Methamphetamine disrupts blood-brain barrier function by induction of oxidative stress in brain endothelial cells

Abstract

Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood-brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. In this study, we provide evidence that METH alters BBB function through direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human brain microvascular endothelial cells (BMVEC) to METH diminishes the tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing the expression of cell membrane-associated tight junction (TJ) proteins. These changes were accompanied by the enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Antioxidant treatment attenuated or completely reversed all tested aspects of METH-induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.

Figure 1

METH induction of BBB leakiness in vivo and in vitro. (A) Four-week old male NOD SCID mice received seven s.c. injections of either METH or sterile 0.9% sodium chloride. BBB permeability was evaluated by administration of the tracer, sodium fluorescein (Na-F) in saline, via i.p. route as described in the Methods. Following perfusion, the content of Na-F in homogenized brain tissue was measured. The results are expressed in ng of Na-F/mg of brain tissue, showing the average ± SEM, n=5, **p<0.001. (B) Barrier function is compromised in METH-treated BMVEC. TEER, an indicator of barrier integrity, was measured (by ECIS) in monolayers untreated or treated with either 50 μM or 250 μM METH. The resistance was measured at 400 Hz in 10 min intervals for the duration of the time shown. Treatments were initiated (arrow) after stable resistance was reached. Each data point is represented as the percent of the mean value ± SEM (n=3).

Figure 2

Altered TJ appearance in METH-treated BMVEC. HCMEC/D3 cells were used for the results shown here. (A) Representative images showing immunofluorescence labeling of occludin depict a continuous pattern for the TJ in untreated monolayers. Discontinuous occluding staining and gap formation are found in monolayers treated with 250 μM METH for 24 hr. The histogram inserts depict values as intensity/pixel from intensity profile lines drawn across the interface (TJ) of adjacent cells. The intensity histograms graphically demonstrate where gaps (double peaks) or discontinuity at the TJ occurs (absent or small peaks). Boxes around areas in images correspond to peaks on densitometry graphs. (B) Based on the intensity line profile, the degree of occludin alteration at the cell junction was evaluated by a semi-quantitative image analysis of multiple cell junctions using the criteria described above. Because the endothelial cells have various contact points with adjacent cells, only one contact point per cell was used in the analysis. The image analysis comparing untreated endothelial cells and METH-treated cells at various times is also shown. The data is presented as the percent of cells displaying 1 of 4 categories: no abnormality, gap formation, discontinuous or discontinuous with gaps (both).

Figure 3

METH downregulates levels of TJ proteins in BMVEC. Shown are representative western blots of occludin (Occ) (A) and claudin-5 (Cld5) (B) from membranous (M) and cytosolic (C) cellular fractions of endothelial cells treated at various time points (6, 12, and 24 hr) and at different concentrations (5, 50 and 250 μM) of METH. The graphs represent densitomet analysis of the band intensity ratio of METH over the untreated control. The results are shown as the mean value ± SD (*p<0.05 versus control), from three independent experiments.

Figure 4

METH generates ROS in the BMVEC. (A) BMVEC were exposed to increasing concentrations of either H₂O₂ (control), METH or the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). In parallel, cells were exposed together with increasing concentration of METH and a fixed concentration of Trolox (25 μM). The results show the fluorescence readings acquired 30 min after the start of treatments. METH generated a dose-dependent increase in the amount of ROS; however in the presence of Trolox, ROS production was significantly inhibited (p<0.005 when compared to METH-exposed cells). Data represent the mean values ± SEM (n=3). (B) Western blot shows occludin levels after 24 hr in untreated BMVEC, Trolox-treated (25 μM), METH-treated (50 μM) and METH with Trolox treated BMVEC. Densitometry values were used to quantify decreases in occludin due to METH treatment (bar graph).

Figure 5

Antioxidant treatment prevented METH-induced barrier dysfunction in vivo and in vitro. Four-week old male NOD SCID mice received seven s.c. injections of either METH, sterile 0.9% sodium chloride, Trolox (50 mg/kg administered i.p every other day) or in combination with METH and Trolox. Na-F administration was used to evaluate BBB permeability. Animals were then perfused to eliminate tracer in the vessels. The data shown in (A) indicate the accumulation of the tracer as ng of Na-F per mg of brain tissue (average ± SEM, n=5). (B) TEER readings are shown in monolayers that were untreated or treated with METH (50 μM), Trolox only (25 μM), or both METH and Trolox. Untreated and Trolox only treated monolayers sustained basal levels of TEER. METH addition produced a drop in TEER, whereas in the presence of Trolox, the effects of METH on TEER were prevented. The resistance was measured at 400 Hz at 10 min intervals for the duration of the experiments. Treatments were initiated (arrow) after stable resistance was reached. The data represent the percent of the mean value ± SEM (n=3).

Figure 6

METH-increased monocyte transendothelial migration via activation of MLCK in endothelial cells. Monolayers of primary BMVEC were left untreated or were exposed to METH at the concentrations shown for 12 hr (A). After treatment removal, monocytes loaded with the cell tracker Calcein-AM, were added to the endothelial monolayers and the relative fluorescence was measured. (B) Co-exposure with the antioxidant Trolox and 250 μM METH, decreased migration in a dose-dependent manner. (C) MLCK inhibitor, ML-7, blocked METH/CCL2-induced monocyte migration across BMVEC monolayers. For panels A–C, the data are represented as the mean ± SEM (n=3) of fold difference. All conditions were compared against the “spontaneous” migration value of untreated cells without chemoattractant. Brackets indicate t-test comparisons for the indicated experimental conditions (*p<0.05). (D) Western blot demonstrates MLC phosphorylation caused by 250 μM METH exposed for 24 hr. Introduction of Trolox at the same time METH was added prevents MLC addition.