Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.

In the adult rodent, stroke induces an increase in endogenous neural progenitor cell (NPC) proliferation in the subventricular zone (SVZ) and neuroblasts migrate towards the ischemic boundary. We investigated the role of stromal cell-derived factor 1a (SDF-1a) in mediating NPC migration after stroke. We found that cultured NPCs harvested from the normal adult SVZ, when they were overlaid onto stroke brain slices, exhibited significantly (Po0.01) increased migration (67.27 25.2 lm) compared with the migration on normal brain slices (29.5729.5 lm). Immunohistochemistry showed that CXCR 4, a receptor of SDF-1a, is expressed in the NPCs and migrating neuroblasts in stroke brain. Blocking SDF-1a by a neutralizing antibody against CXCR 4 significantly attenuated stroke-enhanced NPC migration. ELISA analysis revealed that SDF-1a levels significantly increased (Po0.01) in the stroke hemisphere (43.676.5 pg/mg) when compared with the normal brain (25.27 1.9 pg/mg). Blind-well chamber assays showed that SDF-1a enhanced NPC migration in a dosedependent manner with maximum migration at a dose of 500 ng/mL. In addition, SDF-1a induced directionally selective migration. These findings show that SDF-1a generated in the stroke hemisphere may guide NPC migration towards the ischemic boundary via binding to its receptor CXCR 4 in the NPC. Thus, our data indicate that SDF-1a/CXCR 4 is important for mediating specific migration of NPCs to the site of ischemic damaged neurons.

The migratory behavior of neuroblasts after a stroke is poorly understood. Using time-lapse microscopy, we imaged migration of neuroblasts and cerebral vessels in living brain slices of adult doublecortin (DCX, a marker of neuroblasts) enhanced green fluorescent protein (eGFP) transgenic mice that were subjected to 7 days of stroke. Our results show that neuroblasts originating in the subventricular zone (SVZ) of adult mouse brain laterally migrated in chains or individually to reach the ischemic striatum. The chains were initially formed at the border between the SVZ and the striatum by neuroblasts in the SVZ and then extended to the striatum. The average speed of DCXeGFP-expressing cells within chains was 28.67±1.04 lm/h, which was significantly faster (P < 0.01) than the speed of the cells in the SVZ (17.98 ± 0.57 lm/h). Within the ischemic striatum, individual neuroblasts actively extended or retracted their processes, suggestive of probing the immediate microenvironment. The neuroblasts close to cerebral blood vessels exhibited multiple processes. Our data suggest that neuroblasts actively interact with the microenvironment to reach the ischemic striatum by multiple migratory routes.

Proneuronal basic helix-loop-helix (bHLH) transcription factor, neurogenin 1 (Ngn1), regulates neuronal differentiation during development of the cerebral cortex. Akt mediates proneuronal bHLH protein function to promote neuronal differentiation. Here, we show that recombinant human erythropoietin (rhEPO) significantly increased Akt activity and Ngn1 mRNA levels in neural progenitor cells derived from the subventricular zone (SVZ) of adult rat, which was coincident with increases of neural progenitor cell proliferation, differentiation, and neurite outgrowth. Inhibition of Akt activity by the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) inhibitor, LY294002, abolished rhEPO-increased Ngn1 mRNA levels and the effects of rhEPO on neural progenitor cells. In addition, reducing expression of endogenous Ngn1 by means of short-interfering RNA (siRNA) blocked rhEPO-enhanced neuronal differentiation and neurite outgrowth but not rhEPO-increased proliferation. Furthermore, treatment of stroke rat with rhEPO significantly increased Ngn1 mRNA levels in SVZ cells. These data suggest that rhEPO acts as an extracellular molecule that activates the PI3K/Akt pathway, which enhances adult neural progenitor cell proliferation, differentiation, and neurite outgrowth, and Ngn1 is required for Akt-mediated neuronal differentiation and neurite outgrowth.

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