Article Figures & Data
Figures
- Figure 1.
Noradrenaline increases MCP-1 in astrocytes. A, Enriched cultures of astrocytes (Ast) or microglia (μGlia) were incubated with 0 or 10 μm NA for 24 h, then MCP-1 mRNA levels measured by QPCR. Data are mean ± SE of MCP-1 mRNA levels normalized to values for β-actin and are expressed relative to control (100%) values. **p < 0.001, ***p < 0.0001 versus control; n = 8 replicates per group. B, Astrocytes were incubated with 0–25 μm NA, and MCP-1 levels in the media were assessed after 24 h by ELISA. Data are means ± SE of n = 8 replicates per group. *p < 0.05, **p < 0.001, ***p < 0.0005 versus control. C, Astrocytes were transfected with an pMCP-1 luc reporter (0.2 μg) and Renilla control phRG-TK vector. After 24 h, cells were treated with the indicated concentrations of NA, dbcAMP, or the β-AR agonist Isoproterenol, or (D) 10 μm NA together with varying concentration of the β-AR agonist propranolol or the β2-AR agonist ICI118551. MCP-1 promoter activity was measured after 4 h, and differences in transfection efficiencies were corrected for by normalization to renilla luciferase activity. The data are mean ± SE of n = 3–4 replicates and are relative to the MCP-1 activity measured in nontreated cells. *p < 0.05, **p < 0.005 versus control values.
- Figure 2.
MCP-1 decreases extracellular glutamate. A, Primary neurons were incubated with NMDA (20 μm) in the presence or absence of MCP-1 (10 ng/ml), and after 48 h, the extracellular concentration of glutamate was determined. The data are mean ± SE of n = 10 replicates and is shown relative to values measured in control (Ctrl) cells (100% = 40.5 ± 0.7 μm) §p < 0.05 versus control, *p < 0.05 versus NMDA. B, Primary neurons were incubated with NMDA (20 μm) in the presence of conditioned media from astrocytes treated without (CM-C) or with (CM-NA) NA (75 μm) for 24 h and in the absence or presence of a neutralizing antibody against MCP-1 (10 μg/ml). After 48 h, the extracellular concentration of glutamate was determined. The data are mean ± SE of n = 10 replicates and is shown relative to values measured in CM-C cells (100% = 40.5 ± 0.7 μm) §§§p < 0.0005 versus CM-C, *p < 0.05 versus CM-NA.
- Figure 3.
MCP-1 reduces excitotoxicity. A, Primary neurons were treated for 24 h with the indicated concentrations of MCP-1, then preloaded with Fluo-4 A.M. and incubated with glutamate (200 μm). As a control neurons were incubated with glutamate together with the NMDA-R antagonist MK801 (“MK,” 10 μm). The fluorescence signal was measured after 3 min. The data are mean ± SE of n = 12 replicates and is Ca2+ entry relative to that measured in the presence of glutamate alone. *p < 0.05, **p < 0.005, ***p < 0.0001 versus glutamate alone. B, Primary neurons were incubated with 0 (Ctrl) or 200 μm glutamate and the indicated concentrations of MCP-1, and the cellular ATP levels were measured 24 h later. Data are mean ± SE of n = 12 replicates and is the ATP level relative to control cells. §§§p < 0.0001 versus control; *p < 0.05, **p < 0.005, ***p < 0.0001 versus no MCP-1. C, Primary neurons were incubated with NMDA (20 μm) or glutamate (100 μm) in the presence of the indicated concentrations of MCP-1. After 48 h, neuronal viability was assessed by measurement of LDH in the media. Data are mean ± SE of n = 8 replicates and is the LDH released compared with control (CTL; nontreated cells). §p < 0.05 versus control; §§§p < 0.0005 vs control; **p < 0.005 versus NMDA alone or versus glutamate alone. In control cells, 100% LDH reflected 20 ± 1.4% of total LDH released after 48 h (mean ± SE of n = 15 individual measurements).
- Figure 4.
MCP-1 reduces ischemic damage. A, Primary neurons were preincubated with the indicated concentrations of MCP-1 for 24 h, then exposed to OGD or kept under normoxic conditions [control (Ctrl); open bar], after which the media was replaced with fresh media (containing the same amounts of MCP-1). Neuronal viability was assessed 48 h later by measurement of LDH release. Data are mean ± SE of n = 12 replicates and shown relative to LDH release attributable to OGD alone. §§§p < 0.0005 versus control cells; **p < 0.005, ***p < 0.0001 versus OGD. B, Primary astrocytes growing on transwell membranes were transferred to wells containing primary neurons. After 24 h, the cocultures were treated for a further 24 h with fresh media (none), 10 μm NA (NA), or 10 μm NA and 10 μg/ml of MCP-1 neutralizing antibody (NA+αMCP1). After treatment, the astrocytes were removed, the media replaced, the neurons were exposed OGD, and neuronal viability assessed after 24 h. Control neurons (Ctrl; open bar) were kept normoxic. The data are the mean ± SE on n = 8 replicates and is LDH release relative to OGD treated only cells. ***p < 0.0005 versus control cells; *p < 0.05 versus none; §p < 0.05 versus NA. In control cells, 100% LDH reflected 20 ± 1.4% of total LDH released after 48 h (mean ± SE of n = 15 individual measurements).
