For IL-10, three-way ANOVA indicated significant treatment differences (FCX: = 0.003; STR: = 0.003; HC: = 0.048), strain differences (SN: 0.001; HT: = 0.004; FCX: 0.001; STR: 0.001; RN-1 2HCl CX: 0.001; HC: 0.001; CB: 0.001), and sex differences (CB: = 0.028). brain regions. Cytokine levels were elevated in the brain regions of Hg-treated pnd21 SFvF1 but not of FvSF1 offspring, and SFvF1 females had more brain regions expressing cytokines than the males. At pnd70, the serum IgG, serum antibrain Abs, amounts of brain IgG, and brain cytokine levels of all of the Hg-treated offspring were equivalent to those of their appropriate controls, suggesting that developmental Hg exposure did not induce chronic immunological effects. However, the social behaviors of Hg-exposed SFvF1 offspring at pnd70 were significantly impaired, and SFvF1 females displayed greater decline in social behaviors than males, suggesting that the higher neuroinflammation of SFvF1 females earlier in life is associated with the altered behavior. Thus, developmental Hg exposure induces long-lasting effects on social behavior of offspring, which is dependent on sex and genetics and the induction of neuroinflammation. (Ishitobi from gd8 to pnd21. Control animals were treated with normal drinking water. For the Hg-treated groups, 7 SJL/J dams and 6 FVB dams were used; for the control groups, 7 SJL/J dams and 7 FVB dams were used. SFvF1 and FvSF1 offspring were developmentally exposed to HgCl2 and were evaluated for antibrain Ab levels and altered social behavior. Blood and tissue collection. Serum IgG, serum IgG antibrain Abs, IgG presence in brain sections, and multiple cytokines in the brain were measured at pnd21 and pnd70. Dams and randomly selected pnd21 offspring (one male and one female per litter) at weaning were euthanized by CO2 exposure and assayed. Bloods were collected by cardiac puncture; thereafter, brains were perfused with PBS and then collected. Brains of offspring were excised and dissected to obtain substantia nigra (SN), hypothalamus (HT), frontal cortex (FCX), striatum (STR), cortex (CX), hippocampus (HC), and cerebellum (CB). Thereafter, the brain regions were homogenized immediately. The brain homogenates were sonicated for 1min and then centrifuged at 12,000 g for 30min at 4C. Supernatants, which were for brain IgG and cytokine assays, as described later, were collected and stored at ?80C until use. RN-1 2HCl Bloods were stored at 4C for 24h, and sera were collected after centrifugation at 12,000 g for 10min, and then stored at SMAD9 ?80C until use. HgCl2 exposure was stopped at pnd21. At pnd70, all of the F1 offspring were assayed for sociability and urine protein analysis; thereafter, bloods and brains were harvested as described above. One offspring of each sex was randomly selected from each litter for brain IgG and cytokine assays as described later. In RN-1 2HCl addition, within each strain, sex, and treatment at pnd1, pnd21, and pnd70, RN-1 2HCl respectively, one offspring was selected randomly to make whole brain homogenate as antigens (Ags) for serum antibrain Ab detection as described later. Whole brain homogenates were also made from one pnd21 SJL/J mouse and one pnd21 FVB mouse, respectively. Urine protein analysis. Urine protein analysis of SFvF1 and FvSF1 offspring was performed at pnd70. Mice were first anesthetized with CO2; thereafter, urine RN-1 2HCl was harvested and tested immediately with urinalysis strips (SIEMENS, Tarrytown, NY). Protocol was according to manufacturers instruction. ELISA. ELISA was used to measure levels of serum IgG, serum IgG antibrain Abs, and IgG in brain regions. For measurement of serum IgG, goat anti-mouse (GAM) IgG -chain (Sigma, St Louis, MO) was used as capture Abs. Peroxidase-conjugated GAM IgG whole molecule (Sigma) was used as detection Abs; 3,3?,5,5?-tetramethylbenzidine (Sigma) was used as substrate. One mole of H2SO4 was used to stop the peroxidase-substrate reaction. The ELISA plates were read at OD450 on an ELISA analyzer (Bio-Tek, Winooski, VT). For the analysis of serum IgG antibrain Abs, brains of offspring were homogenized in the presence of homogenization buffer containing 1% NP-40, 50mM Tris-Cl (pH 7.6) (Sigma), 150mM NaCl, 2mM EDTA, 1mM Na-orthovanadate, 5mM NaF, and 10 g/ml of proteinase inhibitor cocktail (Sigma). Brain homogenates from both Hg-treated and water-treated offspring (pnd1, pnd21, and pnd70) and normal FVB and SJL/J mice (pnd21) were used as Ags. Protein concentrations.