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Characterization of the markers of blood-brain barrier disruption in West Nile virus infected mice

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Item Summary

Title: Characterization of the markers of blood-brain barrier disruption in West Nile virus infected mice
Authors: Roe, Kelsey Olds
Keywords: West Nile virus
Issue Date: Aug 2011
Publisher: [Honolulu] : [University of Hawaii at Manoa], [August 2011]
Abstract: Purpose: West Nile virus (WNV) disease is characterized by neuronal loss, neuroinflammation and increased permeability of the blood-brain barrier (BBB). Several in vivo studies demonstrate that BBB disruption and virus replication in the brain correlates with mortality associated with WNV-encephalitis. BBB integrity is maintained through complex interactions between the tight junction proteins (TJP), adherens junction proteins (AJP) of the microvascular endothelial cells (EC). Matrix metalloproteinases (MMPs) are endopeptidases that are known to degrade components of the basal lamina of the BBB as well as multiple TJP, contributing to BBB disruption in several neuroinflammatory diseases. Our previous in vitro studies demonstrated that multiple MMPs secreted by WNV-infected astrocytes degrade TJP of the EC and compromise the integrity of the in vitro BBB model. However, the precise mechanisms underlying BBB disruption in vivo, specifically the kinetics of the morphological changes in tight junctions of the BBB and the relationship of BBB breakdown with virus replication in the brain are unclear. Therefore, the objective of this study was to determine the in vivo effect of WNV infection on the BBB integrity markers in the mouse brain.
Methods: Ten-week old C57BL/6 mice were inoculated via footpad with 100 PFU of WNV and serum, spleen, kidney, and brain tissues were collected at days 2, 4, 6 and 8 after infection. In addition, BBB permeability was assessed using Evan's blue dye at days 6 and 8 after infection. Expression of TJP, AJP and MMP was analyzed using quantitative reverse transcriptase-PCR, ELISA, Western blotting or immunohistochemical staining. A separate group of mice were inoculated via the intracranial route with 100 PFU of WNV, and brains were harvested at day 6 for analysis of TJP, AJP, and MMPs.
Results: Peak peripheral viremia was observed at day 3 after infection and the virus was mostly cleared by day 6. In the brain, WNV was not detected at day 3 after infection, however robust virus replication was observed at day 6 and 8 after infection. Leakage of Evans blue dye into the brain was minimal at day 6 after infection, and became intense at day 8 after infection, suggesting disruption of the BBB. The mRNA levels of TJP (claudin-1, occludin, zonula occludin-1 (ZO-1), and junctioanl adhesion molecule-A (JAM-A)) and AJP (VE-cadherin and β-catenin) in the brain did not alter at any time point after infection. Although there was no significant change in the protein level of TJP or AJP at day 4 after infection, these proteins decreased slightly in the WNV-infected brain at day 6 after infection, which became more pronounced and significant at day 8 after infection. These results were validated using immunostaining of brain sections at day 8 after infection. Similarly, at day 8 after infection, the mRNA and protein expressions of MMP-1,-3, and-9 in WNV-infected brain increased significantly and correlated with high WNV titers in the brain. Assay of MMP-9 in the serum and brain using ELISA further supported the Western blot data and indicated that increased MMPs produced in the brain, but not the serum, correlated with BBB disruption at day 6 and 8 after infection. Following intracranial inoculation of WNV, robust virus replication occurred in the brain, but virus was not detected in the serum. As expected, intracranial inoculation of mice resulted in disruption of the BBB at day 6 after infection, which correlated with decreased junction proteins and increased MMPs Conclusions: In this study, we demonstrate that WNV-induced BBB disruption is correlated with decreases in functionally important TJP and AJP. Additionally we demonstrated that increase in multiple MMP in the brain correlate with TJP degradation and BBB disruption, while MMP production in the periphery does not. Collectively, this study provides new insights into WNV-induced structural and functional changes in the TJ properties of the BBB in vivo and forms a framework for future studies targeting MMPs to block BBB disruption to manage WNV-associated disease sequelae.
Description: M.S. University of Hawaii at Manoa 2011.
Includes bibliographical references.
Appears in Collections:M.S. - Biomedical Sciences (Tropical Medicine)

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