The Effects of Methylene Blue on Autophagy and Apoptosis in MRI-Defined Normal Tissue, Ischemic Penumbra and Ischemic Core

Methylene blue (MB) USP, which has energy-enhancing and antioxidant properties, is currently used to treat methemoglobinemia and cyanide poisoning in humans. We recently showed that MB administration reduces infarct volume and behavioral deficits in rat models of ischemic stroke and traumatic brain injury. This study reports the underlying molecular mechanisms of MB neuroprotection following transient ischemic stroke in rats. Rats were subjected to transient (60-mins) ischemic stroke. Multimodal MRI during the acute phase and at 24hrs were used to define three regions of interest (ROIs): i) the perfusion-diffusion mismatch salvaged by reperfusion, ii) the perfusion-diffusion mismatch not salvaged by reperfusion, and iii) the ischemic core. The tissues from these ROIs were extracted for western blot analyses of autophagic and apoptotic markers. The major findings were: 1) MB treatment reduced infarct volume and behavioral deficits, 2) MB improved cerebral blood flow to the perfusion-diffusion mismatch tissue after reperfusion and minimized harmful hyperperfusion 24hrs after stroke, 3) MB inhibited apoptosis and enhanced autophagy in the perfusion-diffusion mismatch, 4) MB inhibited apoptotic signaling cascades (p53-Bax-Bcl2- Caspase3), and 5) MB enhanced autophagic signaling cascades (p53-AMPK-TSC2-mTOR). MB induced neuroprotection, at least in part, by enhancing autophagy and reducing apoptosis in the perfusion-diffusion mismatch tissue following ischemic stroke.