Caffeine blocks high cholesterol diet-induced extravasation of IgG. (A) Cholesterol-enriched diet increased extravasation of IgG in olfactory bulb and this effect was blocked by caffeine at the dose of 3 mg/day. Caffeine alone had no effect on extravasation of IgG in normal rabbit brain. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 50 μm. (B) Cholesterol-enriched diet significantly increased accumulation of IgG in olfactory bulb and this effect was blocked by caffeine at the dose of 3 mg/day. (C) Neither cholesterol-enriched diet nor caffeine changed significantly plasma levels of IgG. n = 4, *p < 0.05.

Caffeine blocks high cholesterol diet-induced leakage of Evan's blue dye. Cholesterol-enriched diet significantly increased leakage of Evan's blue dye in olfactory bulb (A), cerebral cortex (B) and hippocampus (C), and these effects were blocked by caffeine at the dose of 3 mg/day. n = 2, *p < 0.05.

Double immunostaining of IgG extravasation and the expression of occludin. Cholesterol-enriched diet markedly decreased the immunopositive staining of occludin (green) at the sites where IgG extravasation (red) was present. These effects were blocked by chronic ingestion of caffeine. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 20 μm.

Double immunostaining of IgG extravasation and the expression of GFAP. Cholesterol-enriched diet markedly enhanced the immunopositive staining of GFAP (green) around the sites where IgG extravasation (red) was present. These effects were blocked by caffeine at the dose of 3 mg/day. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 20 μm.

Caffeine blocks high cholesterol diet-induced extravasation of fibrinogen. (A) Cholesterol-enriched diet increased extravasation of fibrinogen in olfactory bulb and this effect was blocked by caffeine at the dose of 3 mg/day. Caffeine alone had no effect on extravasation of fibrinogen in normal rabbit brain. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 50 μm. (B) Cholesterol-enriched diet significantly increased accumulation of fibrinogen in olfactory bulb and this effect was blocked by caffeine at the dose of 3 mg/day. (C) Neither cholesterol-enriched diet nor caffeine changed significantly plasma levels of fibrinogen. n = 4, *p < 0.05.

Caffeine blocks high cholesterol diet-induced down-regulation of tight junction proteins. (A) Schematic draft of a section of olfactory bulb, the green square indicated where the fluorescent images were taken for measures of the expression of occludin and ZO-1. (B) Decreased occludin immunostaining was observed in olfactory bulb from cholesterol-fed rabbits and this effect was blocked by caffeine. Caffeine alone had no effect on occludin immunostaining in normal rabbit brain. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 100 μm. (C) Quantitative data from B shows that high cholesterol diet significantly decreased occludin immunopositive staining in olfactory bulb, and this effect is blocked by caffeine. (D) Cholesterol-enriched diet decreased significantly protein levels of occludin, and these effects were blocked by caffeine at the dose of 3 mg/day. Caffeine alone did not significantly change protein levels of occludin in normal rabbit olfactory bulb (n = 4, *p < 0.05). (E) Decreased ZO-1 immunostaining was observed in olfactory bulb from cholesterol-fed rabbits and this effect was blocked by caffeine. Caffeine alone had no effect on ZO-1 immunostaining in normal rabbit brain. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 100 μm. (F) Quantitative data from E shows that high cholesterol diet significantly decreased ZO-1 immunopositive staining in olfactory bulb, and this effect is blocked by caffeine. (G) Cholesterol-enriched diet decreased significantly protein levels of ZO-1, and these effects were blocked by caffeine at the dose of 3 mg/day. Caffeine alone did not significantly change protein levels of ZO-1 in normal rabbit olfactory bulb. n = 4, *p < 0.05.

Double immunostaining of IgG extravasation and the expression of ZO-1. Cholesterol-enriched diet markedly decreased the immunopositive staining of ZO-1 (green) at the sites where IgG extravasation (red) was present. These effects were blocked by chronic ingestion of caffeine. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 20 μm.

Double staining of IgG extravasation and microglia. (A) In addition to microglia, lectin staining also reveals blood vessels. Cholesterol-enriched diet markedly increased the numbers of microglia (green, arrow heads) and IgG extravasation (red). These effects were blocked by caffeine at the dose of 3 mg/day for 12 weeks. Representative images taken from 2 rabbits in each group with 6 sections from each animal are shown. Bar = 50 μm. (B) In a magnified view of the white boxes as shown in panel A, increased microglia (arrow heads) are co-distributed with perivascular immunopositive IgG staining (arrows) in olfactory bulb from cholesterol-fed rabbits. Bar = 20 μm.