Alternatively-Spliced Extra Domain A of Fibronectin Promotes Acute Inflammation and Brain Injury After Cerebral Ischemia in Mice

Background and Purpose-The fibronectin isoform containing the alternatively spliced extra domain A (EDA(+)-FN) is normally absent from the circulation, but plasma levels of EDA(+)-FN can become markedly elevated in several human pathological conditions associated with inflammation including ischemic stroke. It remains unknown whether EDA(+)-FN contributes to stroke pathogenesis or is simply an associative marker. Several in vitro studies suggest that EDA(+)-FN can activate Toll-like receptor 4, an innate immune receptor that triggers proinflammatory responses. We undertook a genetic approach in mice to investigate the ability of EDA(+)-FN to mediate inflammatory brain damage in a focal cerebral ischemia/reperfusion injury model. Methods-We used genetically modified EDA(+/+) mice, which constitutively express EDA(+)-FN. Extent of injury, neurological outcome, and inflammatory mechanisms were assessed after 1-hour cerebral ischemia/23-hour reperfusion injury and compared with wild-type mice. Results-We found that EDA(+/+) mice developed significantly larger infarcts and severe neurological deficits that were associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of nuclear factor-kappa B, cyclo-oxygenase-2, and inflammatory cytokines tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 in the EDA(+/+) mice compared with wild-type mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA(+/+) mice by treatment with a specific Toll-like receptor 4 inhibitor. Conclusions-These findings provide the first evidence that EDA(+)-FN promotes inflammatory brain injury after ischemic stroke and suggest that the elevated levels of plasma EDA(+)-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients after acute stroke. (Stroke. 2012;43:1376-1382.)