A comparison of the ultrastructure of the cornea of the pre- and post-metamorphic axolotl (Ambystoma mexicanum, Amphibia).

The corneal ultrastructure of the pre- and post-metamorphic stages of the neotenic axolotl Ambystoma mexicanum is examined using light microscopy and both scanning and transmission electron microscopy to reveal whether there are any morphological changes associated with a switch in lifestyle. Although the complement of corneal layers remains the same, there are significant quantitative changes in corneal, epithelial and stromal thickness, epithelial and endothelial cell size and density, and the thickness of Bowman's layer and Desçemet's membrane. Microholes in the epithelium and vertical sutures within the stroma are predominant features in the pre-metamorphic stage but are rarely seen in the post-metamorphic stage. There are also significant quantitative centro-peripheral differences in the thickness of the whole cornea, primarily due to differences in the thickness of the stroma in both metamorphic stages. These changes may reflect the physiological demands on the cornea as it switches from a purely aquatic to an amphibious lifestyle, which includes venturing onto land.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

In vivo and in vitro results of an automated preloaded delivery system for IOL implantation in cataract surgery.

PURPOSE: To compare the corneal tissue trauma after the use of an automated preloaded injector and a manual injector and assess scanning electron microscope (SEM) and atomic force microscope (AFM) features of both injector cartridges. SETTING: Ophthalmology Clinic and Laboratory of Stem Cells and Regenerative Medicine University "G. d'Annunzio" of Chieti-Pescara, Chieti, Italy; DESIGN: Prospective randomized clinical study METHODS: Forty eyes of 40 patients for phacoemulsification were divided into two groups: implantation of intraocular lens was performed with AutonoMe automated delivery system (AutonoMe group: 20 eyes) and Monarch III injector system (Monarch group: 20 eyes). In vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) were performed before surgery, at 1 h, 1 day and 1 month post-operatively. In addition, SEM and AFM were performed on cartridges of both injector systems after injection of the IOL. RESULTS: A greater increase in central corneal thickness and corneal thickness at the incision site were observed in Monarch group versus AutonoMe group 1 h and 1 day post-operatively (p < 0.05). Endothelial cell count loss was significantly higher in Monarch group compared with AutonoMe group (p < 0.05) at 1 and 24 h. AS-OCT showed less endothelial misalignment at 30 days (p < 0.05), and IVCM showed less tunnel inflammation at all time points (p < 0.05) in AutonoMe group compared with Monarch group; roughness analysis at AFM of the AutonoMe cartridge was significantly lower compared to Monarch D cartridge (p < 0.05). CONCLUSIONS: The AutonoMe injector provided less corneal tissue trauma compared with Monarch III injector. The AutonoMe cartridge showed lower roughness at AFM compared to the Monarch D cartridge.

Physiological pressure enhances the formation of tight junctions in engineered and native corneal endothelium.

Cells and tissues are influenced by environmental conditions. In vivo, the corneal endothelium is subjected to hydrostatic intraocular pressure (IOP) and to the hydrokinetic pressure of the moving aqueous humor in the anterior chamber. In this paper, we used a corneal bioreactor to recreate the IOP condition and investigated the effect of the in vivo hydrodynamic environment of corneal endothelial cells on the formation of tight junctions. Native ex vivo corneas and engineered corneal endothelia subjected to pressure showed an increase in ZO-1 expression at the cell periphery. Pressure also improved the corneal transparency of engineered and native corneas. Corneal thickness was accordingly reduced from 926 ±â€¯70 µm to 651 ±â€¯70 µm for the engineered corneal endothelium and from 847 ±â€¯27 µm to 571 ±â€¯23 µm for the native endothelium. These results suggest that the hydrodynamic pressure of the anterior chamber is important for the cell junction integrity of the corneal endothelium.

3D in vitro model for human corneal endothelial cell maturation.

Corneal endothelium is a cellular monolayer positioned on the Descemet's membrane at the anterior cornea, and it plays a critical role in maintaining corneal clarity. Our present study examines the feasibility of utilizing our 3-dimensional (3D) corneal stromal construct, which consists of human corneal fibroblasts (HCF) and their self-assembled matrix, to observe the development and maturation of human corneal endothelial cells (HCEndoCs) in a co-culture model. Three-dimensional HCF constructs were created by growing the HCFs on Transwell membranes in Eagles' minimum essential medium (EMEM) + 10% FBS + 0.5 mM Vitamin C (VitC) for about 4 weeks. HCEndoCs, either primary (pHCEndoC) or cell line (HCEndoCL), were either seeded in chamber slides, directly on the Transwell membranes, or on the 3D HCF constructs and cultivated for 5 days or 2 weeks. The HCEndoCs that were seeded directly on the Transwell membranes were exposed indirectly to HCF by culturing the HCF on the plate beneath the membrane. Cultures were examined for morphology and ultrastructure using light and transmission electron microscopy (TEM). In addition, indirect-immunofluorescence microscopy (IF) was used to examine tight junction formation (ZO-1), maturation (ALDH1A1), basement membrane formation (Laminin), cell proliferation (Ki67), cell death (caspase-3), and fibrotic response (CTGF). As expected, both pHCEndoCs and HCEndoCLs formed monolayers on the constructs; however, the morphology of the HCEndoCLs appeared to be similar to that seen in vivo, uniform and closely packed, whereas the pHCEndoCs remained elongated. The IF data showed that laminin localization was present in the HCEndoCs' cytoplasm as cell-cell contact increased, and when they were grown in the 3D co-culture, the beginnings of what appears to be a continuous DM-like structure was observed. In addition, in co-cultures, ALDH1A1-positive HCEndoCs were present, ZO-1 expression localized within the tight junctions, minimal numbers of HCEndoCs were Ki67-or Caspase-3-positive, and CTGF was positive in both the HCEndoCs cytoplasm and the matrix of the co-culture. Also, laminin localization was stimulated in HCEndoCs upon indirect stimuli secreted by HCF. The present data suggests our 3D co-culture model is useful for studying corneal endothelium maturation in vitro since the co-culture promotes new DM-like formation, HCEndoCs develop in vivo-like characteristics, and the fibrotic response is activated. Our current findings are applicable to understanding the implications of corneal endothelial injection therapy, such as if the abnormal DM has to be removed from the patient, the newly injected endothelial cells will seed onto the wound area and deposit a new DM-like membrane. However, caution should be observed and as much of the normal DM should be left intact since removal of the DM can cause a posterior stromal fibrotic response.

Morphological analysis of corneal findings modifications after death: A preliminary OCT study on an animal model.

The aim of this work was to describe, for the first time, the morphological modifications, in a three-dimensional mode, of the central cornea at different intervals since death. The study design involved the analysis of 30 eyes (15 heads) of female, adult sheep (>2 years) sacrificed at a local slaughterhouse. The eyes, after animal decapitation, were examined in situ, without enucleation. Ocular globes were stored at well-known temperature (within a range of 12-22 °C) and humidity (within a range of 50-60%). The instrumental analysis was executed using a portable spectral-domain OCT (SD-OCT) system (iVue SD-OCT, Optovue Inc, Fremont, CA) calibrated to the corneal mode. OCT imaging was performed at different time-points since death. Pachymetric map, morphological and ultrastructural analysis (epithelium, stroma, and endothelium), were performed for each time-point. After an initial thinning of tissues and an enhancement of epithelial reflectivity, stromal thickness increased from the 2nd up to the 6th hour. Subsequently, a new trend incorneal thinning was observed in association with the appearance ofone or more demarcation lines between the anterior andposterior stroma. After the 12th hour, a recurrence of corneal swelling was detected in association with thedelamination of stromal tissue. Since the 24th hour, the epithelium disappeared in 50% of cases and the anterior chamberdepth progressively decreased. At the 48th hour, various ocular structures showed the onset of putrefaction processes, such as theappearance of hyper-reflective dots in anterior chamber, iridocorneal contact, and the massive vacuolization of theposterior stroma until the total delamination. The portable OCT system is a useful approach for in situ postmortem corneal examination, and it may be potentially applied for the selection of donor cornea in transplantology and for the determination of post-mortem intervals in forensic medicine.

Establishment of a porcine corneal endothelial organ culture model for research purposes.

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas-displaying endothelial cell death rates comparable to those of cultured human corneas-would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called "split corneal buttons" (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.

Two-photon optical microscopy imaging of endothelial keratoplasty grafts.

PURPOSE: To investigate the microstructure of endothelial keratoplasty grafts using two-photon optical microscopy. METHODS: Six endothelial keratoplasty grafts obtained from human donor corneoscleral tissues and prepared by submerged hydrodissection technique were imaged by two-photon optical microscopy. In each graft, two liquid bubbles were created in order to investigate the presence of a conserved cleavage plane regardless of the volume of posterior stroma that remained attached to Descemet's membrane (DM); the first bubble (bubble A) was generated under DM and the second bubble (bubble B) injection was done in order to obtain a layer of deep stroma that kept the two bubbles separated. Six human donor corneoscleral tissues were used as controls. Second harmonic generation and two-photon emitted fluorescence signals were collected from each specimen. RESULTS: Dissection of stroma occurred along the posterior collagen lamellae at variable distance from DM, which ranged between 3 and 16 µm in bubble A and between 23 and 41 µm in bubble B. The residual stroma included, anteriorly, bands of collagen lamellae, and thin bundles of stromal collagen fibrils, posteriorly, which were tightly intertwining with the underlying DM. There was no anatomically distinct plane of separation between these pre-Descemetic stromal collagen bundles and the overlying collagen lamellae with this hydrodissection technique. CONCLUSIONS: Two-photon optical microscopy provided label-free high-resolution imaging of endothelial keratoplasty grafts, showing that the most posterior stroma changes organization at approximately 10 µm above the DM. The pre-Descemetic stromal collagen fibrils form an intertwined complex with DM, which cannot be separated using hydrodissection.

Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses.

PURPOSE: Some forms of keratoplasty assisted by ultrashort-pulse lasers require performing laser cuts close to the endothelium, which requires the knowledge of "safe" values concerning incision depth and pulse energy preserving endothelial cell viability. Our study aims to determine the thresholds for cell death in porcine corneas exposed to ultrashort laser pulses, in terms of laser pulse energy and nearness of the impacts to the endothelium. METHODS: Using a laboratory laser set-up, lamellar cuts were induced while varying pulse energies and distances from the endothelium. A fluorescent staining protocol was used to determine the percentage of surviving endothelial cells. Numerical simulations of the Euler equations for compressible fluids provided pressure level and axial and radial pressure gradient estimates at the endothelium. RESULTS: Ninety percent of the endothelial cells survived when using 16.5 µJ pulses no closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm. The comparison of the observed percentage of surviving cells with the estimates of the shock wave amplitudes and gradients generated by the laser pulses yielded cell death thresholds at amplitudes in the megapascal range, or gradients of the order of 108 Pa/m. CONCLUSIONS: Our results provide limits in terms of pulse energy and distance of the incision from the endothelium within which endothelial cell viability is preserved. Current forms of corneal laser surgery are compatible with these limits. However, these limits will need to be considered for the development of future laser routines working in close proximity to the endothelium.

Morphometry of organ cultured corneal endothelium using Voronoi segmentation.

The endothelial viability of eye-banked corneas must be assessed before transplantation, yet phase-contrast images of the endothelium of corneas stored in an Organ Culture system have a huge variety of endothelial appearance at different focal planes, due to stromal swelling and an evaluation technique that swells the cells and their intercellular spaces to make them visible. This makes them difficult to assess using common edge-based segmentation methodologies that have mostly been developed in vivo using specular microscopy. Additionally, as the endothelial cell architecture has radically different form at apical and basal poles, segmentation of only the apical view cannot describe the cell as a whole. Using custom software, this study instead defines the cell borders using a calibrated Voronoi diagram, a region-based image segmentation technique that uses a point cloud of cell centroids as the only input required to measure the size (polymegathism) and shape (pleomorphism) of the endothelial cells. Measurements included the range and variation in the cell area and density, the number of cell sides, and several novel shape descriptors. A dataset of endothelial cell measurements was compiled from 2000 images of 678 corneas comprising 354,998 cells, and validated by comparison to previous studies from a variety of tissue sources, imaging modalities, and analysis techniques. While there were some differences in cell size variation and pleomorphic composition than seen in some other studies, much of the data was directly comparable, demonstrating fast and accurate endothelial cell morphometry at a large scale for a wide range of routine organ cultured corneas.

Characterization of a corneal endothelium engineered on a self-assembled stromal substitute.

Endothelial dysfunctions are the first indication for allogeneic corneal transplantation. Development of a tissue-engineered posterior cornea could be an alternative to the use of native allogeneic tissues. In this paper, we used the self-assembly approach to form a cellularized stromal substitute that served as a carrier for the engineering of an endothelium. This endothelialized stromal substitute was then characterized using alizarin red staining, histology, scanning and transmission electron microscopy, as well as mass spectrometry and immunodetection of collagens and function-related proteins. We report the engineering of a monolayer of flattened endothelial cells with a cell density of 966 ± 242 cells/mm(2) (mean ± SD). Endothelial interdigitations were present between cells. The stromal fibroblasts deposited a dense and cohesive collagenous matrix. Collagen fibrils had a diameter of 39.1 ± 11.3 nm, and a mean center to center interfibrillar space of 50.9 ± 10.9 nm. The stromal substitute was composed of collagen types I, V, VI and XII, as well as lumican and decorin. Type IV collagen was also present underneath the endothelium. The endothelium expressed both the sodium/potassium (Na(+)/K(-)) ATPase and sodium/bicarbonate (Na(+)/ [Formula: see text] ) cotransporter pumps. These results indicate that the self-assembled stromal substitute is able to support the expression of endothelial cell functionality markers and therefore, is a suitable carrier for the engineering of an endothelium that could be used for the treatment of endothelial dysfunctions.

Dose dependent cytotoxicity of pranoprofen in cultured human corneal endothelial cells by inducing apoptosis.

Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.

Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.

Contact inhibition ubiquitously exists in non-transformed cells that are in contact with neighboring cells. This phenomenon explains the poor regenerative capacity of in vivo human corneal endothelial cells during aging, injury and surgery. This study demonstrated that the conventional approach of expanding human corneal endothelial cells by disrupting contact inhibition with EDTA followed by bFGF activated canonical Wnt signaling and lost the normal phenotype to endothelial-mesenchymal transition, especially if TGFß1 was added. By contrast, siRNA against p120 catenin (CTNND1) also uniquely promoted proliferation of the endothelial cells by activating trafficking of p120 catenin to the nucleus, thus relieving repression by nuclear Kaiso. This nuclear p120-catenin-Kaiso signaling is associated with activation of RhoA-ROCK signaling, destabilization of microtubules and inhibition of Hippo signaling, but not with activation of Wnt-ß-catenin signaling. Consequently, proliferating human corneal endothelial cells maintained a hexagonal shape, with junctional expression of N-cadherin, ZO-1 and Na(+)/K(+)-ATPase. Further expansion of human corneal endothelial monolayers with a normal phenotype and a higher density was possible by prolonging treatment with p120 catenin siRNA followed by its withdrawal. This new strategy of perturbing contact inhibition by selective activation of p120-catenin-Kaiso signaling without disrupting adherent junction could be used to engineer surgical grafts containing normal human corneal endothelial cells to meet a global corneal shortage and for endothelial keratoplasties.

Primary cilia dynamics instruct tissue patterning and repair of corneal endothelium.

Primary cilia are required for several signaling pathways, but their function in cellular morphogenesis is poorly understood. Here we show that emergence of an hexagonal cellular pattern during development of the corneal endothelium (CE), a monolayer of neural crest-derived cells that maintains corneal transparency, depends on a precise temporal control of assembly of primary cilia that subsequently disassemble in adult corneal endothelial cells (CECs). However, cilia reassembly occurs rapidly in response to an in vivo mechanical injury and precedes basal body polarization and cellular elongation in mature CECs neighboring the wound. In contrast, CE from hypomorphic IFT88 mutants (Tg737(orpk)) or following in vivo lentiviral-mediated IFT88 knockdown display dysfunctional cilia and show disorganized patterning, mislocalization of junctional markers, and accumulation of cytoplasmic acetylated tubulin. Our results indicate an active role of cilia in orchestrating coordinated morphogenesis of CECs during development and repair and define the murine CE as a powerful in vivo system to study ciliary-based cellular dynamics.

Histology, histochemistry and ultrastructure of cornea of domestic pigs (Sus scrofa domesticus).

A comprehensive light and ultrastructural examination of the cornea in Domestic Pigs (Sus scrofa domesticus) revealed four distinct layers: the anterior epithelium, corneal stroma, Descemet's membrane and endothelium. Although Bowman's layer was not distinctly identified through histology, histochemical analysis indicated the presence of a rudimentary Bowman's layer, possibly vestigial from evolution. Scanning electron microscopy of the outer corneal surface unveiled two cell types, characterized by micro-projections, with light cells exhibiting shorter, thicker projections compared to dark cells. Examination of the inner surface via scanning electron microscopy demonstrated an endothelial layer devoid of cilia and microvilli, yet faint round to oval elevations were observed, potentially representing cell nuclei. Transmission electron microscopy unveiled that basal cells of the anterior epithelium closely adhered to the basement membrane, featuring half desmosomes along the basal surface. These basal cells extensively interconnected through interdigitations and a few desmosomes. The superficial cell layer consisted of a few rows of closely attached flat cells, forming a leak-proof layer with zona occludens. The outermost cells of this layer displayed fine projections to enhance the surface area, facilitating tear film distribution. At lower magnification, Transmission electron microscopy of the corneal stroma revealed alternating light and dark bands, with light bands representing transverse sections of collagen fibril lamellae and dark bands corresponding to longitudinal or oblique sections. Spindle-shaped keratocytes (fibroblasts) were identified as the primary stromal cells, intermingled between the lamellae, and featured long processes in close contact with neighbouring keratocytes. Overall, the histomorphology of the pig cornea resembles that of the human cornea except indistinct Bowman's membrane. This detailed understanding of the normal corneal structure in pigs hold great significance for biomedical research, providing a valuable reference for studies involving this animal model.

A mouse model of corneal endothelial decompensation using cryoinjury.

PURPOSE: To develop a mouse model of bullous keratoplasty and evaluate the safety and efficacy of cryoinjury-induced corneal endothelial decompensation. METHODS: Transcorneal freezing was performed on the right eye of each mouse. One cycle of cryoinjury was performed in 18 eyes (group A), and three cycles were performed in 17 eyes (group B). Pachymetry and intraocular pressure (IOP) measurements were done preoperatively, as well as at 1, 3, 7, 14, and 21 days after cryoinjury. At each post-cryoinjury time point, three mice from each group were euthanized, and the corneas underwent histology and electron microscopy. RESULTS: In both groups, significant corneal edema was noted at post-cryoinjury day 1, which was maintained throughout the study period. IOP remained within normal range in group A, but increased significantly with time in group B (p=0.011 at day 1, 0.038 at day 3, 0.026 at day 14, and 0.008 at day 21). In group B, serious complications including hyphema (one case), severe iridocorneal adhesion (15 cases), and total cataract (three cases) were detected, while only mild iridocorneal adhesion (four cases) and cataract (three cases) were noted in group A. Live/dead cell assay, hematoxylin and eosin staining, and scanning electron microscopy revealed successful ablation of corneal endothelial cells and absence of regeneration in both groups. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assay showed that apoptosis was mainly confined to the posterior stroma and endothelium in group A, while severe apoptosis was observed throughout all layers of the cornea in group B. CONCLUSIONS: One cycle of cryoinjury was safer than three, while both were equally effective in inducing bullous keratopathy. This cryoinjury mouse model of bullous keratopathy was a consistently reproducible model that can be used for further studies on endothelial cell damage and rescue therapy.

Reproducibility of graft preparations in Descemet's membrane endothelial keratoplasty.

PURPOSE: To assess the reproducibility of manual graft preparation and evaluate the incidence rate and nature of structural anomalies of Descemet's membrane (DM) preventing successful graft preparation in DM endothelial keratoplasty (DMEK). DESIGN: Prospective, single-center, nonrandomized, consecutive case series. PARTICIPANTS: We analyzed 350 corneoscleral buttons from donors aged 18-95 years stored in Optisol-GS or Dulbecco's modified Eagle's medium and used for DMEK surgery in 343 consecutive patients with Fuchs' endothelial dystrophy or pseudophakic bullous keratopathy. METHODS: Residual endothelial cell-DM complexes obtained after successful DM stripping for DMEK and whole donor corneas obtained after unsuccessful DM stripping were examined by transmission electron microscopy and immunohistochemistry. MAIN OUTCOME MEASURES: Accuracy of the cleavage plane between DM and corneal stroma and structural abnormalities of the DM-stroma interface. RESULTS: Uneventful manual separation without any disruption of DM was achieved in 335 of 350 donor corneas (95.7%) by use of a previously established bimanual submerged preparation technique. Correspondingly, the peeled DM specimens revealed a regular and smooth cleavage plane exposing the amorphous interfacial matrix on their anterior surface. Although 8 of 350 donor corneas (2.3%) showed focal adhesions of DM to the corneal stroma and developed isolated tears during stripping, preparation of the graft could be successfully completed. However, 7 of the 350 donor corneas (2.0%) showed extremely strong adhesion and multiple tears of DM, preventing successful preparation of the graft. These specimens revealed either ultrastructural (peg-like interlockings) or biochemical abnormalities (increased staining intensities for adhesive glycoproteins) along the DM-stroma interface. CONCLUSIONS: Using an appropriate technique, manual preparation of grafts for DMEK with reproducible tissue qualities is possible in the vast majority (98%) of donor corneas. Although a relatively rare phenomenon, interindividual variations in DM structure and composition may be responsible for failure of graft preparation in about 2% of donor corneas. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Visual outcome and histological findings following femtosecond laser-assisted versus microkeratome-assisted DSAEK.

BACKGROUND: The aim of this study was to compare the visual outcome of femtosecond laser-assisted Descemet stripping automated endothelial keratoplasty (DSAEK) to microkeratome-assisted DSAEK as well as to contrast precut versus surgeon-cut grafts. Histologic characterization of failed DSAEK grafts was performed in order to correlate ultrastructural changes with graft failures. METHODS: In this case control study, 47 cases of DSAEK were investigated in terms of visual acuity, keratometric astigmatism, spherical equivalent, endothelial cell count, and postoperative complications. We formed three groups: the femtosecondlaser-assisted DSAEK with precut grafts, the microkeratome-assisted DSAEK with precut and with surgeon-cut grafts. Mean follow-up was 6 months. In the case of graft failure, penetrating keratoplasty was performed, and the excised corneal buttons were investigated by light and electron microscopy. RESULTS: Microkeratome-assisted DSAEK lead to better visual outcome than femtosecond laser-assisted DSAEK. Keratometric astigmatism, spherical equivalent and endothelial cell count did not differ significantly between both methods. Precut and surgeon-cut grafts in microkeratome-assisted DSAEK did not show any significant difference regarding all upraised parameters. No definite histological correlate for graft failure following femtosecond laser-assisted DSAEK was found. CONCLUSIONS: Femtosecond laser-assisted DSAEK is not the method of choice, and needs further technical improvement. However, failed femtosecondlaser-assisted DSAEK grafts did not show significant histological changes related to the technique to explain reduced visual acuity. In microkeratome-assisted DSAEK, the preparation time point of the graft does not seem to influence the visual and optical outcome.

c.-61G>A in OVOL2 is a Pathogenic 5' Untranslated Region Variant Causing Posterior Polymorphous Corneal Dystrophy 1.

PURPOSE: The purpose of this study was to investigate the clinical and genetic features of a man and his daughter with posterior polymorphous corneal dystrophy (PPCD), referred to our clinic for Descemet membrane endothelial keratoplasty. No other known relatives were affected. METHODS: Ophthalmic examination and histology, including electron microscopy, were performed. Genetic testing was conducted by means of whole exome sequencing, and variant analysis was achieved by using an internal in silico pipeline. Molecular tests included a dual-luciferase assay. RESULTS: Slowly progressive blurred vision was reported from childhood by the daughter. The father's symptoms started at age 55. Best-corrected visual acuity was reduced in both patients (0.2-0.4). Slit-lamp examination in both patients revealed bilateral corneal clouding with gray endothelial lesions; other family members had no ophthalmological signs. Descemet membrane endothelial keratoplasty was performed uneventfully in both patients. Histology showed thickened Descemet membrane and abnormal endothelium resembling epithelial-like cells. Both patients carried the OVOL2 5' untranslated region NM_021220.4.c.-61G>A variant in the heterozygous state. This change was associated with increased promoter activity and was not present in the unaffected members of the family. CONCLUSIONS: The 5' untranslated region mutation c.-61G>A in OVOL2 has been previously found in 1 individual with PPCD1 and reported as a variant of unknown significance because of insufficient evidence supporting its pathogenicity. Identification of the second family with 2 individuals affected by PPCD1 carrying this change, together with functional data, provides further proofs that it is disease-causing.