This was in marked contrast to nonstressed mice, which significantly gained body weight during the 24-day experimental period (Fig. 1C and D). To examine how CVS affects HPA axis activity we determined CORT levels in urine samples collected weekly. Overall, for the entire experimental period, cumulative urine CORT levels Carfilzomib supplier were significantly

higher in stressed than in nonstressed mice in both females (358 ± 38 ng/mL and 138 ± 17 ng/mL, respectively; p < 0.001) and males (13.7 ± 1.4 ng/mL and 9.26 ± 0.81 ng/mL, respectively; p < 0.01; Fig. 2A). In addition, CORT levels under both basal and stressful conditions were markedly higher in females compared to males (p < 0.001 for each condition; Fig. 2A). These higher CORT levels were observed mainly during the first 3 weeks of the 24-day experimental period; in the fourth

week of stress, CORT levels in stressed mice were not significantly higher than those in nonstressed mice (Fig. 2B). Of note, whereas CORT was found primarily in its free form in the urine of female and male mice (85 and 78% of total CORT, respectively), in the blood it was mostly bound to CORT-binding globulin (92 and 83% of total CORT in females and males, respectively) and was detected at significantly lower concentrations compared with urine CORT. In addition, although to a lesser extent than in the urine, blood CORT levels were significantly higher in females than in males (Fig. 2D and E). Given the overall stress-induced increase

in CORT levels, Pembrolizumab purchase and in light of previous studies [8, 32], we expected stress to induce spleen anomalies and, due to its apparent immunosuppressive activity, attenuate the susceptibility to EAE. To evaluate stress-induced spleen anomalies we measured the spleen weight and number of splenocytes in stressed and nonstressed mice following the 24-day experimental period. To determine stress-induced susceptibility to EAE, we immunized stressed and nonstressed mice with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) following Org 27569 the 24-day experimental period and quantified the severity of EAE-related symptoms. As expected, stressed mice exhibited a significant decrease in splenocyte cell count compared to nonstressed controls (females: 38 × 106 cells compared with 52 × 106 cells; p < 0.01; Supporting Information Fig. 2A. Males: 35 ± 2.37 × 106 cells compared with 62 ± 3.5 × 106 cells; p < 0.001; Supporting Information Fig. 2B), as well as decreased spleen weight (females: 75.0 ± 3.2 mg compared with 97.7 ± 5.7 mg; p < 0.01; Supporting Information Fig. 2C. Males: 71.4 ± 4 mg compared with 95.5 ± 6.2 mg; p < 0.01; Supporting Information Fig. 2D). These differences were not due to overall differences in body weight (e.g. differences resulting from decreased weight gain in stressed mice), as the spleen weight/body weight ratio was also decreased by 15% in stressed mice compared with nonstressed mice (Supporting Information Fig. 2E and F).