Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats.

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

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.

Central versus peripheral thickness in the human cornea explained.

PURPOSE: The human cornea is thicker in the periphery than the center and it has been suggested that this must be due to greater numbers of lamellae in the peripheral corneal stroma. The purpose of this study was to use high-resolution ultrastructural imaging to determine if the greater thickness of the peripheral cornea is due to the presence of more lamellae or if there is some other anatomical explanation. METHODS: In this study, full thickness corneas from three human donors were processed for light microscopy (LM) and transmission electron microscopy (TEM). Images were taken in three distinct stromal regions (anterior, middle, and posterior) from the central and peripheral cornea. Stromal thickness was evaluated by LM while TEM was used to evaluate numbers and thicknesses of lamellae, mean collagen fibril diameter, and mean collagen fibril density. RESULTS: Mean stromal thickness was significantly thinner in the central (415 ± 34 µm) compared to the peripheral (536 ± 29 µm) cornea (P = 0.009). Numbers of lamellae were not significantly different between central (246 ± 14) and peripheral (251 ± 14) cornea. Average lamellar thickness was not different across all regions of the cornea, except for the peripheral posterior where the lamellae were approximately 50 % thicker (P < 0.05). Collagen fibril diameters were larger in the peripheral cornea by approximately 30 % when compared to the central cornea, in all regions (P < 0.01). CONCLUSIONS: This study shows that it is an increase peripheral posterior lamellar thickness, rather than an increase in the number of lamellae, that accounts for the increase in corneal stromal thickness in the periphery of the human cornea. While collagen fibril diameters are greater throughout the peripheral stroma, the lamellae in the mid and anterior peripheral stroma are not thicker than centrally.

Morphogenetic events influencing corneal maturation, development, and transparency: Light and electron microscopic study.

The development of the cornea is a fascinating process. Its dual origin involves the differentiation of surface ectoderm cells and the migration of mesenchymal cells of neural crest origin. This research aimed to demonstrate the morphogenesis of the rabbit cornea from fetal to postnatal life using light- and electron microscopy, and immunohistochemical analysis. There were 27 rabbit embryos and nine rabbits used. The rabbit cornea begins its prenatal development on the twelfth day of gestation. The surface ectoderm differentiates into the corneal epithelium on day 13. Intriguingly, telocytes were visible within the epithelium. The secondary stroma develops on the sixteenth day of gestation by differentiation of keratocytes. At the age of 2 weeks, the lamellae of collagenous fibers become highly organized, and the stroma becomes avascular, indicating that the cornea has become transparent. Bowman's membrane appears on day 23 of pregnancy and disappears on day 30. The Descemet's membrane appears at this time and continues to thicken postnatally. The corneal endothelium appears on the twentieth gestational day as double layer of flattened cells and becomes a single layer of cuboidal cells on day 30. The spaces between the endothelial cells resemble craters. VEGF immunohistochemical expression increases over the course of development, reaching its peak in the first week after birth before decreasing in all corneal layers and becoming negative in the stroma. In conclusion, numerous morphogenetic events contribute to corneal maturation and transparency, allowing the cornea to perform its vital functions.

Comparative study of two re-embedding methods on the ultrastructure of corneal tissue.

PURPOSE: To expand the routine of pathological diagnostics of surgical keratoplasty specimens via transmission electron microscopy. The target was to identify the best re-embedding method for optimal structural preservation of formalin fixed paraffin embedded (FFPE) corneal tissue re-embedded into resin for ultrastructural analysis. BASIC PROCEDURES: Bovine FFPE corneal tissue was re-embedded into resin with either a rapid osmium-free four-hour-method or a four-day-routine-method known from nephropathology, compared with primary resin embedded bovine corneal tissue. The analysis involved the ultrastructure of cytoplasm, the intercellular interfaces of superficial epithelial cells, deepest basal epithelial cells and corneal endothelial cells, cell matrix interfaces, Bowman layer, corneal stroma, its microfibril bundles and Descemet membrane. MAIN FINDINGS: The main observation was the equally reduced preservation status of re-embedded FFPE corneal tissue independent of the used re-embedding method. This extends to the intercellular contacts of superficial epithelial cells and the apical tight junctions of corneal endothelial cells. Hemidesmosomal cell matrix contacts showed less demarcation in re-embedded specimens. Cell matrix interfaces of Bowman layer and Descemet membrane were more clearly bordered in primary resin embedded than re-embedded tissue. In contrast, gap junctions in re-embedded tissue were detected in deepest basal epithelial cells and corneal endothelial cells with comparable preservation to primary resin embedding. Bowman layer, corneal stromal extracellular matrix, its microfibril bundles and Descemet membrane showed equal ultrastructural preservation in all evaluated methods. PRINCIPAL CONCLUSION: Corneal tissue can be successfully analysed with transmission electron microscopy after a rapid osmium-free four hour re-embedding procedure from FFPE material. A comparable morphology with primary resin embedded material can be obtained for gap junctions of deepest basal epithelial cells and corneal endothelial cells, further for Bowman layer, corneal stromal extracellular matrix, its microfibril bundles and Descemet membrane.

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment.

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.

A unique pre-endothelial layer at the posterior peripheral cornea: ultrastructural study.

This study was conducted to investigate the ultrastructure of a unique structures at the anterior side of the endothelium of the posterior peripheral cornea and compare their inner fibers to those of the limbus and sclera. The unique structures at the anterior side of endothelium was referred as a pre-endothelial (PENL) structures in the present manuscript. Ten anonymous-donor human corneoscleral rims (leftover after corneal transplants) were processed for electron microscopy. Semi-thin sections were examined using an Olympus BX53 microscope, and ultrathin sections were studied using a JOEL 1400 transmission electron microscope. A unique PENL structures was identified at the posterior peripheral cornea at a radial distance of approximately 70-638 µm, from the endpoint of Descemet's membrane. The PENL thinned out gradually and disappeared in the center. The contained an electron-dense sheath with periodic structures (narrow-spacing fibers), wide-spacing fibers, and numerous microfibrils. Typical elastic fibers were present in the sclera and limbus but were not observed in the PENL. This study revealed the existence of a new acellular PENL, containing unique fibrillar structures that were unseen in the corneal stroma. From the evidence describe in this paper we therefore suggest that PENL is a distinct morphological structure present at the corneal periphery.

Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function.

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1ß, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

Compaction of very thin corneas from ultraviolet A riboflavin-vitamin E transepithelial cross-linking.

The purpose of the study was to determine the decrease in pachymetry of very thin corneas with advanced keratoconus due to corneal compaction from the ultraviolet-A (UV-A) irradiation phase of transepithelial (epi-on) cross-linking. Twenty removed corneal buttons were obtained from patients who underwent penetrating keratoplasty for advanced keratoconus. Removed corneal buttons selected from among the post-surgical specimens for this study had intact epithelium, no scarring or surgical cautery, endothelial cell density >2500 cells/mm2, and average pachymetry over the measured points of below 400 µm. Corneas were mounted in a Franz chamber. Each epithelial surface was soaked in isotonic riboflavin and D-alpha-tocopheryl polyethylene glycol 1000 succinate (Ribocross® IROMED Group, Italy) for 15 min. Pachymetry was measured at three points over both the shielded and unshielded corneal halves for each corneal button. Surfaces were then washed in saline to remove the Ribocross®. Shields from UV-A irradiation over half of each cornea were then fixed to stand 5 mm above the test corneas. UV-A irradiation using the custom fast cross-linking (CF-CXL) protocol was then performed for the typical 10 ± 1.5 min, for a total energy of 1.08 ± 0.6 J/cm2 after which pachymetry was re-measured. The average percent change in pachymetry was -0.43% ± 0.38% (maximum -1.06%) in the shielded half. Pachymetry change was -6.2% ± 2.2% (maximum 12%) in the cross-linked halves. In conclusion, we estimate that the change in corneal thickness from corneal compaction due to the cross-linking reaction itself was -5.8% ± 2.2%. Scanning electron microscopy of cross-linked corneal segments showed stromal fiber contraction.