CBD Promotes Neuronal Survival and Neurogenesis Figure 2. Effects of CBD on Ab-induced iNOS, GFAP, S100B, and p50/p65 expression in rat astrocytes. Ab-challenged cells (1 mg/ml) were treated with CBD (1029–1027 M) in the presence of PPARa (MK886, 3 mM) or PPARc (GW9662, 9 nM) antagonist. iNOS, GFAP, and S100B expression was evaluated by Western blot 24 h after treatments. NF-kB activation was evaluated 3 h following treatments by Western blot analysis of its component p50 and p65. Figure shows results of Western blot analysis of proteins (A) and densitometric analysis of corresponding bands (B). Results are the mean 6 S.E.M. of four separate experiments. ***p,0.001 vs. control; up,0.5, uu p,0.01, and uuup,0.001 vs. Ab-challenged cells. doi:10.1371/journal.pone.0028668.g002 and p65. Moreover, CBD also counteracted the Ab-induced calbindin down-regulation. All these observed effects displayed by CBD were caused by the selective activation of PPARc (Figure 3). CBD administration inhibited reactive gliosis and rescued neuronal survival in Ab-injected rat hippocampi through PPARc activation In vivo experiments were aimed at assessing the potential modulating role of CBD on reactive gliosis and neuronal survival in rat hippocampi. Results from Nissl staining indicated that Ab injection caused a severe neuronal loss, especially in CA1 area (site of injection), towards vehicle injected animals, while at the same time Ab caused a marked astrocytic activation, as demonstrated by the increased expression of GFAP. According to in vitro results, in vivo 15 consecutive days administration of CBD (10 mg/kg) almost completely rescued CA1 pyramidal neurons integrity and this was accompanied by a massive down-regulation of gliosis entity as shown by a significant decrease of GFAP immunostaining. Once animals were treated with PPARc antagonist (GW9662, 10 mg/ kg) CBD neuroprotective functions resulted completely abolished (Figure 4 A, B). Moreover, our results indicated that CBD treatment restored neurogenesis in dentate gyrus of Ab-injected rat hippocampi through PPARc selective activation. The DG of rat hippocampi processed for DCX immunofluo- rescence revealed that Ab treatment markedly reduced the entity of neurogenesis, as demonstrated by the reduction of DCX- immunopositive nuclei. CBD treatment was able to counteract Ab-induced DCX depletion up to stimulate the basal neurogenesis PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28668