Design
A randomized controlled animal experiment.
Time and setting
Experiments were performed at the Acupuncture Complex Laboratory, Hubei University of Chinese Medicine, China from March 2010 to December 2011.
Materials
A total of 40 clean, healthy, male Sprague-Dawley rats, aged 3 or 4 months and weighing 200–250 g, were purchased from the Experimental Animal Center, Tongji Medical College, Huazhong University of Science & Technology, China (license No. SCXK(E)2004-0007). Protocols were conducted in accordance with Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China[22].
Methods
Establishment and intervention of rat models of Parkinson’s disease
As previously described[23-25], 2 mg/mL rotenone sunflower oil emulsion (2 mg/kg per day) was subcutaneously injected into the neck and back of rats from the Parkinson’s disease model and electroacupuncture groups for 4 consecutive weeks to induce Parkinson’s disease-like symptoms. Rotenone and sunflower oil were purchased from Sigma (St. Louis, MO, USA). Sunflower oil at a dose of 2 mg/mL without rotenone (2 mg/kg per day) was subcutaneously injected into the neck and back of rats from the sham-surgery group. Rats in the blank group were untreated. Rats experiencing tremor, rigor, reduced movement, slow movement and unstable gait were positively identified as having Parkinson’s disease-like symptoms[8].
Electroacupuncture therapy
In accordance with acupoint patterns[26], rat acupoints were localized in combination with comparative anatomy. Rat models in the electroacupuncture group received electroacupuncture at Fengfu (in the posterior region of the neck, directly inferior to the external occipital protuberance) and Taichong (in the groove between the first and second metatarsal bones) acupoints. Rats were subjected to electroacupuncture at Fengfu acupoint at an angle of 45° and at Taichong acupoint at an angle of 90° using a 0.3 mm × 25.0 mm stainless steel needle (Huatuo, Suzhou, China). The depth of needle insertion was 4 mm. G6805-2 electroacupuncture therapeutic apparatus (Shanghai Medical Instruments Co., Ltd., Shanghai, China) was set for a continuous wave and frequency of 2 Hz. The intensity was at the point where rat limbs trembled and could be tolerated.
Electrode conjugation procedure: the positive electrode was connected to the Fengfu acupoint, and the negative electrode was connected to the Taichong acupoint (bilateral Taichong acupoints were selected on alternate days). Electroacupuncture was performed at the same time point every day for 20 minutes, once a day, for 7 days as a treatment course, for 3 consecutive courses. Rats in the blank, sham-surgery and model groups underwent grasping and fixation similar to those in the electroacupuncture group but without acupuncture.
Isolation of substantia nigra of rat midbrain
Rats were intraperitoneally anesthetized with 10% chloral hydrate (35 mg/100 g) and decapitated. The substantia nigra of midbrain was dissociated on ice, placed at –70°C in a nitrogen canister for detection of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA.
Reverse transcription-PCR of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the rat substantia nigra
Total RNA extraction in the substantia nigra: total RNA was extracted by one-step TRIzol method according to RNA extraction kit (Invitrogen Life Technologies, Carlsbad, CA, USA), and then stored at –80°C. Upstream and downstream primers of β-actin, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor were synthesized by Wuhan Guge Biological Technology Co., Ltd., China.
Primer sequence:
Reverse transcription: 2 μL of RNA, 1 μL of upstream primer and 1 μL of downstream primer were added in a 20-μL reverse transcription reaction system. Reverse transcription cDNA kit, Taq enzyme and dNTP were purchased from Toyobo, Shanghai, China.
PCR amplification: 0.125 μL of Takara Taq, 2.5 μL of buffer, 2 μL of dNTP, 2 μL of upstream primer, 2 μL of downstream primer, and 2 μL of cDNA were added in reaction system for amplification. Reaction conditions are as follows: 35 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, followed by 72°C for 10 minutes. All specimens were stored at 4°C.
PCR product detection and data processing: PCR products were determined by 2% agarose gel electrophoresis containing ethidium bromide. Absorbance was monitored by a gel-scanning system (Bio-Rad, Hercules, CA, USA). The absorbance ratio of target gene to β-actin was calculated.
Statistical analysis
The data were analyzed using SPSS 19.0 software (SPSS, Chicago, IL, USA), and expressed as mean ± SD. Multigroup comparison was performed by one-way analysis of variance. Intergroup mean paired comparison was done by independent samples t-test. A value of P < 0.05 was considered statistically significant.
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