Design
An in vitro randomized controlled experiment.
Time and setting
Experiments were performed from August 2010 to July 2011 at the laboratory in Zhuhai Campus of Zunyi Medical College, China.
Materials
A total of 60 male and female Sprague-Dawley rats, 1 day old, weighing 12.31 ± 2.15 g, were provided by the Animal Experimental Center of Sun Yat-sen University, China. All animal experiments were approved by the Experimental Animal Ethics Committee of Sun Yat-sen University, and complied with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[16].
Methods
Culture of primary rat hippocampal neurons
According to previously described methods[6], 1 day old Sprague-Dawley rats were sacrificed and the brain tissue was harvested. The bilateral hippocampi were separated in cold D-Hank’s solution (Guangzhou Whiga Technology Co., Ltd., Guangzhou, Guangdong Province, China), blood vessels and meninges were eliminated, and the specimens were cut into 1-mm3 pieces and digested with 0.25% (w/v) trypsin at 37°C for 15 minutes. The digestion was terminated with DMEM/Ham’s F12 basic culture medium (Gibco, Carlsbad, CA, USA). Cells were dispersed by mechanical trituration, filtered using a 500-mesh filter, and prepared into a single cell suspension. The suspension was centrifuged at 1 000 r/min at room temperature for 10 minutes and added to DMEM/Ham’s F12 culture medium after the supernatant was discarded. Cells were prepared into a 4 × 105/mL single cell suspension. Cells plated on 96-well culture plates, which were previously coated with polylysine, were maintained in a 95% air and 5% CO2 saturated humidified incubator at 37°C. Culture medium consisted of 90% (v/v) DMEM/Ham’s F12, 10% (v/v) fetal bovine serum (Hangzhou Sijiqing, Hangzhou, Zhejiang Province, China), 2 mM glutamine, and 100 U/mL penicillin and streptomycin. At 48 hours, culture medium was replenished with 1 × 10-5 M cytosine arabinoside for an additional 48 hours, to inhibit glial cell proliferation. The medium was changed twice per week. Neurons were identified using Hoechst 33258 staining (Sigma, St. Louis, MO, USA) and the neuron specific antibody anti-NeuN (Sigma)[6]. Specimens were observed under an inverted fluorescence microscope (Olympus, Tokyo, Japan).
Establishment of neuronal apoptosis model
According to previous studies[6, 9], hippocampal neurons cultured for 12 days were randomly divided into three groups, namely a normal control group, SIN-1 group and a SIN-1 + DIDS group (Sigma). Thirty-six wells of cells in each group (same number of cells per well) were used for the identification of neurons, determination of cell survival rate, morphological observation, PARP-1 and AIF immunofluorescence staining and western blot assay. The control group was only cultured with DMEM/Ham’s F12 complete culture medium. The SIN-1 group was exposed to SIN-1 (1.0 mM) for 18 hours to induce apoptosis in vitro and to establish an ischemic neuronal apoptosis model. Neuronal apoptosis was identified using the Hoechst 33258 DNA fluorescence staining kit (Wuhan Boster, Wuhan, Hubei Province, China). Under the inverted fluorescence microscope, Hoechst 33258 staining appeared light blue in control neurons. In apoptotic neurons, Hoechst staining exhibited strong fluorescence in nuclei, and fragmented DNA was evident. In the SIN-1 + DIDS group, 0.1 mM DIDS (Sigma) and 1.0 mM SIN-1 (Sigma) were simultaneously added to the culture medium for 18 hours. The concentration of drugs and intervention methods were used according to a previously described study[6].
Determination of hippocampal neuron survival
According to previously described methods[17], hippocampal neurons cultured in 96-well culture plates were incubated with MTT (Sigma; final concentration 0.5 mg/mL) at 37°C for 4 hours, and then oscillated with dimethyl sulfoxide for 10 minutes, after the culture medium was discarded, to completely dissolve the particles. Cell-free culture medium served as a control. The absorbance value at 570 nm was read using an enzyme labeling instrument (Bio-tek, Hercules, CA, USA).
The survival rate (%) = (absorbance value of experimental group – absorbance value of blank control group)/(absorbance value of normal control group – absorbance value of blank control group) × 100%.
Determination of the rate of hippocampal cell apoptosis
According to previously described methods[6], hippocampal neurons were rinsed with 0.01 M PBS (pH 7.4), fixed with 4% (w/v) paraformaldehyde for 30 minutes, rinsed with PBS three times, blocked with goat serum, and stained with Hoechst 33258 (0.25 μg/mL; Sigma) for 15 minutes. The morphology of cell nuclei was observed under an inverted fluorescence microscope (Olympus), and 200 cells were randomly selected from three fields of view to calculate the percentage of apoptotic cells. Normal cells exhibited oval or circular nuclei and refracted light blue fluorescence, while apoptotic neurons exhibited contracted nuclei and refracted strong blue light fluorescence. The percentage of apoptotic cells represented the rate of apoptosis.
PARP-1 and AIF expression in rat hippocampal neurons by immunohistochemical detection
Based on previously described methods[18-19], PARP-1 and AIF immunofluorescence staining was performed. In brief, cells were rinsed with 0.01 M PBS (pH 7.4), fixed in 4% (w/v) paraformaldehyde for 30 minutes, rinsed with PBS three times, blocked with goat serum, and incubated with rabbit anti-mouse PARP antibody (Sigma; 1:800) and rabbit anti-mouse AIF antibody (Sigma; 1:800) at 4°C overnight, and then with secondary fluorescence agents (FITC and Cy3; Wuhan Boster Biological Technology Ltd., Wuhan, Hubei Province, China) labeled goat anti-rabbit IgG (1:200; Wuhan Boster) for an additional 30 minutes. Cells were observed under a fluorescence microscope (Olympus).
Detection of PARP-1 and AIF protein expression in rat hippocampal neurons by western blot assay
In accordance with previous methods[20-21], hippocampal neurons were rinsed with cold 0.1 M PBS twice and cells were collected in 85°C sampling buffer solution, followed by sodium dodecyl sulfate polyacrylamide electrophoresis, transfer onto polyvinylidene fluoride and blocking with 3% (v/v) fetal bovine serum for 2 hours. The membrane was incubated with rabbit anti-mouse PARP-1 antibody (Sigma; 1:1 000) at 4°C overnight, rinsed, and incubated with rabbit anti-mouse AIF antibody (Sigma; 1:1 000) at 4°C overnight. The membrane was rinsed with triethanolamine-buffered saline containing 0.05% (v/v) Tween-20 three times, 10 minutes each, and finally incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1:500; Wuhan Boster) at room temperature for 2 hours. After rinsing with triethanolamine-buffered saline containing 0.05% (v/v) Tween-20 three times, 5 minutes each, the membrane was subjected to ECL detection of protein.
Statistical analysis
Data were expressed as mean ± SD and analyzed with one-way analysis of variance using SPSS 11.5 statistical software (SPSS, Chicago, IL, USA). Comparison between two groups was performed using the least significant difference t-test. A P value less than 0.05 was considered statistically significant.
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