In a thromboembolic
stroke model after reperfusion by recombinant
tissue plasminogen activator (rt-PA), we aimed to determine whether
therapeutic hypothermia (TH) and
ethanol (EtOH) in combination with low concentration (60 %) of normobaric
oxygen (NBO) enhanced neuroprotection, as compared to using each of these agents alone. We further aimed to elucidate a potential role of the
NADPH oxidase (NOX), phosphorylated
protein kinase B (Akt), and
protein kinase C-δ (PKC-δ) pathway in oxidative stress and neuroprotection. In Sprague-Dawley rats, a focal middle cerebral artery (MCA) occlusion was induced by an autologous
embolus in the following experimental groups: rt-PA treatment alone, rt-PA + NBO treatment, rt-PA + TH at 33 °C, rt-PA + EtOH, rt-PA + NBO + EtOH, rt-PA + NBO + TH, rt-PA + NOX inhibitor, rt-PA + EtOH + NOX inhibitor, or rt-PA + EtOH + Akt inhibitor. Control groups included
sham-operated without
stroke or
stroke without treatment.
Infarct volume and neurological deficit were assessed at 24 h after rt-PA-induced reperfusion with or without treatments. ROS levels, NOX activity, and the
protein expression of NOX subunits p22phox, p47phox,
p67phox, gp91phox, as well as PKC-δ and phosphorylated Akt were measured at 3 and 24 h after rt-PA-induced reperfusion. Following rt-PA in thromboembolic
stroke rats, NBO combined with TH or EtOH more effectively decreased
infarct volume and neurological deficit, as well as
reactive oxygen species (ROS) production than with any of the used monotherapies. NOX activity and subunit expressions were downregulated and temporally associated with reduced PKC-δ and increased p-Akt expression. The present study demonstrated that combining NBO with either TH or EtOH conferred similar neuroprotection via modulation of NOX activation. The results suggest a role of Akt in NOX activation and implicate an upstream PKC-δ pathway in the Akt regulation of NOX. It is possible to substitute EtOH for TH, thus circumventing the difficulties in clinical application of TH through the comparatively easier usage of EtOH as a potential
stroke management.