Background and Purpose-Excess brain extracellular glutamate induced by cerebral ischemia leads to neuronal death, mainly through overactivation of N-methyl-D-aspartate (NMDA) receptors. The cholesterol-lowering drugs statins have been reported to protect from NMDA-induced neuronal death but, so far, the mechanism underlying this protection remains unclear. Because NMDA receptors have been reported to be associated with the cholesterol-rich membrane domains known as lipid rafts, we have investigated the effect of treatments that deplete cholesterol levels on excitotoxicity and on association of NMDA receptors to lipid rafts. Methods-Primary neuronal cultures were pretreated with inhibitors of cholesterol synthesis and cholesterol, and NMDA-induced cell death was determined by measuring release of lactate dehydrogenase. Lipid raft fractions were isolated and Western blots were performed. Results-Treatment with the inhibitors of cholesterol synthesis simvastatin, which inhibits the first step of cholesterol synthesis, or AY9944, which inhibits the last step of cholesterol synthesis, protected neurons from NMDA-induced neuronal death by 70% and 54%, respectively. Treatment with these compounds reduced neuronal cholesterol levels by 35% and 13%, respectively. Simvastatin and AY9944 reduced the association of the subunit 1 of NMDA receptors (NMDAR1) to lipid rafts by 42% and 21%, respectively, and did not change total expression of NMDAR1. Addition of cholesterol reduced neuroprotection by statins and AY9944, and partially reverted the effect of simvastatin on the association of NMDAR1 to lipid rafts. Conclusions-These data demonstrate that reduction of cholesterol levels protects from NMDA-induced neuronal damage probably by reducing the association of NMDA receptors to lipid rafts. (Stroke. 2008;39:1269-1275.)

In general, iron represents a double-edged sword in metabolism in most tissues, especially in the brain. Although the high metabolic demands of brain cells require iron as a redox-active metal for ATP-producing enzymes, the brain is highly vulnerable to the devastating consequences of excessive iron-induced oxidative stress and, as recently found, to ferroptosis as well. The blood–brain barrier (BBB) protects the brain from fluctuations in systemic iron. Under pathological conditions, especially in acute brain pathologies such as stroke, the BBB is disrupted, and iron pools from the blood gain sudden access to the brain parenchyma, which is crucial in mediating stroke-induced neurodegeneration. Each brain cell type reacts with changes in their expression of proteins involved in iron uptake, efflux, storage, and mobilization to preserve its internal iron homeostasis, with specific organelles such as mitochondria showing specialized responses. However, during ischemia, neurons are challenged with excess extracellular glutamate in the presence of high levels of extracellular iron; this causes glutamate receptor overactivation that boosts neuronal iron uptake and a subsequent overproduction of membrane peroxides. This glutamate-driven neuronal death can be attenuated by iron-chelating compounds or free radical scavenger molecules. Moreover, vascular wall rupture in hemorrhagic stroke results in the accumulation and lysis of iron-rich red blood cells at the brain parenchyma and the subsequent presence of hemoglobin and heme iron at the extracellular milieu, thereby contributing to iron-induced lipid peroxidation and cell death. This review summarizes recent progresses made in understanding the ferroptosis component underlying both ischemic and hemorrhagic stroke subtypes.

The studied biomarkers were not sufficient for an accurate differential diagnosis of stroke in the hyperacute setting. Additional discovery of new biomarkers and improvement on laboratory techniques seem necessary for achieving a molecular diagnosis of stroke.