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. 2014 Jan 15;34(3):963–968. doi: 10.1523/JNEUROSCI.2700-13.2014

Figure 2.

Figure 2.

TrkB receptor activation is required for the neuroprotective effects of PBI-05204. A, Representative photomicrographs showing that neurodegeneration induced by transient OGD can be quantitatively rescued by 23 μg/ml PBI-05204 (compare first three panels). Neuroprotection by PBI-05204 is completely blocked by a minimal concentration of K-252a (3 μm) that inhibits the majority of TrkB autophosphorylation (“P-TrkB”) induced by 1 h of treatment with 200 ng/ml BDNF as shown in the representative immunoblot in B. Scale bar, 200 μm. C, In contrast, 3 μm K-252a had no effect on the activation and autophosphorylation of EGFR by 5 or 10 min. of treatment with 200 ng/ml EGF under the identical experimental conditions. D, Quantification of brain slice assays showing that 3 μm K-252a completely blocks the neuroprotection provided by 23 μg/ml PBI-05204 (compare fourth and fifth bars) but has no significant effect on general neuronal health even in OGD-treated brain slices (compare second and third bars). y-Axis shows the percentage increase in numbers of healthy cortical neurons per brain slice relative to the OGD condition. E, Cotransfection with dominant-negative, truncated TrkB.T1 also blocks neuroprotection by 23 μg/ml PBI-05204. Although TrkB.T1 has no effect on control brain slices (light bars), it completely blocks the neuroprotective effects of PBI-05204 (dark bars). F, Finally, the BDNF-neutralizing antibody mAb#9 (Kolbeck et al., 1999) was used as a third independent method to show that disrupting BDNF-TrkB signaling inhibits the neuroprotective activity of 23 μg/ml PBI-05204. mAb#9 was applied to brain slice explants at the concentrations shown (in μg/ml). Significant blockade of the neuroprotective effects of PBI-05204 compared with the preimmune IgG2 isotype control was observed in the concentration range previously reported for mAb#9 (Deogracias et al., 2012). G, Neuronal degeneration induced by transient OGD was rescuable by the pan-caspase inhibitor Boc-D-FMK (100 μm). D–G, Averages of three independent runs are shown; *p < 0.01 by ANOVA followed by Dunnett's post hoc comparison test.