Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer's disease. It represents a serious medical and social issue due to the progressive course and the lack of treatment methods capable of stopping the neurodegenerative process development [1]. According to current estimates, number of patients with PD continues to increase steadily worldwide, due to both the population aging and improved disease diagnosis [2]. The main clinical signs of PD are as follows: bradykinesia, muscle rigidity, resting tremor, and postural instability. These appear themselves when there is a severe nervous system lesion manifested by the death of the substantia nigra dopaminergic (DA-) neurons and the decrease in dopamine (DA) levels in the striatum [3].

The disease process that begins long before the first motor disorder occurs is accompanied by the complex compensatory rearrangement in neurotransmitter systems [4]. DA is one of the most important neurotransmitters of the central nervous system that is involved in regulation of motor activity, learning, motivation, emotional behavior, and reinforcement mechanisms. DA ensures normal functioning of the basal ganglia and maintenance of motor control due to functioning of the nigrostriatal pathway [5].

However, it is difficult to assess the DA-system state based on the DA concentration itself. Important information is provided by studying changes in the levels of its metabolites, reflecting various neurotransmitter metabolism stages. 3,4-Dihydroxyphenylacetic acid (DOPAC) generated under the exposure to monoamine oxidase (MAO) represents one of the main intraneuronal DA metabolism products [6]. The DOPAC concentration reflects the DA synthesis and breakdown intensity, as well as the functional states of the DA-neuron axon terminals [7]. 3-Methoxytyramine (3-МТ) is derived from the DA released into the synaptic cleft. In contrast to DOPAC, this metabolite reflects extracellular metabolic processes and can be an indirect DA release marker. One more important metabolite is homovanillic acid (HVA) representing the DA catabolism end product. The HVA content characterizing the aggregate DA turnover is widely used as an integral indicator of the DA-system activity [6, 7]. Important information is also provided by assessing metabolic coefficients characterizing certain features of the DA turnover and allowing one to better estimate the effects on the DA-system [8].

In addition to the DA-system, PD can be associated with alterations in other monoaminergic systems of the brain [4]. Thus, in PD, special attention is paid to the serotonergic system (5-HT system), involved in regulation of mood, emotional state, cognitive functions, and sleep. The main serotonin (5-HT, 5-hydroxytryptamine) metabolite is 5-hydroxyindoleacetic acid (5-HIAA), the concentration of which reflects the 5-HT metabolic rate [9]. Assessment of the 5-HT and 5-HIAA levels makes it possible to estimate the 5-HT system involvement in the disease process and its possible contribution to the compensatory responses associated with the DA deficiency [9].

Despite significant advances in clinical trials, direct assessment of the dynamic changes in neurochemical processes in the patients’ brain remains extremely difficult. Most brain imaging methods allow one to assess only proxy indicators of the neurotransmitter system functioning, while it is virtually impossible to directly determine concentrations of neurotransmitters and their metabolites in brain tissues of patients with PD. Furthermore, it is difficult to assess earlystage PD due to long prodromal period [10]. In this regard, experimental models of the disease allowing for simulation of the main neurochemical and pathomorphological signs of the disease under controlled conditions are of special importance.

Models based on the 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) neurotoxin administration are among the most widely used. After entering the brain MPTP is converted into MPP+, which is selectively accumulated in DA-neurons, causing damage to these through inhibition of the mitochondrial respiratory complex I [11]. Today, the MPTP-based acute model designs are widely used [12]. Despite their efficacy, these are characterized by the rapid disease process in the brain and do not fully reproduce the slow progressive nature of PD.

In this regard, models of chronic PD course (chronic models) based on administration of the combination of MPTP and probenecid are of great interest. The probenecid administration ensures the more prolonged MPTP effect on the brain’s DA-system through reduction of its renal excretion [13]. In this stidy we used a classical protocol of administering 10 doses of MPTP/probenecid to mice [14], but a single toxin dose was lower (12 mg/kg), which made it possible to simulate earlier stages of the pathological process. Hence, the study aimed to assess the dynamic changes in the levels of DA, 5-HT and their principal metabolites in the striatum of mice with the chronic toxic PD model induced by administration of the combination of MPTP and probenecid.

METHODS

Chronic PD model induced by the course of MPTP/probenecid injections

Male C57BL/6 mice aged 8–10 weeks at baseline with the weight of 22–25 g were used for experiments. The animals were kept 7–8 ones per cage under standard vivarium conditions with ad libitum access to water and standard feed, 12-h light/ dark cycle (light on at 8:00, light off at 20:00). To minimize the animals’ nonspecific responses, these were pre-handled for 2 weeks. After that the animals were randomized to form groups considering such parameters, as the distance travelled and body weight. The distance travelled was assessed using the IC Capture 2.4 (The Imaging Source, LLC, USA) [15] and Minotaur (Neurobotix LLC, Russia) software tools.

Animals (n = 120) were divided into the control (NaCl, n = 60) and expreimental (MPTP, n = 60) groups, then into subgroups (n = 10) based on the time of withdrawal from the experiment: within a 1 week, 1 month, 1.5 months, 2 months, 3 months, and 6 months, respectively.

The combination of MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) (USP, Sigma-Aldrich, USA) and probenecid (USP, Sigma-Aldrich, USA) was used to create a chronic PD model [14, 16]. Thus, experimental animals were intraperitoneally administered probenecid at a dose of 250 mg/kg, and then, after 30 min, these were subcutaneously administered MPTP at a dose of 12 mg/kg. A total of 10 injections were received every 3.5 days (fig. 1).

Animals in the control groups were administered saline at each time point in accordance with a similar scheme. Control and experimental animals were withdrawn from the experiment by cervical dislocation at an appropriate time after the last MPTP/probenecid injection: a 1 week, 1 month, 1.5 months, 2 months, 3 months and 6 months. For further assessment, striatum samples were taken from each animal, frozen in liquid nitrogen, and then stored at –70 °С.

Determination of the levels of dopamine, serotonin, and their metabolites in the striatum

Catecholamines levels in the mouse striatum were determined by chromatography [17]. Homogenization and extraction of brain tissues were accomplished in the 0.1 М НСlO₄ solution supplemented by 250 pМ/mL of 3,4-dihydroxybenzylamine as an internal standard. Samples were centrifuged at 10 000 g for 15 min at 4 °C. The content of catecholamines and their metabolites in the supernatant was determined by highperformance liquid chromatography (ion-pair chromatography) with electrochemical detection using the System Gold liquid chromatography system (Beckman Coulter, Inc., USA) equipped with the Rheodyne 7125 injector (USA) with the 20 µL sampling loop. Test substances were separated using the reversed-phase column Nucleodur C18 Gravity, 4.6 × 250 mm, pore diameter 5 µm (Mashery-Nagel GmbH & Co. KG, Germany). The mobile phase (0.1 М citrate-phosphate buffer pH 3.0, containing 1.1 mМ of octanesulfonic acid, 0.1 mМ of EDTA, and 9% of acetonitrile) with the flow rate of 1 mL/min at the 200 atm pressure was achieved using the System Gold 125 pump (Beckman Coulter, Inc., USA). Measurement was performed using the EC3000 electrochemical detector (RECIPE Chemicals + Instruments GmbH, Germany) equipped with the ClinLab ECD cell, Sputnik model, with the glassy carbon working electrode (+0.85 V) and the Ag/AgCl reference electrode. Sample recording and chromatography data processing were performed using the Multichrom v.1.5 software tool (Ampersand, Russia).

The data about the levels of DA and its metabolites (DOPAC, 3-MT and HVA, as well as 5-HT and 5-HIAA) were obtained based on the analysis results. Fold changes in indicators for each compound at each timepoint was calculated as the ratio of values of the experimental and control groups using MS Excel 2016 (Microsoft, USA). The data were presented as the mean and standard deviation. Significance (p-value, p) was calculated using the nonparametric Mann–Whitney U-test with Statistica for Windows 8.0 (StatSoft, Inc. (2007), STATISTICA (version 8.0. www.statsoft.com, USA). The differences between groups were considered significant at р < 0.05. Metabolic coefficients were calculated as the DOPAC to DA, 3-МТ to DA, HVA to DA, 5-HT to 5-HIAA ratios; the corresponding metabolic coefficients in the control were taken as 100%.

RESULTS

When modeling PD in mice, the state of DA and 5-HT systems was assessed based on the analysis of changes in the levels of DA and its metabolites (DOPAC, 3-МТ, HVA), as well as 5-HT and 5-HIAA in the striatum of mice with chronic PD model. A profile of changes in the levels of these metabolites is provided in tab. 1.

As shown in tab. 1, the most pronounced neurochemical alterations were predictably reported for the DA-system and were largely associated with the levels of DA and its main metabolite (DOPAC). A similar trend of changes was reported for these throughout the experiment, except the timepoint of 1.5 months after the course of MPTP/probenecid injections. Thus, the most pronounced decrease in the levels of these compounds was reported a week after the course of MPTP/ probenecid injections: 2.8-fold for DA and 3.1-fold for DOPAC. It is noteworthy that the DA and DOPAC levels remained low for 3 months and were restored to the control level as late as by month 6 after the end of the course of MPTP/probenecid injections (tab. 1).

When modeling PD, no such rapid changes were reported for the levels of two other DA metabolites (3-МТ and HVA). As for HVA, the decrease in its levels in the striatum of mice was observed within a week and 3 months after the course of MPTP/probenecid injections, and 3-МТ levels did not differ from control values throughout the period of biochemical alteration recording.

Changes in the 5-HT system state in the striatum of mice with the chronic toxic model of PD were far less pronounced (tab. 1). A significant increase in the levels of 5-HT and 5-HIAA was observed within a month, and the decrease in 5-HIAA levels was reported 3 months after the course of MPTP/ probenecid injections.

The analysis of changes in the 5-HT and 5-HIAA turnover throughout the period of alteration recording was conducted. Metabolic coefficients reflecting the dynamic changes in the 5-HT metabolism were calculated (tab. 2), along with the extracellular and intracellular DA metabolism features (tab. 2 and fig. 2). It should be noted that the 5-HT turnover assessment revealed no significant alterations throughout the period of indicator recording, except the timepoint of 3 months, at which the decrease in 5-HT turnover by 69.5% relative to the control was reported.

According to the graph presented in fig. 2, the most pronounced increase in DA turnover compared to the control was reported for 3-МТ/DA and HVA/DA; it persisted for up to 3 months after the course of MPTP/probenecid injections and was back to normal by 6 months. Another trend of changes was reported for DOPAC/DA. The DA turnover acceleration based on this indicator was observed only 1.5 months after the course of MPTP/probenecid injections; it was 134.98% relative to the control. At other timepoints the DA turnover either showed no differences from the control (2 months after the course of MPTP/probenecid injections), or was slightly lower compared to the control values a week, a month, 3 months, and 6 months after the course of MPTP/probenecid injections (within 20% relative to the control).

DISCUSSION

In this study, we assessed the DA- and 5-HT metabolism in the striatum of mice with the chronic PD model allowing one to primarily assess the DA-system state when creating a chronic model of early-stage PD. In our study, we used a protocol based on the well-known scheme of administering 10 doses of the MPTP/probenecid combination [14], but it involved lower single toxin doses, which allow for reproduction of early-stage symptomatic PD with acute administration of the medicine [18]. Such a modified protocol of the classical chronic PD model enables simulation of earlier stages of the disease pathogenesis.

The most pronounced decrease in striatal DA levels was reported a week and a month after the last MPTP/ probenecid injection (by 63.6 and 41.5% relative to the control, respectively) (tab. 1, fig. 2), what can be considered as the development of the phenotype of the PD early clinical or late prodromal stage. A similar decrease in the levels of the main DA-system neurotransmitter in the striatum of patients is typical for early-stage symptomatic PD (Hoehn and Yahr stage 0–1), which can vary between 45.3% based on the recent data [19] and 60–80% reported in earlier studies [20]. Our data are also consistent with the data obtained using other models simulating the early-stage Parkinson-like phenotype in both rodents and primates [18, 21, 22]. It is noteworthy that the decreased DA levels persist for up to 3 months after the course of MPTP/probenecid injections and are restored to the control levels only by 6 months. On the one hand, the observed pattern of changes in DA levels suggests high plasticity of the nigrostriatal system and the ability of surviving neurons to partially compensate the loss of axon terminals of DA-neurons in mice with the MPTP-induced Parkinson-like phenotype [23, 24]. On the other hand, the reported trend of slow DA level increase can result from restoration of function of DA-neurons affected by degeneration [25]. Thus, the period of one week to 3 months after the course of MPTP/probenecid injections can be considered as an analogue of the early symptomatic or late pre-symptomatic PD stage, which opens the prospects for testing and therapy development.

Changes in striatal DOPAC levels largely repeated the trend of changes in DA levels (tab. 1) and were characterized by the decrease throughout 3 months after the course of MPTP/ probenecid injections. It is well known that DOPAC is the main intraneuronal DA metabolite generated under the exposure to MAO after DA reuptake and intracellular metabolism [7]. It has been shown that the decrease in DOPAC levels precedes the death of DA-neurons, being a marker of the early PD pathogenesis [26]. DOPAC is also closely correlated with the state of preservation of DA-axonal terminals; it can be used as a sensitive indicator of the nigrostriatal system functional state [27]. The decrease in DOPAC levels throughout 3 months after the course of MPTP/probenecid injections can be considered as an early manifestation of the DA-system neurodegeneration.

HVA is the DA catabolism end product. HVA levels reflect the aggregate DA metabolism activity. The decrease in HVA levels during the early period after the course of MPTP/probenecid injections corresponds to the period of the maximum DA deficiency and indicates the DA-system dysfunction in the striatum. The detected decrease in HVA levels at the timepoint of 3 months suggests that, despite the onset of compensatory processes and gradual restoration of the DA-system functioning, the DA metabolism remains impaired (tab. 1).

Additional information about the DA-system state is provided by the analysis of metabolic coefficients DOPAC/ DA, 3-МТ/DA, and HVA/DA (tab. 2, fig. 2). These indicators are used in experimental neurochemistry as markers of the intensity of different aspects of the DA turnover [28]. Thus, the DOPAC/DA metabolic coefficient characterizes intracellular DA metabolism, 3-МТ/DA characterizes extracellular DA turnover, and HVA/DA characterizes the aggregate DA turnover. 

The dynamic changes in these indicators throughout the first month after the course of MPTP/probenecid injections are significantly different; there is a decrease in the intracellular DA turnover (based on DOPAC/DA) (89.34 and 87.12%) and a sharp increase in the extracellular DA turnover, suggested by the 3-МТ/DA increase (228.17 and 176.14%) relative to the control at the timepoints of 1 week and 1 month, respectively (tab. 2, fig. 2). Our data are consistent with the independent research data showing that in PD the DA-system functioning is accompanied by the DA metabolism rearrangement and changes in functional activity of surviving neurons [7]. Our findings suggest that the decrease in the intracellular DA turnover can be associated not with the metabolism intensity decrease itself, but with the fact that DA is largely preserved for recirculation and the release into the synaptic cleft to preserve DA transmission. Furthermore, a sharp increase in the 3-МТ/DA and HVA/DA metabolic coefficients indicates the extracellular DA catabolism enhancement (i.e. enhancement during the synaptic signal transmission), suggesting the development of neurodegenerative processes and DA metabolism impairment in the synaptic cleft. Similar alterations are reported in the studies invoving both chronic MPTP/probenecid-induced model and other experimental parkinsonism models, in which the DA content restoration was not accompanied by the complete restoration of its metabolism and was considered as a manifestation of persistent DA-system dysfunction [14, 23]. We should also consider the timepoint of 1.5 months, at which a significant increase in all three metabolic coefficients was revealed and, accordingly, the most pronounced acceleration of DA turnover both in the cell and in the synaptic cleft (tab. 2, fig. 2). These data suggest the development of severe neurodegenerative changes. Such changes in DA metabolism indicators were previously described in works on PD modeling, as well as in studies of patients with various disease stages [7]. It is noteworthy that indicators of extracellular DA turnover and aggregate DA turnover are back to normal only by 6 months after the course of MPTP/probenecid injections, while the DA intracellular turnover remains decreased (by 19.25% relative to the control) as late as half a year after the end of the course of MPTP/probenecid injections, which suggests that there is no complete restoration of the DA turnover intracellular component.

Changes in the 5-HT system were far less pronounced compared to that in the DA-system (tab. 1 and tab. 2). However, a significant increase in 5-HT and 5-HIAA compared to the control a month after the course of MPTP/probenecid injections suggests that the 5-HT neurons are involved in adaptation to DA deficiency in the striatum. Currently, it is believed that the 5-HT system can contribute to the DA-system impairment compensation through enhancement of the basal ganglia activity and the DA release from the surviving axon terminals [29].

CONCLUSIONS

Thus, attention should be paid to the following features of changes occurring in the striatum at early stages of the Parkinson-like phenotype development. First, the data obtained indicate the decrease in the total DA metabolism in both neuronal axonal terminals and the synaptic cleft, which reflects neurodegenerative effects on these structures in the first month after the course of MPTP/probenecid injections. Severe DA-system dysfunction is also indicated by the increase in the levels 5-HT and its metabolite for compensation of DA deficiency in the striatum. Second, changes in the DOPAC/ DA ratio can be considered as a compensatory mechanism aimed to preserve the main function of DA-neurons, i.e. signal transmission through the release of available DA into the synaptic cleft, except for the timepoint of 1.5 months, at which the most severe effects of neurodegenerative factors were reported. Third, we can say that the model obtained makes it possible to extend the window for neurodegenerative alteration manifestations to 3 months, which is a significant advantage over acute and subchronic models and allows for the study of early neurodegeneration stages over a significantly longer time. Fourth, the data obtained once again demonstrate the complex pattern of the effects of neurodegenerative and compensatory processes occurring at the earliest stages of neurodegeneration in PD.