Focal transient cerebral ischemia was induced by MCAO (0.21mm silicone coated suture) for 90 minutes followed by reperfusion as described previously (McCullough et al. 2005 (link)). In aging mice a larger 0.23mm silicone coated suture was utilized to achieve occlusion. Sham animals were subjected to sutures of the same size but the suture was not advanced into the middle cerebral artery. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry (LDF, Moor Instruments Ltd, England) during the surgery as previously described (McCullough et al. 2005 (link)). Only the mice in which CBF in MCA area showed a sharp drop of over 85% of control immediately after MCA occlusion were included.
Neurological deficit was confirmed and scored as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by tail; 2, circling to affected side; 3, unable to bear weight on affected side; and 4, no spontaneous locomotor activity or barrel rolling. Monitoring of physiological variables was performed in companion cohorts for all groups prior to MCAO and 60 minutes after reperfusion as described previously (McCullough et al. 2005 (link)).
In ovariectomized (Ovx) females the ovaries were surgically removed 10 days prior to MCAO as described previously (McCullough et al. 2005 (link)). In E2 treated mice 17β-estradiol was delivered by subcutaneous SILASTIC capsule (0.062 inch inner diameter; 0.125 inch outer diameter) filled with 0.035 ml of 17β-estradiol (180μg/ml; Sigma) (McCullough et al. 2005 (link)) in sesame oil implanted at the time of ovariectomy. Serum 17β-estradiol and levels of the inflammatory marker IL-6 was measured in each group by ELISA (E2: IBL HAMBURG, Hamburg, Germany; IL-6: eBioscience, San Diego, CA). Uteruses of female mice were also weighed at sacrifice to confirm end-organ estrogen effects and ELISA values.
Neurological deficit was confirmed and scored as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by tail; 2, circling to affected side; 3, unable to bear weight on affected side; and 4, no spontaneous locomotor activity or barrel rolling. Monitoring of physiological variables was performed in companion cohorts for all groups prior to MCAO and 60 minutes after reperfusion as described previously (McCullough et al. 2005 (link)).
In ovariectomized (Ovx) females the ovaries were surgically removed 10 days prior to MCAO as described previously (McCullough et al. 2005 (link)). In E2 treated mice 17β-estradiol was delivered by subcutaneous SILASTIC capsule (0.062 inch inner diameter; 0.125 inch outer diameter) filled with 0.035 ml of 17β-estradiol (180μg/ml; Sigma) (McCullough et al. 2005 (link)) in sesame oil implanted at the time of ovariectomy. Serum 17β-estradiol and levels of the inflammatory marker IL-6 was measured in each group by ELISA (E2: IBL HAMBURG, Hamburg, Germany; IL-6: eBioscience, San Diego, CA). Uteruses of female mice were also weighed at sacrifice to confirm end-organ estrogen effects and ELISA values.
