A novel approach to screening for new neuroprotective compounds for the treatment of stroke

Abstract

Despite the significant advances that have been made in understanding the pathophysiology of cerebral ischemia on the cellular and molecular level, only one drug, the thrombolytic tissue plasminogen activator (rt-PA), is approved by the FDA for use in patients with acute ischemic stroke. Therefore, there is a critical need for additional safe and effective treatments for stroke. In order to identify novel compounds that might be effective, we have developed a cell culture-based assay with death being an endpoint as a screening tool. We have performed an initial screening for potential neuroprotective drugs among a group of flavonoids by using the mouse hippocampal cell line, HT22, in combination with chemical ischemia. Further screens were provided by biochemical assays for ATP and glutathione, the major intracellular antioxidant, as well as for long-term induction of antioxidant proteins. Based upon the results of these screens, we tested the best flavonoid, fisetin, in the small clot embolism model of cerebral ischemia in rabbits. Fisetin significantly reduced the behavioral deficits following a stroke, providing proof of principle for this novel approach to identifying new compounds for the treatment of stroke.

Modulation of multiple pathways involved in the maintenance of neuronal function during aging by fisetin

“…Not only did fisetin prevent cell death but it stood out among the flavonoids tested as being able to maintain ATP levels in the presence of IAA. Further studies in the rabbit small clot embolism model of stroke demonstrated that fisetin could significantly improve the behavioral outcome when administered 5 min after the initiation of an embolic stroke [58]. Consistent with our animal data, fisetin was also shown to reduce ischemic damage and infarct volume in the permanent focal middle cerebral artery occlusion model of stroke in rats [73].…”

“…Furthermore, following a single intraperitoneal injection, fisetin was detected in the brains of rats and this correlated with a significant reduction in cerebral damage in a stroke model [73]. Similarly, we have seen significant protection in a rabbit stroke model following intravenous injection of fisetin [58] and we found that oral administration of fisetin could enhance learning and memory in mice [57]. Thus, fisetin is able to affect neuronal function in vivo.…”

“…3). For these studies we used an in vitro ischemia model that we developed for the testing of compounds that might have therapeutic value for the treatment of stroke [58]. This model utilizes the toxin iodoacetic acid (IAA), a well known, irreversible inhibitor of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, in combination with the HT22 nerve cell line.…”

“…Cell lysates were prepared and total ATP levels were measured using a chemiluminescent assay. Adapted from [58] demonstrated that 1 lM fisetin could induce the rapid phosphorylation of CREB and that this phosphorylation was dependent on ERK activation since inhibitors of ERK activation also blocked CREB phosphorylation [57].…”

Long-term treatment of hydrogen-rich saline abates testicular oxidative stress induced by nicotine in mice

“…However, hydrogen, different from normal antioxidants, is electrically neutral and much smaller, so it could easily penetrate blood-testis barrier and enter cells and organelles such as the nucleus and mitochondria. (2) Hydrogen is able to selectively reduce the hydroxyl radical and ONOO-, the most cytotoxic chemicals of ROS, and effectively protect cells, but does not react with other ROS, which possess physiological roles [27].…”