Description of a Novel Phosphodiesterase (PDE)-3 Inhibitor Protecting Mice From Ischemic Stroke Independent From Platelet Function

Figure 1.. Therapeutic treatment with substance V (Sub V) reduces stroke severity without increasing the risk of intracerebral hemorrhages. A, Levels of cAMP in the brain tissue of vehicle-treated mice (0.5% dimethyl sulfoxide) or mice treated with 0.5 mg/kg Sub V at 6, 12, and 24 h after treatment (n=8–10). B, Left, Representative 2,3,5-triphenyltetrazolium chloride staining of 3 corresponding coronal brain sections of vehicle- or 0.5 mg/kg Sub V–treated mice euthanized 24 h after transient middle cerebral artery occlusion (tMCAO; scale bar=10 mm). The infarcts (white) appear smallest in the Sub V–treated mice and this could be confirmed by infarct volumetry (right; n=12–13). C, Bederson score (left) and grip test (right) on day 1 after tMCAO in the 2 mouse groups indicated above (n=12–13). D, Left, Serial coronal T2-weighted gradient echo magnetic resonance images (MRI) show extensive hyperintense (bright) ischemic lesions (arrows) in vehicle-treated mice on day 1 and 7 after tMCAO, whereas the infarctions were smaller in Sub V–treated mice. One representative imaging panel per group is depicted. Right, MRI based infarct volumetry (n=7–8). Statistical significances analyzed by (A) 2-way…

Figure 2.. Substance V (Sub V) shows blood-brain barrier stabilization and antiedematous effects in ischemic stroke. A, Left, Representative corresponding coronal brain sections of vehicle-treated mice and mice treated with 0.5 mg/kg Sub V after the injection of the vascular tracer Evans blue. Brains were analyzed on day 1 after transient middle cerebral artery occlusion (tMCAO; scale bar=10 mm). Vascular leakage was decreased in the cortical and subcortical areas after Sub V treatment. Right, Tissue concentration of Evans blue in the ischemic (ipsi) and contralateral (contra) hemispheres of treated and untreated mice 24 h after tMCAO determined by photometry (n=9–10). B, Edema formation as measured by brain water content in the ischemic (ipsi) and contralateral (contra) hemispheres of vehicle- or Sub V–treated mice 24 h after tMCAO (n=8). C, Left, Claudin-3 protein expression in the ischemic hemispheres of vehicle-treated mice and mice treated with 0.5 mg/kg Sub V on day 1 after tMCAO as determined by immunoblot. Actin was used as loading control. Right, Densitometric quantification of claudin-3 immunoreactivity in the ipsilateral cortex (ci) and ipsilateral basal ganglia (bi) of controls…

Figure 3.. Substance V (Sub V) exerts anti-inflammatory effects in ischemic stroke. A, Intracellular downstream signaling of cAMP in the ipsilesional cortex (ci) and basal ganglia (bi) of vehicle- and Sub V–treated mice was analyzed by immunoblotting 24 h after transient middle cerebral artery occlusion (tMCAO) using antibodies against NF-κB (nuclear factor-κB) p65 and PKA (protein kinase A). The protein phosphorylation was expressed as a relative amount of phosphoprotein to total protein. Actin was used as loading control. In addition to the densitometric quantification, one representative immunoblot is shown (n=4–5). B, Left, Representative immunocytochemical staining against CD11b+ macrophages/microglia and against Ly-6B.2+ neutrophils in the ischemic hemisphere of vehicle-treated mice and mice treated with 0.5 mg/kg Sub V. Scale bar=100 µm. Right, Quantification of CD11b+ macrophages/microglia and Ly-6B.2+ neutrophils per slice in the infarcted hemispheres on day 1 after tMCAO (n=4). C, Relative gene expression of TNF (tumor necrosis factor)-α, IL (interleukin)-1β, and IL-6 in the ischemic cortices (top) and basal ganglia (bottom) of vehicle- and Sub V–treated mice 24 h after tMCAO…

Figure 4.. Substance V (Sub V) has an enhanced neuroprotective effect in ischemic stroke. A, Relative expression of the phosphorylated form of eNOS (endothelial nitric oxide synthase) and AKT (protein kinase B) in the ipsilesional cortex (ci) and basal ganglia (bi) of vehicle- and Sub V–treated mice 24 h after transient middle cerebral artery occlusion (tMCAO) determined by immunoblot and densitometric quantification of the bands (n=4–5). The protein phosphorylation was expressed as a relative amount of phosphoprotein to total protein. Actin was used as loading control. B, Left, Representative brain sections from vehicle-treated and Sub V (0.5 mg/kg)–treated mice 24 h after tMCAO immunolabeled for the neuronal marker NeuN and subjected to TUNEL to depict apoptosis (scale bar=25 µm). Right, The number of TUNEL-positive neurons in the ischemic hemispheres of controls and Sub V–treated mice on day 1 after tMCAO (n=4). C, Left, Caspase-3 and cleaved caspase-3 protein expression in the ipsilesional cortex (ci) and basal ganglia (bi) of vehicle-treated mice and mice treated with 0.5 mg/kg Sub V on day 1 after tMCAO as determined by immunoblot. Right, Densitometric quantification of caspase-3 and…

Figure 5.. Superior therapeutic outcome of substance V (Sub V) treatment in comparison to Cilostazol. A, Left, Representative 2,3,5-triphenyltetrazolium chloride staining of 3 corresponding coronal brain sections of vehicle-, 0.5 mg/kg Sub V–, or 10 mg/kg Cilostazol-treated mice euthanized 24 h after transient middle cerebral artery occlusion (tMCAO; scale bar=10 mm). In comparison to the 2 other groups, the infarcts (white) appear smallest in the Sub V–treated mice and this could be confirmed by infarct volumetry (right; n=11–14). B, Bederson score (left) and grip test (right) on day 1 after tMCAO in the 3 mouse groups indicated above (n=11–14). Statistical significances analyzed by (A) 1-way ANOVA with Bonferroni post hoc test and (B) Kruskal-Wallis test with post hoc Dunn corrections. *P<0.05 and ***P<0.001.