senile cataracts and control populations. Results failed to confirm the reports of others suggesting that ingestion of moderate amounts of seafood containing mercury or excess amounts of milk and milk products represent risk factors for senile cataractogenesis. Several dietary differences were noted lending credence to the prophylatic values of riboflavin enrichment to decrease risk of cataractogenesis and suggesting areas for further study.     

Covalent change in the major intrinsic polypeptide (MIP26K) during cataract development in the streptozotocin-induced diabetic rat

Antisera to synthetic peptides corresponding to residues 229-237, 252-259, and 256-263 have been used to quantitatively bind to the 19.5K, 24.0K, and 26.5K forms of the Major Intrinsic Polypeptide (MIP26K) of lens membrane from the streptozotocin-induced diabetic rat. The binding ratio of anti-229/anti-252 for the 19.5K component, and the binding ratio of anti-252/anti-256 for the 26.5K component, both increase only during the opacification process of the diabetic lens. Together, these results demonstrate that various forms of the MIP26K molecule undergo covalent modification during cataractogenesis of the diabetic rat lens, and that the degree of this change as monitored by binding of the anti-MIP26K peptide sera correlates with severity of the lens opacification.     

The fate of gamma L crystallins in rat lens during diabetic cataractogenesis as determined by a monoclonal antibody

We developed a monoclonal antibody against HPLC purified rat lens gamma L crystallins. This antibody was specific to both the polypeptides (19,000 and 21,000 daltons) which constituted the HPLC gamma L peak. Least reactivity was shown against gamma H (24,000 daltons). This antibody was used as a probe to detect the presence of and quantitate gamma L crystallins in lens soluble, insoluble and urea-insoluble fractions during diabetes. Utilizing a direct binding immunoassay (ELISA) we calculated the absolute quantities of gamma L crystallins present in these fractions. Our results show, in normal animals there was a minimal change in total quantities of gamma L crystallins in soluble fraction from 1 month to 5 months of age, but a slow accumulation of these crystallins in insoluble and urea-insoluble fractions was seen during the same period. Diabetes resulted in a depletion of gamma L crystallins from the soluble fraction, both in terms of relative proportion and absolute quantities. In insoluble and urea-insoluble fractions the relative proportions of these crystallins were increased dramatically up until 60 days followed by a decrease during 90-120 days of diabetes, whereas, the absolute quantities remained more or less steady after reaching the maximum on 60 days. Although the relative proportions of gamma L crystallins in the insoluble fraction seem to be less when compared to urea-insoluble fraction, the total quantity of these crystallins was much higher due to abundance of this fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of prednisolone acetate and indomethacin for maintaining mydriasis during cataract surgery

Preoperative topical nonsteroidal anti-inflammatory drugs such as flurbiprofen and indomethacin have been found to maintain mydriasis during cataract surgery. Steroidal anti-inflammatory drugs are commonly used to treat postoperative inflammation, but their effect on the maintenance of intraoperative mydriasis is unknown. Forty-six patients admitted for elective cataract surgery were randomly assigned to one of three treatment groups and received 1% prednisolone acetate, 1% indomethacin or artificial tears four times before surgery, in addition to standardized preoperative dilating drops and intraoperative epinephrine. Pupillary diameter was measured and the time interval noted five times during the surgery. During surgery the indomethacin group lost significantly less mydriasis than the control group. The mydriasis losses of the prednisolone acetate group were between those of the indomethacin and control groups, but these differences did not reach significance. We conclude that prednisolone acetate is less effective than indomethacin for maintaining mydriasis during cataract surgery.     

Comparison of 1-year surgical outcomes of combined cataract surgery and gonioscopy-assisted transluminal trabeculotomy (GATT) versus cataract surgery and iStent Inject

Graefe's Archive for Clinical and Experimental Ophthalmology - Evaluate the efficacy, safety, and complication rates of phacoemulsification cataract surgery when combined with either...