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Edited by Paul FraserBlood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1␤ (IL-1␤) that is upregulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1␤-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1␤-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1␤ treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1␤-induced calpain activity significantly (p < 0.05). IL-1␤ had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1␤-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1␤-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.The blood-brain barrier (BBB) 2 plays an important role in maintaining the homeostasis of the brain. Blood-brain barrier breakdown and the associated hyperpermeability are hallmark features of several brain pathologies and injuries. The BBB is mainly composed of the cerebral endothelial cells and the tight junctions (TJs) between them (1). TJs between the neighboring endothelial cells include transmembrane TJs, i.e. occludin, claudins, junctional adhesion molecules, etc., and membranebound TJs, i.e. zonula occludens (1). Zonula occludens play an important role in regulating BBB permeability by binding to both transmembrane tight junctions and actin cytoskeleton intracellularly (2). Various mediators of inflammation are shown to modulate BBB breakdown and permeability in a variety of pathologies (3). Blood-brain barrier breakdown and the associated hyperpermeability is the leading cause of brain edema and elevated intracranial pressure followed by decreased perfusion pressure leading to poor clinical outcomes in traumatic brain injury (TBI) (4).I...

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.