**2.3 Analysis of the data**

For the measurements collectively described in this study, values were expressed as an average ± SD or SE. Means were compared by Student's t test, a paired t test, or an ANOVA, depending upon the conditions. Mean values were considered significantly different if the probability of their being the same was 0.05 or less.

#### **2.4 Vertebrate animals**

Plasma membrane vesicles derived from cerebral capillary endothelial cells were isolated from cow brains obtained at a local slaughterhouse immediately after death. The cows were killed for food according to Federal and State laws, and not for the purposes of these experiments. Cultured cerebral capillary endothelial cells originated either from the fresh cow brains described above, or were purchased commercially (Cell Systems Corporation). The middle cerebral artery occlusion procedure *in vivo* was done using rats in the Blood–Brain Barrier Lab at Oregon

#### *Antioxidants*

Health and Science University (OHSU), Portland, OR. OHSU is in full compliance with Federal and State statutes regarding the use and care of vertebrate animals in research. The animal care facility is AAALAC accredited, and all rat maintenance, treatment, recovery, and euthanasia procedures were approved for the reported middle cerebral artery occlusion studies.

To achieve middle cerebral artery occlusion, female Long Evans rats (250–300 grams) were anesthetized with isoflurane inhalant (5% induction, 2% maintenance). Analgesia was provided by administering butorphanol (0.05–2.0 mg/kg SQ ) as needed, and seizures were controlled with diazepam (5 mg/kg) if necessary. Animals were monitored daily by staff veterinarians. Upon completion of the experiments, the rats were euthanized after inhalant anesthetic induction with an intracardiac overdose of sodium pentobarbital, or if symptoms necessitated earlier sacrifice. This method of euthanasia is consistent with requirements of the Panel of Euthanasia of the American Veterinary Medical Association.
