Isoflurane preconditioning protects neurons from male and female mice against oxygen and glucose deprivation and is modulated by estradiol only in neurons from female mice

Abstract

The volatile anesthetic, isoflurane, can protect the brain if administered before an insult such as an ischemic stroke. However, this protective "preconditioning" response to isoflurane is specific to males, with females showing an increase in brain damage following isoflurane preconditioning and subsequent focal cerebral ischemia. Innate cell sex is emerging as an important player in neuronal cell death, but its role in the sexually dimorphic response to isoflurane preconditioning has not been investigated. We used an in vitro model of isoflurane preconditioning and ischemia (oxygen and glucose deprivation, OGD) to test the hypotheses that innate cell sex dictates the response to isoflurane preconditioning and that 17β-estradiol attenuates any protective effect from isoflurane preconditioning in neurons via nuclear estrogen receptors. Sex-segregated neuron cultures derived from postnatal day 0-1 mice were exposed to either 0% or 3% isoflurane preconditioning for 1 h. In separate experiments, 17β-estradiol and the non-selective estrogen receptor antagonist ICI 182,780 were added 24 h before preconditioning and then removed at the end of the preconditioning period. Twenty-three hours after preconditioning, all cultures underwent 2 h of OGD. Twenty-four hours following OGD, cell viability was quantified using calcein-AM fluorescence. We observed that isoflurane preconditioning increased cell survival following subsequent OGD regardless of innate cell sex, but that the presence of 17β-estradiol before and during isoflurane preconditioning attenuated this protection only in female neurons independent of nuclear estrogen receptors. We also found that independent of preconditioning treatment, female neurons were less sensitive to OGD compared with male neurons and that transient treatment with 17β-estradiol protected both male and female neurons from subsequent OGD. More studies are needed to determine how cell type, cell sex, and sex steroids like 17β-estradiol may impact on anesthetic preconditioning and subsequent ischemic outcomes in the brain.

Figure 1

Schematic of the preconditioning and drug treatment paradigm in sex-segregated cortical neuron cultures from postnatal day 0 – 1 day mice. E2, 17β-estradiol; ICI, ICI 182,780; IsoPC, isoflurane preconditioning; OGD, oxygen and glucose deprivation; ShamPC, sham preconditioning.

Figure 2

Isoflurane preconditioning (IsoPC) increased cell survival in male and female cortical neurons, while female neurons were protected from OGD regardless of preconditioning. (A) Mean cell survival of sham preconditioning (ShamPC) and IsoPC-treated male and female cortical neurons following OGD. *p < 0.05 ShamPC vs. IsoPC in male cells, *p < 0.01 ShamPC vs. IsoPC in female cells; n = 11. (B) Sex-independent paired difference analysis of variance (ANOVA) of cell survival outcomes between ShamPC and IsoPC groups. Each line connects cell survival outcomes from the same litter (n = 1); *p < 0.05; n = 11. (C) Preconditioning-independent paired difference ANOVA of cell survival outcomes between male and female cortical neurons. Each line connects the cell survival outcome from the same litter (n=1); *p < 0.001; n = 11.

Figure 3

17β-estradiol (E2) attenuates isoflurane preconditioning (IsoPC)-induced protection from oxygen and glucose deprivation (OGD) in female but not male neurons. (A) IsoPC protects male neurons from subsequent OGD independent of E2. *p < 0.05, ShamPC vs. IsoPC; ⁺⁺p < 0.05, E2 IsoPC vs. vehicle IsoPC; n = 9. (B) E2 attenuates IsoPC protection following subsequent OGD in female neurons. *p < 0.01, ShamPC vs. IsoPC; ⁺p < 0.05, E2 ShamPC vs. vehicle ShamPC; n = 9.