• Himakarnika Alluri, Hayden W Stagg, Rickesha L Wilson, Robert P Clayton, Devendra A Sawant, Madhavi Koneru, Madhava R Beeram, Matthew L Davis, and Binu Tharakan.
• Departments of Surgery and Pediatrics, Texas A&M University Health Science Center College of Medicine and Scott & White Healthcare, Temple, Texas, USA.
• Microcirculation. 2014 Feb 1; 21 (2): 187-95.

ObjectiveMicrovascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this.MethodsRBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively.ResultsOGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly.ConclusionsThese results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.© 2013 John Wiley & Sons Ltd.

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