Animals and experimental design
Sixty adult male Wister rats with body weight 275±25 gm, 9 months age; were purchased from Faculty of Science, Sohag University and were housed in Medical Animal Laboratory in Sohag Faculty of Medicine. Animals allowed free access to food and tap water. Rats were housed in standard cages, at normal light/dark cycle and room temperature. The rats were randomly assigned into 5 groups (n=10 in each group). Control group, rotenone-injected group, rotenone\exercise group, rotenone\L-dopa treated-group and combined (rotenone\L-dopa\exercise) group. The study was approved by Research Ethics Committee considering care and use of laboratory animals (permission number: SOH-IACUC-17050301).
Induction of Parkinsonism
Rats were subcutaneously injected with either vehicle, or rotenone (R8875; 95%, Sigma-Aldrich, USA) 2 mg\kg, for 4 weeks, dissolved in 1ml dimethyl sulfoxide (DSMO) & migylol 812 N (Sigma). L-dopa (L-3, 4 dihydroxyphenylalanine methyl ester hydrochloride, Sigma) was administered at a dose 6 mg\kg\day for 4 weeks.
Exercise protocol and treatments
After induction of PD, rats in exercise group were forced to run in 3-channel treadmill (Heath Life V4000M). Exercise regimen continues for 30 min/day, 5 times a week for 4 weeks. The treadmill speed accelerates beginning with 2 m/min during first 5 minutes, at 3 m/min during second 5 minutes, and then at 5 m/min for the last 20 minutes (11). The efficiency of this exercise protocol was previously assessed by measuring the serum lactate dehydrogenase and creatine phosphokinase in non-published experiment (supplementary figure 1)
Behavioral and motor analysis:
these tests assess motor activity, and behavior of the rats, they were performed at the end of the experiment for all groups, and included.
Open field test (OFT)
OFT was performed according to the previously described method (12). Each animal was then given a score for total locomotor activity; calculated as the sum number of line crosses and rears, a score for exploratory behavior; the sum of the number of central square entries and the duration of time spent in the central square, and the anxiety score is equal to the sum of urination & defecation boli (12).
Object recognition test (ORT)
ORT was performed as described by Walsh and Cummins (13). In order to assess the short-term memory (STM) and long term memory (LTM), four objects used were made of plastic material. The objects and arena were washed with 10% ethanol solution after each trial. Training was conducted by placing each rat for 5 min into the field, where two identical objects (objects A1 and A2) were put in two adjacent sides, 10 cm from the walls. In a STM test a rat given 1.5 h after training, the rats explored the open field for 5 min in the presence of one familiar (A) and one novel (B) object. LTM test given 24 h after training, the same rat explore the field for 5 min in the presence of a familiar object A and a novel object C. Exploration is defined as sniffing or touching the object with the nose and/or forepaws. is calculated by these equations: STM= [B / (A1+B)] 100, and LTM= [C / (A1+C)] 100.
Foot print test
Foot print test was used to measure gait analysis by permitting rats to run in a wood corridor apparatus (65cm× 5 cm×15 cm), which was lined with a pre-cut piece of white paper. Rats were trained to run to the end of the corridor by placing the rats at the far end of the corridor and encouraging them to move towards the end. Training was conducted twice for each rat until the animal could run to the end box without encouragement. For testing, the paws of the animal were painted with four non-toxic water colors as described previously (14). For each animal, the gait was calculated using 4 paw prints; this allowed 5 values-yield/ rat; [front stride length (FSL), front stride width (FSW), hind stride length (HSL), hind stride width (HSW) and overlap (OL)] which were then averaged to provide gait measurements (14, 15).
To assess the motor coordination of the animals, we used an accelerating Rota-rod (Harvard Apparatus, UK). The Rota-rod consisted of a suspended rod, accelerating for 60 s, beginning from 5 rounds per minute (RPM) to reach 15 RPM and continuing at that speed for a further 60 s. A trial was stopped when the rat fell off the Rota-rod or after the complete 120 s. The mean latency time of three trials was taken. Animals were trained for five days to perform the test (16).
After behavioral tests, the rats were anaesthetized with Zoletil (1mg\kg i.p (Vibac Laboratories, Carros, France).
Rats were transcardially perfused with 0.05 M phosphate-buffered saline (PBS). The brain was removed, snap freezed in liquid nitrogen, and kept for 1 hour in - 80°C. Striatum was dissected through multiple manual coronal sections with sharp razor blade. Samples stored in Eppendorf tubes at - 80°C till further analyses.
Total RNA was extracted from 30 mg of tissue samples according to manufacture instructions (RNA Extraction kit (#K0731, Thermo scientific, Lithuania)). They were extracted to measure NRF2, Noq.1, TFAM and using housekeeping GAPDH by real time polymerase chain reaction (real time PCR).
Extracted RNA concentration was quantified using Nanodrop spectrophotometry (Quawell 5000, USA); then 110 ng of total RNA transcribed using RNA reverse transcriptase kits ((#K0251) (Thermo scientific, Lithuania)). Thermal cycler was programmed at 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes and 4°C for 20 hours.
Real time PCR
Prepared cDNA, was used in the qPCR analyzer (Step One, Applied Biosystems, Singapore) using the MAXIMA SYBR Green qPCR Master Mix with the following program: 1 cycle at 95°C for 10 minutes; 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 30 seconds; one cycle at 95°C for 15 seconds, 60°C for 1 minute and 95°C for 15 seconds. The specific primers (table 1) of NRF2, TFAM, NADPH dehydrogenase (NQO1) & housekeeping GAPD were purchased from Metabion international AG. Fold expression was measured according to the relative expression to. the relative expression to housekeeping gene as in the fold = 2-ΔΔct.
Homogenized striatum of the right hemispheres of brain were used to measure the levels of Tyrosine hydroxylase enzyme by ELISA (Tyrosine hydroxylase (TH) rat ELISA kits (#:96791) from Glory Science Co., (Ltd, China) with detection range 0.625- 20 ng\ ml
Statistical package for social sciences (IBM-SPSS), version 24 IBM- Chicago, USA (May 2016) was used for statistical data analysis. Data expressed as mean ± standard deviation (SD), number and percentage. Student t test was used to compare the means between two groups, and one-way analysis of variance (ANOVA) test was used to compare means of more than two groups. Post hoc test was used for individual P value which is considered significant when P < 0.05.