IL-6 and Cognitive Changes Figure 2. Imaging of in vivo superoxide production in brain of young and old animals: role of Nox2 and IL-6. (a) Confocal imaging of in vivo DHE oxidation reveals that superoxide-mediated DHE oxidation is increased in neurons in old mice, and this increase is blocked by the NADPH oxidase inhibitor, apocynin, or by the SOD mimetic, C3. Young (4 mo) and old (24 mo) mice were treated orally for 7 days with the Nox inhibitor, apocynin, or for 21 days with the SOD mimetic, C3. (b) Quantitative analyses of fluorescence in hippocampal CA1 and cortex were performed as described in detail in the methods section. Images were obtained using a 406 WI objective. Values are mean6SEM, n = 3. Significance was determined by ANOVA: for CA1 (*old versus young, P = 0.01;**old versus old+apocynin (P = 0.02) or old+C3 (P = 0.01) and cortex (Ctx) (*old versus young, P = 0.01;**old versus old+apocynin (P = 0.01) or old+C3 (P = 0.006) by Tukey’s. (c) Live animal imaging of DHE oxidation using a GE eXplore OptixTM-MX2 whole animal scanner also demonstrates increased DHE oxidation in brain of old (24 mo) relative to young (4 mo) mice. Fluorescence images of brain through intact skull and skin are shown on the left , and fluorescence intensity from ox-DHE is mapped onto a linear pseudocolor scale. Images to the right are ex vivo scans of the same brains showing greater anatomical detail, including fluorescence from the cortical hemispheres and cerebellum. (d) Repeated injections of IL-6 on two consecutive days (5 mg/kg) in old mice induced Nox2 expression (top images, green), and produced a small increase in DHE oxidation (red). However, repeated injections of IL-6 every 12 h (5 mg/kg, times three) to produce sustained systemic levels resulted in a dramatic increased in ox-DHE fluorescence compared to saline-injected old controls (bottom images). Note that the laser excitation power was decreased from 260 mW in (c) to 100 mW (d) to avoid saturating the camera in the scanner. doi:10.1371/journal.pone.0005518.g002 system[28], a finding we confirmed by RT-PCR in brain of old mice as well (Dugan and Zheng, unpublished). Nox2 requires co- assembly with not only p22phox , but its p47phox and p67phox subunits for activity, whereas Nox4 has been shown to be active in the absence of p47phox and p67phox [29]. Since apocynin and AEBSF block translocation and binding of p47phox to the Nox- p22phox complex, Nox4 is insensitive to these inhibitors, as previously reported[30–32]. Therefore, Nox2 represents the only apocynin-inhibitable NADPH oxidase isoform expressed in aged mouse brain. Collectively, these results suggest that synaptic Nox2 activity is significantly and specifically increased in aged mice. To study the effect of IL-6 on synaptosomal Nox2 activity, we measured NADPH-driven O2 consumption and superoxide production in young mice 16 hours after repeated injection of either saline or IL-6 (5 mg/kg, i.p. on 2 consecutive days). IL-6 injected mice had significantly greater NADPH-dependent oxygen consumption (Fig. 3a, F(1,12) = 5.05, p,0.01) and superoxide production (Fig. 3b, F(1,6) = 15.26, p,0.01) than saline controls. We then evaluated synaptosomes prepared from young and old IL-6-/- mice, and found that compared to old wild-type controls, IL-6-/- mice had an attenuation of age-induced increases in Nox2 O2 consumption (Fig. 3a, (F(1,6) = 15.57, p,0.01) and superoxide production (Fig. 3c, d, F(1,6) = 6.34, p,0.05). In fact, Nox2 activity in old IL-6-/- mice was not significantly different than that observed in young wild-type mice. In Figure 3d we compare the NADPH-triggered superoxide ‘‘burst’’ in synaptosomes isolated from wild type old and IL6-/- old mice using spin-trapping EPR spectroscopy. The superoxide burst produced by synaptosomes from IL6-/- old brain was significantly less than from wild-type synaptosomes, confirming a key role for IL-6 on Nox-induced superoxide production at the synapse. Parvalbumin-positive interneurons are decreased in aged prefrontal cortex and hippocampus Dysfunction of hippocampal mediated processes has been associated with age-related cognitive decline [33]. Spatial learning, PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5518