Stress treatment for 6 h daily for 2 months. Histopathologic analysis indicated that sparse Ab plaques were detected in the brains of TgCRND8 mice at the age of 3 months (Fig. 5B). However, the stress did not increase the number of plaques in either cortex or hippocampus of the brains of the stressed Protein kinase inhibitor H-89 dihydrochloride animals (Fig. 5A). High plaque-load was found in the brains of the animals at 6 months of stressed mice (Fig. 5C), but the number of Ab plaques in either cortex or hippocampus of stressed mice (Fig. 5C) did not exceed that in the non-stressed mice (Fig. 5D). Quantitative analysis also showed no significant difference in plaque load in either cortex or hippocampus in TgCRND8 mice at the ages of 3 (Fig. 5E) and 6 (Fig. 5F) months between the stressed and non-stressed animals.Figure 1. Cross sections of the brains were stained with Thioflavin S staining in TgCRND8 mice at the age of 1 (A), 3 (B) and 6 (C) months. Scale bar = 100 mm. doi:10.1371/journal.pone.0053480.gRestraint stress did not affect Ab levels in hippocampusThe findings of unchanged Bam10-positive Ab deposits were further corroborated by Ab ELISA analysis in hippocampus of theStress Did Not Affect Plaque PathologyFigure 2. Restraint stress activated hypothalamic neurons in TgCRND8 mice. A : Cross sections of the brains stained with c-fos immunohistochemical staining in PVN (A and C) and SON (B and D) of TgCRND8 mice at the age of 4 months undergone restraint stress (A and B) and non-stress treatment (C and D). E: Quantitative analysis of number of c-fos immunoreactive nuclei in SON of stressed and nonstressed TgCRND8 mice. * indicates statistical differences when compared with their age-matched non-stressed controls at p,0.01. Scale bar = 150 mm. doi:10.1371/journal.pone.0053480.gFigure 3. c-fos was induced in oxytotic neurons. A : Double staining of c-fos (red)/oxytocin (green) in paraventricular (PVN). D : Double staining of c-fos (red)/oxytocin (green) in supraoptic nuclei (SON). Scale bar = 75 mm. doi:10.1371/journal.pone.0053480.gbrains. Soluble Ab was first extracted with lysis buffer (SigmaAldrich, Poole, UK) (Fig. 6A and C), and the remaining Ab was pelleted at 100,000 g and extracted with 70 formic acid (Fig. 6B and D). In either lysis buffer or formic acid extractable fraction, neither Ab1?0 nor Ab1?2 was found to be increased in the restraint stress mice when compared to their non-stressed controls.mice despite both their and our groups used the same method to restrict the P88 web movement of animals. Both groups placed the animals in a plastic tube instead of other methods, e.g. taping the limbs of animals to a board or tie them to pads. Differences between the previous reports and the present study include: 1) intensity and duration of restraint, and 2) mouse line of AD model. Indeed,DiscussionDespite an intensive activation of the neurons of hypothalamic PVN and SON and marked increased levels of corticosterone, the stress marker, under restraint stress, this treatment paradigm failed categorically to modify Ab pathology in the brains of TgCRND8 mice with treatment being initiated at the age of either 1 or 4 months. In this study, we applied 1- and 4-month-old TgCRND8 animals since the animals at the age of 1 month were not old enough to have amyloid plaque pathology in cortex and hippocampus, whereas the animals at the age of 4 month had an observable amyloid plaque pathology in their brains. These results indicate that the restraint stress failed to accelerate not only t.Stress treatment for 6 h daily for 2 months. Histopathologic analysis indicated that sparse Ab plaques were detected in the brains of TgCRND8 mice at the age of 3 months (Fig. 5B). However, the stress did not increase the number of plaques in either cortex or hippocampus of the brains of the stressed animals (Fig. 5A). High plaque-load was found in the brains of the animals at 6 months of stressed mice (Fig. 5C), but the number of Ab plaques in either cortex or hippocampus of stressed mice (Fig. 5C) did not exceed that in the non-stressed mice (Fig. 5D). Quantitative analysis also showed no significant difference in plaque load in either cortex or hippocampus in TgCRND8 mice at the ages of 3 (Fig. 5E) and 6 (Fig. 5F) months between the stressed and non-stressed animals.Figure 1. Cross sections of the brains were stained with Thioflavin S staining in TgCRND8 mice at the age of 1 (A), 3 (B) and 6 (C) months. Scale bar = 100 mm. doi:10.1371/journal.pone.0053480.gRestraint stress did not affect Ab levels in hippocampusThe findings of unchanged Bam10-positive Ab deposits were further corroborated by Ab ELISA analysis in hippocampus of theStress Did Not Affect Plaque PathologyFigure 2. Restraint stress activated hypothalamic neurons in TgCRND8 mice. A : Cross sections of the brains stained with c-fos immunohistochemical staining in PVN (A and C) and SON (B and D) of TgCRND8 mice at the age of 4 months undergone restraint stress (A and B) and non-stress treatment (C and D). E: Quantitative analysis of number of c-fos immunoreactive nuclei in SON of stressed and nonstressed TgCRND8 mice. * indicates statistical differences when compared with their age-matched non-stressed controls at p,0.01. Scale bar = 150 mm. doi:10.1371/journal.pone.0053480.gFigure 3. c-fos was induced in oxytotic neurons. A : Double staining of c-fos (red)/oxytocin (green) in paraventricular (PVN). D : Double staining of c-fos (red)/oxytocin (green) in supraoptic nuclei (SON). Scale bar = 75 mm. doi:10.1371/journal.pone.0053480.gbrains. Soluble Ab was first extracted with lysis buffer (SigmaAldrich, Poole, UK) (Fig. 6A and C), and the remaining Ab was pelleted at 100,000 g and extracted with 70 formic acid (Fig. 6B and D). In either lysis buffer or formic acid extractable fraction, neither Ab1?0 nor Ab1?2 was found to be increased in the restraint stress mice when compared to their non-stressed controls.mice despite both their and our groups used the same method to restrict the movement of animals. Both groups placed the animals in a plastic tube instead of other methods, e.g. taping the limbs of animals to a board or tie them to pads. Differences between the previous reports and the present study include: 1) intensity and duration of restraint, and 2) mouse line of AD model. Indeed,DiscussionDespite an intensive activation of the neurons of hypothalamic PVN and SON and marked increased levels of corticosterone, the stress marker, under restraint stress, this treatment paradigm failed categorically to modify Ab pathology in the brains of TgCRND8 mice with treatment being initiated at the age of either 1 or 4 months. In this study, we applied 1- and 4-month-old TgCRND8 animals since the animals at the age of 1 month were not old enough to have amyloid plaque pathology in cortex and hippocampus, whereas the animals at the age of 4 month had an observable amyloid plaque pathology in their brains. These results indicate that the restraint stress failed to accelerate not only t.
