Isoflurane preconditioning neuroprotection in experimental focal stroke is androgen-dependent in male mice

Abstract

Isoflurane preconditioning neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of isoflurane preconditioned male brain and whether androgen effects are androgen receptor dependent were assessed. Male C57BL/6 mice were implanted with flutamide (androgen receptor antagonist), or castrated and implanted with testosterone, dihydrotestosterone, flutamide, letrozole (aromatase inhibitor), or vehicle 7-13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 h with 0% (sham preconditioning) or 1% isoflurane (isoflurane preconditioning) and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion and were evaluated 22 h later for infarct volume. For neurobehavioral outcomes, sham and isoflurane preconditioned castrated male+/-dihydrotestosterone groups underwent 1 h of middle cerebral artery occlusion followed by 9 days of reperfusion. Isoflurane preconditioning neuroprotection relative to infarct volume outcomes were testosterone and dihydrotestosterone dose-specific and androgen receptor-dependent. Relative to long-term neurobehavioral outcomes, front paw sensorimotor function improved in isoflurane preconditioned mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, isoflurane preconditioning improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Our findings suggest that androgen availability during isoflurane preconditioning may influence infarct volume and neurobehavioral outcomes in male mice following experimental stroke.

Fig. 1

Schematic overview of the time line and experimental design for castration, hormone or drug administrations, preconditioning and ischemia. Abbreviations: ArKO, P450 estrogen aromatase knockout; ArWT, P450 estrogen aromatase wild-type; DHT, dihydrotestosterone; IsoPC, isoflurane preconditioning; Sham PC, sham preconditioning.

Fig. 2

Cortical and caudate putamen infarction volumes (% contralateral structure) determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining in male C57BL/6 mice preconditioned for 4 h with 0% (Sham PC) or 1% isoflurane (IsoPC). Preconditioning occurred 24 h before 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. (A) Representative images of TTC stained brains from Sham PC and IsoPC male and castrated (CAST) mice. (B) Effects of IsoPC in male and CAST mice. All values are mean±SEM. * P<0.05.

Fig. 3

Cortical and caudate putamen infarction volumes (% contralateral structure) determined by 2,3,5-triphenyltetrazolium chloride staining in male C57BL/6 mice preconditioned for 4 h with 0% (Sham PC) or 1% isoflurane (IsoPC). Preconditioning occurred 24 h before 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. S.c. testosterone (T) and dihydrotestosterone (DHT) implants were placed in selected groups of male mice concurrently undergoing castration 1 week prior to preconditioning. (A) Effects of different T doses (1.5, 5 mg) in castrated (CAST) mice on the response to isoflurane preconditioning in ischemic mouse brain. (B) Effects of different DHT doses (0.5, 1.5 mg) in castrates on the response to isoflurane preconditioning in ischemic mouse brain. All values are mean±SEM. * P<0.05.

Fig. 4

Cortical and caudate putamen infarction volumes (% contralateral structure) determined by 2,3,5-triphenyltetrazolium chloride staining in male C57BL/6 mice preconditioned for 4 h with 0% (Sham PC) or 1% isoflurane (IsoPC). Preconditioning occurred 24 h before 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. S.c. testosterone (T), dihydrotestosterone (DHT), and flutamide (F) implants were placed in selected groups of male mice concurrently undergoing castration 1 week prior to preconditioning. (A) Effects of isoflurane preconditioning in male and castrated (CAST) mice treated with F (5 mg), an androgen receptor antagonist. (B) Effects of isoflurane preconditioning in T (1.5 mg) or DHT (0.5 mg) replaced castrates treated with F (5 mg). All values are mean±SEM. * P<0.05.

Fig. 5

Cortical and caudate putamen infarction volumes (% contralateral structure) determined by 2,3,5-triphenyltetrazolium chloride staining in male mice preconditioned for 4 h with 0% (Sham PC) or 1% isoflurane (IsoPC). Preconditioning occurred 24 h before 2 h of middle cerebral artery occlusion followed by 22 h reperfusion. S.c. testosterone (T) implants (1.5 mg) and osmotic pumps containing either vehicle (polyethylene glycol) or letrozole (1 mg/kg/day) were placed in selected groups of male mice concurrently undergoing castration 13 days prior to preconditioning. (A) Effect of isoflurane preconditioning on infarct volume in P450 estrogen aromatase knockout (ArKO) and wild-type (ArWT) gonadally intact male mice. (B) Effect of isoflurane preconditioning on infarct volume in castrated (CAST) testosterone-replaced C57BL/6 mice treated with either the aromatase inhibitor, letrozole (L), or its corresponding vehicle (V). All values are mean±SEM. * P<0.05.

Fig. 6

Neurobehavioral assessments in castrated (CAST) male C57BL/6 mice preconditioned for 4 h with 0% (Sham PC) or 1% isoflurane (IsoPC). Preconditioning occurred 24 h before 1 h of MCAO followed by 9 days of reperfusion. A 0.5 mg s.c. dihydrotestosterone (DHT) implant was placed in selected groups of male mice concurrently undergoing castration (CAST) 1 week prior to preconditioning. (A) Neuroscore was assessed one (Day 1) and 3 days (Day 3) after MCAO. No significant differences were observed across treatment groups. (B) Contralateral paw use was tested one day before MCAO (Pre), 3 days (Day 3) and 7 days (Day 7) after MCAO. * P<0.05 compared to same group Pre. (C) Novel object recognition was assessed 7 days post MCAO in each group. The dashed line represents a preference ratio of 0.5, indicating lack of retention of novel object. All values are mean±SEM. * P<0.05 compared to all other groups.