Electroconvulsive seizure-induced VEGF is correlated with neuroprotective effects against cerebral infarction: Involvement of the phosphatidylinositol-3 kinase/Akt pathway Minoru Fujiki ⁎, Eiji Abe, Yasuyuki Nagai, Kong Shiqi, Takeshi Kubo, Keisuke Ishii, Tatsuya Abe, Hidenori Kobayashi Department of Neurosurgery, School of Medicine, Oita University, 1-1, Idaigaoka, Hasama-machi, Oita 879-5593, Japan abstract article info Article history: Received 19 March 2010 Revised 9 July 2010 Accepted 13 July 2010 Available online 21 July 2010 Keywords: Cerebral ischemia Electrical convulsive stimulation Vascular endothelial growth factor (VEGF) PI3K/Akt pathway Neuroprotection The present study demonstrates the cytoprotective effect of electrical convulsive stimulation (ECS) as a potential neuroprotective vascular endothelial growth factor (VEGF) inducer against ischemic insult. Phosphatidylinositol-3 kinase/Akt (PI3K/Akt) is thought to be an important factor that mediates neuroprotection. However, the signaling pathways in the brain in vivo after ECS remain unclear. We measured and compared infarction volumes to investigate the effect of ECS on cerebral infarction induced by permanent middle cerebral artery occlusion in rats. We evaluated the effects of pretreatment with Wortmannin (Wort), a specific PI3K inhibitor of ECS-induced neuroprotection against infarction volumes. To clarify the relationship between PI3K/Akt activation and neuroprotection, we used immunoblot analysis to determine the amounts of p-Akt and VEGF proteins present after ECS with or without Wort treatment. Neuroprotective effects of ECS (pretreatment with a single ECS 6 h before ischemia) were prevented by Wort pretreatment, which indicates that the PI3K/Akt pathway may mediate ECS-dependent protection. ECS induced p-Akt and VEGF and ECS pretreatment enhanced ischemia-induced VEGF, both of which were prevented by Wort pretreatment. These results suggest that a single ECS induces p-Akt and that ECS plays an important role in neuroprotection against the cerebral ischemia through VEGF induction. © 2010 Elsevier Inc. All rights reserved. Introduction Recent studies have demonstrated that vascular endothelial growth factor (VEGF) is a potential neuroprotective factor (Kilic et al., 2006) and that electrical convulsive stimulation (ECS) is a convenient pretreatment strategy for rapid neuronal VEGF induction (Newton et al., 2003). However, the signaling pathways in the rat brain after ECS remain to be defined. Anti-apoptotic activity of Akt is mediated by the activation of the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathways (Gross, 2005). VEGF induction was reported to be involved in cerebral ischemic preconditioning (Kilic et al., 2006). There is strong support for the hypothesis that VEGF mediates neuroprotection via the activation of the PI3k/Akt pathway (Bijur and Jope, 2003; Kilic et al., 2006). We postulated that the ECS-activated PI3K/Akt pathway is upstream of the VEGF up-regulation and that activation of the latter leads to preservation of neuroprotective and neurologic functions. In this study, we used a rat experimental model for focal cerebral ischemia after permanent middle cerebral artery occlusion (MCAO) to investigate the neuroprotective role of ECS. Specifically, we tested the hypothesis that ECS would induce p-Akt and, thereafter, VEGF up-regulation would be involved in neuropro- tection against cerebral ischemia. This is the first report that investigates the role of the PI3K/Akt pathway and VEGF up-regulation in the neuroprotective mechanism of ECS. Materials and methods Animals and experimental protocol All experimental protocols were approved by the Oita University Ethical Review Committee. Male Wistar rats (body weight 290–375 g) were housed at controlled room temperature (24.5–25.0 °C) with a 12/12-h light/dark cycle. The rats had free access to food pellets and tap water. Before MCAO, bilateral ECS was administered via moistened pads on spring-loaded ear clip electrodes using a pulse generator (frequency, 100 pulses/s; pulse width, 0.5 ms; pulse duration, 0.5 s; stimulus current, 55 mA) (Newton et al., 2003). This consistently produced a generalized grand mal seizure with charac- teristic tonic and clonic convulsions. In one experiment, groups of animals received a single shock, and then the ECS–ischemic interval was varied (0, 1, 3, 6, 12, 24, 48, 72 h before ischemia; n = 6 at each interval). Rats in the sham stimulation- Experimental Neurology 225 (2010) 377–383 ⁎ Corresponding author. Fax: + 81 97 586 5869. E-mail address: fujiki@oita-u.ac.jp (M. Fujiki). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.07.010 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr