Descemet Membrane-endothelium Complex Research Articles

Rapid detection of guttae area using aniline blue staining in Fuchs endothelial corneal dystrophy mouse model.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial degeneration resulting in impaired visual acuity. Excessive deposition of extracellular matrix (guttae) on Descemet's membrane (DM) is the hallmark of FECD. We sought to detect the guttae area rapidly using aniline blue (AB) staining in FECD mouse model. FECD mouse model was established via ultraviolet A (UVA) exposure. Masson's trichrome staining was utilized to stain the corneal sections. AB staining was utilized to stain both whole cornea tissues and stripped Descemet's membrane-endothelium complex (DMEC) flat mounts, while immunofluorescence staining of collagen I was employed to stain guttae areas. In Masson's trichrome staining, corneal collagen fibrils were stained blue with AB. The DMEC flat mounts were stained into relative dark blue areas and relative light blue areas using 2% AB staining. The areas of dark blue could almost overlap with collagen I-positive areas, and have an acellular centre and a moderately distinct boundary line with the surrounding corneal endothelial cells. In conclusion, AB staining is a rapid and effective method for the evaluation of the guttae areas in the FECD mouse model.

Thickness of anterior sclera and corneal layers in systemic sclerosis.

To evaluate the thickness of anterior sclera and corneal layers in patients with systemic sclerosis. The present cross-sectional study included 41 patients with systemic sclerosis and 41 age- and gender-matched healthy controls. The study and control groups were compared in terms of the thickness of anterior sclera, corneal epithelium, Bowman's layer, corneal stroma, and Descemet's membrane-endothelium complex. The thickness measurements were obtained using the anterior segment module of spectral-domain optical coherence tomography. The thickness of anterior sclera, corneal epithelium, Bowman's layer, and Descemet's membrane-endothelium complex were similar in the patients with systemic sclerosis and healthy controls (P > 0.05). Total corneal thickness at the apex was 511.1 ± 33.5µm in the systemic sclerosis group and 528.4 ± 29.5µm in the control group (P = 0.015). The corneal stroma was thinner in the systemic sclerosis patients compared to the healthy controls (P = 0.02). The corneal stroma was thinner in the patients with systemic sclerosis compared to that of healthy controls, while the thickness of the anterior sclera was similar in both groups.

Evaluation of corneal sublayers thickness and corneal parameters in patients with Parkinson’s disease

Purpose The aim of this study was to compare the blink rate (BR), tear tests, corneal parameters, and the thickness of each corneal sublayer in patients with Parkinson’s disease (PD) and healthy individuals. Methods A total of 64 eyes from 64 patients with PD and 64 eyes from 64 age- and sex-matched healthy individuals were included in this study. All participants underwent a detailed neurological and ophthalmological evaluation. The severity of disease was measured according to Hoehn–Yahr (H–Y) scale. The blink rate (BR), Schirmer test, and tear break-up time (TBUT) scores were assessed. Corneal parameters were measured using Pentacam. Additionally, the thickness of the corneal epithelium, Bowman layer, stroma, and Descemet membrane-endothelium complex were measured on the central cornea with anterior segment module of spectral domain optical coherence tomography (AS-OCT). Results The BR, Schirmer’s test, TBUT, pachymetric measurements, Bowman layer, and stromal thickness values of the patient group were significantly lower than those of the control group. The disease duration and disease severity were significantly negative correlated with the pachymetric measurements and stromal thickness. Also, the values of TBUT and the score of Schirmer test were significantly positive correlated with the pachymetric measurements and stromal thickness. Conclusions A reduced BR and poor tear quality in the PD, can result in reduced pachymetric measurements and stromal thickness.

Effects of corneal preservation conditions on human corneal endothelial cell culture

The purpose of this study was to investigate the growth capacity of human corneal endothelial cells (HCEnCs) isolated from old donor corneas preserved in 4 different storage conditions. The following conditions were evaluated, A) cold storage (CS) (Optisol GS) for 7 days at 4 °C [n = 6]; B) organ culture (OC) (Cornea Max) for 7 days at 31 °C [n = 6]; C) OC for 28 days at 31 °C [n = 6] and; D) CS for 7 days at 4 °C followed by OC for 28 days at 31 °C [n = 6]. Following preservation, the Descemet membrane-endothelium complex was peeled and digested using Collagenase-Type1 and was subsequently trypsinized before being plated into 2 wells (from each cornea) of an 8-well chamber slide. Media was refreshed every alternate day. The confluence rate (%) was assessed, and overall viability was determined using Hoechst, Ethidium Homodimer and CalceinAM staining. HCEnC-associated markers ZO-1, Na+/K+-ATPase, CD166 (Tag1A3), PRDX-6 (Tag2A12) and proliferative marker Ki-67 were used to analyse the cultures established from each condition. Donor tissues preserved in hypothermia (condition A) resulted in 9.3% ± 4.0% trypan-blue positive cells (TBPCs) hence lower number of HCEnCs was plated. <1% TBPCs were observed in conditions B, C and D. Indicatively, confluence in conditions A, B, C and D was 14.0%, 24.8%, 23.4% and 25.4% respectively (p = 0.9836) at day 1. By day 9, HCEnCs established from all conditions became confluent except cells from condition A (94.2% confluence). All HCEnCs in the 4 conditions were viable and expressed HCEnC-associated markers. In conclusion, OC system has advantages over hypothermic media for the preservation of older donor corneas rejected for corneal transplant and deemed suitable for corneal endothelial cell expansion, with lower TBPCs before peeling and longer period of tissue preservation over hypothermic storage system.

A New Method of Harvesting Descemet Membrane-Endothelium Complex Approaching From the Suprachoroidal Space for Descemet Membrane Endothelial Keratoplasty: An Experimental Animal Study

To report the study in rabbits of a new method of harvesting Descemet membrane-endothelium complex approaching from the suprachoroidal space for Descemet membrane endothelial keratoplasty. Twenty-four eyes of 12 rabbits were used for microscopic analysis, whereas 24 eyes of 12 cats were used for gross photographic analysis. The enucleated eyeballs of the rabbits and cats were incised 360 degrees circumferentially along the equator. Manual separation was performed between the sclera and the underlying connective tissue approaching from the suprachoroidal space in the direction of the central cornea step by step in spiral pattern. After complete separation of the stroma from the Descemet membrane-endothelium-iris complex, it was excised from the globe at the outside of the iris root. The synthetic carrier (soft contact lens) was laid down on the punch block, and then the excised complex was brought over the carrier with the endothelium-iris side upward. Two steps of down punch for the designed graft size were performed. The harvested complexes were processed for vital stains and were evaluated through photography taken under a light microscope and gross close-up. The endothelial cell loss rates were calculated by a computer software. The overall harvest success rate was 89.6% (43 of 48). The overall structural cell loss rate was 1.83 +/- 2.34 by microscopic analysis and 4.68% +/- 1.68% by gross photographic analysis. The new method of en bloc harvesting Descemet membrane-endothelium complex approaching from the suprachoroidal space could provide minimal damage to the endothelium during harvesting, and it can be carried and passed through an injector.

Computer analysis of corneal innervation density using a novel double stain in rat corneal whole mounts.

Computerised morphometry and a double stain technique were utilised to examine the corneal nerves in whole mounts. This novel stain combines the nonspecific acetylcholinesterase (NsAchE) and gold chloride (AuCl) procedures to enhance staining contrast and facilitate computerised detection of corneal nerves. Fresh rat corneas were dissected, and the Descemet's membrane-endothelium complex was removed. Then the corneas were fixed in 4% paraformaldehyde with 50 mM Na-K phosphate buffer (pH 7.2) and 8% sucrose for 30 min. They were rinsed and stained singly with NsAchE or AuCl, or were double stained using NsAchE followed by AuCl. Between NsAchE and AuCl staining the corneas were stored frozen in OCT compound at -70 degrees C. Flat mounts of whole corneas were photographed before and after the second staining. Measurable stromal innervation density (mean +/- S.D.) in age-matched corneas stained with AuCl (3.90 +/- 0.36 mm/mm2) was not significantly different from that of NsAchE stained corneas. However, double staining compared with NsAchE staining of the same corneas revealed a 48 +/- 27% increase in demonstrable innervation density of the subepithelial nerve plexus (7.95 +/- 0.86 mm/mm2 vs 5.52 +/- 1.31 mm/mm2, respectively). Improved visualisation of epithelial nerves and their fine ramifications (leashes) was also obtained by double staining. This novel combination of 2 procedures enhances the detection of corneal nerves for analysis by computerised morphometry and provides a more representative estimate of total corneal innervation density.

Improved gold chloride procedure for nerve staining in whole mounts of rat corneas.

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryoprotective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at -70 C in O.C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO4-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO4-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO4-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O.C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced non-specific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohistochemical procedures where the reaction products would be obscured by background staining.

Lysosomal enzyme activities of the bovine corneal endothelium.

A quantitative study was carried out on the lysosomal enzyme activities of the bovine corneal endothelium-Descemet's membrane preparation. The corneal endothelium and Descemet's membrane were peeled off together. Cathepsin D was assayed using hemoglobin as substrate; N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, acid phosphatase, and alpha-mannosidase were also examined using p-nitrophenyl derivatives as substrate. The proportions of N-acetyl-beta-D-glucosaminidase, cathepsin D, and beta-glucuronidase of the Descemet's membrane-endothelium complex were particularly high: 11.5%, 12.6%, and 12.5% of the whole cornea, respectively. Corneal endothelial cells also showed high activities of acid phosphatase and alpha-mannosidase (3.8%, and 5.0% of the whole cornea, respectively), while the protein and DNA contents were 0.5% and 0.5% in the complex. Lysosomal enzyme activities in the complex were also compared with those in other ocular tissues and were determined by the same methods at the same time.