IL-1β treatment induces dose and time dependent increase in monolayer hyperpermeability.

<p>In Panel A, IL-1β treatment at doses 10, 50 and 100 ng/mL for 2 hours are shown to significantly increase BBB permeability compared to the control group (n = 4; <i>p</i><0.05). Panel B indicates significant increase in IL-1β induced BBB permeability at 2, 3 and 4 hours compared to the control (n = 4; <i>p</i><0.05). Monolayer permeability is expressed as a percentage control of FITC-dextran-10 kDa fluorescent intensity, plotted on the Y-axis. Data are expressed as mean ± % SEM. ‘*a’ indicates significant increase compared to the control group.</p

Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition - Fig 9

<p>Melatonin pretreatment attenuates TBI-induced BBB hyperpermeability studied by Evans blue dye extravasation method (Panel A). Pictorial representation of the brain tissue from various groups is shown in Panel 9B. Sham injury group was used as the baseline for all comparisons. Melatonin (10 μg/gram body weight of the animal) pretreatment significantly attenuated TBI-induced Evans blue leakage into the extravascular tissue space (<i>p</i><0.05). Animals were divided into sham (n = 6), vehicle + sham (n = 6), vehicle + TBI (n = 5) and melatonin + TBI (n = 6). Data are expressed as ng/brain cortex ± SEM. ‘*’ indicates statistical significance. ‘a’ indicates significant increase compared to the sham injury/vehicle + sham injury group and ‘b’ indicates significant decrease compared to the vehicle + TBI group.</p

MMP-9 inhibitor 1 and melatonin pretreatment attenuates IL-1β treatment- induced MMP-9 activity.

<p>MMP-9 inhibitor 1 (n = 4) and melatonin (n = 5) pretreatment attenuated IL-1β treatment-induced MMP-9 activity in RBMECs. MMP-9 activity is expressed as relative fluorescence units (RFU), plotted on the Y-axis. Data are expressed as mean ± SEM. ‘*a’ indicates significant increase compared to the control group; ‘*b’ indicates significant decrease compared to the IL-1β (10 ng/mL; 2 hours) treatment group. <i>p</i><0.05 was considered statistically significant.</p

IL-1β treatment does not induce cell death.

<p>IL-1β (10 ng/mL; 2 hours) treatment had no effect on cell viability (n = 5). Hydrogen peroxide (used as a positive control) treatment decreases cell viability significantly (<i>p<</i>0.05). Data are expressed as mean ± % SEM. ‘*’ indicates statistical significance. ‘*a’ indicates significant decrease compared to the control group.</p

Knockdown of MMP-9 by siRNA attenuates IL-1β treatment-induced monolayer hyperpermeability.

<p>Monolayer permeability is expressed as percentage flux of FITC-dextran-10 kDa fluorescence intensity, plotted on the Y-axis. Data are expressed as mean ± % SEM. ‘*a’ indicates significant increase compared to the control group; ‘*b’ indicates significant decrease compared to the IL-1β (10 ng/mL; 2 hours) treatment group. siRNA transfected groups were compared to control siRNA transfected group (n = 4; p<0.05).</p

GM6001, MMP-9 inhibitor 1 and melatonin pretreatment attenuates IL-1β treatment-induced monolayer hyperpermeability.

<p>Panel A indicates the effect of GM6001 (broad-spectrum MMP inhibitor; n = 4); while Panels B and C employ MMP-9 specific inhibitors: MMP-9 inhibitor 1 (n = 4) and melatonin (n = 6) pretreatment on IL-1β (10 ng/mL; 2 hours)—induced monolayer hyperpermeability. Monolayer permeability is expressed as a percentage control of FITC-dextran-10 kDa fluorescence intensity, plotted on the Y-axis. Data are expressed as mean ± % SEM. ‘*a’ indicates significant increase compared to control group; ‘*b’ indicates significant decrease compared to the IL-1β treated group. <i>p</i><0.05 was considered statistically significant.</p

MMP-9 inhibitor 1 and melatonin pretreatment protects against IL-1β treatment-induced loss of ZO-1 junctional integrity.

<p>IL-1β (10 ng/mL; 2 hours) treatment-induced ZO-1 junctional disruption (white arrows) was decreased on pretreatment with MMP-9 inhibitor 1 (n = 4) and melatonin (n = 4).</p

IL-1β treatment does not induce ZO-1 mRNA or protein expression.

<p>IL-1β (10 ng/mL; 2 hours) treatment neither induces ZO-1/MMP-9 mRNA expression (n = 3) nor alter ZO-1 protein expression (n = 4). RT-PCR data plotted on the Y-axis are expressed as relative expression of ZO-1 normalized to GAPDH. Data are represented as mean ± SEM.</p