It has been previously demonstrated that 17β-estradiol (E 2 ) inhibits the response of microglia, the resident brain macrophages, to acute injuries in specific brain regions. We here show that the effect of E 2 in acute brain inflammation is widespread and that the hormone reduces the expression of inflammatory mediators, such as MCP-1, MIP-2 and TNF-α, induced by LPS, demonstrating that microglia are a direct target of estrogen action in brain. Using the APP23 mice, an animal model of

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... 's Disease reproducing chronic neuroinflammation, we demonstrate that ovary ablation (ovx) increases microglia activation at β-amyloid (Aβ) deposits and facilitates the progression of these cells towards a highly reactive state. Long-term administration of E 2 reverts the effects of ovx and decreases microglia reactivity as compared to control animals. In this animal model, these events do not correlate with a reduced number of Aβ deposits. Finally, we show that E 2 inhibits Aβ-induced expression of scavenger receptor-A in macrophage cells, providing a mechanism for the effect of E 2 on Aβ signalling observed in the APP23 mice. Altogether, our observations reveal a substantial involvement of endogenous estrogen in neuroinflammatory processes and provide novel mechanisms for hormone action in the brain. believed to be mediated by the activation of the endogenous estrogen receptors-α (ERα) (10, 11, 12), a ligand-dependent transcriptional factor expressed in several cell types including monocyte-macrophage cells (13). In cell-based assays E 2 was shown to inhibit the metabolic activation of monocytes-macrophages induced by inflammatory stimuli by inhibiting the expression of inflammatory mediators, such as the inducible form of nitric oxide (iNOS) and tumor necrosis factor-α (TNF-α) (14, 15, 16). However, it is still unknown whether expression of inflammatory molecules is regulated by E 2 also in vivo and whether the anti-inflammatory activity of E 2 is present in brain areas with low neuronal expression of ERs. In addition, despite the debated role of E 2 as a protective drug against brain degenerative disorders associated with the menopause (17), little is known on the activity of E 2 on chronic brain inflammation. Activation of microglia cells, the resident macrophage cells of the brain, is a hallmark for neurodegenerative diseases, such as Alzheimer's Disease (AD), Parkinson Disease (PD) and amyotrophic lateral sclerosis (ALS). In these disorders microglia Material and Methods Animals. The study was conducted according the guidelines of the Institutional Animal Care Committee of University of Milan. Hormone and Lipopolysaccharide treatments. Female Sprague-Dawley rats or C57/B6 mice (Charles River Breeding Laboratories) were ovariectomized (ovx) at 6 weeks of age. For acute hormonal treatments, 3 weeks after ovx animals were injected subcutaneously with vehicle (purified corn oil) or 50 µg/kg 17β-estradiol (E 2 ; Sigma, Milan, Italy) 6 h before the injection of Escherichia coli Lypopolysaccharide (LPS; serotype 0.111:B4 from Sigma) (for rats: 10 µg LPS in 3 µl of saline; for mice: 1 µg LPS in 1 µl) in the 3 rd cerebral ventricle (icv) according to specific stereotaxic coordinates (for rats: bregma, -4 mm; lateral, 1 mm; depth, 5 mm; for mice: bregma, -0.25 mm; lateral, 1mm; depth, 2.25 mm) and using a Hamilton Syringe rotated on the coronal plane of about 3° from the orthogonal position, as previously described (10). Chronic estrogen replacement settings: E 2 was administered for 6 weeks by implanting pellets (Innovative Research of America, Sarasota, FL, USA) releasing for 21 days 0.025 mg/day for rats and 0.010 mg/day for mice of placebo or E 2 . Pellets were replaced at the end of the 3 rd week. No alteration in animal behaviour or body temperature and no acute-phase proinflammatory proteins in plasma were detected after LPS icv injection (data not shown). Effect of estrogen withdrawal or replacement was evaluated in each animal using the uterotrophic assay, a standard test for measuring estrogen activity (data not shown). The blood levels of E 2 achieved by pellet release were 20-35 pg/ml, as measured in a subset on mice/rats (data not shown). Experiments were repeated at least two times; each experimental group consisted of at least 5 animals. number of Congo Red-positive plaques surrounded by Mac-1 immunoreactive microglia were counted and subdivided into inflammatory ("I") or hypertrophic ("HI") plaques on the basis of microglia morphological appearance. All three levels of APP23 brain were analysed. Data on the APP23 mice refer only to margin 1; margins 2 and 3, although showing a lower number of plaques, had a similar inflammatory profile as that of level 1. Cell culture RAW 264.7 cells were purchased from ATCC (Manassas, USA) and grown in DMEM medium+10%FBS (ATCC) supplemented with 2 g/L sodium carbonate, 0.11 g/L sodium Maggi A, Ciana P, Belcredito S, Vegeto E 2004 Estrogens in the nervous system: mechanisms and nonreproductive functions. Annu Rev Physiol 66:291-313 3. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 388:507-525 4.