EFFECTS OF ESTROGEN AND RALOXIFENE ON NEUROGLIA NUMBER AND MORPHOLOGY IN THE HIPPOCAMPUS OF AGED FEMALE MICE D.-L. LEI, a,b,c J. M. LONG, d J. HENGEMIHLE, a J. O’NEILL, b K. F. MANAYE, b D. K. INGRAM a AND P. R. MOUTON a * a Laboratory of Experimental Gerontology, Gerontology Research Cen- ter, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Balti- more, MD 21224, USA b Department of Biophysics and Physiology, Howard University School of Medicine, Washington, DC 20059, USA c Department of Anatomy and Neurobiology, Xiangya Medical College, Central South University, Changsha, Hunan 410078, China d School of Medicine, University of California at San Diego, La Jolla, CA 92093, USA Abstract—Hormone replacement therapy with the gonadal steroid estrogen or synthetic agents such as raloxifene, a selective estrogen receptor modulator, may affect cellular function in brains of postmenopausal women. In vitro studies suggest that 17 estradiol and raloxifene can alter the micro- glial and astrocyte expression of immuno-neuronal modula- tors, such as cytokines, complement factors, chemokines, and other molecules involved in neuroinflammation and neu- rodegeneration. To directly test whether exogenous 17 es- tradiol and raloxifene affect the number of glial cells in brain, C57BL/6NIA female mice aged 20 –24 months received bilat- eral ovariectomy followed by s.c. placement of a 60-day re- lease pellet containing 17 estradiol (1.7 mg), raloxifene (10 mg), or placebo (cholesterol). After 60 days, numbers of mi- croglia and astrocytes were quantified in dentate gyrus and CA1 regions of the hippocampal formation using immunocy- tochemistry and design-based stereology. The results show that long-term 17 estradiol treatment in aged female mice significantly lowered the numbers of astrocytes and micro- glial cells in dentate gyrus and CA1 regions compared with placebo. After long-term treatment with raloxifene, a similar reduction was observed in numbers of astrocytes and micro- glial cells in the hippocampal formation. These findings indi- cate that estrogen and selective estrogen receptor modula- tors can influence glial-mediated inflammatory pathways and possibly protect against age- and disease-related neuropathology. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: 17 estradiol, microglia, astrocytes, stereology, hippocampal formation, raloxifene. In the past decade, 30 –35 million American women born in the generation after World War II entered menopause, a natural condition of female aging in which reductions in plasma levels of gonadal steroids lead to a variety of affective, metabolic, and physiological changes. The effi- cacy of hormone replacement therapy (HRT) using estro- gen alone, in combination with progestins, or with selective estrogen receptor modulators (SERMs) has been well doc- umented to reduce risk of heart disease and decrease the incidence of osteoporosis (Welty, 1996). Recently, the bal- ance between risk and benefit for HRT has been shifted by evidence from the Woman’s Health Initiative showing that the standard estrogen-progestin combination poses greater risks for breast cancer and coagulopathies than benefits from reduced risks for colon cancer and osteopo- rosis (Women’s Health Initiative, 2002). Also important to this debate are the SERMs, a second class of compounds that binds selectively to  and  estrogen receptors. An example is the SERM raloxifene (RAL; Evista, Eli Lilly Co., Indianapolis, IN, USA), which mimics the effects of estro- gen on retarding bone loss and improving lipid metabolism (Gluck and Maricic, 2002; Boyack et al., 2002), but carries a dramatically reduced risk of cancer through its action as a potent antagonist at estrogen receptors in breast and uterine tissue (Bramlett and Burris, 2002). Epidemiological evidence suggests an inverse rela- tionship exists between duration of HRT and the risk of Alzheimer’s disease (AD) in postmenopausal women (Henderson et al., 1994; Paganini-Hill and Henderson, 1994; Ohkura et al., 1995; Kawas et al., 1997; Henderson, 1997; Kampen and Sherwin, 1998; Sherwin, 1997; Simp- kins et al., 1997; Baldere-schi et al., 1999; Resnick and Maki, 2001; for recent reviews, see Gandy and Duff, 2000; Brinton, 2001). Recent randomized clinical trials of HRT report positive effects in the prevention of AD-type demen- tia, rather than improvement in cognitive performance of patients with probable AD (Henderson et al., 1994; Hen- derson, 1997; Mulnard et al., 2000; Wang et al., 2000). These clinical trials are supported by results of animal studies in female rodents showing that 17 estradiol (E2) has neuroprotective effects against excitotoxicity (Woolley and McEwen, 1994; Brinton et al., 2000), oxidative dam- age (Green et al., 1997; Behl and Manthey, 2000), apo- ptosis (Garcia-Segura et al., 1998, 1999; Liu et al., 2001); acute brain injury (Garcia-Estrada et al., 1993, 1999; Gar- cia-Segura et al., 1996); cerebral ischemia (Sudo et al., 1997; Green et al., 2001; Dubal et al., 2001); neuroglial activation (Dodel et al., 1999; Mor et al., 1999; Mouton et al., 2001); AD-type neurodegeneration (Mouton et al., *Corresponding author. Tel: +1-410-643-4901; fax: +1-410-558- 8323. E-mail address: moutonpe@grc.nia.nih.gov (P. R. Mouton). Abbreviations: AD, Alzheimer’s disease; B6, C57BL/6NIA; CA1, sector 1 cornu ammon; DG, dentate gyrus; E2, 17 estradiol; GFAP, glial fibrillary acidic protein; HRT, hormone replacement therapy; Mac-1, complement 3 receptor; OVX, ovariectomy; PBS, phosphate-buffered saline; PLA, placebo; RAL, raloxifene; SERM, selective estrogen re- ceptor modulator. Neuroscience 121 (2003) 659 – 666 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00245-8 659