Purpose. To compare corneal flaps made with a femtosecond (FS) laser with those made with a mechanical microkeratome (MM) in rabbits, measuring early postoperative inflammation and apoptosis and late postoperative adhesion strength. methods. Study 1 involved four treatment groups: one with flaps made only with the FS laser (FS group), one with flaps made with the FS laser followed by excimer laser ablation (FS+LASIK), one with flaps made with the MM, and one with flaps made with the MM followed by excimer laser ablation (MM+LASIK). The eyes were analyzed by histology and TUNEL staining for apoptosis at 4 and 24 hours. Study 2 involved two reference groups: an FS group and an MM group. Adhesion strength was measured with a tension meter 1 and 3 months later. results. Study 1: Inflammatory cell infiltration in the central cornea was significantly greater in the FS group than in the MM group at 4 and 24 hours (P < 0.05) and was significantly greater in the FS+LASIK group than in the MM+LASIK group at 24 hours (P < 0.05). Infiltration at the peripheral interface was significantly greater in the FS group than in the MM group and was significantly greater in the FS+LASIK group than in the MM+LASIK group at 24 hours (P < 0.05). Study 2: At 1 and 3 months, 126.7 and 191.3 grams of force (gf) were needed to detach the flaps in the FS group, compared with 65 and 127.5 gf in the MM group, respectively. The grams of force needed was significantly higher in the FS group than in the MM group at 3 months (P < 0.05). conclusions. The FS laser produces greater corneal stromal inflammation than the MM early postoperatively without any increase in apoptosis and stronger flap adhesion late postoperatively. Therefore, it may require stronger anti-inflammatory drugs to be administered.

Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na⁺/K⁺ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation’s in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

Purpose. To investigate the effects of mitomycin C (MMC) on the number of keratocytes and the proliferation of fibroblasts after photorefractive keratectomy (PRK) and exposure to ultraviolet B (UV-B) irradiation. methods. The right eyes of New Zealand White rabbits in Groups 1, 2, and 3 (n = 18 each) underwent PRK to correct −10 diopters with 5 mm optical zone. Sponges soaked with 0.02% MMC were applied to the right eyes of Group 1 rabbits for 2 minutes. Antibiotic ointment was applied daily to all rabbits until the epithelium healed completely, after which 0.02% MMC eye drops were applied twice daily to the right eyes in Group 2 until 4 weeks after PRK. Three weeks after PRK, the right eyes of all the remaining rabbits were exposed to 100 mJ/cm² C UV-B radiation. Corneal haziness was assessed biomicroscopically using the Fantes scale every 3 weeks. Six eyes of each group were each enucleated 3, 6, and 12 weeks after PRK, and tissue specimens were stained with hematoxylin and eosin and with TUNEL stain. The tissues were evaluated immunohistochemically with antibody to α-smooth muscle actin (SMA). Cellular changes in the anterior stroma and epithelial basement membrane were evaluated by electron microscopy. results. Corneal haze was observed after PRK and was aggravated by UV-B irradiation. A single intraoperative application of MMC immediately after PRK induced opacity and apoptosis of keratocytes. Twelve weeks after PRK, MMC significantly reduced corneal haze, the number of keratocytes, apoptotic cells, and fibroblasts, even after UV-B irradiation. Relatively large numbers of apoptotic and SMA-positive cells were found only in PRK-treated, non-MMC treated rabbits (Group 3), even after 12 weeks. Three weeks after PRK, dying stromal cells showed cell shrinkage, and chromatin condensation was observed in all treated groups by electron microscopy. Twelve weeks after PRK, fewer keratocytes and inflammatory cells were observed just beneath the epithelial layer in Group 1 than in any of the other groups. conclusions. MMC is a potent inhibitor of corneal haze induced by PRK. MMC reduced the number of keratocytes and fibroblasts after PRK and UV-B irradiation. Although MMC would improve the clinical results of PRK, it has significant toxicity on corneal keratocytes, which did not disappear until 3 months after PRK.

PURPOSE: To evaluate the clinical efficacy and safety of femtosecond laser-assisted arcuate keratotomy (FS-AK) versus toric intraocular lens (IOL) implantation for correcting astigmatism in patients with cataract. METHODS: A retrospective chart review was performed. All patients had senile cataracts with corneal astigmatism (range: +1.00 to +3.00 diopters [D]) before cataract surgery. Twenty-five patients agreed to undergo toric IOL implantation (the toric IOL group). Twenty-three patients did not agree to undergo toric IOL implantation despite astigmatism; however, these patients were not satisfied with their remaining astigmatism following cataract surgery and requested astigmatism correction using FS-AK (the FS-AK group). Visual acuity and intraocular pressure were evaluated, and automated refraction, keratometry, and slit-lamp examinations were performed at 1 day, 1 week, 1 month, and 5 months after surgery. RESULTS: Refractive astigmatism decreased in both groups. The mean preoperative and postoperative refractive cylinders were 1.71 ± 1.15 and 0.78 ± 1.06 D, respectively, in the FS-AK group ( P < .001) and 1.67 ± 0.13 and 0.83 ± 0.097 D, respectively, in the toric IOL group ( P < .001). There were no statistically significant differences between groups at any time during the follow-up period. CONCLUSIONS: FS-AK is a fast, customizable, adjustable, precise, and safe procedure for reducing refractive errors in patients with residual astigmatism after cataract surgery. The results of this procedure are comparable to the toric IOL. [ J Refract Surg. 2015;31(9):574–578.]

Purpose: To examine the effects of nerve growth factor (NGF) on apoptosis and inflammation in the diabetic cornea. Methods: To investigate the effects of NGF on glucose-induced apoptosis, we stained human corneal epithelial cells (HCECs) for annexin-V and propidium iodide (PI), and measured expression of cleaved caspase-3 and the Bcl-2–associated X protein (BAX). Moreover, to examine the effects of NGF on inflammation, we quantified the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using multiplex cytokine analysis, and analyzed nuclear factor-κB (NF-κB) activation and NF-κ-B inhibitor α (IκBα) degradation using Western blot analysis. To investigate the effects in vivo, we induced diabetes in male Sprague–Dawley rats using streptozotocin. The rats were divided into three groups: control, diabetic control, and diabetic NGF; topical NGF was applied three times daily for 3 weeks. We used the TUNEL assay to detect apoptosis in corneal tissue, and immunohistochemistry to identify cleaved caspase-3 and IL-1β. Results: In HCECs, high glucose concentration (≥25 mM) led to reactive oxygen species (ROS) generation, apoptosis, and the release of inflammatory cytokines. Nerve growth factor markedly reduced ROS activation, annexin-PI–positive cells, and levels of cleaved caspase-3, BAX, IL-1β, and TNF-α. In the diabetic rat cornea, apoptosis and inflammation were enhanced, as were levels of cleaved caspase-3 and IL-1β. These responses were markedly reduced by NGF. Conclusions: Apoptosis and inflammation are enhanced in the diabetic cornea; NGF attenuates these responses—both in vivo and in vitro. Therefore, NGF therapy may represent a novel approach for the management of diabetic keratopathy.

Purpose: To evaluate the effect of diquafosol on corneal epithelium in a dry eye model using Transwell culture and a scopolamine-induced dry eye rat model. Methods: Desiccation stress induced in an in vitro dry eye model using human corneal epithelial cells was used, and the cells were incubated with or without diquafosol media diluted at 1:100. Reactive oxygen species (ROS) generation was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was analyzed, and levels of phosphorylated Erk1/2, phosphorylated p90RSK, phosphorylated Akt, IκB-α, and NF-κB-p65 were determined. Levels of IL-1β, TNF-α, IL-6, IL-8, and GM-CSF were quantified. To investigate the in vivo effects of diquafosol, we induced dry eye in Wistar rats using scopolamine hydrobromide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol; topical DIQUAS was applied four times daily for 28 days. We used immunohistochemistry to detect the levels of phosphorylated Erk1/2, phosphorylated p90RSK, and IL-1β, and used the TUNEL assay in corneal tissue. Results: The distribution of highly fluorescent dichlorofluorescein and the proportion of annexin V- and PI-positive cells decreased in the diquafosol medium. Diquafosol increased the levels of phospho-Erk1/2, phospho-90RSK, phospho-Akt, and IκB-α, whereas it significantly decreased the levels of NF-κB-p65, IL-1β, and TNF-α. In vivo, apoptosis was enhanced in dry eye group. This response was markedly reduced and the level of phosphorylated p90RSK and phosphorylated ERK1/2 were upregulated and IL-1β was downregulated by DIQUAS. Conclusions: Diquafosol treatment reduced intracellular ROS levels, apoptosis, and inflammation, all of which were increased in the dry eye model through desiccation.