* Abstract Results Results Introduction Introduction Abstract Ginkgo biloba Prevents Transient Global Ischemia-Induced Delayed Hippocampal Neuronal Death Through Antioxidant and Anti-inflammatory Mechanism Jatin Tulsulkar and Zahoor A. Shah Department of Medicinal & Biological Chemistry, University of Toledo College of Pharmacy and Pharmaceutical Sciences, Health Sciences Campus, Toledo, OH I Abstract Results Results 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. 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 pretreatment protected neurons in the hippocampus against global ischemia. Immunofluorescence-based studies demonstrated that the vehicle group had comparatively lower expression levels of heme oxygenase 1 (HO1), nuclear factor erythroid 2-related factor 2 (Nrf2), and vascular endothelial growth factor (VEGF) which was attenuated in EGb 761 pretreated groups. In addition, increased number and expression of activated astrocytes and microglia in the vehicle group was observed to be comparatively lower in EGb 761 pretreated group. These results suggest that the neuroprotective effect of EGb 761 is associated with activation and upregulation of HO1/Nrf2 pathway and VEGF, and by downregulation of inflammatory mediators such as astrocytes and microglia. Figure 1: Effect of EGb761 and its components on Hippocampal neuronal survival A B * Figure 5: Effect of EGb 761 on astrocytes activation in CA-1 region of hippocampus. Figure 6: Effect of EGb 761 on microglial activation. Percent of cell death DAPI Astrocytes Overlay DAPI CD11B Overlay DAPI TUNEL Merged Merged (60X) B Overlay Sham Sham Sham BB GB GA TFM Vehicle Sham EGb 761 Vehicle Vehicle Vehicle Figure 1: Fig. 1. Detection of delayed hippocampal neuronal cell death in CA-1 region by TUNEL assay and EGb 761 neuroprotection. (A) Hippocampal section showing 4 boxes (570 µm X 680 µm) in CA1 subregion were selected for counting TUNEL positive cells (B) TUNEL labeling (green)and DNA staining with blue (DAPI). Quantitative analysis of different CA-1 regions showed that only EGb 761 significantly protected hippocampal neurons. Data are expressed as means±SEM; p<0.05 vs. vehicle; magnifications, 10X for DAPI, TUNEL and merged photograps, 60X for magnified view. EGb 761 EGb 761 EGb 761 10 µm 10 µm Introduction Introduction Abstract BB Figure 5: EGb 761 reduces astrocytes expression in CA-1 region of hippocampus. Figure 6: EGb reduces microgllial expression in CA-1 region of hippocampus. Conclusion The standardized EGb 761 extract is one of the most effective and commonly used nutraceuticals in the market. In Europe, EGb 761 is prescribed for multiple problems related to mental health and overall wellbeing. Numerous studies, including ours, have shown the neuroprotective effects of EGb 761 and its constituents in different models of ischemia, and the mechanism of action has been attributed to hemeoxygenase 1 (HO1), Nrf2 and vascular endothelial growth factor (VEGF) (Shah et al., 2011, Nada). However, there are very few studies that have shown protective mechanism of EGb 761 in tBCCAO induced delayed hippocampal neuronal death. After the transient global ischemia, reperfusion process brings a surge in inflammatory markers leading to activation of microglial and astrocytic cells in addition to enhanced oxidative stress in the hippocampus (Chan SA et.al. 1998). Of late, targeting these inflammatory markers in conjunction with oxidative stress has become a major focus of therapeutic strategies. In this study, we hypothesized that pretreatment with EGb 761 and its bioactive compounds [bilobalide (BB); Ginkgolide A (GA); Ginkgolide B (GB); Terpene Free material (TFM)] could protect against delayed neuronal cell death in the hippocampus after 8min-tBCCAO. Furthermore, we investigated for the first time the molecular mechanism of protection offered by EGb 761 in hippocampus of mice by studying the expression pattern and localization of HO1, Nrf-2, VEGF, and other inflammatory markers. GB Figure 3: NRF2 induction in CA-1 region of hippocampus by Egb 761 We found that only EGb 761 pretreatment prevents delayed neuronal death (DND) induced by 8-minutes of global ischemia. However, other components of EGb 761 (BB, GA, GB and TFM) were not observed to protect hippocampal DND. We observed that EGb 761 enhanced the expression of HO1, Nrf2 and VEGF. Interestingly, we have for the first time shown that HO1 is co-localized in endothelial cells of the hippocampus. Furthermore, we showed that EGb 761 pretreatment reduces activation of astrocytes and microglia during global ischemia. GA DAPI Nrf2 Overlay TFM Sham 10 µm Figure 2: HO-1 induction in the CA-1 region of hippocampus in EGb 761 treated group. Vehicle DAPI HO1 Overlay A Sham EGb 761 10 µm References Figure 3: Egb 761 induces expression of Nrf 2 in ca-1 region of hippocampus Methods Vehicle Shah, Z. A., et al., 2011. Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection. Neuroscience. 180, 248-55. Chan SA, Reid KH, Schurr A, Miller JJ, Iyer V, Tseng MT (1998) Fosphenytoin reduces hippocampal neuronal damage in rat following transient global ischemia. Acta Neurochir (Wien) 140:175-180. Zhen G, Dore S (2007) Optimized protocol to reduce variable outcomes for the bilateral common carotid artery occlusion model in mice. J Neurosci Methods 166:73-80. Figure 4: VEGF expression in CA-1 region of hippocampus by EGB 761 Male C57BL/6 mice were administered orally with 10% polyethylene glycol (PEG) (Vehicle) and test drugs [Egb 761 (100mg/kg), BB (6mg/kg), GA (6mg/kg), GB (6mg/kg), TFM (10mg/kg)] 7 days prior to tBCCAO and survived for 7 days to study the delayed neuronal cell death in hippocampus. TUNEL assay and immunohistochemistry: Sections were de-paraffinized in xylene and in graded series of ethanol; incubated with 20 µg/ml of proteinase K; incubated with equilibrium buffer for 10 min and then incubated in the mixture of terminal deoxynucleotidyl transferase (TrdT) and equilibration buffer in a humidified chamber at 37oC for 1 hour and DAPI was mounted on slides. Double staining: Sections were first incubated in bovine serum albumin to block non-specific binding and then with primary antibodies to CD31 (1:100, endothelial cell marker, mouse monoclonal, Millipore, Temecula CA); VEGF and HO1 overnight at 4oC. Cells were washed in PBS and incubated in anti mouse IgG and anti rabbit IgG. Quantitative analysis: Analysis of dead neurons for TUNEL assay were quantified by counting the green fluorescent cells in 4 boxes of 570 µm X 680 µm each, applied approximately at the center of CA-1 subregion. Counting was performed by an investigator blinded to the study design. Quantification of immunofluorescent images was carried out using image J software. EGb 761 DAPI VEGF Overlay A 10 µm Sham DAPI CD-31 HO1 Merge B Vehicle Acknowledgements 10 µm Figure 2: Induction and co-localization of HO-1 in endothelial cells EGb 761 Support contributed by: NIH-National Center for Complimentary and Alternative Medicine (R00AT004197) to ZAS. EGb 761 extract and components from Dr. Willmar Schwabe Pharmaceuticals, Germany are gratefully acknowledged. 10 µm DAPI VEGF CD-31 Overlay B 10 µm Figure 4: EGb 761 induces VEGF in CA-1 region of hippocampus.