IL-6 and Cognitive Changes Figure 6. Old IL-6-/- mice or old mice treated with the SOD mimetic, C3, exhibit preserved spatial working memory. (a) Old IL-6-/- mice show improved learning performance on the Novel Object task. Old (22 month) C57BL6 and IL-6-/- mice (on a C57B6 background) were tested for habituation to each of three novel objects, and IL-6-/- mice showed significantly better performance than wild-type mice (statistics shown in Results). (b) Barne’s maze escape latency (time to find the darkened chamber) showed a statistically significant decrease in latency in old IL-6-/- mice but wild- type mice (statistics included in results section). (c) Preservation of spatial learning and memory on the Morris water maze by chronic treatment with C3. Young (6 M), old (24 M), and old-C3-treated (24 M) male mice were tested on components of the Morris water maze as previously described[27]. Graph shows performance on the probe trial component for the specific set of male mice used in the current study. Data from the probe component of the trial are shown for the immediate (1 h) recall, and delayed (24 h) recall. C3 significantly preserved performance at both time-points. Data are mean6SEM, and were analyzed by 2-Way ANOVA followed by Tukey’s post-hoc test, n = 13 young, n = 10 old, n = 12 old-C3. doi:10.1371/journal.pone.0005518.g006 than wild-type mice (Figure 6a). Indeed, overall exploration was higher in IL-6 deficient mice, but there was no floor effect limiting habituation in wild-type mice. Thus, these data suggest greater object recognition memory formation in IL-6 deficient mice. In the Barnes maze test, latencies to locate the escape chamber over the first 4 days of training were analyzed. This is the phase of the experiment where the strategy for escaping is developing and there is typically a significant decrease in escape latencies observed across this time in young mice. In this case, the aged wild-type mice did not display a significant decrease in escape latencies across the first 4 trials (F(3,27) = 1.4, P = 0.25); whereas the IL-6 deficient mice did (F(3,27) = 5.0, P,0.01) Fig. 6b). This also suggests greater spatial memory consolidation in IL-6-/- mice. To rule out possible differences in motor ability between genotypes we evaluated locomotor activity and found no baseline differences between genotypes. Rescue of PV-interneurons in hippocampus was observed not only in IL-6 deficient mice, but in mice treated long-term treatment with an SOD mimetic (C3). Therefore, to explore the relationship between preservation of PV-interneurons and hippocampal forms of memory in C3-treated mice, we analyzed data from the probe component of the Morris water maze for the specific mice used for the PV-interneuron counts. Mice were tested on the Morris water maze as previously described[27]. The number of platform crossings over the area where the removed platform had been located was measured (higher score is better performance). Old males had significantly fewer platform crossings than young males, indicating that spatial memory is reduced in old males (Fig. 6c), On the other hand, aged C3-treated mice performed significantly better than wild-type old mice, showing that treatment with the SOD mimetic preserves spatial memory in aged mice. Data were analyzed by 2-way ANOVA and Tukey’s post-hoc test. Discussion Increased plasma IL-6 is an independent risk factor for many diseases associated with aging, including type 2 diabetes, atherosclerosis, hypertension, frailty, and cardiovascular ischemia, and is a significant predictor of delirium, psychosis, stroke, transition from mild cognitive impairment (MCI) to Alzheimer’s disease (AD)[3,7], and progression of AD in older individuals[2,3]. Recent studies also suggest that even in healthy older adults without MCI or AD, circulating IL-6 is inversely correlated with cognitive performance, and that there is a linear relationship between elevated plasma IL-6 and poor cognitive function. Because of this consistent association between higher plasma IL- 6 and adverse health outcomes, IL-6 was named ‘‘the gerontol- ogist’s cytokine’’ by William Ershler, who speculated that this cytokine regulates a key aging pathway in man[39]. However, the question of whether IL-6 is primarily a marker or is mechanis- tically integral to age-related nervous system dysfunction has not been established. We found that expression of Nox2, the respiratory burst oxidase, was significantly increased in neurons throughout the hippocampus and cortex in aged mouse brain, as documented by immunocytochemistry, Western blot analysis, and mRNA (data not shown). Increased Nox2 protein was associated with enhanced Nox2 activity and superoxide production in neurons and in synaptosomes, and in fact, Nox2 appeared to be the major source of increased cytosolic and synaptosomal superoxide production in old animals. This was supported by results showing that systemic administration of the Nox inhibitor, apocynin reduced superoxide production in neurons in vivo to levels similar to those in young animals. Moreover, two chemically distinct Nox inhibitors, apocynin and AEBSF, fully blocked the age-related increase in Nox2 activity (O2 consumption) in synaptosomes prepared from old animals, and apocynin reduced synaptosomal superoxide production to levels in young synaptosomes, as well. Our data further indicated that Nox2 is tonically active in neurons in vivo, as evidenced by ongoing neuronal superoxide production in normal awake mice which was blocked not only by apocynin, but by oral treatment with the SOD mimetic, C3. Nox2 was also found to be constitutively active in synaptosomes; Nox2-dependent O2 con- sumption and superoxide production in synaptosomes was PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5518