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Forced exercise activates the NrF2 pathway in the striatum and ameliorates motor and behavioral manifestations of Parkinson's disease in rotenone-treated rats

Forced exercise activates the NrF2 pathway in the striatum and ameliorates motor and behavioral... Background: Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by progressive loss of nigrostriatal dopaminergic neurons leading to dopamine depletion and problems of movement, emotions, and cog- nition. While the pathogenesis of PD is not clear, damage of dopaminergic neurons by oxygen-derived free radicals is considered an important contributing mechanism. This study aimed to evaluate the role of treadmill exercise in male Wister rats as a single treatment and as an aid-therapy with L-dopa for rotenone-induced PD. To study the role of the Nrf2- ARE pathway as a mechanism involved in exercise-associated improvement in rotenone-induced PD in rats. Method: Animals were divided into 5 groups, (Control, rotenone, rotenone\exercise, rotenone\L-dopa, and rote- none\exercise\L-dopa (combination)groups). After the PD induction, rats in the rotenone\exercise and combination groups were daily treadmill exercised for 4 weeks. Results: Treadmill exercise significantly improved behavioral and motor aspects of rotenone-induced PD. When treadmill exercise was introduced as a single intervention, it amended most behavioral aspects of PD, gait fully cor- rected, short-term memory, and motor coordination. Where L-dopa corrected locomotor activity and motor coordina- tion but failed to improve short-term memory and only partially corrected the gait of rotenone-treated rats. When treadmill exercise was combined with L-dopa, all features of PD were corrected. It was found that exercise upregu- lated some of its associative genes to Nrf2 pathways such as TFAM, Nrf2 and NQO.1 mRNA expression. Conclusion: This study suggests that forced exercise improved parkinsonian like features by activating the Nrf2 pathway. Keywords: Parkinson, Rotenone, Behavioral tests, Exercise, Nrf2. TFAM, Noq1 Background Parkinson’s disease (PD) is a common neurodegenerative *Correspondence: motamed71111@gmail.com; amany_abdelhameed@med. disorder characterized by persistent loss of nigrostriatal sohag.edu.eg Dina M. Monir and Motamed E. Mahmoud are equally contributed dopaminergic neurons leading to depletion of dopamine Department of Physiology, Faculty of Medicine, Sohag University, and consequent problems of movement, emotions, and Sohag 82524, Egypt cognition. While many factors contribute to the patho- Department of Animal Behavior and Husbandry (Genetics, Breeding, and Production), Faculty of Veterinary Medicine, Sohag University, genesis of PD, damage of dopaminergic neurons by Sohag 82524, Egypt oxygen-derived free radicals is considered an important Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Monir et al. Behav Brain Funct (2020) 16:9 Page 2 of 12 contributing mechanism [1, 2]. Rotenone is a potent in the Medical Animal Laboratory in Sohag Faculty of inhibitor of complex I (NADH: ubiquinone oxidoreduc- Medicine. Animals were allowed free access to food and tase) of the mitochondrial electron transport chain. This tap water. Rats were housed in standard cages, at normal allows for accumulation and overproduction of reac- light/dark cycle and room temperature. The rats were tive oxygen species (ROS) which eventually leads to cell randomly divided into 5 groups (n = 10 in each group); damage [3, 4]. Chronic rotenone exposure in rats causes control group, rotenone-injected group, rotenone\exer- both neuropathological findings and behavioral symp - cise group, rotenone\L-dopa treated-group, and com- toms of PD [5]. The rotenone model mimics the grad - bined (rotenone\L-dopa\exercise) group. The study was ual progression of PD as observed in humans. Systemic approved by Research Ethics Committee considering the mitochondrial inhibition by rotenone leads to selective care and use of laboratory animals (permission number: nigrostriatal degeneration [6]. SOH-IACUC-17050301). Treadmill exercise is indicated as a physical therapy to improve motor symptoms in patients with PD. Exer- Induction of Parkinsonism cise can improve and alleviate memory loss in elderly Rats were subcutaneously injected with either vehicle or patients, and decrease the risk of developing PD. The rotenone (R8875; 95%, Sigma-Aldrich, USA) 2 mg/kg, for neuroprotective potentials of exercise are great, but the 4 weeks. Rotenone solution was prepared as a 50 × stock underlying mechanisms remain a debatable issue. Evi- in 100% dimethylsulfoxide (DMSO) then in a medium- dence suggests that exercise neuroprotection is due to its chain triglyceride, miglyol 812  N (Sigma) to obtain a neurotrophic effects, as exercise increases the availability final concentration of 2 mg/mL rotenone in 98% miglyol of several neurotrophic factors [7]. Long-term exercise 812 N, 2% DMSO. The prepared solution was stored in an benefits brain functioning by increasing the blood and amber septa vial to be protected from light and inverted oxygen flow to the brain, mobilizing growth factors that many times before each injection to eliminate the possi- promote neurogenesis and synaptic plasticity, releasing of bility of settling [12]. L-dopa (L-3, 4 dihydroxyphenylala- neurotransmitters, such as dopamine (DA), noradrena- nine methyl ester hydrochloride, (Sigma) was dissolved line, serotonin, and glutamate and consequently improve in normal saline and was administered at a dose 6 mg/kg/ manifestations of the disease either at the motor or cog- day, injected intraperitoneally for 4 weeks. nitive. levels in Alzheimer’s animal model [8]. Exercise increases the antioxidant status in the stria- Exercise protocol and treatments tum of animals and protects against neurological oxida- After induction of PD, rats in the exercise group were tive challenges. The nuclear factor erythroid-derived forced to run in a 3-channel treadmill (Heath Life 2-like 2 (Nrf2)-antioxidant response element (ARE) V4000M). The exercise regimen continues for 30  min/ signaling pathway, a major cellular defense mechanism day, 5 times a week for 4  weeks. The treadmill speed against oxidative stress. Exercise activates Nrf2 in human accelerates beginning with 2  m/min during the first skeletal muscles and mouse heart [9]. The Nrf2-ARE 5 min, at 3 m/min during the second 5 min, and then at signaling pathway appears to be a strong mechanism for 5  m/min for the last 20  min [13]. The efficiency of this exercise-induced neuroprotection [10]. The activation of exercise protocol was previously assessed by measuring the Nrf2 gene activates genes that encode for antioxidant the serum lactate dehydrogenase and creatine phospho- enzymes within the cells like heme oxygenase and NADH kinase in a non-published experiment (Additional file  1: quinone oxidoreductase (NQO1). Also, Nrf2 activates the Fig. S1). mitochondrial transcription factor A (TFAM) which reg- ulates for mitochondrial DNA (mtDNA) replication [11]. Behavioral and motor analysis Therefore, in this study, we investigated whether tread - These tests assess motor activity, and behavior of the rats, mill exercise as a single therapeutic intervention and as they were performed at the end of the experiment for all add-on therapy with L-dopa improve manifestations of groups and included; PD in rotenone-treated rats. Also, we assessed the Nrf2 pathway as a possible mechanism activated by treadmill Open field test (OFT) exercise. OFT was performed according to the previously described method [14]. The apparatus was a squared Materials and methods plastic arena (114 × 114 × 44 height cm) its floor was Animals and experimental design divided into smaller squares (19 × 19 cm ) with a central Fifty-adult male Wistar rats with body weight averaged square (38 × 38 cm ) each rat was put gently in the center 275 ± 25 gm, 9  months aged; were purchased from the of the arena and was left for 5  min moving and explor- Faculty of Science, Sohag University, and were housed ing the field. A video camera was fixed at the top of the M onir et al. Behav Brain Funct (2020) 16:9 Page 3 of 12 arena to record the activity of the rat which was scored after they complete 120  s. The mean latency time of the by a specialist who was blind to the experimental groups. three trials was taken. Animals were trained for five days Each animal was then given a score for total locomotor to perform the test [17]. activity; calculated as the sum number of line crosses and rears, a score for exploratory behavior; the sum of the Tissue Sampling number of central square entries and the duration of time After behavioral tests, the rats were anesthetized with spent in the central square, and the anxiety score is equal Zoletil (1 mg\kg i.p (Vibac Laboratories, Carros, France). to the sum of urination and defecation boli [14]. Rats were transcardially perfused with 0.05 M phosphate- buffered saline (PBS). The brain was removed, separated Object recognition test (ORT) into right and left hemispheres, snap-frozen in liquid ORT was performed as described by Walsh and Cummins nitrogen, and kept for 1  h at − 80  °C. The striatum was [15]. To assess the short-term memory (STM) and long- dissected through multiple manual coronal sections with term memory (LTM), four objects used were made of plas- a sharp razor blade and was collected with the help of two tic material. The objects and arena were washed with a 10% Dumont No. 5 forceps [18]. Samples then were stored in ethanol solution after each trial. The training was conducted Eppendorf tubes at − 80 °C until further analyses. by placing each rat for 5 min into the open field apparatus, where two identical objects (objects A1 and A2) were put in (1) RNA extraction two adjacent sides, 10 cm from the walls. In the STM test, Total RNA was extracted from 30  mg of tissue samples a rat was given 1.5  h after training; the rats explored the according to manufacture instructions (RNA Extraction open field for 5 min in the presence of one familiar (A) and kit (#K0731, Thermo Scientific, Lithuania)). They were one novel (B) object. LTM test was done 24  h after train- extracted to measure Nrf2, NOQ.1, TFAM, and using ing, the same rat explored the field for 5 min in the presence housekeeping GAPDH by real-time polymerase chain of a familiar object A and a novel object C. Exploration is reaction (real-time PCR). defined as sniffing or touching the object with the nose and/ or forepaws. The time (T) which is spent by each rat explor - ing the different objects was measured as TA1, TB, and (2) Reverse transcription TC. These times were used for calculation of STM = [TB / Extracted RNA concentration was quantified using Nan - (TA1 + TB)] 100, and LTM = [TC / (TA1 + TC)] 100. odrop spectrophotometry (Quawell 5000, USA); then 110 ng of total RNA transcribed using RNA reverse tran- Foot print test scriptase kits ((#K0251) (Thermo Scientific, Lithuania)). Footprint test was used to measure gait analysis by The thermal cycler was programmed at 25 °C for 10 min, permitting rats to run in a wood corridor apparatus 37 °C for 120 min, 85 °C for 5 min, and 4 °C for 20 h. (65 × 5 × 15 cm ), which was lined with a pre-cut piece of white paper. Rats were trained to run to the end of (3) Real‑time PCR the corridor by placing the rats at the far end of the cor- Prepared cDNA, was used in the qPCR analyzer (Step ridor and encouraging them to move towards the end. One, Applied Biosystems, Singapore) using the MAX- The training was conducted twice for each rat until the IMA SYBR Green qPCR Master Mix with the following animal could run to the end box without encouragement. program: 1 cycle at 95  °C for 10  min; 40 cycles of 95  °C For testing, the paws of the animal were painted with four for 15  s, 60  °C for 30  s and 72  °C for 30  s; one cycle at non-toxic watercolors as described previously [16]. For 95  °C for 15  s, 60  °C for 1  min and 95  °C for 15  s. The each animal, the gait was calculated using 4 paw prints; specific primers (Table  1) of Nrf2, TFAM, NADPH dehy- this allowed 5 values-yield/rat; [front stride length (FSL), drogenase (NQO1) & housekeeping GAPDH were pur- front stride width (FSW), hind stride length (HSL), hind chased from Metabion international AG, Germany. Fold stride width (HSW) and overlap (OL)] which were then −ΔΔct expression (2 ) was calculated according to the rela- averaged to provide gait measurements [15]. tive expression of housekeeping gene GAPDH. Rota‑rod test To assess the motor coordination of the animals, we used (4) Tyrosine hydroxylase an accelerating Rota-rod (Harvard Apparatus, UK). The Homogenized striatum of the right hemisphere of all rat Rota-rod consisted of a suspended rod, accelerating for brains was used to measure the levels of tyrosine hydrox- 60 s, beginning from 5 rounds per minute (RPM) to reach ylase enzyme by ELISA (tyrosine hydroxylase (TH) rat 15 RPM, and continuing at that speed for a further 60 s. ELISA kits (#:96,791) from Glory Science Co., (Ltd, A trial was stopped when the rats fell off the Rota-rod or China) with a detection range 0.625- 20 ng\ ml. Monir et al. Behav Brain Funct (2020) 16:9 Page 4 of 12 Table 1 Forward and reverse primers for real time PCR Gene name Forward primer Reverse primer Access number References NRF2 5′ CAC ATC CAG ACA GAC ACC AGT-3′ 5′ CTA CAA ATG GGA ATG TCT CTGC-3′ NM_031789 [19] TFAM 5′AGT TCA TAC CTT CGA TTT TC-3′ 5′ TGA CTT GGA GTT AGC TGC -3` NM_031326.1 [20] NQO1 5′CAG CGG CTC CAT GTACT-3′ 5′ GAC CTG GAA GCC ACA GAA G-3′ NM_017000 [19, 21] GAPDH NM_017008 [19] 5′CAG GCA TAT GGT GGT CCA TAGAG-3′ 5′ TCA TGG GAT CCA CCT GCA GC-3′ NRF2 nuclear factor erythroid 2 (NFE2)-related factor 2, NQO1 quinone oxidoreductase 1, TFAM Mitochondrial transcriptional factor A, GAPDH glyceraldehyde-3- phosphate dehydrogenase Statistical analysis different between both groups. To sum, rotenone injec Statistical package for social sciences (IBM-SPSS), version tion decreased exploration and locomotion scores in 24 IBM- Chicago, USA (May 2016) was used for statistical the open field test. data analysis. Data expressed as mean ± standard devia- It was noticed that after injection of rotenone, rats tion (SD), number, and percentage. A one-way analysis of practiced 5 times/week a treadmill exercise spent less variance (ANOVA) test was used to compare the means time in the central arena and increased number of line of the analyzed groups. Post hoc test (LSD type) was used crossings in the open-field test compared to the non- for multiple comparisons between each group and the exercised rotenone group (p = 0.0004). Similarly, after other. P-value was considered significant when P < 0.05. rotenone-administration, in rats treated with L-dopa and L-dopa co-treatment with exercise practice recov Results ered the locomotor and exploratory activities after Treadmill exercise, L‑dopa and their combination improved rotenone treatment. However, L-dopa\rotenone and the exploration, and locomotion in rotenone‑treated groups combined exercise\L-dopa\rotenone groups showed (Fig. 1) significantly higher locomotor scores than the exercise\ After 4  weeks of daily administration of rotenone, rats rotenone group, (p = 0.0009). There was an insignificant spent less time in locomotion and exploration of open difference regarding exploration (p = 0.50) in-between field environment compared to the vehicle-injected the three treatment groups. Also, there was no signifi group (p = 0.00007, Fig .  1). But the anxiety scores cant change concerning the anxiety score between the in terms of urination and defecation times were not five groups under the study. Fig. 1 Open field test effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). N = 10 rats\group. The analysis was done by ANOVA test. P-value < 0.05, *compare to control G, # in comparison with the rotenone G M onir et al. Behav Brain Funct (2020) 16:9 Page 5 of 12 Treadmill exercise alone and in combination with L‑dopa Treadmill exercise alone and in combination with L‑dopa improved STM in the rotenone‑treated groups (Fig. 2) fully corrected the gait, while L‑dopa alone caused a partial There was a significant decrease in STM of the rote - correction of gait analysis none-treated group compared to the control group Rotenone injection caused a significant gait impairment (p = 0.02). No significant difference as regard LTM in in comparison to the control rats in terms of increased either group. overlap (OL) distance (p = 0.001), shortened both stride L-dopa alone didn’t improve STM in the rote- length; hind (HSL) and front (FSL) steps, and similarly none treated rats (p = 0.23) while, exercise alone or significant wide base, as detected by increased FSW and its combination with L-dopa has produced significant HSW (p = 0.03, Table 2). improvements in the STM in comparison with the rote- Treadmill exercise alone and exercise /L-dopa treat- none-injected group (p = 0.03). Meanwhile, LTM was ment corrected OL distance induced by rotenone injec- insignificantly changed among all groups (p = 0.38). tion (p = 0.001). Additionally, increased the FSL and 300 *$ *# STM control Grotenone Grotenone\exercise G rotenone\L-dopa Gcombined G LTM controlrotenone Grotenone\exercise G rotenone\L-dopa Gcombination G Fig. 2 Object recognition test, the effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks) on STM (Fig a) and LTM (Fig b). The analysis was done by ANOVA test. P-value is considered significant when P < 0.05, n = 10 rat in each group. *compare to control group, in comparison with the rotenone treated group. STM short term memory, LTM long term memory Mean in sec Mean in sec Monir et al. Behav Brain Funct (2020) 16:9 Page 6 of 12 Table 2 Eec ff ts of exercise, L-dopa and their combination on gait measured by Foot print test (overlap, front stride width, front stride length, hind stride width and hind stride length in cm) Group Control G Rotenone G Rotenone\ exercise G Rotenone\L‑ dopa G Combined G P‑ value by ANOVA OL 1.50 ± 0.3 2.10 ± 0.45 1.40 ± 0.3 1.35 ± 0.2 1.55 ± 0.3 0.0008 # # # *P = 0.001 P = 0.001 P = 0.0001 P = 0.003 FSW 4.95 ± 1.23 5.95 ± 0.83 3.40 ± 0.99 5.20 ± 0.91 4.15 ± 0.8 0.006 # # *P = 0.02 P = 0.007 P = 0.12 P = 0.009 FSL 11.89 ± 1.8 9.70 ± 1.65 13.5 ± 3.2 13.1 ± 1.44 11.1 ± 2.5 0.002 # # # *P = 0.01 P = 0.001 P = 0.006 P = 0.003 HSW 6.15 ± 0.78 7.00 ± 0.88 4.55 ± 1.2 6.15 ± 0.78 5.65 ± 0.7 0.001 # # *P = 0.004 P < = 0.001 P = 0.08 P = 0.002 HSL 12.65 ± 1.6 10.1 ± 1.46 12.8 ± 1.53 13.5 ± 1.24 11.7 ± 2.5 0.001 # # # *P= 0.02 P=0.002 P=0.001 P=0.03 P value is calculated by ANOVA test, and is considered significant if < 0.05. n = 10 in each group. Data was expressed as mean ± SD OL overlap, FSW front stride width, FSL front stride length, HSW hind stride width, HSL hind stride length *significant when compared to control group. significant when compared to rotenone group HSL (p = 0.0009), and decreased the HSW and FSW compared to the control group (p = 0.00008). Rats in all (p = 0.0008). L-dopa treatment improved the asymmetri- groups exhibited significant increases in the latency time cal gait and caused an elongation in the stride length, to fall, in comparison to the rotenone-injected group and a decrease in OL in the rotenone-injected group (p = 0.001). However, the L-dopa and the exercise/L- (p = 0.001), while it did not affect HSW (p = 0.08) and dopa-treated group showed a longer latency time than FSW (p = 0.12). To sum, treadmill exercise alone and in the exercise\rotenone group (p = 0.02). Therefore, forced combination with L-dopa treatment fully corrected the exercise, L-dopa treatment, and their combination pro- gait of rotenone-injected rats. longed the latency to fall in the Rota-rod test (Fig. 3). Treadmill exercise, L‑dopa, and their combination prolonged the latency time to fall in the Rota‑rod test Eec ff ts of treadmill exercise, L‑dopa and their combination (Fig. 3) on tyrosine hydroxylase (TH) levels in the striatum There was a significant decrease in latency time to fall Rotenone administration in rats significantly reduced after 4  weeks of a daily injection of rotenone in rats TH levels (to 40% of the control value) in the corpus *#$ *#$ *# controlr G otenone Grotenone\exercise rotenone\L-dopa combined G G G rota-rod test Fig. 3 Rota-rod test, the effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP daily for 4 weeks), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). P-value was calculated by ANOVA test and was considered significant if < 0.05. n = 10 in each group. *significant when # $ compared to the control group. significant when compared to the rotenone-treated group. Significant when compared to rotenone\exercise G latency me to fall (sec) M onir et al. Behav Brain Funct (2020) 16:9 Page 7 of 12 striatum when compared to the vehicle-injected group This study showed that the administration of rotenone (P = 0.00006). Whereas, forced exercise and their increased Nrf2 expression in the corpus striatum, such combined applications caused a significant increase upregulation was augmented by forced exercise, dra- in striatum TH levels which increased up to 62% and matically increased by the combination of treatment of 78% of normal) when compared to the rotenone- L-dopa and forced exercise, but was not affected by single injected group (P = 0.001). L-dopa alone didn’t signif- treatment of L-dopa (Fig. 5a). icantly increase the striatal TH level when compared to the rotenone group (P = 0.17). Besides, the tyrosine Eec ff ts of treadmill exercise, L‑dopa and their combination hydroxylase level in the L-dopa\rotenone group was on target genes of Nrf2 in the striatum significantly lower than that of the exercise\ rotenone Rotenone significantly increased expression of NQO1 in and the combined group (P = 0.09, Fig . 4). the rotenone group compared to control (P = 0.0009) as shown in Fig. 5b. Exercise produced a significant increase Eec ff ts of treadmill exercise, L‑dopa and their combination in NQO1 mRNA expression level when compared to the on Nrf2 expression in the striatum rotenone group (P = 0.001). However, the L-dopa treat- Rotenone caused a significant increase of Nrf2 mRNA ment reduced NQO1 mRNA expression in the rote- expression in the striatum of rats in comparison to the none-injected group (P = 0.001). Whereas when exercise vehicle-injected group (P = 0.02). performed with L-dopa treatment did not change NQO1 Forced exercise alone and in combination upregulated mRNA expression compared to the rotenone group Nrf2 mRNA expression in corpus striatum when com- (P = 0.49), but decreased NQO1 mRNA expression when pared to the rotenone-injected group (P = 0.0009). How- compared to the exercise\rotenone group (P = 0.003). ever, treatment with L-dopa did not affect Nrf2 mRNA Additionally, there was a significant increase in NQO1 expression (P = 0.62). Moreover, Nrf2 mRNA expres- expression in the exercise\L-dopa\rotenone group com- sion was lower in the L-dopa\rotenone group compared pared to the L-dopa\rotenone group (P = 0.001) (Fig. 5b). to the exercise\rotenone group (P = 0.00). Interestingly, Consistently, treadmill exercise caused a signifi - the combination between forced exercised and L-dopa cant increase in TFAM mRNA expression after injec- treatment showed a significant increase in Nrf2 mRNA tion of rotenone and when practiced in combination expression in comparison to the exercise\rotenone with L-dopa treatment compared to rotenone group group (P = 0.03) and L-dopa\rotenone group (P = 0.001). (P < 0.001) and L-dopa\rotenone (P = 0.0009 and = 0.01, *#$ *# *$ *$ ROTENONE\ ROTENONE\L- COMBINATION G CONTROL G ROTENONE G DOPA G EXERCISE G tyrosine hydroxylase in striatal ssue Fig. 4 Striatal tyrosine hydroxylase measured by ELISA, effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg, IP daily for 4 weeks), and their combination on tyrosine hydroxylase level in rotenone-treated rats (4 weeks, 2 mg\kg, SC). P-value is obtained by ANOVA and is considered # $ significant if < 0.05. *significant in comparison to the control group, significant when compared to the rotenone group, significant when compared to the rotenone exercise group. n = 10 in each group concentraon (ng\mg ssue) Monir et al. Behav Brain Funct (2020) 16:9 Page 8 of 12 (See figure on next page.) Fig. 5 Expression levels of genes by PCR in the corpus striatum, effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP daily for 4 weeks), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). a Nrf2 gene, b expression level of NADPH dehydrogenase (NQO1) mRNA, and c expression level of TFAM mRNA, P-value is calculated by ANOVA test, and is considered significant if < 0.05. n = 10 rats in each group. *Significant # $ when compared to control G. significant when compared to the rotenone group. Significant when compared to the rotenone\exercise group the results of this study were in line with Madiha et  al. successively). Similar to NQO1 expression, L-dopa treat- [29] their data showed significantly impaired walking pat - ment did not affect TFAM mRNA expression in the rote - tern and shortened stride length in rotenone-treated rats. none-injected group (P = 0.73). There was a significant Treadmill exercise significantly increased explora - increase in TFAM mRNA expression in the combination tion and locomotion in rotenone-treated rats. Treadmill group when compared to the exercise\rotenone group exercise corrected the gait impairment in rotenone- (P = 0.05), (Fig. 5c). treated rats; the asymmetrical patterns were corrected Overall, our results demonstrated that 4 weeks of daily by a significant reduction in overlap, accompanied by treatment with rotenone increased Nrf2-related genes, a significant increase in stride length and a significant NQO1 and TFAM, expression in the corpus striatum. decrease in stride width. Treadmill exercise significantly Such expression was enhanced by forced exercise and improved short-term memory novel object recognition was not affected by a single treatment of L-dopa (Fig.  5b, in the rotenone-treated group. Tyrosine hydroxylase c). levels significantly increased in the brain after treadmill exercise. Exercise also improved motor coordination as Discussion it increased latency time to fall on Rota-rod significantly. Parkinson’s disease (PD) is a common neurodegenerative The beneficial effects of the treadmill and other means disease, about 1% above the age of 65  years suffer from. of exercise in improving motor coordination (prolong In PD, there is a progressive degeneration of dopaminer- latency time to fall on Rota-rod) were also reported by gic neurons of the substantia nigra of the midbrain [22]. Shin et  al. and Lee et  al. [30, 31], in the rotenone model This results in the characteristic motor impairment and of PD. In line with our study, Chen et  al. [32] reported the extra motor manifestation of the disease. Rotenone a significant improvement of the Rota-rod test in addi - is known to induce PD in rats by targeting dopaminergic tion to a significant gait improvement (improved overlap, neurons [23]. It acts by inhibiting complex I of the mito- stride length, and base width) with treadmill exercise but chondrial electron transport chain and causing accumu- in 6-OHDA model PD. lation of reactive oxygen species (ROS) and subsequently, When L-dopa was administrated in rotenone-treated cell damage. Age-related mitochondrial dysfunction and rats, a significant improvement regarding exploration and oxidative stress have been strongly involved in the patho- locomotion was found. Motor co-ordination significantly physiology of PD [24, 25]. improved ever more than exercise alone. The gait showed In this study, rotenone 2 mg\kg\day was injected by the significant partial improvement, the asymmetrical pat - subcutaneous route for 4 weeks to induce rotenone-Par- tern was significantly corrected, and the stride length sig - kinson’s disease rat model. The rotenone-treated group, nificantly increased, meanwhile, short-term memory did showed a significant decrease in exploration and loco - not significantly improve. Tyrosine hydroxylase levels did motion as regards to the open field test, gait impairment; not significantly increase in the brain. Our results agree (asymmetrical foot pattern, shortened stride length, and with Allam et  al. [33] who found a significant increase widened base), a significant decline in motor coordina - in locomotor activity and exploration with L-dopa treat- tion was observed by shortened latency time on Rota- ment of rotenone-treated rats in the open field test, rod. Cognitive function impairment of PD also existed; and Sgroi et  al. [34] who reported a significant increase as a significant decrease in novel object preference in the in locomotion and motor co-ordination with 8  mg\kg short-term memory test. Tyrosine hydroxylase levels in L-dopa treatment for ten days in 6-OHDA model of PD. the striatum significantly decreased in rotenone treated When L-dopa treatment was accompanied by treadmill rats. Similar to our study, Vijayalakshmi et  al. [26] and exercise, there was a significant increase in exploration Valdez et al. [27] reported a significant decrease in loco - and locomotion behavior in the open field. A significant motion in the Open field test in rotenone-treated Wis - increase in the latency time of the Rota-rod test was ter rats. von Wrangel et  al. [28], and Cannon et  al. [12] found indicating improved motor coordination. There reported that rotenone-treated rats showed significant was a significant gait improvement with L-dopa and exer - impairment in Rota-rod and significant decline in tyros - cise co-treatment, the asymmetrical pattern was fully ine hydroxylase in the striatum. As regard footprint test, M onir et al. Behav Brain Funct (2020) 16:9 Page 9 of 12 Monir et al. Behav Brain Funct (2020) 16:9 Page 10 of 12 corrected, with a significant increase in stride length and As regard expression of TFAM, this is the first study a significant decrease in stride width. Short-term mem - which investigated its expression in rotenone-treated ory significantly improved. Tyrosine hydroxylase levels in rats, Rotenone downregulated TFAM expression which the brain were significantly increased. may underlie the instability in the mtDNA and conse- In our study, we investigated some genes of the Nrf2- quent damage of the dopaminergic neuron. This is evi - ARE pathway as a possible mechanism involved in the denced by the decreased level of tyrosine hydroxylase treadmill exercise effects on the brain. Exercise causes measured in the striatum. Noteworthy, Chen et al. [39] in an increase in O2 consumption with an increase in ROS their study on post mortem human parkinsonian patients production, especially H2O2. Oxidative stress leads to found that expression of TFAM in substantia nigra is inhibition of KEAP-1, dissociation of Nrf2 from KEAP- lower and their mtDNA is less stable in comparison to 1 in the cytoplasm, migrates into the nucleus, and acti- age-matched elderly control subjects. vates the Nrf2-ARE [35]. Nrf2 activates anti-oxidant gene As regards our study results, treadmill exercise acti- expression with increased productions of detoxifying vated the Nrf2 pathway in the striatum of the rote- enzymes: Heme oxygenase, and NADPH quinone oxi- none-treated rat. This activation could be one of the doreductase (NQO.1). Activation of Nrf2 together with mechanisms involved in treadmill exercise beneficial PCG1α, which also is activated by exercise, caused an effects on the rotenone-treated model of PD. Few stud - increase in mitochondrial transcription factor A (TFAM) ies which measured TFAM expression in animal models expression in the striatum. TFAM is a nuclear factor e.g. Aguiar et  al. [40] reported that exercise significantly that controls the replication of mitochondrial DNA (mt increased Nrf2 and TFAM in the striatum of 6-OHDA DNA). The upregulation of TFAM increases the number hemiparkinsonism. Other studies reported TFAM acti- of copies and packaging of mtDNA. Also, adequate levels vation with exercise in different pathological conditions of TFAM are required to prevent mtDNA release into the other than parkinsonism. For instance, Lashgarie et  al. cytoplasm and initiate an inflammatory response [11]. [11] reported that treadmill exercise increased TFAM This study measured the rate of expression of Nrf2, expression in the heart of nicotine sensitized rats, which TFAM, and Nqo.1 in the striatum of rotenone-treated attenuated mitochondrial-mediated damage in the myo- rats. Rotenone downregulated expression of the TFAM cardium induced by nicotine. in the rotenone group while upregulated Nrf2 and Nqo1 in comparison to the control rats. Treadmill exercise showed a significant increase in Nrf2, TFAM, and NQO.1 Conclusion in the striatum in the exercise\rotenone group in com- In this study, treadmill exercise increased tyrosine parison to control and rotenone groups. L-dopa\rotenone hydroxylase and activated the Nrf2 pathway and some of group showed an insignificant change in the expression its associated genes however L-dopa could not. Where of these genes. However, the exercised \L-dopa\rotenone L-dopa corrected locomotion, exploration, and motor co- group, showed a significant increase in Nrf2, TFAM, and ordination but failed to improve short-term memory and Nqo.1 expression, when compared to both control and only partially corrected the gait of rotenone-treated rats. rotenone group. When treadmill exercise was combined with L-dopa, all Whether rotenone increases or decreases the expres- the behavioral motor and non-motor aspects of PD were sion of Nrf2 and NQO1 is a matter of debate. Many corrected. studies [36] found that rotenone in in-vivo and in-vitro studies downregulated the expression of these genes and enhanced apoptosis of the neurons in the nigros- Recommendation triatal tissue which was reversed by administration of Exercise is highly recommended and is a mandatory danshensu herbal extract. Similarly, Cui et al. [37] found approach for the treatment of parkinsonism in humans. decreased expression of NRF2 and NQO1 proteins meas- Further studies are needed to explore the importance of ured by western blotting in rotenone treated rats which expression of the TFAM gene as part of the Nrf2 pathway were corrected with pretreatment with curcumin. On the in protecting against mitochondrial induced oxidative contrary, Wei et  al. [38] found that rotenone increased damage. NRF2 and NQO1 expression in the striatum which was further enhanced by ellagic acid. They measured the Supplementary information NRF2 protein in the cytoplasm and in the nucleus of the Supplementary information accompanies this paper at https ://doi. org/10.1186/s1299 3-020-00171 -9. cell indicating that the expression of these transcription factors was increased in the nucleus. M onir et al. Behav Brain Funct (2020) 16:9 Page 11 of 12 5. Ferris CF, Marella M, Smerkers B, Barchet TM, Gershman B, Matsuno-Yagi A, Additional file1: Figure S1. The effects of exercise on serum levels of CPK et al. A phenotypic model recapitulating the neuropathology of Parkin- and LDH in control groups, * if the differences were significantly different son’s disease. Brain Behav. 2013;3(4):351–66. from the control group. 6. Bezard E, Yue Z, Kirik D, Spillantini MG. Animal models of Parkinson’s dis- ease: limits and relevance to neuroprotection studies. Movement Disord. 2013;28(1):61–70. Abbreviations 7. Lee NY, Lee DK, Song HS. Eec ff t of virtual reality dance exercise on the ARE: Antioxidant response elements; DA: Dopamine; NQO1: NAD(P) H quinone balance, activities of daily living, and depressive disorder status of Parkin- oxidoreductase 1: NAD(P)H dehydrogenase; Nrf2: Nuclear factor erythroid 2 son’s disease patients. J Phys Ther Sci. 2015;27(1):145–7. (NFE2)-related factor 2; PA: Physical activity; PGC1α: Peroxisome-proliferator- 8. Um HS, Kang EB, Leem YH, Cho IH, Yang CH, Chae KR, et al. Exercise activated receptor-γ coactivator-1α; ROS: Reactive oxygen species; TFAM: training acts as a therapeutic strategy for reduction of the pathogenic Mitochondrial transcriptional factor A; OL: Overlap; FSL: Forward stride length; phenotypes for Alzheimer’s disease in an NSE/APPsw-transgenic model. FSW: Forward stride width; HSL: Hind stride length; HSW: Hind stride width. Int J Mol Med. 2008;22(4):529–39. 9. Li T, He S, Liu S, Kong Z, Wang J, Zhang Y. Eec ff ts of different exercise dura- Acknowledgements tions on Keap1-Nrf2-ARE pathway activation in mouse skeletal muscle. The authors would like to thank the staff of the central laboratory in Sohag Free Radical Res. 2015;49(10):1269–74. Medical Faculty for their cooperation all over the period of practical work. 10. Hinson VK, Goetz CG, Leurgans S, Fan W, Nguyen T, Hsu A. Reducing dosing frequency of carbidopa/levodopa: double-blind crossover study Authors’ contributions comparing twice-daily bilayer formulation of carbidopa/levodopa AA selected the point of research, MM and DM equally contributed to the (IPX054) versus 4 daily doses of standard carbidopa/levodopa in stable practical part of the study, IR collected the data and shared in the analysis of Parkinson disease patients. Clin Neuropharmacol. 2009;32(4):189–92. the behavioral tests, all authors shared in the writing of the research with final 11. Lashgari AA, Azarbayjani M-A, Peeri M, Nasehi M. The Eec ff t of Short- revision from OG and AA. All authors read and approved the final manuscript. Term Treadmill Exercise on the Expression Level of TFAM in the Heart of Nicotine-Sensitized Rats. Archives of Advances in Biosciences. 2020;11(1). Funding 12. Cannon JR, Tapias V, Na HM, Honick AS, Drolet RE, Greenamyre JT. A The authors declare that there is no special funding taken for this research. highly reproducible rotenone model of Parkinson’s disease. Neurobiol Dis. 2009;34(2):279–90. Availability of data and materials 13. Shin JH, Kang KW, Kim JH, Chin JY, Kim NY, Park SH, et al. Treadmill The datasets used and/or analyzed during the current study are available from exercise-induced E/e’ elevation as a predictor of cardiovascular event in the corresponding author on request. end-stage renal disease on peritoneal dialysis. Korean J Intern Med. 2016. 14. Walsh RN, Cummins RA. The Open-Field Test: a critical review. 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Front Behav Neurosci. 2014;8:331. lished maps and institutional affiliations. 35. Vargas-Mendoza N, Morales-González Á, Madrigal-Santillán EO, Madrigal- Bujaidar E, Álvarez-González I, García-Melo LF, et al. Antioxidant and adap- tative response mediated by Nrf2 during physical exercise. Antioxidants. 2019;8(6):196. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

Forced exercise activates the NrF2 pathway in the striatum and ameliorates motor and behavioral manifestations of Parkinson's disease in rotenone-treated rats

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Abstract

Background: Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by progressive loss of nigrostriatal dopaminergic neurons leading to dopamine depletion and problems of movement, emotions, and cog- nition. While the pathogenesis of PD is not clear, damage of dopaminergic neurons by oxygen-derived free radicals is considered an important contributing mechanism. This study aimed to evaluate the role of treadmill exercise in male Wister rats as a single treatment and as an aid-therapy with L-dopa for rotenone-induced PD. To study the role of the Nrf2- ARE pathway as a mechanism involved in exercise-associated improvement in rotenone-induced PD in rats. Method: Animals were divided into 5 groups, (Control, rotenone, rotenone\exercise, rotenone\L-dopa, and rote- none\exercise\L-dopa (combination)groups). After the PD induction, rats in the rotenone\exercise and combination groups were daily treadmill exercised for 4 weeks. Results: Treadmill exercise significantly improved behavioral and motor aspects of rotenone-induced PD. When treadmill exercise was introduced as a single intervention, it amended most behavioral aspects of PD, gait fully cor- rected, short-term memory, and motor coordination. Where L-dopa corrected locomotor activity and motor coordina- tion but failed to improve short-term memory and only partially corrected the gait of rotenone-treated rats. When treadmill exercise was combined with L-dopa, all features of PD were corrected. It was found that exercise upregu- lated some of its associative genes to Nrf2 pathways such as TFAM, Nrf2 and NQO.1 mRNA expression. Conclusion: This study suggests that forced exercise improved parkinsonian like features by activating the Nrf2 pathway. Keywords: Parkinson, Rotenone, Behavioral tests, Exercise, Nrf2. TFAM, Noq1 Background Parkinson’s disease (PD) is a common neurodegenerative *Correspondence: motamed71111@gmail.com; amany_abdelhameed@med. disorder characterized by persistent loss of nigrostriatal sohag.edu.eg Dina M. Monir and Motamed E. Mahmoud are equally contributed dopaminergic neurons leading to depletion of dopamine Department of Physiology, Faculty of Medicine, Sohag University, and consequent problems of movement, emotions, and Sohag 82524, Egypt cognition. While many factors contribute to the patho- Department of Animal Behavior and Husbandry (Genetics, Breeding, and Production), Faculty of Veterinary Medicine, Sohag University, genesis of PD, damage of dopaminergic neurons by Sohag 82524, Egypt oxygen-derived free radicals is considered an important Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Monir et al. Behav Brain Funct (2020) 16:9 Page 2 of 12 contributing mechanism [1, 2]. Rotenone is a potent in the Medical Animal Laboratory in Sohag Faculty of inhibitor of complex I (NADH: ubiquinone oxidoreduc- Medicine. Animals were allowed free access to food and tase) of the mitochondrial electron transport chain. This tap water. Rats were housed in standard cages, at normal allows for accumulation and overproduction of reac- light/dark cycle and room temperature. The rats were tive oxygen species (ROS) which eventually leads to cell randomly divided into 5 groups (n = 10 in each group); damage [3, 4]. Chronic rotenone exposure in rats causes control group, rotenone-injected group, rotenone\exer- both neuropathological findings and behavioral symp - cise group, rotenone\L-dopa treated-group, and com- toms of PD [5]. The rotenone model mimics the grad - bined (rotenone\L-dopa\exercise) group. The study was ual progression of PD as observed in humans. Systemic approved by Research Ethics Committee considering the mitochondrial inhibition by rotenone leads to selective care and use of laboratory animals (permission number: nigrostriatal degeneration [6]. SOH-IACUC-17050301). Treadmill exercise is indicated as a physical therapy to improve motor symptoms in patients with PD. Exer- Induction of Parkinsonism cise can improve and alleviate memory loss in elderly Rats were subcutaneously injected with either vehicle or patients, and decrease the risk of developing PD. The rotenone (R8875; 95%, Sigma-Aldrich, USA) 2 mg/kg, for neuroprotective potentials of exercise are great, but the 4 weeks. Rotenone solution was prepared as a 50 × stock underlying mechanisms remain a debatable issue. Evi- in 100% dimethylsulfoxide (DMSO) then in a medium- dence suggests that exercise neuroprotection is due to its chain triglyceride, miglyol 812  N (Sigma) to obtain a neurotrophic effects, as exercise increases the availability final concentration of 2 mg/mL rotenone in 98% miglyol of several neurotrophic factors [7]. Long-term exercise 812 N, 2% DMSO. The prepared solution was stored in an benefits brain functioning by increasing the blood and amber septa vial to be protected from light and inverted oxygen flow to the brain, mobilizing growth factors that many times before each injection to eliminate the possi- promote neurogenesis and synaptic plasticity, releasing of bility of settling [12]. L-dopa (L-3, 4 dihydroxyphenylala- neurotransmitters, such as dopamine (DA), noradrena- nine methyl ester hydrochloride, (Sigma) was dissolved line, serotonin, and glutamate and consequently improve in normal saline and was administered at a dose 6 mg/kg/ manifestations of the disease either at the motor or cog- day, injected intraperitoneally for 4 weeks. nitive. levels in Alzheimer’s animal model [8]. Exercise increases the antioxidant status in the stria- Exercise protocol and treatments tum of animals and protects against neurological oxida- After induction of PD, rats in the exercise group were tive challenges. The nuclear factor erythroid-derived forced to run in a 3-channel treadmill (Heath Life 2-like 2 (Nrf2)-antioxidant response element (ARE) V4000M). The exercise regimen continues for 30  min/ signaling pathway, a major cellular defense mechanism day, 5 times a week for 4  weeks. The treadmill speed against oxidative stress. Exercise activates Nrf2 in human accelerates beginning with 2  m/min during the first skeletal muscles and mouse heart [9]. The Nrf2-ARE 5 min, at 3 m/min during the second 5 min, and then at signaling pathway appears to be a strong mechanism for 5  m/min for the last 20  min [13]. The efficiency of this exercise-induced neuroprotection [10]. The activation of exercise protocol was previously assessed by measuring the Nrf2 gene activates genes that encode for antioxidant the serum lactate dehydrogenase and creatine phospho- enzymes within the cells like heme oxygenase and NADH kinase in a non-published experiment (Additional file  1: quinone oxidoreductase (NQO1). Also, Nrf2 activates the Fig. S1). mitochondrial transcription factor A (TFAM) which reg- ulates for mitochondrial DNA (mtDNA) replication [11]. Behavioral and motor analysis Therefore, in this study, we investigated whether tread - These tests assess motor activity, and behavior of the rats, mill exercise as a single therapeutic intervention and as they were performed at the end of the experiment for all add-on therapy with L-dopa improve manifestations of groups and included; PD in rotenone-treated rats. Also, we assessed the Nrf2 pathway as a possible mechanism activated by treadmill Open field test (OFT) exercise. OFT was performed according to the previously described method [14]. The apparatus was a squared Materials and methods plastic arena (114 × 114 × 44 height cm) its floor was Animals and experimental design divided into smaller squares (19 × 19 cm ) with a central Fifty-adult male Wistar rats with body weight averaged square (38 × 38 cm ) each rat was put gently in the center 275 ± 25 gm, 9  months aged; were purchased from the of the arena and was left for 5  min moving and explor- Faculty of Science, Sohag University, and were housed ing the field. A video camera was fixed at the top of the M onir et al. Behav Brain Funct (2020) 16:9 Page 3 of 12 arena to record the activity of the rat which was scored after they complete 120  s. The mean latency time of the by a specialist who was blind to the experimental groups. three trials was taken. Animals were trained for five days Each animal was then given a score for total locomotor to perform the test [17]. activity; calculated as the sum number of line crosses and rears, a score for exploratory behavior; the sum of the Tissue Sampling number of central square entries and the duration of time After behavioral tests, the rats were anesthetized with spent in the central square, and the anxiety score is equal Zoletil (1 mg\kg i.p (Vibac Laboratories, Carros, France). to the sum of urination and defecation boli [14]. Rats were transcardially perfused with 0.05 M phosphate- buffered saline (PBS). The brain was removed, separated Object recognition test (ORT) into right and left hemispheres, snap-frozen in liquid ORT was performed as described by Walsh and Cummins nitrogen, and kept for 1  h at − 80  °C. The striatum was [15]. To assess the short-term memory (STM) and long- dissected through multiple manual coronal sections with term memory (LTM), four objects used were made of plas- a sharp razor blade and was collected with the help of two tic material. The objects and arena were washed with a 10% Dumont No. 5 forceps [18]. Samples then were stored in ethanol solution after each trial. The training was conducted Eppendorf tubes at − 80 °C until further analyses. by placing each rat for 5 min into the open field apparatus, where two identical objects (objects A1 and A2) were put in (1) RNA extraction two adjacent sides, 10 cm from the walls. In the STM test, Total RNA was extracted from 30  mg of tissue samples a rat was given 1.5  h after training; the rats explored the according to manufacture instructions (RNA Extraction open field for 5 min in the presence of one familiar (A) and kit (#K0731, Thermo Scientific, Lithuania)). They were one novel (B) object. LTM test was done 24  h after train- extracted to measure Nrf2, NOQ.1, TFAM, and using ing, the same rat explored the field for 5 min in the presence housekeeping GAPDH by real-time polymerase chain of a familiar object A and a novel object C. Exploration is reaction (real-time PCR). defined as sniffing or touching the object with the nose and/ or forepaws. The time (T) which is spent by each rat explor - ing the different objects was measured as TA1, TB, and (2) Reverse transcription TC. These times were used for calculation of STM = [TB / Extracted RNA concentration was quantified using Nan - (TA1 + TB)] 100, and LTM = [TC / (TA1 + TC)] 100. odrop spectrophotometry (Quawell 5000, USA); then 110 ng of total RNA transcribed using RNA reverse tran- Foot print test scriptase kits ((#K0251) (Thermo Scientific, Lithuania)). Footprint test was used to measure gait analysis by The thermal cycler was programmed at 25 °C for 10 min, permitting rats to run in a wood corridor apparatus 37 °C for 120 min, 85 °C for 5 min, and 4 °C for 20 h. (65 × 5 × 15 cm ), which was lined with a pre-cut piece of white paper. Rats were trained to run to the end of (3) Real‑time PCR the corridor by placing the rats at the far end of the cor- Prepared cDNA, was used in the qPCR analyzer (Step ridor and encouraging them to move towards the end. One, Applied Biosystems, Singapore) using the MAX- The training was conducted twice for each rat until the IMA SYBR Green qPCR Master Mix with the following animal could run to the end box without encouragement. program: 1 cycle at 95  °C for 10  min; 40 cycles of 95  °C For testing, the paws of the animal were painted with four for 15  s, 60  °C for 30  s and 72  °C for 30  s; one cycle at non-toxic watercolors as described previously [16]. For 95  °C for 15  s, 60  °C for 1  min and 95  °C for 15  s. The each animal, the gait was calculated using 4 paw prints; specific primers (Table  1) of Nrf2, TFAM, NADPH dehy- this allowed 5 values-yield/rat; [front stride length (FSL), drogenase (NQO1) & housekeeping GAPDH were pur- front stride width (FSW), hind stride length (HSL), hind chased from Metabion international AG, Germany. Fold stride width (HSW) and overlap (OL)] which were then −ΔΔct expression (2 ) was calculated according to the rela- averaged to provide gait measurements [15]. tive expression of housekeeping gene GAPDH. Rota‑rod test To assess the motor coordination of the animals, we used (4) Tyrosine hydroxylase an accelerating Rota-rod (Harvard Apparatus, UK). The Homogenized striatum of the right hemisphere of all rat Rota-rod consisted of a suspended rod, accelerating for brains was used to measure the levels of tyrosine hydrox- 60 s, beginning from 5 rounds per minute (RPM) to reach ylase enzyme by ELISA (tyrosine hydroxylase (TH) rat 15 RPM, and continuing at that speed for a further 60 s. ELISA kits (#:96,791) from Glory Science Co., (Ltd, A trial was stopped when the rats fell off the Rota-rod or China) with a detection range 0.625- 20 ng\ ml. Monir et al. Behav Brain Funct (2020) 16:9 Page 4 of 12 Table 1 Forward and reverse primers for real time PCR Gene name Forward primer Reverse primer Access number References NRF2 5′ CAC ATC CAG ACA GAC ACC AGT-3′ 5′ CTA CAA ATG GGA ATG TCT CTGC-3′ NM_031789 [19] TFAM 5′AGT TCA TAC CTT CGA TTT TC-3′ 5′ TGA CTT GGA GTT AGC TGC -3` NM_031326.1 [20] NQO1 5′CAG CGG CTC CAT GTACT-3′ 5′ GAC CTG GAA GCC ACA GAA G-3′ NM_017000 [19, 21] GAPDH NM_017008 [19] 5′CAG GCA TAT GGT GGT CCA TAGAG-3′ 5′ TCA TGG GAT CCA CCT GCA GC-3′ NRF2 nuclear factor erythroid 2 (NFE2)-related factor 2, NQO1 quinone oxidoreductase 1, TFAM Mitochondrial transcriptional factor A, GAPDH glyceraldehyde-3- phosphate dehydrogenase Statistical analysis different between both groups. To sum, rotenone injec Statistical package for social sciences (IBM-SPSS), version tion decreased exploration and locomotion scores in 24 IBM- Chicago, USA (May 2016) was used for statistical the open field test. data analysis. Data expressed as mean ± standard devia- It was noticed that after injection of rotenone, rats tion (SD), number, and percentage. A one-way analysis of practiced 5 times/week a treadmill exercise spent less variance (ANOVA) test was used to compare the means time in the central arena and increased number of line of the analyzed groups. Post hoc test (LSD type) was used crossings in the open-field test compared to the non- for multiple comparisons between each group and the exercised rotenone group (p = 0.0004). Similarly, after other. P-value was considered significant when P < 0.05. rotenone-administration, in rats treated with L-dopa and L-dopa co-treatment with exercise practice recov Results ered the locomotor and exploratory activities after Treadmill exercise, L‑dopa and their combination improved rotenone treatment. However, L-dopa\rotenone and the exploration, and locomotion in rotenone‑treated groups combined exercise\L-dopa\rotenone groups showed (Fig. 1) significantly higher locomotor scores than the exercise\ After 4  weeks of daily administration of rotenone, rats rotenone group, (p = 0.0009). There was an insignificant spent less time in locomotion and exploration of open difference regarding exploration (p = 0.50) in-between field environment compared to the vehicle-injected the three treatment groups. Also, there was no signifi group (p = 0.00007, Fig .  1). But the anxiety scores cant change concerning the anxiety score between the in terms of urination and defecation times were not five groups under the study. Fig. 1 Open field test effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). N = 10 rats\group. The analysis was done by ANOVA test. P-value < 0.05, *compare to control G, # in comparison with the rotenone G M onir et al. Behav Brain Funct (2020) 16:9 Page 5 of 12 Treadmill exercise alone and in combination with L‑dopa Treadmill exercise alone and in combination with L‑dopa improved STM in the rotenone‑treated groups (Fig. 2) fully corrected the gait, while L‑dopa alone caused a partial There was a significant decrease in STM of the rote - correction of gait analysis none-treated group compared to the control group Rotenone injection caused a significant gait impairment (p = 0.02). No significant difference as regard LTM in in comparison to the control rats in terms of increased either group. overlap (OL) distance (p = 0.001), shortened both stride L-dopa alone didn’t improve STM in the rote- length; hind (HSL) and front (FSL) steps, and similarly none treated rats (p = 0.23) while, exercise alone or significant wide base, as detected by increased FSW and its combination with L-dopa has produced significant HSW (p = 0.03, Table 2). improvements in the STM in comparison with the rote- Treadmill exercise alone and exercise /L-dopa treat- none-injected group (p = 0.03). Meanwhile, LTM was ment corrected OL distance induced by rotenone injec- insignificantly changed among all groups (p = 0.38). tion (p = 0.001). Additionally, increased the FSL and 300 *$ *# STM control Grotenone Grotenone\exercise G rotenone\L-dopa Gcombined G LTM controlrotenone Grotenone\exercise G rotenone\L-dopa Gcombination G Fig. 2 Object recognition test, the effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks) on STM (Fig a) and LTM (Fig b). The analysis was done by ANOVA test. P-value is considered significant when P < 0.05, n = 10 rat in each group. *compare to control group, in comparison with the rotenone treated group. STM short term memory, LTM long term memory Mean in sec Mean in sec Monir et al. Behav Brain Funct (2020) 16:9 Page 6 of 12 Table 2 Eec ff ts of exercise, L-dopa and their combination on gait measured by Foot print test (overlap, front stride width, front stride length, hind stride width and hind stride length in cm) Group Control G Rotenone G Rotenone\ exercise G Rotenone\L‑ dopa G Combined G P‑ value by ANOVA OL 1.50 ± 0.3 2.10 ± 0.45 1.40 ± 0.3 1.35 ± 0.2 1.55 ± 0.3 0.0008 # # # *P = 0.001 P = 0.001 P = 0.0001 P = 0.003 FSW 4.95 ± 1.23 5.95 ± 0.83 3.40 ± 0.99 5.20 ± 0.91 4.15 ± 0.8 0.006 # # *P = 0.02 P = 0.007 P = 0.12 P = 0.009 FSL 11.89 ± 1.8 9.70 ± 1.65 13.5 ± 3.2 13.1 ± 1.44 11.1 ± 2.5 0.002 # # # *P = 0.01 P = 0.001 P = 0.006 P = 0.003 HSW 6.15 ± 0.78 7.00 ± 0.88 4.55 ± 1.2 6.15 ± 0.78 5.65 ± 0.7 0.001 # # *P = 0.004 P < = 0.001 P = 0.08 P = 0.002 HSL 12.65 ± 1.6 10.1 ± 1.46 12.8 ± 1.53 13.5 ± 1.24 11.7 ± 2.5 0.001 # # # *P= 0.02 P=0.002 P=0.001 P=0.03 P value is calculated by ANOVA test, and is considered significant if < 0.05. n = 10 in each group. Data was expressed as mean ± SD OL overlap, FSW front stride width, FSL front stride length, HSW hind stride width, HSL hind stride length *significant when compared to control group. significant when compared to rotenone group HSL (p = 0.0009), and decreased the HSW and FSW compared to the control group (p = 0.00008). Rats in all (p = 0.0008). L-dopa treatment improved the asymmetri- groups exhibited significant increases in the latency time cal gait and caused an elongation in the stride length, to fall, in comparison to the rotenone-injected group and a decrease in OL in the rotenone-injected group (p = 0.001). However, the L-dopa and the exercise/L- (p = 0.001), while it did not affect HSW (p = 0.08) and dopa-treated group showed a longer latency time than FSW (p = 0.12). To sum, treadmill exercise alone and in the exercise\rotenone group (p = 0.02). Therefore, forced combination with L-dopa treatment fully corrected the exercise, L-dopa treatment, and their combination pro- gait of rotenone-injected rats. longed the latency to fall in the Rota-rod test (Fig. 3). Treadmill exercise, L‑dopa, and their combination prolonged the latency time to fall in the Rota‑rod test Eec ff ts of treadmill exercise, L‑dopa and their combination (Fig. 3) on tyrosine hydroxylase (TH) levels in the striatum There was a significant decrease in latency time to fall Rotenone administration in rats significantly reduced after 4  weeks of a daily injection of rotenone in rats TH levels (to 40% of the control value) in the corpus *#$ *#$ *# controlr G otenone Grotenone\exercise rotenone\L-dopa combined G G G rota-rod test Fig. 3 Rota-rod test, the effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP daily for 4 weeks), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). P-value was calculated by ANOVA test and was considered significant if < 0.05. n = 10 in each group. *significant when # $ compared to the control group. significant when compared to the rotenone-treated group. Significant when compared to rotenone\exercise G latency me to fall (sec) M onir et al. Behav Brain Funct (2020) 16:9 Page 7 of 12 striatum when compared to the vehicle-injected group This study showed that the administration of rotenone (P = 0.00006). Whereas, forced exercise and their increased Nrf2 expression in the corpus striatum, such combined applications caused a significant increase upregulation was augmented by forced exercise, dra- in striatum TH levels which increased up to 62% and matically increased by the combination of treatment of 78% of normal) when compared to the rotenone- L-dopa and forced exercise, but was not affected by single injected group (P = 0.001). L-dopa alone didn’t signif- treatment of L-dopa (Fig. 5a). icantly increase the striatal TH level when compared to the rotenone group (P = 0.17). Besides, the tyrosine Eec ff ts of treadmill exercise, L‑dopa and their combination hydroxylase level in the L-dopa\rotenone group was on target genes of Nrf2 in the striatum significantly lower than that of the exercise\ rotenone Rotenone significantly increased expression of NQO1 in and the combined group (P = 0.09, Fig . 4). the rotenone group compared to control (P = 0.0009) as shown in Fig. 5b. Exercise produced a significant increase Eec ff ts of treadmill exercise, L‑dopa and their combination in NQO1 mRNA expression level when compared to the on Nrf2 expression in the striatum rotenone group (P = 0.001). However, the L-dopa treat- Rotenone caused a significant increase of Nrf2 mRNA ment reduced NQO1 mRNA expression in the rote- expression in the striatum of rats in comparison to the none-injected group (P = 0.001). Whereas when exercise vehicle-injected group (P = 0.02). performed with L-dopa treatment did not change NQO1 Forced exercise alone and in combination upregulated mRNA expression compared to the rotenone group Nrf2 mRNA expression in corpus striatum when com- (P = 0.49), but decreased NQO1 mRNA expression when pared to the rotenone-injected group (P = 0.0009). How- compared to the exercise\rotenone group (P = 0.003). ever, treatment with L-dopa did not affect Nrf2 mRNA Additionally, there was a significant increase in NQO1 expression (P = 0.62). Moreover, Nrf2 mRNA expres- expression in the exercise\L-dopa\rotenone group com- sion was lower in the L-dopa\rotenone group compared pared to the L-dopa\rotenone group (P = 0.001) (Fig. 5b). to the exercise\rotenone group (P = 0.00). Interestingly, Consistently, treadmill exercise caused a signifi - the combination between forced exercised and L-dopa cant increase in TFAM mRNA expression after injec- treatment showed a significant increase in Nrf2 mRNA tion of rotenone and when practiced in combination expression in comparison to the exercise\rotenone with L-dopa treatment compared to rotenone group group (P = 0.03) and L-dopa\rotenone group (P = 0.001). (P < 0.001) and L-dopa\rotenone (P = 0.0009 and = 0.01, *#$ *# *$ *$ ROTENONE\ ROTENONE\L- COMBINATION G CONTROL G ROTENONE G DOPA G EXERCISE G tyrosine hydroxylase in striatal ssue Fig. 4 Striatal tyrosine hydroxylase measured by ELISA, effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg, IP daily for 4 weeks), and their combination on tyrosine hydroxylase level in rotenone-treated rats (4 weeks, 2 mg\kg, SC). P-value is obtained by ANOVA and is considered # $ significant if < 0.05. *significant in comparison to the control group, significant when compared to the rotenone group, significant when compared to the rotenone exercise group. n = 10 in each group concentraon (ng\mg ssue) Monir et al. Behav Brain Funct (2020) 16:9 Page 8 of 12 (See figure on next page.) Fig. 5 Expression levels of genes by PCR in the corpus striatum, effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP daily for 4 weeks), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). a Nrf2 gene, b expression level of NADPH dehydrogenase (NQO1) mRNA, and c expression level of TFAM mRNA, P-value is calculated by ANOVA test, and is considered significant if < 0.05. n = 10 rats in each group. *Significant # $ when compared to control G. significant when compared to the rotenone group. Significant when compared to the rotenone\exercise group the results of this study were in line with Madiha et  al. successively). Similar to NQO1 expression, L-dopa treat- [29] their data showed significantly impaired walking pat - ment did not affect TFAM mRNA expression in the rote - tern and shortened stride length in rotenone-treated rats. none-injected group (P = 0.73). There was a significant Treadmill exercise significantly increased explora - increase in TFAM mRNA expression in the combination tion and locomotion in rotenone-treated rats. Treadmill group when compared to the exercise\rotenone group exercise corrected the gait impairment in rotenone- (P = 0.05), (Fig. 5c). treated rats; the asymmetrical patterns were corrected Overall, our results demonstrated that 4 weeks of daily by a significant reduction in overlap, accompanied by treatment with rotenone increased Nrf2-related genes, a significant increase in stride length and a significant NQO1 and TFAM, expression in the corpus striatum. decrease in stride width. Treadmill exercise significantly Such expression was enhanced by forced exercise and improved short-term memory novel object recognition was not affected by a single treatment of L-dopa (Fig.  5b, in the rotenone-treated group. Tyrosine hydroxylase c). levels significantly increased in the brain after treadmill exercise. Exercise also improved motor coordination as Discussion it increased latency time to fall on Rota-rod significantly. Parkinson’s disease (PD) is a common neurodegenerative The beneficial effects of the treadmill and other means disease, about 1% above the age of 65  years suffer from. of exercise in improving motor coordination (prolong In PD, there is a progressive degeneration of dopaminer- latency time to fall on Rota-rod) were also reported by gic neurons of the substantia nigra of the midbrain [22]. Shin et  al. and Lee et  al. [30, 31], in the rotenone model This results in the characteristic motor impairment and of PD. In line with our study, Chen et  al. [32] reported the extra motor manifestation of the disease. Rotenone a significant improvement of the Rota-rod test in addi - is known to induce PD in rats by targeting dopaminergic tion to a significant gait improvement (improved overlap, neurons [23]. It acts by inhibiting complex I of the mito- stride length, and base width) with treadmill exercise but chondrial electron transport chain and causing accumu- in 6-OHDA model PD. lation of reactive oxygen species (ROS) and subsequently, When L-dopa was administrated in rotenone-treated cell damage. Age-related mitochondrial dysfunction and rats, a significant improvement regarding exploration and oxidative stress have been strongly involved in the patho- locomotion was found. Motor co-ordination significantly physiology of PD [24, 25]. improved ever more than exercise alone. The gait showed In this study, rotenone 2 mg\kg\day was injected by the significant partial improvement, the asymmetrical pat - subcutaneous route for 4 weeks to induce rotenone-Par- tern was significantly corrected, and the stride length sig - kinson’s disease rat model. The rotenone-treated group, nificantly increased, meanwhile, short-term memory did showed a significant decrease in exploration and loco - not significantly improve. Tyrosine hydroxylase levels did motion as regards to the open field test, gait impairment; not significantly increase in the brain. Our results agree (asymmetrical foot pattern, shortened stride length, and with Allam et  al. [33] who found a significant increase widened base), a significant decline in motor coordina - in locomotor activity and exploration with L-dopa treat- tion was observed by shortened latency time on Rota- ment of rotenone-treated rats in the open field test, rod. Cognitive function impairment of PD also existed; and Sgroi et  al. [34] who reported a significant increase as a significant decrease in novel object preference in the in locomotion and motor co-ordination with 8  mg\kg short-term memory test. Tyrosine hydroxylase levels in L-dopa treatment for ten days in 6-OHDA model of PD. the striatum significantly decreased in rotenone treated When L-dopa treatment was accompanied by treadmill rats. Similar to our study, Vijayalakshmi et  al. [26] and exercise, there was a significant increase in exploration Valdez et al. [27] reported a significant decrease in loco - and locomotion behavior in the open field. A significant motion in the Open field test in rotenone-treated Wis - increase in the latency time of the Rota-rod test was ter rats. von Wrangel et  al. [28], and Cannon et  al. [12] found indicating improved motor coordination. There reported that rotenone-treated rats showed significant was a significant gait improvement with L-dopa and exer - impairment in Rota-rod and significant decline in tyros - cise co-treatment, the asymmetrical pattern was fully ine hydroxylase in the striatum. As regard footprint test, M onir et al. Behav Brain Funct (2020) 16:9 Page 9 of 12 Monir et al. Behav Brain Funct (2020) 16:9 Page 10 of 12 corrected, with a significant increase in stride length and As regard expression of TFAM, this is the first study a significant decrease in stride width. Short-term mem - which investigated its expression in rotenone-treated ory significantly improved. Tyrosine hydroxylase levels in rats, Rotenone downregulated TFAM expression which the brain were significantly increased. may underlie the instability in the mtDNA and conse- In our study, we investigated some genes of the Nrf2- quent damage of the dopaminergic neuron. This is evi - ARE pathway as a possible mechanism involved in the denced by the decreased level of tyrosine hydroxylase treadmill exercise effects on the brain. Exercise causes measured in the striatum. Noteworthy, Chen et al. [39] in an increase in O2 consumption with an increase in ROS their study on post mortem human parkinsonian patients production, especially H2O2. Oxidative stress leads to found that expression of TFAM in substantia nigra is inhibition of KEAP-1, dissociation of Nrf2 from KEAP- lower and their mtDNA is less stable in comparison to 1 in the cytoplasm, migrates into the nucleus, and acti- age-matched elderly control subjects. vates the Nrf2-ARE [35]. Nrf2 activates anti-oxidant gene As regards our study results, treadmill exercise acti- expression with increased productions of detoxifying vated the Nrf2 pathway in the striatum of the rote- enzymes: Heme oxygenase, and NADPH quinone oxi- none-treated rat. This activation could be one of the doreductase (NQO.1). Activation of Nrf2 together with mechanisms involved in treadmill exercise beneficial PCG1α, which also is activated by exercise, caused an effects on the rotenone-treated model of PD. Few stud - increase in mitochondrial transcription factor A (TFAM) ies which measured TFAM expression in animal models expression in the striatum. TFAM is a nuclear factor e.g. Aguiar et  al. [40] reported that exercise significantly that controls the replication of mitochondrial DNA (mt increased Nrf2 and TFAM in the striatum of 6-OHDA DNA). The upregulation of TFAM increases the number hemiparkinsonism. Other studies reported TFAM acti- of copies and packaging of mtDNA. Also, adequate levels vation with exercise in different pathological conditions of TFAM are required to prevent mtDNA release into the other than parkinsonism. For instance, Lashgarie et  al. cytoplasm and initiate an inflammatory response [11]. [11] reported that treadmill exercise increased TFAM This study measured the rate of expression of Nrf2, expression in the heart of nicotine sensitized rats, which TFAM, and Nqo.1 in the striatum of rotenone-treated attenuated mitochondrial-mediated damage in the myo- rats. Rotenone downregulated expression of the TFAM cardium induced by nicotine. in the rotenone group while upregulated Nrf2 and Nqo1 in comparison to the control rats. Treadmill exercise showed a significant increase in Nrf2, TFAM, and NQO.1 Conclusion in the striatum in the exercise\rotenone group in com- In this study, treadmill exercise increased tyrosine parison to control and rotenone groups. L-dopa\rotenone hydroxylase and activated the Nrf2 pathway and some of group showed an insignificant change in the expression its associated genes however L-dopa could not. Where of these genes. However, the exercised \L-dopa\rotenone L-dopa corrected locomotion, exploration, and motor co- group, showed a significant increase in Nrf2, TFAM, and ordination but failed to improve short-term memory and Nqo.1 expression, when compared to both control and only partially corrected the gait of rotenone-treated rats. rotenone group. When treadmill exercise was combined with L-dopa, all Whether rotenone increases or decreases the expres- the behavioral motor and non-motor aspects of PD were sion of Nrf2 and NQO1 is a matter of debate. Many corrected. studies [36] found that rotenone in in-vivo and in-vitro studies downregulated the expression of these genes and enhanced apoptosis of the neurons in the nigros- Recommendation triatal tissue which was reversed by administration of Exercise is highly recommended and is a mandatory danshensu herbal extract. Similarly, Cui et al. [37] found approach for the treatment of parkinsonism in humans. decreased expression of NRF2 and NQO1 proteins meas- Further studies are needed to explore the importance of ured by western blotting in rotenone treated rats which expression of the TFAM gene as part of the Nrf2 pathway were corrected with pretreatment with curcumin. On the in protecting against mitochondrial induced oxidative contrary, Wei et  al. [38] found that rotenone increased damage. NRF2 and NQO1 expression in the striatum which was further enhanced by ellagic acid. They measured the Supplementary information NRF2 protein in the cytoplasm and in the nucleus of the Supplementary information accompanies this paper at https ://doi. org/10.1186/s1299 3-020-00171 -9. cell indicating that the expression of these transcription factors was increased in the nucleus. M onir et al. Behav Brain Funct (2020) 16:9 Page 11 of 12 5. Ferris CF, Marella M, Smerkers B, Barchet TM, Gershman B, Matsuno-Yagi A, Additional file1: Figure S1. The effects of exercise on serum levels of CPK et al. A phenotypic model recapitulating the neuropathology of Parkin- and LDH in control groups, * if the differences were significantly different son’s disease. Brain Behav. 2013;3(4):351–66. from the control group. 6. Bezard E, Yue Z, Kirik D, Spillantini MG. Animal models of Parkinson’s dis- ease: limits and relevance to neuroprotection studies. Movement Disord. 2013;28(1):61–70. Abbreviations 7. Lee NY, Lee DK, Song HS. Eec ff t of virtual reality dance exercise on the ARE: Antioxidant response elements; DA: Dopamine; NQO1: NAD(P) H quinone balance, activities of daily living, and depressive disorder status of Parkin- oxidoreductase 1: NAD(P)H dehydrogenase; Nrf2: Nuclear factor erythroid 2 son’s disease patients. J Phys Ther Sci. 2015;27(1):145–7. (NFE2)-related factor 2; PA: Physical activity; PGC1α: Peroxisome-proliferator- 8. Um HS, Kang EB, Leem YH, Cho IH, Yang CH, Chae KR, et al. Exercise activated receptor-γ coactivator-1α; ROS: Reactive oxygen species; TFAM: training acts as a therapeutic strategy for reduction of the pathogenic Mitochondrial transcriptional factor A; OL: Overlap; FSL: Forward stride length; phenotypes for Alzheimer’s disease in an NSE/APPsw-transgenic model. FSW: Forward stride width; HSL: Hind stride length; HSW: Hind stride width. Int J Mol Med. 2008;22(4):529–39. 9. Li T, He S, Liu S, Kong Z, Wang J, Zhang Y. Eec ff ts of different exercise dura- Acknowledgements tions on Keap1-Nrf2-ARE pathway activation in mouse skeletal muscle. The authors would like to thank the staff of the central laboratory in Sohag Free Radical Res. 2015;49(10):1269–74. Medical Faculty for their cooperation all over the period of practical work. 10. Hinson VK, Goetz CG, Leurgans S, Fan W, Nguyen T, Hsu A. Reducing dosing frequency of carbidopa/levodopa: double-blind crossover study Authors’ contributions comparing twice-daily bilayer formulation of carbidopa/levodopa AA selected the point of research, MM and DM equally contributed to the (IPX054) versus 4 daily doses of standard carbidopa/levodopa in stable practical part of the study, IR collected the data and shared in the analysis of Parkinson disease patients. Clin Neuropharmacol. 2009;32(4):189–92. the behavioral tests, all authors shared in the writing of the research with final 11. Lashgari AA, Azarbayjani M-A, Peeri M, Nasehi M. The Eec ff t of Short- revision from OG and AA. All authors read and approved the final manuscript. Term Treadmill Exercise on the Expression Level of TFAM in the Heart of Nicotine-Sensitized Rats. Archives of Advances in Biosciences. 2020;11(1). Funding 12. Cannon JR, Tapias V, Na HM, Honick AS, Drolet RE, Greenamyre JT. A The authors declare that there is no special funding taken for this research. highly reproducible rotenone model of Parkinson’s disease. Neurobiol Dis. 2009;34(2):279–90. Availability of data and materials 13. Shin JH, Kang KW, Kim JH, Chin JY, Kim NY, Park SH, et al. Treadmill The datasets used and/or analyzed during the current study are available from exercise-induced E/e’ elevation as a predictor of cardiovascular event in the corresponding author on request. end-stage renal disease on peritoneal dialysis. Korean J Intern Med. 2016. 14. Walsh RN, Cummins RA. The Open-Field Test: a critical review. 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Front Behav Neurosci. 2014;8:331. lished maps and institutional affiliations. 35. Vargas-Mendoza N, Morales-González Á, Madrigal-Santillán EO, Madrigal- Bujaidar E, Álvarez-González I, García-Melo LF, et al. Antioxidant and adap- tative response mediated by Nrf2 during physical exercise. Antioxidants. 2019;8(6):196. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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