The recombinant mouse interferon-γ (IFN-γ proteins were purchased from R&D Systems; Minneapolis, MN). The recombinant mouse LCN2 protein was prepared, as previously described (16). In brief, the recombinant mouse LCN2 protein was expressed as a glutathione S-transferase (GST) fusion protein in the BL21 strain of E. coli, which does not synthesize siderophore. The protein was purified by using glutathione-Sepharose 4B beads (GE Healthcare, Princeton, NJ). All other chemicals, unless otherwise stated, were obtained from Sigma Chemical Co. (St. Louis, MO). The mouse primary astrocyte and microglia cultures were prepared from the brains of 0-to 3-day-old ICR mice (Samtako Co., Osan, Korea), as previously described (16). Primary cultures of dissociated cerebral cortical neurons were prepared from embryonic day 20 (E20) ICR mice, as described previously (17,18). The purity of the glial or neuronal cultures was determined by immunocytochemical staining, using antibodies against microglia-, astrocyte-, or neuron-specific markers. Animals used in the current research were acquired and cared for in accordance with guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was approved by the Institutional Review Board of the Kyungpook National University School of Medicine.

To prepare astrocyte-conditioned media (ACM), primary astrocytes prepared from the brains of 0~3-day-old ICR mice were cultured at the density of 1.5×10⁶ cells in 100 mm plates in DMEM, supplemented with 10% FBS for 24 hr. Primary astrocyte cultures were treated with the recombinant LCN2 protein (10µg/ml) or left untreated for 24 hr. Cells were then washed twice with PBS, and cultured in fresh DMEM for an additional 24 hr. The ACM was then collected, centrifuged at 1,000 rpm for 5 min to remove cell debris, and stored at -80℃ until further analysis.

Cell migration was determined by using a 48-well Boyden chamber (NeuroProbe, Gaithersburg, MD), according to the manufacturer's instructions. ACM was placed into base wells separated from the top wells by polyvinylpyrrolidone-free polycarbonate filters (8µm pore size; 25×80 mm; Neuro-Probe). Cells were harvested by trypsinization, resuspended in DMEM, and added to the upper chamber at a density of 1×10⁴ cells/well. Cells were incubated at 37℃ under 5% CO₂ for 48 hr. At the end of the incubation, non-migrating cells on the inner side of the membrane were removed with a cotton swab. Migrated cells on the underside of the membrane were fixed with methanol for 10 min and stained with Mayer's Hematoxylin (Dakocytomation, Glostrup, Denmark) for 20 min. Photomicrographs of five random fields were taken (Olympus CK2; Tokyo, Japan), and cells were enumerated to calculate the average number of cells that had migrated. All migrated cells were counted, and the results were presented as the mean±SD of triplicates.

The morphological analysis of neuronal cells was performed by using fluorescence microscopy (Olympus BX50). Cells were blocked with 1% BSA in PBS-Tween 20 for 10 min and incubated in PBS containing 3% BSA and mouse anti-microtubule-associated protein-2 (MAP2) antibody (1:600 dilution; Promega). After two washes in PBS-Tween 20, cells were incubated with anti-mouse IgG-fluorescein isothiocyanate (FITC)-conjugated secondary antibody (BD Biosciences, San Jose, CA). Neuronal processes were quantified as previously described, but with a slight modification (19). In brief, the total number of neuronal process that was longer than one cell body diameter was counted. The number of neuronal process was determined from a minimum of five randomly chosen microscopic fields containing at least 200 cells.

Zebrafish embryos were collected from pair mating, raised at 28.5℃ in egg water, and staged according to hours post-fertilization (hpf) and morphological criteria as previously described (20). Wild-type AB zebrafish line was used for this study. Plasmids used in this study were constructed by MultiSite Gateway recombination cloning system (Invitrogen). To generate her4-5' entry clone, her4 promoter from the pBS-her4 plasmid (21) was ligated into the multiple-cloning site of p5E-MCS entry vector (22). The lcn2-middle entry clone (16), EGFP-middle entry clone (pME-EGFP), EGFP-3' (p3E-EGFP) and polyA-3' (p3E-polyA) entry clones (22) were used. For the generation of her4:lcn2:egfp construct, LR reaction was performed with her4-5' entry, lcn2-middle entry, and egfp-3' entry clones according to the Gateway LR reaction manual (Invitrogen). For the generation of her4:egfp construct, LR reaction was performed with her4-5' entry, egfp-middle (pME-EGFP), and polyA-3' (p3E-polyA) (22) entry clones. The final plasmids were prepared using the EndoFree plasmid kit (Qiagen, Valencia, CA) and injected into one-cell stage embryos at a concentration of 30 ng/µl in 0.1 M KCl solution containing 0.05% phenol red. For immunocytochemistry, embryos were fixed in AB Fix (4% paraformaldehyde, 8% sucrose, 1× PBS) overnight at 4℃, embedded in 1.5% agarose/30% sucrose, and frozen in 2-methyl butane chilled by immersion in liquid nitrogen. Transverse sections (10µm) were collected by using a cryostat microtome, and sequentially stained with mouse monoclonal antibody against HuC/D (1:20 dilution; Molecular Probes, Eugene, OR) and Alexa Fluor 568-conjugated goat anti-mouse IgG (Molecular Probes). The relative migration of neuronal cells of zebrafish embryos was quantified by comparing the number of neurons located in the medial position with the number of neurons in the lateral margin of the spinal cord. Ten spinal cord sections for each embryo were quantified.

All data were presented as mean±SD from three or more independent experiments, unless stated otherwise. Statistical comparisons between different treatments were done by either a Student's t-test or one-way ANOVA with Dunnett's multiple-comparison tests by using the SPSS version 14.0K program (SPSS Inc., Chicago, IL). Differences with a value of p<0.05 were considered to be statistically significant.