1A,B). Quiescent, ramified microglia cells continuously monitor their surrounding environment through filopodia expansion and retraction.19 As shown by time-lapse microscopy, primary microglia in culture expresses fine-structured filopodial processes which continuously reorganize while probing their microenvironment (Fig. 1C). However, upon addition of NH4Cl (5 mmol/L) the cells retract within seconds their filopodia to a length of about 50% of that found in untreated control cells (Fig. 1D). This retraction was accompanied by a reduction of the cell diameter by about 30% as compared

with the control condition (Fig. Pifithrin�� 1D). Microglia are a major phagocytosing cell type that removes cell debris and pathogens in the brain.15, 16 Brain dysfunction in neurodegenerative diseases is frequently associated with increased phagocytotic activity, EPZ 6438 which may represent another surrogate marker for microglia activation.15, 16, 19 As shown in Fig. 2, ammonia inhibited phagocytosis in a subset of microglial cells, thereby reducing overall phagocytosis to

approximately 65% of the control condition (Fig. 2A). However, in phagocytosing microglia cells, the number of phagocytosed fluorescent latex beads per cell was not significantly affected (Fig. 2B,C). These findings may reflect the known heterogeneity of microglia in the brain. Upon activation, microglia increase the expression of the ionized calcium-binding adaptor molecule-1 (Iba-1).18 This protein transduces calcium signals for reorganization of the cytoskeleton, thereby allowing for morphology changes and cell migration.18 As shown by immunofluorescence analysis (Fig. 3A), NH4Cl induced Iba-1 up-regulation in cultured microglia in a time- and triclocarban concentration-dependent manner (Fig. 3A-D). Significant Iba-1 up-regulation occurred 6 hours after NH4Cl (5 mmol/L) treatment (Fig. 3A) but not at 1 or 3 hours of exposure (data not shown). Ammonia concentrations of 5 mmol/L (6 hours) and 1 mmol/L (20 hours), respectively, were sufficient to induce a significant Iba-1 up-regulation

to approximately 1.25 or 1.5-fold of control. However, Iba-1 messenger RNA (mRNA) expression was not up-regulated by NH4Cl (5 mmol/L) after 6 hours (0.65 ± 0.12-fold of control, n = 3) or 20 hours after treatment (0.70 ± 0.07-fold of control, n=4 [P = 0.02]). Microglia activation is frequently accompanied by an increased formation of ROS and an induction of iNOS.13, 14, 16 As shown in Table 1, NH4Cl increased microglial ROS production significantly in a time- and dose-dependent manner as measured by DCF-fluorescence. Pretreatment of microglia with apocynine (300 μmol/L, 30 minutes pretreatment) completely abolished the NH4Cl (5 mmol/L, 6 hours)-induced DCF-fluorescence increase suggestive for an activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by ammonia (0.96 ± 0.05-fold of apocynine-treated controls, n = 4).