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.

Precise thickness measurements of Bowman's layer, epithelium, and tear film.

PURPOSE: To visualize corneal microstructure such as tear film, epithelium, and Bowman's layer in three dimensions with spectral domain optical coherence tomography (SDOCT) exhibiting 1.3 µm axial resolution at 100,000 A-scans/s. This enables measurement of epithelial and Bowman layer thickness across an area of 8.4 mm × 8.4 mm and measuring the tear film thickness at the central cornea. METHODS: We designed a high-performance SDOCT system, which uses a broad bandwidth TiSapph Laser and a high-speed complementary metal-oxide-semiconductor detector technology, providing a resolution in tissue of 1.3 µm and an acquisition speed of 100,000 A-scans/s. Such speed and resolution is a prerequisite if precise anatomy is to be determined. The high resolution gives access to corneal microstructure such as the epithelium layer as well as the boundaries of Bowman's layer and stroma. Even more interestingly, the tear film can be distinguished on the surface of the cornea. The Bowman's layer and epithelial thickness for both eyes of nine subjects have been measured out of which two subjects underwent photorefractive keratectomy treatment. RESULTS: Three-dimensional volumes of the human cornea have been recorded in vivo at an A-scan rate of 100,000 scans/s. Epithelial thickness was measured to be 55.8 ± 3.3 µm and Bowman's layer thickness 18.7 ± 2.5 µm in normal eyes. Epithelial thickness in the eyes after refractive surgery was measured to be 68.2 ± 5.0 µm. The Bowman layer was degenerated in these eyes. The average tear film thickness of four eyes was 5.1 ± 0.5 µm. CONCLUSIONS: Using a high-performance SDOCT system with high-imaging speed and ultrahigh resolution, we produced precise thickness maps of the epithelium and for the first time of the Bowman's layer. Such a system will give insight into high-fidelity three-dimensional corneal microstructure helping to precisely plan refractive surgery. It may furthermore yield new perspectives on studying and understanding tear film dynamics.

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.

Mapping of Corneal Layer Thicknesses With Polarization-Sensitive Optical Coherence Tomography Using a Conical Scan Pattern.

Purpose: We demonstrate segmentation and mapping of corneal layers (epithelium, Bowman's layer, and stroma) across the entire cornea (limbus to limbus), using additional contrast provided by polarization-sensitive optical coherence tomography (PS-OCT) and analyze the reproducibility of the procedure. Methods: A custom built PS-OCT system operating at 1045 nm central wavelength with conical scanning was used for image acquisition. Conical scanning allows for almost perpendicular beam incidence on the corneal surface and provides good signal quality over the entire field of view. Epithelium, Bowman's layer, and stroma were segmented using the additional contrast provided by PS-OCT. Thickness maps were computed and analyzed in sectors. Both eyes of 20 healthy volunteers were imaged at least three times to test this method and to quantify reproducibility. Results: Thickness maps of the epithelium show significant (P < 0.001) superior thinning and an inferior thickening. Bowman's layer appears homogeneous within the central 7 to 8 mm diameter of the cornea and gets thinner toward the periphery until this layer disappears between 4 and 5.5 mm eccentricity from the center. Intersubject variations of the measured thicknesses of epithelium (coefficient of variation [CV] ∼8%), Bowman's layer (CV∼25%), and stroma (CV∼10%) were observed. Very good reproducibility of thickness measurements of epithelium (CV < 3%), Bowman's layer (CV < 5%), and stroma (CV < 2%) was found. Furthermore, a significant correlation (P < 0.001) between layer thicknesses of the right and left eyes of the same subject was found. Conclusions: PS-OCT with conical scanning is a feasible approach for determining thickness maps of corneal layers on a large field of view with high reproducibility.

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.

Age-related thinning of Bowman's layer in the human cornea in vivo.

PURPOSE: To determine the thickness of Bowman's layer (BL) in vivo in a healthy population and to determine its variation with age. METHODS: Eighty-two subjects aged 15 to 88 years with clear, healthy corneas were examined bilaterally with laser scanning in vivo confocal microscopy (IVCM). Bowman's layer thickness was determined from IVCM images of anterior and posterior BL boundaries. For a given eye, BL thickness was averaged across four central locations by two independent observers. In addition, central corneal thickness was measured by time-domain optical coherence tomography. RESULTS: A significant negative correlation of BL thickness with age was found in right eyes (Pearson r = -0.579, P < 0.0001) and in left eyes (r = -0.558, P < 0.0001). Linear regression analysis yielded a decline in BL thickness of 0.06 µm per year. In 41 older subjects (mean age, 64.4 years), BL thickness was significantly thinner (mean ± SD, 8.6 ± 1.7 µm in right eyes) than that in 41 younger subjects (mean age, 31.6 years) (mean ± SD, 10.7 ± 1.6 µm in right eyes) (P < 0.001). No correlation of corneal thickness with age or of BL thickness with corneal thickness was observed. Strong intereye correlations in BL thickness (r = 0.771, P < 0.0001) and corneal thickness (r = 0.969, P < 0.001) were found. CONCLUSIONS: Bowman's layer thins with age in the normal cornea, losing one-third of its thickness between the ages of 20 and 80 years. In vivo measurement of BL thickness by IVCM could aid in clinical assessment and planned treatments of the anterior cornea.

Topographic thickness of Bowman's layer determined by ultra-high resolution spectral domain-optical coherence tomography.

PURPOSE: To characterize the thickness profile of the corneal epithelium and the Bowman's layer across the horizontal meridian. METHODS: Forty-four eyes of 22 healthy subjects were investigated in this study. Ultra-high resolution anterior segment spectral domain-optical coherence tomography (SD-OCT) was used to assess the topographic thickness of the epithelium and the Bowman's layer across the cornea. Thicknesses at five locations, including the center, midperiphery, and periphery close to the limbus, on both the nasal and the temporal sides along the horizontal meridian, were analyzed. RESULTS: Mean epithelial thickness at the central cornea was 52.5 ± 2.4 µm. It increased gradually from the center to the periphery (P < 0.001). There was no significant difference between the nasal side and the temporal side for epithelial thickness. The central Bowman's layer thickness was 17.7 ± 1.6 µm, and it remained constant from the center to the midperiphery (P > 0.05). However, thicknesses at the nasal and temporal periphery, 20.0 ± 1.9 µm and 19.8 ± 2.2 µm, respectively, were significantly greater than the central and midperipheral thicknesses (P < 0.001). Nasal and temporal thicknesses were similar on either side of the center. CONCLUSIONS: The epithelium and the Bowman's layer were not evenly distributed across the horizontal meridian of the cornea. SD-OCT provided useful information about topographic thickness of the different corneal layers in vivo.

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.

Morphological evaluation of normal human corneal epithelium.

PURPOSE: The human corneal epithelium is usually described as a 50-µm-thick layer of regular stratified squamous non-keratinized cells with a thickness of 5-7 cells. The purpose of this study is systemically to revisit the histopathological appearance of 100 corneas. METHODS: 5-µm-thick sections of corneas from 100 consecutively selected paraffin-embedded eyes were stained with hematoxylin-eosin and Periodic Acid-Schiff (PAS). All specimens were evaluated by light microscopy. The eyes were enucleated from patients with choroidal melanoma. Corneas were considered to be normal. RESULTS: Ninety of 100 eyes were evaluated. The central epithelial, stromal and total corneal thickness was measured as 36.0 µm, 618 µm and 651µm, respectively, with a variation coefficient from 0.21 to 0.22. Pathological appearances were found in 27% of corneas, including thickened basement membrane and alterations in Bowman's membrane. No intraepithelial microcysts, as found in Meesmann corneal dystrophy, were observed. CONCLUSION: The total corneal thickness was higher than reported in in vivo studies and with a wider variation. This may be an effect of uncontrolled swelling and dehydration during preparation. The high number of pathological observations suggests that 'normal' eyes harbour and potentially accumulate considerable pathology.