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. 2007 Oct 10:1173:117-25.
doi: 10.1016/j.brainres.2007.07.061. Epub 2007 Aug 9.

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

Pamela Maher  1 , Karmen F Salgado, Justin A Zivin, Paul A Lapchak
Affiliations

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

"V体育ios版" Pamela Maher et al. Brain Res. .

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. VSports手机版.

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V体育官网入口 - Figures

Figure 1
Figure 1. Dose Dependence of IAA Toxicity
HT22 cells were treated with increasing doses of IAA for 2 hr. % survival was confirmed by microscopy and measured after 24 hr by the MTT assay. Similar results were obtained in four independent experiments. * indicates a significant difference between control and IAA-treated cells (p < 0.001; ANOVA followed by Bonferroni’s test)
Figure 2
Figure 2. Flavonoids Protect from IAA Toxicity in HT22 Cells
HT22 cells were treated with 20 μM IAA (HT22) for 2 hr alone (Ct) or in the presence of 10 μM 3 hydroxyflavone (HF), 10 μM 3,6 dihydroxyflavone (3,6 DHF), 10 μM 3,7 dihydroxyflavone (3,7 DHF), 10 μM galangin, 10 μM baicalein, 10 μM kaempferol, 10 μM luteolin, 10 μM fisetin or 10 μM quercetin. The flavonoids were also included in the fresh medium added after the 2 hr treatment with IAA (I/R). In other studies, the flavonoids at the same doses were added only to the fresh medium added after the 2 hr IAA treatment (R only). % survival was confirmed by microscopy and measured after 24 hr by the MTT assay. Similar results were obtained in 3–5 independent experiments. All flavonoid treatments provided significant protection (p < 0.001; ANOVA followed by Bonferroni’s test) from IAA toxicity.
Figure 3
Figure 3. Flavonoids Maintain ATP and GSH Levels in Response to Chemical Ischemia
HT22 cells were treated with 20 μM IAA for 2 hr followed by 2 hr in fresh medium alone (Ct) or in the presence of 10 μM 3 hydroxyflavone (HF), 10 μM 3,6 dihydroxyflavone (3,6 DHF), 10 μM 3,7 dihydroxyflavone (3,7 DHF), 10 μM galangin, 10 μM baicalein, 10 μM kaempferol, 10 μM luteolin, 10 μM fisetin, 10 μM quercetin or 10 μM resveratrol. Total ATP (A) and GSH (B) levels were measured by chemical and chemiluminescent assays, respectively as described in Methods. Similar results were obtained in 3–5 independent experiments. * indicates a significant difference between IAA-treated cells (Ct) and cells treated with IAA + flavonoids (p < 0.001; ANOVA followed by Bonferroni’s test) ** indicates a significant difference between IAA + fisetin and IAA + other flavonoids (p < 0.001; ANOVA followed by Bonferroni’s test)
Figure 4
Figure 4. Flavonoids Induce the Expression of Nrf2, the ARE-specific transcription factor, and HO-1, a phase II detoxification protein
HT22 cells were untreated (Ct) or treated with 10 μM fisetin, 10 μM 3 hydroxyflavone (HF), 10 μM 3,6 dihydroxyflavone (3,6 DHF), 10 μM 3,7 dihydroxyflavone (3,7 DHF), 10 μM galangin, 10 μM baicalein, 10 μM quercetin, 10 μM luteolin, 10 μM kaempferol or 10 μM resveratrol for 2 hr (Nrf2) or 24 hr (HO-1). Nuclei were prepared (Nrf2) and equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with Nrf2 antibodies or cell lysates were prepared and equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with HO-1 antibodies. Immunoblotting with anti-actin is shown as a loading control. Similar results were obtained in 3 independent experiments.
Figure 5
Figure 5. Behavioral Improvements in the Rabbit SCEM Following Fisetin Treatment
The control curve (dotted line) has a P50 value of 1.06 ± 0.15 mg (n = 19). Fisetin treatment (50 mg/kg IV) initiated 5 minutes following embolization increased the P50 value to 2.53 ± 0.55 mg (n = 19, *P < 0.05) (dark solid line). The dark circles ● represent the raw data from the control group and the triangles ▲ represent the raw data for the fisetin-treated group. A normal animal for a specific clot weight is represented by a symbol plotted at 0% on the y-axis, whereas an abnormal animal for a specific clot weight is represented by a symbol plotted at 100% on the y-axis.
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