Brain Research, Volume 1173, 10 October 2007, Pages 117-125
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.
Stroke is the leading cause of adult disability and the third leading cause of death in the US. Worldwide, approximately 6 million people died of stroke in 2005, with a projected increase over the next decade of 12% (Ingall, 2004). Ischemic stroke occurs when the normal blood supply to the brain is disrupted, usually due to artery blockage by a blood clot, thereby depriving the brain of oxygen and metabolic substrates and hindering the removal of waste products (for review see Lapchak and Araujo, 2007). The nerve cell damage caused by cerebral ischemia results in functional impairment and/or death. Despite the significant advances that have been made in understanding the pathophysiology of cerebral ischemia on the cellular and molecular level, only one drug, recombinant tissue-type plasminogen activator (rt-PA), is approved by the FDA for use in patients with ischemic stroke (Green and Shuaib, 2006). Unfortunately, the utilization of rt-PA is limited by its short time window of efficacy and its potential to cause intracerebral hemorrhage (NINDS rt-PA trial) (Lapchak, 2002a, Lapchak, 2002b, Lyden and Zivin, 1993). Thus, there is a critical need for additional safe and effective treatments for stroke.
To date, most of the compounds that have been tested in clinical trials for the treatment of ischemic stroke were chosen based on mechanisms proposed to underlie nerve cell death in stroke (Green and Shuaib, 2006). In order to identify novel compounds that might be effective for the treatment of stroke, a new approach is needed. Rather than taking a mechanism-based approach, we have chosen to utilize a cell culture-based assay with death being an endpoint as a screening tool. In this way we can identify potential neuroprotective compounds that act at multiple sites in the cell death pathway and therefore could provide a better chance at protecting the cells.
Most cell culture models of stroke utilize primary cortical cultures that are exposed to some form of hypoxia/glucose deprivation (e.g. Arthur et al., 2004, Munns et al., 2003). While these assays are clearly closer to the in vivo condition than assays utilizing neuronal cell lines, they have a number of problems which make them difficult to use for the routine screening of potential neuroprotective compounds. The most serious problem is that the conditions needed to kill a fixed percentage of cells are highly variable from experiment to experiment. In order to circumvent these problems, we used the mouse hippocampal cell line, HT22, in combination with chemical ischemia as an initial screen for potential neuroprotective drugs for the treatment of stroke. Further screens were provided by biochemical assays for ATP and glutathione, the major intracellular antioxidant, as well as for the long-term induction of antioxidant proteins.
In the first test of this approach we decided to focus on flavonoids, polyphenolic compounds widely distributed in fruits and vegetables and therefore regularly consumed in the human diet (for reviews see Heim et al., 2002, Middleton et al., 2000, Ross and Kasum, 2002). Flavonoids were historically characterized on the basis of their antioxidant and free radical scavenging effects. However, more recent studies have shown that flavonoids have a wide range of activities that could make them particularly effective as neuroprotective agents for the treatment of stroke. First, flavonoids can protect nerve cells from oxidative stress-induced death by both acting as a direct antioxidant and by maintaining high levels of glutathione (GSH), the major intracellular antioxidant (Ishige et al., 2001). Second, multiple studies have shown that certain flavonoids can induce the activity and expression of phase II detoxification proteins (Hanneken et al., 2006, Hou et al., 2001, Maher and Hanneken, 2005b, Maher, 2006, Myhrstad et al., 2002, Valerio et al., 2001). The phase II detoxification proteins include enzymes associated with glutathione biosynthesis and metabolism and redox sensitive proteins such as heme oxygenase 1 (HO-1) (Hayes and McLellan, 1999). By inducing the expression of antioxidant defense enzymes, these flavonoids have the potential to have long lasting effects on cellular function. Finally, flavonoids have been shown to induce neurite outgrowth (Sagara et al., 2004), reduce inflammation (Read, 1995), improve cerebral blood flow (Curin et al., 2006), prevent platelet aggregation (Curin et al., 2006) and enhance cognition (Maher et al., 2006), all properties that could have additional benefits for the treatment of stroke. Indeed, the phenylpropanoid chlorogenic acid was recently shown to improve behavioral performance in a rabbit embolic stroke model (Lapchak, 2007). Finally, in animal studies, flavonoids have generally shown low levels of toxicity over a wide range of doses.
In order to test the hypothesis that in vitro assays can be used to identify compounds that might be useful for the treatment of stroke, we used our cell culture-based assays to identify flavonoids with neuroprotective activities. Based on the results of these screens, we then tested the best flavonoid, fisetin, in the small clot embolism model of cerebral ischemia in rabbits.
In order to induce chemical ischemia, we used the compound iodoacetic acid (IAA), a well known, irreversible inhibitor of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (Winkler et al., 2003) in combination with the HT22 mouse hippocampal cell line. IAA has been used in a number of other studies to induce ischemia in nerve cells (Rego et al., 1999, Reiner et al., 1990, Reshef et al., 1997, Sigalov et al., 2000, Sperling et al., 2003) but not previously as a screen for
The above data show that an in vitro neuroprotection screen using a neuronal cell line and chemical ischemia in combination with biochemical assays targeting key metabolic changes associated with ischemic stroke in vivo can be used to identify novel neuroprotective compounds that are effective against stroke in an animal model. Although in this study only a limited screen of potential neuroprotective compounds was carried out for the purpose of providing proof of principle for this approach, in
Flavonoids were from Alexis (San Diego, CA), Sigma/Aldrich (St. Louis, MO) or Indofine Chemical Co. (Hillsborough, NJ). All other chemicals were from Sigma.
Fetal calf serum (FCS) and dialyzed FCS (DFCS) were from Hyclone (Logan, UT). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Invitrogen (Carlsbad, CA). HT-22 cells (Davis and Maher, 1994, Maher and Davis, 1996) were grown in DMEM supplemented with 10% FCS and antibiotics.
Cell viability was determined by a modified version of
