Establishment of rat model of Parkinson's disease
60 SPF male SD rats weighting 180~220g and aging about 8weeks were purchased from the Laboratory animal center of Xinjiang Medical University. Five rats were fed in a cage. The animals were maintained in an air-conditioned room (23±3℃,relative humidity 40-70%) on a 12-h light/dark cycle with free access to water and food. The animals were randomly divided into four groups(sham operation group, model group, sham operation+SAE group, model+SAE group),with 15 rats in each group.
Before the surgery the rats were given introperitoneal anaesthesia with 0.15mL/100g chloral hydrate (10% solution). Sterile operation was strictly executed during the surgery. The model group received slowly injection of 5μL of 6-OHDA (dissolved with normal saline containing 1% ascorbic acid, concentration of 2μg/μL)for each point into the right SNC(substantia nigra,compact part) and VTA(ventral tegmental area) on the rat stereolocator. After pinhole pressing to stop bleeding, the scalp was sutured and disinfected with iodine,the rats were injected with penicillin of 50,000 units intraperitoneally to prevent infection.
Sham operation group received injection of 5μL normal saline containing 1% ascorbic acidin each point of the right SNC and VTA.Sham operation+SAE group and model+SAE group received skilled aerobic exercise two weeks after the surgery.
Skilled aerobic exercise
The training started from the third week after modeling, the sham operation+SAE group and model+SAE group received SAE, which was training running on wheels with irregular spacing steps of OOOOXOX pattern (Figure1), O represented steps and X represented missing steps with interval of 1.3cm. At beginning of the training, rats were placed on regular running wheels (Figure2),Rats were trained at initial speed of 2m/min, then increased the speed by 1 or 2m/min daily until the speed reached 8m/min. In the second week, the rats of SAE groups were trained on irregular running wheels, and the initial speed was still 2m/min, and the speed of the wheels was gradually increased for 8m/min without tension of rats, defecation and urination were monitored as signs of acute stress response. The sham operation group and the model group received training on normal wheels with free speed. The training lasted 20min a day, 5 days a week, for 4 weeks. The wheels` diameter were 34.4cm and the width of each step was 0.3cm.
Behavior test of experimental animals
Cylinder experiment
Rats were placed in a transparent resin-glass bucket (20cm of diameter and 30cm of height) on day 7, 14 and 28 after modeling injury. The contact times of rats` left forelimbs on cylinders in 3min were observed and counted, and the ratio of left forelimbs contacting with cylinders was calculated with the formula of , a means the times of rats` left forelimb contacting with cylinders; b means the times of rats` bilateral forelimbs alternatedly contacting with the cylinders; c means the the times of rats` both forelimbs simultaneously contacting with the cylinder; d means the the times of rats` right forelimbs contacting with the cylinder.
Rotation experiment induced by APO
Rats of four groups were intraperitoneally injected with apmorphine of 0.05mg/kg respectively on day 7, 14 and 28 after modeling injury, to induce the unilateral levorotation, and the rotation number of rats was recorded within 30min.
To detect the expression of tyrosine hydroxylase (TH) in the substantia nigra with immunohistochemistry
After fixation, embedding, and section, the substansubstania nigra tissues were soaked in xylene for dewaxing, hydration, and antigen repair. The endogenous peroxidase was soaked in 3%H2O2 deionized water to remove endogenous peroxidase. The diluted primary antibody (TH 1:170) was soaked overnight in a refrigerator at 4℃, and the horseradish peroxidase labeled secondary antibody was dropped and incubated at room temperature for 20 min. DAB for 3-10 min, tap water stops for color, hematoxylin is redyed for 3 min, dehydrated, transparent and neutral gum seals, and films are observed and taken under a microscope.
The expression levels of Bip/Grp78 and CHOP protein in the substantia nigra and FNDC5 protein in the quadriceps femoris of the rats` left hind limbs were detected by WB
After grinding with liquid nitrogen, about 100mg of the samples were added into a precooled 1.5ml centrifuge tube, and then 400μL RIPA lysis solution (RIPA:PMSF: Broad-spectrum phosphatase inhibitor =100:1:1) was added, fully mixed, and placed at 4℃ for 60 min. Supernatant was collected at 12000 RPM and centrifuged at 4℃ for 15 min. Protein concentration was determined by BCA method. The samples were loaded with 5× SDS-Page loading buffer (containing β -mercaptoethanol), heated with boiling water at 100℃ for 5min, and the proteins were fully denaturated before being stored in a refrigerator at -80℃. According to the formula, 15%, 12%, 8% separated glue and 5% concentrated glue. Adding sample: Marker 9L of pre-dyed protein, 50g of sample protein per well. Electrophoresis: at a constant pressure of 80V, bromophenol blue was transferred to the separation glue, and at a constant pressure of 100V for 90 min, the electrophoresis was stopped when bromophenol blue reached the bottom. After sdS-PAGE electrophoresis, the PVDF membrane was soaked in methanol for 10 s and rinsed in distilled water for 1 min. Then the polyacrylamide gel, filter paper and the treated PVDF membrane were soaked in the Transfer Buffer for 10 min to prepare the "sandwich" of membrane Transfer. Bip/Grp78 and Actin were treated with 0.45μm PVDF membrane, CHOP,FNDC5 and β-actin were treated with 0.22μm PVDF membrane, and the membrane transfer time of CHOP,FNDC5 and β-actin was 60min, and Bip/Grp78 was 80min. After membrane transfer, the PVDF membrane was washed with water for 3 times, 5 min/ time. Use sealing solution containing 5% skim milk powder to seal the transfer film for 1 h, and then wash it with TBST 3 times, 5 min/ time. TBST diluted primary antibody, β-actin (1:1000), CHOP (1:500), Bip/Grp78 (1:500),、FNDC5(1:500) were added. Incubate overnight at 4℃ in a shaker. Rinse 1×TBST for 3 times, 5min/ time. Suitably diluted secondary antibody (1:5000) was added and incubated at room temperature for 1 h. Rinse 1×TBST for 3 times, 5min/ time. DAB chromogenic solution A and B were mixed and 2 mL of DAB chromogenic solution was added to the membrane. ChemiScope Mini chemiluminescence instrument was used for detection and photography. Image J Image analysis software was used for analysis. Each experiment was repeated three times.
Irisin in serum was detected with ELISA kit
After the rats were anesthetized by intraperitoneal injection of chloral hydrate, the abdominal wall was cut open and the abdominal aorta was revealed,then 3ml of blood was extracted from the abdominal aorta into the anticoagulant tube. Put it on ice and centrifuge it at 5000rpm for 10min as soon as possible. Then the serum was sucked out. The detection was executed following the instructions of Irisin ELISA kitstrictly. There were 6 rats in each group and 3 multiple holes for each rat. There were blank holes, standard holes and sample holes. Each well in the working plate was added with sample of 100μL, then incubated in an incubator at 37℃ for 90min,the holes were filled with biotinylated antibody working solution, then incubated for 1h at a constant temperature again. Then enzyme conjugate working solution of 100μL was added into each well after the plate was washed sufficiently, incubated for 30min at a constant temperature. Substrate solution of 90μL was added, and incubated for 15min avoid light. Stop solution of 50μL was added to each well, then the blue color quickly changed to different shade of yellow. When the reaction stopped, the absorbance (OD) of wave length at 450nm was measured by a microplate reader. The concentration of each sample was calculated by standard curve.
Data Statistics
All data were expressed as mean±standard deviation (x̄ ± s). SPSS19.0 software was used for statistical analysis. One-way anova was used for statistical analysis: P <0.05 indicated significant difference. GraphPad Prism 6.0 was used to assiste in graphing.