Accumulating evidence indicates that glial cells, i.e., astrocytes and microglia, are actively involved in the pathogenesis of Alzheimer's disease. 1,2) Thus one promising target of preventive or therapeutic intervention in Alzheimer's disease is suppression of the proinflammatory activity of activated glial cells in the brains of Alzheimer's disease patients. 3, 4) Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is a phenolic compound present in a variety of plants. We recently reported that

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... ng-term administration of ferulic acid for 4 weeks protects mice against learning and memory deficits induced by centrally administered b-amyloid (Ab) 1-42 . 5) In that report, we found that the Ab 1-42 -induced increases in immunoreactivities of glial fibrillary acidic protein (GFAP), the astrocyte marker, and interleukin-1b in the hippocampus are also suppressed by pretreatment with ferulic acid. In the present study, we further characterize the effects of long-term administration of ferulic acid on the activation of microglial cells induced by centrally administered Ab 1-42 in mice. MATERIALS AND METHODS Materials Male ICR mice weighing 18-26 g at the beginning of experiments (Myung-Jin, Inc., Seoul, Korea) were used in all experiments. The mice were housed 5 per cage in a room maintained at 22Ϯ1°C with an alternating 12-h light-dark cycle. Food was available ad libitum. Ab 1-42 (American Peptide Company, U.S.A.) and Ab 42-1 (Bachem, Switzerland) were prepared as stock solutions at a concentration of 37 mg/ml in sterile 0.1 M phosphate-buffered saline (PBS) (pH 7.4) and aliquots were stored at Ϫ20°C until use. Ferulic acid (Sigma) was dissolved each day in tap water at a concentration of 0.006% (w/v). Assay with HPLC with electrochemical detection showed 11% loss of ferulic acid (0.006% w/v) in tap water in 24 h at room temperature. Experimental Design Mice were allowed free access to normal drinking water or ferulic acid solution for 4 weeks. Subsequently, Ab 1-42 was administered via intracerebroventricular injection, and at various time points after the Ab 1-42 injection the brains were analyzed immunohistochemically. Control animals were injected with Ab 42-1 . Intracerebroventricular Injection of Ab b 1-42 The administration of Ab 1-42 was performed according to the procedure established by Laursen and Belknap. 6) Briefly, each mouse was injected at the bregma with a 50-ml Hamilton microsyringe fitted with a 26-gauge needle that was inserted to a depth of 2.4 mm. The injection volume was 5 ml. Immunocytochemistry At various time points after injection of Ab 1-42 , mice were transcardially perfused and postfixed for 4 h in 4% paraformaldehyde. Brains were cryoprotected in 30% sucrose, sectioned coronally (45 mm) on a freezing microtome, and collected in a cryoprotectant for storage at Ϫ20°C. Floating sections of brains were processed as described previously. 5) After 3 10-min rinses in PBS, sections were placed in the cryoprotectant, preincubated for 30 min in 0.1 M PBS with 1% bovine serum albumin and 0.2% Triton X-100, and incubated overnight with the following primary antisera: anti-rat interferon (IFN)-g (1 : 500, R&D); and anti-rat OX-42 (1 : 500, Harlan Sera-Labs). On the following day, the sections were incubated for 1 h in biotinylated rabbit and goat secondary antibody obtained from Vector Laboratories. After incubation with the Vector Elite ABC kit, antigens were detected with 3,3-diaminobenzidine tetrahydrochloride (DAB) as the chromogen. RESULTS Inhibition of Ab b 1-42 -Induced Increase in OX-42 Immunoreactivity by Ferulic Acid To examine whether central Ab 1-42 injection induces microglial activation, OX-42 immunoreactivity, a marker of activated microglia, was examined. OX-42 immunoreactivity was markedly enhanced 8 h after injection of Ab 1-42 (Fig. 1b) , and returned to basal levels at 1 d (data not shown). In animals pretreated with ferulic acid for 4 weeks, the Ab 1-42 -induced increases in hippocampal OX-42 immunoreactivity were effectively blocked (Fig. 1c) . Inhibition of Ab b 1-42 -Induced Increase in IFN-g g Immunoreactivity by Ferulic Acid Interestingly, the time Flavonoids and monophenolic compounds have been well described in recent years as antioxidants and scavengers of reactive oxygen and nitrogen species. In the present study, we aimed to characterize the effects of longterm administration of ferulic acid on the centrally administered b b-amyloid peptide (Ab b) 1-42 -induced activation of microglial cells in mice. Ab b 1-42 increased the immunoreactivity of OX-42, a microglial marker, and interferong g in the hippocampus at 8 h after the intracerebroventricular injection. The effects were suppressed by long-term (4-week) pretreatment with ferulic acid. This inhibition of microglial cell activation may underlie the beneficial effects of long-term administration of ferulic acid on Ab b 1-42 -induced toxicity in vivo. Key words b-amyloid peptide (Ab); microglia; endothelial nitric oxide synthase; ferulic acid; Alzheimer's disease 120 Notes