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

Microvascular hyperpermeability that occurs at the level of the blood‐brain barrier (BBB) often leads to vasogenic brain edema following traumatic/ischemic brain injury. At a cellular level, tight junction proteins (TJPs) regulate the functions of the blood‐brain barrier. Zonula occludens‐1 (ZO‐1) is an important TJ associated protein that binds to the transmembrane TJPs and actin cytoskeleton intracellularly and plays an important role in maintaining the integrity of the blood‐brain barrier. The pro‐inflammatory cytokine, interleukin‐1β (IL‐1β) as well as the proteolytic enzyme, matrix metalloproteinase‐9 (MMP‐9) play major role in promoting brain edema following trauma/ischemia. Recent studies indicate that melatonin a pineal hormone also acts as an endogenous inhibitor of MMP‐9. Our objective was to test if melatonin will attenuate IL‐1β‐induced BBB hyperpermeability via MMP‐9 inhibition in vitro, and also protect the BBB in a mouse model of mild traumatic brain injury. Rat brain microvascular endothelial cells (RBMEC) grown as monolayers on Transwell inserts or chamber slides were exposed to melatonin prior to IL‐1β treatment. Monolayer permeability was studied using FITC‐dextran permeability assay. Tight junction integrity and cytoskeletal organization were studied utilizing ZO‐1 immunofluorescence and rhodamine phalloidin staining of f‐actin, respectively. Effect of IL‐1β on cell viability was studied using Calcein AM assay. MMP‐9 activity was measured fluorometrically. ZO‐1 protein and gene expressions were studied by western blotting and RT‐PCR respectively. The effect of melatonin against BBB hyperpermeability was studied in a mouse controlled cortical impact model of traumatic brain injury. Acute IL‐1β (10 ng/mL; 2 hours)‐induced BBB hyperpermeability was significantly attenuated by pretreatment with melatonin (10 μg/mL; 1 hour) and MMP‐9 inhibitor‐1 (MMP‐9 specific inhibitor; 5 nM; 1 hour). Melatonin attenuated IL‐1β‐induced MMP‐9 activity, loss of ZO‐1 junctional integrity and f‐actin stress fiber formation. IL‐1β treatment had no effect on ZO‐1 protein or gene expression or on cell viability. Melatonin attenuated TBI‐induced BBB hyperpermeability in a mouse model of TBI. In conclusion, these results suggest that, one of the protective effects of melatonin against BBB hyperpermeability occurs due to enhanced BBB integrity via MMP‐9 inhibition. Melatonin treatment provided protection against BBB hyperpermeability in a mouse model of TBI suggesting its potential as a therapeutic agent against brain edema when established in humans.

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