Physical activity upregulates endothelial nitric oxide synthase (eNOS), improves endothelium function, and protects from vascular disease. Here, we tested whether voluntary running would enhance neovascularization and long-term recovery following mild brain ischemia. Wild-type mice were exposed to 30 minutes of middle-cerebral artery occlusion (MCAo) and reperfusion. Continuous voluntary running on wheels conferred long-term upregulation of eNOS in the vasculature and of endothelial progenitor cells (EPCs) in the spleen and bone marrow (BM). This was associated with higher numbers of circulating EPCs in the blood and enhanced neovascularization. Moreover, engraftment of TIE2/LacZ-positive BM-derived cells was increased in the ischemic brain. Four weeks after the insult, trained animals showed higher numbers of newly generated cells in vascular sites, increased density of perfused microvessels and sustained augmentation of cerebral blood flow within the ischemic striatum. Moreover, running conferred tissue sparing and improved functional outcome at 4 weeks. The protective effects of running on angiogenesis and outcome were completely abolished when animals were treated with a NOS inhibitor or the antiangiogenic compound endostatin after brain ischemia, and in animals lacking eNOS expression. Voluntary physical activity improves long-term stroke outcome by eNOS-dependent mechanisms related to improved angiogenesis and cerebral blood flow.

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16^INK4ais an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemiain vivo. Loss of p27^Kip1, another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen–glucose deprivationin vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number (∼1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell deathin vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Small ubiquitin-like modifier (SUMO)2/3 but not SUMO1 conjugation is activated after transient cerebral ischemia. To investigate its function, we blocked neuronal SUMO2/3 translation through lentiviral microRNA delivery in primary cortical neurons. Viability was unaffected by SUMO2/3 silencing unless neurons were stressed by transient oxygen–glucose deprivation (OGD). Both 15 and 45 minutes of OGD were tolerated by control microRNA-expressing neurons but damaged >60% of neurons expressing SUMO2/3 microRNA. Damaging OGD (75 minutes) increased neuronal loss to 54% (control microRNA) and to 99% (SUMO2/3 microRNA). This suggests that activation of SUMO2/3 conjugation is an endogenous neuroprotective stress response.

The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 β-estradiol in two models for oxidative stress-independent apoptotic neuronal cell death with that in necrotic neuronal cell death in primary neuronal cultures derived from rat hippocampus, septum, or cortex. Neuronal apoptosis was induced either by staurosporine or ethylcholine aziridinium (AF64A), as models for necrotic cell death glutamate exposure or oxygen–glucose deprivation (OGD) were applied. Long-term (20 hr) pretreatment (0.1 μm17 β-estradiol) was neuroprotective in apoptotic neuronal cell death induced by AF64A (40 μm) only in hippocampal and septal neuronal cultures and not in cortical cultures. The neuroprotective effect was blocked by the estrogen antagonists ICI 182,780 and tamoxifen and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. In glutamate and OGD-induced neuronal damage, long-term pretreatment was not effective. In contrast, short-term (1 hr) pretreatment with 17 β-estradiol in the dose range of 0.5–1.0 μmsignificantly reduced the release of lactate dehydrogenase and improved morphology of cortical cultures exposed to glutamate or OGD but was not effective in the AF64A model. Staurosporine-induced apoptosis was not prevented by either long- or short-term pretreatment. The strong expression of the estrogen receptor-α and the modulation of Bcl proteins by 17 β-estradiol in hippocampal and septal but not in cortical cultures indicates that the prevention of apoptotic, but not of necrotic, neuronal cell death by 17 β-estradiol possibly depends on the induction of Bcl proteins and the density of estrogen receptor-α.