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.

Graft Edge Reflection of a Tightly Scrolled Roll Using Endoillumination as a Simple Method for Determining Graft Orientation in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To describe a simple finding that can be used to determine donor tissue orientation in Descemet membrane endothelial keratoplasty (DMEK). This involves the appearance of a highly reflective round curved line from an overlapping graft edge within the anterior chamber using light from an endoilluminator. We here name this Kobayashi sign (K-sign). METHODS: Twelve consecutive eyes from 12 patients who underwent DMEK were evaluated for the presence of a K-sign. The presence of Berrospi sign (B-sign), a double-line reflection from the peripheral curls of the Descemet membrane (DM) roll that has been reported to be useful for correct graft orientation, was also evaluated. RESULTS: Of 12 cases, 3 donors showed a loosely scrolled roll soon after DM donor insertion; all 3 of these showed a positive B-sign only when the endoilluminator was used. Nine donors showed a tightly scrolled DM roll without a B-sign; among these 9 donors, a K-sign was visible in 7 cases only when the endoilluminator was used. The remaining 2 cases with a tight scroll configuration showed no K-sign even with the use of endoillumination, indicating that the roll edge was located completely downward; rotation of the roll using a small jet of fluid from paracentesis revealed K-sign in these cases. CONCLUSIONS: This technique is useful for determining the correct orientation of a tightly scrolled DM roll, subsequently enabling rapid DMEK surgery. For loosely scrolled DM roll, endoillumination enhanced B-sing is proven useful.

In-vivo Three-dimensional Characteristics of Bowman's Layer and Endothelium/Descemet's Complex Using Corneal Microlayer Tomography in Healthy Subjects.

Purpose: To characterize the three-dimensional (3D) thickness profile and age-related changes of Bowman's layer (BL), and endothelium/Descemet's membrane (En/DM) complex among healthy individuals using Corneal Microlayer Tomography (CML-T), and to describe its repeatability and accuracy.Methods: Sixty-six eyes of 41 healthy volunteers; 27 eyes (< 40 years old), and 39 eyes (>40 years old) were imaged using HD-OCT. Automatic and manual segmentation of the corneal layers was performed, and 3D thickness maps were generated, using custom-built CML-T software. A regional analysis of mean thickness parameters between the 2 age groups was performed. A regression analysis was used to assess the correlation between age, and thickness maps. Intraclass Correlation Coefficients (ICC), Coefficients of Variation (COV), and Bland-Altman plots were used to assess the reliability of the repeated measurements in 198 locations.Results: CML-T successfully mapped the BL and En/DM in all included eyes. Thickness maps showed a significant increase in corneal thickness (CT), BL thickness (BMT), and En/DM complex thickness (DMT) toward the periphery with a mean difference 28 µm (p < .001), 1.1 µm (p < .001), and 1.4 µm (p < .001), respectively. There was a strong correlation between age and central DMT (r = 0.61; p < .001), while there was no correlation between age and both CT, and BMT. ICC values ranged from 0.9 (BMT) to 0.997 (DMT), and from 0.808 (BMT) to 0.979 (CT) for intraoperator repeatability of manual measurements, and the accuracy of auto matic measurements, respectively. COV values were lower than 7.5% in all cases.Conclusion: CML-T is a novel tool that can generate 3D-thickness maps of both BL and En/DM. CT, BMT, and DMT increase toward the periphery in healthy corneas. DMT increases with aging, while BMT does not. We also report excellent repeatability, accuracy and good agreement between automatic and manual measurements.

A comparative study of the elastic fibre system within the mouse and human cornea.

The cornea relies on its organised extracellular matrix for maintaining transparency and biomechanical strength. Studies have identified an elastic fibre system within the human posterior cornea, thought to allow for slight deformations in response to internal pressure fluctuations within the eye. However, the type of elastic fibres that exist within the cornea and their roles remain elusive. The aim of this study was to compare the distribution and organisation of the elastic fibres within the posterior peripheral mouse and human cornea, and elucidate how these fibres integrate with the trabecular meshwork, whilst characterising the distribution of their main likely components (fibrillin-1, elastin and type VI collagen) in different parts of the cornea and adjacent sclera. We identified key differences in the elastic fibre system between the human and mouse cornea. True elastic fibres (containing elastin) were identified within the human posterior peripheral cornea. Elastic fibres appeared to present as an extensive network throughout the mouse corneal stroma, but as fibrillin-rich microfibril bundles rather than true elastic fibres. However, tropoelastin staining indicated the possibility that true elastic fibres had yet to develop in the young mice studied. Differences were also apparent within the anatomy of the trabecular meshwork. The human trabecular meshwork appeared to insert between the corneal stroma and Descemet's membrane, with elastic fibres continuing into the stroma from the trabecular meshwork anterior to Descemet's membrane. Within the mouse cornea, no clear insertion point of the trabecular meshwork was seen, instead the elastic fibres within the trabecular meshwork continued into Descemet's membrane, with the trabecular meshwork joining posterior to Descemet's membrane.

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.

Evaluation of Total Corneal Thickness and Corneal Layers With Spectral-Domain Optical Coherence Tomography.

PURPOSE: To evaluate total corneal thickness and corneal layers in healthy young adults using spectral-domain optical coherence tomography and to describe its repeatability and reproducibility. METHODS: Eighty-six eyes from 86 healthy volunteers were prospectively and consecutively enrolled. Manual measurements of central corneal thickness (CCT) and central thickness of epithelium, Bowman's layer, stroma, and the Descemet-endothelium complex were performed using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany). To assess the reliability of the repeated measurements, intraclass correlation coefficients and coefficients of variation were used. RESULTS: Mean CCT, epithelium, Bowman's layer, stroma, and Descemet-endothelium values were 555.50 ± 29.64, 54.60 ± 4.25, 16.70 ± 1.73, 467.51 ± 28.91, and 16.74 ± 1.66 µm, respectively. The intraclass correlation coefficients ranged from 0.746 (Bowman's layer) to 0.999 (CCT and stroma) and from 0.483 (Bowman's layer) to 0.995 (CCT) and 0.998 (stroma) for intraobserver repeatability and interobserver reproducibility, respectively. The measurements showed coefficients of variation lower than 11% in all cases. CONCLUSIONS: This study establishes a normal database for corneal thickness and all its layers in healthy young adults with spectral-domain optical coherence tomography. This device exhibited a high degree of intraobserver repeatability and interobserver reproducibility for all regions except Bowman's layer.

Preloaded donor corneal lenticules in a new validated 3D printed smart storage glide for Descemet stripping automated endothelial keratoplasty.

PURPOSE: To design and validate the efficacy of three-dimensional (3D) printed smart storage glide (SSG) which is capable of preserving and delivering posterior lenticules for Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Laboratory investigation (A) was followed by clinical validation (B). Unsuitable corneas for transplantation (n=20) were used for study A. These tissues were cut using a standard microkeratome, punched and loaded into the SSG and preserved for 7 days in transport media. Endothelial cell density (ECD), Trypan blue and Alizarin red staining for endothelial morphology, thickness measurements and glucose uptake, cell apoptosis and immunostaining post preservation were analysed. For study B, clinical grade corneas (n=14) were preloaded in SSG and grafted in patients with indications of Fuchs' dystrophy (n=8), pseudophakic bullous keratopathy (n=3), posterior polymorphous dystrophy (n=2), and previous DSAEK failure (n=1). Standard DSAEK included descemetorhexis under air and bimanual delivery of the tissue under infusion of buffered saline solution through an anterior chamber maintainer placed at the 12 o'clock position. Main outcome measures for study B were less surgical time, best spectacle-corrected visual acuity (BSCVA), speed of visual recovery, and ECD. RESULTS: For study A, an average ECD loss was 2.30±3.21%, thickness increased by 30.80±20.85% and one-third of glucose was utilised during the preservation phase. Alizarin red showed hexagonality of the cells. Cell apoptosis was not observed and expression of ZO-1 was noted on the preserved tissues. In study B, 25% ECD loss was observed after 6 months. BSCVA improved to 20/25 or better within 3 months after DSAEK. Mean surgical time recorded was 21 min. CONCLUSIONS: This paper describes the development, design, laboratory and clinical validation of a 3D printed SSG which helps to store and deliver posterior lenticules, therefore allowing transportation of quality-controlled precut tissues.

The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer).

BACKGROUND: The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemet's layer (Dua's layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM. METHODS: The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34. RESULTS: EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemet's membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL. CONCLUSIONS: This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.

Human corneal anatomy redefined: a novel pre-Descemet's layer (Dua's layer).

PURPOSE: To define and characterize a novel pre-Descemet's layer in the human cornea. DESIGN: Clinical and experimental study. PARTICIPANTS: We included 31 human donor sclerocorneal discs, including 6 controls (mean age, 77.7 years). METHODS: Air was injected into the stroma of donor whole globes (n = 4) and sclerocorneal discs (n = 21) as in the clinical deep anterior lamellar keratoplasty procedure with the big bubble (BB) technique. The following experiments were performed: (1) creation of BB followed by peeling of the Descemet's membrane (DM); (2) peeling off of the DM followed by creation of the BB, and (3) creation of the BB and continued inflation until the bubble popped to measure the popping pressure. Tissue obtained from these experiments was subjected to histologic examination. MAIN OUTCOME MEASURES: Demonstration of a novel pre-Descemet's layer (Dua's layer) in the human cornea. RESULTS: Three types of BB were obtained. Type-1, is a well-circumscribed, central dome-shaped elevation up to 8.5 mm in diameter (n = 14). Type-2, is a thin-walled, large BB of maximum 10.5 mm diameter, which always started at the periphery, enlarging centrally to form a large BB (n = 5), and a mixed type (n = 3). With type-1 BB, unlike type-2 BB, it was possible to peel off DM completely without deflating the BB, indicating the presence of an additional layer of tissue. A type-1 BB could be created after first peeling off the DM (n = 5), confirming that DM was not essential to create a type-1 BB. The popping pressure was 1.45 bar and 0.6 bar for type-1 BB and type-2 BB, respectively. Histology confirmed that the cleavage occurred beyond the last row of keratocytes. This layer was acellular, measured 10.15 ± 3.6 microns composed of 5 to 8 lamellae of predominantly type-1 collagen bundles arranged in transverse, longitudinal, and oblique directions. CONCLUSIONS: There exists a novel, well-defined, acellular, strong layer in the pre-Descemet's cornea. This separates along the last row of keratocytes in most cases performed with the BB technique. Its recognition will have considerable impact on posterior corneal surgery and the understanding of corneal biomechanics and posterior corneal pathology such as acute hydrops, Descematocele and pre-Descemet's dystrophies. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Handheld slit beam techniques to facilitate DMEK and DALK.

PURPOSE: To describe techniques for using a handheld slit beam to identify the position of detached Descemet membrane in Descemet membrane endothelial keratoplasty (DMEK) and deep anterior lamellar keratoplasty (DALK). METHODS: The Eidolon model 510L handheld slit lamp (Eidolon Optical LLC) was used during DMEK cases to identify the orientation of inserted grafts in the anterior chamber before unrolling. In DALK, the beam was used to identify the presence of a big bubble. RESULTS: By using the slit beam technique, a DMEK surgeon can know which side of the partially scrolled graft is endothelium and decide whether to turn it over or inject an air bubble under it. In DALK, the technique allows the surgeon to verify if a big bubble has formed while looking through moderately opacified stroma after air injections. CONCLUSIONS: These slit beam techniques should prove useful to lamellar keratoplasty surgeons and improve patient outcomes.

Large diameter descemet membrane endothelial keratoplasty in buphthalmic eyes.

PURPOSE: To report the outcomes of Descemet membrane endothelial keratoplasty (DMEK) using a large diameter graft in the management of endothelial decompensation in buphthalmic eyes. METHODS: Four eyes of 4 adults (1 man, 3 women) with bullous keratopathy and buphthalmos secondary to congenital glaucoma were treated with DMEK using posterior lamellar grafts with diameters ranging from 10 to 12 mm. The mean age was 31 (±9) years (range, 20-38 years). Mean follow-up time was 13.5 (±7.5) months (range, 6-24 months). Main outcome measures were best-corrected visual acuity (BCVA), intraocular pressure (IOP), pachymetry, endothelial cell density, and complications after surgery. RESULTS: In all eyes, there was improved corneal clarity with decrease in pachymetry. The final postoperative BCVA improved in most eyes. There was no significant change in IOP, with 3 eyes needing additional antiglaucoma medication. Endothelial cell loss ranged from 37% to 42%. Postoperative complications were early partial graft detachment in 2 eyes, one resolving spontaneously without intervention and the other requiring a rebubbling, and cataract formation requiring phacoemulsification in 1 eye. CONCLUSIONS: DMEK using a large or even full diameter Descemet membrane graft may be an effective treatment for bullous keratopathy in buphthalmic eyes. Partial graft detachment after surgery may be the main complication. Postoperative IOP control is mandatory, and BCVA may vary with ocular comorbidity unrelated to the transplanted cornea.

Reorientation of inverted endothelial graft during Descemet stripping automated endothelial keratoplasty.

We describe a technique during Descemet stripping automated endothelial keratoplasty for reorientation of an inverted endothelial graft. This technique allows the inversion to be predictably corrected with minimal manipulation and trauma to the endothelium, thereby reducing the risk of primary failure and subsequent repeat of the transplant.

How normal is the transparent cornea? Effects of aging on corneal morphology.

PURPOSE: To ascertain the effects of aging on corneal morphology and to illustrate the morphologic diversity of the different layers in the normal cornea as seen by in vivo confocal microscopy (IVCM). DESIGN: Observational cross-sectional study. PARTICIPANTS: A total of 150 healthy subjects, evenly distributed over 5 age categories, comprising 75 men and 75 women. METHODS: Both transparent corneas (n = 300) of all subjects were examined in duplicate by white light IVCM (Confoscan 4, NIDEK Technologies, Albignasego, Padova, Italy). After reviewing the IVCM examinations for morphologic variations of the corneal layers, we selected the 8 most common features to illustrate the morphologic diversity. Subsequently, all 600 IVCM examinations were assessed for the presence of these features. We used binary logistic regression analyses to assess the age-relatedness of each feature. MAIN OUTCOME MEASURES: Age distribution of bright superficial epithelial cells, dendriform cells, alterations characteristic of epithelial basement membrane dystrophy (EBMD), tortuous stromal nerves, stromal microdots in the anterior stroma, folds in the posterior stroma, opacification of Descemet's membrane, and corneal guttae. RESULTS: Four features were found characteristic of the aging cornea: stromal microdots in the anterior stroma (P<0.0001), folds in the posterior stroma (P<0.0001), opacification of Descemet's membrane (P<0.0001), and corneal guttae (P<0.0001). Alterations characteristic of EBMD were found in 3% of all eyes and only detected in subjects aged ≥40 years, suggesting age-relatedness (P = 0.09). Other features, such as bright superficial epithelial cells (n = 38, 13%), dendriform cells (n = 42, 14%), and tortuous stromal nerves (n = 115, 38%), were age-independent. We also found a novel phenotype of corneal endothelium in 4 normal eyes of 2 subjects, which we coined "salt and pepper endothelium." We could not establish whether this novel phenotype represented a morphologic variant of normal endothelium, an early stage of a known corneal endothelial disorder, or a completely new disease entity. CONCLUSIONS: Knowledge of the common morphologic variations of the corneal layers and the effects of aging on corneal morphology as seen by IVCM increases our understanding of corneal degenerative disorders and is essential to detect corneal pathology. Our finding of a novel phenotype of corneal endothelium emphasizes the morphologic diversity of this optically transparent tissue.

Anatomy and physiology of the cornea.

The importance of the cornea to the ocular structure and visual system is often overlooked because of the cornea's unassuming transparent nature. The cornea lacks the neurobiological sophistication of the retina and the dynamic movement of the lens; yet, without its clarity, the eye would not be able to perform its necessary functions. The complexity of structure and function necessary to maintain such elegant simplicity is the wonder that draws us to one of the most important components of our visual system.