In an effort to elucidate the molecular mechanisms underlying cerebral vascular alteration after stroke, the authors measured the spatial and temporal profiles of blood-brain barrier (BBB) leakage, angiogenesis, vascular endothelial growth factor (VEGF), associated receptors, and angiopoietins and receptors after embolic stroke in the rat. Two to four hours after onset of ischemia, VEGF mRNA increased, whereas angiopoietin 1 (Ang 1) mRNA decreased. Three-dimensional immunofluorescent analysis revealed spatial coincidence between increases of VEGF immunoreactivity and BBB leakage in the ischemic core. Two to 28 days after the onset of stroke, increased expression of VEGF/VEGF receptors and Ang/Tie2 was detected at the boundary of the ischemic lesion. Concurrently, enlarged and thin-walled vessels were detected at the boundary of the ischemic lesion, and these vessels developed into smaller vessels via sprouting and intussusception. Three-dimensional quantitative analysis of cerebral vessels at the boundary zone 14 days after ischemia revealed a significant (P < 0.05) increase in numbers of vessels (n = 365) compared with numbers (n = 66) in the homologous tissue of the contralateral hemisphere. Furthermore, capillaries in the penumbra had a significantly smaller diameter (4.8 +/- 2.0 microm) than capillaries (5.4 +/- 1.5 microm) in the homologous regions of the contralateral hemisphere. Together, these data suggest that acute alteration of VEGF and Ang 1 in the ischemic core may mediate BBB leakage, whereas upregulation of VEGF/VEGF receptors and Ang/Tie2 at the boundary zone may regulate neovascularization in ischemic brain.

Background and Purpose-We tested the hypothesis that sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, promotes functional recovery and neurogenesis after stroke. Methods-Male Wistar rats were subjected to embolic middle cerebral artery occlusion. Sildenafil (Viagra) was administered orally for 7 consecutive days starting 2 or 24 hours after stroke onset at doses of 2 or 5 mg/kg per day. Ischemic rats administered the same volume of tap water were used as a control group. Functional outcome tests (foot-fault, adhesive removal) were performed. Rats were killed 28 days after stroke for analysis of infarct volume and newly generated cells within the subventricular zone and the dentate gyrus. Brain cGMP levels, expression of PDE5, and localized cerebral blood flow were measured in additional rats. Results-Treatment with sildenafil significantly (PϽ0.05) enhanced neurological recovery in all tests performed. There was no significant difference of infarct volume among the experimental groups. Treatment with sildenafil significantly (PϽ0.05) increased numbers of bromodeoxyuridine-immunoreactive cells in the subventricular zone and the dentate gyrus and numbers of immature neurons, as indicated by ␤III-tubulin (TuJ1) immunoreactivity in the ipsilateral subventricular zone and striatum. The cortical levels of cGMP significantly increased after administration of sildenafil, and PDE5 mRNA was present in both nonischemic and ischemic brain. Conclusions-Sildenafil increases brain levels of cGMP, evokes neurogenesis, and reduces neurological deficits when given to rats 2 or 24 hours after stroke. These data suggest that this drug that is presently in the clinic for sexual dysfunction may have a role in promoting recovery from stroke.

The mechanisms underlying cerebral microvascular perfusion deficit resulting from occlusion of the middle cerebral artery (MCA) require elucidation. We, therefore, tested the hypothesis that intravascular fibrin deposition in situ directly obstructs cerebral microcirculation and that local changes in type 1 plasminogen activator inhibitor (PAI-1) gene expression contribute to intravascular fibrin deposition after embolic MCA occlusion. Using laser-scanning confocal microscopy (LSCM) in combination with immunofluorescent staining, we simultaneously measured in three dimensions the distribution of microvascular plasma perfusion deficit and fibrin(ogen) immunoreactivity in a rat model of focal cerebral embolic ischemia (n ϭ 12). In addition, using in situ hybridization and immunostaining, we analyzed expression of PAI-1 in ischemic brain (n ϭ 13). A significant ( p Ͻ 0.05) reduction of cerebral microvascular plasma perfusion accompanied a significant ( p Ͻ 0.05) increase of intravascular and extravascular fibrin deposition in the ischemic lesion. Microvascular plasma perfusion deficit and fibrin deposition expanded concomitantly from the subcortex to the cortex during 1 and 4 hr of embolic MCA occlusion. Threedimensional analysis revealed that intravascular fibrin deposition directly blocks microvascular plasma perfusion. Vascular plugs contained erythrocytes, polymorphonuclear leukocytes, and platelets enmeshed in fibrin. In situ hybridization demonstrated induction of PAI-1 mRNA in vascular endothelial cells in the ischemic region at 1 hr of ischemia. PAI-1 mRNA significantly increased at 4 hr of ischemia. Immunohistochemical staining showed the same pattern of increased PAI-1 antigen in the endothelial cells. These data demonstrate, for the first time, that progressive intravascular fibrin deposition directly blocks cerebral microvascular plasma perfusion in the ischemic region during acute focal cerebral embolic ischemia, and upregulation of the PAI-1 gene in the ischemic lesion may foster fibrin deposition through suppression of fibrinolysis.

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