Ginkgo biloba prevents transient global ischemia-induced delayed hippocampal neuronal death through antioxidant and anti-inflammatory mechanism

Abstract

We have previously reported neuroprotective properties of Ginkgo biloba/EGb 761® (EGb 761) in transient and permanent mouse models of brain ischemia. In a quest to extend our studies on EGb 761 and its constituents further, we used a model of transient global ischemia induced delayed hippocampal neuronal death and inflammation. Mice pretreated with different test drugs for 7 days were subjected to 8-min bilateral common carotid artery occlusion (tBCCAO) at day 8. After 7 days of reperfusion, mice brains were dissected out for TUNEL assay and immunohistochemistry. In situ detection of fragmented DNA (TUNEL staining) showed that out of all test drugs, only EGb 761 (13.6% ± 3.2) pretreatment protected neurons in the hippocampus against global ischemia (vs. vehicle, 85.1% ± 9.9; p<0.05). Immunofluorescence-based studies demonstrated that pretreatment with EGb 761 upregulated the expression levels of heme oxygenase 1 (HO1), nuclear factor erythroid 2-related factor 2 (Nrf2), and vascular endothelial growth factor (VEGF) as compared to the vehicle group. In addition, increased number of activated astrocytes and microglia in the vehicle group was observed to be significantly lower in the EGb 761 pretreated group. Together, these results suggest that EGb 761 is a multifunctional neuroprotective agent, and the protection is in part associated with activation of the HO1/Nrf2 pathway, upregulation of VEGF and downregulation of inflammatory mediators such as astrocytes and microglia.

Fig. 1

EGb 761 pretreatment protects hippocampal neurons. Mice were pretreated with test drugs for 7 days and subjected to eight minutes of global ischemia at day 8. Mice brains isolated after 7 days of global ischemia were fixed in 4% paraformaldehyde and paraffin fixed for TUNEL assay. (A) Coronal hippocampal sections (4μm) showing 4 boxes (570 μm × 680 μm each/mice) in CA1 subregion were selected for counting TUNEL positive cells (B) TUNEL positive cells are stained fluorescent green, and the nuclei are stained blue (DAPI). The box in the merged photographs represents the area selected for magnified view (60X). Sham (n = 4), no death of neurons; vehicle (n = 6), significantly higher neuronal death was observed; EGb 761 (n = 7) pretreated group, significantly restored neuronal survival. Other components BB (n = 5), GA (n = 4), GB (n = 5) and TFM (n = 4), no protection was observed. (C) Quantitative analysis of CA-1 subregions showed that only EGb 761 significantly protected hippocampal neurons. Data are expressed as means ± SEM; 10X magnification for DAPI, TUNEL and merged photographs; 60X for magnified view; ^# vs. Sham; * vs. vehicle; p < 0.05.

Fig. 2

EGb 761upregulates HO1 in the CA-1 region of the hippocampus. Hippocampal sections (paraffin) of mice pretreated with EGb 761 for 7 days and subjected to eight-minutes of global ischemia were used in this immunofluorescence assay. (A) HO1 expression was visualized by immunofluorescent staining with specific rabbit polyclonal antibody for HO1, followed by secondary anti rabbit IgG antibody (red). DNA was stained blue (DAPI). (B) Robust upregulation of HO1 expression was observed in the hippocampus of mice pretreated with EGb 761 compared to vehicle. (C) Localization of HO1 was evaluated by double staining with CD-31 (endothelial cell marker) monoclonal antibody followed by secondary anti rat IgG (green). The arrows in the figure show overlapping of CD-31(green) on HO1 (red), thereby giving out yellow fluorescence. HO1 was observed to be expressed by endothelial cells. Magnified view, 60X; ^# vs. Sham; * vs. vehicle; p < 0.05.

Fig.3

EGb 761 upregulates Nrf2 in the CA-1 region of the hippocampus. (A) Hippocampal sections (paraffin) of mice pretreated with EGb 761 for 7 days and subjected to 8-minutes of global ischemia were used in this immunofluorescence assay. Nrf2 expression was visualized by immunofluorescent staining by a specific rabbit polyclonal antibody for Nrf2, followed by secondary anti rabbit IgG (red). DNA was stained blue (DAPI). (B) Nrf2 expression was observed to be increased in the hippocampus of mice pretreated with EGb 761 but not in vehicle group (B). Magnified view, 60X; * vs. vehicle; p < 0.05.

Fig. 4

EGb 761 upregulates VEGF expression in the CA-1 region of the hippocampus. Hippocampal sections (paraffin) of mice pretreated with EGb 761 for 7 days and subjected to 8-minutes of global ischemia were used in this immunofluorescence assay. (A) VEGF expression was visualized by immunofluorescent staining with a specific rabbit polyclonal antibody followed by secondary anti rabbit IgG (red), and DNA was stained blue (DAPI). (B) VEGF expression was increased in EGb 761 pretreated mice as compared to vehicle group. (C) Double staining for VEGF localization was visualized by a specific anti rabbit polyclonal antibody followed by secondary anti rabbit IgG (red) and CD-31 mouse monoclonal antibody for endothelial cell marker followed by secondary anti-rat IgG (green). The arrows in the figure show overlapping of CD-31 (green) on HO1 (red), thereby giving out yellow fluorescence. VEGF was observed to be expressed in endothelial cells. Magnified view, 60X; * vs. vehicle; p < 0.05.

Fig. 5

Effect of EGb 761 on astrocyte activation. (A and B) Activated astrocytes (stained with rabbit polyclonal antibody directed against astrocytic activation (GFAP) were higher in the hippocampus of the vehicle-treated group compared to basal levels in the sham group. EGb 761 pretreated group followed by global ischemia showed fewer activated astrocytes compared to vehicle group. Counting of active astrocytes was performed in a 60X field and expressed as activated cells/field. Magnified view, 60X; ^# vs. Sham; * vs. vehicle; p < 0.05

Fig. 6

Effect of EGb 761 on microglial activation. (A and B) Activated microglial cells (stained with mouse monoclonal antibody against microglial activation (Cd11b) were higher in the hippocampus of the vehicle-treated group compared to basal levels in the sham group. EGb 761 pretreated group followed by global ischemia showed fewer number of activated microglia cells compared to vehicle group. Counting of active microglial cells were performed in a 60X magnified view and expressed as activated cells/field. Magnified view, 60X; ^# vs. Sham; * vs. vehicle; p < 0.05.