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Modulators Reduce Microglia Reactivity in Vivo After Peripheral Inflammation

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Modulators Reduce Microglia Reactivity in Vivo After Peripheral Inflammation 219 Selective oestrogen receptor (ER) modulators reduce microglia reactivity in vivo after peripheral inflammation: potential role of microglial ERs Silvia Tapia-Gonzalez, Paloma Carrero, Olga Pernia, Luis M Garcia-Segura and Yolanda Diz-Chaves Instituto Cajal, CSIC, Avenida Doctor Arce 37, E-28002 Madrid, Spain (Correspondence should be addressed to L M Garcia-Segura; Email: [email protected]) Abstract It has been previously reported that the neuroprotective addition, raloxifene had per se a moderate pro-inflammatory hormone oestradiol reduces microglia inflammatory activity. activity in the brain of control female rats and its anti- The objective of this study was to test whether two selective inflammatory activity was partially impaired in female animals oestrogen receptor modulators, tamoxifen and raloxifene, after 1 month of deprivation of ovarian hormones. Spots of modulate in vivo the activation of microglia induced by the oestrogen receptor (ER)-a immunoreactivity were detected peripheral administration of lipopolysaccharide (LPS). Acti- in the soma and cell processes of microglia. Treatment with vation of microglia was assessed in the white matter of the LPS, oestradiol or tamoxifen induced an increase of ER-a cerebellum using immunoreactivity for major histocompat- immunoreactive spots in the perikaryon of microglia, while ability complex-II. Oestradiol, tamoxifen and raloxifene oestradiol antagonized the effect of LPS. The results indicate decreased microglia activation induced by LPS in male and that some oestrogenic compounds decrease brain inflam- ovariectomized female rats, although the doses of oestradiol mation by a mechanism that may involve ERs expressed by that were effective in decreasing microglia reactivity were not microglia. The findings support the potential therapeutic role the same in both sexes. Tamoxifen reduced microglia of oestrogenic compounds as protective anti-inflammatory activation in all experimental groups at all doses tested agents for the central nervous system. (0.5–2 mg/kg b.w.) while raloxifene lost its anti-inflam- Journal of Endocrinology (2008) 198, 219–230 matory activity at the higher dose tested (2 mg/kg b.w). In Introduction 2006) and brain injury (Barreto et al. 2007). These actions may be highly relevant for the neuroprotective actions of the The central nervous system is a target for the ovarian hormone. Activation of microglia is a normal response from hormone oestradiol, a key regulator of reproductive neural tissue to cope with infections and neurodegeneration physiology. Oestradiol acts in different regions of the brain and is oriented to protect neural tissue. However, exaggerated and the spinal cord controlling neuroendocrine secretions, and chronic activation of microglia may lead to neurotoxicity reproductive behaviours and non-reproductive events, modu- and may be detrimental for neural tissue (Thomas 1992, lating synaptic function and plasticity, and affecting mood and Streit et al. 1999, Polazzi & Contestabile 2002, Depino cognition (Fink et al. 1996, McEwen et al. 1997, Woolley et al. 2003, Kim & de Vellis 2005, Marchetti & Abbracchio 2007). In addition, oestradiol is a neuroprotective factor that 2005, Minghetti 2005, Suzumura et al. 2006, Block et al. promotes neuronal survival and tissue integrity in different 2007). Therefore, the regulation of the response of microglial experimental models of neurodegeneration (Garcia-Segura cells to inflammation may represent a therapeutic approach to et al. 2001, Maggi et al. 2004, Suzuki et al. 2006). control neurodegeneration. Recent studies suggest that at least part of the neuropro- Oestrogen receptors (ERs) are candidate targets for possible tective effects of oestradiol may be exerted by reducing brain neuroprotective and anti-inflammatory therapies based on inflammation acting on microglia. Oestradiol has been shown oestrogen actions. The activity of ERs is regulated by their to inhibit the expression of inflammatory mediators by association with transcriptional cofactors that have a tissue- or microglia in vitro (Bruce-Keller et al. 2000, 2001,Drew& cell-specific expression (Klinge 2000, McKenna & O’Malley Chavis 2000, Vegeto et al. 2001, Baker et al. 2004, Dimayuga 2002, Belandia & Parker 2003). This association depends on the et al. 2005) and to reduce microglia reactivity in vivo in three-dimensional conformation of the receptors (Norris et al. different models of brain inflammation (Vegeto et al. 2003, 1999), which is modified by different oestrogenic ligands Journal of Endocrinology (2008) 198, 219–230 DOI: 10.1677/JOE-07-0294 0022–0795/08/0198–219 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 06:34:44AM via free access 220 S TAPIA-GONZALEZ and others . Oestrogenic compounds reduce brain inflammation (Brzozowski et al.1997, Paige et al.1999, Pike 2006). Therefore, vehicles. Some animals were injected with oestrogenic several ER ligands, known as selective ER modulators compounds and the vehicle for LPS and some animals (SERMs), are able to exert tissue- and cellular-specific received injections of LPS and the vehicle for oestrogenic inductions or repressions of the activity of ERs, acting as ER compounds. The oestrogenic compounds tested were: agonists in some tissues and as antagonists in others. SERMs 17b-oestradiol (E2758, Sigma; 50, 250, 500 and 700 mg/kg may therefore represent an alternative to oestradiol for the b.w.), tamoxifen (T5648, Sigma; 0.5, 1 and 2 mg/kg b.w.) and treatment or prevention of neurodegenerative disorders (Cyr raloxifene (R1402, Sigma; 0.5, 1 and 2 mg/kg b.w). The et al.2000, Dhandapani & Brann 2002, Littleton-Kearney et al. oestrogenic compounds were dissolved in 40 ml/mg dimethyl- 2002, Kimelberg et al.2003, Murphy et al.2003, Brinton 2004, sulphoxide (Sigma) in 0.9% NaCl (pH 7.4) and were injected DonCarlos et al.2007, Prokai & Simpkins 2007), since they may at doses that in previous studies resulted in neuroprotective have oestrogenic neuroprotective actions in the brain and effects (Picazo et al. 2003, Ciriza et al. 2004). absence of oestrogenic actions, or even anti-oestrogenic effects, in other tissues, avoiding the peripheral risks associated with Tissue fixation and immunohistochemistry oestrogen therapy.A previous study has shown that two SERMs of clinical use in humans, tamoxifen and raloxifene, are able to Four days after the second injection of LPS, animals were reduce microglia activation in vitro (Suuronen et al.2005). In this deeply anaesthetized with pentobarbital (50 mg/kg, Normon study, we have assessed the anti-inflammatory effects of 17b- Veterinary Division, Madrid, Spain) and perfused through the oestradiol, tamoxifen and raloxifene, in an in vivo model of left cardiac ventricle, first with 50 ml saline solution (0.9% microglia activation induced by the peripheral administration of NaCl) and then with 250 ml fixative solution (4% the bacterial endotoxin lipopolysaccharide (LPS). Our findings paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains indicate that 17b-oestradiol, tamoxifen and raloxifene exert were removed and immersed for 4–6 h at 4 8C in the same anti-inflammatory actions on central nervous system microglia fixative solution and then rinsed with phosphate buffer. in response to peripheral inflammation and suggest that part of Sagittal sections of the cerebellum, 50 mm thick, were these effects may be exerted directly on microglial cells obtained using a vibratome (VT 1000S, Leica Microsystems, expressing ERa. Wetzlar, Germany). Immunohistochemistry was carried out in free-floating sections under moderate shaking. The endogenous peroxidase activity was quenched for 10 min at room temperature in a Materials and Methods solution of 3% hydrogen peroxide in 30% methanol. After several washes in 0.1 M phosphate buffer (pH 7.4), containing Animals and experimental treatments 0.3% BSA, 0.3% Triton X-100 and 0.9% NaCl (washing buffer), Wistar albino male and female rats from the animal colony of sections were incubated overnight at 4 8C with one of the Complutense University were kept in a 12:12 h light/dark following primary antibodies: a mouse monoclonal antibody for schedule and received food and water ad libitum. Animals were major histocompatability complex-II (MHC-II; MRC-OX6, handled in accordance with the guidelines presented in the MCA46G, Serotec, Bicester, UK; diluted 1:300); a mouse UFAW Handbook on the Care and Management of monoclonal antibody for OX42 (MCA275G, Serotec; diluted Laboratory Animals (Poole 1999) and following the European 1:300); a mouse anti-rat lysosomal enzyme (anti CD68, ED1, Union guidelines (Council Directive 86/609/EEC). Experi- Serotec; diluted 1:1000); a rabbit polyclonal antibody for ERa mental procedures were approved by our institutional animal (MC20, sc 542, raised against an epitope mapping at the use and care committee. Special care was taken to minimize C-terminus of ERa of mouse origin, Santa Cruz Bio- suffering and to reduce the number of animals used to the technology,Madrid, Spain; diluted 1:300) or a rabbit polyclonal minimum required for statistical accuracy. Females were antibody for ERb (51-7900, Zymed, Barcelona, Spain; diluted bilaterally ovariectomized under halothane anaesthesia 1:500). Primary antibodies were diluted in washing buffer (Fluothane, AstraZeneca Farmace´utica, Madrid, Spain) to containing 3% normal goat serum. To amplify the immuno- eliminate the influence of cycling changes in ovarian
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