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.

Cleavage plane after liquid-bubble preparation of Descemet's membrane.

PURPOSE: To investigate the ultrastructure of the cleavage plane of human cornea after liquid-bubble-prepared tissue for Descemet's membrane endothelial keratoplasty (DMEK). METHODS: Experimental study with scanning electron microscopy (SEM) and block-face SEM of 18 corneal specimens. Fresh human research donor corneoscleral discs (n = 12) were prepared with liquid-bubble technique or examined as untreated controls (n = 3). In addition, Descemet's membrane samples, n = 3, were obtained in DMEK surgery. RESULTS: The cleavage plane after liquid-bubble Descemet's membrane (DM) preparation was consistently located between interfacial matrix and posterior stromal collagen lamellae, providing a largely smooth surface exposing the amorphous interfacial zone without any significant amounts of adherent stromal remnants. No demarcation of a distinct pre-DM layer could be detected. CONCLUSION: The DMEK graft preparation performed by liquid-bubble technique showed a smooth cleavage plane and could not reveal any demarcation of a distinct pre-DM layer.

Collagen Remodeling Plays a Pivotal Role in Endothelial Corneal Dystrophies.

Purpose: To elucidate the collagen structure in the Descemet membrane (DM) of the human cornea and to characterize its rearrangement in patients with endothelial corneal dystrophies. Methods: Corneas from nine human donors and dystrophic DMs removed from 16 affected eyes of 13 patients by endothelial keratoplasty (DMEK) were investigated using a correlative RT-qPCR and label-free two-channel multiphoton microscopy (MPM) setup. Although collagen formation was visualized by second harmonic generation, the cellular structure was determined by autofluorescence. Results: The DM of the human donor cornea was characterized by a consistent pattern of fine hexagonal collagen structures that form a supportive scaffold for the endothelial cells. Accordingly, network-forming collagens (8A1 and 8A2) but less fibrillar collagens (only 1A2) were expressed. DMEK resulted in significant (P < 0.0001) improvement of best-corrected visual acuity. In the removed dystrophic DMs, MPM analyses revealed collagen rearrangement in addition to loss of endothelial cells and the development of guttae. MPM analyses of the whole patient's DM demonstrated this collagen remodeling in its entirety and facilitated correlation to Scheimpflug corneal tomography. In most DMs a unique honeycomb collagen network was identified, with distinct bundles surrounding the guttae and correlating with expression of fibrillar collagens (1A1). Conversely, some DMs showed either reduced collagen on MPM and RT-qPCR analysis or diffuse thickening and storage of extracellular matrix. Conclusions: The collagen structure of the DM and its adaptive remodeling in endothelial corneal dystrophies has been characterized for the first time here and will facilitate individual therapeutic approaches.

Ex vivo excimer laser ablation of cornea guttata and ROCK inhibitor-aided endothelial recolonization of ablated central cornea.

PURPOSE: To determine whether excimer laser ablation of guttae is a viable strategy for removal of diseased tissue in Fuchs' endothelial corneal dystrophy (FECD) on excised human Descemet membranes and whether an excimer laser-created wound on healthy human corneas ex vivo is recolonized with corneal endothelial cells. METHODS: Descemet membranes of FECD patients and corneal endothelium of normal human corneas were ablated ex vivo using an excimer laser licensed for glaucoma surgery. Specimens were kept in cell culture medium supplemented with 10 µm of rho-kinase inhibitor ripasudil. Corneal endothelial cell regeneration was observed using light and electron scanning microscopy. Furthermore, the whole corneal samples were evaluated by haematoxylin/eosin staining and immunohistochemical analysis using antibodies against Na+ /K+ -ATPase. RESULTS: Guttae and corneal endothelium could be ablated with an excimer laser without total ultrastructural damage to the Descemet membrane or stroma. Nearly complete endothelial wound closure was accomplished after 26-38 days in treated corneas. Light and electron scanning microscopy suggested the establishment of a layer of flat endothelial cells. Additionally, Na+ /K+ -ATPase expression could only be observed on the inner side of the Descemet membrane. CONCLUSION: Our proof of concept study demonstrated that excimer lasers can be used to ablate diseased tissue from excised FECD Descemet membranes ex vivo. Additionally, corneal endothelial cells recolonize a previously ablated endothelial area in healthy human corneas ex vivo under treatment with ripasudil. Thus, our results are the first experimental basis to further investigate the feasibility of an excimer laser ablation as a graftless FECD treatment option.

Ultrastructural findings in graft failure after Descemet membrane endothelial keratoplasty (DMEK) and new triple procedure.

To investigate factors that influence graft failure after Descemet membrane endothelial keratoplasty (DMEK) based on transmission electron microscopy results.Retrospective observational case series.This single center study included 16 eyes of 16 patients with penetrating keratoplasty (n = 14) or repeat DMEK (n = 2) following graft failure after DMEK. The main outcome measures were ultrastructural changes in the explanted graft on transmission electron microscopy, best-corrected visual acuity, and central corneal thickness.The mean preoperative and postoperative best-corrected visual acuity was 1.01 ±â€Š0.54 logMAR and 0.56 ±â€Š0.37 logMAR. The mean central corneal preoperative and postoperative thickness was 667 ±â€Š187 µm and 511 ±â€Š42 µm. Visual acuity and central corneal thickness improved significantly (P = .001/P = .003) after repeat surgery. Electron microscopy showed that 3 of 14 corneas showed upside down transplantation, and 3 corneas had pigmented cells or pigment granules at the Descemet-stroma interface. Further, 9 of 16 specimens showed a posterior collagenous layer deposited onto the Descemet membrane (average thickness 5.1 ±â€Š6.2 µm; ranged 0.65-20 µm); this did not correlate significantly with the time between the original and repeat keratoplasty. Of 16 original grafts, 7 showed ultrastructural anomalies of the Descemet membrane, but one excised cornea showed no Descemet membrane pathologies.The majority of eyes with graft failure after DMEK showed ultrastructural changes in the Descemet membrane. It is crucial to assess donor tissue quality and to conduct graft marking before surgery to avoid immediate or delayed graft failure after DMEK. Nevertheless, repeat keratoplasty provided significant improvement in central corneal thickness and visual acuity.

Descemet's Membrane Biomimetic Microtopography Differentiates Human Mesenchymal Stem Cells Into Corneal Endothelial-Like Cells.

PURPOSE: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell-based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane-like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells. METHODS: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level. RESULTS: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins. CONCLUSIONS: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

Aspectos qualitativos das rupturas e afinamentos da membrana de Descemet no exame histopatológico / Qualitative aspects of ruptures and thinnings of Descemet's membrane in histopathological examination

Resumo Objetivo: Descrever os diferentes tipos de rupturas e afinamentos da membrana de Descemet encontrados no exame histopatológico, questionando uma possível relação entre eles. Métodos: Estudo observacional, transversal, retrospectivo e descritivo de botões corneanos provenientes de ceratoplastia penetrante, durante o período escolhido de forma aleatória de quatro anos: 2006, 2010, 2014 e 2015. A coloração foi realizada com Hematoxilina-eosina (HE). Após o preparo, os tecidos foram examinados com microscópio óptico pelos autores. Selecionamos apenas os casos de rupturas ou afinamento da membrana de Descemet no exame histopatológico, e classificamos os diferentes tipos desses achados. Resultados: As rupturas encontradas foram classificadas em total ou comum, parcial, fratura e bisel. Os afinamentos foram divididos em generalizado, localizado e extensivo. Conclusão: Apresentamos várias nuances das rupturas e dos afinamentos da membrana de Descemet no exame histopatológico. Os achados sugerem, considerando apenas aspectos mecânicos, uma possível relação entre afinamento e ruptura como causa e efeito.

Abstract Objective: To describe the different types of ruptures and thinning of Descemet's membrane found in the histopathological examination, questioning a possible relationship between them. Methods: Observational, transversal, retrospective and descriptive study of corneal buttons from penetrating keratoplasty during the randomly chosen period of four years: 2006, 2010, 2014 and 2015. The staining was performed with hematoxylin-eosin (HE). After preparation, the authors examined the tissues with an optical microscope. We selected only the cases of rupture or thinning of Descemet's membrane in histopathological examination and classified the different types of these findings. Results: The ruptures found were classified as total or common, partial, fracture and bevel. The thinnings were divided into generalized, localized and extensive. Conclusion: We presented several nuances of Descemet's membrane ruptures and thinning in histopathological examination. Considering only mechanical aspects, the findings suggest a possible relationship between thinning and rupture as cause and effect.

Effectiveness of curvilinear approach in dissection of Descemet's membrane: first 500 cases - factors influencing graft preparation.

PURPOSE: To confirm the reproducibility of manual graft preparation using curvilinear forceps and evaluate the incidence and type of structural abnormalities of Descemet's membrane (DM) preventing successful grafts preparation. METHODS: Five hundred corneo-scleral buttons were prepared. Factors such as endothelial cell number before preparation, donor age, post-mortem time, time in culture, pigmentation of the trabecular meshwork and preparation characteristics of the fellow eye were analysed. According to the preparation characteristics, three groups were formed: A, uncomplicated; B, complicated preparation with stripping from the contralateral side; and C, failure of preparation. Three failed grafts were examined by transmission electron microscopy (TEM). RESULTS: Using curvilinear forceps, manual separation of DM was achieved without any adverse effects in 457 of 500 corneas (91.4%). In 32 corneas (6.4%) with micro-tears during preparation, stripping from the opposite side was possible. However, 11 of the 500 corneas (2.2%) showed extremely strong adhesion leading to multiple tears of DM and preventing successful preparation of the graft. Endothelial cell number, donor age, post-mortem time, time in culture and pigmentation of the trabecular meshwork showed no significant correlation with failure to successfully obtain a DM graft. Complicated graft preparations of one eye showed a highly significant correlation with complicated graft preparations in the fellow eye. TEM analysis of failed grafts showed abnormal invasion of stromal parts into the DM, cell accumulation and pigmentation in the DM plane. CONCLUSION: Using curvilinear forceps for dissecting of the graft shows valid and reproducible results in the vast majority (97.8%) of donor corneas.

In vivo confocal microstructural analysis of corneas presenting Kayser-Fleischer rings in patients with Wilson's disease.

PURPOSE: To evaluate microstructural differences between corneas with and without Kayser-Fleischer rings in age-matched subjects with Wilson's disease with neurological symptoms, using confocal laser scanning microscopy. METHODS: The study included 12 subjects with Wilson's disease with neurological symptoms. Twelve corneas presented clinically with classic Kayser-Fleischer rings, visible on slit lamp examination; the other 12 served as controls. The subjects underwent a comprehensive clinical examination. Microstructural analysis using confocal laser scanning microscopy evaluated increased corneal thickness, decreased number of cells, increased debris or specific deposits, and unusual microstructures. RESULTS: Clinically, the subjects with Kayser-Fleischer rings had similar corneal findings and normal intraocular pressure; two had typical sunflower cataracts and decreased visual acuity. The control eyes all presented normal visual acuity, intraocular pressure, and corneal appearance. The microstructural analysis demonstrated similar findings in all the affected corneas. Compared with the control corneas, there were fewer keratocytes in the anterior stroma (17.380 vs. 22.380/mm3). Round, "hollow" dark areas were observed between the keratocytes; these were universal and similar in appearance in all affected corneas and all cornea layers. In the peripheral posterior stroma, there were dust-like, bright, granular deposits that tended to increase in number and density toward Descemet's membrane, masking the peripheral endothelium. The control corneas presented a normal microstructure apart from dust-like granular deposits in the periphery. CONCLUSIONS: In vivo confocal microscopy is a useful tool for evaluating the corneal microstructure when a Kayser-Fleischer ring is clinically present. The ring consists of granular, bright particles that increase in density toward Descemet's membrane, and is associated with a decreased number of keratocytes and peculiar dark, round areas in all stromal layers, probably a sign of corneal damage. When the ring is not visible in subjects with Wilson's disease, changes to the corneal microstructure are insignificant.

In vivo confocal microstructural analysis of corneas presenting Kayser-Fleischer rings in patients with Wilson's disease / Análise microestrutural confocal in vivo de córneas apresentando anel de Kayser-Fleischer em pacientes com doença de Wilson

ABSTRACT Purpose: To evaluate microstructural differences between corneas with and without Kayser-Fleischer rings in age-matched subjects with Wilson's disease with neurological symptoms, using confocal laser scanning microscopy. Methods: The study included 12 subjects with Wilson's disease with neurological symptoms. Twelve corneas presented clinically with classic Kayser-Fleischer rings, visible on slit lamp examination; the other 12 served as controls. The subjects underwent a comprehensive clinical examination. Microstructural analysis using confocal laser scanning microscopy evaluated increased corneal thickness, decreased number of cells, increased debris or specific deposits, and unusual microstructures. Results: Clinically, the subjects with Kayser-Fleischer rings had similar corneal findings and normal intraocular pressure; two had typical sunflower cataracts and decreased visual acuity. The control eyes all presented normal visual acuity, intraocular pressure, and corneal appearance. The microstructural analysis demonstrated similar findings in all the affected corneas. Compared with the control corneas, there were fewer keratocytes in the anterior stroma (17.380 vs. 22.380/mm3). Round, "hollow" dark areas were observed between the keratocytes; these were universal and similar in appearance in all affected corneas and all cornea layers. In the peripheral posterior stroma, there were dust-like, bright, granular deposits that tended to increase in number and density toward Descemet's membrane, masking the peripheral endothelium. The control corneas presented a normal microstructure apart from dust-like granular deposits in the periphery. Conclusions: In vivo confocal microscopy is a useful tool for evaluating the corneal microstructure when a Kayser-Fleischer ring is clinically present. The ring consists of granular, bright particles that increase in density toward Descemet's membrane, and is associated with a decreased number of keratocytes and peculiar dark, round areas in all stromal layers, probably a sign of corneal damage. When the ring is not visible in subjects with Wilson's disease, changes to the corneal microstructure are insignificant.

RESUMO Objetivo: Avaliar, ao nível microestrutural, através de microscopia confocal in vivo a lazer, 12 córneas com anel de Kayser-Fleischer visível ao exame da lâmpada de fenda e compará-las com 12 córneas clinicamente normais de indivíduos com idades correspondentes aos pacientes com doença de Wilson e sintomas neurológicos. Métodos: O estudo incluiu 12 indivíduos com doença de Wilson e sintomas neurológicos (24 córneas). Doze córneas apresentavam clinicamente o anel clássico de Kayser-Fleischer e as outras 12 serviram como controle. Todos os pacientes foram submetidos a um exame clínico abrangente e a uma análise microestrutural subsequente utilizando microscopia confocal in vivo de varredura a laser. Os principais resultados observados foram: aumento da espessura da córnea, diminuição do número de células, aumento de resíduos/depósitos específicos e microestrutura atípica. Resultados: Clinicamente, todos os indivíduos com anel de Kayser-Fleischer (12 olhos) apresentaram achados similares da córnea e pressão intraocular normal. Dois indivíduos também apresentaram uma catarata de girassol típica e diminuição da acuidade visual. Todos os olhos do grupo controle apresentaram acuidade visual, pressão intraocular e aparência corneana normais. A microscopia confocal in vivo com varredura a laser revelou achados semelhantes em todas as córneas afetadas. O número de ceratócitos no estroma anterior era menor, 17.380/mm3 (22.380/mm3 no grupo controle), e entre eles foram identificadas áreas escuras arredondadas "vazias". Essas zonas escuras eram generalizadas e similares em todas as córneas examinadas e em todas as camadas da córnea. No estroma posterior periférico, havia presença de depósitos granulares brilhantes e com aparência de pó que tendiam a aumentar em número e densidade no sentido da membrana de Descemet, mascarando o endotélio periférico. As córneas controle apresentaram estrutura normal, com exceção de depósitos granulares com aparência de pó na periferia. Conclusões: A microscopia confocal in vivo é uma ferramenta útil para a avaliação da microestrutura da córnea quando o anel de Kayser-Fleischer está clinicamente presente. O anel é constituído de partículas granulares brilhantes com densidade aumentada no sentido da membrana de Descemet. Sua presença está associada com uma diminuição do número de ceratócitos e com áreas circulares escuras "peculiares" em todas as camadas estromais, que representam, provavelmente, um sinal de dano da córnea. Quando o anel não está clinicamente visível, a estrutura da córnea in vivo encontra-se insignificantemente alterada.

Scanning electron microscopy assessment of the Descemet membrane interface during DMEK graft preparation.

We set out to determine microscopic characteristics of the Descemet membrane interface during Descemet membrane endothelial keratoplasty (DMEK) graft preparation. Ten corneas were partially prepared, preserving half of the Descemet membrane attached to the stroma to enable visualisation of the Descemet-stroma interface. This tissue was prepared for viewing with a scanning electron microscope. The Descemet-stroma interface was categorised into three regions: centre, mid-periphery and periphery. We classified adhesions in these regions as either minor thread-like adhesions or major bridge-like adhesions with stromal detachments. We found a region-specific differentiation of the Descemet-stroma morphology. The presence of minor (P = 0,0001) and major (P = 0,0001) adhesions at the explored regions of the Descemet-stroma interface were found to be statistically significant. Fibrotic linear adhesions were predominant in the centre and mid-periphery, whereas the larger bridge-like adhesions were found mainly in the periphery. In addition, we observed a positive correlation between the size of the adhesions and the presence of ruptures in the underlying stromal bed. Viewing of the Descemet-stroma interface with electron microscopy reveals morphological differences between the centre of a graft and its periphery. These findings are of potential clinical relevance in terms of developing a better understanding of tissue behaviour during graft preparation.

Structural, ultrastructural, and morphometric study of the zebrafish ocular surface: a model for human corneal diseases?

PURPOSE: A morphological and morphometric study of the adult zebrafish ocular surface was performed to provide a comprehensive description of its parts and to evaluate its similarity to the human. MATERIALS AND METHODS: The eyes of adult zebrafish were processed for light, transmission and scanning electron microscopy, and for immunohistochemical stain of corneal nerves; a morphometric analysis was also performed on several morphological parameters. RESULTS: The corneal epithelium was formed by five layers of cells. No Bowman's layer could be demonstrated. The stroma consisted of lamellae of different thickness with few keratocytes. The Descemet's membrane was absent as the flat and polygonal endothelial cells directly adhered to the deepest corneal lamella. The immunohistochemical stain of neurofilaments failed to demonstrate corneal nerve fibers. The conjunctival epithelium was stratified, overlying the stroma formed by a subepithelial and a deep layer, this latter connected to the scleral cartilage. In the peripheral cornea and in the conjunctiva, many goblet and rodlet cells were observed. The morphometric analysis showed that the peripheral cornea epithelium was thicker when compared to the other parts of the ocular surface, with smaller superficial cells. Desmosomes and hemidesmosomes in the conjunctiva were significantly fewer in number than the other parts of the ocular surface. The stroma was thinner in the conjunctiva than in the cornea, while corneal lamellae were thicker in the intermediate stroma. CONCLUSIONS: The zebrafish ocular surface showed significant differences compared to the human, such as the absence of Bowman's layer, Descemet's membrane and corneal nerve fibers, the reduced stromal thickness, and the presence of rodlet cells. On the basis of these original findings, it is suggested that the use of the zebrafish as a model for studying normal or pathological human corneas should be undertaken with particular caution.

Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus.

Purpose: To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus. Methods: Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic). Results: In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen. Conclusions: Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

Role of dextran in maintaining adhesive and stiffness properties of prestripped DMEK lenticules.

PURPOSE: To investigate the adhesive and stiffness properties of prestripped Descemet membrane endothelial keratoplasty (DMEK) lenticules in different preservation conditions (with and without dextran). METHODS: The study included 3 conditions: (C1) tissues collected from tissue culture media (TCM), stripped and preserved in TCM; (C2) tissues collected from transport media (TM) (TCM supplemented with 6% dextran T-500), stripped and preserved in TM; and (C3) tissues collected from TCM, stripped and preserved in TM. Using a hinge, 9.5-mm stripped DMEK lenticules were restored back on the stroma and preserved for 4 days at room temperature (RT) in different conditions as above. Nine tissues, 3 from each condition, were used to check the adhesive (fibronectin, laminin, and vitronectin) and elastic properties (fibrillin, elastin, and collagen VI) using different antibodies. Six tissues, 2 from each condition, were used to check the stiffness properties after preservation using atomic force microscopy (AFM) nanoindentation method. RESULTS: On the Descemet membrane, fibronectin was strongly expressed in C2 and C3, whereas laminin was intense in C2 postpreservation. Vitronectin was expressed in all the conditions. Elastic proteins were not expressed in either of the conditions apart from collagen VI, which was expressed on the posterior stroma. Atomic force microscopy showed higher stiffness in C3 and an insignificant but lower rigidity in C2 as compared to C1. CONCLUSIONS: The tissues from C2 showed expression of adherent proteins and lower stiffness. Dextran may be suitable in preservation of DMEK grafts before and after preparation. Less stiff tissues may help reduce manipulations required in the recipient eye during DMEK surgery.

Influence of Ultrastructural Corneal Graft Abnormalities on the Outcome of Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To investigate if ultrastructural alterations in the Descemet membrane (DM) are correlated with the clinical outcome after Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Retrospective cohort study. METHODS: setting: Institutional, single-center. STUDY POPULATION: One hundred and twelve residual DM specimens obtained after DM stripping. MAIN OUTCOME MEASURES: Incidence of ultrastructural abnormalities in transmission electron microscopy, graft detachment rate, graft failure rate, best-corrected visual acuity (BCVA), endothelial cell density (ECD), and central corneal thickness (CCT). Examination dates were on the day before DMEK and 1, 3, 6, and 12 months after surgery. RESULTS: Abnormalities in the ultrastructure of DM were found in 16 of 112 specimens (14%) (abnormal DM group), comprising deposits of long-spacing collagen, fine filaments (proteoglycans), a posterior collagenous layer, pseudoexfoliative material, and guttae. The secondary graft failure rate was significantly higher in the abnormal DM group compared with the normal DM group (P = .001). There was a trend for an increased graft detachment rate in the abnormal DM group (11/16) compared with the normal DM group (42/96) (P = .103). There was no significant difference in mean CCT and ECD after surgery. Mean CCT in the eyes with graft failure in the abnormal DM group at the last follow-up before regrafting was 850 µm, indicating endothelial failure with stromal edema. CONCLUSION: This study reveals a correlation between ultrastructural alterations of DM in donor corneas and the graft failure rate after DMEK. Thus, graft failure after DMEK not only is determined by surgical trauma and postoperative events but may also be influenced by intrinsic, graft-specific features.

Instrument to Enhance Visualization of Descemet Membrane During Graft Preparation for DMEK Surgery.

PURPOSE: Descemet membrane (DM) endothelial keratoplasty has improved outcomes of corneal transplantation in patients with corneal endothelial disease. However, the procedure has been criticized for jeopardizing donor tissue during graft preparation. Standardization of this procedure may provide a way toward minimizing tissue loss. For this purpose, we propose the use of a novel tool. METHODS: Computerized numerical control milling was used to create a blunt instrument, which was used to remove endothelial cells within a defined area in the periphery of donor corneas. Trypan blue was used to stain denuded DM. Graft preparation was continued as per our standard protocol. Transmission electron microscopy was performed on the treated area, and endothelial cell counts were obtained. RESULTS: Use of the modified procedure resulted in delineation of a peripheral band of denuded DM, which readily stained with trypan blue. This provided increased visibility of DM during subsequent steps. Transmission electron microscopy confirmed that no structural deficits of DM were induced. Mean endothelial cell loss (±SD) at 24 hours after preparation was 63 (±130) cells per square millimeter in the group prepared with the use of the new instrument (n = 7), versus 116 (±107) cells per square millimeter in the group prepared without the new instrument (n = 7; P = 0.45). CONCLUSIONS: The device presented here enhances visualization of DM during creation of the peripheral margin for subsequent lifting of the margin and stripping of the graft. This may increase success rates and shorten preparation times and learning periods for DM preparation. DM ultrastructure and endothelial cells were not negatively affected.

The Ultrastructures and Mechanical Properties of the Descement's Membrane in Fuchs Endothelial Corneal Dystrophy.

Fuchs endothelial corneal dystrophy (FECD), is the most common corneal endothelial dystrophy, and contributes up to 50% of all corneal transplantations performed in developed countries. FECD develops in Descemet's membrane (DM) and possibly alters the mechanical properties and internal structures in this basal lamina. In this work, the morphology and mechanical properties of FECD-DMs are studied by transmission electron microscopy (TEM) and quantitative dynamic atomic force microscopy (QD-AFM) at nano scale. Pathological wide-space collagens that are typical of FECD display different mechanical properties in that they are softer than the remaining tissue both for dehydrated- and fully hydrated samples. Additionally, the hydration level has major influence on the mechanical properties. These findings could help to further understand the structural changes in FECD, and possibly be useful for further characterization of the disease, the diagnosis and assessment or even pathologic analysis.

Feasibility and safety of porcine Descemet's membrane as a carrier for generating tissue-engineered corneal endothelium.

The aim of this study was to evaluate the feasibility and safety of porcine Descemet's membrane (DM) as a carrier for the generation of tissue-engineered corneal endothelium by analyzing porcine endogenous retroviruses (PERVs) and the α-gal epitope. The morphology of porcine and human DM was observed by hematoxylin and eosin staining and scanning electron microscopy. Immunohistochemical staining was used to investigate the location of α-gal epitopes on porcine DM used for xenotransplantation. The porcine DM was treated with ethylene glycol diglycidyl ether (EDGE) for 2 weeks, and then the PERV gene sequences in porcine DM and DM-EDGE were detected by polymerase chain reaction (PCR) and real-time PCR, respectively. The porcine DM had tight basement membrane morphology, which was similar to human DM in terms of thickness. No positive immunohistochemical staining of the α-gal epitope was detected in porcine DM. PERV expression of pol, gag, env-A and env-B was noted in porcine DM, but in DM-EDGE it was completely degraded. Based on structural, immunological and etiological studies, porcine DM may be an ideal and viable carrier for the generation of tissue-engineered corneal endothelium.

Big bubble deep anterior lamellar keratoplasty: the collagen layer in the wall of the big bubble is unique.

PURPOSE: In big bubble (BB), deep anterior lamellar keratoplasty intracorneal injection of air separates Descemet's membrane (DM) and the pre-Descemet's layer (Dua's layer [DL]) to create a type 1 BB. We tested the hypothesis that air injection after excision or ablation of DL will fail to produce a BB. METHODS: Nine human sclero-corneal discs were used. Three served as controls. In three, a type 1 BB was created, the bubble wall was excised and more air injected in an attempt to create another BB. In three samples, the DM was removed and 22µ of posterior cornea were ablated by phototherapeutic keratectomy (PTK). Air was injected to induce formation of a BB. Tissue from these experiments was subjected to light and electron microscopy. RESULTS: In all three control eyes, a type 1 BB (DL + DM) was obtained. Air injection after excision of the type 1 BB wall in three samples failed to produce another BB. Following PTK of DL, injection of air failed to create a BB in all three samples. Multiple points of air leak from the deep stroma were observed in all six samples. Light and electron microscopy showed a clear distinction between the ablated and non-ablated areas of cornea. CONCLUSIONS: This study supports the hypothesis that a BB cannot be created once the DL is excised or ablated. This adds to evidence that DL is unique. It also demonstrates that DL is not a random separation of deep stroma of the cornea.

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.