miR-339 Promotes the Recovery of the Bone Marrow Mesenchymal Stem Cells (BMSCs)-Induced Corneal Epithelium Damage

This study intends to identify the expression profiles of micoRNAs during the recovery of damaged corneal epithelium induced by BMSCs. Differential expressions of miRNA after damage of corneal epithelium stimulated by BMSCs were analyzed based on micro-array and validated by qRT-PCR. The miRNA’s effect on cell proliferative and apoptotic activity was evaluated through transfection of plasmid with over presentation of miRNA and inhibitor of miRNA. miR-339 was significantly down-regulated in the process of recovery of the damaged corneal epithelium induced by BMSCs. Importin 13 and EGF expression was reduced after transfection of plasmid with over presentation of miR-339, which were reversed by transfection of the inhibitor of miR-339. Importin 13 was a target of miR-339. The cell proliferation and apoptosis could be restrained by miR-339 through regulation of the expression of Importin 13. In conclusion, the damaged corneal epithelium induced by BMSCs could be recovered by miR-339 through restraining Importin 13 expression, indicating that it might be a novel target for amelioration of corneal epithelium damage.

Dysregulation of Receptor for Advanced Glycation End Products (RAGE) Expression as a Biomarker of Keratoconus

Background. Because of the implications of Receptor for Advanced Glycation End Products (RAGE) in keratoconus (KC), we describe a differential expression of RAGE transcripts and proteins in corneal tissues and tears of KC and healthy patients. Methods. Using a case-controlled study, corneal epitheliums and tears of KC and healthy subjects were obtained during corneal collagen cross-linking and photorefractive keratectomy (PKR) and during usual consultations. Quantitative reverse transcription (RT-qPCR) and Western-Blot were performed to analyze RAGE transcripts and proteins’ expression in corneal tissues and tears. Results. One hundred and six patients were included in this study. The characteristics of the patients were as follows: 56 KC (25 corneal epithelium and 31 tears) and 50 control subjects (25 corneal epithelium and 25 tears). Transcripts of RAGE, HMGB1, and S100 family ligands were quantified by RT-qPCR, identifying a significantly higher expression of RAGE and HMGB1 in the healthy group than in the KC group ( p = 0.03 and 0.04, respectively). Western Blot showed a significantly higher fl-RAGE expression in KC corneal epithelium than control ( p < 0.001 ) and lower s-RAGE expression in KC tears than control ( p = 0.04 ). Conclusions. Linked with the inflammatory process occurring in KC pathophysiology, we propose for the first time that the RAGE expression (total and truncated forms of receptor and ligands) in KC corneal tissues and tear samples provides viable biomarkers.

Activity-Dependent Neuroprotective Protein (ADNP)-Derived Peptide (NAP) Counteracts UV-B Radiation-Induced ROS Formation in Corneal Epithelium

The corneal epithelium, the outermost layer of the cornea, acts as a dynamic barrier preventing access to harmful agents into the intraocular space. It is subjected daily to different insults, and ultraviolet B (UV-B) irradiation represents one of the main causes of injury. In our previous study, we demonstrated the beneficial effects of pituitary adenylate cyclase-activating polypeptide (PACAP) against UV-B radiation damage in the human corneal endothelium. Some of its effects are mediated through the activation of the intracellular factor, known as the activity-dependent protein (ADNP). In the present paper, we have investigated the role of ADNP and the small peptide derived from ADNP, known as NAP, in the corneal epithelium. Here, we have demonstrated, for the first time, ADNP expression in human and rabbit corneal epithelium as well as its protective effect by treating the corneal epithelial cells exposed to UV-B radiations with NAP. Our results showed that NAP treatment prevents ROS formation by reducing UV-B-irradiation-induced apoptotic cell death and JNK signalling pathway activation. Further investigations are needed to deeply investigate the possible therapeutic use of NAP to counteract corneal UV-B damage.

Anti-inflammatory and anti-apoptotic effects of N-acetylcysteine in diabetic rat corneal epithelium

AIM: To characterize the anti-inflammatory and anti-apoptotic effects of N-acetylcysteine (NAC) in streptozotocin (STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells (HCECs) exposed to a high-glucose environment. METHODS: HCECs were incubated in 0, 5, 50 mmol/L glucose medium, or 50 mmol/L glucose medium with NAC for 24h. Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group. We characterized receptor for advanced glycation end-products (RAGE) expression using immunofluorescence, and interleukin (IL)-1β and cleaved caspase-3 (CCAP-3) expression using immunohistochemistry. Circulating tumor necrosis factor (TNF)-α concentration was measured by ELISA and cleaved poly-ADP ribose polymerase (PARP) concentration was quantified by Western blotting. Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining. RESULTS: Diabetic rats had higher expression of RAGE (2.46±0.13 fold), IL-1β, and CCAP-3 in apoptotic cells of their corneas than control rats. The expression of RAGE (1.83±0.11 fold), IL-1β, and CCAP-3, and the number of apoptotic cells, were reduced by topical NAC treatment. HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α (310±2.00 pg/mL) and cleaved PARP (7.43±0.56 fold), and more extensive apoptosis than cells in 50 mmol/L glucose medium. However, the addition of NAC reduced the concentrations of TNF-α (153.67±2.31 pg/mL) and cleaved PARP (5.55±0.31 fold) and the number of apoptotic cells. CONCLUSION: NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

A new approach for quantifying epithelial and stromal thickness changes after orthokeratology contact lens wear

The aim of the study was to develop an automatic segmentation approach to optical coherence tomography (OCT) images and to investigate the changes in epithelial and stromal thickness profile and radius of curvature after the use of orthokeratology (Ortho-K) contact lenses. A total of 45 right eyes from 52 participants were monitored before, and after one month of, uninterrupted overnight Ortho-K lens wear. The tomography of their right eyes was obtained using optical OCT and rotating Scheimpflug imaging (OCULUS Pentacam). A custom-built MATLAB code for automatic segmentation of corneal OCT images was created and used to assess changes in epithelial thickness, stromal thickness, corneal and stromal profiles and radii of curvature before, and after one month of, uninterrupted overnight wear of Ortho-K lenses. In the central area (0–2 mm diameter), the epithelium thinned by 12.8 ± 6.0 µm (23.8% on average, p < 0.01) after one month of Ortho-K lens wear. In the paracentral area (2–5 mm diameter), the epithelium thinned nasally and temporally (by 2.4 ± 5.9 µm, 4.5% on average, p = 0.031). The stroma thickness increased in the central area (by 4.8 ± 16.1 µm, p = 0.005). The radius of curvature of the central corneal anterior surface increased by 0.24 ± 0.26 mm (3.1%, p < 0.01) along the horizontal meridian and by 0.34 ± 0.18 mm (4.2%, p < 0.01) along the vertical meridian. There were no significant changes in the anterior and posterior stromal radius of curvature. This study introduced a new method to automatically detect the anterior corneal surface, the epithelial posterior surface and the posterior corneal surface in OCT scans. Overnight wear of Ortho-K lenses caused thinning of the central corneal epithelium. The anterior corneal surface became flattered while the anterior and posterior surfaces of the stroma did not undergo significant changes. The results are consistent with the changes reported in previous studies. The reduction in myopic refractive error caused by Ortho-K lens wear was mainly due to changes in corneal epithelium thickness profile.

Effect of Platelet-Rich Plasma on Corneal Epithelial Healing after Phototherapeutic Keratectomy: An Intraindividual Contralateral Randomized Study

Purpose. To assess the effect of platelet-rich plasma (PRP) on the healing response of the corneal epithelium in eyes undergoing phototherapeutic keratectomy (PTK). Methods. We prospectively examined 20 eyes of 10 patients undergoing bilateral PTK for granular corneal dystrophy or band keratopathy. Patients were randomly assigned to start topical administration of PRP ophthalmic suspension (PRP group) or artificial tears (control group) 4 times daily for 2 weeks. Immediately, 1, and 2 days, and 1 week after PTK, we quantitatively measured the staining area of the corneal epithelium, using slit-lamp photography. We also determined the subjective symptoms and the satisfaction, using the visual analogue system (VAS). Results. The staining area in the PRP group was significantly smaller than that in the control group on days 1 and 2 (Wilcoxon signed-rank test, p = 0.022 and p = 0.017 , respectively), but not on day 7 ( p = 0.317 ). The recovery rate of the corneal epithelium in the PRP group was significantly higher than that in the control group on days 1 and 2 ( p = 0.022 and p = 0.017 , respectively), but not on day 7 ( p = 0.317 ). We found no significant differences in pain ( p = 0.139 ), foreign body sensation ( p = 0.108 ), epiphora ( p = 1.000 ), or satisfaction ( p = 0.295 ), between the two groups. Postoperative complications did not occur in any of the eyes in the study. Conclusions. The PRP treatment was effective for enhancing corneal epithelial recovery in the early postoperative period, without significant adverse events, in post-PTK-treated eyes, suggesting that it may hold promise as one of the treatment options for treating such postsurgical patients.

An Analysis of the Progression of Conjunctivalisation after Transplantation of Cultivated Corneal Epithelium

Purpose. To analyse the recurrence of superficial neovascularisation after previous corneal surface reconstruction with cultivated corneal epithelial cells. Materials and Methods. Forty-eight eyes underwent autologous transplantation of cultivated corneal epithelium to treat partial or total limbal stem cell deficiency caused by chemical or thermal injury. The carrier for the epithelial sheets was a denuded amniotic membrane. Follow-up was conducted for up to 120 months. Recurrent revascularisation (measured in terms of clock hours affected) was evaluated with slit-lamp examination and the support of confocal microscopy. Results. During the long-term observation, only 7 eyes had stable epithelia with no neovascularisation from the conjunctiva. Nineteen eyes developed pathologic vessels in 1 quadrant, with additional 4 eyes developing them in 2 quadrants. Twelve patients developed subtotal or total conjunctivalisation of the corneal surface. They were referred for second cultivated epithelium transplantation (3 patients), allogenic keratolimbal transplantation (7 patients), or keratoprosthesis (2 patients). Six patients withdrew consent. The use of confocal scans of up to 100 µm in resolution enabled the detection of pathologic microvasculature originating from the conjunctiva and the exclusion of stromal vascular ingrowth. Conclusions. Local ingrowth of the conjunctiva is a common complication after the transplantation of cultivated epithelial cells. Severe and progressive vascularisation inevitably leads to graft failure. However, if local ingrowth stops before reaching the central cornea, the treatment even with this complication can be considered a success.

Impact of Different Oxygen Supply Methods on the Healing of Corneal Epithelial Wound and the Level of Acetylcholine

Purpose. To investigate the impact of different oxygen supply methods on corneal epithelial wound healing and acetylcholine level during wound healing. Methods. We randomly divided 75 rabbits into three groups: A, B, and C, with 25 rabbits in each group. The central corneal epithelium was removed from all eyes of the rabbits using a 5 mm trephine. Group A rabbits were given low flow oxygen (3 L/min; concentration: 33%) for 2 h per day through goggles. Group B rabbits were given low flow oxygen (3 L/min; concentration: 33%) for 2 h per day via oxygen masks for inhalation. Group C rabbits healed naturally. The area of healed corneal epithelium and acetylcholine content in corneal epithelium were determined at 12 h, 24 h, and 36 h after injury. Results. At 12 h, 24 h, and 36 h after injury, the healing area of corneal epithelium in the three groups was in the order group A > group B > group C ( P < 0.05 ). At all timepoints, the acetylcholine level in corneal epithelium was in the order of group A > group B > group C ( P < 0.05 ). In all three groups, the acetylcholine content in corneal epithelium showed the order 12 h > 24 h > 36 h ( P < 0.05 ). There was a correlation between acetylcholine expression and the area of unhealed corneal epithelium, and the correlation coefficients of groups A, B, and C were 0.80, 0.83, and 0.85 respectively. Conclusion. Increasing oxygen concentration through inhalation or via goggles can promote corneal epithelial wound healing, but increasing local oxygen concentration of the eye showed a better effect. Acetylcholine may play an important role in the early process of corneal epithelial wound healing.

Key Role of Staphylococcal Fibronectin-Binding Proteins During the Initial Stage of Staphylococcus aureus Keratitis in Humans

ObjectivesStaphylococcus aureus is one of the main causes of bacterial keratitis in humans. This study was aimed at investigating the mechanisms of S. aureus adhesion to the human corneal epithelium involved during the initial stage of infectious keratitis.MethodsHuman corneas stored in a specific active storage machine that restores a normal pluristratified epithelium were used to assess S. aureus adhesion level to intact and injured tissues using immunostaining. S. aureus adhesion to immobilized fibronectin was measured in microtiter plate. Internalization of S. aureus clinical isolates recovered from keratitis was assessed on human corneal epithelial HCE-2 cells.ResultsSuperficial corneal injury unmasked fibronectin molecules expressed within the human corneal epithelium. S. aureus adhesion level was increased by 117-fold in the area of injured epithelium (p &lt; 0.0001). The deletion of staphylococcal fnbA/B genes decreased by 71% the adhesion level to immobilized fibronectin (p &lt; 0.001). The deletion of fnbA/B genes and the incubation of the corneas with anti-fibronectin blocking antibodies prior to the infection significantly reduced the S. aureus adhesion level to injured corneal epithelium (p &lt; 0.001). Finally, S. aureus clinical isolates triggered its internalization in human corneal epithelial cells as efficiently as the 8325-4 wt.ConclusionS. aureus was almost unable to bind the intact corneal epithelium, whereas a superficial epithelial injury of the corneal epithelium strongly increased S. aureus adhesion, which is mainly driven by the interaction between staphylococcal fibronectin-binding proteins and unmasked fibronectin molecules located underneath the most superficial layer of the corneal epithelium.