Application of adipose-derived stem cells on scleral contact lens carrier in an animal model of severe acute alkaline burn.

PURPOSE: To evaluate the therapeutic effect of human adipose-derived stem cells (hASCs) overlaid on a scleral contact lens (SCL) carrier in a rabbit model of ocular alkaline burn. MATERIALS AND METHODS: After inducing alkaline burn in 11 New Zealand white rabbits, hASCs cultured on SCLs were placed on the right eye of 5 rabbits, SCLs without cells were used in 5, and no treatment was applied in 1 eye. Each eye was examined and photographed for corneal vascularization, opacities, and epithelial defect in week 1, 2, and 4 after surgery. After 1 month, rabbits were killed and the corneas were removed and cut in half for electron and light microscopy examination. RESULTS: Human adipose-derived stem cells were attached to SCL surface and confluent easily. Human adipose-derived stem cells on SCL eyes showed smaller epithelial defect, less corneal opacity, corneal neovascularization relative to SCL eyes. Both groups showed no symblepharon. However, the cornea in the untreated eye was melted in 2 weeks and developed severe symblepharon. CONCLUSION: Human adipose-derived stem cells on SCL can reduce inflammation and corneal haziness in severe ocular alkaline burn injury in rabbits.

Corneal light transmission and roughness after refractive surgery.

PURPOSE: To determine the relation between the corneal light transmission measurements and the epithelial surface properties in hen corneas after different refractive surgery techniques photorefractive keratectomy, laser in situ keratomileusis, and laser-assisted subepithelial keratomileusis, and a group with only epithelial corneal removal (deepithelialization). METHODS: Five groups of hen corneas with different treatments and a control group were analyzed at 30 days. Direct transmittance and corneal light scattering were measured by a scatterometer developed by our group. Quantitative and systematic measurements of external and internal roughness and epithelium thickness were assessed using standard techniques developed for quantitative analysis of microphotographs of the corneal epithelium. RESULTS: Data analysis revealed that the roughness in the epithelial surface was associated with the corneal light transmission. The direct transmittance of light showed a significant correlation with the epithelial roughness in the control (r = -0.99, p < 0.05) and photorefractive keratectomy (r = -0.99, p < 0.05) groups. However, there was no relation between the epithelial thickness and the corneal light transmission measurements. CONCLUSIONS: The experimental results suggested that the roughness of the epithelial surfaces is related to the light transmission in the cornea.

Induction of controlled wound healing with PMMA segments in the deep stroma in corneas of hens.

PURPOSE: Polymethylmethacrylate (PMMA) segments are normally used in additive surgery to treat both corneal ectasia post laser-assisted in situ keratomileusis and keratoconus. The aim of this work was to develop an experimental animal model to induce wound healing in the deep stroma in corneas of hens. METHODS: PMMA segments were implanted in the right eyes of 40 adult hens without suture in the wound incision. Animals were randomized for 5 time points to histopathology study (1, 3, 15, 30, and 90 days) being clinically evaluated during the experiment. RESULTS: Thirty-four eyes (85%) had a successful clinical outcome and intraoperative mistakes occurred in 6 eyes (15%), including anterior chamber perforation resulting in abscess (1), excess of lamellar dissection with segment migration (3), and peripheral incisions evolving with neovascularization (2). At 24 hours, all the epithelial injuries were completed in integrity, but a persistent stromal incision, with a fish mouth form, was observed until day 15. Corneal edema disappeared at the fifth day. Haze, keratocyte cell death, keratocyte proliferation, myofibroblast differentiation, and new matrix production were observed in length around the segment. CONCLUSIONS: Wound healing was induced in the deep corneal stroma by means of PMMA segment implantation in a new animal model developed in hens.

Does mitomycin C cause toxicity in the cornea after photorefractive keratectomy? A comparative wound-healing study in a refractive surgery animal model.

PURPOSE: In this study, we investigated the wound-healing process after photorefractive keratectomy with mitomycin C (MMC) in hen corneas. In addition, we evaluated the synergistic effect of ethanol and MMC. METHODS: Forty-eight adult hens were divided into 3 groups: A: ethanol-assisted debridement plus MMC; B: mechanical debridement plus MMC; and C: mechanical debridement (MMC-untreated control). Photorefractive keratectomy was performed, and the animals were followed up for up to 60 days. Epithelial healing was measured with fluorescein. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end-labeling assay and proliferation was measured by BrdU incorporation. Both myofibroblast differentiation and collagen deposition were evaluated by immunofluorescence and histology. RESULTS: Epithelial wound closure was similar in all 3 groups (P > 0.05). Significant reduction in haze was observed in groups A and B compared with C (P < 0.01), but there was no difference between groups A and B (P > 0.05). Furthermore, there was no difference in the number of apoptotic cells between groups. Proliferation was delayed in both groups A and B compared with C (P < 0.01), but groups A and B did not differ significantly (P > 0.05). Myofibroblasts, cellular density, and collagen deposition were lower in both groups A and B compared with C (P < 0.01), but they were not significantly different from each other (P > 0.05). CONCLUSIONS: Topical application of MMC in hen corneas reproduces the wound healing observed in humans by reducing haze, keratocyte proliferation, myofibroblast differentiation, and new collagen deposition. Synergistic cytotoxic effects of ethanol and MMC were not observed.

Nerve growth factor promotes corneal epithelial migration by enhancing expression of matrix metalloprotease-9.

PURPOSE: Nerve growth factor (NGF) is a neuropeptide essential for the development, survival, growth, and differentiation of corneal cells. Its effects are mediated by both TrkA and p75 receptors. Clinically relevant use of NGF was introduced to treat neurotrophic ulcerations in patients. Herein, we examine the mechanisms by which NGF enhances epithelial wound healing both in vivo and in vitro. METHODS: An animal model using adult hens was implemented for the in vivo experiments. Laser ablation keratectomy was performed and animals were observed for up to 7 days. Epithelial healing was measured with fluorescein. In addition, proliferation was measured using BrdU incorporation and both TrkA and matrix metalloprotease-9 (MMP-9) expression were measured by immunohistochemistry (IHC) and Western blot (WB). In vitro experiments were carried out with telomerase-immortalized human corneal epithelial cells (HCLE). The rate of proliferation was measured using a colorimetric assay and BrdU incorporation. Real-time migration was evaluated with an inverted microscope. MMP-9 expression was evaluated by immunocytochemistry (ICC), WB, zymography, and RT-PCR. Finally, beta-4 integrin (ß4) expression was assessed by ICC and WB. RESULTS: Faster epithelial healing was observed in NGF-treated corneas compared with controls (P < 0.01). These corneas showed increased proliferation, TrkA upregulation, and enhanced MMP-9 presence (P < 0.01). In vitro, faster spreading and migration were observed in response to NGF (P < 0.01). Enhanced proliferation, as well as enhanced TrkA and MMP-9 expression, and decreased ß4 levels were observed after adding NGF (P < 0.01). CONCLUSIONS: NGF plays a major role during the epithelial healing process by promoting migration, a process that is accelerated by cell spreading. This effect is mediated by both the upregulation of MMP-9 and cleavage of ß4 integrin.

Ocular mucosal CD11b+ and CD103+ mouse dendritic cells under normal conditions and in allergic immune responses.

Steady state dendritic cells (DC) found in non-lymphoid tissue sites under normal physiologic conditions play a pivotal role in triggering T cell responses upon immune provocation. CD11b+ and CD103+ DC have received considerable attention in this regard. However, still unknown is whether such CD11b+ and CD103+ DC even exist in the ocular mucosa, and if so, what functions they have in shaping immune responses. We herein identified in the ocular mucosa of normal wild-type (WT) and Flt3-/- mice the presence of a CD11b+ DC (i.e., CD11c+ MHCII+ CD11b+ CD103- F4/80+ Sirp-a+). CD103+ DC (i.e. CD11c+ MHCII+ CD11b low CD103+ CD8a+ DEC205+ Langerin+) were also present in WT, but not in Flt3-/- mice. These CD103+ DC expressed high levels of Id2 and Flt3 mRNA; whereas CD11b+ DC expressed high Irf4, Csfr, and Cx3cr1 mRNA. Additionally, the functions of these DC differed in response to allergic immune provocation. This was assessed utilizing a previously validated model, which includes transferring specific populations of exogenous DC into the ocular mucosa of ovalbumin (OVA)/alum-primed mice. Interestingly, in such mice, topical OVA instillation following engraftment of exogenous CD11b+ DC led to dominant allergic T cell responses and clinical signs of ocular allergy relative to those engrafted with CD103+ DC. Thus, although CD11b+ and CD103+ DC are both present in the normal ocular mucosa, the CD11b+ DC subset plays a dominant role in a mouse model of ocular allergy.

Efficacy and safety of short-duration topical treatment with azithromycin oil-based eyedrops in an experimental model of corneal refractive surgery.

PURPOSE: To assess the tolerance and side effects of azithromycin eyedrops at the ocular surface after corneal refractive surgery in an experimental animal model. METHODS: The effect of azithromycin eyedrops was evaluated in hen corneas that underwent laser-assisted in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK) surgery in 1 eye, using the fellow eye (not manipulated) as a control. Animals were treated bid 3 days prior to surgery and 3 days after surgery with T1225 1.5% azithromycin eyedrops or saline eyedrops (balanced salt solution), or were left untreated as a control. Clinical course and cell biology (apoptosis, proliferation, and differentiation) measurements were assessed. RESULTS: Infections were present in the following proportions of corneas operated on by LASIK: 0% treated with azithromycin, 60% treated with BSS, and 30% untreated. No corneal abscess or keratitis were present in any PRK or unmanipulated corneas. Conjunctival edema and redness were less prevalent in LASIK-operated eyes treated with azithromycin than in BSS-treated or untreated eyes and were not observed in any PRK or unmanipulated corneas. In PRK-operated eyes treated with azithromycin, a decrease was observed in the apoptosis and an increase in the stromal proliferation. There were no differences in these parameters for LASIK and unmanipulated eyes. CONCLUSIONS: Topical administration of T1225 oil-based azithromycin eyedrops was well tolerated in both unmanipulated hen corneas and those treated with corneal refractive surgery (PRK and LASIK). T1225 demonstrated a potent antibiotic effect after LASIK treatment.