Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke

Background: The link between early blood-brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Sle(x)) where the latter is known to bind to activated endothelium via E-or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wildtype (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T-1 maps were acquired prior to and following contrast injection. In addition to measuring P-and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa). Results: MRI confirmed similar infarct sizes and T-1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (K-gd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P < 0.03). In the P selectin KO mice,. Delta T-1 stroke -Delta T-1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLe(X)) group compared to Gd-DTPA, indicative of sLe(X) mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P < 0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P < 0.001) thereby supporting the observed increase in K-gd permeability in stroke brain of WT compared to KO mice. Conclusion: P-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.