Cribed previously [37]. Hippocampal tissue was homogenized in Trisbuffered saline (20 mM Tris and 137 mM NaCl, pH 7.6) supplemented with protease inhibitors. The formic acid-soluble Ab was collected, and neutralized with 1 M Tris buffer (pH 11). The levels of Ab1?0 and Ab1?2 peptides were analyzed using human b Amyloid Ab1?0 and Ab1?2 Ab colorimetric sandwichImmunohistochemistryFor immunofluorescent staining, cross sections of the brain were incubated overnight at room temperature with primary antibodies (Bam-10, 1:3000, Sigma-Aldrich, c-fos, 1:3000, Chemicon andStress Did Not Affect Plaque PathologyELISA kits (Wako Pure Chemical Industries Ltd., Japan) according to the manufacturer’s instructions.Statistical analysisAll data were given as means 6 standard error of the mean (SEM). Data distribution was evaluated and student t test was then used to test the difference between non-stressed and stressed groups. A value of p,0.05 was considered statistically significant.Table 1. The number of Thioflavin 18325633 S-positive plaques/section in the brains of TgCRND8 mice at the age of 1, 3 or 6 monthold.Age of animals 1 month-old 3 month-old 6 month-oldNumber of plaques in the brain/section 0 1964.5Results Age-related Ab deposition in the brain of TgCRND8 miceConsistent with previous studies [32], no Ab plaque was found in either cortex or hippocampus of TgCRND8 at the age of 1 month (Fig. 1A, Table 1), and Ab plaques could be observable in either cortex (arrow Fig. 1B, Table 1) or hippocampus (arrow head in Fig. 1B, Table 1) at the 24272870 age of 3 months. It was found that Ab plaques increased with age and abundant plaques were observed in cortex (arrows in Fig. 1C, Table 1) or hippocampus at the age of 6 months (arrow heads in Fig. 1C, Table 1).doi:10.1371/journal.pone.0053480.tRestraint stress activated hypothalamic neurons in TgCRND8 miceTo determine whether the restraint treatment induces stress response on TgCRND8 mice, we examined whether the neurons in hypothalamus was activated by restraint stress treatment. Consistent with previous study [38?0], few c-fos, if any, could be observed in both paraventricular (PVN) (Fig. 2C) and SON (Fig. 2D and E) of TgCRND8 mice. However, stress Title Loaded From File induced intensive c-fos expression in the two nuclei (Fig. 2A and B, respectively). Quantitative analysis also showed a significant difference in the number of c-fos immunoreactive nuclei in SON between the stressed and non-stressed animals (Fig. 2E). Colabeling of c-Fos and oxytocin in PVN (Fig. 3A ) and SON (Fig. 3E ) further confirmed that c-fos was induced in the oxytocin-neurons of hypothalamus. The findings corroborated that restraint stress was able to induce stress response in hypothalamus of the experimental mice.Restraint stress increased the plasma corticosterone level of TgCRND8 miceThe stress response by the restraint treatment was further confirmed by the increased plasma corticosterone level of TgCRND8 mice. The basal levels of circulating corticosterone in TgCRND8 mice were 69.2612.5 ng/ml at the age of 3 Title Loaded From File monthold and 146.5632.1 ng/ml at the age of 6 month-old (Fig. 4). After the 6 h-2 m stress, the plasma corticosterone levels in TgCRND8 mice significantly increased to 341.1657.3 and 409676.3 ng/ml, respectively (Fig. 4).Restraint stress did not influence cortical and hippocampal amyloid plaque loadsTo test whether behavioral stress directly affects the onset and progression of Ab pathology, TgCRND8 mice at the age of 1 or 4 months were subjected to restraint.Cribed previously [37]. Hippocampal tissue was homogenized in Trisbuffered saline (20 mM Tris and 137 mM NaCl, pH 7.6) supplemented with protease inhibitors. The formic acid-soluble Ab was collected, and neutralized with 1 M Tris buffer (pH 11). The levels of Ab1?0 and Ab1?2 peptides were analyzed using human b Amyloid Ab1?0 and Ab1?2 Ab colorimetric sandwichImmunohistochemistryFor immunofluorescent staining, cross sections of the brain were incubated overnight at room temperature with primary antibodies (Bam-10, 1:3000, Sigma-Aldrich, c-fos, 1:3000, Chemicon andStress Did Not Affect Plaque PathologyELISA kits (Wako Pure Chemical Industries Ltd., Japan) according to the manufacturer’s instructions.Statistical analysisAll data were given as means 6 standard error of the mean (SEM). Data distribution was evaluated and student t test was then used to test the difference between non-stressed and stressed groups. A value of p,0.05 was considered statistically significant.Table 1. The number of Thioflavin 18325633 S-positive plaques/section in the brains of TgCRND8 mice at the age of 1, 3 or 6 monthold.Age of animals 1 month-old 3 month-old 6 month-oldNumber of plaques in the brain/section 0 1964.5Results Age-related Ab deposition in the brain of TgCRND8 miceConsistent with previous studies [32], no Ab plaque was found in either cortex or hippocampus of TgCRND8 at the age of 1 month (Fig. 1A, Table 1), and Ab plaques could be observable in either cortex (arrow Fig. 1B, Table 1) or hippocampus (arrow head in Fig. 1B, Table 1) at the 24272870 age of 3 months. It was found that Ab plaques increased with age and abundant plaques were observed in cortex (arrows in Fig. 1C, Table 1) or hippocampus at the age of 6 months (arrow heads in Fig. 1C, Table 1).doi:10.1371/journal.pone.0053480.tRestraint stress activated hypothalamic neurons in TgCRND8 miceTo determine whether the restraint treatment induces stress response on TgCRND8 mice, we examined whether the neurons in hypothalamus was activated by restraint stress treatment. Consistent with previous study [38?0], few c-fos, if any, could be observed in both paraventricular (PVN) (Fig. 2C) and SON (Fig. 2D and E) of TgCRND8 mice. However, stress induced intensive c-fos expression in the two nuclei (Fig. 2A and B, respectively). Quantitative analysis also showed a significant difference in the number of c-fos immunoreactive nuclei in SON between the stressed and non-stressed animals (Fig. 2E). Colabeling of c-Fos and oxytocin in PVN (Fig. 3A ) and SON (Fig. 3E ) further confirmed that c-fos was induced in the oxytocin-neurons of hypothalamus. The findings corroborated that restraint stress was able to induce stress response in hypothalamus of the experimental mice.Restraint stress increased the plasma corticosterone level of TgCRND8 miceThe stress response by the restraint treatment was further confirmed by the increased plasma corticosterone level of TgCRND8 mice. The basal levels of circulating corticosterone in TgCRND8 mice were 69.2612.5 ng/ml at the age of 3 monthold and 146.5632.1 ng/ml at the age of 6 month-old (Fig. 4). After the 6 h-2 m stress, the plasma corticosterone levels in TgCRND8 mice significantly increased to 341.1657.3 and 409676.3 ng/ml, respectively (Fig. 4).Restraint stress did not influence cortical and hippocampal amyloid plaque loadsTo test whether behavioral stress directly affects the onset and progression of Ab pathology, TgCRND8 mice at the age of 1 or 4 months were subjected to restraint.
