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. 2005 Apr 27;25(17):4319–4329. doi: 10.1523/JNEUROSCI.5200-04.2005

Figure 3.

Figure 3.

PAR-1 activation induces astrocyte proliferation. A, Images of BrdU (FITC) and GFAP (Texas Red) immunofluorescence show that all BrdU-containing cells are immunoreactive for GFAP. B, Fluorescent image analysis of cortical astrocytes labeled for BrdU in control condition or after treatment with TFLLR (30 μm) suggests that application of TFLLR increases the number of BrdU-positive cells only in wild-type (wt), but not in PAR1-/-, astrocytes. C, Proliferation is shown as the percentage change in the number of BrdU-positive cells compared with control (mean ± SEM; *p < 0.05; #p < 0.01; unpaired t test; n = 10). D, Cortical astrocytes were treated with TFLLR (30 μm) or forskolin (50 μm) for 12, 24, or 48 h. Five micrograms of protein were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with anti-GFAP antibody. The application of TFLLR did not significantly modify the protein level of GFAP (n = 3). In this experiment, forskolin treatment served as a positive control (*p < 0.05; n = 3). The immunoblot shown is representative of three independent experiments; the results of densitometry are tabulated. E, A monolayer of astrocytes was wounded by scratching and treated with serum, TFLLR (30 μm), or buffer, all in the presence of AraC (see Materials and Methods). Photomicrographs show the migration of astrocytes to the wound area at 0 h (e1, e3) and at 24 h (e2, e4) after TFLLR (e1, e2) or 10% FBS (e3, e4). F, There was no significant difference (p > 0.05; unpaired t test) in wound closure (percentage of area recovered after 24 h of treatment) between vehicle and TFLLR in the absence of serum. Serum treatment served as a positive control and strongly stimulated repopulation of the injured area (p < 0.001; unpaired t test). Data shown are percentage of area recovered (n = 10-12). N.S., Not significant. G, TFLLR or vehicle was injected into the right striatum (anterior, 0.9 mm; lateral, 2.0 mm; and ventral, 2.5 mm from bregma) in a volume of 0.5 μl over 5 min using a Hamilton syringe (33 gauge needle). The animal received one intraperitoneal injection of 50 μg/g BrdU immediately after surgery and the nevery 12 h for 5 d, for a total of 11 injections. Animals were perfusion fixed at 5 d. GFAP and BrdU immunohistochemistry was performed on brain sections (20 μm) 5 d after injection. With this procedure, the cytoplasm of astrocyte cells was stained red (g1, g2) and the nuclei of proliferating cells were stained green (g2, g3). H, Cell counts were performed in five fields of five independent slices per animal (separated by a distance of 50 μm around the injection site). The number sign indicates a significant difference from PBS/BSA injection. All experimentation and analysis were performed blind. A total of three TFLLR-treated mice plus three PBS-0.1% BSA-treated control animals were analyzed. Error bars represent SEM.