Human anterior lens capsule epithelial cells contraction

Purpose: Human anterior lens epithelial cells, attached to surgically isolated capsules, were found to contract upon stimulation. The purpose of this study was to characterize these contractions, which create gaps between cells, and to assess the underlying physiological mechanisms and their possible association with cataract formation. Methods: Lens capsules obtained during cataract surgery were stained with fluorescent dye Fura‐2. Its fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in cell morphology and cytosolic free Ca²⁺concentrations ([Ca²⁺]_i) in response to pharmacological stimulation by acetylcholine (ACh) and to mechanical stimulation by flow of saline or direct contact. Results: Epithelial cells contracted in approximately a third of preparations when stimulated by either ACh application, fluid movement or direct mechanical contact. Contractions started either before or at best simultaneously with the rise in [Ca²⁺]_i. Contractions also occurred when there was hardly any change in [Ca²⁺]_i upon application of physiological saline alone. The probability of contractions occurring did not differ significantly among cortical, nuclear and combined cortical + nuclear cataract. Conclusions: This study provides the evidence that contractions of the anterior lens epithelial cells take place in significant portion of human lens anterior capsule postoperative preparations after non‐specific stimulation. Contractions are at least partially independent of changes in [Ca²⁺]_i. They can be mechanically induced, are localized and reversible and have a fast response and did not differ among different types of cataract. Physiological and clinical significance of this phenomenon remains to be elucidated.     

疏水丙烯酸酯囊袋张力环的制作及其对晶状体上皮细胞生长和后囊膜混浊的抑制作用

目的研制以疏水丙烯酸为材料直角矩形设计的囊袋张力环(capsule tension ring,CTR),对其生物相容性进行鉴定,观察其在体外对人晶状体上皮细胞(human lens epithelial cells,HLEC)的生长抑制作用及在兔眼中抑制后囊膜混浊(posterior capsule opacification,PCO)的效果。方法将甲基丙烯酸苯乙酯和丙烯酸苯乙酯按一定比例聚合成疏水丙烯酸酯,进一步加工成直角矩形设计、闭合直径10.5mm、宽0.5mm、高0.2mm的开放式CTR,并对其进行细胞毒性试验、热原试验、眼刺激试验等生物相容性测试。取白内障术中前囊膜进行原代HLEC培养,将第2代细胞接种于CTR内进行细胞实验,观察其对细胞的移行作用。动物实验选用青紫兰兔,对照组4只仅接受白内障超声乳化吸出术;实验组4只接受白内障超声乳化同时植入CTR,观察术后炎症反应及前、后囊膜混浊情况。结果细胞毒性为1级,热原试验中试品个体升温均在0.6℃以下,且升温总值为0.1℃,眼刺激试验无异常,证明该材料有较好的生物相容性。体外细胞实验中,所有环均未见有细胞在环上生长或越过环向外生长。兔眼实验中,所有术眼术后均出现不同程度的前房渗出,1～2周基本吸收,术后1个月观察发现实验组兔眼的后囊膜均保持清晰,对照组兔眼的后囊膜出现不同程度的PCO。结论兔眼白内障术中植入疏水丙烯酸材料CTR可以降低PCO的发生。     

Expression of matrix metalloproteinases of human lens epithelial cells in the cultured lens capsule bag

AIM OF PURPOSE: To observe the different expression of matrix metalloproteinases (MMPs) between pre- and postoperation of sham cataract surgery in vitrohuman lens capsule bag model from the same donor eye in order to investigate a possible role of MMPs in posterior capsule opacification (PCO).Methods: Sham cataract surgeries were performed in six human donor eyes. Immunohistochemical staining was used to detect the expression of MMP-2 and -9 of human lens epithelial cells (LECs) on the anterior capsulorhexis. LEC migration on posterior capsule of human lens from the same donor eye was observed in a modified capsule bag model without pin. Total MMP-2 and -9 protein production were determined by enzyme-linked immunosorbent assay at days 2, 10, 20, and 30 postoperation, respectively. RESULTS: MMP-2 and -9 could not be detected immunohistochemically on the anterior capsulorhexis preoperation of cataract. Lens epithelia cells at the equator began to migrate by day 4. A confluent monolayer of lens epithelia cells was present on the posterior capsule at day 20. Total MMP-2 and -9 protein production increased with time with maximum levels reached on day 30. CONCLUSION: MMP-2 and -9 were showed to be upregulated following sham cataract surgery. MMP expression could play an important role in PCO.     

Human lens cells in culture. I. Isolation of adult lens epithelial cells from lens capsule preparations and reactivation of nucleus-containing fiber cells

BACKGROUND: Bovine lens epithelial cells in culture revealed a high sensitivity against micromolar concentrations of linoleic acid. To prove the assumption that unsaturated free fatty acids are risk factors for cataractogenesis, human lens cell lines are needed. Furthermore, the reactivation of nucleus-containing fiber cells to mitotic growth may hint at their role in after cataract genesis. MATERIAL AND METHODS: Epithelium-capsule-preparations obtained by capsulorhexis were cultured in serum containing medium. Subculturing of these adult human lens epithelial cells was done by trypsinization. Fiber cell bundles from the equator region of a fetal human lens were transferred into culture medium. Aggregates of nucleus containing fiber cells were isolated from floating fiber cell bundles by trypsinization. Subculturing and cryoconservation of suitable cell lines. RESULTS: Primary culture of epithelium-capsule-preparations results in flattening, migration and proliferation of adult human lens epithelial cells. Nucleus containing fiber cells were reactivated to mitotic growth after adhesion to a suitable substratum. Established cell lines were received from adult human lens epithelial cells and fetal human fiber cells after repeated subculturing. CONCLUSIONS: Lens-capsule-preparations available from cataract surgery are well suited for the isolation of human lens cell lines, which were needed for testing cytotoxicity of drugs and for tracing of cataractogenic risk factors. The finding that nucleus containing fiber cells from the equator of human lenses can be reactivated to proliferating cells let us suppose, that these cells, which can not be removed easily from the posterior lens capsule, contribute to the after cataract formation.     

Low-intensity ultraviolet A irradiation of the lens capsule to remove lens epithelial cells during cataract surgery. Effectiveness and safety

PURPOSE: To assess the effectiveness and safety of low-intensity ultraviolet A (UVA) irradiation in removing lens epithelial cells (LECs) during cataract surgery and compare them with those of mechanical polishing and no treatment. SETTING: Eyecove Ophthalmology Clinics, Pune, India. METHODS: This prospective randomized double-masked study consisted of preoperative screening of 36 patients, of which 30 met the inclusion criteria and were recruited. The patients had routine cataract surgery. A bean-shaped capsulorhexis was performed. After the nucleus and cortex were removed, the capsular bag was irradiated from inside with low-intensity UVA in 1 group. A second group had mechanical polishing, and a third group was not treated. A small flap of the anterior capsule was removed in each patient. The flap was stained and mounted in a Fuchs-Rosenthal chamber. For estimation of effectiveness, the area of capsule covered with epithelial cells was estimated by examination under a light microscope. One day postoperatively, an examination was performed to assess the safety of each technique. RESULTS: The area of the capsule from which the LECs were removed was significantly larger in the UVA-irradiation group than in the mechanical-polishing group (P = .001) and the no-treatment group (P = .001). There was no significant difference between the mechanical-polishing and no-treatment groups (P>.05). The area of the capsule flap that was covered with LECs was significantly less in the UVA-irradiation group than in the mechanical-polishing group (P = .017) and the no-treatment group (P = .001). The mechanical-polishing group and no-treatment group were not significantly different from each other (P>.05). Corneal edema was significantly less in the UVA-irradiation group than in the mechanical-polishing group (P<.001) and no-treatment group (P = .012). No patient in the UVA-irradiation group had postoperative lid edema; 8 patients in each of the other 2 groups had lid edema. The difference was statistically significant (P<.0001). Pupil size was significantly larger in the UVA-irradiation group than in the mechanical-polishing group and no-treatment group; the difference was significant (both P = .0001). There was no significant difference in pupil size in the mechanical-polishing group and no-treatment group. No significant difference was observed between the 3 groups in visual acuity, conjunctival edema, anterior chamber flare, and intraocular pressure. CONCLUSION: Ultraviolet A irradiation of the capsular bag was effective and safe in removing LECs from the anterior capsule during cataract surgery.     

Removal of lens epithelial cells to delay anterior capsule-intraocular lens adherence

To test the theory that removing lens epithelial cells at the time of cataract extraction with intraocular lens (IOL) implantation or refractive lens exchange might decrease the rate at which the anterior capsule becomes adherent to the lens optic postoperatively, we performed the technique in approximately 200 eyes that were considered likely to require postoperative IOL exchange. In 4 eyes that had an IOL exchange procedure 6 to 12 weeks after the primary procedure, the anterior capsule was nonadherent or weakly adherent to the lens optic.     

葛根素对培养的晶状体上皮细胞氧化损伤的保护作用

目的 探讨过氧亚硝基阴离子(ONOO-)对培养的兔晶状体上皮细胞(LEC)造成的影响以及葛根素(Pur)的对抗作用.方法 为实验研究.原代培养兔LEC,将其第3或第4代用作实验.实验分为:(1)对照组:加入无热源生理盐水200 μl.(2)ONOO-组:加入ONOO-200 μl使之终浓度为0.5 mmol/L.(3)Pur组:同时加入5 μg/ml ONOO- 和10μg/ml的Pur.各组加入试剂总容积为200 μl,继续培养24 h.收集细胞分别用免疫荧光技术检测ONOO-的标记物硝基酪氨酸(NT)在LEC中的抗原表达变化;免疫印迹法检测晶状体组织中NT蛋白表达变化;光学显微镜观察细胞形态;基因组DNA电泳、流式细胞仪和Fas/FasL免疫组织化学染色检测细胞凋亡.采用单因素方差分析和q检验对数据进行分析.结果 实验6～24 h期间,对照组LEC呈现绿色荧光;ONOO-组:LEC颜色逐渐由黄绿色转变为橘黄色荧光;Pur组LEC颜色逐渐由淡绿色转变为淡黄色和淡黄绿色荧光;对照组NT蛋白质表达极微弱;ONOO-组:实验各期均有NT蛋白质表达,且呈由弱到强的趋势(A值为77.22±2.44,145.00±3.94,235.78±5.97).Pur组实验6 h表达微弱(A值为72.78±2.64),12 h表达增强(A值为84.94±3.01),但至24 h表达强度减弱(A值为74.44±3.00);计算机图像分析表明,实验6、12及24 h,3组NT蛋白质平均表达水平比较,差异有统计学意义.对照组细胞形态和基因组DNA电泳正常,流式细胞仪检查可见极轻微凋亡,但细胞膜和细胞浆均未见Fas/Fas L的表达;ONOO-组出现了明显的细胞形态改变,基因组DNA电泳可见典型的"梯形条带",随时间延长细胞凋亡逐渐加重(q=78.12,82.76,69.98,P＜0.01)并可见Fas/Fas L的表达;而Pur组细胞形态、基因组DNA电泳以及流式细胞仪检查大致正常,未见Fas/Fas L的表达.结论 葛根素可减轻ONOO-所致培养的LEC凋亡.Fas/Fas L信号转导途径可能影响并加强了ONOO-介导的LEC的凋亡过程.     

Selective and specific targeting of lens epithelial cells during cataract surgery using sealed- capsule irrigation

We describe the technique of sealed capsule irrigation, which aims to reduce posterior capsule opacification and better control lens epithelial cell activity.     

Fatty acid cytotoxicity to human lens epithelial cells

Data obtained with the neutral red cytotoxicity assay reveal that human lens epithelial cells in culture are highly sensitive to low micromolar concentrations of unsaturated, cis-configured fatty acids in the following order: arachidonic acid>linolenic acid=linoleic acid=oleic acid, whereas the saturated fatty acids are much less effective. Though the cytotoxic effects of the unsaturated fatty acids could not be discerned from effects of their oxidation products, the fact that oleic acid is equally cytotoxic as linoleic acid or linolenic acid as well as previously reported findings with bovine lens epithelial cells support the idea that the unsaturated fatty acid molecules directly account for the cytotoxicity and not their products of lipid peroxidation. Bleb formation and cell retraction are early morphological signs of fatty acid-induced lens cell damage. These cellular alterations are accompanied by an aggregation of intermediate filaments in a first step, whereas the disorganization of microfilaments occurs at a later time and only at higher fatty acid concentrations. Measurements of protein-, RNA- and DNA-synthesis turned out to be much less sensitive parameters for the fatty acid-induced damage of lens cells. The uptake rate of linoleic acid by human lens cells is relatively high (4.35 fmol sec(-1) per 1000 cells), 30 and 50% higher as compared with diploid human embryonal lung fibroblasts and chemically transformed mouse fibroblasts, respectively. Saturation kinetics in combination with competition between linoleic acid, oleic acid and palmitic acid on one hand and ineffectiveness of trypsin and DIDS treatment on the other hand hint at cytoplasmic fatty acid binding proteins as receptors with high binding affinity (5.55 micromol l(-1), calculated for the linoleic acid-albumin complex) to be involved in the fatty acid uptake in human lens cells. Cellular fatty acid uptake is mainly influenced by the albumin concentrations present in physiological solutions. Albumin determinations in aqueous humor from 177 cataract patients reveal an age-dependent, statistically significant albumin rise with average values below 2 micromol l(-1) up to the age of 40 years to about 4 micromol l(-1) at the age between 80 and 90 years with single values up to 10 micromol l(-1). Using physiological fatty acid mixtures it is demonstrated that fatty acid-induced lens cell damage is strongly increased by elevated albumin concentrations found in aqueous humor of the elderly, who already have cataracts. Free fatty acid induced lens cell damage as a possible cause for age-dependent cataracts as well as a molecular link between systemic diseases such as diabetes and cataract formation is discussed.     

端粒酶活性在兔后囊膜混浊晶状体上皮细胞中的表达及意义

目的 探讨端粒酶活性在兔后囊膜混浊晶状体上皮细胞中的表达及意义.方法 对新西兰大白兔11只(22只眼)行超声乳化晶状体吸除术法,术后3个月所有实验动物的晶状体后囊膜均已混浊.20只眼分别取赤道部囊膜、混浊后囊膜组织,采用端粒重复序列扩增-聚丙烯酰胺凝胶电泳法定性和端粒重复序列扩增-酶联免疫吸附定量法检测其晶状体上皮细胞端粒酶活性.两者端粒酶活性比较采用成组设计t检验.结果 以HepG2细胞作为端粒酶检测阳性对照,兔晶状体赤道部囊膜组织、后囊膜均可检测到端粒酶活性,表现为自50 bp开始多个模糊梯度条带状电泳图谱,其吸光度A450-690值分别为0.85±0.23、0.67±0.19,两者比较差异有统计学意义(t=2.526,0.021;P＜0.05).结论 在兔后囊膜混浊晶状体上皮细胞中存在端粒酶活性,其活性较晶状体赤道部囊膜上皮细胞低.     

MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule

PURPOSE: The development of posterior capsule contraction following cataract surgery is caused by the activity of residual lens epithelial cells. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are essential for cell migration and cell mediated contraction following wound healing. The authors investigated whether inhibiting MMP activity can reduce lens epithelial cell migration and as a result, lead to a reduction in cell mediated capsule contraction. METHODS: Human donor lens capsules were cultured and treated with a broad spectrum MMP inhibitor, Ilomastat (GM6001). MMP-2 and MMP-9 production were determined by ELISA. Cell migration onto the posterior capsule and capsule contraction were digitally measured. RESULTS: MMP inhibition significantly reduced lens epithelial cell migration onto the posterior capsule (p<0.05), and a reduction in capsule contraction was observed (p<0.05). CONCLUSIONS: Ilomastat significantly reduced lens epithelial cell migration onto the posterior capsule surface and inhibited capsule contraction. MMP inhibition may have a role in the therapeutic treatment of posterior capsule opacification.     

Anterior capsule opacification and lens epithelial outgrowth on the intraocular lens surface after curettage

PURPOSE: To evaluate the long-term difference in lens epithelial cell (LEC) outgrowth on the anterior surface of a hydrogel intraocular lens (IOL) after curettage of the entire or one half of the circumference of the anterior capsule. SETTING: Department of Ophthalmology, University of Vienna, Austria. METHODS: Forty eyes with senile cataract only were randomly assigned to Group A, which had curettage of the entire anterior capsule, or Group B, which had curettage of the nasal half of the anterior capsule. Rentsch capsule curettes (Geuder) were used, a straight one for the nasal half and a bent model for the temporal half. One surgeon performed all standardized procedures with a temporal clear corneal incision, phacoemulsification, and in-the-bag implantation of a hydrogel IOL. Two years after surgery, the anterior surface of the IOL was examined by specular microscopy in a double-blinded fashion, and LEC outgrowth was graded semiquantitatively. Anterior capsule opacification (ACO) was also graded semiquantitatively. RESULTS: In Group A, grade 2 ACO was observed in 53% of patients and grade 1 ACO in 47%. Similar results were achieved in Group B (59% and 41%, respectively). Two years after IOL implantation, the typically circumferential monolayer outgrowth of LECs on the hydrogel IOL surface was present in 80% in Group A and 60% in Group B. The ongrowth was less dense in the other IOLs; however, no significant differences between the groups were observed. CONCLUSIONS: Mechanical removal of residual LECs with a Rentsch capsule curette from the entire or from one half of the anterior capsule did not reduce LEC outgrowth 2 years after IOL implantation. Furthermore, the ACO grade was not significantly different. Lens epithelial cell proliferation in the germinative region and consecutive migration might be the cause of this outgrowth.     

Shape of lens epithelial cells after intraocular lens implantation

PURPOSE: To evaluate the morphological behavior of lens epithelial cells (LECs) after human cataract surgery with implantation of a poly(methyl methacrylate) (PMMA) or silicone intraocular lens (IOL). SETTING: Department of Ophthalmology, Fujita Health University, Banbuntane Hotokukai Hospital, Nagoya, Aichi, Japan. METHODS: Morphological observations of LECs in the patients with IOLs were made by light and transmission electron microscopy. The LECs were from 4 areas: (1) the region below the anterior capsule, touching the IOL; (2) the area between region 1 and the equatorial region; (3) the equatorial region; and (4) the central equatorial region and of the posterior capsule not touching the IOL. Case 1 had implantation of a single-piece IOL with a PMMA optic and haptics. Case 2 had a 3-piece IOL with a PMMA optic and polypropylene haptics. Case 3 had a 3-piece IOL with a silicone optic and polypropylene haptics. Areas 1 and 4 could not be observed in Case 2. RESULTS: The major difference between the patient with a PMMA IOL (Case 1) and the patient with a silicone IOL (Case 3) was that among the 4 areas observed, collagen fibers were present only in area 1 in Case 1 but in areas 2 or 3 as well in Case 3. CONCLUSIONS: Fibrous collagen fibers appeared in regions in which LECs adhered and there was capsule contact with the IOL optic. In addition fibrous collagen fibers appeared in more areas in the eye with the silicone IOL than in that with the PMMA IOL, perhaps because IOLs with silicone optics move slightly while in the capsular bag.     

前囊膜划伤诱导小鼠晶状体上皮细胞早期生长反应因子-1的表达

目的 观察前囊膜划伤对小鼠晶状体上皮细胞早期生长反应因子-1(early growth response factor-1,Egr-1)表达的影响.方法 36只成年雄性昆明小鼠,随机分为6组,制作小鼠晶状体前囊膜划伤模型.采用Western blot和逆转录聚合酶链反应(RT-PCR)观察前囊膜划伤后不同时间(0,0.5,0.75,1,2,5 h)晶状体上皮细胞Egr-1蛋白和mRNA的表达变化.结果 在正常未划伤的晶状体上皮细胞,Egr-1 mRNA低表达,划伤后0.5 h表达即增强,并在2 h时达到峰值,随后逐渐减弱.正常未划伤的晶状体上皮细胞Egr-1蛋白表达微弱.划伤后0.5 h,Egr-1蛋白表达即显著增强,并在2 h时达到峰值,随后开始减弱.结论 前囊膜划伤可诱导小鼠晶状体上皮细胞Egr-1 mRNA和蛋白的表达.