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Ishankhodjaev Tokhir Mukhitdinovich, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Senior researcher, Laboratory of Metabolomics, Uzbekistan Academy of Sciences E-mail: tokhir50@gmail.com Zainutdinov Bokhodir Ravilovich, Acad. O. A. Sadykov Institute of Bioorganic Chemistry Senior researcher, Laboratory of Metabolomics, Uzbekistan Academy of Sciences E-mail: t.saatov@yandex.ru
Mustafakulov Mukhammadjon Abduvaliyevich, Junior researcher, Laboratory of Metabolomics, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences E-mail: mmustafakulov@bk.ru Ibragimov Zafar Zakirdjanovich, Senior researcher, Laboratory of Metabolomics, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences E-mail: zafar-biolog@mail.ru
Saatov Talat Saatovich, Professor, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Head of laboratory, Laboratory of Metabolomics, Uzbekistan Academy of Sciences E-mail: t.saatov@yandex.ru
STUDY ON SOME PARAMETERS OF LIPID METABOLISM IN THE CEREBRAL TISSUES OF RATS WITH THE ROTENONE - INDUCED MODEL OF PARKINSON'S DISEASE
Abstract. The work was initiated to study the role of lipids, lipid peroxidation processes and antioxidant enzymes of the cerebral tissues of rats with the rotenone-induced model of Parkinson's disease. An attempt to find correlations between the behavioral performance of rats and changes in lipid compositions of the brain regions within the various periods of experimentally induced Parkinson's disease was made. Changes in the behavioral performance of the animals were established to take place on the 2nd day after administration of rotenone, to increase peaking on the 4th day, and to decrease on the 9th day. The changes in the behavioral performance of the animals with the neurodegenerative disease in question was found to correlate with changes in lipid composition of their cerebral tissues within the various periods of the experimentally induced neurodegenerative disease.
Keywords: neurodegeneration, Parkinson's disease, behavioral tests, corpus striatum, lipids, phospholipids, cholesterol, lipid peroxidation, antioxidant enzymes.
Introduction. Today, degenerative nerve diseases, also called neurodegenerative diseases, are figuring larger among underlying causes of work decrement and mortality increase. However, regardless of the enormous aggregation of data on the factors underlying the onset of the diseases, understanding of mechanisms accounting for the processes is far from perfect. The extracellular deposition of amyloid-beta protein followed by formation of the senile plaques inducing apoptosis, causing
pathological activation of the microglia and initiation of the immune inflammation is suggested as a key aspect [1; 2; 3]. At the moment, the role of sphingomyelin cycle in apoptosis and a neurodegenerative disease's onset is under discussion [4; 5; 6]. Meanwhile, there is no the single viewpoint of the root cause for neurodegenerative diseases ultimately resulting in the death of brain cells. In our opinion, a disorder in lipid metabolism causing secondary changes in the biochemistry ofamyloid-beta
protein and tau protein, as well as in reactions of oxidative stress, is a potential cause for the onset of neurodegenerative diseases.
In the view of the aforesaid, the work was initiated to study the role of lipids, lipid peroxidation processes and antioxidant system of the cerebral tissues in the rotenone-induced model of Parkinson's disease.
Materials and methods
The experiment was conducted on the outbred rats weighing 250-300g kept on a standard diet. Prior to administration of rotenone, all rats were subjected to the cognitive tests [7; 8]. For the purposes of experiment, all animals were divided into three groups, to name the group including intact animals (IG), the exposure group (EG) consisting of animals receiving rotenone in the vegetable oil intraperitoneally in the dose of 2.5 mg per kg of animal's weight for 7 days and the control group consisting (CG) of animals intraperitoneally receiving the vegetable oil. The McGraw Stroke Index Scale was used to evaluate the neurological status of the animals; behavioral activity was assessed be means of the open field test on the 2nd, 4th and 9th day after rotenone administration. Materials for biochemical and histological investigation from the striatum were taken on the 12th day after rotenone administration. The Bligh and Dyer method was used for total lipid extraction [9]. Fractionation of lipids
Confirming the presence of PD symptoms, our findings in the open field test are consistent with the literature data reporting an elongation of the latent period in adaptation of animals to new conditions, and a reduction in the distance traveled in the playpen and as a consequence, the number of the line crossings, of the squares transversed and of the frequency of hear-dipping.
Our findings demonstrated that the main PD symptoms, such as retardation of motion and reduction in its frequency, as well as postural instability and unsteadiness of gait manifested as late as on the 2nd day after rotenone administration. The accessory ones, the rigidity, in particular, appeared on the 4th day; as to the resting tremor, the symptom could be seen in 10% of the animals on the late PD stages.
was performed by the thin-layer chromatography according to M. Kates [10]. Phospholipid quantification was performed as described elsewhere [11]. The cholesterol was determined as described elsewhere [12]. The total protein was determined with modified Lowry method according to Hartree [13]. Initial levels of the substrates reactive with thiobarbituric acid were determined as described elsewhere [14]. Activity ofantioxidant enzymes, such as catalase, superoxide dismutase and glutathione reductase was estimated as describes elsewhere [15; 16; 17]. Cary 60 spectrophotometer (Agilent Technologies, USA) was used to make optical measurements. Student's t-test was used to process the data.
Results and discussion
Prior to Parkinson's disease (PD) modeling, behavioral performance of animals was tested to evaluate main PD symptoms, such as hypokinesia, bradykinesia and oligokinesia, postural instability, unsteadiness of gait, and the accessory ones, to name muscle rigidity and tremor, which are not always present in experimental PD [18]. Total symptom intensity was scored (from 1 to 18), as well [19].
The 7-day administration of rotenone was demonstrated to cause some changes in the behavioral performance of the animals in the open field test (Table 1, Fig. 1).
The dynamics of behavioral performance in the animals early in the course of the rotenone - induced model of Parkinson's disease seems to be a result of changes in lipid compositions of dopamine receptors' (DR) rafts and synaptic compartments with the functional properties strongly determined by the lipid compositions of the nerve cell membranes.
In the view of the above, the lipid composition, lipid peroxidation and antioxidant enzymes' activity in the brain of animals from the control and the exposure group were studied. Table 2 demonstrated changes in lipid composition of the brain tissues of rats with the rotenone - induced model of Parkinson's disease on the 2nd, 4th and 9th day of rotenone administration.
Table 1. - Behavioral performance of animals in the open field test
Behavioral activity Groups of animals
Intact (n = 5) Control (n = 5) Exposure (n = 15)
Latent period (s) 52.3 ± 15.3 44.5 ± 20.3 90.6 ± 25.6
Distance traveled (m) 13.4 ± 7.8 12.5 ± 8.5 4.3 ± 2.1
Line crossings (n) 19.2 ± 2.0 17.3 ± 1.6 4.6 ± 1.1**
Squares transversed (n) 54.5 ± 16.4 52.3 ± 8.3 8.6 ± 4.2**
Groomings (n) 2.5 ± 0.5 2.4 ± 0.3 1.6 ± 0.2
Frequency of head-dipping 25.2 ± 5.4 20.3 ± 6.3 7.4 ± 3.1*
** statistically significant differences, significance level p < 0.05; * significance level p > 0.05.
Figure 1. Changes in behavioral activity of rats according to the McGraw Stroke Index Scale on the 2nd, 4th and 9th day after administration of rotenone: x axis -scores in the cognitive test, y axis - days of experiment
As our findings demonstrated, on the 2nd day after rotenone administration concentrations of cholesterol, lysophos-pholipids and phosphatidic acid (PA) tended to increase while those of sphingomyelin (SPH) fractions, phosphatidylserine (PS) and phosphatidylinositol (PI) appeared to decrease. Total phospholipids changed insignificantly. On the 4th day after rotenone administration, the tendency above seemed to preserve, but the range of changes was more significant than of those seen on the 2nd day (Table 2). Thus, cholesterol, ly-sophospholipids and PA can be seen to increase by 8%, 50% and 14%, respectively, both SPH and PS decreased by 15%, while PI reduced by 14%. There was 8% reduction in the total phospholipids.
Table 2.- Lipid composition of striatum regions of the rat brain on the 2nd, 4th and 9th day after rotenone administration
Phospholipids and lipids Control group Exposure group (n=15)
Time after administration 2nd day 4th day 9th day
^g of rotenone/g of tissue ^g of rotenone/g of tissue ^g of rotenone/g of tissue ^g of rotenone/g of tissue
Lysophospholipids 10.9 ± 0.5 12.5 ± 0.6 16.4 ± 0.3** 13.1 ± 0.4**
SPH 131.2 ± 3.8 120.7 ± 3.3 111.5 ± 3.2** 118.1 ± 3.1**
PC 589.38 ± 28.6 571.6 ± 25.6 554.0 ± 21.3 560.0 ± 20.6
PS 197.4 ± 9.5 177.7 ± 6.5 167.8 ± 5.6** 156.4 ± 5.1**
PI 91.7 ± 4.8 85.3 ± 3.2 78.9 ± 2.5 82.5 ± 3.1
PE 506.6 ± 22.4 491.4 ± 20.3 476.2 ± 15.6 486.3 ± 19.1
Cardiolipin 61.2 ± 3.1 60.0 ± 3.5 55.1 ± 2.5 58.1 ± 2.1
PA 15.4 ± 0.8 16.9 ± 1.0 17.6 ± 1.1 17.2 ± 1.2
Total phospholipids 1604.2 ± 32.6 1536.1 ± 30.3 1477.4 ± 35.2** 1491.8 ± 36.3*
TC (mg/g of tissue) 19.3 ± 0.3 19.8 ± 0.2 21.9 ± 0.1** 21.0 ± 0.3
Abbreviations: SPH - sphingomyelin, PC - phosphatidylcholine, PS - phosphatidylserine, PI - phosphatidylinositol, PE - phosphatidylethanolamine, PA - phosphatidic acid, TC - total cholesterol
** statistically significant differences, significance levelp < 0.05; * significance level p > 0.05.
Cholesterol was found to increase by 6% on the 9th day after rotenone administration. As compared to those in the control animals, concentrations of lysophospholipids and PA were increased, while those of SPH, PS and PI appeared to reduce, but their absolute values were lower than those observed on the 4th day after rotenone administration. Less significant changes in lipid composition of the brain tissues could be attributed to lower accessibility of the substrates by phospholipases and LPO.
The role of cholesterol, phospholipids and lipid peroxidation in the functioning of the nerves remains obscure. Some authors demonstrated the crucial role of cholesterol, phospho-lipids and other components in the neuroplasticity, neurility
and apoptosis of the nerve cells [20; 21; 22]. According to Isakina [23], concentrations of PI and PE reduced by 30.3 and 8.4%, respectively, in the stimulated nerve as compared with those in the non-stimulated one, and the data could be the evidence for close relationship between lipid components of nerve cell membranes and neurility.
The changes we have found in phospholipids seem to affect the neuroplasticity of the synaptic part and neurility and to be a cause for changes in the behavioral performance of the animals with the rotenone-induced Parkinson's disease. LPO and antioxidant enzymes are known to have a significant effect on the lipid composition of cell membranes. With that in mind, we studied LPO products and activity of some
antioxidant enzymes in the brain regions of interest within the various periods of rotenone administration. Changes in
LPO parameters and activity of antioxidant enzymes in the
%
regions of interest on the 2nd, 4th and 9th day after administration of rotenone (in %) can be seen in (Fig. 2).
250'
200'
150'
G-E>10 0 ■
50
I
L
i>
I
M DA C atalase SOD
an gr
■
2nd day
4th day
9 th day
Figure 2. Changes in LPO parameters and activity of antioxidant enzymes in the striatum regions of interest on the 2nd, 4th and 9th day after administration of rotenone (in percentage of control)
MDA - malondialdehyde;
SOD - superoxide dismutase;
GR -glutathione reductase;
CG - control group.
The LPO activation could be seen as late as on the 2nd day after rotenone administration with more significant stimulation on the 4th day and slight reduction on the 9th day. Presumably, this can be explained by lower accessibility of the substrates to LPO. In the context of reduction in total phospholipids upon experimental induction of Parkinson's disease, levels of phospholipids rich with the unsaturated fatty acids seem to reduce too.
According to Isakina [23], upon nerve stimulation LPO products increased, while concentrations of diene conjugates and malondialdehyde are 20.4 and 27.7% higher, respectively, than the control ones. This can be the evidence for the fact that certain levels of LPO products is constantly present in the nerve tissue, and only a concentration threshold crossing results in neurodegenerative diseases.
In accordance with our findings, changes in lipid composition of the brain regions of interest appear as late as on the early stages of experimentally induced PD. Intensification of the changes seems to have a significant effect on the sig-
nal reception and transduction by the nerve cells to result in changes ofbehavioral performance of the animals with experimentally induced PD. The increase in the levels of cholesterol in the cerebral tissues upon the PD model of interest seems to be associated with its unique property to impact and maintain the microviscosity of cell membranes.
Thus, levels of cholesterol and phospholipids, as well as LPO activity and the one of antioxidant system are the most significant factors having an impact on physical properties of cell membranes facilitating optimal conditions for the receptor and synaptic parts of neurons. Changes in the parameters upon experimentally induced model of the disease seem to be a cause for changes in the behavioral activity of the animals.
Conclusions
To sum up, concentrations of cholesterol and phospholipids, as well as activity of the LPO and antioxidant system are the factors producing the most significant effect on physical properties of cell membranes facilitating optimal conditions for the function of receptor and synaptic parts of a neuron. Changes of the parameters generated upon experimentally induced neurodegenerative disease in animals seem to be a cause for changes in their behavioral performance.
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