Anterior Capsule Specimens Research Articles

Downregulation of AMPK dependent FOXO3 and TFEB involves in the inhibition of autophagy in diabetic cataract

ABSTRACT Purpose Autophagy plays a crucial role in intracellular quality control of crystalline lens and AMPK has regulatory effect on autophagy. However, whether AMPK regulated autophagy is involved in diabetic cataract (DC) progression remains unknown. This study aims to investigate the AMPK-FOXO3 and AMPK-TFEB induced autophagy activity in DC patients. Materials and Methods First, anterior capsule specimens from DC and age-related cataract (ARC) patients were obtained to compare the expression difference of autophagy-related genes. The phosphorylation levels of AMPK, AKT, and mTOR and the expression of FOXO3 and TFEB were measured. Then, human lens epithelial cells (LECs, SRA 01/04) were cultured with 30 mM or 5.5 mM glucose, and AMPK activator (AICAR) and inhibitor (Compound C) were applied to further investigate the regulatory role of AMPK on autophagy. Results Compared with ARC patients, the expression of autophagy-related genes ATG5, FYCO1, ATG8, ATG12, Beclin1, and ULK1 in anterior capsules LECs of DC patients were significantly down-regulated. Meanwhile, AMPK and AMPK-dependent transcription factors, FOXO3 and TFEB were also inhibited. Similar results were found in high glucose (HG) treated SRA 01/04 model. Notably, this down-regulation of autophagy activity was rescued by AICAR in vitro, which was manifested by inhibition of AKT and mTOR phosphorylation and up-regulation of FOXO3, TFEB, Beclin1 and LC3B-II expression. Conclusions Down-regulation of AMPK-FOXO3 and AMPK-TFEB induced autophagy activity was found in both LECs of anterior capsule from DC patients and SRA 01/04 cells under HG condition, which may be the underlying mechanism of DC formation. Thus, targeting AMPK-induced autophagy may be a potential therapeutic approach for diabetic cataract.

Effect of intraoperative trypan blue on lens epithelial cells – Histomorphological analysis

IntroductionTrypan Blue is an acid azo dye commonly used as a stain to distinguish viable from non-viable cells. It is a vital stain used intra operatively during cataract surgery to stain the external surface of the anterior lens capsule for better visualization. AimTo analyze the histomorphological effects of trypan blue on Lens Epithelial cells and the Basement Membrane on direct exposure by staining the internal surface of the anterior lens capsule during Small Incision Cataract Surgery. MethodsAnalytical cross sectional case control study. Anterior capsule specimens of 14 Patients undergoing small incision cataract surgery at Department of Ophthalmology, Govt Medical College Hospital, Thrissur were studied. Two specimens of anterior capsule taken from the same eye form the case and control. Control specimen (sample A) was removed first, after the routine external staining with trypan blue 0.06% (w/v) for 10 seconds. The stain was washed off by balanced salt solution in every case. Then trypan blue was injected under the remaining anterior capsule and case (Test) specimen (sample B) was obtained after direct contact of trypan blue to the internal surface (lens epithelial cells) for 1 minute. Histomorphological (qualitative and quantitative) examination of both specimens done. ResultsQualitative data analysis was done by EPI INFO software.v.7. Intactness of LECs throughout the length was statistically significant in Sample A (p = 0.000027). Partial and complete detachment of Lens Epithelial Cells, degeneration, and nuclear smudging were significantly higher in Sample B. Qualitative analysis of the basement membrane showed significant edema of the basement membrane in sample B. Basement membrane splitting observed in sample B was not statistically significant. Quantitative data analyzed using independent t test. There was a statistically significant decrease in cell density in sample B with p value less than 0.05. DiscussionOur study demonstrated that direct staining of the internal surface of anterior capsule with trypan blue affected LECs and the basement membrane. There were reduction in cell density, irreversible degeneration of Lens Epithelial Cells and basement membrane edema. Hence treating the internal surface of capsular bag with trypan blue may reduce incidence of Posterior capsular opacification.

Transmission electron microscopy study of undescribed material at the anterior lens capsule in exfoliation syndrome.

In the present study, we aimed to examine the anterior lens capsule using transmission electron microscopy (TEM) and compare the findings in patients with and without exfoliation syndrome (XFS). Eighteen patients with senile cataract, including 10 with XFS, were included. Anterior capsule specimens were obtained from patients during phacoemulsification through continuous curvilinear capsulorrhexis, and were examined via TEM. In the XFS group, in addition to the typical fibrillar material above the basement membrane of the lens capsule, another unknown, microgranular, electron-dense, unbound material was observed beneath the lens epithelium. Larger formations appeared to detach from the surface of the epithelial cells, and oval or crescent-shaped structures of an electron-denser material were less commonly observed on the apical side of the epithelium. Membranous structures were occasionally attached to epithelial cells that often exhibited thicker or ruptured cell membranes on their apical-free side, along with deposits of electron-dense material. Degenerative lesions of various severities were observed at the epithelium in both groups. The anterior lens capsule epithelium in patients with XFS exhibits a highly irregular and rough margin on its free side, with unbound material over its membrane, which probably contributes to loose contact with the underlying lens and leads to different clinical behaviors in XFS eyes during cataract surgery.

Ultrastructural effects of silicone oil on the clear crystalline lens of the human eye

To evaluate light and electron microscopic changes of the anterior capsule and its epithelium after clear lens extraction of vitrectomized myopic eyes with silicone oil tamponade. This prospective, controlled, non-randomized, interventional study included 20 anterior lens capsular specimens that were excised during combined clear lens extraction and silicone oil removal from previously vitrectomized highly myopic patients with silicone oil tamponade for previous retinal detachment surgeries. The specimens were examined via light microscopy and electron microscopy and compared with 20 anterior capsule specimens removed during clear lens extraction of non-vitrectomized highly myopic eyes. Light microscopic examination of clear lens anterior capsule specimens of vitrectomized myopic eyes filled with silicone oil showed relatively more flat cells with irregular outline of lens' epithelial cells with wide intercellular spaces, deeply stained nuclei, and multiple intracytoplasmic vacuoles. Scanning electron microscopy revealed collagenous surfaces filled with multiple pits, depressions, and abnormal deposits. Transmission electron microscopy revealed lens epithelial cells with apoptotic changes, many cytoplasmic vacuoles, and filopodia-like protrusions between lens epithelial cells and the capsule. Epithelial proliferation and multilayering were also observed. silicone oil may play a role in the development of apoptotic and histopathological changes in clear lens epithelial cells. Clarity of the lens at the time of silicone oil removal does not indicate an absence of cataractous changes. We found justification of combined clear lens extraction and silicone oil removal or combined phacovitrectomy when silicone oil injection is planned, but further long-term studies with larger patient groups are required.

Histological comparison between the anterior capsule integrity in femtosecond laser-assisted capsulotomy and traditional continuous curvilinear capsulotomy in cataract surgeries

Purpose To compare the histological edge structures of anterior lens capsule specimens obtained from laser-assisted cataract surgery capsulotomies using two different laser platforms (LenSx post-soft-fit and Victus II) with that of traditional continuous curvilinear capsulorhexis (CCC) using scanning electron microscopy (SEM). Setting I-Care Center, Alex Eye Center, Faculty of Medicine, Alexandria University, Alexandria, Egypt. Design Prospective comparative series. Patients and methods Anterior capsule specimens following CCC (n=15) or femtosecond laser-assisted capsulotomy using two platforms (LenSx, n=15 and Victus, n=15) were studied by SEM. Irregularity of the capsule edge was quantified using two parameters (angular second moment and contrast) using ImageJ software. The clinical features and laser parameters were correlated with angular second moment and contrast. SEM images were analyzed for the coefficient of variation (CoV) of pixilation along the capsule edge and homogeneity using gray-level co-occurrence matrix analysis. Subjective analysis for cut-surface irregularities and assessment for complications and anomalies, such as tags and misdirected laser shots, were also made. Results The femtosecond laser-assisted capsulotomy edge surfaces created by both laser platforms showed marked irregularity compared with the smoother edge of the CCC. The angular second moment and contrast measures for both lasers differed significantly from those obtained for CCC (P Conclusion Laser capsulotomies are approaching the smoothness of manual capsulorhexis. The LenSx post-soft-fit platform shows less anomalies and smaller difference for the CoV and homogeneity metrics compared with the Victus II platform. In the manual group, there is dishomogeneous thickness along the capsulorhexis edge, while in the femtosecond laser samples the cuts are more homogeneous regarding thickness throughout the whole capsulorhexis edge.

Apoptosis Factors of Lens Epithelial Cells Responsible for Cataractogenesis in Vitrectomized Eyes with Silicone Oil Tamponade

BackgroundThe aim of this study was to determine the expression of apoptotic factors Bax, Bcl-2, and Caspase-3 in lens epithelial cells (LECs) from cataracts secondary to pars plana vitrectomy with silicone oil (SO) tamponade. We also investigated the impact of SO emulsification on the expression of apoptotic factors.Material/MethodsAnterior capsulotomy specimens of 20 eyes in 20 patients with cataract secondary to SO tamponade (Group 2), were collected. Another 20 eyes of 20 patients with age-related cataract (Group 1) were recruited as controls. The anterior capsule specimens were obtained from the patients during cataract surgery, frozen and later analyzed with respect to immunohistochemical stains of Bax, Bcl-2, and Caspase-3 using a confocal microscope.ResultsAge, sex, and laterality did not show any difference between the 2 groups. There was a greater increase in Bax and Caspase-3 expression in LEC in Group 2 than in Group 1 (PBax<0.0001, PCaspase-3<0.0001). The Bcl-2 expression decreased in Group 2, although the difference was not significant (P=0.616). The changes of apoptosis factors are not associated to SO emulsification (PBax=0.354, PBcl-2=0.728, PCaspase-3=0.939).ConclusionsThe caspase-3-dependent apoptosis of LECs increased in complicated cataract patients with SO endotamponade. The Bax played a critical role in regulating apoptosis of LECs in vitrectomized eyes with SO tamponade. The SO emulsification had no significant impact on the expression of apoptosis factors.

Comparison of the anterior capsulotomy edge created by manual capsulorhexis and 2 femtosecond laser platforms: Scanning electron microscopy study

To compare the scanning electron microscopy (SEM) features of the anterior capsule edge created by continuous curvilinear capsulorhexis (CCC) and femtosecond laser-assisted capsulotomy using angular second moment and contrast. King Khaled Eye Specialist Hospital, Riyadh, Kingdom of Saudi Arabia. Prospective comparative series. Anterior capsule specimens following CCC (n = 10) or femtosecond laser-assisted capsulotomy using 2 platforms (Lensx, n = 9, and Victus, n = 10) were studied by SEM. Irregularity of the capsule edge was quantified using 2 parameters (angular second moment and contrast) using ImageJ software. The clinical features and laser parameters were correlated with angular second moment and contrast. By SEM, the femtosecond laser-assisted capsulotomy edge surfaces created by both lasers showed marked irregularity compared with the smooth edge of the CCC. The angular second moment and contrast measures for both lasers differed significantly from those obtained for CCC (P<.001). There was no between-laser difference in angular second moment and contrast measures. The angular second moment showed only a weak negative correlation with increasing laser power, whereas contrast showed a weak positive correlation with increasing power. Both laser platforms created an irregular capsulotomy edge. The angular second moment and contrast quantified capsule edge irregularities for further comparisons. These measures could be used to quantify efforts to reduce capsule-edge irregularity from femtosecond laser-lens capsule interaction.

Primate lens capsule elasticity assessed using Atomic Force Microscopy

The purpose of this project is to measure the elasticity of the human and non-human primate lens capsule at the microscopic scale using Atomic Force Microscopy (AFM). Elasticity measurements were performed using AFM on the excised anterior lens capsule from 9 cynomolgus monkey (5.9–8.0 years), 8 hamadryas baboon (2.8–10.1 years), and 18 human lenses (33–79 years). Anterior capsule specimens were obtained by performing a 5 mm continuous curvilinear capsulorhexis and collecting the resulting disk of capsular tissue. To remove the lens epithelial cells the specimen was soaked in 0.1% trypsin and 0.02% EDTA for 5 min, washed, and placed on a Petri dish and immersed in DMEM. Elasticity measurements of the capsule were performed with a laboratory-built AFM system custom designed for force measurements of ophthalmic tissues. The capsular specimens were probed with an AFM cantilever tip to produce force–indentation curves for each specimen. Young’s modulus was calculated from the force–indentation curves using the model of Sneddon for a conical indenter. Young’s modulus of elasticity was 20.1–131 kPa for the human lens capsule, 9.19–117 kPa for the cynomolgus lens capsule, and 13.1–62.4 kPa for the baboon lens capsule. Young’s modulus increased significantly with age in humans ( p = 0.03). The age range of the monkey and baboon samples was not sufficient to justify an analysis of age dependence. The capsule elasticity of young humans (<45 years) was not statistically different from that of the monkey and baboon. In humans, there is an increase in lens capsule stiffness at the microscale that could be responsible for an increase in lens capsule bulk stiffness.

Thinning of the anterior capsule associated with congenital aniridia

Purpose To report the unusual finding of intraoperative fragility of the anterior capsule in some patients with congenital aniridia and determine the histopathologic etiology of this finding. Setting Cincinnati Eye Institute, Cincinnati, Ohio, USA. Methods Anterior lens capsule specimens were obtained from aniridic and nonaniridic patients during cataract surgery. The intraoperative behavior of each capsule was noted, after which the specimens were submitted for histopathologic evaluation. Results All anterior capsule specimens from the nonaniridic patients were of normal thickness. Some, but not all, anterior capsule specimens from the aniridic patients were remarkably thin. Thin capsules were associated with extreme intraoperative fragility. Conclusion Greater awareness of anterior capsule fragility in some aniridic patients may reduce the risk of capsule complications and lead to safer surgical outcomes.

Detection of herpes simplex virus in pseudoexfoliation syndrome and exfoliation glaucoma.

The pathogenesis of pseudoexfoliation syndrome (PEX) remains unknown. An infection, possibly viral, is one of the proposed pathogenetic mechanisms. This study examines the presence of herpes simplex virus (HSV) and varicella-zoster virus (VZV) in iris and anterior capsule specimens of PEX and non-PEX patients. Iris and anterior capsule specimens were obtained from 64 patients with PEX (study group, SG) and 61 patients without PEX (control group, CG). The presence of HSV and VZV DNA was evaluated with a polymerase chain reaction (PCR). Herpes simplex virus type I was detected significantly more often in iris specimens from the SG (13.79%), compared to those from the CG (1.75%). Varicella-zoster virus DNA was not detected in any of the examined specimens. Results imply a possible relationship between HSV type I and PEX, although no aetiological role of HSV infection in PEX pathogenesis can be established. Results also advocate against any association between VZV and PEX.

Pyruvate modulates antioxidant status of cultured human lens epithelial cells under hypergalactosemic conditions.

Lens epithelial cells are the metabolic unit of the lens and antioxidant enzymes are mainly concentrated here. The purpose of this study was to maintain human lens epithelial cells (HLEC) in culture and examine the status of antioxidant enzymes (glutathione peroxidase (GSHPx), catalase (CAT), glutathione-S-transferase (GST)), lipid peroxidation product malondialdehyde (MDA) and glutathione (GSH) levels in these cells under normal as well as hypergalactosemic (30 mM galactose) conditions. Further, effect of pyruvate, a physiological antioxidant has also been evaluated on these parameters. For conducting experiments, anterior capsule specimens obtained from fresh cadaver eyes from eye bank were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal calf serum. Upon confluency, the cells were subcultured in three separate flasks containing DMEM alone (normal group), DMEM + 30 mM D-galactose (control group), DMEM + 30 mM D-galactose + 5 mM pyruvate (test group) and incubated for 24 or 72 h. These cells were observed under the phase contrast microscope for any morphological changes and harvested for the estimation of various antioxidant parameters. Our results show significant weakened antioxidant defense in HLEC when incubated in the presence of galactose as compared to normal. Addition of pyruvate significantly modulated levels of GSH, MDA, GSHPx, CAT and GST.

Cytokeratin in lens epithelial cells and its effect on anterior lens capsule opacification

Postoperative lens capsule opacification is a complication of extracapsular cataract extraction. To avoid this problem, we aspirated the epithelial cells lining the internal surface of the capsular bag. Anterior capsule specimens collected three and six years after the first surgical intervention (extracapsular cataract extraction with intercapsular implantation of a poly[methyl methacrylate] intraocular lens) and in vitro studies of epithelial cell cultures revealed the presence of intracellular and extracellular cytokeratin. After several years, the cytokeratin layers were arranged in a pattern analogous to that of liquid crystalline phases and many nonhuman extraocular tissues.

Observations on lens epithelial cells and their removal in anterior capsule specimens.

We studied the morphology and removal of lens epithelial cells in the following ways. In 49 senile cataractous eyes with a mean (+/- SD) age of 69 +/- 9 years, mean (+/- SD) cell density was 3574 +/- 512 cells/mm2 and mean (+/- SD) cell size was 281 +/- 48 micron 2. With age there was a significant decrease in cell density and an increase in cell size. Immediately after each of the following procedures was performed on the central anterior capsule during routine cataract surgery, the capsule was removed and observed. Procedures used were (1) aspiration, (2) aspiration with ultrasound, (3) instant single cryopexy application, (4) double cryopexy, and (5) double cryopexy followed by aspiration with ultrasound. Each procedure could destroy or remove lens epithelial cells completely. The procedure that we recommend is single cryopexy application followed by aspiration with ultrasound.