(−)-Epicatechin (EC) is protective against transient middle cerebral artery occlusion (MCAO)-induced cerebral damage in wild-type (WT) mice. (A) A schematic diagram showing the pretreatment protocol of EC administration (90 minutes before MCAO) in WT mice. (B–D) EC was administered 90 minutes before 90-minute MCAO and 24 hours of reperfusion. (B) Infarct volumes were significantly smaller than those of vehicle-treated (control; n=10) mice at EC doses of 30 (n=10), 15 (n=9), and 5 (n=10), but not 2.5 mg/kg (n=12). (C) Neurologic deficit at 24 hours was significantly less severe in the mice that were administered 30 mg/kg EC than in the vehicle-treated mice. (D) No significant differences in cerebral blood flow (CBF), as monitored by laser-Doppler flowmetry, were observed among the mouse groups receiving vehicle or EC (n=5/group). (E) A schematic diagram showing the posttreatment protocol of EC administration (3.5 and 6 hours after MCAO) in WT mice. (F) WT mice were subjected to 90 minutes of MCAO, and EC (30 mg/kg) was administered orally after 3.5 hours (n=10) or 6 hours (n=8). Quantification showed that mice posttreated with EC at 3.5 hours had significantly smaller infarct volumes than did vehicle-treated mice (n=10), but no protection was observed in mice posttreated at 6 hours. (G) After 72 hours of reperfusion, neurologic deficit scores of 3.5-hour posttreated mice, but not of 6-hour posttreated mice, were significantly smaller than those of the vehicle-treated mice. Data represent means ± s.e.m.; ^*P<0.05; ^**P<0.01; NS, not significant.

Epicatechin (EC) treatment reduces N-methyl-aspartate (NMDA)-induced acute excitotoxicity in mice. (A) A schematic diagram showing the pretreatment protocol of EC administration in wild-type (WT) mice injected with NMDA. (B, C) Mice were pretreated with either vehicle or 30 mg/kg EC (n=12/group) before being administered a unilateral intrastriatal injection of 15 nmol NMDA. At 48 hours after NMDA injection, mice were killed and brains were processed for lesion volume analysis. (B) Representative photographs of Cresyl Violet-stained brain sections of mice that were pretreated with vehicle (left) or EC (right) followed by a unilateral intrastriatal NMDA injection. (C) Bar graph showing the volume of brain damage induced by NMDA. The brain lesion volume was attenuated in the EC-treated group compared with the vehicle-treated group. Values are reported as means ± s.e.m. ^*P<0.03 versus vehicle-treated group.

Neuronal protection by epicatechin (EC). Neurons were grown for 24 hours in culture medium alone (vehicle) or in the presence of tert-butyl hydroperoxide (t-BuOOH; 60 μmol/L) or H₂O₂ (60 μmol/L) with or without EC (100 μmol/L). (A) Neuronal viability was assessed by the MTT assay; the absorbance at 570 nm is shown. (B) Neuronal cell death was assessed by the level of lactate dehydrogenase (LDH) released into the cell culture medium; the absorbance at 490 nm is shown. (C) Caspase-3/7 activity was determined by the amount of fluorescent substrate formed and is shown as percentage of control. ^*P<0.05, ^**P<0.01, ^***P<0.001 versus vehicle control; ^†P<0.05 versus the corresponding t-BuOOH or H₂O₂ control. Values shown are the mean±s.e.m. of three batches of cells.

Effects of epicatechin (EC) on heme oxygenase 1 (HO1) and Nrf2 protein expression and nuclear accumulation in cortical neurons. (A, B, left panels) Western blots showing HO1 and actin expression in (A) cortical neurons treated for 6 hours with Neurobasal medium alone or with serial concentrations of EC and (B) cortical neurons treated with 100 μmol/L EC for 15 minutes to 6 hours. The expression of actin (used as a loading control) was unaffected. (A, B, right panels) The bar graphs show the ratio of density captured from the HO1 bands to that of actin. Values shown are means±s.e.m. for three independent blots. (C, left panel) The effect of EC on primary cortical neurons incubated for the times shown (minutes) with serum-free B27 minus antioxidant-supplemented medium alone or that containing EC (100 μmol/L). Nuclear and cytoplasmic samples were analyzed by Western blot with antibodies to Nrf2 and actin. The actin expression level was unchanged. (C, right panel) The bar graph shows the ratio of density captured from the Nrf2 band to that of actin. ^*P<0.05 versus control. (D) Cortical neurons incubated under basal conditions or treated with EC for 30 minutes were stained with antibodies to Nrf2 (red) and neuronal marker NeuN (green). The merged image shows colocalization, depicting the presence of Nrf2 in the nucleus.

The protective effect of epicatechin (EC) is lost in Nrf2^−/− and HO1^−/− mice. (A) A schematic diagram showing the pretreatment protocol of EC administration (90 minutes before middle cerebral artery occlusion (MCAO)) in Nrf2^−/− and HO1^−/− mice. (B, C) Nrf2^−/− and HO1^−/− mice and their wild-type (WT) littermates were treated with vehicle (n=10 WT, 7 HO1^−/−, and 8 Nrf2^−/−) or 30 mg/kg EC (n=11 WT, 7 HO1^−/−, and 8 Nrf2^−/−) 90 minutes before MCAO. At 24 hours after reperfusion, mice were tested for neurologic deficits and killed; their brains were collected and sectioned for TTC staining (note that the values for the WT mice are those from Figure 1 and are shown again here for comparison). (B) Quantification analysis revealed that the infarct volumes of EC-treated WT mice were significantly smaller than those of vehicle-treated WT mice. The infarct volumes of EC-treated Nrf2^−/− and HO1^−/− mice were not significantly different from those of their vehicle-treated counterparts, but were significantly higher than those of the EC-treated WT mice. (C) Similarly, the neurologic deficit scores of EC-treated Nrf2^−/− and HO1^−/− mice at 24 hours after MCAO were significantly higher than those of the EC-treated WT mice and not significantly different from those of their vehicle-treated counterparts. ^*P<0.05 compared with EC-treated WT controls; NS, not significant. (D) Neurons were grown for 24 hours in serum-free medium with B27 supplement minus antioxidant in the presence and absence of tert-butyl hydroperoxide (t-BuOOH; 60 μmol/L) or H₂O₂ (60 μmol/L), with or without EC (100 μmol/L), SnPPIX (10 μmol/L), or combinations thereof. Cell viability was assessed with the MTT assay. ^***P<0.001 versus vehicle; ^†P<0.05, ^††P<0.01 versus t-BuOOH or H₂O₂; ^‡‡‡P<0.001 versus t-BuOOH+EC or H₂O₂+EC. Values shown are the means ± s.e.m. of three batches of cells. (E) Primary postnatal cortical neurons isolated from HO1^−/− or Nrf2^−/− mice were incubated for 24 hours in Neurobasal medium alone (control) or in the presence or absence of t-BuOOH (60 μmol/L), EC (100 μmol/L), or combinations thereof. Cell viability was assessed with the MTT assay. ^***P<0.001 versus control; NS, not significantly different versus t-BuOOH.