Journal: Iranian Biomedical Journal

Article Title: The Lentiviral Vector Pseudotyped by Modified Rabies Glycoprotein Does Not Cause Reactive Gliosis and Neurodegeneration in Rat Hippocampus

doi: 10.29252/.23.5.324

Figure Lengend Snippet: Schematic representation and frequency of green fluorescent protein (GFP)-expressed neurons in CA1 and CA3 of the rat hippocampus one week after injection of the lentiviral vector into CA1. Upper part, left: a coronal section of the rat brain stained by hematoxylin and eosin. Upper part, LV-RG: double-staining of the hippocampal cells of the vector-injected rats by anti-GFP and anti-NeuN monoclonal antibodies. Upper part, control: staining of the hippocampal cells of the saline-injected rats by anti-neuronal Nuclear protein (NeuN) monoclonal antibody. Lower part: frequency of neurons in CA1 and CA3 regions of rat hippocampus, one week after delivery of the lentivector or saline into CA1. No significant difference was found in the frequency of NeuN-positive cells between control and the vector-injected groups in both regions. LV-RG: the HIV-1-based lentiviral vector pseudotyped by the modified rabies glycoprotein, FUG-B2. Scale bar: 100 µm. Magnification: 20 , 400 × 350 µm 2

Article Snippet: Materials The monoclonal rabbit anti-green fluorescent protein (GFP), mouse anti-neuronal nuclear protein (NeuN), mouse monoclonal anti-glial fibrillary acidic protein (GFAP), and secondary Texas Red-X conjugated goat anti-mouse IgG antibodies were purchased from Merck, Millipore (USA).

Techniques: Injection, Plasmid Preparation, Staining, Double Staining, Bioprocessing, Control, Saline, Modification

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: DNA sequences of the primers used for traditional and real time RT-PCR

Article Snippet: The sections were incubated with rabbit polyclonal anti-LCN2 antibody (1:50 dilution; Santa Cruz), or mouse monoclonal anti-GFAP antibody (1:500 dilution; Biogenex) at 4 °C overnight.

Techniques: Amplification

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: The effect of LCN2-treated ACM on the morphology of astrocytes and microglia. ACM was prepared after the treatment of primary astrocytes with LCN2 (10 μg/ml) for 24 h. The addition of LCN2-treated ACM (ACM-LCN2) induced morphological changes in primary astrocytes and primary microglia cultures after 24 h (A). Primary astrocytes were stained with GFAP antibody (original magnification, ×400), followed by the incubation with anti-mouse IgG-FITC-conjugated secondary antibody. Primary microglia were stained with the peroxidase-labeled isolectin B4 (original magnification, ×100), followed by incubation with diaminobenzidine tetrahydrochloride. Scale bars, 25 μm. The length of the longest process in each astrocyte or the percentage of ramified microglia was assessed by examining several randomly chosen microscopic fields (B). The results are the means ± S.D. (n = 3). *, p < 0.05; compared with the untreated ACM control (ACM-None).

Article Snippet: The sections were incubated with rabbit polyclonal anti-LCN2 antibody (1:50 dilution; Santa Cruz), or mouse monoclonal anti-GFAP antibody (1:500 dilution; Biogenex) at 4 °C overnight.

Techniques: Staining, Incubation, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: JAK2/STAT3 and IKK/NF-κB mediated LCN2 up-regulation of CXCL10 and GFAP expression in astrocytes. Astrocytes were pretreated with the recombinant LCN2 protein (10 μg/ml) for 24 h prior to the treatment with phorbol 12-myristate 13-acetate (100 μg/ml), ATP (3 mm), or IFN-γ (50 units/ml) for 30 min. Astrocytes were also exposed to phorbol 12-myristate 13-acetate (PMA), ATP, or IFN-γ for 30 min without LCN2 pretreatment (A). Alternatively, astrocytes were treated with LCN2 for 0.5–24 h for the time kinetics analysis (B). The levels of phosphorylated STAT3 (pSTAT3 at Ser727 or Tyr705) or total STAT3 protein were then evaluated by Western blot analysis. The results are one representative of more than three independent experiments. Alternatively, astrocytes were pretreated with AG490 (JAK2/STAT3-specific inhibitor, 50 μm) or piceatannol (JAK1/STAT1-specific inhibitor, 50 μm) for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h. The secreted CXCL10 protein was measured by specific ELISA (C). The results are the means ± S.D. (n = 3). *, p < 0.001 compared with the treatment without inhibitors. Astrocytes were pretreated with AG490 for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) for 24 h. The expression of GFAP protein levels was assessed by Western blot analysis, respectively (D). After astrocytes were treated with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 1 h, an EMSA analysis of the nuclear extracts was conducted by using a 32P-labeled NF-κB oligonucleotide probe (E). Binding specificity was determined by the supershift assay using antibody against p65 (p65 Ab) or its coincubation with an unlabeled probe containing the NF-κB binding sequence (cold probe) to compete with the labeled oligonucleotide. The results are one representative of more than three independent experiments. Primary astrocytes were pretreated with pyrrolidine dithiocarbamate (PTDC, NF-κB-specific inhibitor; 0–10 μm) for 30 min prior to their treatment with the recombinant LCN2 protein (10 μg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 h (F). The concentration of nitrite in the culture media was measured by the Griess reagent. The results are the means ± S.D. (n = 3). *, p < 0.001 compared with the LCN2 or LPS/IFN-γ treatment alone. The astrocytes were pretreated with SC-514 (IKK-specific inhibitor, 10 μm) for 30 min prior to the treatment with the recombinant LCN2 protein (10 μg/ml) for 8–24 h. The expression of GFAP at mRNA or protein levels after 8 or 24 h was then assessed by RT-PCR or Western blot analysis, respectively (G).

Article Snippet: The sections were incubated with rabbit polyclonal anti-LCN2 antibody (1:50 dilution; Santa Cruz), or mouse monoclonal anti-GFAP antibody (1:500 dilution; Biogenex) at 4 °C overnight.

Techniques: Expressing, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, Sequencing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Role of LCN2 in astrocyte migration and CXCL10 induction in cortical stab wound injury model. Cortical stab wound injury was performed with LCN2 wild-type (LCN2+/+) or LCN2-deficient mice (LCN2−/−) (A, upper). At 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against GFAP. The asterisk indicates stab wound injury site. The boxes indicate 200-μm × 200-μm squares placed for cell counting. Immunohistochemistry results showed that GFAP-positive cells in the peri-injury region were observed in both LCN2+/+ and LCN2−/− mice. A significant decrease in the number of GFAP-positive cells was observed in the immediate vicinity of injury site in LCN2−/− mice (A, lower panel). The results are one representative of more than three independent experiments. Scale bars, 200 μm. The values are the means ± S.D. from three different animals and six independent sections/animal. *, p < 0.05 compared with LCN2−/− mice in the same counting area; #, p < 0.05 between the values indicated. The mRNA levels of lcn2 (upper panel) and CXCL10 (lower panel) in LCN2+/+ and LCN2−/− mice were examined at 2 days after cortical stab wound injury (B). RNA was isolated from the injury site in the cortex and subjected to real time PCR. The injury-induced CXCL10 expression was completely abrogated in LCN2−/− brain as compared with LCN2+/+ littermates. GAPDH was used as a control in the real time PCR. The results are the means ± S.D. (n = 3). *, p < 0.05 compared with uninjured LCN2+/+ mice; **, p < 0.05 compared with injured LCN2+/+ mice; #, p < 0.05 compared with uninjured LCN2−/− mice.

Article Snippet: The sections were incubated with rabbit polyclonal anti-LCN2 antibody (1:50 dilution; Santa Cruz), or mouse monoclonal anti-GFAP antibody (1:500 dilution; Biogenex) at 4 °C overnight.

Techniques: Migration, Cell Counting, Immunohistochemistry, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Essential role of LCN2 in reactive astrocytosis and CXCL10 induction in LPS-induced mouse neuroinflammation models. LCN2+/+ or LCN2−/− were injected with LPS intracortically (A) or icv (B). After 2 dpi, the mice were sacrificed, and cryosections were immunostained with antibodies against LCN2 (green, upper) or GFAP (red, middle). The nuclei were counterstained with DAPI (blue, lower). The asterisk indicates the injection site. A significant decrease in both LCN2 and GFAP expression was observed in LCN2−/− mice. The results are one representative of more than three independent experiments. Scale bars, 100 μm. Quantification of the GFAP-positive cells was done in the cortex (A, upper graph) or hippocampus (B, upper graph). The values are the means ± S.D. from three different animals and five independent sections per animal. The mRNA levels of lcn2, CXCL10, and GFAP in LCN2+/+ and LCN2−/− mice were examined by real time PCR of cortical tissue around the injection site (A, lower graph) or hippocampus (B, lower graph) at 2 days after intracortical or icv injection with LPS, respectively. LCN2−/− mice exhibited markedly lower levels of CXCL10 and GFAP as compared with LCN2+/+ littermates. *, p < 0.05; compared with wild-type LPS-injected mice (LCN2+/+) at the same inflammation model. At 2 days after icv injection of LCN2+/+ mice with LPS, hippocampus was immunostained with antibodies against LCN2 (green) or GFAP (red) (C). The nuclei were counterstained with DAPI (blue). A merged image is shown in the lower right panel. The arrowheads indicate colocalization of LCN2 and GFAP (yellow). Cell bodies and processes of astrocytes in hippocampus were stained with LCN2 antibody. The results are representative of more than three independent experiments. Scale bars, 20 μm.

Article Snippet: The sections were incubated with rabbit polyclonal anti-LCN2 antibody (1:50 dilution; Santa Cruz), or mouse monoclonal anti-GFAP antibody (1:500 dilution; Biogenex) at 4 °C overnight.

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

doi: 10.1074/jbc.M111.299248

Figure Lengend Snippet: Schematic diagram depicting the promotion of CNS cell migration by LCN2-induced CXCL10 (A) and the possible pathway through which LCN2 induces astrocyte migration and morphological changes (B). A, LCN2 up-regulates CXCL10 expression in the multiple cell types in the CNS, such as astrocytes, microglia, neurons, and endothelial cells. Astrocyte-derived CXCL10 acts in a paracrine or autocrine manner to promote cell migration in the inflammatory scene. CXCL10 secreted by other cell types may play a similar role. The lcn2 receptor and CXCL10 receptor (CXCR3) are widely expressed in glia, endothelial cells, and neurons. B, LCN2 up-regulates CXCL10 and GFAP expression in reactive astrocytes through JAK2/STAT3 and NF-κB pathways. Although LCN2-up-regulated CXCL10 promotes cell migration, GFAP induction may lead to morphological changes observed in reactive astrocytosis. Based on the microarray analysis, LCN2 induces the up-regulation of IL-6 and down-regulation of PIAS3, thereby facilitating the STAT3 pathway (dotted line). NO production, downstream of the NF-κB, may cooperate with the STAT3 pathway to induce GFAP expression. NO has been shown to induce GFAP expression in astrocytes (bold dotted line) (54). Moreover, IL-6 previously induced GFAP expression through the STAT3 pathway (96). Other pathways may also participate in astrocyte migration and morphological change under the current conditions.

Article Snippet: The sections were incubated with rabbit polyclonal anti-LCN2 antibody (1:50 dilution; Santa Cruz), or mouse monoclonal anti-GFAP antibody (1:500 dilution; Biogenex) at 4 °C overnight.

Techniques: Migration, Expressing, Derivative Assay, Microarray