ABSTRACT
Blood-brain barrier breakdown and associated vascular hyperpermeability leads to vasogenic edema in traumatic brain injury (TBI). Tight junctions maintain blood-brain barrier integrity; their disruption in TBI holds significant promise for diagnosis and treatment. A controlled cortical impactor was used for TBI in mouse studies. Blood was collected 1 h after injury and sent for antibody microarray analysis. Twenty human subjects with radiographic evidence of TBI were enrolled and blood collected within 48 h of admission. Control subjects were individuals with nontrauma diagnoses. The subjects were matched by age and gender. Enzyme-linked immunosorbent assays were performed on each TBI and control sample for tight junction-associated proteins (TJPs), inflammatory markers, and S100ß. Plasma was used to conduct in vitro monolayer permeability studies with human brain endothelial cells. S100ß and the TJP occludin were significantly elevated in TBI plasma in both the murine and human studies. Monolayer permeability studies showed increased hyperpermeability in TBI groups. Plasma from TBI subjects increases microvascular hyperpermeability in vitro. TJPs in the blood may be a potential biomarker for TBI.
ABSTRACT
BACKGROUND: The integrity of the blood-brain barrier (BBB) is paramount in limiting vasogenic edema following traumatic brain injury (TBI). The purpose of this study was to ascertain if quetiapine, an atypical antipsychotic commonly used in trauma/critical care for delirium, protects the BBB and attenuates hyperpermeability in TBI. METHODS: The effect of quetiapine on hyperpermeability was examined through molecular modeling, cellular models in vitro and small animal models in vivo. Molecular docking was performed with AutoDock Vina to matrix metalloproteinase-9. Rat brain microvascular endothelial cells (BMECs) were pretreated with quetiapine (20 µM; 1 hour) followed by an inflammatory activator (20 µg/mL chitosan; 2 hours) and compared to controls. Immunofluorescence localization for tight junction proteins zonula occludens-1 and adherens junction protein ß-catenin was performed. Human BMECs were grown as a monolayer and pretreated with quetiapine (20 µM; 1 hour) followed by chitosan (20 µg/mL; 2 hours), and transendothelial electrical resistance was measured. C57BL/6 mice (n = 5/group) underwent mild to moderate TBI (controlled cortical impactor) or sham craniotomy. The treatment group was given 10 mg/kg quetiapine intravenously 10 minutes after TBI. The difference in fluorescence intensity between intravascular and interstitium (ΔI) represented BBB hyperpermeability. A matrix metalloproteinase-9 activity assay was performed in brain tissue from animals in the experimental groups ex vivo. RESULTS: In silico studies showed quetiapine thermodynamically favorable binding to MMP-9. Junctional localization of zonula occludens-1 and ß-catenin showed retained integrity in quetiapine-treated cells as compared with the chitosan group in rat BMECs. Quetiapine attenuated monolayer permeability compared with chitosan group (p < 0.05) in human BMECs. In the animal studies, there was a significant decrease in BBB hyperpermeability and MMP-9 activity when compared between the TBI and TBI plus quetiapine groups (p < 0.05). CONCLUSION: Quetiapine treatment may have novel anti-inflammatory properties to provide protection to the BBB by preserving tight junction integrity. LEVEL OF EVIDENCE: level IV.
Subject(s)
Antipsychotic Agents/pharmacology , Blood-Brain Barrier/metabolism , Brain Injuries, Traumatic/physiopathology , Endothelial Cells/physiology , Quetiapine Fumarate/pharmacology , Tight Junctions/metabolism , Animals , Brain/blood supply , Cells, Cultured , Chitosan/pharmacology , Computer Simulation , Disease Models, Animal , Electric Impedance , Humans , Intravital Microscopy , Male , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Microvessels/diagnostic imaging , Models, Molecular , Permeability/drug effects , Rats , Tight Junctions/drug effects , Zonula Occludens-1 Protein/metabolism , beta Catenin/metabolismABSTRACT
Loss of microvascular endothelial barrier integrity leads to vascular hyperpermeability and vasogenic edema in a variety of disease processes including trauma, ischemia and sepsis. Understanding these principles gives valuable information on pathophysiology and therapeutic drug development. While animal models of traumatic and ischemic injuries are useful to understand vascular dysfunctions associated with such injuries, in vitro barrier integrity assays are reliable and helpful adjuncts to understand the cellular and molecular changes and signaling mechanisms that regulate barrier function. We describe here the endothelial monolayer permeability assay and transendothelial electrical resistance (TEER) measurement as in vitro methods to test changes in microvascular integrity and permeability. These in vitro assays are based on either the measurement of electrical resistance of the monolayer or the quantitative evaluation of fluorescently tagged molecules (e.g., FITC-dextran) that pass through the monolayer when there is damage or breakdown.
