Accumulating evidence indicates that the ideal therapeutic window for anti-inflammatory treatment targeting neurotoxic microglial activation may be early in the neurodegenerative process 
, highlighting a role for prevention. Microglial NADPH oxidase has been implicated in the progressive nature of AD through the chronic production of ROS in response to Aβ and/or neuron damage and the amplification of pro-inflammatory factors, such as TNFα. Here, we used an in vivo/in vitro
approach to test the hypothesis that inhibition of NADPH oxidase reduces microglia-mediated neuropathology (neuroinflammation, oxidative stress, and neuron damage) and behavioral symptoms (learning and memory deficits) associated with AD. Specifically, using the hAPP(751)SL
transgenic mouse model of AD, the ability of chronic administration (4 month) of two NADPH oxidase inhibitors (apocynin and DM) to prevent toxic microglial activation, reduce plaque size, preserve neuron function, and attenuate cumulative learning and memory deficits was tested.
Importantly, this study also addressed the utility of the hAPP(751)SL
transgenic mouse model for testing anti-inflammatory compounds. The hAPP(751)SL
mice over-express human APP Swiss and London mutations, with elevated expression in neurons throughout the brain, pronounced expression in the hippocampus, and little expression the periphery 
. Amyloid depositions occur as plaques and begin at 3 to 4 months in hAPP(751)SL
mice, with accumulation in the hippocampus commencing at 7 months 
. Using this defined window of plaque deposition, we sought to prevent neuropathology and behavioral deficits by administering the drugs early, from 4–8 months of age, before significant damage had occurred. Despite the high level of Aβ, accumulation of Aβ protein deposits, and behavioral deficits associated with this model 
, we found that at 8 months hAPP(751)SL
mice showed little evidence of neuroinflammation and oxidative stress in saline control animals, as TNFα protein, lipid peroxidation, protein nitration, and NADPH oxidase activation were low, making reduction by any inhibitors improbable. We were intrigued by these findings, as there is a well established link between Aβ and neuroinflammation/oxidative stress 
. Further, post-mortem analysis of AD brains reveal microglia clustered around plaques combined with high levels of oxidative stress and neuroinflammation 
, including activation of NADPH oxidase 
. However, recent reports reveal distinct differences in murine AD models when compared to the human disease that are consistent with our findings. For example, the activation of complement, which is absent in mouse models and present in human disease, has been strongly implicated in the cross-species difference in neuroinflammation 
. Yet, recent reports employing a slightly different murine model, aged (14 month) R1.40 mice, show that NADPH oxidase is activated in these aged mice and that this response can be modified by ibuprophin 
. While this study reported significant effects on plaque load, microglial activation, and indicators of oxidative stress that were modified by ibuprophin 
, the effect on cytokines, neuron damage, and behavioral deficits were not discussed. Thus, it remains possible that with significant aging (perhaps at 14 months) there would be more pronounced evidence of NADPH oxidase activation and neuroinflammation in the hAPP(751)SL
However, despite the lack of evidence for NADPH oxidase-induced pathology in vivo and the consequent inability of either compound tested to regulate the enzyme complex's low function in hAPP(751)SL mice, apocynin (and not DM) treatment reduced microglial number () and Aβ plaque size () in vivo. While in vitro analysis employed the use of immature cells and cell lines, the data revealed that apocynin had no effect on microglia cell death (), nor microglial increases in neuron-glial cultures treated with LPS or Aβ (). Together, these results suggest that the reduction of microglia number in vivo may not be due to direct effects of apocynin on microglial number, but may instead occur through effects on the deposition, such as APP processing/amyloidgenesis, Aβ aggregation, Aβ transport, or degradation of Aβ. Rather, we speculate that perhaps the reduction in microglial number by apocynin may be driven by the reduced plaque size.
The reduction in cortex and hippocampus plaque size conferred by apocynin could be the consequence of a number of processes, including plaque phagocytosis, deposition, degradation, or APP processing and transport. As loss of microglial phagocytic function has been implicated as a key component to the development of plaques and AD progression 
, we next tested the ability of apocynin to regulate Aβ fibril phagocytosis. Our results indicate that treatment of primary microglia cultures with high doses (2 µM) of unlabeled, fibrilized Aβ for 24 hours reduced the ability of microglia to phagocytize fluorescent Aβ after the unlabeled ligand was washed away, supporting that high levels of Aβ may reduce microglial phagocytosis. However, our data also indicate that apocynin does not modify Aβ phagocytosis in any of the conditions tested. Therefore, the reduction in plaque size observed in hAPP(751)SL mice with apocynin treatment are independent the microglial functions tested here.
Another interesting finding emphasized by this work is the disconnect between plaque size and memory deficits in the hAPP(751)SL mice. While apocynin was able to reduce plaque size () and microglial number () in vivo
, there were no significant effects on behavior or synaptic density (synaptophysin staining). This was unexpected, as apocynin has been shown to protect against behavioral deficits linked to chronic brain hypoxia 
and presumably the behavior loss in hAPP(751)SL mice is due to Aβ deposition. As neuronal damage and behavioral deficits in the hAPP(751)SL model peak around 14 months of age, aging may again be necessary to acquire more AD-relevant pathology for this model. Alternatively, it is also possible that a reduction of greater than 50% of the plaque size is necessary to impact synaptic plasticity and behavior.
The in vitro
component of this study demonstrated that both DM and apocynin attenuate Aβ-induced extracellular superoxide (O2•-
) production in primary microglia cultures () and protect against Aβ-induced toxicity in cortical mixed neuron-glia cultures (), as expected. In addition, further in vitro
analysis with apocynin including several functional positive controls revealed that apocynin reduced H2
production () and LPS-induced cytokine production (), demonstrating its established anti-inflammatory properties as expected. Furthermore, apocynin and associated metabolites readily reach the brain, where they have demonstrated properties such as NADPH oxidase inhibition, neuroprotection, and anti-inflammatory properties in other CNS disease models, such as hypoxia 
. This further supports the premise that NADPH oxidase activation was not present in hAPP(751)SL mice at this time.
In summary, apocynin treatment for 4 months in hAPP(751)SL mice reduced plaque size and microglial number, resulting in brains that resembled younger mice. In vitro analysis confirmed that apocynin reduced Aβ toxicity in mixed cortical neuron-glia cultures, and H2O2, O2•-, and TNFα production in primary microglia cultures. However, in vivo analysis revealed no effects for apocynin on synaptophysin (indicative of subleathal neuronal damage) or behavioral measures of learning and memory. In fact, upon further analysis, it was apparent that 8 month old hAPP(751)SL mice presented low levels of neuroinflammation and oxidative stress, which not surprisingly, was unaffected by apocynin. Additional in vitro study indicated that apocynin failed to affect microglial death, proliferation, and phagocytosis, indicating that the microglia number and plaque size reduction in vivo likely occur through unknown mechanisms that are independent of apocynin's anti-inflammatory characteristics. Together, these findings suggest that apocynin is a unique NADPH oxidase inhibitor with anti-β amyloid traits, supporting its possible use as a novel and preventative therapeutic compound for early AD.