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.
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).
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.
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).
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).
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.
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).
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).
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.
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.
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
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).
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.
The author declared no conflict of interest.
This manuscript does not contain any stud- ies with human subjects performed by any of the authors.
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.