Identification of the preoperative and perioperative factors that predict postoperative endothelial cell density after Descemet membrane endothelial keratoplasty: A retrospective cohort study.

Low postoperative endothelial-cell density (ECD) plays a key role in graft failure after Descemet-membrane endothelial keratoplasty (DMEK). Identifying pre/perioperative factors that predict postoperative ECD could help improve DMEK outcomes. This retrospective study was conducted with consecutive adult patients with Fuchs-endothelial corneal dystrophy who underwent DMEK in 2015-2019 and were followed for 12 months. Patients underwent concomitant cataract surgery (triple-DMEK) or had previously undergone cataract surgery (pseudophakic-DMEK). Multivariate analyses assessed whether: patient age/sex; graft-donor age; preoperative ECD, mean keratometry, or visual acuity; triple DMEK; surgery duration; surgical difficulties; and need for rebubbling predicted 6- or 12-month ECD in the whole cohort or in subgroups with high/low ECD at 6 or 12 months. The subgroups were generated with the clinically relevant threshold of 1000 cells/mm2. Surgeries were defined as difficult if any part was not standard. In total, 103 eyes (95 patients; average age, 71 years; 62% women) were included. Eighteen eyes involved difficult surgery (14 difficult graft preparation or unfolding cases and four others). Regardless of how the study group was defined, the only pre/perioperative variable that associated significantly with 6- and 12-month ECD was difficult surgery (p = 0.01, 0.02, 0.05, and 0.0009). Difficult surgery also associated with longer surgery duration (p = 0.002). Difficult-surgery subgroup analysis showed that difficult graft dissection associated with lower postoperative ECD (p = 0.03). This association may reflect endothelial cell loss due to excessive graft handling and/or an intrinsic unhealthiness of the endothelial cells in the graft that conferred unwanted physical properties onto the graft that complicated its preparation/unfolding.

The impact of biomechanics on corneal endothelium tissue engineering.

The integrity of innermost layer of the cornea, the corneal endothelium, is key to sustaining corneal transparency. Therefore, disease or injury causing loss or damage to the corneal endothelial cell population may threaten vision. Transplantation of corneal tissue is the standard treatment used to replace malfunctioning corneal endothelial cells. However, this surgery is dependent upon donor tissue, which is limited in supply. Hence, tissue engineers have attempted to construct alternative transplantable tissues or cell therapies to alleviate this problem. Nevertheless, the intrinsic non-dividing nature of corneal endothelial cells continues to foil scientists in their attempts to yield large numbers of cells in the laboratory for use in such novel therapies. Interestingly, the contribution of the biomechanical properties of the underlying extracellular matrix (ECM) on cell division, tissue development and maintenance has been extensively investigated in other many cell types. However, the impact of biomechanics on corneal endothelial cell behaviour is relatively unexplored. Here, we describe contemporary tissue engineering solutions aimed at circumventing donor tissue scarcity. We review the ECM structure and biomechanical features of corneal endothelial cells. We discuss the alterations of ECM in endothelial disease development and progression and point out the role of ECM in developing a tissue-engineered corneal endothelium. We highlight the main biomechanical cues, including topographical and mechanical features, that impact cellular behaviors. Finally, we discuss the influence of biomechanical cues on cell and tissue development, and how corneal endothelial cells response to individual biomechanical stimuli in tissue engineering, which have implications for designing an engineered endothelium and maintaining cell function.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Evaluation of reconstructed human corneal endothelium sheets made with porcine Descemet's membrane in vitro and in vivo.

PURPOSE: To identify the feasibility of reconstructing corneal endothelial sheets by seeding non-infected monoclonal human corneal endothelial cells (HCECs) onto porcine Descemet's membrane (DM) and verifying the function in vitro and in vivo. METHODS: Denuded porcine DM was decellularized for haematoxylin and eosin staining, and DNA was removed via incubation with ethylene glycol diglycidyl ether (EGDE). The physical properties of the incubated DMs were evaluated and compared to those of unincubated DMs. The non-infected monoclonal HCECs were examined by chromosome analysis and the cell proliferation was evaluated by BrdU-labelling. Then HCECs at passage 30 were then seeded on the DM and cultured for approximately 5 days. The cell growth, density and expression of the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), the tight-junction-associated protein zonula occludens (ZO-1) and acetylated alpha tubulin were examined by electron microscopy and immunocytochemistry to compare HCECs cultivated on porcine DM and those cultured in vitro. Cells on the reconstructed HCEC sheets were labeled with DiI, and the sheets were subsequently transplanted into cat eyes via DM endothelial keratoplasty (DMEK). The corneal transparency, thickness, anterior segment, and HCEC density were monitored in vivo, and the corneal endothelial cell morphology and histological structure were examined ex vivo 98 days after surgery. RESULTS: No significant differences were observed in the elongation at break of the DMs and the thickness of the DMs incubated with EGDE compared to those of the unincubated DMs (P > 0.05). Results of chromosome analysis shown the number of the HCEC cell line was still 46 and no abnormal chromosome structure was found. BrdU-labelling shown the HCECs stopped proliferating after 5 days and the cells formed a single layer. The cells transferred to porcine DM formed tight connections with the substrate and generated layers of hexagonal cells on day 5. Adjacent cells cultivated on DM were closely attached to each other, tightly adhered to the porcine DM and expressed the Na+/K+-ATPase, ZO-1 protein and acetylated alpha tubulin, as did HCECs cultured in vitro. In addition, the HCEC density on DMs was 3020.14 ± 52.30 cells/mm2. After surgery, the corneas gradually became transparent, and the thickness decreased to 525.33 ± 56.23 µm at day 98 after the transplantation, while the control corneas showed consistent oedema during the monitoring period. The HCEC density was 2521.60 ± 78.24 cells/mm2 in vivo 98 days after transplantation. The histological results showed that the DiI-labeled cells were dense in the transplanted area and had a hexagonal or polygonal morphology and a normal ultrastructure; adjacent cells were closely attached to each other and tightly adhered to the porcine DM. CONCLUSIONS: Seeding non-infected monoclonal HCECs on porcine DM could reconstruct functional corneal endothelial sheets. These results may help uncover new applications for tissue-engineered endothelium in endothelial keratoplasty.

Integrin: Basement membrane adhesion by corneal epithelial and endothelial cells.

Integrins mediate adhesion of cells to substrates and maintain tissue integrity by facilitating mechanotransduction between cells, the extracellular matrix, and gene expression in the nucleus. Changes in integrin expression in corneal epithelial cells and corneal endothelial cells impacts their adhesion to the epithelial basement membrane (EpBM) and Descemet's membrane, respectively. Integrins also play roles in assembly of basement membranes by both activating TGFß1 and other growth factors. Over the past two decades, this knowledge has been translated into methods to grow corneal epithelial and endothelial cells in vitro for transplantation in the clinic thereby transforming clinical practice and quality of life for patients. Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membrane expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.

Preloading Trifolded Grafts for Descemet Membrane Endothelial Keratoplasty Affects Scroll Formation.

PURPOSE: The trifolded, endothelium-in approach to Descemet membrane endothelial keratoplasty (DMEK) facilitates tissue insertion into the anterior chamber. We hypothesized that preloading the trifolded donor grafts in a cartridge for 48 hours before insertion would induce biomechanical changes that decrease their scrolling tendency compared with those loaded immediately before insertion. METHODS: Ten Descemet membrane donor grafts, peeled and cut to 8.0 mm, were prepared by a single eye bank technician. Each graft was trifolded and pulled into a DMEK cartridge and stored for 48 hours. They were then pulled with microforceps into a petri dish filled with balanced salt solution. A video was recorded of the graft becoming a scroll over a 2-minute period. Each graft, serving as its own control, was then trifolded, pulled into the cartridge, and the process repeated. Images from 1, 5, 10, 60, and 120 seconds were extracted from video recording of the procedures. Scroll width was analyzed by graders masked to group assignment. A paired t test was used to determine differences in scroll width at each time point between the 48-hour and instant trifolding conditions. RESULTS: All grafts scrolled after removal from the cartridge into balanced salt solution. We measured a significant difference at all time points 1 through 120 seconds (4.02 preloaded vs. 2.91-mm instant trifold, P = 0.035). CONCLUSIONS: Preloading DMEK grafts in a trifolded configuration for 48 hours reduces the scrolling tendency of Descemet membrane for at least 2 minutes.

Microkeratome-assisted ultrathin Descemet's stripping automated endothelial keratoplasty: A randomized trial comparing single-pass versus double-pass technique.

Purpose: To compare the outcomes of two techniques, for preparation of microkeratome-assisted ultrathin grafts for Descemet's stripping automated endothelial keratoplasty (DSAEK). Methods: The study involved 20 eyes of 20 patients with pseudophakic bullous keratopathy, randomized into two groups. Group 1 eyes underwent microkeratome-assisted DSAEK using the single-pass technique for lenticule preparation, whereas group 2 eyes underwent microkeratome-assisted DSAEK using the double-pass technique. Patients were followed up till 6 months, postoperatively. Best-corrected visual acuity (BCVA) at final follow-up was considered as the primary outcome measure, whereas graft thickness (GT) contrast sensitivity and endothelial cell loss were considered as the secondary outcome measures. A P value of <0.05 was considered as statistically significant. Results: Baseline characteristics of two groups were comparable. The mean central GT was comparable in both groups at 6 months follow-up [group 1: 98 ± 24.46 µm, group 2: 129 ± 31.46 µm (P = 0.18)]. Both groups fared equally in terms of BCVA (P = 0.33). Contrast sensitivity was significantly better in group 1 eyes (P = 0.045). A statistically significant negative correlation was found between postoperative BCVA and postoperative GT (R = -0.728, P = 0.016). The percentage endothelial cell loss was slightly higher in group 2 eyes, although not statistically significant. Two eyes in group 2 experienced complications during lenticule preparation. None of the eye experienced any complication in the postoperative period. Conclusion: Both techniques provided grafts with comparable thickness and endothelial cell loss and were associated with comparable BCVA, at final follow-up visit. The contrast sensitivity was, however, better in eyes receiving grafts prepared with the single-pass technique.

Minimizing Endothelial Cell Loss Caused by Orientation Stamps on Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: To quantify endothelial cell loss (ECL) caused by orientation stamps on prestripped and preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts, and to examine a method for reducing ECL using a smaller stamp. METHODS: Ten prestripped and 10 preloaded DMEK grafts were prepared with S-stamps. Ten additional preloaded DMEK grafts were prepared with both an S-stamp and a smaller F-stamp in different paracentral areas of the graft. The footprint of each stamp was measured using ink on cardstock. DMEK grafts were stored in viewing chambers filled with 20 mL of Optisol-GS for 3 days at 4°C. ECL was quantified using Calcein-AM staining and FIJI Weka Segmentation. RESULTS: S-stamps on prestripped DMEK grafts contributed an average ECL of 1.1% ± 0.5% (range: 0.6%-2.2%) toward total graft damage, whereas S-stamps on preloaded DMEK grafts contributed approximately twice that amount (average ECL: 2.0% ± 0.7%, range: 1.3%-3.1%, P = 0.004). Overall ECL for prestripped grafts (average: 7.1% ± 3.3%, range: 3.3%-13.7%) and preloaded grafts (average: 11.3% ± 4.2%, range: 6.9%-19.4%) was similar to previous reports. The footprint of the S-stamp was approximately 45% larger than that of the F-stamp. In 10 preloaded grafts marked with both stamps, the S-stamp caused an average ECL of 1.9% ± 0.6% (range: 1.2%-3.2%), whereas the smaller F-stamp caused an average ECL of 1.0% ± 0.2% (range: 0.8%-1.4%, P = 0.0002). CONCLUSIONS: Loss of endothelial cells associated with graft-stamping was greater in preloaded tissue than in prestripped tissue and was less with a smaller F-stamp than with a larger S-stamp. Using a smaller stamp could help minimize ECL in prestripped and preloaded DMEK grafts.

Comparison of preservation and transportation protocols for preloaded Descemet membrane endothelial keratoplasty.

BACKGROUND/AIMS: Descemet membrane endothelial keratoplasty (DMEK) preparation is technically demanding and is a limiting factor for uptake of this kind of surgery. Supply methods that simplify the procedure for surgeons are key to increasing uptake. This study compares two different shipping protocols for DMEK. METHODS: An 8.5 mm DMEK graft was punched, marked and loaded for transportation in two different conditions: (A) endothelium trifolded inwards in organ culture conditions (n=7) and (B) endothelium rolled outwards in hypothermic conditions (n=7). Tissues were shipped from Italy to the UK, then analysed for orientation, endothelial cell density, denuded areas, cell mortality, triple viability staining (Hoechst/ethidium homodimer/calcein AM (HEC)), immunolocalisation of ZO-1 and Na/K-ATPase proteins, visualisation of actin filaments using phalloidin and histological analysis using H&E on paraffin-embedded sections. RESULTS: All tissues clearly showed the mark used for graft orientation. After shipping in condition A, there was an increase in cell mortality of 8.1% and in denuded areas of 22.4%, whereas for condition B there was an increase in cell mortality of 14.2% and in denuded areas of 34.3% after shipping. HEC staining revealed areas of viable cells and apoptotic cells, with large denuded areas found in the periphery for condition B and within folds for condition A. CONCLUSIONS: Prestripped preloaded DMEK grafts retained sufficient viable cells for transplantation, with condition A (endothelium-in) offering the advantage of greater flexibility of use due to a longer shelf-life. HEC analysis provides further detailed information as to the status of DMEK grafts and should be used in future similar studies.

Improving the success rate of human corneal endothelial cell cultures from single donor corneas with stabilization medium.

Main objective of this study was to improve the success rate of human corneal endothelial cell (hCEC) cultures from single donor corneas. We could show that the use of stabilization medium prior to cell isolation may have a positive effect on the success rate of hCEC cultures from single research-grade donor corneas by allowing growth of otherwise possibly not successful cultures and by improving their proliferative rate. hCEC were obtained from corneo-scleral rims of 7 discarded human research-grade cornea pairs. The Descemet membrane-endothelium (DM-EC) sheets of each pair were assigned to 2 experimental conditions: (1) immediate cell isolation after peeling, and (2) storage of the DM-EC sheet in a growth factor-depleted culture medium (i.e. stabilization medium) for up to 6 days prior to cell isolation. hCEC isolated by enzymatic digestion were then induced to proliferate on pre-coated culture plates. The success rate of primary cultures established from single donor corneas were higher for DM-EC sheets kept in stabilization medium before cell isolation. All cultures (7/7) initiated from stabilized DM-EC sheets were able to proliferate up to the third passage, while only 4 out of 7 cultures initiated from freshly peeled DM-EC sheets reached the third passage. In addition, for the 4 successful paired cultures we observed a faster growth rate if the DM-EC sheet was pre-stabilized prior to cell isolation (13.8 ± 1.8 vs 18.5 ± 1.5 days, P < 0.05). Expression of the phenotypical markers Na+/K+-ATPase and ZO-1 could be shown for the stabilized cultures that successfully proliferated up to the third passage.

[Density of the endotheliocytic layer of the cornea of the eyeball as a function of age].

This article presents the results of measuring the number of corneal endotheliocytes in a unit area of descemet membrane surface in 546 volunteers of different ages. The average values of the density of the corneal posterior epithelium for the age intervals 40-49, 50-59, 60-69, 70-79, 80 years and older are shown, a constant decrease in the number of endotheliocyte cells as the number of years lived increases.

Evaluation and Quality Assessment of Prestripped, Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: To determine graft quality and feasibility of Descemet membrane endothelial keratoplasty (DMEK) grafts that are prestripped and preloaded into injectors by eye bank technicians before shipping to surgeons. METHODS: DMEK grafts (n = 31) were prepared from donor corneas and preloaded into Straiko Modified Jones tubes and set inside viewing chambers filled with 20 mL of Optisol-GS. Preloaded grafts were evaluated using specular microscopy and slit-lamp biomicroscopy. Endothelial cell loss (ECL) was captured by vital dye staining and quantified using FIJI. A subset of preloaded tissues was subjected to a shipping validation and 5-day storage assay. Fourteen additional DMEK grafts (not preloaded) were examined to quantify damage resulting from prestripping alone. RESULTS: Specular microscopy was able to be performed for all preloaded tissues. Average ECL for preloaded tissues quantified by vital dye staining and FIJI after overnight storage was 16.8% ± 5.9%, and differed from slit-lamp ECL estimation by an average of 5.3% ± 3.6%. The average damage caused by prestripping alone was 9.3% ± 5.9%, and it was significantly less than that of preloaded tissues (P < 0.01). Average ECL for preloaded tissues subjected to round-trip shipping events was 18.5% ± 12.4%, and ECL for tissues stored at 4°C for 5 days after preloading was 13.1% ± 9.5%. CONCLUSIONS: It is possible to prepare, evaluate, and ship DMEK grafts loaded inside a glass carrier and viewing chamber. The ability to evaluate tissues after processing allows for adherence to the Eye Bank Association of America Medical Standards, and for surgeons to receive the most accurate tissue information.

Restoration of Mitochondrial Integrity, Telomere Length, and Sensitivity to Oxidation by In Vitro Culture of Fuchs' Endothelial Corneal Dystrophy Cells.

PURPOSE: Fuchs' endothelial corneal dystrophy (FECD), a degenerative disease of the corneal endothelium that leads to vision loss, is a leading cause of corneal transplantation. The cause of this disease is still unknown, but the implication of oxidative stress is strongly suggested. In this study, we analyzed the impact of FECD on mitochondrial DNA (mtDNA) integrity and telomere length, both of which are affected by the oxidative status of the cell. METHODS: We compared the levels of total mtDNA, mtDNA common deletion (4977 bp), and relative telomere length in the corneal endothelial cells of fresh Descemet's membrane-endothelium explants and cultured cells from healthy and late stage FECD subjects. Oxidant-antioxidant gene expression and sensitivity to ultraviolet A (UVA)- and H2O2-induced cell death were assessed in cultured cells. RESULTS: Our results revealed increased mtDNA levels and telomere shortening in FECD explants. We also found that cell culture restores a normal phenotype in terms of mtDNA levels, telomere length, oxidant-antioxidant gene expression balance, and sensitivity to oxidative stress-induced cell death in the FECD cells compared with the healthy cells. CONCLUSIONS: Taken together, these results bring new evidence of the implication of oxidative stress in FECD. They also show that FECD does not evenly affect the integrity of corneal endothelial cells and that cell culture can rehabilitate the molecular phenotypes related to oxidative stress by selecting the more functional FECD cells.

Engineered Basement Membranes for Regenerating the Corneal Endothelium.

Basement membranes are protein-rich extracellular matrices (ECM) that are essential for epithelial and endothelial tissue structure and function. Aging and disease cause changes in the physical properties and ECM composition of basement membranes, which has spurred research to develop methods to repair and/or regenerate these tissues. An area of critical clinical need is the cornea, where failure of the endothelium leads to stromal edema and vision loss. Here, an engineered basement membrane (EBM) is developed that consists of a dense layer of collagen IV and/or laminin ≈5-10 nm thick, created using surface-initiated assembly, conformally attached to a collagen I film. These EBMs are used to engineer a corneal endothelium (CE) that mimics the structure of Descemet's membrane with a thin stromal layer, toward use as a graft for lamellar keratoplasty. Results show that bovine and human CE cells form confluent monolayers on the EBM, express ZO-1 at the cell-cell borders, and achieve a density of ≈1600 cells mm-2 for 28 and 14 d, respectively. These results demonstrate that the technique is capable of fabricating EBMs with structural and compositional properties that mimic native basement membranes and that EBM may be a suitable carrier for engineering transplant quality CE grafts.

The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet's Membrane Components.

PURPOSE: To clarify the adherent properties of cultured human corneal endothelial cell (cHCEC) subpopulations (SPs). METHODS: Each SP was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell-surface markers. Binding abilities of HCEC SPs were examined by adding the cells to culture plates immobilized with collagens, laminins, or proteoglycans, and then centrifuging the plates. Adhered cells were then evaluated by phase-contrast microscopy. RESULTS: The cHCECs were bound to laminin-511, laminin-411, and Type-IV collagen in a concentration-dependent manner, yet weakly bound to Perlecan, Agrin, and TSP-1. Comparison of the influence of cell-suspension vehicles on cHCEC attachment showed that cells suspended in Opti-MEM-I or Opeguard-MA were bound to laminin, yet no binding was observed in cells suspended in BSS-Plus. Next, we compared the adherent properties of HCEC SPs. Both the fully differentiated, mature cHCEC SP and the epithelial-to-mesenchymal-transitioned (EMT)-phenotype SP were found to attach to laminin- or collagen-coated plates. Interestingly, the binding properties to laminins differed among those SPs. Although the level of cells adhered to the laminin-411-coated plate was the same among the cHCEC SPs, the fully differentiated, mature cHCEC SP was significantly more tightly bound to laminin-511 than was the EMT-phenotype SP. CONCLUSIONS: The findings of this study suggest that the binding ability of cHCECs to major Descemet's membrane components is distinct among cHCEC SPs, and that Opti-MEM-I and Opeguard-MA are useful cell-suspension vehicles for cell-injection therapy.

In Vivo Imaging and Morphometry of the Human Pre-Descemet's Layer and Endothelium With Ultrahigh-Resolution Optical Coherence Tomography.

PURPOSE: To visualize in vivo and quantify the thickness of the posterior corneal layers: the acellular pre-Descemet's layer (PDL), Descemet's membrane (DM), and endothelium (END) in healthy subjects, using ultrahigh-resolution optical coherence tomography (UHR-OCT). METHODS: A research-grade, 800-nm UHR-OCT system with 0.95-µm axial resolution in corneal tissue was used to image in vivo the posterior cornea in healthy subjects. The system offers approximately 98 dB sensitivity for 680 µW optical power incident on the cornea and 34,000 A-scans/s image acquisition rate. This study comprised 20 healthy subjects, aged 20 to 60 years. The thickness of the PDL, DM, and END layers was measured both with a custom, automatic segmentation algorithm and manually. RESULTS: The boundaries and structure of the posterior corneal layers were clearly visible in the UHR-OCT images. The average thickness was measured to be 6.6 ± 1.4 µm (PDL), 10.4 ± 2.9 µm (DM), and 4.8 ± 0.4 µm (END), which agrees well with published data from ex vivo studies. Both the END and DM thickness showed minor spatial variations, whereas the PDL showed up to 2× thickness change for different locations on the same cross-sectional corneal image or over the entire imaged region of the cornea. CONCLUSIONS: Our data indicate that all three layers of the posterior cornea can be clearly visualized in vivo and their thicknesses measured precisely with UHR-OCT. Although the PDL thickness showed large spatial variations, the thickness of the DM and END layers was consistent over the entire imaged region of the cornea.

Preloaded Tissues for Descemet Membrane Endothelial Keratoplasty.

INTRODUCTION: To determine the feasibility of preloading endothelial tissues for Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Laboratory investigation. METHODS: setting: Institutional. PARTICIPANTS: Twenty human donor corneas unsuitable for transplantation with endothelial cell density in a range of 1600-2700 cells/mm(2). INTERVENTION: The endothelium was punched, stripped (8.5 mm diameter) and manually tri-folded with the endothelial side inward. The excised membranes were gently moved in a 2.2 intraocular lens (IOL) cartridge and pulled further in the funnel using 25 G end-grasping forceps. The cartridge was filled with transport media (TM) (sealed at its funnel and back entrance with a stopper) and the tissue was preserved for 4 days at room temperature in the bottles containing TM. MAIN OUTCOME MEASURES: Success rate of preparation, processing time, endothelial cell loss (ECL), and active metabolism. RESULTS: The tissues were peeled and loaded successfully in all cases. Average stripping and loading time was 20 and 4.5 minutes, respectively. ECL after preservation was 4.35% with 3.55% (± 5.89%) mortality and 7.80% (± 14.12%) uncovered areas. A total of 0.55 (± 0.26) mg/mL of glucose was consumed by the cells showing active metabolism. CONCLUSIONS: Tri-folded (endothelium-in) DMEK grafts can be preloaded using TM in an IOL cartridge and stored up to 4 days with limited endothelial damage. Direct injection of TM should be avoided because of the presence of bovine serum, but the tissue can be washed using balanced salt solution and gently injected. Alternatively, the graft can be easily delivered using a bimanual pull-through technique. Preloading DMEK grafts will simplify the surgery with reproducibility, reduced surgical time, and reduced tissue wastage, cost, and logistical requirements.

Transplantation of cultured rhesus monkey vascular endothelial cells to allogeneic cornea concomitant with stripping of Descemet's membrane.

CONTEXT: In cases of damaged corneal endothelium cells (CECs) of the eye, transplantation of cultured vascular endothelial cells (VECs) may be a viable method to restore transparency. AIMS: To evaluate the viability of replacing damaged primate CECs with cultured allogeneic VECs. SUBJECTS AND METHODS: Rhesus monkey VECs (RMVECs) were cultured and proliferating cells were labeled with bromodeoxyuridine (BrdU) in vitro. RMs of the experimental group (n = 6) underwent manual Descemettt membrane stripping with transplantation of RMVECs labeled with BrdU; those in the control group received manual Descemetnt membrane stripping without transplantation. Postoperative evaluations included the transparency and appearance of the corneal graft; distribution and ultrastructural changes of RMVECs on the inner surface of the cornea using scanning and transmission electron microscopy, and immunohistological identification of BrdU. RESULTS: At 90 days postsurgery, the corneal grafts of the monkeys in the experimental group retained better transparency than those of the controls, without corneal neovascularization or bullous keratopathy. A layer of cells with positive BrdU staining was found on the posterior surface of the treated corneas in the experimental group, while there was no VEC structure in corneal grafts from the monkeys of the control group. CONCLUSIONS: RMVECs can grow on the posterior surface of the cornea without Descemet's membrane. Cultured and transplanted RMVECs appeared similar in ultrastructure. VECs can provide a barrier to maintain corneal dehydration and transparency to some extent.

Distrofia polimorfa posterior, presentación de un caso y análisis de la literatura / Posterior polymorphous dystrophy, case report and literature review

CASO CLÍNICO: La distrofia polimorfa posterior (DPP) es una distrofia corneal rara de transmisión autosómica dominante. Las estructuras corneales afectadas en esta distrofia son la membrana de Descemet y el endotelio. Se presenta el caso clínico de una mujer de 47 años sin antecedentes de importancia, con hallazgos típicos de DPP (lesiones vesiculares y en banda a nivel del endotelio y Descemet posterior). DISCUSIÓN: Encontramos que las manifestaciones clínicas en nuestra paciente son muy similares a los casos reportados en otras poblaciones. La ausencia de antecedentes heredofamiliares no descarta el diagnóstico de DPP puesto que esta enfermedad generalmente cursa asintomática

CASE REPORT: Posterior Polymorphous Dystrophy (DPP) is a rare posterior corneal dystrophy that is genetically transmitted as autosomal dominant. Corneal structures affected in this dystrophy are Descemet membrane and the endothelium. A case is presented on a 47 years old woman with no relevant history, with typical findings of DPP (vesicular and band lesions at the endothelium and posterior Descemet). DISCUSSION: To our knowledge there are no reported cases of DPP in Latin-American patients in the literature. The clinical manifestations in our patient were found to be very similar to the cases reported in other populations

On the Cornea of Healthy Merino Sheep: A Detailed Ex Vivo Confocal, Histological and Ultrastructural Study.

Our study performed qualitative and quantitative studies on the corneal ultrastructure of healthy female Merino sheep of ages 4 months and 6 years old from the Argentinean Pampa. The corneas were evaluated using ex vivo laser-scanning confocal microscopy, light microscopy and transmission electron microscopy. Those studies allowed us to obtain detailed images of the corneal layers as well as quantitative data of the cellular and sub-basal nerve densities in the cornea from sheep of different ages. The density of the corneal cells was significantly different in the anterior versus the posterior epithelium and stroma. Moreover, the density of the epithelial, stromal cells and endothelial cells, as well as the sub-basal nerve density were significantly lower in adult than in young animals. Our work provided a wide-ranging description of the corneal ultrastructure of healthy female Merino sheep, which adds to the current knowledge about the ophthalmological aspects of this species and undoubtedly benefits veterinarians.