Pre-treatment with L-NAME, to inhibit NOS activity, did not significantly alter superoxide anion production.

<p>Regional microvessels were isolated and incubated with or without 30 μmol/L of L-NAME for 30 minutes at 37°. Following this incubation, microvessels were incubated with 50 μmol/L dihydroethidium at 37° for 15 minutes. Microvessels were homogenized in methanol and intracellular superoxide anions quantified by HPLC-based fluorescence. Intracellular superoxide anions were normalized to mg protein of each sample (n = 6). Data are represented as mean ± SEM.</p

Levels of the antioxidant enzyme, MnSOD are decreased in the cortical and hippocampal microvessels.

<p>(A) Brain region microvessels were isolated and tissue homogenates were analyzed by Western blot analyses using anti- CuZnSOD (n = 9), ECSOD (n = 9), MnSOD (n = 10), and catalase (n = 4) antibodies. A representative image is shown. Densitometry analyses were performed normalizing against the loading control, Actin, for (B) MnSOD, (C) CuZnSOD, (D) ECSOD, and (E) catalase. Data are presented as mean ± SEM (P<0.05 as compared to cerebellum microvessels).</p

Intracellular superoxide anions are increased in the microvessels isolated from the cortex and hippocampus.

<p>Regional microvessels were isolated and incubated with 50 μmol/L dihydroethidium at 37° for 15 minutes. Microvessels were homogenized in methanol and intracellular superoxide anions quantified by HPLC-based fluorescence. Intracellular superoxide anions were normalized to mg protein of each sample (n = 12–13). Data are represented as mean ± SEM (P<0.05 as compared to cerebellum microvessels).</p

cGMP levels were not different between microvessels isolated from the cerebellum, cortex, or hippocampus.

<p>Regional microvessels were isolated and cGMP analyzed using a commercially available immunoassay.</p

Protein levels of NOX-2 and NOX-4 were significantly increased in cortex and hippocampus microvessels.

<p>(A) Brain region microvessels were isolated, homogenized, and analyzed via Western blot analyses. Membranes were probed with anti-NOX-2 (n = 7) and anti-NOX-4 (n = 7–8) antibodies. A representative image is shown. (B) Densitometric analysis of NOX-2 and (C) NOX-4 levels were perfomed and normalized against Actin as a loading control. Data are presented as mean ± SEM (P<0.05).</p

BH₄ bioavailability was decreased in cortical and hippocampal microvessels as compared to microvessels from the cerebellum.

<p>Biopterin levels, BH₄ and BH₂, were analyzed by HPLC. (A) Total biopterin levels were unchanged between microvessels isolated from the three brain regions. (B) BH₄ levels were significantly lower in the hippocampal microvessels as compared to the cerebellum. (C) BH₂, the oxidized product of BH₄, was significantly higher in the cortical microvessels as compared to the cerebellum. (D) The ratio of BH₄:BH₂, indicative of the bioavailable levels of BH₄, is significantly lower in both the cortical and hippocampal microvessels as compared to the cerebellum. Data is presented as mean ± SEM (n = 7–9, P<0.05).</p

Protein levels of COX-1 and PGI-2S did not differ between brain region microvessels.

<p>(A) Isolated brain region microvessels were homogenized and analyzed by Western blot analyses using anti- COX-1 (n = 11) and PGI2S (n = 8) antibodies. A representative image is shown. Densitometry analyses were performed normalizing against Actin, a loading control, for (B) COX-1 and (C) PGI2S. Data are presented as mean ± SEM.</p