Neuroprotective Effects of Pramipexole in Parkinson's Disease

Med. J. Cairo Univ., Vol. 84, No. 2, March: 193-202, 2016 www.medicaljournalofcairouniversity.net

Induced by Rotenone in Mice

NAJLA'A K.M. AL-SHAIBANI, M.Sc.*; ABD EL-AZIM ASSI, M.D.*; RASHA B. ABD EL-LATIEF, M.D.* and

DALIA A.H. ELSERS, M.D.**

The Departments of Pharmacology* and Pathology**, Faculty of Medicine, Assiut University, Egypt

Abstract Introduction

Background: Pramipexole (PPX), an agonist for Dopamine PARKINSON'S Disease (PD) is one of the leading (DA), has been used to treat both early and advanced Parkin- causes of neurologic disability in elderly [1] . Its son's Disease (PD). pathological hallmark is the specific and progres- Objectives: This study was done to evaluate the neuro- sive degeneration of dopaminergic neurons in the protective effect of PPX in a DA neuron degeneration model Substantia Nigra Pars compacta (SNpc) [2] which of PD induced by rotenone. results in extrapyramidal motor dysfunction ac- Methods: Thirty six male mice were used and divided companied by progressive impairment of autonomy, into three equal groups. The first group, the control group, mood, and cognitive functions [3-5] . Although the received only the vehicle (sunflower oil) for a period of 7 weeks (49 days). The second group received rotenone (2mg/kg; mechanisms of neurodegeneration in PD are not

IP) dissolved in sunflower oil daily for 49 days. The third fully understood, oxidative stress, excessive free- group received a combined treatment of rotenone (2mg/kg, radical formation, environmental toxins, and/or IP) and PPX (1mg/kg, IP) daily for 49 days. Behavioral tests endogenous neurotoxins may be involved in the were performed a day prior to drug administration and then once weekly along the duration of drugs or vehicle adminis- progressive loss of nigrostriatal Dopamine (DA) tration. At the end of the 49 days all animals were sacrificed neurons [6,7] . and their midbrains were subjected to immunohistochemical analysis for dopaminergic neurons staining for Anti-Tyrosine Rotenone is widely used as an insecticide and

Hydroxylase (TH) antibodies. Midbrain tissues were also fish poison. Several studies reported that chronic isolated for biochemical measurements. exposure to rotenone causes highly selective ni-

Results: Behavioral tests revealed that PPX counteracted grostriatal dopaminergic degeneration that is asso- the rotenone effect in mice locomotor activity and catalepsy. ciated with neurochemical, behavioral and neuro- Immunohistochemistry results showed that PPX inhibited the pathological features of PD [8,9] . Previous studies rotenone-induced loss of TH-immunopositive neurons in the found that rotenone causes a partial inhibition of substantia nigra pars compacta. Biochemical measurements demonstrated that PPX treatment significantly reversed the mitochondrial complex i which in turn leads to

rotenone-induced decrease in midbrain DA level. Interestingly, increased oxidative stress [8,10,11] . Rotenone may PPX ameliorate the rotenone-induced increase in the malon- induce superoxide production in cultured microglia dialdehyde and nitric oxide levels as well as the decrease in [12].The increased production of ROS may result total antioxidant capacity. in oxidative damage to the proteins and DNA of Conclusion: The results indicate the beneficial effect of neural cells and cause the cells to undergo dysfunc- PPX against the dopaminergic neurodegeneration induced by tion and eventually lead to necrosis or apoptosis chronic IP administration of rotenone. This neuroprotective effect seems to be mediated by inhibition of rotenone induced [8] . Moreover, chronic rotenone administration can oxidative stress and nitric oxide overproduction and through lead to significant injury to the nigrostriatal system, maintenance of the cellular antioxidant status. mediated by increased generation of nitric oxide

[13]. ROS, especially superoxide and hydroxyl Key Words: Parkinson's disease – Rotenone – Pramipexole

– Neuroprotection – Oxidative stress. radicals, can interact with nitric oxide to form peroxynitrite [12,14] . The potent oxidant peroxyni-

trite may directly oxidize DA and lead to the Correspondence to: Dr. Najla'a K.M. Al-Shaibani,

[15,16] The Department of Pharmacology, Faculty of Medicine, dopaminergic neurons damage . Nitric oxide

Assiut University, Egypt fosters dopamine depletion and is associated with

193 194 Neuroprotective Effects of PPX in PD Induced by Rotenone in Mice

neurotoxic processes underlying PD [17] . Therefore, Material and Methods

strategies that protect cells at the level of the I- Drugs and chemicals: mitochondria appear to have protective potential

in PD [18] . Rotenone and pramipexole were obtained from

Sigma-Aldrich (China). Rotenone was dissolved

Pramipexole (PPX) is a synthetic amino- in sunflower oil. Pramipexole was dissolved in benzothiazole derivative. It has been shown to be saline to obtain the necessary doses. Mouse dopa-

a selective and specific full DA receptor agonist mine ELISA kit was obtained from (Chongqing

with high affinity and selectivity for the DA D2 Biospes Co., Ltd., China). Total antioxidant capac-

receptor subfamily, and particularly the D3 receptor ity kit was obtained from (Biodiagnostic, Egypt).

subtype. Pramipexole is a non-ergot dopamine Tyrosine hydroxylase antibody kit was obtained

agonist with actions similar to those of bromocrip- from (Novus USA). Power-StainTM 1.0 poly HRP

tine. PPX is used successfully in the treatment of DAB kit was obtained from (Genemed Biotechnol-

Parkinson's disease [19] . Previous studies reported ogies, USA). 2-Thiobarbituric acid was obtained that PPX protects against MPTP, 6-hydroxy- from (MP Biomedicals Inc., France). Malondial-

dopamine, levodopa, glutamate and metamphet- dehyde bis-(dimethyllacetal) was obtained from

amine-induced neurodegeneration [20-25] . (Merk, Germany).

Various studies were performed to clarify the II- Animals:

possible neuroprotective mechanism of PPX. Many Thirty six (36) male mice of average weight

authors reported that PPX neuroprotective effect 25-35g were purchased from Assiut University

is directed to specific sites on dopamine neurons Animal House from March 2014 to January 2015.

and receptors, which is an action that it does not Mice were housed in groups in clean capacious

share with bromocriptine and pergolide suggested macrolene cages (up to 4 per cage) under standard

that PPX, like other dopamine agonists, reduces laboratory conditions including aerated room with ± dopamine synthesis turnover in mice during repeat- suitable temperature (25 5ºC) maintained at good

ed injections of amphetamine and, thus, reduces light. All experiments were carried out in accor-

the free radical formation during this process [26- dance with the National Institutes of Health Guide

29] . This is considered to contribute to the neuro- for the Care and Use of Laboratory Animals. The protective effect of PPX. Other investigators re- protocol of the research was approved by the Ethical ported that PPX attenuates intracellular processes Committee for Research at Assiut University. Mice

such as the mitochondrial transition pore opening were divided into 3 groups. Each group contains

that is associated with programmed cell death 12 animals as follows: [30,31] . Du and colleagues indicated that PPX stim- ulates a mesencephalic-derived neurotrophic activ- Group 1: Control:

ity [32] . Pan and colleagues reported that PPX has Mice received the vehicle (sunflower oil 4ml/kg, biological regulatory effects on dopaminergic neu- IP) daily for a period of 7 weeks (49 days).

ron-associated genes, which may explain both the Group 2: Rotenone treated: slower decline of imaged dopamine transporter

Mice received rotenone (2mg/kg, IP) dissolved and the neuroprotective effect [33] . Further, Izumi and colleagues concluded that PPX has a neuro- in sunflower oil (1mg/2ml) daily for a period of 7

weeks (49 days). protective effect and protects dopaminergic neurons

from glutamate neurotoxicity by the reduction of Group 3: Rotenone plus PPX: intracellular dopamine content, independently of Mice received rotenone (2 mg/kg, IP) and PPX dopamine D2-like receptor activation [24] . In vitro (1 mg/kg, IP) daily for a period of 7 weeks (49 and in vivo studies of PPX in Parkinson disease days). models show that it decreases the phosphorylation

of alpha-synuclein, which may contribute to its III- Behavioral assesment: neuroprotective properties [34] . Behavioral tests were performed a day prior to

In view of the putative neuroprotective effects drug administration and then once weekly along

of PPX reported elsewhere, the present study was the duration of the experiments (7 weeks). All mice groups were subjected to the following behavioral designed to evaluate the possible neuroprotective

tests: effects of pramipexole in rotenone-induced PD in

mice and to clarify the possible mechanism under- - Locomotor activity. The locomotor activity

lying the evaluated protective effects. of the tested animals was quantitatively estimated Najla'a K.M. Al-Shaibani, et al. 195

by using an activity cage (Model 7401, Ugo basile, Malondialdehyde (MDA), an end product of

Comerio, Italy). The activity was monitored con- lipid peroxidation, is a major indicator of oxidative

tinuously for a period of 5 minutes. Locomotion stress and its level was determined in tissue homo-

was expressed in terms of total photo beam crossing genates spectrophotometrically by use of thiobar-

counts per 5 minutes per animal [35] . bituric acid reactive substances method previously

described by Ohkawa et al., [39] . - Exploratory behavior (rearing). When placed

in a clear cylinder, animals will engage in explor- - Determination of the midbrain nitrite level:

atory behavior, including rearing. During rearing Nitric oxide was measured as its stable oxidative behavior, the forelimbs will contact the wall of the metabolite nitrite. Serum nitrite concentration was cylinder. For this test, the animals was placed in assayed by using Griess reagents as described by a clear plexiglass cylinder (height = 30cm, diameter Green et al., [40] . = 20cm) for 5 minutes. To be classified as a rear, the animal had to raise forelimbs above shoulder - Determination of midbrain total antioxidant ca- level and make contact with the cylinder wall with pacity: either one or both forelimbs. Removal of both The determination of the anti-oxidative capacity forelimbs from the cylinder wall and contact with is performed by the reaction of antioxidants in the the table surface was required before another rear sample with a defined amount of exogenously was scored [36] . provide hydrogen peroxide (H 2O2). The antioxi-

- Catalepsy. Each animal was hung by the paws dants in the sample eliminate a certain amount of

on a vertical grid. The vertical grid apparatus is the provided hydrogen peroxide. The residual H 2O2

an open box of 8cm X 55cm X 5cm, set vertically. is determined colorimetrically by an enzymatic

The back side of the vertically standing box is reaction which involves the conversion of 3, 5,

made of a wire mesh of 0.8cm X 0.8cm, the front dichloro-2-hydroxy benzensulphonate to a colored

side is open and the other sides are made of black product [41] . plexiglass. For stability, the bottom of the apparatus

has a 5cm extension to the front. In the experiment, V- Immunohistochemistry:

each mouse was carefully placed inside the appa- Sections from mid brain were trimmed after

ratus at 3cm from the top, facing upward, and was fixation in 10% formalin for 8 hours, dehydrated,

left free to turn around and climb down. The trials and paraffin embedded. Cutting of blocks was

were videotaped. The videos were replayed for nearly at the same level. Four gm sections were

recording the total time taken for the mouse to mounted on positively-charged Super frost slides make a turn, climb down and reach the floor by (Thermo Scientific, Menzel, Braunschweig, Ger-

its forepaw [37] . many). The sections were deparaffinized in xylene

for 30 minutes, rehydrated in descending graded

IV- Biochemical estimations: alcohol up to distilled water. Antigen retrieval was

done by 10mm sodium citrate buffer solution, pH After performance of behavioral tests, animals

6.0 for 12 minutes. The sections were incubated were sacrificed by decapitation. Midbrain tissues

were obtained from each mouse for biochemical with monoclonal mouse antihuman antibody against measurements. The midbrain was rinsed in ice- tyrosine hydroxylase protein at 1:50 dilution (Code

cold saline, blotted carefully and weighed. Then no. NB100-2051, Novus USA). The resulting im-

the midbrain was placed in a glass homogenizer mune complex was detected by use of HRP DAB

containing 2ml saline. After homogenization, by kit (Cat. no. 52-0017, Genemed Biotechnologies,

using motor-driven Teflon pestle (Glas-Col, USA), USA) at room temperature and following the in-

the homogenate was centrifuged for 10 minutes at structions attached with the kit. Negative and 1000rpm and the supernatant was used for estima- positive controls were included in each staining

tion of dopamine and Malondialdehyde (MDA) series. Mouse substantia nigra was used as positive

levels as well as total antioxidant capacity. controls.

Evaluation of Tyrosine Hydroxylase (TH)- - Determination of midbrain dopamine level: positive neurons: The positivity for TH was detected Dopamine was estimated by use of Enzyme- as brownish staining of the cytoplasm of neurons linked Immune-Sorbent Assay (ELISA) technology [42] . The total numbers of TH-stained neurons were as described by Cavalcanti et al., [38] . counted independently by two observers (Dr. Dalia

- Determination of Midbrain Malondialdehyde Elsers and Dr. Najla) blinded to the group under

(MDA) Level: a light microscope (40X magnification). The mean 196 Neuroprotective Effects of PPX in PD Induced by Rotenone in Mice

of number of neurons in 5 fields was presented This reduction started on the day 21 and reached

[43] . its maximum on the day 49 (Table 3).

2- Midbrain dopamine level: VI- Statistical analysis: The data were collected, tabulated and statisti- Chronic IP rotenone administration was associated with significant (p<0.01) decrease in mid- cally analyzed, using a personal computer with brain dopamine level in comparison to vehicle- "Statistical Package for the Social Sciences" treated group. Simultaneous administration of PPX (SPSS), version 19, (SPSS Inc., Chicago Illinois and rotenone was associated with a highly signif- USA) for windows and GraphPad Prism version icant elevation of midbrain dopamine level as 5.00 for Windows, (Graph Pad Software, San compared with rotenone only group with p<0.01 Diego California USA). The quantitative data were ± (Table 4). presented in the form of mean standard error of

mean (S.E.). The significance of differences was 3- Immunohistochemistry: determined by an analysis of variance (ANOVA). Tyro sine-Hydroxyl ase (TH) Immunohis- Further statistical analysis for post hoc comparisons was performed using the Least Significant Differ- tochemical examinations demonstrated a signifi- ence (LSD) test. The difference considered statis- cant (p<0.01) decrease in number of TH–immu-

tically significant at p<0.05 and highly significant nopo-sitive neurons in rotenone treated group in

at p<0.01. comparison to vehicle treated control group. How-

ever, concurrent administration of pramipexole

with rotenone was associated with a significant Results (p<0.01) increase of TH-immunopositive neurons

number as compared to rotenone treated group 1- Behavioral assesment: Fig. (1A-D). • Locomotor activity:

The locomotor activity of mice received roten- 4- Oxidative stress parameters: one was significantly lower than that of vehicle- • Midbrain Malondialdehyde Level (MDA): treated control group with p<0.01. This reduction Induction of PD in mice was associated with a started on day 14 of our experiment and continued significant increase in midbrain MDA level when till the day 49 when the least activity was recorded. compared with vehicle-treated control group with However, mice received PPX plus rotenone showed p>0.01. However, concomitant administration of a significant increase in spontaneous locomotor PPX and rotenone was associated with significant activity in comparison to rotenone-only group with decrease in midbrain MDA level as compared to p<0.01. This increase was observed on the day 28 rotenone only treated group with p>0.01 Fig. (2A). and continued till the day 49 (Table 1).

• Midbrain nitrite level:

• Exploratory behavior (rearing): Chronic IP administration was associated with

Rotenone treated group showed a significant highly significant increase in midbrain nitrite (the reduction in exploratory behavior in comparison major metabolite of nitric oxide) level when com- to vehicle-treated control group with p<0.01. This pared with control group with p>0.01. In contrast, reduction started at the day 28 and continued till administration of PPX in concomitant with rotenone the day 49 when least activity was recorded. On was associated with significant decrease in midbrain the day 28, treated group with PPX plus rotenone nitrite level as compared to rotenone only treated showed a significant increase in exploratory be- group with p>0.05 Fig. (2B). havior in comparison to rotenone only-treated

group with p<0.01 (Table 2). • Midbrain total antioxidant capacity:

Induction of Parkinsonian-like disorder by use

• Catalepsy: of rotenone was associated with highly significant

On performance of the grid test, rotenone treated decrease in midbrain total antioxidant capacity

group showed a significant increase in descending when compared with vehicle-treated control group

time latency as compared to vehicle-treated control with p>0.01. However, concomitant administration

group with p<0.01, this increase was noticed on of PPX with rotenone was associated with a sig-

the day 14. However, PPX treated group showed nificant increase in midbrain total antioxidants

a significant decrease in descending latency time capacity as compared to rotenone only treated

as compared to rotenone-treated group with p<0.01. group with p>0.05 Fig. (2C). Najla'a K.M. Al-Shaibani, et al. 197

(A, X40) (B, X40)

(C, X40) (D, X100)

Fig. (1): Expression of Tyrosine Hydroxylase (TH) in studied groups. (A) Shows TH-positive neurons in normal mice (control

group). (B) Demonstrates decreased number of TH-positive neurons in the rotenone group. (C,D) Show increased

number of TH-positive neurons in comparison with the rotenone group and nearly similar to control group.

5000 600 150 ** ** ## 4000 # #

400 100 ** 3000

2000 200 50 Mid brain MDA Mid brain nitrite 1000 level (1.tmol/g w.wt) level (i.tmol/g w.wt) capacity (mmol/g w.wt) Mid brain total Antioxidant 0 0 0

(A) (B) (C)

Control Rotenone PPX + Rotenone

Fig. (2): Effects of rotenone and Pramipexole (PPX) on: Mid brain Malondialdehyde (MDA) level (A), mid brain nitrite level

(B), and mid brain total antioxidant capacity (C). Animals were treated with vehicle (control), rotenone (2mg/kg), or

rotenone (2mg/kg) and PPX (1mg/kg). Data represent mean with S.E. of 6 mice analyzed using one way ANOVA

followed by LSD test.

**: p>0.01 highly significant difference as compared to vehicle control group.

# : p>0.05 significant difference as compared to rotenone group.

##: p>0.01 highly significant difference as compared to rotenone group.

198 Neuroprotective Effects of PPX in PD Induced by Rotenone in Mice

Table (1): Effect of pramipexole on rotenone-induced alteration in locomotor activity.

Locomotor activity (frequency)

Day of treatment Treatment

1 7 14 21 28 35 42 49 ± ± • Control 269.6±20.9 204.0± 17.1 253.4 21.2 211.7±20.3 243.0± 10.8 226.5 14.2 220.0±7.4 171.7± 10.6 ± * * * * * ± * • Rotenone 218.8±20.6 185.5 12.2 176.6± 16.8 a 104.3± 10.5 c 74.3±5.8 c 96.5±6.8 c 64.8±3.8 c 69.8 4.6 c

• Pramipexole 215.6±9.5 160.8± 16.1 a 169.0± 17.2a 127.1 ±9.9a 135.6±9.3#a 154.3± 10.0#a 143.3±7.3#a 132.7±9.4#a

+ Rotenone

Animals were treated with vehicle (control), rotenone (2mg/kg), or rotenone (2mg/kg) and PPX (1mg/kg). ± Data represent mean S.E. of (6-8) mice.

Analyzed using two way ANOVA followed by LSD test.

*p<0.05 significant as compared to control of respective week. #p<0.05 significant as compared to rotenone treated group of respective week. st th 14th st 28th th h (a, b, c, d, e, f, g) p<0.05 significant as compared to 1 7 21 35 42t respectively day observations of same treatment group.

Table (2): Effect of pramipexole on rotenone-induced alteration in exploratory behavior.

Rearing (frequency)

Day of treatment Treatment

1 7 14 21 28 35 42 49 ± ± ± ± ± • Control 28.5 2.9 27.6 1.7 25.4 1.5 19.9±0.6 20.4±0.8 20.7 1.0 20.6 1.1 21.3±0.9 * * * * • Rotenone 26.1 ± 1.6 29.8± 1.9 23.8± 1.7b 17.8± 1.3 c 8.1±0.7 d 7.5±0.6 d 7.4±0.7 d 7.0±0.4 d

• Pramipexole + Rotenone 31.1 ±2.8 24.5± 1.7a 21.9±0.9a 19.7± 1.5a 23.0± 1.8#a 21.2±2.0#a 25.4± 1.2#a 23.2± 1.0#a

Animals were treated with vehicle (control), rotenone (2mg/kg), or rotenone (2mg/kg) and PPX (1mg/kg). ± Data represent mean S.E. of (6-8) mice.

Analyzed using two way ANOVA followed by LSD test.

Table (3): Effect of pramipexole on rotenone-induced alteration in descending latency off a vertical grid (catalepsy).

Descending latency (sec)

Day of treatment Treatment

1 7 14 21 28 35 42 49

• Control 9.4±0.8 8.6±0.7 8.3±0.7 8.1±0.5 7.2±0.4 9.0±0.9 8.5±0.7 7.8±0.7 * * ± ± * • Rotenone 9.5±0.8 9.9±0.4 10.6±0.9* 14.0±0.8 c 12.0±0.7* 19.4±0.7 e 36.4 3.1 *f 36.8 1.7 f

• Pramipexole + Rotenone 9.1±0.9 8.6±0.7 10.0±0.7 9.8±0.7# 11.7±.06#b 10.3±0.6# 9.2±0.8#e 9.5±0.8# ± ± • Rasagiline + Rotenone 10.0 1.0 11.3±0.5 10.3±0.5 10.0±0.4# 10.0±0.8# 11.0±0.9# 10.1 1.0# 9.7±0.8#

Animals were treated with vehicle (control), rotenone (2mg/kg), rotenone (2mg/kg) and PPX (1mg/kg). ± Data represent mean S.E. of (6-8) mice.

Analyzed using two way ANOVA followed by LSD test.

*p<0.05 significant as compared to control of respective week. #p<0.05 significant as compared to rotenone treated group of respective week. st th 14th st 28th 35th 42th (a, b, c, d, e, f, g) p<0.05 significant as compared to 1 7 21 respectively day observations of same treatment group.

Table (4): Effect of pramipexole on rotenone-induced alteration Discussion in midbrain dopamine level.

Midbrain dopamine level Parkinson's Disease (PD) a neurodegenerative Group µ ( g/g w.wt) disorder for which no preventive or long-term effective treatment strategies are available. Epide- Control 1.67±0.16

Rotenone 0.69±0.06** miologic studies have failed to identify specific

Pramipexole + Rotenone 1.77±0.09## environmental, dietary or lifestyle risk factors for ± Data represent mean S.E. of (6-8) mice. PD [44] . However, oxidative stress in the SN is the Analyzed using one way ANOVA followed by LSD test. most broadly accepted hypothesis for the etiopa- **: Highly significant (p>0.01) difference from vehicle control group. ##: Highly significant (p>0.01) difference from rotenone group. thology of PD [45] . Najla'a K.M. Al-Shaibani, et al. 199

Rotenone is well known complex i inhibitor Concerning oxidative stress, the present study

that reproduces features of Parkinson's disease [36] . revealed that chronic rotenone administration

In the present study we induced effective progres- caused elevation in MDA and nitrite levels as well

sive PD manifestations that can be seen from the as decline in total antioxidant capacity in midbrain behavioral, biochemical and immunohistochemical region. The significant elevation in the level of examination results. Behavioral assessment re- MDA (a marker of lipid peroxidation, which indi-

vealed that chronic intraperitoneal administration cates increased free radical production with con-

of rotenone causes loss of spontaneous locomotor sequent attack on membrane lipids) was in accor-

dance with several in vitro and in vivo rotenone activities, decline in exploratory behavior and catalepsy. These results coincide with the observa- models consistently demonstrate an increase in lipid peroxidation [48,51,56,57] . The level of nitrite tion of other investigators [9,35,37,46] . Biochemical examination showed that chronic rotenone admin- which is the major nitric oxide metabolite was also increased; this increase is most likely due to ele- istration caused a significant decline in the level vated nitric oxide production due to induction of of neurotransmitter dopamine in midbrain region. the inducible form of Nitric Oxide Synthase (iNOS) This study result was further supported by earlier [58] . The finding of elevated nitrite level was con- and more recent study reports showing decline in sistent with findings of Bashkatova et al., [59] and the neurotransmitter DA level following rotenone Abdel-Salam et al., [51] . Moreover, the increase in exposure [35,47,48] . Immunnohistochemical exam- oxidative stress induced by rotenone was also ination showed that systemic administration of demonstrated by decreased total antioxidant capac- rotenone resulted in nigrostriatal cell damage and ity, suggesting consumption of these scavenger decreased tyrosine hydroxylase immunoreactivity molecules by free radical excess. This decline in in the SN, this result was consistent with results total antioxidant capacity was in line with the of previous studies [25,49-51] . However, other re- observations recorded by Kaur et al., [57] and searchers have found that the effect of systemic Abdel-Salam et al., [51] . administration of rotenone in rodents is widespread

[52] . The damage inflicted by rotenone on the brain The present study revealed that simultaneous

is thus may not limited to the nigrostriatal pathway. daily administration of PPX with rotenone inhibited

Nevertheless, our results demonstrate that chronic the remarkable increase in the mid brain Malond-

intraperitoneal administration of rotenone causes ialdehyde and nitrite levels as well as the decrease

nigrostriatal dopaminergic degeneration that is in mid brain total antioxidant capacity. These results

associated with biochemical and behavioral features suggesting decreased oxidative and nitrosative

of PD. stress on administration of PPX. These findings

were further supported by previous and recent

Pramipexole (PPX), a non-ergot dopamine re- findings showing that PPX possess antioxidant

properties [20,23-25,60-64] . Furthermore, our results ceptor agonist, has been used in the management

were related with that of Uberti et al., [65] who of Parkinson's disease, alone or as an adjunct to demonstrated that PPX inhibited generation of levodopa therapy in more advanced stages of the H2O2-induced reactive oxygen species in SH- disease [53] . In the present study PPX administration produces a significant beneficial effect on locomo- SY5Y neuroblastoma cell in a DA receptor independent way. Similar findings were obtained by tor impairment induced by rotenone as it prevented Ferrari-Toninelli et al., [66] reported that both S the loss of spontaneous locomotor activities, the (–) and R (+) pramipexole enantiomers are mito- decline in exploratory behavior and the increase chondria-targeted antioxidants and suggested that in cataleptic behavior; this finding is consistent the antioxidant, neuroprotective activity of these with the findings of Gad El-Hak et al., (2012) [37] . drugs is independent of both the chiral 6-propyl Moreover, PPX counteracted the decrease in the amino group in the pramipexole molecule and the level of dopamine neurotransmitter in midbrain DA receptor stimulation. region; this result is consistent with that of Ander-

[54] son et al., (2001) . Furthermore, PPX prevented In conclusion, these results suggest that PPX

the loss of dopaminergic neurons in the SNpc of has neuroprotective effect against the dopaminergic rotenone-treated mice; this result is agreed with neurodegeneration induced by chronic IP adminis-

that of Inden et al., (2009) [25] and Gad El-Hak et tration of rotenone. This neuroprotective effect

al., (2012) [37] . These findings are related with that may be mediated by inhibition of rotenone induced

of Kim et al., (2015) [55] who suggested that PPX oxidative stress and nitric oxide overproduction

may protect DA neurons from the damaging effect and through maintenance of the cellular antioxidant

of 6-hydroxydopamine (6-OHDA). status. 200 Neuroprotective Effects of PPX in PD Induced by Rotenone in Mice

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