The normal corneal epithelium is a nonkeratinized, stratified,

Effects of Nicergoline on Corneal Epithelial Wound Healing in Rat Eyes Su-Young Kim, 1 Jun-Sub Choi, 2 and Choun-Ki Joo 1,2 PURPOSE. To investigate the effect of nicergoline on corneal epithelial wound healing in rats. METHODS. One hundred Sprague-Dawley male rats were divided into two groups, the control group and the nicergoline-treated group, for 2 weeks. Corneal wound healing was evaluated by fluorescein staining after epithelial debridement. Nerve growth factor (NGF) protein and NGF mrna were measured in rat corneas by ELISA and RT-PCR. NGF concentration of lacrimal gland was also evaluated by means of ELISA. Immunofluorescent staining was performed in rat corneas. RESULTS. The corneal wound healing rate was increased in nicergoline-treated rats compared with control rats after debridement. Twenty-four hours after epithelial debridement, corneal NGF protein and NGF mrna levels were higher in the nicergoline-treated group than in the control group. Immunofluorescent staining showed that NGF staining was stronger in nicergoline-treated corneas than in control corneas 24 hours after epithelial debridement. In addition, NGF concentrations in lacrimal glands of the nicergoline-treated group were significantly higher than in the control group 24 hours after epithelial debridement. CONCLUSIONS. Nicergoline accelerated wound healing in rat corneas. The promoting effect of nicergoline in corneal wound healing is likely to be related to increased NGF in corneas and lacrimal glands. (Invest Ophthalmol Vis Sci. 2009;50:621 625) DOI:10.1167/iovs.08-2037 The normal corneal epithelium is a nonkeratinized, stratified, squamous epithelium that consists of basal cells, wing cells, and superficial cells. Intact corneal epithelium is essential for corneal transparency and the maintenance of visual acuity. Clinically, persistent epithelial defects can induce stromal ulcer or opacity and can lead to decreased visual acuity. Photorefractive keratectomy (PRK), laser in situ keratomileusis (LASIK), and epi-lasik are refractive surgeries for increased uncorrected visual acuity. Before applying the laser procedure, PRK and epi-lasik require removal of the epithelium. Disruption of the epithelium activates the wound healing and remodeling process. Rapid wound healing is important for increased visual acuity in patients who undergo PRK and epi- From the 1 Department of Ophthalmology and Visual Science and the 2 Laboratory of Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea. Supported by the Ministry of Education and Human Resources Development (MOE), the Ministry of Commerce, Industry and Energy (MOCIE), and the Ministry of Labor (MOLAB) through the fostering project of the Lab of Excellency. Submitted for publication March 17, 2008; revised August 14, 2008; accepted December 22, 2008. Disclosure: S.-Y. Kim, None; J.-S. Choi, None; C.-K. Joo, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked advertisement in accordance with 18 U.S.C. 1734 solely to indicate this fact. Corresponding author: Choun-Ki Joo, Department of Ophthalmology, Kangnam St. Mary s Hospital, College of Medicine, The Catholic University of Korea, #505, Banpo-Dong, Seocho-Gu, Seoul, Korea; ckjoo@catholic.ac.kr. LASIK and for patients with healing defects of the corneal epithelium (chemical burn, neurotrophic ulcer, diabetes mellitus, abuse of topical anesthetics, limbal cell deficiency). Nerve growth factor (NGF) is a neurotrophin that plays a role in the development and differentiation of neuronal tissues. 1 Recent evidence has demonstrated that NGF was identified in cornea, conjunctiva, tears, and lacrimal gland. 2 5 In addition, one study reported human corneal epithelium expressed NGF receptors (TrkA). 6 Various studies report that NGF promotes corneal healing and is important for maintaining corneal epithelial integrity. 7 9 Nicergoline (10a-methoxy-1,6-dimethylergoline-8 -methanol- 5-bromonicotinate; Sermion [Biogenesis AntiAging, Fish Hoek, South Africa]) is an ergoline derivative known to cross the blood-brain barrier and is now widely and safely used to treat cognitive impairment from stroke and degenerative dementia. 10,11 In vivo studies have shown that nicergoline treatment induces significant increases in the NGF levels within the frontal region of the brain and supports cholinergic neurons, increasing the content of NGF and brain-derived neurotrophic factor in the brain of aged rats. 12,13 The objective of this study was to investigate the positive effects of nicergoline on corneal wound healing in rat eyes through NGF function. MATERIALS AND METHODS Animals One hundred ten eyes from 100 Sprague-Dawley male rats (weight range, approximately 250 300 g) were used. The animals were divided into two groups of 50 rats each. In the control group, rats were housed with food and water. In the treatment group, rats were treated with nicergoline (10 mg/kg/d) for 2 weeks. Nicergoline was reconstituted in 0.1 N (normality) HCl 3.0 ml and diluted with water. In the control group, 0.1 N HCl 3.0 ml and the same amount of water, but not nicergoline, was administered through a gavage feeding needle. In Vivo Animal Model of Corneal Epithelial Wound Healing Rats were deeply anesthetized by intraperitoneal injection of 50 mg/kg tiletamine plus zolazepam (Zoletil; Virbac, Carros, France) and 15 mg/kg xylazine hydrochloride (Rompun; Bayer, Leuverkeusen, Germany). The eye was stabilized by grasping the conjunctiva with forceps, and the epithelium was scraped with a microblade (Sharpoint 78 6900; Surgical Specialties, Reading, PA) under a microscope. All procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wound size was determined by staining with 1% fluorescein and photographing at the initial wound and 24, 48, and 72 hours after debridement. The area of the corneal scrape wound was quantified from the photographs by using a computer-assisted image analyzer. The healing rate was calculated by the following formula: initial wound size wound area at each time point. Protein Extraction and ELISA for NGF Detection from Cornea and Lacrimal Gland The cornea was trephined with a 4.0-mm diameter trephine before epithelial debridement and 24, 48, and 72 hours after wounding (n Investigative Ophthalmology & Visual Science, February 2009, Vol. 50, No. 2 Copyright Association for Research in Vision and Ophthalmology 621

622 Kim et al. IOVS, February 2009, Vol. 50, No. 2 FIGURE 1. Corneal epithelial wound healing in (A) control rat and (B) nicergoline-treated rat. Slit lamp photographs of fluorescein staining of corneal epithelial defects at the initial wound and at 24, 48, and 72 hours after debridement. 5 animals/time point/group). Excised cornea was frozen at 80 C and homogenized in lysis buffer consisting of 100 mm Tris-HCl (ph 7.0), 2% BSA, 1 M NaCl, 4 mm EDTA, 2% Triton X-100, 0.1% sodium azide, and the protease inhibitors. Homogenates were centrifuged at 14,000g for 30 minutes. Resultant supernatants were used for the NGF assay. NGF levels were measured before wounding and 24, 48, and 72 hours after wounding. NGF levels were measured by an ELISA development system (Duoset; R&D Systems, Minneapolis, MN) for rat and mouse. The rat lacrimal gland was excised 24 hours after epithelial debridement, and the NGF level of the lacrimal gland was measured according to the same method (n 10 animals per group). Before ELISA, total protein was measured using the BCA protein assay kit by the Bradford method in cornea and lacrimal gland. 14 The ratio of NGF to total protein (pg/mg) was calculated. Semiquantitative RT-PCR Examination of NGF Gene Expression NGF mrna was determined by RT-PCR. Total RNA was extracted from homogenates of cornea in each group (n 5). Cornea was trephined with a 4.0-mm diameter trephine 24 hours after debridement. The cornea was homogenized in reagent (Trizol; Gibco BRL, Grand Island, NY). Single-strand cdna was synthesized by a first-strand synthesis system for RT-PCR (Superscript III; Invitrogen, Carlsbad, CA) and a random primer and was used as a template for PCR. PCR experiments were normalized to -actin gene expression. PCR amplification was performed with primer (sense, 5 -caacaggactcacaggagca-3 ; antisense, 5 -cacacacgcaggctgtatct-3 ) for NGF mrna. The conditions were 5-minute hot start at 94 C, followed by 30 cycles of denaturation for 1 minute at 94 C, annealing for 1 minute at 58 C, and extension for 1 minute at 72 C. Amplified products were separated by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining. To investigate the relative expression of NGF, band densities were measured with densitometric analysis (Image Master VDS 2.0; Pharmacia Biotech Inc., San Francisco, CA). Immunofluorescence Staining Five rats in each group were used for immunofluorescent staining. Rat corneas 24 hours after debridement were embedded in optical cutting temperature compound (Sakura Finetek, Torrance, CA), frozen, and cut into cryosections (6 m). Frozen sections were thawed, dehydrated, and fixed in cold methanol/acetone (1:1) at 30 C for 10 minutes. Sections were incubated with blocking solution (normal goat serum; Zymed, South San Francisco, CA) for 30 minutes. Immunofluorescent staining was performed with affinity-purified rabbit polyclonal antibodies against NGF (M20; Santa Cruz Biotechnology, Santa Cruz, CA) according to a previously reported method. 15,16 Primary antibodies against NGF were used at a dilution of 1:200 and were incubated for 2 hours at room temperature. Secondary antibodies (Alexa-Fluor 555 [red] conjugated goat anti-rabbit IgG [1:5000]; Molecular Probes, Eugene, OR) were then applied and incubated in a dark chamber for 1 hour, followed by counterstaining with DAPI (4 6-diamidino-2-phenylindole, dihydrochloride; 1:1000) for 5 minutes. After washing with PBS, antifade mounting medium (Gel Mount; Biomedia Corp., Foster City, CA) and a coverslip were applied. Staining was evaluated under an epifluorescence microscope (IX71-F22FL; Olympus, Tokyo, Japan) and photographed with a digital camera (DP70-Set2; Olympus). Statistical Analysis Data were compared among groups and were expressed as mean SD. Statistical analysis was carried out with repeated-measures ANOVA; the significance value was P 0.05. RESULTS Effect of Nicergoline on Corneal Epithelial Wound Healing Quantitative analysis revealed that the healed area was 3.98 1.9 mm 2 in the control group and 5.69 2.6 mm 2 in the FIGURE 2. Corneal epithelial wound healing (healed area) in control group and nicergoline-treated group. Wound healing in the nicergoline-treated group was significantly higher than in the control group (*P 0.05; n 20 per group; ANOVA repeated measure).

IOVS, February 2009, Vol. 50, No. 2 Nicergoline and Corneal Wound Healing 623 FIGURE 3. Corneal NGF concentrations after epithelial debridement (NGF/total protein). Nicergoline treatment increased corneal NGF concentrations compared with the control group (P 0.001; n 5 per group; ANOVA repeated measure). nicergoline-treated group and that the defect area was 11.12 2.8 mm 2 in the control group and 9.83 3.1 mm 2 in the nicergoline-treated group 24 hours after epithelial debridement. At 48 hours, the healed areas of the control and nicergoline groups were 9.24 2.5 mm 2 and 12.20 2.4, and the defect areas of the control and nicergoline groups were 7.39 3.9 mm 2 and 3.09 2.7 mm 2, respectively. At 72 hours, the healed areas of the control and nicergoline-treated groups were 13.26 2.2 mm 2 and 13.34 1.8 mm 2, and the defect areas of the control and nicergoline-treated groups were 3.37 0.8 mm 2 and 1.95 1.6 mm 2. Nicergoline-accelerated wound healing compared with the control group (P 0.05) (Figs. 1, 2). Corneal and Lacrimal NGF Protein Corneal NGF levels are shown in Figure 3. NGF levels in the nicergoline-treated group was significantly higher than in the control group (P 0.001). Immunofluorescent stain showed that NGF staining was stronger in the nicergoline-treated cornea than the control cornea (Fig. 4). The lacrimal NGF concentration (NGF/total protein) of the nicergoline-treated group was significantly higher than that of the control group 24 hours after epithelial debridement (P 0.001; Fig. 5). NGF mrna by Semiquantitative RT-PCR Semiquantitative RT-PCR using normalization to -actin showed that the mean levels of gene expression for NGF in the nicergoline-treated group were 1.61 0.09-fold compared with the control group (Fig. 6). The corneas of the nicergolinetreated group expressed more NGF mrna than those of the FIGURE 5. NGF concentrations of lacrimal gland (NGF/total protein) 24 hours after epithelial debridement. *NGF concentrations were significantly increased in the nicergoline-treated group compared with the control group (P 0.001; n 10 per group, ANOVA repeated measure). control group 24 hours after epithelial debridement (P 0.001). DISCUSSION Corneal epithelium, keratocytes, and endothelium produce NGF in humans and rats. 7 NGF accelerates the corneal epithelial proliferation through its high-affinity receptor, TrkA. 2,3 NGF may play an important role in corneal nerve sensitivity by its release of several neuropeptides and its trophic effect on the peripheral nervous system. 17 20 Corneal nerve damage or impairment of sensory innervation of the cornea induced decreased mitotic rates in the corneal epithelium and reduced acetylcholine, 21,22 which led to a persistent epithelial defect, stromal ulcer. Topically applied exogenous NGF facilitated corneal epithelial healing and improved corneal sensitivity in patients with neurotrophic ulcer. 8 Nicergoline is effective at restoring the age-related deficit of acetylcholine and at increasing the release of acetylcholine in the rat brain. 23 Increased NGF was measured in the basal FIGURE 4. Immunofluorescent stain of NGF in (A) control cornea and (B) nicergoline-treated cornea 24 hours after epithelial debridement. DAPI was used as nuclear staining (blue). Original magnification, 400.

624 Kim et al. IOVS, February 2009, Vol. 50, No. 2 Refractive surgery such as LASIK or PRK leads to corneal nerve destruction. After LASIK, neurotrophic keratopathy happened in some case reports. 26,27 In one study, tear NGF concentration appeared to correlate with corneal sensation and ocular surface dryness after PRK and LASIK. 28 It is possible that nicergoline can accelerate corneal epithelial wound healing in PRK and can stimulate corneal nerve regeneration in LASIK or PRK mediated by increased levels of NGF in lacrimal gland or corneal tissue. At present, we are conducting a trial of nicergoline in patients with corneal epithelial disease, corneal ulcer, refractive surgery, and diabetes. In the future, the types of cells that increase NGF by nicergoline and the molecular mechanism responsible for corneal healing by nicergoline should be investigated. In conclusion, nicergoline stimulated corneal epithelial wound healing in rat corneas. The increased NGF by nicergoline could be a major contributor to the positive effect in corneal wound healing. Acknowledgments The authors thank Ji Eun Oh, Ji Hyoun Woo, and Sung Hyoun Cho for technical expertise (Clinical Research Laboratory, Uijeongbu St. Mary s Hospital, Korea) and Jae Ho Kim (The Cernsan Foundation for Eye Research, Seoul, Korea) for advice and scientific support of this work. References FIGURE 6. Semiquantitative RT-PCR analysis of RNA extracted from cornea 24 hours after epithelial debridement. NGF expression in the corneas of the nicergoline-treated group was 1.61 0.09-fold and was compared with the mean of the control group (*P 0.001; n 5 per group; ANOVA repeated measure). forebrain of old nicergoline-treated rats. 13 In our study, the epithelial healing rate of nicergoline-treated rats was faster than of control rats. ELISA, immunohistochemistry, and RT-PCR results showed that the corneal NGF concentration in the nicergoline-treated group was higher than in the control group. Oral nicergoline has an advantage over direct topical NGF application in that topical NGF is expensive and requires preparation. NGF was present in lacrimal gland, tears, and cornea, and the neurotrophins released from the apical side of acini may appear in tears. 4 The NGF released from the lacrimal gland could secrete into the tears. The nerves in the cornea and lacrimal gland are interconnected as a morphofunctional unit and feedback. The problem of the circuit could lead to dry eye and corneal epithelial disease. In our study, lacrimal gland NGF concentrations in the nicergoline-treated group were significantly higher than in the control group. 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