Imipramine and amitriptyline ameliorate the rotenone model of Parkinson’s disease in rats

Accepted Manuscript Imipramine and Amitriptyline Ameliorate the Rotenone Model of Parkinson’s Disease in Rats Esraa A. Kandil, Noha F. Abdelkader, Bahia M. El-Sayeh, Samira Saleh PII: DOI: Reference:

S0306-4522(16)30279-2 http://dx.doi.org/10.1016/j.neuroscience.2016.06.040 NSC 17187

To appear in:

Neuroscience

Accepted Date:

23 June 2016

Please cite this article as: E.A. Kandil, N.F. Abdelkader, B.M. El-Sayeh, S. Saleh, Imipramine and Amitriptyline Ameliorate the Rotenone Model of Parkinson’s Disease in Rats, Neuroscience (2016), doi: http://dx.doi.org/ 10.1016/j.neuroscience.2016.06.040

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Imipramine and Amitriptyline Ameliorate the Rotenone Model of Parkinson's Disease in Rats Esraa A. Kandil*, Noha F. Abdelkader, Bahia M. El-Sayeh, Samira Saleh Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

Corresponding author: Esraa A. Kandil Postal address: Faculty of Pharmacy, Kasr El Aini St, 11562 Cairo, Egypt Fax number: +202-23628426 Mobile number: +201007072720 E-mail: [email protected]

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List of abbreviations 5-HT

Serotonin

AMI

Amitriptyline

BDNF

Brain derived neurotrophic factor

CAT

Catalase

CREB

cAMP-response element-binding protein

DMSO

Dimethyl sulfoxide

DA

Dopamine

IMI

Imipramine

iNOS

Inducible nitric oxide synthase

MDA

Malondialdehyde

NA

Noradrenaline

PD

Parkinson's disease

RT-PCR Reverse transcription polymerase chain reaction SN

Substantia nigra

SOD

Superoxide dismutase

TCA

Tricyclic antidepressant

TNF-α

Tumor necrosis factor alpha

TH

Tyrosine hydroxylase

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Abstract Amitriptyline, a commonly prescribed tricyclic antidepressant to parkinsonian patients, specifically showed a significant delay in dopaminergic therapy initiation and improvement in motor disability in parkinsonian patients. Moreover, it was recently shown that amitriptyline has neuroprotective properties; however, the mechanisms underlying this effect in Parkinson's disease are not fully understood. The current study aimed to investigate the possible neuroprotective mechanisms afforded by amitriptyline in the rotenone model of Parkinson's disease and to assess whether another tricyclic antidepressant member, imipramine, shows a corresponding effect. Rats were allocated into seven groups. Four groups were given either saline, dimethyl sulfoxide, amitriptyline or imipramine. Three rotenone groups were either untreated or treated with amitriptyline or imipramine. Rats receiving rotenone exhibited motor impairment in open field and rotarod tests. Additionally, immunohistochemical examination revealed dopaminergic neuronal damage in substantia nigra. Besides, striatal monoamines and brain derived neurotrophic factor levels were declined. Furthermore, oxidative stress, microglial activation and inflammation were evident in the striata. Pretreatment of rotenone groups with amitriptyline or imipramine prevented rotenone-induced neuronal degeneration and increased striatal dopamine level with motor recovery. These effects were accompanied by restoring striatal monoamines and brain derived neurotrophic factor levels, as well as reducing oxidative damage, microglial activation and expression of proinflammatory cytokines and inducible nitric oxide synthase. The present results suggest that modulation of noradrenaline and serotonin levels, up-regulation of neurotrophin, inhibition of glial activation, anti-oxidant and antiinflammatory activities could serve as important mechanisms underlying

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the neuroprotective effects of the used drugs in the rotenone model of Parkinson's disease. Key words: Amitriptyline; imipramine; neuroprotection; Parkinson's disease; rotenone.

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Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized primarily by motor abnormalities; however, many patients also suffer from non-motor symptoms that deleteriously influence their quality of life (Cummings, 1992). Depression is the most debilitating and sustained non-motor symptom affecting almost half of PD patients (Ravina et al., 2007). Depressive symptoms can appear in all PD stages, but often precede the onset of motor symptoms by several years (Weintraub and Stern, 2005). It is usually accompanied by greater motor disability, reduced quality of life, and increased mortality (Akerud et al., 2001); thus, many of PD patients take antidepressants on a daily basis (Ravina et al., 2007). Interestingly, the tricyclic antidepressants (TCAs) have been shown to be superior in treating PD-related (Menza et al., 2009). Besides, they could improve PD motor symptoms via their anticholinergic action (Rehavi et al., 1977) as well as alleviating sleep problems commonly encountered in PD by their sedative effect (Versiani et al., 1999). In this context, antidepressants from various classes have the ability to modulate vital signaling pathways implicated in cell survival and plasticity (Drzyzga et al., 2009), and to elicit neuroprotection in multiple animal models of neurodegenerative diseases (Duan et al., 2008; Chung et al., 2010; Chadwick et al., 2011; Chung et al., 2011). Remarkably, TCAs show distinctive neuroprotective properties. It is worth mentioning that both imipramine (IMI) and amitriptyline (AMI) up-regulate hippocampal brain derived neurotrophic factor (BDNF) expression (Peng et al., 2008; Jang et al., 2009) which is essential for neurogenesis, maturation, differentiation and survival of neuronal cells (Lee et al., 2002). Recently it was reported that AMI, a commonly prescribed TCA to PD patients (Chen et al., 2007), specifically postponed ϱ 

the need to initiate dopaminergic therapy in a cohort of early PD patients, proposing its disease-modifying properties (Paumier et al., 2012). Thereafter, an experimental study using 6-hydroxydopamine rat model of PD verified that AMI attenuates motor deficits and preserves dopaminergic neurons in substantia nigra (SN) (Paumier et al., 2015). Increased trophic support elicited by AMI within the nigrostriatal system is a likely contributor to the dopaminergic neuron protection; however, the pattern and magnitude of such action suggest that additional mechanisms are involved (Paumier et al., 2015). Thus, the current study was directed to explore further mechanisms underlying the neuroprotective effect of AMI in the rotenone model of PD, and to assess whether another TCA member, IMI, possesses comparable neuroprotective effect to AMI. Experimental procedures Animals One hundred sixty eight adult male Wistar rats (200-270 g) were obtained from Modern Veterinary Office for Laboratory Animals, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt. They were acclimatized for one week in the animal house of Faculty of Pharmacy, Cairo University before any experimental procedures and were allowed standard rat chow and water ad libitum. Rats were housed under constant temperature (25 ± 2°C), humidity (60 ± 10%), and a 12/12-h light/dark cycle. The investigation complies with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and is approved by the Ethics Committee of Faculty of Pharmacy, Cairo University (Permit Number: PT 699).

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Chemicals and drugs Amitriptyline and imipramine were provided by Al-Kahira Pharmaceuticals (Cairo, Egypt) and Alexandria Pharmaceuticals (Alexandria, Egypt), respectively. Rotenone and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), respectively. All Other chemicals were of the highest purity and analytical grade. Experimental design In the present study, AMI and IMI were daily administered for 35 consecutive days beginning 2 weeks prior to rotenone administration to account for the delay in the therapeutic efficacy of antidepressants (Malberg and Blendy, 2005; Martinez-Turrillas et al., 2005; Paumier et al., 2015). On the 15 th day from the start of the experiment, rats in appropriate groups received rotenone or its solvent DMSO every other day for 3 weeks for a total of 11 injections (Fig 1). Rats were divided into 7 groups consisting of 24 rats each. Group I received saline (1 ml/kg, i.p.) for 35 days and served as control. Group II received 11 subcutaneous injections of 10% DMSO (0.2 ml/kg) every other day starting from the 15th day. Animals of groups III & IV received daily AMI (5 mg/kg, i.p.) (Paumier et al., 2015) and IMI (10 mg/kg, i.p.) (Daniel et al., 1981) for 35 consecutive days dissolved in physiological saline, respectively. Groups V - VII received 11 subcutaneous injections of rotenone (1.5 mg/kg) (Abdelkader et al., 2014) every other day starting from the 15th day dissolved in DMSO. Moreover, groups VI & VII were daily treated 1 h before rotenone injection with AMI and IMI as previously described, respectively. All rats were screened for motor impairment using the open field and rotarod tests, 24 h after the last rotenone injection. At the end of

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the observation period, animals in each group were further divided into 3 sets, then were sacrificed by cervical dislocation under anesthesia and the brain was carefully removed and washed with ice-cold saline. Dissection of each brain was performed on an ice-cold glass plate for separation of both striata. Thereafter in the first set both striata were homogenized in ice-cold saline for the evaluation of monoamines' level. In the second set, both striata were homogenized in ice-cold saline for the estimation of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and tumor necrosis factor-α (TNF-α). The last set was conducted to assess inducible nitric oxide synthase (iNOS) and BDNF expression as well as immunohistological alterations in the striata and substantia nigra of animals representing each group. Measured parameters Open field test A square wooden box measuring 80 x 80 x 40 cm with red walls and white smooth polished floor divided by black lines into 16 equal squares 4 x 4 was used. Each rat was placed gently in the central area of the open field and allowed to freely explore the area for 3 min. The floor and walls were cleaned after testing each rat to eliminate possible bias due to odors left by previous rats. A video camera was fixed on the top of the box to record movement and behavior of rats for later off-line analysis, namely ambulation, rearing, grooming and immobility time (Walsh and Cummins, 1976). Rotarod test Animals were placed in the orientation opposite to that of a rotating rod (3 cm diameter, 90 cm height, and 10 rpm). Before experimentation, rats were trained for 3 days to remain on the stationary and rotating rod (3 sessions, 1 min). After accomplishing the open field test, animals were

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evaluated for a period of 5 min and the time spent by each rat on the rod was recorded (Jones and Roberts, 1968). Estimation of striatal monoamines' level Striatal noradrenaline (NA), dopamine (DA) and serotonin (5-HT) levels were quantified by the ELISA technique using purchased kits from Eagle biosciences (Nashua, USA), USCN Life Science Inc. (Wuhan, China) and Cusabio (Wuhan, China), respectively, according to the manufacturer’s protocol. The assays apply the competitive enzyme immunoassay technique using a monoclonal antibody specific for each monoamine and a monoamine-Horseradish Peroxidase (HRP) conjugate. Standards or samples were incubated together with the corresponding monoamine-HRP conjugate in pre-coated plates for 1 h. After washing, wells are incubated with a substrate for HRP enzyme. Finally, a stop solution is added to stop the reaction, and the intensity of the color was measured at 450 nm using a microplate reader (BioTek Elisa Reader ELx808). The results were expressed as ng/mg protein. Estimation of striatal oxidative stress biomarkers Malondialdehyde level Striatal MDA level was estimated using the Biodiagnostic colorimetric kit (Cairo, Egypt) according to the method described by Ohkawa et al., (1979). MDA forms a pink colored complex with thiobarbituric acid, in acidic medium at 95 °C for 30 min. The absorbance was measured at 534 nm and the results were expressed as nmol/mg protein. Superoxide dismutase activity Striatal SOD activity was estimated using the Biodiagnostic colorimetric kit (Cairo, Egypt) according to the method of Marklund and Marklund (1974). SOD activity was determined by monitoring its inhibitory effect on pyrogallol autoxidation, at alkaline pH < 9.5, through ϵ 

scavenging the formed superoxide anion at 420 nm. One unit of SOD activity is defined as the amount of enzyme causing 50% inhibition of pyrogallol autoxidation. The results were expressed as U/mg protein. Catalase activity Striatal CAT activity was assessed using the Biodiagnostic colorimetric kit (Cairo, Egypt) according to the method described by Aebi (1984). The decrease in absorbance following the decomposition of hydrogen peroxide by CAT was measured at 510 nm. One unit of CAT activity is defined as the amount of enzyme that decomposes 1 µM hydrogen peroxide per min at 25 °C. The results were expressed as U/mg protein. Estimation of striatal inflammatory biomarkers Tumor necrosis factor- α level Striatal TNF-α level was quantified by the ELISA technique using commercial TNF-α ELISA Kit (Elabscience, Wuhan, China) according to manufacturers’ prescripts. Samples were added to the microtiter plate wells coated with the antibody specific to TNF-α. A biotin conjugated antibody specific for this antigen was added to the wells following the removal of any unbound substances. After washing, an avidin conjugated HRP was added to the wells. Thereafter, tetramethylbenzidine was added to all wells for color development and the enzyme-substrate reaction was terminated by the addition of sulfuric acid solution. Color intensity, which is proportional to the amount of TNF-α bound, was measured at 450 nm using a microplate reader (BioTek Elisa Reader ELx808). The results were expressed as pg/mg protein. Inducible nitric oxide synthase protein expression The expression of striatal iNOS protein was assessed using the Western blot assay. After protein solutions were extracted from striatal tissues, equal amounts of proteins were loaded onto a sodium dodecyl  ϭϬ 

sulfate polyacrylamide gel electrophoresis, which allows separation of proteins according to their molecular weight. After electrophoresis, proteins were transferred to nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA). Blocking of non-specific binding sites was then achieved by placing the membranes in a 5% skim milk. Membranes were then incubated with the anti-iNOS primary antibody solution (1:1000; Sigma-Aldrich, St. Louis, MO, USA) overnight on a roller shaker at 4°C. Subsequently they were washed and incubated with the horseradish peroxidase-conjugated secondary antibody (1:2000; Fluka, St. Louis, MO, USA). Finally, the blots were developed with enhanced chemiluminescence detection reagents (Amersham Biosciences, Arlington Heights, IL). The amount of iNOS protein was quantified by densitometric analysis using a scanning laser densitometer (GS-800 system, Bio-Rad, Hercules, CA, USA). Results were expressed as arbitrary units after normalization with ß-actin protein expression. Estimation of striatal brain derived neurotrophic factor gene expression Striatal BDNF gene expression was assessed using real time-PCR (RT-PCR) technique. Total RNA was extracted from striatal tissue using SV Total RNA Isolation system (Promega, Madison, WI, USA) and the purity of obtained RNA was verified spectrophotometrically at 260/280 nm. The extracted RNA was then reverse transcribed into complementary DNA using RT-PCR kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s procedure. Quantitative RT-PCR was performed using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) as described by the manufacturer. The sequences of primers were as follows: BDNF, F: 5′-ACC CTG AGT TCC ACC AGG TG-3′, R: 5′TGG GCG CAG CCT TCA T-3′, and ß-actin, F: 5′-CGT TGA CAT CCG  ϭϭ 

TAA AGA CCT C-3′, R: 5′-TAG GAG CCA GGG CAG TAA TCT-3′. After the quantitative RT-PCR run, the relative expression of target gene was obtained using the 2−∆∆CT formula using ß-actin as a housekeeping gene (Livak and Schmittgen, 2001). Assessment of dopaminergic neuron density and microglial activity Immunohistochemistry technique was used to assess dopaminergic neuron density in SN as well as microglial activity in the nigrostriatal system. The brain pieces were processed into paraffin blocks, thereafter, 4 µm sections were prepared on positively charged glass slides. Endogenous peroxidase activity was quenched by first incubating the specimens in 3% hydrogen peroxide. The specimens were then incubated with primary monoclonal anti-tyrosine hydroxylase (TH) antibody (diluted 1:1000; Sigma-Aldrich, St. Louis, MO, USA) or anti-CD68 antibody (diluted 1:1000; Dako, Carpinteria, CA, USA), followed by sequential incubations with biotinylated link antibody and peroxidaselabelled streptavidin (Dako, Carpinteria, CA, USA). Labeling was then revealed by diaminobenzidine chromogen. Slides were counterstained with hematoxylin, dehydrated, cover slipped and examined through the light electric microscope [Olympus CX21, Tokyo, Japan]. The number of TH-positive cells in the SN as well as CD68-positive cells in the SN and striatum was counted microscopically (magnification x40) using the Leica Qwin 500 Image Analyzer (Leica Microsystems, Wetzlar, Germany) from 4 randomly selected fields in each section. The results were presented as percentage of TH-positive cells or CD68-positive cells with respect to the control (Noor et al., 2012). Estimation of striatal protein content

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In order to express the units of measured parameters per mg protein, protein content was quantified according to the method described by Lowry et al. (1951). Statistical analysis All data were checked for normality as well as homogeneity of variance prior to ANOVA analyses using Kolmogorov-Smirnov and Bartlett’s tests, respectively. Datasets that met the assumptions for parametric analysis were analyzed using one-way ANOVA followed by Tukey-Kramer multiple comparisons test and were expressed as mean ± S.E.M. For behavioral and iNOS experiments, data did not fulfill the assumption for normality and homogeneity of variance; thus were analyzed using Kruskal-Wallis nonparametric one-way ANOVA followed by Dunn’s multiple comparisons test and were expressed as median and range. A probability level of less than 0.05 (p < 0.05) was accepted as statistically significant. Statistical analysis was performed using GraphPad Prism software version 5 (San Diego, CA, USA). Results There was no change in any of the tested parameters between control rats and those that received DMSO, AMI and IMI alone. Amitriptyline and imipramine attenuated rotenone-induced alterations in rats’ motor performance in the open field along with motor coordination in the rotarod tests Rotenone produced a significant deterioration in the motor performance and coordination of rats as compared with the control group (p < 0.001) (Fig 2). Pretreatment with AMI significantly reversed the decrease in ambulation, rearing and falling time as compared with the rotenone group (test statistic = 50.9, 47.3 and 85.2, p < 0.001, 0.001 and 0.05, respectively). While it significantly lessened the immobility time  ϭϯ 

(test statistic = 105.4, p < 0.001) as compared with the rotenone group. Similarly, pretreatment with IMI displayed a significant elevation in ambulation, rearing and falling time as compared with the rotenone group (p < 0.001, 0.001 and 0.01). Amitriptyline and imipramine attenuated rotenone-induced alterations in striatal monoamines' level Rotenone significantly decreased the striatal levels of NA, DA and 5-HT as compared with the control group (F(6,35) = 57.6, 91.7 and 62.8, respectively, p < 0.001) (Fig 3). Pretreatment with AMI or IMI significantly reversed striatal monoamines' level back to their normal values (p < 0.001). Amitriptyline and imipramine attenuated rotenone-induced alterations in striatal oxidative stress biomarkers Rotenone group exhibited a significant surge in striatal MDA level (F(6,44) = 18.3, p < 0.001) along with a significant decline in striatal SOD and CAT activities (F(6,42) = 21.5 and F(6,39) = 11.2, p < 0.001 and 0.01, respectively) as compared with the control group (Fig 4). Pretreatment with AMI or IMI significantly decreased the level of MDA (p < 0.001 and 0.01, respectively), while significantly restored the activities of SOD and CAT as compared with the rotenone group (p < 0.001). Amitriptyline and imipramine downregulated rotenone-induced alterations in striatal inflammatory biomarkers Repeated subcutaneous injection of rotenone significantly upregulated striatal iNOS protein expression as compared with the control group (test statistic = 37.3, p < 0.01) (Fig 5A). Pretreatment with AMI or IMI downregulated the elevated iNOSexpression, yet this was not statistically significant as compared with the rotenone group (p > 0.05).  ϭϰ 

In addition, rotenone significantly elevated striatal TNF-α level in comparison to the control group (F(6,38) = 48.9, p < 0.001) (Fig 5B). Pretreatment with AMI or IMI significantly reversed such increment in TNF-α level as compared with the rotenone group (p < 0.001). Amitriptyline and imipramine downregulated rotenone-induced alterations in striatal brain derived neurotrophic factor gene expression Striatal BDNF gene expression was significantly reduced after rotenone administration as compared with the control group (F(6,38) = 44.4, p < 0.001). However, pretreatment with AMI or IMI significantly amended such alteration in BDNF gene expression as compared with the rotenone treated animals (p < 0.001) (Fig 5C). Amitriptyline and Imipramine mitigated rotenone-induced alterations in dopaminergic neurons of substantia nigra Rotenone regimen resulted in a significant loss of dopaminergic neurons in the SN, indicated by significantly decreased tyrosine hydroxylase-immunoreactive neurons as compared with the control group (F(6,105) = 95, p < 0.001) (Fig 6). Administration of AMI or IMI significantly alleviated rotenone-induced dopaminergic neurons degeneration as shown by increased tyrosine hydroxylaseimmunoreactive neurons as compared with the rotenone group (p < 0.001); however, still significant from the control group (p < 0.001). Amitriptyline and Imipramine mitigated rotenone-induced alterations in striatal and substantia nigra microglial activation Rotenone induced notable microglial activation in rat striatum (Fig 7) and SN (Fig 8), determined by a significant increase in CD-68 immunoreactive neurons in comparison to the control rats (F(6,105) = 215.3 and 222.6, respectively, p < 0.001). Pretreatment with AMI or IMI

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resulted in a significant suppression of microglial activation in both brain regions as compared with the rotenone group (p < 0.001); yet both groups were significant from the control group (p < 0.001).

Discussion The novel findings of the present investigation reveal that the tricyclic antidepressant IMI protected the vulnerable dopaminergic neurons against a neurotoxic insult with a comparable efficacy to AMI in the rotenone model of PD. Neuroprotection is based on the noted preservation of the dopaminergic neurons in SN as well as the improvement of rats' motor function in the behavioral tests. The restoration of striatal monoamines' level, inhibition of oxidative stress, attenuation of microglial activation and modulation of inflammation along with promotion of neurogenesis by BDNF enhancement could serve as important mechanisms underlying the neuroprotective effects afforded by AMI and IMI in the rotenone model of PD in rats. In the present work, repeated exposure of rats to rotenone resulted in impaired locomotor activity in the open field test, in addition to diminished muscle coordination and balance on the rotarod apparatus, which is in agreement with previous studies (Betarbet et al., 2000; Sindhu et al., 2005; Abdelkader et al., 2014). These observations indicate that this neurotoxin more likely imitate the signs of PD (Sharma et al., 2016). Such deterioration in the motor performance is a consequence to the observed dopaminergic neuronal death in SN along with obvious decrease in DA level in the striatum. These results are in line with former studies (Betarbet et al., 2000; Alam and Scmidt, 2002). Herein, treatment with AMI and IMI attenuated the rotenone-induced dopaminergic neuronal loss in SN along with restoration in the striatal DA level; effects

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supported by a previous finding that TCA delayed the need for initiating dopaminergic therapy in PD patients (Paumier et al., 2012). Moreover, AMI prevented the degeneration of nigrostriatal dopaminergic neurons as well as the motor deficits in the 6-hydroxydopamine rat model of PD (Paumier et al., 2015). Similarly, an enhancement in rats’ motor performance and coordination in the open field and rotarod tests, respectively, was evident herein following treatment with both drugs. Restoration of striatal NA and 5-HT instigated by AMI and IMI treatment in this work perhaps could account partly for the preferential effects of both drugs on the lesioned dopaminergic neurons. Both drugs increase the extracellular level of NA and 5-HT by blocking their reuptake (Frommer et al., 1987; Tatsumi et al., 1997). Inevitably, changes in neurotransmitters homeostasis is well documented to occur in PD (Sharma et al., 2016). Loss of 5-HT markers in striatal and extra-striatal tissues in PD has been reported both clinically and experimentally; indicating the crucial role of serotonergic system in PD pathology (Tohgi et al., 1993; Kish et al., 2008; Lesemann et al., 2012). Moreover, the level of NA decreases during the progression of PD (Höglinger et al., 2004). Herein, repeated rotenone administration significantly decreased striatal levels of 5-HT and NA alongside DA as reported by Sharma et al. (2016). It is worth mentioning that enhanced noradrenergic activity may modulate the severity of motor symptoms and influence the progression of dopaminergic neurodegeneration in PD (Isaias et al., 2011). NA displays a compensatory activity by binding to dopaminergic receptors in the striatum (Newman-Tancredi et al., 1997; Cornil et al., 2002); hence moderately compensates the loss of dopaminergic innervation due to degeneration of SN (Isaias et al., 2011). In addition, NA has a neuroprotective role in SN via suppressing pro-inflammatory molecules, elevating anti-inflammatory mediators and scavenging reactive oxygen  ϭϳ 

species, which contribute to dopaminergic neuronal damage (Cornil et al., 2002; Feinstein et al., 2002; Isaias et al., 2011). Similarly, 5-HT exhibits neuroprotective potential as evidence suggests that a reciprocal modulatory interaction exists between 5-HT and BDNF, where 5-HT increases BDNF production, while BDNF promotes the development and function of serotonergic neurons (Martinowich and Lu, 2008; Sharma et al., 2016). Indeed, neurotrophic factors are essential for the survival, differentiation and maintenance of neurons in the central nervous system (Gorski et al., 2003). Of all the neurotrophic factors, BDNF is of particular significance for nigrostriatal dopaminergic neurons vulnerable to degeneration in PD (Allen et al., 2013). Antidepressants, specifically TCAs, elicit their therapeutic action, in part, by increasing BDNF expression in rat brains, which further confirms the neuroprotective potential of such drugs (Jang et al., 2009; Sharma et al., 2016). In the present findings, treatment with AMI or IMI counteracted the rotenoneinduced reduction of BDNF in the striatum. In agreement, recent studies reported that AMI increased the expression of BDNF in the nigrostriatal system and retarded the progression of its dopaminergic neuronal loss in the 6-hydroxydopamine rat model of PD (Paumier et al., 2015). In addition, AMI augmented hippocampal and striatal BDNF levels in mouse models of multiple systems atrophy and Huntington's disease, respectively (Valera et al., 2014; Cong et al., 2015). While treatment of maternally deprived rats with IMI increased BDNF level in the amygdala (Réus et al., 2013). It has been reported that antidepressants induce BDNF transcriptional activation mediated by cAMP-response elementbinding protein (CREB) in the rat brain (Thome et al., 2000). In support, Cong et al. (2015) found that AMI treatment raised BDNF expression locally via activation of CREB in the mice striatum. Binding of BDNF to  ϭϴ 

tyrosine kinase receptor B (TrkB) initiates tyrosine autophosphorylation in its cytoplasmic domain, leading to activation of intracellular signaling such as the mitogen-activated protein kinase, the phospholipase C gamma and the phosphatidylinositol-3-kinase pathways. These signals are involved in neuronal survival as well as plasticity (Yoshii and Constantine-Paton, 2010). Finally, further evidence has shown that AMI and IMI directly bind TrkB and possess marked neurotrophic activity (Jang et al., 2009). It is well established that oxidative stress is implicated in the pathogenesis of PD (Jenner et al., 1992). Mitochondrial dysfunction as well as DA metabolism generate excessive free radicals that overwhelms endogenous antioxidants; resulting in oxidative burden in the dopaminergic neurons (Henchcliffe and Beal, 2008). Indeed, dopaminergic neurons are vulnerable to oxidative damage due to their reduced antioxidant capacity and high content of oxidizable dopamine and lipids (Greenamyre et al., 1999). In the present work, rotenone exposure decreased the antioxidants SOD and CAT along with a significant elevation in MDA signifying free radical production in the striatum; effects which are in accordance with preceding investigations (Bashkatova et al., 2004; Kaur et al., 2011; Zaitone et al., 2012). However, treatment with AMI or IMI attenuated increased MDA level and restored reduced SOD and CAT activities in the striatal tissues. These antioxidant effects can be related to upregulation of antioxidant enzymes and downregulation of oxidants (Sharma et al., 2016). Similar to the present work, Zafir et al. (2009) showed that treatment with IMI augmented the cellular antioxidant status in restraint-induced oxidative stress in rat brains. Furthermore, AMI up-regulated SOD gene expression and activity (Li et al., 2000; Kolla et al., 2005). It is worth mentioning that the prevention of lipid peroxidation by AMI and IMI could preserve  ϭϵ 

membrane integrity through protecting membrane phospholipids against oxidative damage (Zafir et al., 2009; Behr et al., 2012). Accordingly, the improvement of the antioxidant status by both drugs could partially contribute to their observed neuroprotective effects in the existing work (Behr et al., 2012; Sharma et al., 2016). Reactive oxygen species initiate inflammatory pathways, hence extending the deleterious milieu for vulnerable dopaminergic neurons (Taylor et al., 2013). In the present findings, rotenone activated microglial cells in the nigrostriatal system and increased the expression of striatal TNF-α and iNOS, which is in accordance with previous experimental (Sherer et al., 2003; Zhou et al., 2007; Madathil et al., 2013) and clinical studies (Hunot et al., 1996; Hirsch et al., 1998). In PD, microglial activation outcomes the release of a wide array of neurotoxic mediators including pro-inflammatory cytokines, reactive oxygen species and nitric oxide that impact neurons to undergo neurodegeneration (Block et al., 2007). On the other hand, treatment with AMI or IMI significantly suppressed the neurotoxic-induced microglia activation as well as TNF-α and iNOS elevation. In parallel, a previous study explored that IMI inhibits the activation of microglia and astrocytes and consequently reduces TNF-α and nitric oxide production in lipopolysaccharidestimulated microglia and astrocyte cultures (Hwang et al., 2008). Besides, AMI was shown to impede the release of TNF-α and interleukin-1β from mixed glial and microglial cultures stimulated with lipopolysaccharide (Obuchowicz et al., 2006). Such anti-inflammatory effect might be attributed to inhibition of nuclear factor kappa-B and p38 mitogenactivated protein kinases hence suppressing the transcriptional activation of multiple inflammatory genes (Hwang et al., 2008). These findings suggest that AMI and IMI may debit at least some of their

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neuroprotective efficacy to their antioxidant and anti-inflammatory properties. In conclusion, the present study demonstrates that the tricyclic antidepressants IMI and AMI have comparable neuroprotective effects in the rotenone model of PD in rats. Their beneficial actions might be accredited to their ability to modulate not only DA, but also NA and 5HT levels in striatum, as well as upregulating BDNF expression, which is a vital trophic factor for dopaminergic neurons survival.In addition, they mitigated oxidative stress, microglial activation and inflammation in the dopaminergic neurons. These changes will eventually target multiple factors underlying PD pathogenesis. Furthermore, AMI and IMI have the advantage of being safe and well-tolerated antidepressant drugs that are already used clinically in PD. Therefore, it is suggested that AMI and IMI may provide a more effective strategy to subside both neurodegeneration and depression in PD, thereby alleviating motor and non-motor symptoms in PD and improving patients’ quality of life.

Acknowledgments The authors are grateful to Dr. Hebat Allah Amin, Department of Pathology, Egyptian Forensic Medicine Authority, Ministry of Justice, Cairo, Egypt for the kind help in immunohistochemistry. The authors are also thankful to Dr. Omnia M. Abo-Elazm, Department of Biostatistics and Cancer Epidemiology, National Cancer institute, Cairo University, Egypt for the kind help in statistical analysis.

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Figure captions Fig 1. Experimental design Fig 2. Amitriptyline and imipramine attenuated rotenone-induced alterations in rats’ motor performance in the open field along with motor coordination in the rotarod tests Each bar with vertical line represents the median and range of 20-24 rats per group; *** significantly different from control group at p < 0.001, # significantly different from rotenone group at p < 0.05, ## significantly different from rotenone group at p < 0.01, ### significantly different from rotenone group at p < 0.001 using Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparisons test. Fig 3. Amitriptyline and imipramine attenuated rotenone-induced alterations in striatal monoamines' level Each bar with vertical line represents the mean ± S.E.M. of 6 rats per group; *** significantly different from control group at p < 0.001, ### significantly different from rotenone group at p < 0.001 using One-Way ANOVA followed by Tukey-Kramer multiple comparisons test. Fig 4. Amitriptyline and imipramine attenuated rotenone-induced alterations in striatal oxidative biomarkers Each bar with vertical line represents the mean ± S.E.M. of 6-8 rats per group; ** significantly different from control group at p < 0.01, *** significantly different from control group at p < 0.001, ## significantly different from rotenone group at p < 0.01, ### significantly different from  ϯϯ 

rotenone group at p < 0.001 using One-Way ANOVA followed by Tukey-Kramer multiple comparisons test. Fig 5. Amitriptyline and imipramine downregulated rotenoneinduced alterations in striatal inflammatory biomarkers and brain derived neurotrophic factor gene expression (A) Striatal iNOS protein expression was analyzed using Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparisons; each bar with vertical line represents the median and range of 6-8 rats per group. (B) Striatal TNF-α level and (C) striatal BDNF gene expression were analyzed using one-way ANOVA followed by Tukey-Kramer multiple comparisons test; each bar with vertical line represents the mean ± S.E.M. of 6-8 rats per group. * Significantly different from control group at p < 0.05, ** significantly different from control group at p < 0.01, *** significantly different from control group at p < 0.001, ### significantly different from rotenone group at p < 0.001. Fig 6. Amitriptyline and imipramine mitigated rotenone-induced alterations in dopaminergic neurons of substantia nigra (A) Immunohistochemical staining demonstrated the loss of TH-positive dopaminergic neurons in the SN after rotenone administration. AMI and IMI treatment ameliorated rotenone-induced reduction in the number of SN TH-positive dopaminergic neurons (photomicrographs captured at ×20 magnification). (B) Number of TH-positive dopaminergic neurons in the SN. Data are presented as percentage of control group; *** significantly different from control group at p < 0.001, ### significantly different from rotenone group at p < 0.001 using One-Way ANOVA followed by Tukey-Kramer multiple comparisons test (magnification x40). Fig 7. Amitriptyline and imipramine mitigated rotenone-induced striatal microglial activation  ϯϰ 

(A) Striatal CD68 immunohistochemical staining revealed microglial activation after rotenone administration as denoted by an increase in CD68 positive cells. AMI and IMI treatment attenuated the rotenone-induced rise in CD-68 positive cells in striatum (photomicrographs captured at ×10 magnification). (B) Number of CD68-positive cells in the striatum. Data are presented as percentage of control group; *** significantly different from control group at p < 0.001, ### significantly different from rotenone group at p < 0.001 using One-Way ANOVA followed by Tukey-Kramer multiple comparisons test (magnification x40). Fig 8. Amitriptyline and imipramine mitigated rotenone-induced midbrain microglial activation (A) SN CD68 immunohistochemical staining showed microglial activation after rotenone administration as indicated by an increase in CD-68 positive cells. AMI and IMI treatment attenuated the rotenone-induced rise in CD-68 positive cells in SN (photomicrographs captured at ×10 magnification). (B) Number of CD68-positive cells in the SN. Data are presented as percentage of control group; *** significantly different from control group at p < 0.001, ### significantly different from rotenone group at p < 0.001 using One-Way ANOVA followed by Tukey-Kramer multiple comparisons test (magnification x40).

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Highlights •

Imipramine and amitriptyline abate neurodegeneration in Parkinson's disease.

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Modulation of monoamines and neurotrophin levels contributes to the neuroprotection.

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Antioxidant and anti-inflammatory properties underlie the drugs' protective effects.



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