Design

A randomized, observational, controlled animal experiment.

Time and setting

This experiment was performed at the Department of Pharmacology, Anhui Medical University, China between May 2010 and July 2011.

Materials

Drugs and reagents

BHDF containing total astragalosides 51%, total paeony glycoside 12%, and carthamus tinctorius flavonoids 8% was provided by Hefei Qi-xing Medicine and Technology Co., Ltd. (Solid powder, Lot No. 20090504, Hefei, Anhui Province, China).

Ginkgo biloba extract (tablets; 2.4 mg/pill; approval No. GYZZ Z20028024; lot No. 080201) was provided by the Herb Production Factory of Ningbo City (Ningbo, Zhejiang Province, China).

Animals

Ninety-six healthy adult male Sprague-Dawley rats aged 6–8 weeks and weighing 250–320 g were used. The animals were obtained from the Experimental Animal Center of Anhui Medical University (License No. SCXK (Wan) 2005-001). The animals were housed in clear plexiglas cages with stainless steel wire lids and filter tops, in a temperature-controlled (20–24°C) room. Sprague-Dawley rats were maintained on a 12-hour light/dark cycle (lights on at 7:00 a.m.). Rats were tested during the light phase of the light/dark cycle. All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[31].

Methods

In vivo experiments

Establishment of the rat middle cerebral artery occlusion model: Rats were anesthetized with an intraperitoneal injection of chloral hydrate (10% (w/v), 0.3 mL/100 g), then fixed in a supine position. After removing the fur around the neck, a midline incision was performed at the cervical region. The middle cerebral artery occlusion model was performed as previously described in detail with slight modifications^[32-34]. With the tip rounded by heating near a flame, a length of 20.0–22.0 mm of 3-0 surgical monofilament nylon suture was inserted through the right common carotid arteries into the internal carotid artery until it blocked the origin of the middle cerebral artery. Rats were subjected to 2 hours of focal ischemia followed by 22 hours of reperfusion and were allowed to survive for 24 hours. The suture was not advanced to occlude the middle cerebral artery in the sham group. Rectal temperature was maintained at 37°C with air conditioning throughout the surgical procedure. Finally, animals were allowed to regain consciousness and were maintained at a room temperature of 25°C with free access to food and water. Neurological symptoms were examined and only rats demonstrating circling contralateral to the middle cerebral artery occlusion, detection of hemiplegia and abnormal posture^[35] were included in the study. All rats were euthanized 24 hours after ischemia.

Drug treatment: BHDF (50, 100, 200 mg/kg; reference to the pre-experiment) or Ginkgo biloba extract      (100 mg/kg) were administered to the appropriate treatment group by gavage for 7 days (once a day). Drugs were administered 7 days prior to undergoing middle cerebral artery occlusion. Animals in the sham group and the ischemia/reperfusion group were administered the same volume (1 mL/100 g) of 0.9% (w/v) saline by gavage.

Measurement of infarct volume: The rats were given an overdose of chloral hydrate and decapitated. Brains were quickly removed, rinsed in cold PBS, placed at –80°C for 3 minutes and sliced into 2 mm thick coronal sections. Tissue sections were incubated for 30 minutes at 37°C in a solution of 2% (w/v) 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO, USA) in PBS in the dark. The borders of the infarct in each brain slice were outlined and the area quantified using NIH image J software. To correct for brain swelling, the infarct area was determined by subtracting the area of undamaged tissue in the left hemisphere from that of the intact contralateral hemisphere. Infarct volume was calculated by integrating the infarct areas for all slices of each brain^[36]. Infarct volume ratio was calculated according to the following formula: Infarct volume ratio = (Σ infarct area × thickness)/(Σ brain slices area × thickness) × 100%.

Preparation of brain samples: middle cerebral artery occluded rats were anesthetized and perfused through the heart, first with PBS, and then with PBS containing 4% (w/v) paraformaldehyde. Rat brains were removed rapidly, and then fixed with 10% (v/v) formalin and embedded in paraffin blocks. Sections from infracted area of each group were sliced into 4-μm-thick slices using a microtome (RM-2145; Leica Slicer, Solms, Germany) for hematoxylin-eosin^[37-38] and immunohistochemical staining.

Immunohistochemical staining: Immunohistochemical staining was used to determine the protein expression of NF-κB, TNF-α and nestin. A commercially available avidin-biotin-peroxidase complex staining kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Paraffin-embedded cerebral tissue blocks were sectioned at 4 μm. Sections were deparaffinized in xylene and dehydrated through graded ethanol, then washed thrice with PBS for 5 minutes. The sections were treated with 3% (v/v) H₂O₂ for 10 minutes at room temperature to quench endogenous peroxidase activity. After blocking with 5% (w/v) normal goat serum in PBS for 30 minutes, they were then incubated in primary antibodies for 1 hour at 37°C. The primary antibodies were rabbit polyclonal antibodies (1:200): anti-NF-κB/p65 (sc-8008), anti-TNF-α (sc-1350) and anti-nestin (sc-20978), all purchased from Beijing Zhongshan Biological Technology Co., Ltd. (Beijing, China). PBS was used to replace the antibody in the negative control group. After three washes in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (1:1 000; Beijing Zhongshan Biological Technology Co., Ltd.) for 30 minutes, followed by incubation with streptavidin- biotin complex for 60 minutes at 37°C. Ten non- overlapping random fields of view of each section from each group were captured using a microscope (Nikon 80i, Tokyo, Japan). Images were analyzed using the Jetta 801D morphological image analysis system (Version 6.0; Nanjing, Jiangsu Province, China). The parameters remained unchanged for each treatment group. The positive track point was set as the brown localization expressed in the cytoplasm or membrane. The total number of positive cells in each visual field was calculated as follows: number of positive cells/total cells × 100%.  

In vitro experiments

HT22 hippocampal neuronal cell cultures: HT22 cells (provided by Nanfang Medical University, Guangzhou, Guangdong Province, China), a murine hippocampal cell line, were plated at a density of 10⁵–10⁶ cells per well in plates. The cells were cultured in Dulbecco’s modified Eagle’s medium (ThermoScientific, Barrington, IL, USA) containing 10% (v/v) fetal calf serum to ensure normal neuronal development and survival, and medium was replaced every 2 days. Cells were incubated at 37°C in a 5% CO₂ and 95% air humidified atmosphere until the day of the experiment. 

Oxygen-glucose deprivation model: HT22 hippocampal neurons in the logarithmic growth phase were used for all experiments. Sodium dithionite (1 mM; Wuxi Zhanwang Chemical Reagent Co., Ltd., Wuxi, Jiangsu Province, China) was used to induce oxygen-glucose deprivation in all experiments^[39-40]. HT22 hippocampal neuronal cells were randomly divided into six groups: (1) Control group (no oxygen-glucose deprivation): cells were cultured with Dulbecco’s modified Eagle’s medium, (2) Model group (oxygen-glucose deprivation alone, with no treatment): cells were cultured for 24 hours in fresh medium containing sodium dithionite (1 mM), without glutamate, (3) BHDF groups: cells were treated with BHDF (10, 30, 100 mg/L) containing Dulbecco’s modified Eagle’s medium, and then incubated for    24 hours. The supernatant was then removed and replaced with fresh medium containing sodium dithionite (1 mM) without glutamate for an additional  24 hours, (4) Ginkgo biloba extract group: cells were treated with 100 mg/L Ginkgo biloba extract, and the same method was performed as for the BHDF groups, except that BHDF was replaced with Ginkgo biloba extract.

Assessment of cell supernatant: Supernatants were collected to measure lactate dehydrogenase release and malondialdehyde content. Lactate dehydrogenase release was used to measure neurotoxicity^[41]. Lactate dehydrogenase is a cytoplasmic enzyme released from cells with compromised cell membranes. Malondialdehyde can induce cell damage in various ways and levels reflect the content of free radicals produced by lipid peroxidation^[41]. Lactate dehydrogenase activity and malondialdehyde content were detected using a colormetric reaction, which was read at an absorbance of 440 and 532 nm, respectively, using a microplate reader (Haimen Boyang, Haimen, Jiangsu Province, China). The assays were performed using commercially available kits (Nanjing Jiancheng Biological Company, Nanjing, Jiangsu Province, China). Both assays were conducted according to the manufactures’ instructions.

The MTT assay: Cell survival was assayed using the microculture tetrazolium method^[42]. Approximately 10⁵ to 10⁶ cells/well were dispensed within 96-well culture plates in 10 μL volumes. In each of the above cultured wells, 10 μL of MTT working solution (Sigma) was added to every well, and then incubated continuously for 4 hours. All culture medium supernatant was removed from the wells and then each of the plates was replaced with 100 μL of dimethyl sulfoxide (Sigma). The absorbance value of each well was measured using a microplate reader (Haimen Boyang) at 550 nm. Cell survival rate was calculated according to the following formula: Cell survival rate = absorbance value of observed group/absorbance value of control group × 100%.

Nuclear staining for the assessment of apoptosis: For quantification of apoptosis-related DNA fragments, we used Hoechst 33342 (Shanghai Shize Biological Technology Co., Ltd., Shanghai, China) to determine and detect apoptotic cell death, but not necrotic cell death^[43]. At the end of oxygen-glucose deprivation treatment, the supernatants were removed. HT22 cells from each well were fixed with a 1:3 mixture of acetic acid and methanol at 4°C for 20 minutes. The supernatants were removed. After three rinses with PBS, cells were stained with 10 μg/mL Hoechst 33342 (Sigma) for 20 minutes at room temperature. Cultures were then washed three times with PBS and mounted in an anti-fluorescein fading medium (Amresco, Solon, OH, USA). Slides were visualized under a fluorescent microscope (Nikon). Experiments were repeated at least three times using separate batches of cultures. Apoptotic cells in each group were quantified by counting the mean number of positive cells per six fields at 200 × magnification using an inverted microscope. The apoptotic ratio was equal to the number of apoptotic cells/total cells × 100%.

Flow cytometry analysis of apoptosis: After cells were treated and collected, HT22 cells were digested with trypsin (2.5 g/L; Difco, Franklin Lakes, NJ, USA) and centrifuged at 1 500 r/min for 10 minutes, and then the supernatant was discarded. Cells were rinsed with PBS three times and fixed with 70% (v/v) ice-cold ethanol in the dark at 4°C overnight. On the following day, neurons were centrifuged at 1 500 r/min for 10 minutes, washed twice with PBS and adjusted to a concentration of 1 × 10⁶ cells/mL. After RNase (0.5 mL, 1 mg/mL in PBS; Sigma) was added, the specimens were stained with propidium iodide (50 mg/L; Sigma) in the dark at 4°C for 30 minutes, and then subjected to flow cytometry analysis using the FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). Experiments were repeated at least three times using separate batches of cultures.

Statistical analysis

All data were expressed as mean ± SD, and were analyzed using SPSS 15.0 for Windows software (SPSS, Chicago, IL, USA). One-way analysis of variance and Student’s t-test were used for comparisons between groups. A level of P < 0.05 was considered to be statistically significant.