Evaluation of antioxidants and argpyrimidine in normal and cataractous lenses in north Indian population

AIM: To assess the level of glutathione, thioltransferase, and argpyrimidine in nuclear and cortical cataractous lenses as well as in the clear lenses in the north Indian population. METHODS: Human cataractous lenses were collected from the patients who underwent extracapsular cataract extraction surgery; clear lenses were collected from the freshly donated eye bank eyes. Antioxidant molecules such as glutathione and thioltransferase enzyme activity were measured; simultaneously in these lenses a blue fluorophore argpyrimidine, an advanced glycation end (AGE) product level was assessed using high performance liquid chromatography (HPLC). RESULTS: The protein concentration was found to be present at higher levels in the control lenses compared to cataract lenses. A significant decrease in the glutathione level was observed in the nuclear cataractous lenses compared to cortical cataractous (P=0.004) and clear lenses (P≤0.005), but no significant change in the level of antioxidant enzyme thioltransferase was observed. Further, argpyrimidine a blue fluorophore (AGE) was found to be significantly higher in the nuclear cataract (P=0.013) compared to cortical cataract lenses. CONCLUSION: Antioxidants such as glutathione significantly decrease in age-related nuclear and cortical cataract and an AGE, argpyrimidine are present at significantly higher levels in nuclear cataract.     

The Study of G6PD in Erythrocyte and Lens in Senile and Presenile Cataract

The G6PD activity of erythrocytes in 113 male patients with senile and presenile cataract and 86 controls, and G6PD activity of lens in 30 patients with senile cataract and 42 controls were reported. The cataractous group had higher frequency of G6PD deficiency and lower average G6PD level in erythrocytes and lenses, but with out statistical significance. The frequency of G6PD deficiency of erythrocytes in presenile cataractous group was higher than that of senile cataractous group but with no statistical significance too. However, the average G6PD level of erythrocytes in presenile cataractous group was lower than that of senile cataractous group and with statistical significance (P<0.05). The G6PD activity of lenses only presenile in the cortex and have a positive correlation with that of erythrocytes. There was a case with deficiency of G6PD both of erythrocytes and cataractous lenses in both eyes. The results indicate that the deficiency of G6PD might be one of the cataractous pathogenetic factor for presenile cataract. Measurement of G6PD activity of erythrocytes among population might be of significance in finding the risk factor for cataract.     

The Biological Study of the Cultured Human Lens Epithelial Cells in Vitro

The human lens epithelial cells (HLE) cultured in vitro was established in normal and cataractous lenses. The biological feature, histological characteristics and the ultrastructure of the cultured HLE cells were investigated. The results reveal that the proliferative capacity of the culutured HLE cells is reversely proportional to the donour age; the cultured HLE cells has the limited proliferative capacity in vitro. The relieve of the contact inhibition is the effective trigger of the HLE cell proliferation. This research work is possible to make a basis for the further study of the regeneration of the lens and the roles of the lens epithelial cells in the development of the cataract. Eye Science 1994; 10:27-31.     

Assessment of Sulf hydryl Group in Individual Rat Lens Protein Subunits During Galactose Cataract Development

A specific reagent DACM [N-( 7-Dimethylamino-4-methyl-3-coumarinyl) maleimide] is used to study the -SH groups in lens proteins of normal and galactose cataractous rats. DACM when reacts readily with -SH groups form strong fluorescent adducts. The two -dimensional electrophoresis with DACM pre-labeled proteins is a simple and sensitive method for detecting -SH groups of protein subunit. In the present study, based on IEF/SDS-PAGE electrophoretically characterized soluble crystallins, describes specific changes in -SH groups of protein subunit during the development of galactose cataract. The contents of -SH groups of crystallins are progressively decreased in cataractous (5 ) lens, the reduction of -SH content in α-and β-crystallin portein subunits of the normal and cataractous lens proteins is also noticeable. Eye Science 1994; 10: 21-26.     

Lens Epithelial Cell Proliferation and Cell Density in Human Age—relat

Purpose: To discuss the potential effect of the lens epithelial cell proliferation inage-related cataract.Methods: In vitro cell proliferation was assayed by MTT method to evaluate the lensepithelial cell density, index, and proliferation capacity in normal lens and all kinds ofage-related cataract. Capsulotomy specimens from all kinds of patients who underwentcataract phacoemulsification extraction surgery were compared with the lens epithelialspecimens from non-cataract lenses of Eye Bank eyes.Results:Lens epithelial cell density of central anterior capsule(LECD) in female normallens was higher than that in male, LECD in nuclear cataract (> NⅢ) was higher thanthat in normal lens, but in the mature cortical cataract, LECD was lower. Mitotic indexof three kinds of age-related cataracts in vivo had no statistical difference, neither didcell proliferation capacity of cultivated cells in vitro.Conclusion: The individual difference of lens epithelial cell density and proliferationcapacity in vivo may be an     

αB-crystallin malondialdehyde, superoxide dismutase, and lutathione peroxidase changes in X-ray irradiated rat lens

AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray irradiation to the head with rest of the body protected. The exposure dose ranged from 2 to 25 Grays (Gy). The cataract status were examined by slit lamp and rated with “four-grade systems” post-irradiation. The lens MDA level, and the activities of SOD and GPx were measured in a short-term experiment post-irradiation, and αB-crystallin protein levels were quantified. RESULTS: The lenses of normal control and the X-ray irradiated groups with the dose up to 10 Gy remained transparent throughout the experiment. The lens first appeared tiny scatters, and even lamellar opacities in the posterior capsule 45 days post-irradiation with the dose of 15 Gy, and progressed slowly to the advance stage of cataract; while, for the higher dose (25 Gy), the opacity of lens appeared much earlier, and progressed more rapidly to mature stage of cataract within 1 month. At the end of the observation (90 days post-irradiation), almost all lenses became complete opacity with the higher dose (25 Gy). The degree of lens opacity was rated accordingly. The lens MDA level was increased, and SOD and GPx activities were decreased with a dose-dependent manner post-irradiation. The αB-crystallin protein level was decreased dose-dependently at the end point of observation. CONCLUSION: Oxidative events and αB-crystallin may play important roles in the pathogenesis of cataract in X-ray irradiated rat lens.     

Pigment Epithelium-derived Factor in Cataractous Aqueous Humor and Lens Epithelial Cells

Purpose: To study the characteristics of PEDF in cataractous aqueous humor and its expression in human lens epithelium. Methods: The PEDF concentration in the aqueous humor was measured by enzyme -linked immunosorbent assay in senile (130cases) and congenital (18cases) cataract patients who underwent cataract phacoemulsification extraction surgery. Anterior lens capsular specimens were obtained from these patients to count lens epithelial cells (LEC) density. The Lens Opacities Classification System Ⅲ was used to classify the senile cataracts as cortical, nuclear, posterior subcapsular and mixed types of opacity, and quantitative analysis of the nuclear opacities was performed by Pentacam Scheimpflug imaging system. Anterior lens capsular specimens from another senile (10cases) and congenital (10cases) cataract were collected for immunofluorescence with polyclonal antibodies specific to human pigment epithelium -derived factor (PEDF). Results:The mean aqueous level of PEDF was(178. 9±87. 5)ng/ml, and there was negative linear correlation of PEDF level and age (r=0. 811, P<0. 001). In senile cases, the aqueous PEDF concentration decreased with increasing nuclear opacities (r=0. 447, P < 0.01) , and the mean PEDF level in nuclear cataract was significantly lower than that in posterior subcapsular opacity (P < 0.01) . PEDF immunostaining was detected in LEC of all capsular specimens. Conclusion : The PEDF level in human aqueous humor is related to age, types of cataracts and lens opacity. PEDF also express in human LEC. The study results suggest PEDF may regulate and/or protect LEC by paracrine and autocrine, and lack of PEDF may play a role in cataractogenesis.     

【In Press】 Descemet stripping automated endothelial keratoplasty in phakic eyes: incision modification reducing cataract formation

AIM: To evaluate the efficacy of a technical modification to reduce the incidence of traumatic cataract induced by Descemet stripping automated endothelial keratoplasty (DSAEK) performed in phakic eyes. METHODS: A retrospective cohort study. The records of all patients with a clear crystalline lens and endothelial failure that underwent modified DSAEK at our insitution were reviewed. In this modification, in order to avoid inadvertent touch of the insertion forceps against the exposed crystalline lens while passing across the anterior chamber, the incision sites were shifted from the standard 9 and 3 o’clock positions, superiorly to the 10 and 2 o’clock position respectively. Formation of typically traumatic, anterior subcapsular cataract in these patients was compared to that observed in a cohort including all the patients with a clear crystalline lens and endothelial failure that underwent conventional DSAEK at our institution. RESULTS: The study group included 49 eyes following modified DSAEK and the control group included 35 eyes following DSAEK with conventional incision sites. Anterior subcapsular cataract occurring 4 mo or less postoperatively was identified in 2 of 49 (4%) eyes in the study group and 7 of 35 (20%) eyes in the control group. The rates of traumatic cataract were significantly higher in the control group in comparison to the study group (P=0.03, RR=4.9, 95%CI 1.08-22.1). CONCLUSION: Traumatic cataract formation following phakic DSAEK may be avoided with a simple modification to the position of the incision sites.     

Descemet stripping automated endothelial keratoplasty in phakic eyes: incision modification reducing cataract formation

AIM: To evaluate the efficacy of a technical modification to reduce the incidence of traumatic cataract induced by Descemet stripping automated endothelial keratoplasty (DSAEK) performed in phakic eyes. METHODS: A retrospective cohort study. The records of all patients with a clear crystalline lens and endothelial failure that underwent modified DSAEK at our insitution were reviewed. In this modification, in order to avoid inadvertent touch of the insertion forceps against the exposed crystalline lens while passing across the anterior chamber, the incision sites were shifted from the standard 9 and 3 o’clock positions, superiorly to the 10 and 2 o’clock position respectively. Formation of typically traumatic, anterior subcapsular cataract in these patients was compared to that observed in a cohort including all the patients with a clear crystalline lens and endothelial failure that underwent conventional DSAEK at our institution. RESULTS: The study group included 49 eyes following modified DSAEK and the control group included 35 eyes following DSAEK with conventional incision sites. Anterior subcapsular cataract occurring 4mo or less postoperatively was identified in 2 of 49 (4%) eyes in the study group and 7 of 35 (20%) eyes in the control group. The rates of traumatic cataract were significantly higher in the control group in comparison to the study group (P=0.03, RR=4.9, 95%CI 1.08-22.1). CONCLUSION: Traumatic cataract formation following phakic DSAEK may be avoided with a simple modification to the position of the incision sites.     

Carbamylation-induced inactivation of glyceraldehydes 3-phosphate dehydrogenase and thioltransferase in bovine lens

AIM: To investigate whether potassium cyanate can inactivate glyceraldehydes 3-phosphate dehydrogenase (GAPDH) and thioltransferase (TTase) in bovine lens. METHODS: Fresh intact bovine lenses were incubated with 100mmol/L potassium cyanate(KCNO) for 7 and 12 days respectively. Then all lens were incubated in 50mmol/L DMEM solution. The proteins in the water-soluble fractions from the normal control and the cyanate-modified lens were extracted. The activity of GAPDH and TTase in the water-soluble fraction after incubation at 37℃ was measured by spectrophotometer. RESULTS: GAPDH activity was significantly lower in the cyanate-modified lens proteins than that of the normal control(P <0.01), and considerably diminished in protein incubated with 100mmol/L potassium cyanate for 12 days. There were statistically significant differences in the activity of TTase between the normal control lenses and the carbamylated lenses incubated for 7 days(P <0.05) and 12 days(P <0.01). However, there was no statistical difference between the samples incubated with 100mmol/L KCNO for 7 and 12 days (P =0.19296). CONCLUSION: This study provides evidence to show carbamylation is able to inactivate GAPDH and TTase in bovine lenses. This may have implications for the susceptibility of lenticular GAPDH and TTase to carbamylation, and also for the research on pathogenesis of cataract.     

An unusual central retinal dystrophy associated with ichthyosis vulgaris

A number of ichthyosis syndromes may have retinal abnormalities such as the retinitis pigmentosa-like diffuse rod-cone dystrophy in Refsum’s syndrome and the maculopathy in Sjögren-Larsson syndrome. We present two sisters who have an unusual, almost identical, bilaterally symmetric central retinal dystrophy associated with ichthyosis vulgaris in the absence of other systemic disorders. We believe that this dystrophy has not been previously described in patients with any of the known varieties of ichthyosis.     

CPC/BMP复合材料眼毒性的实验研究

目的通过观察CPC/BMP复合材料在动物实验中对眼球形态的影响,并检测其体外细胞毒性,评价其对眼的毒性。方法将CPC/BMP复合材料及对照组单纯CPC材料分别植入18只新西兰兔右、左眼眶骨缺损区,每日观察动物眼外观有无异常。使用植入眼眶后0天、3天和3周的CPC/BMP复合材料、单纯CPC材料的浸提液、Medpor材料浸提液、单纯培养液、0.64%苯酚稀释液培养L929小鼠肺成纤维细胞和人角膜缘上皮细胞,孵育1天、3天时,MTT法检测吸光度(OD值),并计算细胞相对增值率,判定细胞毒性。结果 CPC/BMP复合材料及单纯CPC材料植入眼眶骨缺损区术后可见实验动物出现一过性结膜充血。各时间点CPC/BMP复合材料、单纯CPC材料、Medpor材料及空白对照组的OD值差异无统计学意义(P>0.05),细胞相对增值率≥80%,细胞毒性≤1级。结论 CPC/BMP复合材料未见明显眼毒性,符合眼眶内植入材料的生物学要求。     

Resolution of macular oedema in occult choroidal neovascularization under oral Sorafenib® treatment

Purpose: To report on the effect of oral nexavar (Sorafenib^®) treatment in one patient with neovascular age‐related macular degeneration (AMD) and advanced renal cell cancer (RCC). Methods: After two intravitreal injections of bevacizumab (1.25 mg) for occult choroidal neovascularization (CNV) in AMD, the patient was started on oral Sorafenib^® (400 mg twice daily) treatment for RCC. Results: Visual acuity (VA) was 20/80 in the left eye and optical coherence tomography (OCT) demonstrated persistent central thickening to 251 µm after bevacizumab. After 6 weeks of oral Sorafenib^® treatment, VA had increased to 20/70 and a significant decrease in retinal thickness to 208 µm was observed on OCT. The patient remained stable during a further 3 months of follow‐up. Conclusions: Resolution of macular oedema and stabilization of VA under oral treatment with the multikinase inhibitor Sorafenib^® was observed. This observation warrants further investigation.     

Combination of AIBP,apoA-I,and Aflibercept Overcomes Anti-VEGF Resistance in Neovascular AMD by Inhibiting Arteriolar Choroidal Neovascularization

PurposeAnti-VEGF resistance represents a major unmet clinical need in the management of choroidal neovascularization (CNV). We have previously reported that a combination of AIBP, apoA-I, and an anti-VEGF antibody overcomes anti-VEGF resistance in laser-induced CNV in old mice in prevention experiments. The purpose of this work is to conduct a more clinically relevant study to assess the efficacy of the combination of AIBP, apoA-I, and aflibercept in the treatment of anti-VEGF resistance of experimental CNV at different time points after laser photocoagulation.MethodsTo understand the pathobiology of anti-VEGF resistance, we performed comprehensive examinations of the vascular morphology of laser-induced CNV in young mice that are highly responsive to anti-VEGF treatment, and in old mice that are resistant to anti-VEGF therapy by indocyanine green angiography (ICGA), fluorescein angiography (FA), optical coherence tomography (OCT), and Alexa 568 isolectin labeled choroid flatmounts. We examined the efficacy of the combination therapy of AIBP, apoA-I, and aflibercept intravitreally delivered at 2, 4, and 7 days after laser photocoagulation in the treatment of CNV in old mice.ResultsLaser-induced CNV in young and old mice exhibited cardinal features of capillary and arteriolar CNV, respectively. The combination therapy and the aflibercept monotherapy were equally effective in treating capillary CNV in young mice. In old mice, the combination therapy was effective in treating anti-VEGF resistance by potently inhibiting arteriolar CNV, whereas aflibercept monotherapy was ineffective.ConclusionsCombination therapy of AIBP, apoA-I, and aflibercept overcomes anti-VEGF resistance in experimental CNV in old mice by inhibiting arteriolar CNV.     

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways differently regulate retinal pigment epithelial cell-mediated collagen gel contraction

Retinal pigment epithelial (RPE) cell-mediated extracellular matrix contraction is believed to contribute to developing proliferative vitreoretinopathy. It has been shown that platelet-derived growth factor (PDGF) and its intracellular signaling pathway, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K), are mainly involved in this process. The aim of this study is to investigate how these downstream signaling pathways are related to RPE-mediated collagen gel contraction. We performed the gel contraction assay to evaluate the effect of PDGF in cultured ARPE-19 cells under the presence or absence of PD98059, MAPK inhibitor or wortmannin, PI3K inhibitor. Experiments treated with neutralizing antibody for various subtypes of integrin were also performed and the effect on PDGF-induced gel contraction was investigated. Expression changes of integrin alpha1, alpha2 and beta1 after PDGF stimulation was evaluated using quantitative real-time PCR and flow cytometry. The results showed that PDGF up-regulated ARPE-19 cell-mediated gel contractile activity. PDGF-induced collagen gel contraction was attenuated under presence of PD98059, wortmannin, or neutralizing antibody for integrin alpha1, alpha2, or beta1, all of which are critical subset for binding with type I collagen. The expression of integrin alpha1 and alpha2 was increased after PDGF stimulation in both real-time PCR and flow cytometry, however beta1 expression was not increased. PD98059 significantly attenuated integrin alpha1 and alpha2 expressions. However, wortmannin did not have the same effect. In conclusion, PDGF promotes ARPE-19 cell-mediated gel contraction via both MAPK and PI3K. This was probably due to an increased expression of integrin alpha1 and alpha2, which is mediated by MAPK, but not by PI3K. PI3K may regulate collagen gel contraction by another mechanism other than the up-regulation of integrin expression.     

热休克蛋白及其在眼部疾病中作用的研究进展

热休克蛋白(HSP)是生物体在应激状态下合成的一类高度保守的蛋白,具有分子伴侣、协同免疫、抗凋亡等生物学功能。近来随着对热休克蛋白研究的不断深入,HSP在眼病的发病机制、防治方面的作用已受到广泛关注。     

Immunohistochemical study on IRBP-induced EAU

We studied the pathogenesis of IRBP-induced experimental autoimmune uveoretinitis (EAU) by immunohistochemical detection of various immune cells using specific monoclonal antibodies to their surface markers and adhesion molecules. The following results were obtained: (1) During each stage of EAU, CD4 positive T cells predominated over CD8 positive T cells in the retina and the uvea. (2) One day prior to the clinical onset of disease, la positive cells began to appear in the ciliary body. (3) LFA-1 and ICAM-1 were expressed on intraocularly infiltrating cells. (4) ICAM-1 was also expressed on endothelial cells of uveal and retinal vessels. In addition, ICAM-1 was expressed on ciliary body epithelium and retinal pigment epithelium, a finding which may be associated with the breakdown of the blood-ocular barrier. In conclusion, the expression of LFA-1 and ICAM-1 in EAU demonstrates that adhesion molecules, such as these, play an important role in inflammatory ocular disease in vivo.     

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.     

Epigallocatechin-3-gallate reduces retinal ischemia/reperfusion injury by attenuating neuronal nitric oxide synthase expression and activity

Retinal ischemia/reperfusion (IR) injury causes profound tissue damage, especially retinal ganglion cell death. The aims of the study were twofold: (1) to investigate the benefits of epigallocatechin-3-gallate (EGCG), the major catechin found in tea, after IR challenge, and (2) to elucidate the mechanism of EGCG inhibition of nitric oxide synthase (NOS) expression. Wistar female rats were divided into four groups: normal control, EGCG with sham operation, retinal IR, and EGCG with IR groups. EGCG (50mg/kg) was administered by intraperitoneal injection 30 min before the experiment. IR injury to a rat's retina was induced by raising intraocular pressure to 150 mmHg for 60 min. With EGCG pretreatment, retinal ganglion cell death from IR was reduced by approximately 10% 3 days afterward. EGCG significantly downregulated IR-induced glial fibrillary acidic protein expression. EGCG treatment also reduced TUNEL-positive cells after IR in the inner retina as well as IR-induced lipid peroxidation. Histological analyses showed fewer neuronal NOS and nicotinamide adenine dinucleotide phosphate diaphorase-positive cells in the retina after IR with EGCG administration. Therefore, EGCG is effective in protecting retinal ganglion cells from IR challenge by ameliorating retinal nitrosactive stress and by regulating cell death through apoptotic pathways.     

EGCG诱导人视网膜色素上皮细胞凋亡作用的研究

目的：探讨表没食子儿茶素没食子酸酯(EGCG)对人视网膜色素上皮细胞(ARPE-19)凋亡的影响及其机制。

方法：体外培养ARPE-19,分别采用0、40、80、160μg/mL EGCG处理。处理预定时间后分别用hoechst 33258染色法检测细胞凋亡形态学变化; 流式细胞仪检测细胞凋亡率; 实时荧光定量RT-PCR和Western blotting检测细胞凋亡相关因子B淋巴细胞瘤-2基因(bcl-2)、BCL2-Associated X的蛋白质(Bax)、胱天蛋白酶-3(caspase-3)和p53的表达。

结果：hoechst 33258染色结果发现ARPE-19随着EGCG药物浓度的增加,凋亡细胞数量逐渐增多,可见明显的凋亡小体; 流式细胞仪结果显示随着EGCG药物浓度的升高,凋亡率逐渐增高,40、80、160μg/mL凋亡率分别为4.95%±0.071%、11.75%±0.075%和21.25%±0.919%与对照组(2.8%±1.556%)相比有差异(P<0.01),呈现出药物浓度依赖性; 实时荧光定量PCR和Western blot结果表明EGCG能明显上调凋亡促进因子Bax、caspase-3和p53的mRNA和蛋白表达,同时下调凋亡抑制因子bcl-2的表达,均呈现浓度依赖性。

结论：EGCG能明显诱导ARPE-19发生凋亡,其机制与抑制bcl-2的表达,增强Bax、caspase-3和p53的表达有关。