Carmen Martinez-Garcia

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 (β4) 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 β4 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 β4 integrin.

Experimental Model of Laser in situ Keratomileusis in Hens

PURPOSE To develop an experimental animal model of laser in situ keratomileusis (LASIK) in hens. METHODS One hundred adult hens underwent a 60-microm corneal flap followed by -4 diopter excimer laser ablation. Surgical technique, clinical course, and pathology correlations are presented. RESULTS Sixty percent of animals had a successful LASIK outcome according to clinical observations. Pathology showed the integrity of Bowmans layer and homogeneous flap in all eyes with good clinical outcomes. Flap complications such as buttonholes (5%), free-flaps (2%), and slipped flaps (3.4%) occurred with the surgical technique. Corneal abscess (2.9%) was also present. Loss of transparency was noted in 26.4%, which correlates with epithelial ingrowth detected by light histology study. CONCLUSIONS An experimental animal model of LASIK was successfully developed in adult hens. This model could be useful in the study of wound healing and pharmacological modulation after LASIK in an animal with Bowmans layer.

Oleanolic Acid Controls Allergic and Inflammatory Responses in Experimental Allergic Conjunctivitis

Pollen is the most common aeroallergen to cause seasonal conjunctivitis. The result of allergen exposure is a strong Th2-mediated response along with conjunctival mast cell degranulation and eosinophilic infiltration. Oleanolic acid (OA) is natural a triterpene that displays strong anti-inflammatory and immunomodulatory properties being an active anti-allergic molecule on hypersensitivity reaction models. However, its effect on inflammatory ocular disorders including conjunctivits, has not yet been addressed. Hence, using a Ragweed pollen (RWP)-specific allergic conjunctivitis (EAC) mouse model we study here whether OA could modify responses associated to allergic processes. We found that OA treatment restricted mast cell degranulation and infiltration of eosinophils in conjunctival tissue and decreased allergen-specific Igs levels in EAC mice. Th2-type cytokines, secreted phospholipase A2 type-IIA (sPLA2-IIA), and chemokines levels were also significantly diminished in the conjunctiva and serum of OA-treated EAC mice. Moreover, OA treatment also suppressed RWP-specific T-cell proliferation. In vitro studies, on relevant cells of the allergic process, revealed that OA reduced the proliferative and migratory response, as well as the synthesis of proinflammatory mediators on EoL-1 eosinophils and RBL-2H3 mast cells exposed to allergic and/or crucial inflammatory stimuli such as RWP, sPLA2-IIA or eotaxin. Taken together, these findings demonstrate the beneficial activity of OA in ocular allergic processes and may provide a new intervention strategy and potential therapy for allergic diseases.

Scleral changes induced by atropine in chicks as an experimental model of myopia

To determine the effects of intravitreal atropine on scleral growth in the form‐deprived chick as an experimental model of myopia.

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.