Caffeine protects against MPTP-induced blood-brain barrier dysfunction in mouse striatum

Abstract

The blood-brain barrier (BBB) is important physiologically. Pathologically, BBB disruption has been implicated in a wide spectrum of neurological disorders including Parkinson's disease (PD). Recent studies indicate that caffeine is protective against PD, but by poorly understood mechanisms. Using a MPTP neurotoxin model of PD we tested the hypothesis that the protective actions of caffeine were because of, at least in part, preventing MPTP-induced BBB dysfunction. FVB mice were pre-treated with caffeine (10 mg/kg, i.p.) or saline for 7 days prior to initiation of neurotoxin treatments; during the 7 days of neurotoxin treatment, caffeine or saline continued to be administered 10 min before each dose of MPTP (20 mg/kg, i.p.). Striatum (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, tight junction protein expression levels, integrity of dopaminergic neurons, and activation of astrocytes and microglia using immunostaining, immunoblotting and real-time PCR techniques. We found that caffeine blocked MPTP-induced decreases in numbers of tyrosine hydroxylase-positive dopaminergic neurons, increases in leakage of Evan's blue dye and FITC-albumin in striatum but not in cerebral cortex or hippocampus, decreases in levels of the tight junction proteins occludin and ZO-1, and increases in reactive gliosis. Our results suggest that caffeine might protect against PD and PD-like features in animal models, in part, by stabilizing the BBB.

Figure 1. Caffeine attenuated MPTP-induced loss of tyrosine hydroxylase (TH) positive dopaminergic neurons in striatum

(A) MPTP decreased markedly TH immunoreactivity in striatum, and this effect was attenuated by caffeine. Bar = 100 μm. (B) MPTP decreased significantly (p < 0.01) TH protein levels in striatum, and this effect was blocked significantly (p < 0.01) by caffeine. (n = 4, **p < 0.01)

Figure 2. Caffeine attenuated MPTP-induced gliosis

(A) MPTP significantly (p < 0.001) increased GFAP immunoreactivity in striatum and this effect was blocked significantly (p < 0.001) by caffeine. The upper left panel of four photomicrographs was representative of findings observed in 3 animals with 6 sections taken from each animal. ***p < 0.001, Bar = 50 μm. (B) In addition to microglia, lectin staining also revealed blood vessels. Amoeboid-shaped microglia (arrow heads) were observed only in striatum from MPTP treated animals; not in control or caffeine-treated animals. Representative images were taken from 3 animals; 6 sections were taken from each animal. Bar = 50 μm.

Figure 3. Caffeine blocked MPTP-induced increases in striatum FITC-albumin leakage

(A) MPTP markedly increased FITC-albumin leakage in striatum, and this effect was blocked by caffeine. (B) Neither caffeine nor MPTP increased FITC-albumin leakage in hippocampus. Bar = 100 μm (n = 4)

Figure 4. Caffeine blocked MPTP-induced increases in striatum Evan's blue dye leakage

(A) MPTP-induced increases (p < 0.01) in leakage of Evan's blue dye in striatum were decreased significantly (p < 0.01) by caffeine. Neither caffeine nor MPTP changed significantly Evan's blue dye leakage in cerebral cortex (B) or in hippocampus (C). (n = 6, **p < 0.01)

Figure 5. Caffeine blocked MPTP-induced decreases in the expression of ZO-1

(A) MPTP decreased significantly (p < 0.01) ZO-1 immunostaining in striatum, and this effect was blocked significantly (p < 0.01) by caffeine. Bar = 20 μm. (B) MPTP decreased significantly (p < 0.01) ZO-1 protein levels in striatum, and this effect was blocked significantly (p < 0.01) by caffeine. (C) Neither MPTP nor caffeine changed significantly ZO-1 mRNA levels in striatum. (n = 4, **p < 0.01)

Figure 6. Caffeine blocked MPTP-induced decreases in occludin expression levels

(A) MPTP decreased significantly (p < 0.05) occludin immunostaining in striatum, and this effect was blocked significantly (p < 0.05) by caffeine. Bar = 20 μm. (B) MPTP decreased significantly (p < 0.05) occludin protein levels in striatum, and this effect was blocked significantly (p < 0.05) by caffeine. (C) Neither MPTP nor caffeine changed significantly occludin mRNA levels in striatum. (n = 4, *p < 0.05).

Figure 7. Caffeine attenuated MPTP-induced increases in in situ gelatinolytic activity and increases in the expression of MMP9

(A) MPTP increased in situ gelatinolytic activity in striatum and this effect was attenuated by caffeine. Bar = 100 μm. (B) MPTP increased significantly (p < 0.05) mRNA levels of MMP-9 in striatum and this effect was blocked significantly (p < 0.05) by caffeine. (n = 4, *p < 0.05) (C) MMP-9 co-localized (see arrow heads) with GFAP in striatum of MPTP-treated mice.