Adverse effect of cyclosporin A on barrier functions of cerebral microvascular endothelial cells after hypoxia-reoxygenation damage in vitro - PubMed (original) (raw)

Adverse effect of cyclosporin A on barrier functions of cerebral microvascular endothelial cells after hypoxia-reoxygenation damage in vitro

Shinya Dohgu et al. Cell Mol Neurobiol. 2007 Nov.

Abstract

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood-brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan's blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.

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Figures

Fig. 1

Fig. 1

Time course of Na-F (A) and EBA (B) permeability in MBEC4 monolayers after exposure to H/R (4 h/1 h). Permeability studies were performed at 0, 1, 3, 5, and 7 days after H/R (4 h/1 h). Values are means ± SEM (n = 11–15). *P < 0.05, **P < 0.01, significantly different from controls. The inset in panel A shows the time course of cell viability in MBEC4 monolayers after H/R (4 h/1 h). Results are expressed as percentage of cell viability in the control monolayer. Values are means ± SEM (n = 9–15)

Fig. 2

Fig. 2

Rhodamine 123 accumulation in MBEC4 cells after H/R damage. P-gp function was evaluated at 7 days after H/R (4 h/1 h). Results are expressed as percentage of controls (0.67 ± 0.18 nmol/mg protein). Protein concentrations of the control and H/R-treated MBEC4 cells were 0.34 ± 0.02 mg and 0.30 ± 0.02 mg, respectively. Values are means ± SEM (n = 12). **P < 0.01, significantly different from control MBEC4 cells

Fig. 3

Fig. 3

Effects of CsA on Na-F (A) and EBA (B) permeability in MBEC4 monolayers after H/R damage. MBEC4 cells were cultured with normal medium for 7 days after H/R (4 h/1 h). Permeability studies were performed after exposure to CsA (5 μM) for 12 h. Permeability coefficients of Na-F in each vehicle-treated group were 1.91 ± 0.10 × 10−4 and 1.94 ± 0.11 × 10−4 cm/min for control and H/R-treated MBEC4 cells, respectively. Permeability coefficients of EBA for each corresponding vehicle-treated group were 0.50 ± 0.03 × 10−4 and 0.52 ± 0.04 × 10−4 cm/min for control and H/R-treated MBEC4 cells, respectively. Results are expressed as percentage of each corresponding vehicle-treated group. Values are means ± SEM (n = 11–12). **P < 0.01, significantly different from each corresponding vehicle group. (##P < 0.01; significant difference between control and H/R-treated MBEC4 cells, when exposed to CsA

Fig. 4

Fig. 4

Effect of CsA on rhodamine 123 accumulation in MBEC4 cells after H/R damage. MBEC4 cells were cultured with normal medium for 7 days after H/R (4 h/1 h). P-gp function was evaluated after exposure to CsA (2 and 5 μM) for 12 h. Results are expressed as percentage of each corresponding vehicle (control MBEC4 cells, 2.72 ± 0.38 nmol/mg protein and H/R-treated MBEC4 cells, 2.41 ± 0.34 nmol/mg protein). Protein concentrations of the control MBEC4 cells after treatment with vehicle, CsA 2 μM and 5 μM were 0.05 ± 0.01 mg, 0.07 ± 0.01 mg, and 0.07 ± 0.01 mg, respectively. Protein concentrations of the H/R treated MBEC4 cells after treatment with vehicle, CsA 2 μM and 5 μM were 0.07 ± 0.01 mg, 0.07 ± 0.01 mg, and 0.06 ± 0.01 mg, respectively. Values are means ± SEM (n = 14–16). *P < 0.05, **P < 0.01, significant difference from each corresponding vehicle. (##P < 0.01; significant difference between control and H/R-treated MBEC4 cells, when exposed to CsA (2 μM). ††P < 0.01; significant difference between control and H/R-treated MBEC4 cells when exposed to CsA (5 μM)

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