Introduction
Female animals are less susceptible to ischemic brain injury than males 1 . However, the sex difference in ischemic brain injury is abolished by ovariectomy and restored by 17b-estradiol, suggesting that protection is mediated in part via ovarian estrogen. Estradiol is also produced locally in brain via the P450 aromatase, and aromatase gene deletion exacerbates brain injury in female mice 2 . Moreover, P450 aromatase is induced in brain after ischemia specifically in cortical astrocytes 3 . However, it is not clear if P450 aromatase is differentially expressed in male vs. female cortical astrocytes, and if this contributes to the observed sex difference in response to ischemia. We tested the hypothesis that female astrocytes are less susceptible to ischemic injury than male astrocytes, in part due to higher aromatase expression in female vs. male astrocytes.
Methods
Primary cultured cortical astrocytes were prepared from 1–3-day old male and female rat pups separately and grown to confluency in estrogen-free medium. Confluent monolayers (10–14 days in vitro) were incubated under 1% oxygen in glucose free medium for 6 hours (oxygen-glucose deprivation, OGD), and then returned to normoxia and glucose-containing medium for 24 hours. Cell death was assayed by propidium iodide (PI) staining, and surviving cells were identified by calcein-AM. Aromatase activity was measured in male and female astrocytes using a radiometric technique that quantifies the incorporation of tritium from [1b-3 H]androstenedione into 3H-labeled water. To determine the role of aromatase in astrocyte cell death, male and female cultures were treated with aromatase inhibitor Arimidex (100 nM) and aromatase product 17b-estradiol (10 nM).
Results
Female cortical astrocytes sustained significantly less cell death after 6-hour OGD compared to male astrocytes (19±4%, n=13 compared to 41±5%, n=7, respectively, p<0.05). Estradiol treatment reduced cell death in both male (from 41±5% to 18±8%, n=7, p<0.05) and female astrocytes (from 19±4% to 10±3%, n=6, p<0.05). Transfer of culture medium from male to female cells had no effect on the magnitude of cell death. However, media transfer from female to male cells significantly reduced cell death from 41±5% (n=7) to 4±1% (n=3). Arimidex abolished the difference in OGD-induced cell death between male and female astrocytes (40±2%, n=10 in female astrocytes compared to 42±1%, n=10 in male cells, p>0.05). Aromatase activity was higher in female (25±5 fmoles 3H2O/mg/hr, n=11) compared to male astrocytes (13±3 fmoles 3H2O/mg/hr, n=15, p<0.05).
Conclusions
We conclude that astrocytes isolated from neonatal male vs. female cortex exhibit marked differences in response to oxygen-glucose deprivation, likely due to higher aromatase expression and estradiol formation in female cells.