Evaluation of Betaine Neuroprotective Effects on 6-Hydroxydopamine-Induced hemi-Parkinsonism in Male Wistar Rats

Document Type : Physiology- Pharmacology-Biochemistry -Toxicology


1 Section of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran,

2 Section of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran,Tehran, Iran

3 Section of Anatomy and Emberiology, Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

4 Division of Biochemistry, School of Veterinary Medicine, Lorestan University, Khorram Abad, Lorestan, Iran


BACKGROUND: Parkinson's disease (PD) is one of the prevalent debilitating neurodegenerative disor- ders. Accordingly, researchers are working on methods to modify PD progression. Previously, the neuro- protective effects of betaine, as a methyl donor agent in homocysteine metabolism, have been demonstrated in animal models of chronic cerebral hypoperfusion and memory deficits.
OBJECTIVES: It was aimed to investigate the neuroprotective effects of betaine in an animal model of PD.
METHODS: In male Wistar rats under two-week course of oral betaine administration (50, 100, and 200 mg/kg per day), the behavioral, biochemical, and histological evaluations were conducted one week follow- ing unilateral nigral 6-OHDA injection.
RESUTLS: Betaine administration with dose of 200 mg/kg, one week before and after 6-OHDA lesioning, was associated with a meaningful reduction in the plasma levels of homocysteine (Hcy) in comparison with the control and sham groups (P < 0.05). Our evaluations revealed a remarkable improvement in motor asymmetry induced by apomorphine in the rats under treatment of betaine 200 mg/kg. Moreover, in this group, a significant decrease of malondyaldehyde (MDA) concentrations was detected in the brain tissues, as well as a significantly diminished neuronal cell loss (percent) in substantia nigra pars compacta (P < 0.05). The results of 50 and 100 mg/kg betaine groups were not significant.
CONCLUSIONS: Altogether, our findings indicate the antioxidant neuroprotective effects of betaine in this animal model of PD and it is in concordance with betaine properties in decreasing the plasma levels and possible neurotoxic effects of Hcy.


Article Title [Persian]

ارزیابی اثرات محافظتی بتائین بر پارکینسون یکطرفه القاء شده توسط 6- هیدروکسی دوپامین در رت های نر نژاد ویستار

Authors [Persian]

  • بهروز رحمانی 1
  • مرتضی زنده دل 2
  • وهاب باباپور 2
  • جواد صادقی نژاد 3
  • مسعود علیرضائی 4
1 بخش فیزیولوژی، گروه علوم پایه، دانشکده دامپزشکی، دانشگاه تهران، تهران، ایران
2 بخش فیزیولوژی، گروه علوم پایه، دانشکده دامپزشکی، دانشگاه تهران، تهران، ایران
3 بخش آناتومی و جنین شناسی، گروه علوم پایه، دانشکده دامپزشکی، دانشگاه تهران، تهران، ایران
4 بخش بیوشیمی، دانشکده دامپزشکی، دانشگاه لرستان، خرم آباد، لرستان، ایران
Abstract [Persian]

زمینة  مطالعه:  بیماری پارکینسون یکی از بیماری های ناتوان کننده و شایع با منشاء تخریب نورونی می باشد. بر این اساس،‌‌ امروزه محققان به دنبال یافتن روش های درمانی مؤثر در جلوگیری از روند پیشرفت این بیماری هستند. در تحقیقات به عمل آمده اثرات محافظتی بتائین (به عنوان عامل دهنده گروه متیل در مسیر متابولیسم هموسیستئین) در مدل های تحقیقاتی کاهش مزمن خونرسانی مغز  و اختلال حافظه، نشان داده شده است.
هدف: این مطالعه با هدف بررسی اثرات محافظتی بتائین در مدل حیوانی بیماری پارکینسون یکطرفه، شکل گرفته است.
روش کار: سویهدر این پژوهش ارزیابی های رفتاری، بیوشیمیایی و بافت شناسی بر روی رت های نر نژاد ویستار که تحت تجویز دو هفته ای بتائین به صورت خوراکی با دوزهای روزانه 50، 100 و 200 mg/kg بودند به دنبال تزریق نوروتوکسین 6- هیدروکسی دوپامین در ناحیه متراکم جسم سیاه نیمکره چپ (به منظور القاء پارکینسون یکطرفه) انجام شد.
نتایج: مراحل تجویز بتائین در حیوانات با دوز روزانه 200 mg/kg از یک هفته قبل تا یک هفته پس از تزریق استریوتاکسیک 6- هیدروکسی دوپامین، با کاهش معنادار سطوح پلاسمایی هُموسیستئین نسبت به حیوانات گروه های شم و کنترل همراه بود. در این گروه، نسبت به گروه کنترل، کاهش معناداری در تحلیل داده های استخراج شده از آزمون رفتاری چرخش، ملاحظه شد. همچنین، بررسی های بیوشیمیایی بافت مغزی حیوانات، که به منظور ارزیابی میزان استرس اکسیداتیو نورون ها در اثر تزریق نوروتوکسین 6- هیدروکسی دوپامین به عمل آمد، نشانگر کاهش معنادار غلظت های مالون دی آلدئید در گروه بتائین 200 mg/kg نسبت به گروه های شم و کنترل بود. در نهایت، درصد نورون های تخریب شده ناحیه متراکم جسم سیاه نیمکره چپ در حیوانات گروه بتائین 200 mg/kg با کاهش معناداری نسبت به گروه کنترل، همراه بود (05/0 > P). در بررسی های مشابه انجام شده در گروه های بتائین 50 و 100 mg/kg تغییرات معناداری نسبت به گروه کنترل، ملاحظه نگردید.
 نتیجهگیری نهایی: یافته های این پژوهش بیانگر اثرات آنتی اکسیدانی و محافظتی بتائین در مدل تحقیقاتی پارکینسون یکطرفه می باشد که با ویژگی بتائین در کاهش سطوح پلاسمایی هُموسیستئین و در نتیجه کاهش اثرات سمی احتمالی هُموسیستئین هم خوانی دارد.

Keywords [Persian]

  • بیماری پارکینسون
  • بتائین
  • هُموسیستئین
  • مالون دی آلدئید
  • 6- هیدروکسی دوپامین


After Alzheimer's disease, Parkinson's dis- ease (PD) is the second most-prevalent neu- rodegenerative disorder, which affects ap- proximately 2-3% of humans more than 65 years of age (Poewe et al., 2017). Clinically, the core presentation of PD is bradykinesia in combination with rigidity and/or rest tremor (Postuma et al., 2015). Besides, the majority of PD patients are suffering the non-motor problems including sleep disorders, cogni- tive impairment, mood and affect disorders, autonomic dysfunction, and sensory symp- toms (Chaudhuri & Schapira,  2009).

From  the  pathophysiological  view,   PD is mainly characterized by dopaminergic neurodegeneration in substantia nigra pars compacta (SNpc) and  dopamine  depletion  in striatum (Golpich et al., 2015; Poewe  et al., 2017). The dopaminergic neurodegen- eration in PD is caused by different under- lying mechanisms. In this regard, oxidative stress (OS) performs an important role. OS   is a result of a disequilibrium between the produced levels of reactive oxygen species (ROS) and the reactive intermediates detox- ifying system’s ability which can lead to cell injury (Dias et al., 2013). It has been illus- trated that, nigral dopaminergic neurons with high levels of dopamine and Fe2+ are vul- nerable to OS. Since dopamine is an unstable molecule, its auto-oxidation forms quinones and free radicals that result in OS. In addi- tion, metals, oxygen, and several enzymes such as tyrosinase can catalyze dopamine auto-oxidation. Moreover, dopamine me- tabolism can be mediated by other enzymes such as monoamine oxidase (MAO-A and MAO-B) and catechol-o-methyl transferase (COMT) in the catecholaminergic neurons. In PD with  progressing  of neuronal   degen-



eration, MAO-B located in glial cells will increase and become a predominant enzyme in dopamine metabolism (Dias et al., 2013; Fowler et al., 1997). During dopamine deg- radation by MAO-B, the produced hydrogen peroxide can react with Fe2+ leading to for- mation of hydroxyl radicals and increasing the occurrence of OS (Hermida-Ameijeiras  et al., 2004).

It has been shown that, under OS condi- tions, oxidative damages take place at the various compartments of cells generating lipid peroxidation, protein oxidation, DNA and RNA oxidation; subsequently, resulting in cell death. In addition, OS may be linked to other processes such as mitochondrial dys- function, neuroinflammation, excitotoxisity, and nitrosative stress as the cascade of con- sequences associated with cell injury in PD (Ambrosi et al., 2014; Blesa et al., 2015) . The link between OS and PD progression is the theoretical base of developing the animal models of PD with toxins such as 6-OHDA, MPTP, rotenone, paraquat in which the tox- ins induce oxidative stress (Dias et al., 2013). Another agent that  can induce  cellular OS is homocysteine (Hcy), a non-essential amino acid with sulfhydryl  group.  Hcy  is an intermediate product derived during me- thionine metabolism that can pass across blood brain barrier (BBB). Increased plas- ma levels of  Hcy,  hyperhomocysteinemia, is considered as a risk factor for  a number of peripheral and cardio cerebrovascular dis- eases, cancers and neurodegenerative disor- ders (Ganguly & Alam, 2015; Zhang et al., 2015). Hcy can increase production of ROS along with inhibiting the enzymatic  activ- ity of antioxidants (Lehotsky et al., 2014; Skovierova et al., 2016). Although in several




studies it has been reported that 10-30% of PD patients have presented with hyperhomo- cysteinemia, it is not clear whether Hcy is a causative agent of PD onset or not. In this respect, according to the neurotoxic prop- erties of Hcy, there  are several  suggestions to hypothesize that Hcy may contribute to pathophysiology of PD (Obeid et al., 2009; Sharma et al., 2015; Skovierova et al., 2016; Zoccolella et al., 2010).

Trimethylglycine, which is known as beta- ine, can transfer its methyl group to Hcy with the aid of the enzyme betaine homocysteine methyl transferase (BHMT). During this re- action with formation of dimethylglycine from betaine, Hcy is remethylated to methi- onine similar to another reaction pathway that catalyzes Hcy by methionine synthase using 5-methyltetrahydrofolate.  Because  BHMT is expressed mostly in liver and kidney, Hcy metabolism by the use of betaine takes place mainly in mentioned tissues (Imbard et al., 2015; Ueland et al., 2005). Considering beta- ine’s effect on reduction of Hcy level, it has been hypothesized that administration of be- taine can protect against hazardous effects of Hcy on cell damage. In this respect, previous animal studies have shown that the adminis- tration of betaine reduced plasma Hcy level; subsequently prevents oxidative stress in dif- ferent tissues such as brain, cerebellum, liv- er, kidney, testis and ovary (Alirezaei, 2015; Alirezaei, et al., 2015; Alirezaei, et al., 2012; Kim et al., 2009; Tan et al., 2015).

In consideration of PD importance as one of the prevalent debilitating disorders, many research studies are conducted to  identify  the methods for modifying the trajectory of PD progression. In this respect, according to the above mentioned neuroprotective effects of betaine and probable role of Hcy in PD, this study is conducted with the main aim  of

examining the effects of betaine administra- tion in an animal model of PD induced by 6-OHDA, as neurotoxin. In this regard, be- taine effects will be under study in relation with plasma Hcy levels as well as evaluating brain lipid peroxidation and histological al- terations within SNpc.

Material and Methods:


The homocysteine ELIZA kit was prepared from MyBioSource® (MBS703069, USA). Lipid peroxidation assay kit provided from Nalondi™ (NS-15022, Navand Salamat Co. Iran). Betaine, 6-hydroxy dopamine hydro- chloride (6-OHDA), apomorphine hydro- chloride and ascorbic acid were obtained from Sigma Chemical, St. Louis, Mo., USA. Betaine solved in water with concentra- tions of 1, 2 and 4% and stored at 8 °C to perform gavage in different doses. 6-OHDA was solved in saline and 0.2 % ascorbic acid and used freshly. Apomorphine hydrochlo- ride was solved in water with concentration of 0.05% and the fresh solvent was  adminis-

tered intraperitoneally (IP).

Experimental design

Adult male Wistar rats weighing between 190 and 240 grams were provided from the animal house of Faculty of Veterinary Medi- cine, University of Tehran, Tehran, Iran. An- imals were treated in accordance with Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press). The rats were left in a colony room with con- trolled temperature under a 12:12 hours of light and dark cycle. The animals were per- mitted to have ad libitum access to food and tap water. This study was approved by the Institutional Animal Ethics Committee, Fac- ulty of Veterinary Medicine, University of Tehran, Tehran, Iran; no. 750600/6/12.  Drug




administration, surgery, behavioral  testing and blood and brain sampling were carried out between 08:00  and  17:00  hours.  After at least one week of keeping the animals in the colony room, a total number of 40 rats with less than 30 net rotations following in- traperitoneal administration of apomorphine hydrochloride (0.5 mg/kg), were included in our study. The animals were randomly divid- ed into five equal groups (n = 8) in which  the treated groups were administered betaine or water (vehicle of betaine) by daily gavage for two weeks. In this way, 6-OHDA lesion- ing was conducted one week after initiation of oral gavage. Our studied groups were as follows: a sham group, a control group ga- vaged daily with water, a 50 mg/kg betaine group, a 100 mg/kg betaine group and a 200 mg/kg betaine group which received betaine in mentioned doses by performing daily ga- vage. One hour after the last gavage, the an- imals were under behavioral assessment by rotation test and about 2 h after the behavior- al testing, rats were deeply anesthetized by intraperitoneal injection of ketamine/xyla- zine (100 mg/kg and 5 mg/kg, respectively) in order to collect blood samples via the left ventricle after performing thoracotomy. Bio- chemical evaluations of four obtained blood samples in each group were conducted to identify Hcy plasma levels. In order to mon- itor the OS, five brains in each animal group were removed from the skulls, and immedi- ately frozen and stored at -80 °C to measure malondialdehyde (MDA) concentrations. In addition, the brains of three anesthetized rats in each group were prepared for histological assessment by intracardiac formalin 10% perfusion.

6-OHDA lesioning

Intranigral injection of 6-OHDA in the left brain  hemisphere  was  performed  via stereo-

taxic surgery in order to conduct 6-OHDA le- sioning. After anesthetizing of male Wistar rat with ketamine  (100 mg/kg IP) and xylazine  (5 mg/kg IP), the animal was placed on the stereotaxic  apparatus  (Stoelting,  USA)  with a mounted 30-gauge needle, which was at- tached to a 10-µl Hamilton syringe. During 5 min, 8 µg 6-OHDA in 5 µl of saline and 0.2 % ascorbic acid was infused into the left SNpc.  In our study, the coordinates of injection area were AP -4.8 mm, ML -2.2 mm from bregma fontanel and DV -7.8 mm from the surface of dura matter based on the Paxinos and Watson atlas (Paxinos & Watson, 2007).

Behavioral testing

One week following the 6-OHDA lesion- ing in laboratory room, each animal was kept in a plexiglass cylindrical container with 33 cm diameter and 35 cm height. After 10 min of  habituation,  apomorphine   hydrochlo- ride was administered intraperitoneally (0.5 mg/kg) and 1 min later, full rotations were counted at 10 min intervals for 1 h. As de- scribed previously, the positive scores were assigned to the full rotations contralateral to the 6-OHDA lesioned side (right turns) and the negative scores were assigned to the full rotations ipsilateral to the damaged brain hemisphere (left turns). At last,  net number of rotations was defined by subtracting the mentioned values (Fujita et al., 1996; Rezaei & Alirezaei, 2014).

Measurement of Hcy  concentration

Within 30 min after blood sampling through left ventricle, in order to prevent the release of red blood cells Hcy, the plasma samples were collected by centrifuging EDTA blood for 15 min at 1000 ˟ g. Immediately, the plasma samples were stored at -80 °C until the Hcy level measurement. The  Hcy  concentrations of plasma were quantitatively determined according to   the  method   described   by  rat




Hcy ELISA kit (My Bio Source, Cat # MBS703069) in which the biochemical tech- nique is based on Hcy antibody-Hcy antigen interactions and a horseradish peroxidase (HRP) colorimetric detection  system.  The Hcy concentrations were expressed as micro- moles per liter (µmol/l) of plasma.

Evaluation of lipid  peroxidation

Lipid peroxidation as an indicator of oxi- dative stress in cells can be detected by mon- itoring of its naturally occurring product, namely MDA (Tsikas, 2017). In this regard, we measured the MDA levels in homog- enized brain tissue by the use of lipid per- oxidation assay kit (Nalondi™, Iran)  based on the manufacturer’s instructions. In brief, forebrain and brainstem (1g), containing the mesencephalon, were thawed and manually homogenized in RIPA buffer (Gibco, USA), and the protease inhibitor PMSF (Gibco, USA). Debris was removed by centrifuga- tion and the supernatant was recovered and used for evaluation of MDA concentration and protein content. By utilizing thiobarbi- turic acid (TBA) at high temperatures, the MDA content in supernatant reacted to TBA and produced a pink to red derivative quan- tified by colorimetric method at the wave- length of 530–540 nm. The  concentrations  of MDA were expressed as nanomoles per milligram of tissue protein (nmol/mg pro- tein) (Hamzyan Olia et al., 2017). In addi- tion, total protein concentration in the brain homogenate was determined by BCA protein quantification kit (DNAbiotech Co.  Iran).

Histological study

Following formalin perfusion, the  brains of rats were removed from the skulls and post-fixed in 10% formalin. The fixed brain tissues were cut coronally from the brain re- gion 3.2 to 4.2 mm anterior of the interaural line  containing  substantia  nigra  (Paxinos &

Watson, 2007). These tissue slices were rou- tinely processed for light microscopy and then embedded in paraffin. By means of a microtome (Leica Microsystems, Wetzlar, Germany) the paraffin-embedded blocks were cut into the 5 µm coronal sections and three  slides  were  selected  with  at least 100

µm interval thickness. Afterwards, sections were deparaffinized by xylene (Merck, Ger- many), rehydrated through a graded ethanol series; eventually stained by 0.1 % cresyl vi- olet solution (Sigma-Aldrich, St. Louis, Mo, USA). The slides were observed under light microscope equipped with camera (YS2-T, Nikon, Tokyo, Japan) and ImageJ software (NIH, Maryland, USA).

Only Nissl stained neurons with  a  nucle- us, which contains visible nucleolus, within SNpc region were counted by a trained ob- server who was blind to the groups' status. By comparing the ipsilateral and contralateral cell count difference to the contralateral cell count in each rat, the percentage of  neuronal  cell loss was determined and considered as sample data to perform analysis (Browne et al., 2017).

Statistical analysis

To analysis of the extracted data between groups, the one-way analysis of variance (ANOVA) bootstrap method was used with Tukey’s post hoc test to do multiple compar- isons. Our extracted data presented the num- ber of rotations induced by apomorphine as behavioral testing. We also showed the lev- els of plasma Hcy in parallel to betaine ther- apy in rats and MDA levels in brain tissue  as a lipid peroxidation marker. Moreover, the percentage of neuronal cell loss under the effects of neurotoxin 6-OHDA or its vehicle was analyzed as histological evaluation. All the mentioned data were expressed as   mean

± SEM and the significance level was set at  P < 0.05.





There was no significant weight loss and mortality detected in any of the groups after operating gavage nor stereotactic  surgery.

Rotation numbers

The effect of betaine administration on behavioral presentation was evaluated by an- alyzing the net number of rotations induced by apomorphine over one hour rotation test- ing. There was no significant rotation di- versity between groups during behavioral assessment at the initiation of our animal re- search, while the relevant statistical analysis one week after stereotactic surgery was no- ticeable. Following 6-OHDA lesioning, apo- morphine induced significant contralateral rotations in the animals of the control group (P < 0.0001) and in the rats, which were un- der treatment of betaine with doses  50 mg/ kg (P = 0.002), and 100 mg/kg (P < 0.0001) during  comparison  with  the  sham  group. A significant reduction in rotations was re- corded from the rats under two weeks’ oral administration of 200 mg/kg betaine in com- parison with the control (P < 0.0001), betaine

50 mg/kg (P = 0.001), and betaine 100 mg/ kg (P < 0.0001) groups (F = 14.86). All data summarized in Fig. 1.

Biochemical evaluations

Treatments of rats with betaine  200 mg/  kg significantly decreased the plasma levels of Hcy as compared to the sham and control groups with p values of 0.028 and 0.017 re- spectively (Fig. 2). The non-significantly reduced plasma Hcy  levels  were  detected  in animals under administration of betaine with doses 50 and 100 mg/kg in comparison with the rats in sham and control groups    (F

= 5.43).

In terms of the measuring MDA levels in brain tissues, the statistical analysis revealed that in sham group and in animals of treat- ment group with betaine 200 mg/kg, the MDA levels that were high significantly de- creased as compared to the control and be- taine groups with doses  of 50 and 100 mg/ kg (F = 142.53, P < 0.0001). Non-significant upper MDA concentrations  were  detected  in the brain tissues of the rats in betaine 200 mg/kg group versus sham group (Fig.  3).






Figure 1. Net number of rotations (mean ± SEM) in experi- mental groups (n = 8) within one hour following IP injection of apomorphine 0.5 mg/kg, one week after stereotactic sur- gery. * Treatment with 200 mg/kg of betaine significantly associated with decreased net rotation numbers versus the control, betaine 50 mg/kg, and betaine 100 mg/kg groups (P < 0.05).






Figure 2. Homocysteine (Hcy) concentrations of plasma (Mean ± SEM) among our studied groups. * The animals under treatment of two-week course of betaine with dose of 200 mg/kg represented a significant reduction in the plasma levels of Hcy (µmol/l) as compared to the animals in sham (P = 0.028) and control (P = 0.017) groups, without betaine administration.










Figure 3. Brain tissue malondyaldehyde (MDA) concen- trations (Mean ± SEM) in experimental groups. * The in- tranigral injection of 6-OHDA in animals within the control group and the animals under treatment of betaine with doses of 50 and 100 mg/kg resulted in significantly elevated brain tissue MDA concentrations in comparison with the sham group (P < 0.05). ** MDA levels (nmol/mg protein) in the brain tissues of the rats under treatment of betaine 200 mg/ kg, in spite of 6-OHDA administration, were associated with a remarkable reduction in comparing with the control, and betaine 50 and 100 mg/kg groups (P < 0.05).



Histological examination

In consideration of histological assessment, our findings yield that the percentage of neu- ronal cell loss was significantly increased, under the effect of neurotoxin 6-OHDA, in the control, betaine 50 mg/kg, and betaine 100 mg/kg groups in comparison with the sham group (P < 0.0001). In the betaine 200 mg/kg group, the percentage of neuronal loss was meaningfully diminished  as  compared to the control and betaine groups with dos- es 50 mg/kg and 100 mg/kg (F = 55.68, P < 0.0001). However, the percentage of neuro- nal cell loss in rats with treatment of betaine 200 mg/kg was non-significantly higher than that of sham group (Fig. 4).


Previously, the neuroprotective effects of betaine have been shown in the animal mod- els for chronic cerebral hypoperfusion and memory  deficits  (Chai  et  al.,  2013;  Nie etal., 2016). Here, we demonstrated the neu- roprotective efficacy of betaine with dose of 200 mg/kg, but not 50 and 100 mg/kg, in an animal model of PD induced by 6-OHDA. Within our research, we evaluated the beta- ine effects from multiple perspectives com- prising behavioral, biochemical and histo- logical aspects.







Figure 4. The percent of neuronal loss in SNpc (Mean ± SEM) was determined by comparing the difference be- tween Nissl- stained neurons number of ipsilateral and contralateral sides of 6-OHDA injection to the neurons number of contralateral side. * Under influence of unilat- eral neurotoxin administration, the neuronal cell loss per- cent was significantly increased in the control, and betaine 50 mg/kg and 100 mg/kg animals in comparison with the rats in the sham group, which were unilaterally under the injection of vehicle (P < 0.05). ** A significant reduction of neuronal cell loss percent was detected in animals under treatment of betaine with dose of 200 mg/kg versus the control group (P < 0.05).



A  prominent  number  of  neurons   with- in SNpc are dopaminergic (Ip et al., 2017; Nair-Roberts et al., 2008). Destructing these dopaminergic neurons by administration of neurotoxins can, to some extent, mimic the PD pathophysiology, which has been pro- cessed as an animal model of PD in diverse research (Fabricius et al., 2017). In this view, unilateral intranigral injection  of  6-OHDA, as the most commonly used neurotoxin in rodents, exhibits prominent motor asymme- try caused by unilateral nigral dopaminergic neuronal loss and dopamine depletion in stri- atum. Unilateral dopamine depletion in stria-



tum subsequently alters the striatal dopamine D1 and D2 receptors sensitivity, unilaterally. The motor asymmetry, which is developed  by change in sensitivity of mentioned recep- tors, can be assessed by inspecting the num- ber of rotations under exposure of dopamine agonists that stimulate striatal dopamine re- ceptors either directly (i.e. apomorphine) or indirectly (i.e. amphetamine) (Konieczny et al., 2017). In this research, while performing rotation test by apomorphine one week after unilateral, left side, intranigral injection of 6-OHDA, a significantly decreased rotation number has been observed in rats with two weeks treatment of betaine 200 mg/kg com- pared to the control group (Fig. 1). The ame- lioration of turning behavior can be ascribed to the probable neuroprotective properties of betaine, in mentioned dose, against the neu- rodegenerative effects of 6-OHDA. On the one hand, the possible neuroprotection in- duced by betaine eventuated in preserving a more number of dopaminergic neurons that can lead to maintaining left nigrosteriatal do- pamine at a level in which the sensitivity of striatal dopamine receptors was not different enough from the contralateral side to gener- ate marked turning behavior, under stimula- tion of apomorphine.

Moreover, our histological evaluations, carried out in the control group, showed the partial lesioning of Nissl-stained neurons in SNpc. In this regard, the histological study revealed a significant reduction in  neuro-  nal loss percentage in betaine 200 mg/kg group in comparison with the control group (Fig. 4). As mentioned before, it has been demonstrated  that  the  prominent  number of Nissl-stained neurons within SNpc are dopaminergic and 6-OHDA, as a selective catecholaminergic neurotoxin, has toxic ef- fects  on  dopaminergic  cells  (Konieczny  etal., 2017; Nair-Roberts et al., 2008). In rel- evance, since we obtained more number of intact Nissl-stained neurons along with at- tenuated rotational behavior in betaine 200 mg/kg group, it can be concluded that beta- ine may protect the dopaminergic neurons toward neurotoxic effects of  6-OHDA.

As hinted above, several lines of evidence indicate that OS has an important role in in- ducing dopaminergic neuronal degeneration in PD and in animal model of PD under the influence of 6-OHDA. OS can induce cel- lular damages at the various compartments generating lipid peroxidation, protein, DNA and RNA oxidation (Blesa et al., 2015; Dias et al., 2013). During lipid  peroxidation, MDA can be formed, as secondary products, among producing the many different alde- hydes. Measuring MDA tissue levels, as a convenient biomarker for lipid peroxidation, has been widely used in different research (Ayala et al., 2014). Accordingly, in the pres- ent study, we evaluate the MDA levels in homogenized brain tissue with respect to the monitoring of OS, induced by neurotoxin. Our findings significantly revealed the de- creased levels of MDA in betaine 200 mg/kg group in comparison with the control group which can show the possible antioxidant role of betaine, with depicted dose, against OS induced by 6-OHDA. This result is in con- cordance with our mentioned behavioral and histological evaluations.

With taking into account Hcy properties which lead to cell damage via different path- ways such as inducing OS, and the influence of betaine on reducing hyperhomocystein- emia, it has been hypothetized that betaine can protect cells by detracting the Hcy levels (Alirezaei et al., 2015). Analysis of the ex- tracted data in the present research indicated a  significant  reduction  in  concentrations of




plasma Hcy in animals under administration of betaine with dose of 200 mg/kg as com- pared to the control and sham groups. Sta- tistically significant decreased levels of Hcy, under exposure of betaine, were associated with a meaningful reduction of MDA con- centrations in brain tissues as well as a signifi- cantly diminished neuronal cell loss percent- age in SNpc and remarkable  improvement  in motor asymmetry recorded during behav- ioral testing. However, as pointed out above, the findings of clinical trials concerning the influence of Hcy on pathophysiology of PD are controversial, several researches have verified a close relationship between hyper- homocysteinemia and PD (Xie et al., 2017; Zoccolella et al., 2010). Likewise, the results of our study on this model of PD highlighted the role of Hcy in PD pathogenesis.

Altogether, the extracted data in the pres- ent manuscript postulates the antioxidant neuroprotective effects of betaine in our ap- plied model of PD. Here, betaine represents the neuroprotective properties along with detracting the plasma levels of Hcy. In this view, by consideration of Hcy toxic effects,  it is suggested that betaine applies the neu- roprotection through effects on Hcy metab- olism. Nevertheless, it is not clear whether betaine performs its neuroprotective role solely via Hcy methylation or through addi- tional pathways from Hcy metabolism. This issue remains to be determined in the future research. Moreover, in order to have a better outline, the possible neuroprotective effects of betaine on underlying mechanisms of PD progression, in addition to OS, should be un- der evaluation.



The authors of this manuscript would like to  acknowledge  the  physiology   laboratory


and the animal house in Faculty of Veteri- nary Medicine, University of Tehran, Iran for cooperation.

Conflicts of Interest

The author declared no conflict of interest.

Informed Consent

This manuscript does not contain any stud- ies with human subjects performed by any of the authors.

Human and Animal Rights

All experiments were executed according to the Guide for the Care and Use of Lab- oratory Animals and were approved by the Institutional Animal  Ethics Committee.

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