Ovarian Steroids Mediate Sex Differences in Alcohol Reward After Brain Injury in Mice

Abstract

Intoxication is a leading risk factor for injury, and TBI increases the risk for later alcohol misuse, especially when the injury is sustained in childhood. Previously, we modeled this pattern in mice, wherein females injured at postnatal day 21 drank significantly more than uninjured females, while we did not see this effect in males. However, the biological underpinnings of this sex difference have remained elusive. In this study, we utilize this preclinical model and traditional endocrine manipulations to assess the effect of perinatal sex steroids on post-injury ethanol response. We found that perinatal androgen administration and adult ovariectomy prevented the development of conditioned place preference to ethanol in females, while there was not an effect of gonadectomy either developmental time point on the severity of axonal degeneration. Finally, although TBI increased the number of microglia in males, there was no corresponding effect of gonadectomy, which suggests that males exhibit prolonged neuroinflammation after brain injury irrespective of circulating sex steroids. Taken together, our results indicate a potential role for ovarian sex steroids in the development of greater alcohol preference after a juvenile TBI in female mice.

Keywords: alcohol; androgens; organizational effects; ovaries; sex steroids; traumatic brain injury.

FIGURE 1

Timeline of experimental procedures and tissue responses to endocrine manipulation. (A) Male and female mice underwent neonatal endocrine manipulations at PND 4 (T or oil for females, GDX or control surgery for males). TBI or sham surgery was conducted at PND 21. Additional endocrine manipulations were conducted at PND 60 (females underwent GDX or control surgery, males that underwent early life control surgery had either GDX or control surgery in adulthood, and males that underwent early life GDX had a control surgery). Females (control/sham n = 9, control/TBI n = 9, GDX/sham n = 8, GDX/TBI n = 8, T + GDX/sham n = 9, T + GDX/TBI n = 10), Males (control/sham n = 7, control/TBI n = 7, neonatal GDX/sham n = 7, neonatal GDX/TBI n = 7, adult GDX/sham n = 8, adult GDX/TBI n = 8). All mice were tested for conditioned place preference beginning PND 80. (B) Uterine and ovarian tissue masses and body mass in female mice and (C) epididymal, seminal vesicle and testicular tissue masses and body mass in male mice. An asterisk (*) indicates statistically significant difference from the relevant control group within each sex, ANOVA p < 0.05. Data are presented as mean ± SEM.

FIGURE 2

Conditioned place preference to ethanol. Percent change of conditioned place preference to alcohol from baseline in (A) female and (B) male mice. An asterisk (*) indicates statistically significant difference from control/TBI females, ANOVA p < 0.05. Data are presented as mean ± SEM.

FIGURE 3

Axon degeneration. Axonal damage scores based on silver staining in (A) female and (B) male mice. An asterisk (*) indicates statistically significant difference from sham injured mice, ANOVA p < 0.05. Boxplots show representative range (minimum and maximum), first quartile, median, and third quartile data for each group. Representative photomicrographs are shown for (C) female sham, (D) female TBI, (E) male sham, and (F) male TBI. Scale bar = 500 μm, inset scale bar = 200 μm.

FIGURE 4

Iba1-positive microglia. (A) Bar graphs depicting mean (± SEM) microglial cell counts per mm² in brain regions important for alcohol reward in male and female mice. (B) representative photomicrographs of Iba1 immunohistochemistry in male mice. An asterisk (*) indicates significant main effect of injury, ANOVA p < 0.05. Scale bar = 100 μm, inset scale bar = 20 μm.