The Role of Estriol and Estrone in Keratoconic Stromal Sex Hormone Receptors

Keratoconus (KC) is a progressive corneal thinning disease that manifests in puberty and worsens during pregnancy. KC onset and progression are attributed to diverse factors that include: environmental, genetics, and hormonal imbalances; however, the pathobiology remains elusive. This study aims to determine the role of corneal stroma sex hormone receptors in KC and their interplay with estrone (E1) and estriol (E3) using our established 3D in vitro model. Healthy cornea stromal cells (HCFs) and KC cornea stromal cells (HKCs), both male and female, were stimulated with various concentrations of E1 and E3. Significant changes were observed between cell types, as well as between males and females in the sex hormone receptors tested; androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) using Western blot analysis. E1 and E3 stimulations in HCF females showed AR, PR, and ERβ were significantly upregulated compared to HCF males. In contrast, ERα and ERβ had significantly higher expression in HKC’s females than HKC’s males. Our data suggest that the human cornea is a sex-dependent, hormone-responsive tissue that is significantly influenced by E1 and E3. Therefore, it is plausible that E1, E3, and sex hormone receptors are involved in the KC pathobiology, warranting further investigation.

Evaluation of Ocular Surface Epithelial and Stromal Thicknesses in Psoriasis Using Anterior Segment Optical Coherence Tomography

Purpose: To evaluate the epithelial and stromal thicknesses of conjunctiva and cornea in psoriatic patients with anterior segment optical coherence tomography (AS-OCT),Methods: In this cross-sectional study, 61 patients with psoriasis and 42 age-matched, healthy individuals were enrolled. The epithelial and stromal thicknesses of both inferotemporal bulbar conjunctiva and central cornea were measured using AS-OCT. Results: Both the Tear break-up time and Schirmer-1 test values were significantly lower in the psoriasis group compared with the controls (p<0.05). The epithelial thickness of conjunctiva and cornea did not differ between psoriasis and control groups. (p>0.05) The central corneal stroma was significantly thicker in the psoriasis group (p=0.04). PASI was positively correlated with the thickness of central cornea stroma (r=0.442, p=0.006) in the nail psoriasis group. Conclusions: Psoriasis is not associated with altered epithelial thicknesses of the cornea and conjunctiva. It is accompanied by the stromal thickening of the cornea without conjunctival stromal involvement.

Atomic Force Microscopy for the Evaluation of Corneal Surface Roughness after Femtosecond Laser Flap Creation and Excimer Ablation.

Introduction: It is well known that the femtosecond laser lamellar cut has some degree of roughness. Nevertheless, as in femtosecond laser assisted LASIK (FS-LASIK), an excimer LASIK ablation is performed, the post-ablation stromal bed should show a marked decrease in roughness. We decided to compare, using atomic force microscopy (AFM), the roughness of the corneal stromal bed, after a Femtosecond lasers device (FS) flap was created with or without an excimer myopic ablation.Methods: Using 6 freshly enucleated porcine eyes, we created in every eye a flap using a femtosecond laser. Additionally in 3 eyes an excimer laser ablation to correct -3 diopters (D) was made. AFM imaging of the remaining corneal stroma was performed. Ten different square areas of 20μm x 20μm at the central area of the stroma of each corneal sample were studied. The roughness parameters used was the root-mean-square (RMS) deviation from a perfectly flap plane.Results: The RMS deviation were 360 ± 120 nm in femtosecond laser only, and 110 ± 20 nm in those cases where excimer is also involved (p<0.0001).Conclusions: Our results show that the roughness of the surface treated with excimer is clearly lower than in the group with no excimer ablation, thus the application of laser excimer after a flap creation by femtosecond laser flap creation may soften the nano-irregularities created by this technique.

Ultrastructural Analysis of Rehydrated Human Donor Corneas After Air-Drying and Dissection by Femtosecond Laser

Purpose: To evaluate the efficiency of femtosecond laser (FSL) incision of rehydrated human donor corneas after air-drying and its effects on corneal structure.Methods: We compared the rehydrated and fresh-preserved corneas by microscopy following Victus-Tecnolas FSL treatment for straight-edge anterior lamellar keratoplasty (ALK). The corneas were dehydrated at room temperature under a laminar-flow hood.Results: To obtain the horizontal cut in rehydrated corneas, we increased the FSL pulse energy to 1.2 μJ from 0.80 μJ applied for the fresh corneas and obtained a clear-cut separation of the lamellar lenticule cap from the corneal bed. Light microscopy showed regular arrangement of stromal collagen lamellae, with spaces in between the fibers in the corneal stroma in the fresh and the rehydrated corneas, but the uppermost epithelial layers in the rehydrated corneas were lost. Transmission electron microscopy (TEM) revealed no signs of thermal or mechanical damage to the corneal structure. The epithelial basal membrane and Bowman's layer maintained their integrity. The epithelial basal layer and cells were separated by large spaces due to junction alteration in the rehydrated corneas. There were gaps between the lamellar layers in the stroma, especially in the rehydrated corneas. Keratocytes displayed normal structure in the fresh corneas but were devoid of microorganules in the rehydrated corneas. Minor irregularities were observed in the vertical incision and the horizontal stroma appeared smooth on scanning electron microscopy.Conclusion: The corneal stroma of rehydrated corneas maintained morphology and integrity, while corneal cellular components were generally altered. When corneas are intended for FSL-assisted ALK, effective stromal bed incision is best achieved at a laser power higher than that currently adopted for fresh corneas.

Continuous administration of voriconazole enhances therapeutic effect for recalcitrant fungal keratitis

Purpose To evaluate the therapeutic effect of incorporating continuous administration of voriconazole in the treatment of recalcitrant fungal keratitis. Methods In this prospective case study, 5 consecutive patients (5 eyes) with fungal keratitis were treated with a standard protocol after the failing maximal conventional medical treatment. The protocol involved continuous lavage of the ulcer with 1% voriconazole through an irrigator for 2 h, twice a day, combined with local and systemic antifungals. Visual acuity, slit lamp findings of the ulcer, and fungal hyphae density by confocal microscope were documented, respectively. Results In 4 patients, the clinical symptoms and slit lamp examination were significantly improved after only 3 days of treatment. The hyphae were shown to decrease in number and morphologically fragmented in corneal stroma by confocal microscopy. After the infection was controlled, 2 cases required further keratoplasty. In one case, the treatment was deemed ineffective and a conjunctival flap had to be created to help control the infection. In all 5 patients, the best spectacle-corrected visual acuity had improved after treatment. With more than 3 months of follow-up, no recurrence of infection was seen in any cases. Conclusion Our treatment protocol demonstrated improvement in the treatment of clinically resistant fungal keratitis. Continuous lavage of voriconazole is easy to be implemented and well-tolerated by patients. Modification of the current protocol should be further explored to optimize the therapeutic effectiveness in future.

Small-Molecule Induction Promotes Corneal Endothelial Cell Differentiation From Human iPS Cells

Purpose: Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach.Methods: Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules.Results: We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na+/K+-ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs.Conclusion: We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases.

A Clinical Case of Fitting Scleral Lens for Correction of Pellucid Marginal Degeneration

Background. Pellucid Marginal Degeneration (PMD) is a rare bilateral degenerative corneal disease. It causes corneal ectasia with bilateral, clear, inferior (typically 4 o’clock to 8 o’clock), peripheral corneal thinning. It usually affects about 80% of corneal stroma, which leads to the corneal ectasia above the thinning area as well as provokes the irregular astigmatism and visual impairment that are difficult to correct. Purpose. To study the possibilities of correction and social rehabilitation in a patient with PMD wearing scleral lenses. Materials and methods. The study included a patient with PMD, signs of corneal dysfunction and narrow-angle subcompensated glaucoma in the right eye, and PMD of the cornea resulted in a corneal leukoma – terminal glaucoma in the left eye. In addition to standard ophthalmic methods, we performed corneal topography and optical coherence tomography (OCT). For intraocular pressure measurement, ICARE IOP tonometer was used in several areas of the intact peripheral cornea. Results. Prior to lens fitting, UCVA in the right eye amounted to 0.06. Scleral lens helped achieve a high visual acuity of 0.9–1.0. The lens was well-tolerated by the patient. Conclusion. Scleral lenses may be a good choice for patients with irregular cornea caused by corneal dystrophy. Not only they are easy to use and have a good visual effect, but they also help patients with social rehabilitation.

Melt electro-written scaffolds with box-architecture support orthogonally-oriented collagen

Melt Electro-Writing (MEW) is a state-of-the-art technique that supports fabrication of 3D, precisely controlled and reproducible fiber structures. A standard MEW scaffold design is a box-structure, where a repeat layer of 90° boxes is produced from a single fiber. In 3D form (i.e., multiple layers), this structure has the potential to mimic orthogonal arrangements of collagen, as observed in the corneal stroma. In this study, we determined the response of human primary corneal stromal cells and their deposited fibrillar collagen (detected using a CNA35 probe) following 6-weeks in vitro culture on these box-structures made from poly(ε-caprolactone). Comparison was also made to glass substrates (topography-free) and electrospun poly(ε-caprolactone) fibers (aligned topography). Cell orientation and collagen deposition were non-uniform on glass substrates. Electrospun scaffolds supported an excellent parallel arrangement of cells and deposited collagen to the underlying architecture of aligned fibers, but there was no evidence of bidirectional collagen. In contrast, MEW scaffolds encouraged the formation of a dense, interconnected cellular network and deposited fibrillar collagen layers with a distinct orthogonal-arrangement. Collagen fibrils were particularly dominant through the middle layers of the MEW scaffolds’ total thickness and closer examination revealed these fibrils to be concentrated within the pores’ central regions. With the demand for donor corneas far exceeding the supply - leaving many with visual impairment - the application of MEW as a potential technique to recreate the corneal stroma with spontaneous, bidirectional collagen organization warrants further study.