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. 2017 Nov 8;37(45):10998–11020. doi: 10.1523/JNEUROSCI.3874-16.2017

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Copyright © 2017 the authors 0270-6474/17/3710998-23$15.00/0

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Figure 3. — Levels of pregnenolone and progesterone and its major metabolites were analyzed by GC-MS/MS in the ipsilateral (left, ischemic) hemisphere and in plasma of young adult male and diestrus female mice at different times (hours) after MCAO (for an extended steroid profiling, see Table 4). A, In males, levels of pregnenolone were increased significantly in the ipsilateral hemisphere at 6 h after MCAO (F_(5,46) = 3.0, p < 0.05) and slightly in plasma at 4 and 6 h (F_(5,37) = 8.3, p < 0.001). As in males, plasma levels of pregnenolone were weakly but significantly increased in females at 4 and 6 h (F_(5,28) = 10.7, p < 0.001). Analysis by 2-way ANOVA (sex × time) indeed revealed a significant effect of sex (F_(5,81) = 11.7, p < 0.001). B, In males, progesterone was significantly increased at 4 and 6 h after MCAO both in the ipsilateral hemisphere (F_(5,46) = 9.67, p < 0.0001) and in plasma (F_(5,35) = 11.4, p < 0.0001). Analysis by 2-way ANOVA (sex × time) of brain progesterone levels showed a significant effect of time (F_(5,80) = 7.94, p < 0.0001) and a significant interaction between time and sex (F_(5,80) = 3.20, p < 0.05). Similarly, 2-way ANOVA analysis of plasma progesterone levels revealed a significant effect of time (F_(5,60) = 12.4, p < 0.0001), a significant interaction between time and sex (F_(5,60) = 4.64, p < 0.001), and also an effect of sex (F_(1,60) = 17.8, p < 0.0001). C, In the male ipsilateral hemisphere, levels of 5α-DHP increased continuously between 1 and 6 h after MCAO, and they were still elevated at 24 h (F_(5,45) = 7.33 p < 0.0001). Analysis by 2-way ANOVA (sex × time) of brain 5α-DHP levels showed a significant effect of time (F_(5,79) = 7.05, p < 0.0001), of sex (F_(1,79) = 158.7, p < 0.0001) and a significant interaction between time and sex (F_(5,79) = 5.80, p < 0.0001). D, Levels of 3α,5α-THP in brain and plasma were higher in males compared with females. This observation was supported by 2-way ANOVA (sex × time) for brain (effect of sex: F_(1,80) = 33.7, p < 0.0001) and plasma (effect of sex: F_(1,62) = 29.1, p < 0.0001). E, In males, levels of 20α-DHP were increased significantly in brain (F_(5,42) = 9.0, p < 0.0001) and in plasma (F_(5,34) = 9.60, p < 0.0001) at 4 and 6 h after MCAO. No significant changes over time were observed in females. A two-way ANOVA (sex × time) revealed a significant effect of sex for plasma levels of 20α-DHP (F_(1,61) = 30.5, p < 0.0001). Results are presented as means ± SEM (n = 5–10 per group) and were analyzed by one-way ANOVA followed by Newman–Keuls multiple-comparisons tests. ***p < 0.001, **p < 0.01, *p < 0.05 compared with the control (CTL).