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. 2010 Apr 14;30(15):5437–5450. doi: 10.1523/JNEUROSCI.5169-09.2010

Figure 2.

Figure 2.

NRG1 stimulates microglial survival, proliferation, and chemotaxis in vitro. Survival of microglia in vitro was assessed by incubating cultured microglia for 3 d in serum-free medium, which was supplemented with increasing doses of NRG1 (0.5–10 nm) or GM-CSF (1 nm) (as a positive control). a, b, Representative wells are shown in which microglia are identified by Iba1 immunostaining after no treatment (a) or addition of 10 nm NRG1 (b). NRG1 promoted microglial survival, and erbB2 inhibition using PD168393 (INH, 10 μm) or mAb 7.16.4 (AB, 4 μg/ml) blocked this effect. c, Quantification of microglial numbers is shown. d–f, Cultured microglia were incubated for 1 day in serum-free medium with (e) or without (d) the addition of NRG1. DAPI staining (blue) demonstrated nuclei, and TUNEL (yellow) revealed those undergoing apoptosis. Treatment with NRG1 (10 nm) significantly decreased apoptosis, an effect that was prevented when microglia were treated with the erbB inhibitor PD168393 in combination with NRG1 (quantification in f). g–i, Proliferation was assessed by incubating microglia in medium supplemented with 5% FBS for 3 d and pulse-labeling with BrdU (yellow). NRG1 treatment significantly increased the proportion of BrdU-positive microglial nuclei, an effect that was erbB2-dependent. j–l, The effects of NRG1 on microglial migration were studied using a Boyden chamber. The addition of NRG1 to the lower well of the chamber (k) increased microglial migration to the inner membrane surface compared with control (j). Inhibition of erbB2 blocked this action. I, Quantification of migrated cells. Scale bars: 100 μm. Error bars represent ± SEM (three to five independent experiments). The statistical tests used were one-way ANOVA with Bonferroni post hoc analysis for all comparisons except for the proliferation experiment in which the data were not normally distributed and ANOVA on ranks with Dunn's post hoc test was used instead. *p < 0.05, **p < 0.005. CON, Control; INH, inhibitor (PD168393); AB, neutralizing antibody (7.16.4).