Design
A randomized controlled animal experiment.
Time and setting
Experiments were performed from June 2011 to February 2012 in the Laboratory of Neurology, Medical Transformation Center, Bethune First Hospital of Jilin University, China.
Materials
Eighteen clean, healthy, male Wistar rats, aged 8– 10 weeks and weighing 280–320 g, were provided by the Animal Experimental Center of Jilin University (license No. SCXK (Ji) 2003-0002). All rats were fed in separate cages at 21 ± 2°C, 30–35% humidity under a 12-hour day/night cycle, and allowed free access to food and water. All experimental disposals were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[36].
Methods
Establishment of a rat model of Alzheimer’s disease
Alzheimer’s disease models were established according to the methods described by Nabeshima et al [37]. In brief, amyloid β protein (1–40) polypeptide (ANASPEC Inc., San Jose, CA, USA) was dissolved in a 60 μM solution containing 35% acetonitrile and 1% trifluoroacetate acid (Wanxingda Chemical Co. Ltd., Jinan, Shandong Province, China), and stored in the Alzet micro-injection pump (Alzet, Cupertino, CA, USA). Rats were anesthetized by subcutaneous injection of ketamine (100 mg/mL, 100 mg/kg), xylazine (20 mg/mL, 2.5 mg/kg) and acetylpromazine (2.5 mg/mL, 2.5 mg/kg; Wanxingda Chemical Co., Ltd.,), and fixed in a brain stereotactic instrument (Ruiwode Life Science Co., Ltd., Shenzhen, Guangdong Province, China). After 75% ethanol disinfection, a 2.5-cm-long sagittal incision was made along the midline, exposing the skull. The coordinates of needle entry point were defined (anterior 0.3 mm to the bregma, ventral 3.6 mm, left 1.1 mm[37]), and a hole was then drilled at the skull surface. After drilling, the dura mater was punctured, and the cannula was placed and inserted through the entry points into the left lateral ventricle. The cannula was connected via the catheter and syringe pump, and the pump was placed on the rat back skin. The wound was sutured, and the rats were released from the brain stereotactic instrument and housed in the controllable incubator at 37°C for 60 minutes. After that, rats were fed in separate breeding cages and received daily injections of 300 pmol amyloid β protein via syringe pump, for 14 consecutive days.
Estrogen receptor β intervention
Prior to connection of the syringe pump, rats in the model group were injected with PBS into the left ventricle through the catheter; rats in the estrogen receptor β group were injected with lentiviral-CMV-estrogen receptor β plasmid (25 µL, 108 TU/mL; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and rats in the short hairpin RNA group were injected with lentiviral-CMV-estrogen receptor β short hairpin RNA plasmid (25 µL, 108 TU/mL; Shanghai Jikai Gene Chemical Technology Co., Ltd., Shanghai, China).
Morris water maze test for assessing behavioral changes in rats
Before modeling and 14 days after syringe pump implantation, the behaviors of Alzheimer’s disease rats were detected according to Liu’s method[38]. In brief, a circular water tank 150 cm in diameter and 60 cm in height was filled with water to a depth of 30 cm, at 26 ± 1°C. There were four equidistant points on the tank wall, which was divided into four quadrants, and a small platform located in the center of one quadrant, 2 cm below the water. Starting from the western point, rats were placed on the platform for 30 seconds and then entered the water tank; a video camera was fixed on the top of water tank to record the time it took rats to find the platform (escape latency) and the route of rats swimming in search of the platform. All data were automatically recorded by a computer. The maximum recording time is 200 seconds.
Real-time quantitative PCR detection of tumor necrosis factor α and interleukin-1β mRNA expression
All rats were killed under anesthesia after behavioral tests, the brains were harvested after cardiac reperfusion, and fresh left hippocampal tissues were collected. According to the Finch’s method[39], tumor necrosis factor α mRNA and interleukin-1β mRNA expression was determined by PCR as follows: Trizol (Sigma, St. Louis, MO, USA) was added to extract total RNA. The extracted RNA content was assayed using a type UV-240 ultraviolet spectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China); the A260/A280 ratio of total RNA was 1.80–2.00. Total RNA (2 μg) was reverse transcribed into cDNA using a Superscript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). The synthesized cDNA was subjected to real-time quantitative PCR using a SYBR Green PCR Kit (Beijing TransGen Biotech Co., Ltd., Beijing, China) as follows: 1 μL of specific primer (0.5 μL upstream primer and 0.5 μL downstream primer), 12.5 μL of 2 × SYBR Green QPCR Master Mix, 2.5 μL of diluted cDNA and 9 μL of nuclease-free PCR-grade water were mixed and denaturated at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 1 minute and 72°C for 30 seconds. PCR product SYBR green fluorescence was detected, with β-actin as the internal reference for quantitative product concentration.
The sequences of specific primers for tumor necrosis factor α and interleukin-1β mRNAs were obtained from GenBank, designed using Primer Premier 5.0 software (Premier, Vancouver, Canada), and synthesized in the Shanghai Institute of Biological Engineering, China. Specific sequences are shown in Table 1.
Western blot assay for estrogen receptor β, Akt and amyloid β protein expression in the hippocampus
The rat hippocampus was lysed with lysis buffer containing protease inhibitors on ice for 30 minutes, homogenized for 30 seconds, and centrifuged at 12 000 r/min for 20 minutes (centrifugal radius 6 cm). After the supernatant was discarded and lysate was diluted, the total protein concentration was detected using the BCA method[39]. Total protein (15 μg) in each sample was mixed with 4 μL of 6 × loading buffer, degenerated using boiling water for 5 minutes, and subjected to 10% SDS-PAGE discontinuous gel electrophoresis at 80 V for 40 minutes and at 110 V for 90 minutes, followed by 200 mA electrical membrane transfer for 60–90 minutes. Samples were blocked with calf serum (Boster, Wuhan, Hubei Province, China) and skim milk for 2 hours, rinsed with TBST six times (10 minutes each), and incubated with rabbit anti-estrogen receptor β, Akt and amyloid β protein polyclonal antibody (1:2 000; Santa Cruz Biotechnology) at 4°C overnight. The next day, samples were rinsed with TBST six times (10 minutes each) and incubated with horseradish peroxidase-labeled goat anti-rabbit IgM (1:200; Santa Cruz Biotechnology) at room temperature for 2 hours. ECL chemiluminescence was used to visualize protein bands. The absorbance of the scanned bands was determined using Image J (National Institutes of Health, Rockville, MD, USA). The results are expressed as the ratio of the target protein band absorbance to that for β-actin.
Immunofluorescence staining for detection of amyloid β protein expression in the hippocampus
According to the methods of Liu et al [38], rat hippocampal slices were stained for amyloid β protein. In brief, brain tissue frozen slices were permeated with 0.5% Triton X-100 (Hufeng Biotechnology Co., Ltd., Shanghai, China) at room temperature for 20 minutes, rinsed with 0.1 M PBS for 5 minutes, three times. After being blocked with 10% calf serum at room temperature for 1 hour, samples were rinsed with 0.1 M PBS for 5 minutes, three times, incubated with rabbit anti-amyloid β protein polyclonal antibody (1:200; Santa Cruz Biotechnology) at 4°C; rinsed with 0.1 M PBS for 5 minutes, three times, and then incubated with goat anti-rabbit IgG-FITC (1:2 000; Santa Cruz Biotechnology) diluted with 3% bovine serum albumin for 1 hour at room temperature. Samples were then rinsed with 0.1 M PBS for 5 minutes, three times, and mounted with fluorescence quenching agent. Under a confocal laser scanning microscope (Olympus, Tokyo, Japan), five visual fields were selected from each slice under 100 × magnification. The fluorescent products were quantified and the intensity of fluorescence was measured using Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA) software.
Statistical analysis
Measurement data are expressed as mean ± SD and grouped data are represented by relative values. Statistical processing was performed using SPSS 17.0 software (SPSS, Chicago, IL, USA). Intergroup differences were compared using analysis of variance followed by q test, with a = 0.05 as the standard.
Author contributions: Zhu Tian and Qun Liu designed and evaluated the study. Yang Zhao and Sheng Bi performed data analysis. Zhu Tian drafted the manuscript. All authors were involved in performing the experiments. All authors approved the final version of the paper.
Conflicts of interest: None declared.
Ethical approval: The experiment was approved by the Animal Ethics Committee of Jilin University in China.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application disputations.
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