Endothelial Descemet Membrane Research Articles

Descemet membrane adhesion strength is greater in diabetics with advanced disease compared to healthy donor corneas

Descemet membrane endothelial keratoplasty (DMEK) is an increasingly popular surgical procedure for treating ocular diseases that require a corneal transplant. Previous studies have found that tissue tearing during surgical preparation is more likely elevated in eyes from donors with a history of diabetes mellitus. To quantify these potential differences, we established an experimental technique for quantifying the force required to separate the endothelium-Descemet membrane complex (EDM) from stroma in human donor corneal tissue, and we assessed differences in adhesion strength between diabetic and non-diabetic donor corneas. Transplant suitable corneas were obtained from 23 donors 50–75 years old with an average preservation to assay time of 11.5 days. Corneas were classified from a medical records review as non-diabetic (ND, n = 9), diabetic without evidence of advanced disease (NAD, n = 8), or diabetic with evidence of advanced disease (AD, n = 10). Corneas were sectioned into 3 mm wide strips and the EDM peeled from the stroma. Using the force-extension data obtained from mechanical peel testing, EDM elastic peel tension (TE), elastic stiffness (SE), average delamination tension (TD), and maximum tension (TMAX) were calculated. Mean TE, SE, TD, and TMAX values for ND corneas were 0.78 ± 0.07 mN/mm, 0.37 ± 0.05 mN/mm/mm, 0.78 ± 0.08 mN/mm, and 0.94 ± 0.17 mN/mm, respectively. NAD values did not differ significantly. However, AD values for TE (1.01 ± 0.18 mN/mm), TD (1.09 ± 0.21 mN/mm), and TMAX (1.37 ± 0.24 mN/mm) were greater than ND and NAD corneas (P < 0.05). SE did not differ significantly between groups. These findings provide proof of the concept that chronic hyperglycemia from diabetes mellitus results in a phenotypically more adhesive interface between Descemet membrane and the posterior stroma in donor corneal tissue. Results of this study provide a foundation for further investigations into the impact of diabetes on the posterior cornea, eye banking, and keratoplasty.

Impact of Donor Age on Corneal Endothelium-Descemet Membrane Layer Scroll Formation.

To correlate corneal endothelium-Descemet membrane (EDM) layer parameters of scroll tightness with donor age, endothelial cell density (ECD), and history of diabetes. Endothelium-Descemet membrane layer scrolls were harvested from 26 corneoscleral buttons using the SCUBA technique by a cornea-fellowship trained ophthalmologist masked to donor age. Two independent outcome parameters were used to characterize the scrolling severity of successfully harvested tissue: scroll width and tendency for EDM scroll formation (referred to as scroll rating on a 1-4 scale: incomplete scroll formation to tightly scrolled). Mean donor age was 59 ± 17 (15-69) years. Mean ECD of EDM scroll was 2,451 ± 626 (range: 1,307-3,195) cells per square millimeter. Using stepwise linear regression, a significant correlation was found between scroll width and donor age (R=0.497, P<0.05). Additionally, a significant inverse correlation was found between scroll width and ECD (R=-0.605, P<0.05). There was no statistically significant correlation between a donor history of diabetes and the parameters of scrolling tendency. Our data suggest that using older donors reduces EDM scroll tightness.

Comparison of Sulfur Hexafluoride 20% versus Air Tamponade in Descemet Membrane Endothelial Keratoplasty

To compare clinical outcomes using 20% sulfur hexafluoride (SF6) versus 100% air as a tamponade for graft attachment in Descemet membrane endothelial keratoplasty (DMEK). Retrospective, comparative, interventional case series. Pseudophakic patients with Fuchs' endothelial dystrophy or pseudophakic bullous keratopathy that underwent DMEK using either 20% SF6 (group 1; 42 eyes) or 100% air (group 2; 39 eyes) tamponade between April 2010 and August 2011. A bimanual infusion technique was used to introduce and position the donor endothelium-Descemet membrane graft tissue. Outcome measures were analyzed at the following time points: before surgery, 3 and 6 months after surgery, and at yearly intervals up to at least 3 years. Corrected distance visual acuity (CDVA), manifest refraction, pachymetry, central endothelial cell count (cECC), complications, and rebubbling rates. Three years after surgery, mean CDVA improved from 0.48±0.45 logarithm of the minimum angle of resolution (logMAR) to 0.04±0.23 in group 1 (P < 0.001) and from 0.67±0.45 logMAR to 0.09±0.13 logMAR in group 2 (P < 0.001). The percentage of eyes with CDVA of 20/25 or more was 85.71% (36/42 eyes) in group 1 and 82.05% (32/39 eyes) in group 2 (P = 0.43). Mean preoperative cECCs and at last follow-up were: group 1, 2525±338 cells/mm(2) and 1758 ± 398 cells/mm(2) (mean cell loss, 30±11%; P = 0.008); and group 2, 2492±204 cells/mm(2) and 1678±373 cells/mm(2) (mean cell loss, 32±13%; P = 0.008). Endothelial cell loss was similar in both groups (P = 0.65). Intracameral air reinjection was needed in 1 patient in group 1 (2.38%) and in 5 patients in group 2 (12.8%). The rebubbling rate was significantly higher in group 2 (P = 0.004). No episodes of immunologic graft rejection were documented. Although clinical outcomes and corneal endothelial cell loss were similar in both groups, tamponade with 20% SF6 yielded a significantly lower incidence of graft detachments that may warrant its routine use in DMEK. Longer-term, randomized studies are needed to recommend this approach fully.

Regional assessment of energy-producing metabolic activity in the endothelium of donor corneas.

We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

Simple, inexpensive, and effective injector for descemet membrane endothelial keratoplasty.

We describe an inexpensive, simple, and effective endothelium-Descemet membrane (EDM) graft injector assembled from regular operating room supplies in Descemet membrane endothelial keratoplasty (DMEK). To assemble the injector, standard intravenous tubing was cut approximately 2 inches from the Luer lock end, leaving a steep bevel. The cut end of the tubing was firmly wedged bevel up and advanced into the back of an Alcon IOL B cartridge. The Luer lock end of the tubing was then attached to a 5- or 10-mL syringe filled with BSS Plus. The EDM graft was then placed into a Petri dish filled with BSS. After the graft was sucked into an injector with bevel-side up under the surgical microscope, the graft was then inserted into the anterior chamber with the injector through the main incision in the superotemporal quadrant. In seven eyes of seven patients with Fuchs endothelial corneal dystrophy treated with DMEK using our injector, clear attached grafts and improved visual acuity were achieved. This simple, inexpensive, and effective injector is a safe and viable device to facilitate this part of DMEK surgery.

Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.

The cornea remains in a state of deturgescence, maintained by endothelial cell Na+/K+ ATPase and by tight junctions between endothelial cells that limit entrance of fluid into the stroma. Fuchs’ endothelial corneal dystrophy (FECD) was initially described by Fuchs in 1910 as a combination of epithelial and stromal edema in older patients. It manifests as bilateral, albeit asymmetric, central corneal guttae, corneal edema, and reduced vision. When edema is severe, the corneal epithelium can detach from its basement membrane, creating painful bullae on the anterior surface of the cornea. The course of this dystrophy can be further accelerated after intraocular surgery, specifically cataract extraction. Pseudophakic bullous keratopathy (PBK) is endothelial cell loss caused by surgery in the anterior chamber. If the corneal endothelium is damaged during surgery, the same spectrum of symptoms as found in FECD can develop. In the nineteenth century, penetrating keratoplasty was the only surgical procedure available for isolated endothelial disease. In the 1960s, Dr. José Barraquer described a method of endothelial keratoplasty using an anterior approach via laser-assisted in situ keratomileusis (LASIK) flap. In 1999, Melles and colleague described their technique of posterior lamellar keratoplasty. Later, Melles et al. started to change host dissection using simple “descemetorhexis” in a procedure known as Descemet’s stripping endothelial keratoplasty. Following the widespread adoption of Descemet’s stripping automated endothelial keratoplasty, the Melles group revisited selective Descemet’s membrane transplantation and reported the results of a new procedure, Descemet’s membrane endothelial keratoplasty (DMEK). Recently, some eye banks have experimented with the preparation of DMEK/Descemet’s membrane automated endothelial keratoplasty donor tissue that may help the surgeon avoid the risk of tissue loss during the stromal separation step. Recently, the authors described a new bimanual technique for insertion and positioning of endothelium–Descemet membrane grafts in DMEK.Electronic supplementary materialThe online version of this article (doi:10.1007/s40123-014-0022-y) contains supplementary material, which is available to authorized users.

Bimanual Technique for Insertion and Positioning of Endothelium–Descemet Membrane Graft in Descemet Membrane Endothelial Keratoplasty

To describe a new bimanual technique for insertion and positioning of endothelium-Descemet membrane (EDM) grafts in Descemet membrane endothelial keratoplasty (DMEK). Prospective, noncomparative, consecutive interventional case series, including 15 pseudophakic eyes of 15 patients treated with DMEK at the Instituto de Microcirugia Ocular, Barcelona, Spain. A bimanual infusion technique was used to introduce and position donor's EDM. Partial tamponade was achieved with 20% sulfur hexafluoride (SF6). Intraocular manipulation time of EDM grafts, rebubbling rate, and endothelial cell density were evaluated. Six months postoperatively, mean uncorrected visual acuity and best spectacle-corrected visual acuity (BSCVA) improved from 20/100 to 20/50 and from 20/80 to 20/25, respectively (P < 0.001). Six eyes (40%) had ≥ 20/20 BSCVA, and 13 (86%) eyes had ≥ 20/30 BSCVA. Mean donor cell density decreased from 2690 ± 302 to 1998 ± 621 cells per square millimeter, representing a mean cell loss of 26% ± 20%. EDM was stripped successfully in all cases. Mean intraocular manipulation time of donor's EDM (interval between main incision closure and final EDM positioning) was 4.10 ± 0.5 minutes. Intracameral air reinjection was needed in 1 case (6.6%) with a partial peripheral graft detachment. No episodes of immunologic graft rejection were documented. Insertion and positioning of EDM grafts in DMEK was successfully achieved using this bimanual infusion technique and 20% SF6 partial tamponade, with a low intraocular manipulation time and very low postoperative rebubbling rates, regardless of the preservation method and the donor's age. However, prospective comparative studies are required to demonstrate the long-term safety compared with other approaches.

Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm(2). The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.

Split-Cornea-Transplantation - ein neues Konzept gegen Spendermangel

Penetrating keratoplasty is at present the gold standard for corneal transplantation, but tremendous progress has been made in recent years in improving lamellar keratoplasty techniques, such as deep anterior lamellar keratoplasty (DALK) and Descemet membrane endothelial keratoplasty (DMEK). The purpose of this report is to describe split-cornea transplantation by combining DALK and DMEK as a novel concept to reduce donor shortage. The study consists of a PUBMED literature review and our own clinical results. Splitting of a single donor cornea into an anterior part (including epithelium, its basement membrane, Bowman layer, and stroma) for use in a DALK procedure in a patient with anterior-stromal disease (e. g., keratoconus) and into a posterior part (endothelium-Descemet membrane layer) for use in a DMEK procedure in a patient with endothelial disease (e. g., Fuchs endothelial dystrophy) can reduce the need for corneal donor tissue by around 50 %. A short-term 6-months follow-up has revealed good visual and refractive outcomes with low complication rates and acceptable endothelial cell loss. Split-cornea transplantation by combining DALK and DMEK surgeries is a promising concept to reduce donor shortage in corneal transplantation surgery in the future.

Split Cornea Transplantation for 2 Recipients: A New Strategy to Reduce Corneal Tissue Cost and Shortage

To evaluate the feasibility of using a single donor cornea for 2 recipients by combining deep anterior lamellar keratoplasty (DALK) and Descemet's membrane endothelial keratoplasty (DMEK) surgeries on the same day. Single-center, nonrandomized, prospective, interventional case series. Twelve consecutive donor corneas were scheduled for split cornea transplantation combining DALK for a keratoconus patient and DMEK for a Fuchs' endothelial dystrophy patient on the same surgery day. First, a big-bubble DALK procedure was performed for the keratoconus eye. When bare Descemet's membrane was prepared successfully requiring no conversion to penetrating keratoplasty (PK), then during surgery the donor, endothelium-Descemet's membrane layer was removed and stored for subsequent DMEK in a second patient, and the remaining anterior lamella of the donor cornea was used to complete the DALK surgery. Afterward, a DMEK procedure was performed on the second patient with Fuchs' endothelial dystrophy, grafting the stored endothelium-Descemet's membrane layer of the original donor button. Success of using a single donor cornea for 2 recipient eyes, best spectacle-corrected visual acuity (BSCVA), and complication rates within 6 months follow-up. A single donor cornea could be used for 2 recipients in 10 of 12 donor buttons (83%). In 2 cases (17%), the DALK procedure had to be converted to PK requiring a full-thickness corneal graft. Therefore, 10 donor corneas (45%) could be saved. Six months after surgery, mean BSCVA was 20/35 (range, 20/50-20/25) in 10 eyes that underwent successful DALK, 20/50 (range, 20/63-20/40) in 2 eyes that underwent conversion from DALK to PK, and 20/31 (range, 20/50-20/16) in 10 eyes that underwent DMEK. Postoperative complications after DALK included Descemet's folds in 3 eyes (30%) and epitheliopathy in 2 eyes (20%). After DMEK, partial graft detachment occurred in 5 eyes (50%) and was managed successfully with intracameral air reinjection. All corneas remained clear up to 6 months after surgery. Split use of donor corneal tissue for combined DALK and DMEK procedures in 2 recipients on the same surgery day is a promising strategy to reduce donor shortage and cost in corneal transplantation surgery in the future.

A Method to Confirm Correct Orientation of Descemet Membrane During Descemet Membrane Endothelial Keratoplasty

Descemet membrane endothelial keratoplasty is a new technique for the replacement of diseased corneal endothelium with healthy donor endothelium. During this procedure, manipulation of the donor endothelium-Descemet membrane layer within the recipient anterior chamber can be associated with loss of the correct anterior-posterior orientation. Herein, we describe a simple method to keep the correct donor orientation during transplantation by marking the edge of the endothelium-Descemet membrane layer. Interventional case series. Donor discs were created by stripping of the endothelium-Descemet membrane layer from corneoscleral buttons. Before completion of stripping, 3 circular marks were set in an identifiable order at the edge of the donor disc. After removal of the recipient Descemet membrane, the donor graft was inserted into the anterior chamber, unfolded, and attached to the posterior corneal stroma with an air bubble. Correct anterior-posterior orientation of the graft was identified by the clockwise order of the 3 marks. The marks allowed identification of the anterior-posterior orientation of the endothelium-Descemet membrane layer after unfolding in the anterior chamber and attachment to the recipient stroma in all cases. In 4 of 25 patients, orientation was upside down after unfolding of the roll, requiring inversion of the refolded donor graft. Marking the edges of the endothelium-Descemet membrane layer helps to keep the correct orientation without adverse effects on donor detachment or corneal clarity during and after Descemet membrane endothelial keratoplasty.

Technique for Preparation of the Corneal Endothelium-Descemet Membrane Complex for Transplantation

Replacing diseased corneal endothelium with a preparation of Descemet membrane carrying functional endothelium and no stroma may be a feasible method for treating corneal endothelial decompensation. To obtain a viable donor of a Descemet membrane endothelium disc, we modified the Descemet membrane stripping technique and monitored the percentage of endothelial damage to the donor tissue preparation. Forty-eight human corneas were used. Cornea buttons were mounted on an artificial anterior chamber, endothelial side up. Endothelia were stained with alizarin red, examined under the microscope, and photographed at 5 different sites (microscope, x100; digital magnification, x2.83). A 6 x 7-mm rectangular piece of endothelium-Descemet membrane complex was obtained using a Grieshaber microsurgical knife and Kelman-McPherson forceps. Digital photographs of endothelia were analyzed with a computer, and the percentage of endothelial damage was calculated. Specimens were processed for hematoxylin-eosin staining. Forty of 48 endothelium-Descemet membrane preparations (83.3%) were complete peels with minimal endothelial damage. Endothelial damage before and after the surgery was 1.57 +/- 2.11% and 2.61 +/- 1.77%, respectively. Eight preparations (16.7%) failed because of tearing. Multiple hematoxylin-eosin-stained sections showed the presence of endothelium with intact Descemet membrane and no stromal tissue. We modified the technique of Melles and obtained a sheet of Descemet membrane and endothelium with minimal endothelial damage and with no remaining stroma observed. This simple technique can be used to obtain the endothelium-Descemet membrane complex in minutes. It may be useful for corneal endothelium transplantation.

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.