Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial–mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

Purpose: The corneas preserved in bioreactor (BR) had been shown to have not only a better endothelial viability, but also a more differentiated and stratified epithelium than corneas preserved in organoculture. Purpose: By using BR, we would analyse the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration. Methods: Five pairs of corneas from body donation to Science were used with a death‐to‐collection time < 20 h. A 3‐ to 5‐mm‐wide conjunctival flange was kept intact. Five patterns were set up by fully mechanical removal of 1, 2, or 3 epithelia: C‐L + Conj+, C‐L‐Conj+, C‐L + Conj‐, C + L‐Conj‐, C‐L‐Conj‐ (control) n = 2 for each pattern. The limbus was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21 mmHg, 2.5 μl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analysed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer. Results: No epithelium was reconstituted in the controls. In the other 4 models including the C‐L‐Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression. Conclusions: In this BR model, conjunctival epithelial cells allowed the regeneration of a typical corneal epithelium in model C‐L‐Conj+. The corneal epithelium can also migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia (including the conj) contain stem cells or progenitors capable of migrating throughout the cornea and restoring the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti‐angiogenic barrier rather than the loss of limbic stem cells.

PurposeQuality of the endothelial transplant is a critical parameter in the success of Descemet Membrane Endothelial Keratoplasty (DMEK) and in the graft survival. The peeling techniques, preservation methods and operator’s skill level need to be experimentally assessed and validated, as they are key elements influencing graft quality. The most reliable method of quality evaluation of the endothelial transplant is the triple staining with Hoechst‐Calcein AM‐Ethidium (HEC) allowing to determine the total number of viable endothelial cells. However, the test HEC has defects: (1) Unspecific fluorescence of the same filter than that Calcein AM prevents an accurate viability analysis; (2) Incompatibility with immunofluorescence (IF) which could provide additional information. The aim of this study is to develop technical tips to overcome these defects.MethodsTwo strategies were employed to improve Calcein AM staining: 1. Increase the specific fluorescence intensity by changing the concentration of Calcein AM and diluent; 2. Decrease unspecific fluorescence by adding the fluorescence quencher Trypan Blue (TB). In order to combine IF after the test HEC, an extensive wash in PBS was performed. In total, 19 human corneas had been used.ResultsCalcein AM at 4µM diluted in OptiMEM increased fluorescence intensity by 3‐fold (p = 0.0017, n = 5) compared to conventional staining at 2 µM in PBS. Trypan Blue visibly decreased the unspecific fluorescence of Calcein, reduced the inter‐operator (n = 6) variability of cell count by 42% (p = 0.0027, n = 10) and reduce analysis time by 40% (p = 0.0002, n = 10). To perform IF after HEC test, prolonged washing in PBS was an effective method to remove residual Calcein fluorescence and allow imaging using FITC/Alexa 488 filter.ConclusionsThis study provides effective technical tips for optimising assessment of the endothelial viability and for being able to perform IF after the test HEC.