INTRODUCTION:

Parkinson’s disease (PD) could be a destroying neurological infection causing tremor, inflexibility, bradykinesia and stride impedance.¹Agreeing to WHO, PD is characterized as unremitting dynamic neurodegenerative clutter of guileful onset, characterized by the nearness of overwhelmingly engine symptomatology, bradykinesia, rest, tremor, inflexibility and postural unsetting influences. It is additionally related with a differing qualities of non-motor indications, which at the side late-onset engine side effects (such as postural insecurity and falls, solidifying of stride, discourse and gulping troubles).²

PD torments 53 million individuals and brought about in around 103,000 passing all inclusive. Its top age of onset is within the 60s (run is 35 to 85 a long time), and the course of the aliment ranges between 10 and 25 a long time. Familial clustering of autosomal overwhelmimg and latent shapes of PD contain 5% of cases. These are characterized by an prior age of onset (regularly some time recently age 50 a long time) and a longer course than the more common place “sporadic” PD. Chance components incorporate a positive family history, male sexual orientation, head damage, introduction to pesticides, utilization of well water and provincial living. Components connected to a diminished rate of PD incorporate coffee drinking, smoking, utilize of non-steroidal anti-inflammatory drugs.³

The essential side effects of parkinson’s infection result from incredibly diminished movement of dopamine emitting cells caused by cells caused by cell passing within the pars compact a locale of the Substantia nigra. There are five major pathways with in the brain interfacing other brain regions with the basal ganglia. These are known as the engine, oculo-motor, associative, limbic and orbitofrontal circuits, with names showing the most projection zone of each circuit.

6-OHDA utilizes the same catecholamine transport framework as dopamine. This prepare will lead to exceptionally particular harm through oxidative stretch to dopaminergic neurons. Because it neurotoxic to the brain, 6-OHDA must be managed by intracerebral or intraventricular infusions since it is incapable to cross the blood brain obstruction. The required specificity is accomplished by sterotaxically focusing on 6-OHDA to the Substatina nigra, the ventral tegmental range, the nigrostrital tract or the striatum. The test parkinsonism show related with 6-OHDA has been delivered in numerous species, counting mice, rodent, cat and non- humans primates. The foremost commonly used rat for 6-OHDA parkinsonism demonstrate may be a rodent, dueto the well built up stereotaxic procedures and sensible fetched. As their name indicates kinase inhibitors are designed to inhibit protein kinases.⁴

Kinases are proteins that catalyze the expansion of phosphate bunches to other proteins. This phosphorylation actuates or deactivates the target proteins, serving as an or off flag for cellular capacities such as atomic intuitive, flag transduction and localization inside the cell of undeeded proteins.⁵

MATERIALS AND METHODS:

Animals:

Healthy, male wistar rats (180-220g) were obtained from the central animal house facility from J.S.S College of Pharmacy, Ootacamund, Tamilnadu. The animals were kept in a well large spacious, hygienic polypropylene cages during the course of the experimental period. The animals were fed with water and rat feed ad libitum. All the experiments were performed after obtaining prior approval from CPCSEA and IAEC. The animals were housed in suitable environmental conditions. Approval No: JSSCP/IAEC/M.PHARM/PH.COLOGY/02/2016-17. Animals are divided into four groups each group has six animals Group l- vehicle control, Group ll-6 OHDA, Group-lll-6 OHDA+L-DOPA(6mg/kg), Group lV-6 OHDA+ Imatinib (10mg/kg).

Surgical Procedure⁶:

Animals were anaesthatised with ketamine (100mg/kg ip), xylaxine (15mg/kg im) and then placed in a sterling stereotaxic apparatus. The scalp was retracted and unilateral holes were drilled in the skull above the injection site. 6-OHDA lesions were made in an identical manner expect that vehicle alone was injected. Three weeks after the lesion, the animals ability to rotate in response to apomorphine (0.5mg/kg, s.c) was tested. Contralateral rotations induced by apomorphine were measured only animals showing at least 7 turns/min in both tests were included in this study. After the induction of parkinsonism (3week) the animals were treated with L-DOPA (6mg/kg), dose of imatinib (10mg/kg s.c) according to the groups of the animal for 14 days.

BEHAVIOURAL EVALUATION:

Circling behavior:

At the end of the treatment period the animals were tested for circling behavior. Circling behavior was induced by 0.5 mg/kg apomorphine respectively. The animals were observed for 10 minutes period for counting circling behavior. During observational period the animals were not disturbed. The number of full and counter clockwise turns were observed for ten minutes among different groups. The total number of circle for ten minutes were recorded and compared with control groups.⁷

Grip strength:

Rotarod has been used to evaluate muscle grip strength by testing the ability of rats to remain on revolving rod. The apparatus has a horizontal rough metal rod of 3 cm diameter attached to a motor with variable speed. This 70cm long rod was divided into four sections by wooden partitions. First rotarod apparatus was turned on then selected 20 rpm as an appropriate speed. Each rat was given five trials before the actual readings was taken. The animal was placed individually one by one on the rotating rod. The fall of time was noted when animal falls from the rotating rod and then the fall off time of animals were compared in treated group.^{8, 9, 10}

Catatonia:

The major clinical symptoms of parkinson’s disease includes difficulty to move and change the posture (akinesia and rigidity) and tremors. So by this parameter we could observe the severity of catatonia as followed Stage I-Rat moves normally when placed on table =(Score-0) Stage II- Rat moves only when touched/pushed=(Score-0.5) Stage III-Rat placed on the table with front paws set at least on a 3cm high block fails to correct the posture in 10sec=(Score-0.5 for each paw total score-1) Stage IV- Rat fails to remove when front paws are placed alternately on 9cm block=(score-1 for each paw total score-2) Thus for a single rat maximum possible score would be 3.5 revealing total catatonia.^{11, 12}

BIOCHEMICAL EVALUATION:

Estimation of total protein Lowry’s method¹³:

Extraction of protein from sample-Extraction was carried out with buffer used for the enzyme assay.500 mg of the brain sample was weighed and homogenized with 5-10ml of the buffer. Then homogenized was centrifuged and the supernatant was used for the protein estimation.

Estimation of glutathione reductase level ^{14, 15}:

GSH was measured enzymatically by the method described by Owen. The striatal were homogenized in ice cold per chloric acid (0.2 M) containing 0.01% EDTA. The homogenate was centrifuged at 10, 000rpm at 40C for 10 min. The enzymatic reaction was started by adding 200µl of clear supernatant in a spectrophotometric cuvette containing 500µl of 0.3 mm reduced NADPH, 100µl of 6mM DTNB and 10µl of 25units/ml glutathione reductase(all reagents freshly prepared in phosphate buffer at Ph 7.5). The absorbance was measured over a period of 3min at 412 nm at 300C. The GSH levels was determined by comparing the change of absorbance ΔA of standard GSH.

Estimation of Lipid peroxidation^{16, 17}:

Briefly, tissue homogenate (0.2 mL), 0.2 mL of 8.1% SDS, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% TBA were added. The volume of the mixture was made up to 4 ml with distilled water and then heated at 95°C on a water bath for 60 min using glass balls as condenser. After incubation the tubes were cooled to room temperature and final volume was made to 5 ml in each tube. 5 ml of butanol: pyridine (15:1) mixture was added and the contents were vortexed thoroughly for 2 min. After centrifugation at 3000 rpm for 10 min, the upper organic layer was taken and its OD was taken at 532 nm against an appropriate blank without the sample The levels of lipid peroxides can be expressed as n moles of TBARS /mg protein using an extinction coefficient of 1.56×105 ML cm-1.

Estimation of the rat brain calcium level¹⁸:

The brain samples were immediately weighed on aluminum foil and then dried at115 0C for 20 h and weighed again. Dry samples were then ashed in a silica crucible by gradual heating up to 550 0C for 24 h. The ash was dissolved in a variable volume of 3 M nitric acid to ensure optimum concentrations of analytes for atomic absorption measurements.

The concentration of calcium present in the supernatant was determined by atomic absorption spectroscopy. The standards of different Ca2+ concentrations (i.e., 1, 1.5, 2 and 2.5 μg/ml) were prepared from stock standard. The standards and samples were read against the blank solution420nm. The absorbance of samples, standards and blank were noted. The concentration of calcium in the different groups brains were calculated by reading from the standard curve.

RESULTS:

Effect of imatinib of rotational behavior in rats:

In this study the circling behavior of the animals after receiving the vehicle control, standard and the test group were observed in 6OHDA induced parkinson’s model. The result of the study is given in the table Two weeks after intrastriatal injection of 6OHDA rats exhibited rotational behavioral towards the opposite side of the lesion (contralateral rotation) after apomorphine administration significant (P<0.001) increase in the number of apomorphine induced rotation were observed in 6OHDA lesioned group compared to vehicle control When compared with the 6-OHDA control the number of contralateral turns in 10mins was significantly (P<0.001) reduced for L-DOPA and Imatinib.

Fig1: Effect of Imatinib on quantification of circling behavior in rats

Values are mean± SEM n=6 in each group *** P<0.001 when compared with vehicle control group

###P<0.001 when compared with 6OHDA controlOne way ANOVA followed by Bonferroni multiple comparison test

Fig2:Effect of Imatinib in experimental groups by using rotarod apparatus

values are mean ± SEM n=6 in each group, ***P<0.001 when compared with vehicle control group

### P<0.001 when compared with 6 OHDA control, One way ANOVA followed by Bonferroni multiple comparisons test.

Effect of Imatinib of experimental groups by using rotarod apparatus:

In the study the retention time of the animals by rotarod apparatus after receiving of vehicle, standard and the test were observed in 6OHDA induced parkinson’s model. The results of the study are given in table Rats exhibited altered behavioral activity after intrastriatal injection of 6OHDA and significantly (P<0.001) decreased the retention time were observed in 6OHDA lesioned group compared to vehicle control when compared with the control the retention time was significantly increased for L-DOPA and Imatinib(P<0.001)treated group.

Effect of Imatinib in experimental groups on cataleptic activity of experimental groups:

In the study the catalepsy scores were calculated after receiving of vehicle standard and the test compounds in 6OHDA induced parkinson’s model. The results of the study are given in table significant (P<0.001) reduction in the cataleptic score were observed in 6OHDA lesioned group compared to vehicle control. When compared with the 6 OHDA control there were significant reduction in the cataleptic score for L-DOPA and Imatinib (P<0.001) treated group.

Fig3:Effect of Imatinib of experimental groups on cataleptic activity

Values are mean ±SEM n= 6 in each group***P<0.001 when compared with vehicle control group

###P<0.001 when compared with 6OHDA control One way ANOVA followed by Bonferroni multiple comparisons test

Effect of Imatinib of experimental groups of total protein concentration in brain:

In this study the total protein contents were determined from the rat brain by biurette method after receiving vehicle, standard and the test compounds in 6OHDA induced parkinson’s model. The result of the study is given in table significant (P<0.001) decrease in the total protein concentration is observed in 6 OHDA lesioned group compared to vehicle control. When compared with the 6OHDA control the dopamine concentration was significantly increased for L DOPA and Imatinib (P<0.001) treated groups.

Fig4:Effect of Imatinib in experimental groups on total protein concentration

Values are mean ±SEM n=6 in each group *** P<0.001 when compared with vehicle control group

### P<0.001 when compared with 6OHDA control One way ANOVA followed by Bonferroni multiple comparison test

Effect of Imatinib in experimental groups on reduced glutathione:

In the study reduced glutathione levels were determined in vehicle, standard and the test compounds treated rat brain in 6Ohda induced parkinson’s model. The results of the study is given in the table significant (P<0.001) decrease in the glutathione is observed in 6OHDA lesioned group compared to vehicle control when compared with the 6 OHDA control the dopamine concentration was significantly increased for L DOPA and Imatinib (P<0.001).

Fig 5:Effect of experimental groups in reduced glutathione

Values are mean ±SEM n=6 in each group***P<0.001 when compared with vehicle control group

###P<0.001 when compared with 6 OHDA control One way ANOVA followed by Bonferroni multiple comparisons test

Effect of Imatinib in experimental groups on lipid peroxidation level:

In the study MDA levels were determined in the rat brain in vehicle, standard and the test compounds treated groups in 6 OHDA induced parkinson’s model. The results of the study are given in table significantly (P<0.001) increase in the MDA levels is observed in 6 OHDA lesioned group compared to vehicle control. When compared with the 6 OHDA control the dopamine concentration was significantly increased for L DOPA and Imatinib (P<0.001).

Fig 6:Effect of Imatinib of experimental groups on lipid peroxidation

Values are mean ±SEM n=6 in each group *** P<0.001 when compared with vehicle control group

###P<0.001 when compared with 6OHDA One way ANOVA followed by Bonferroni multiple comparisons test.

Fig 7:Effect of Imatinib on brain calcium in rat brain

values are mean ±SEM n=6 in each group ***P<0.001 when compared with vehicle control group

### P<0.001 when compared with 6OHDA controlOne way ANOVA followed by Bonferroni multiple comparisons test

Effect of Imatinib on calcium level in rat brain:

The brain calcium level was significantly increased in 6-OHDA control when compared with vehicle control L-DOPA and Imatinib treated group significantly decreased the brain calcium level when compared to 6-OHDA control group given in table significant (P<0.001) increase in the calcium levels is observed in 6-OHDA lesioned group compared to vehicle control. When compared with the 6-OHDA control the dopamine concentration was significantly reduced for L-DOPA and Imatinib (P<0.001) treated groups.

DISCUSSION:

Anti Parkinson agents used in PD treatment partially relive the symptoms of this disease, but they are not able to block dopaminergic neurodegeneration thus the disease continues to progress. Currently there is a great demand for new therapies that prevent neuronal death. New therapeutic strategies for PD must identify compounds that are neuroprotective and able to cross the BBB to produce the desired effects without causing adverse side effects. ¹⁹

The pharmacology of Imatinib in 6-OHDA induced PD was analysed. The fourteen days treatment with the tyrosine kinase inhibitor led to positive results with its preclinical anti parkinson’s efficacy. In this study different groups of animals were treated with vehicle control 6-OHDAcontrol, L-DOPA, Imatinib (10mg/kg).

In this study apomorphine induced circling behavior was demonstrated. Apomorphine is a mixed (D1 andD2) dopamine receptor agonist that does not share metabolic pathways with L-DOPA and presumably acts by direct stimulation of dopamine receptor. ²⁰ In this study the circling controversial to the lesion side following the administration of dopamine agonist result from stimulation of dopamine receptor. The 6-OHDA control rats showed a greater level of circling behavior and other treatment group including L-DOPA and Imatinib treated rats might be replenishing dopamine or might have protected dopaminergic neurons in substantia nigral. Further it could presumably suggest the confirmation of nigral lesion in all the treatment groups. In this study it was observed that Imatinib decreased the number of contralateral rotations of lesioned rats, suggestive of a neuroprotective effect.²¹

Rotarod experiment demonstrated the impairment in the motor function and coordination in parkinson’s rats. 6OHDA control group showed less retention time on the rotating rod when compared to control suggesting impairment in their ability to integrate sensory input with appropriate motor commands to balance their posture. Lack of motor co-ordination and maintenance of normal limb posture has been reported in PD condition. The treatment with imatinib in rats increase the retention time when compare to control rats. This evaluation revealed the efficacy of tyrosine kinase inhibitor in increasing muscle tone and thus could co relate with possible action on CNS.

In the present study 6-OHDA induced group shown increase in catalepsy and imatinib reversed the catalepsy induced by 6-OHDA induction.

In this study it was found Imatinib caused a pronounced increase in dopamine levels in Subtantia nigra regions of 6-OHDA induced rats and it could be a result of protection of dopaminergic neurons by the drug L-DOPA treatment hiked the dopamine levels and it is easily demonstrated by its high concentration after treatment. The beneficial roles in retaining dopamine levels demonstrated the protection of nigral neuron by imatinib.

It was found that protein concentration reduced significantly in 6 OHDA induced group where as the test drug could increase that total protein concentration in 6 OHDA lesioned rats.

In this study different in vivo antioxidant levels were estimated and determined whether the drug treatment could elevate or suppress the natural antioxidant enzymes intracellularly in Substantia nigral region. With respect to this study the finding showed that the Imatinib treatment could maintain the normal range of natural antioxidant enzymes in brain tissue. This has given us knowledge of possible role of antioxidant enzymes in protecting the mitochondrial activities and reduces in vivo oxidative stress in neurons.

The intracellular mitochondrial calcium levels were assessed in all treatment groups. From that result, it was clear that imatinib could significantly reduced intracellular calcium levels and retain the mitochondrial activity.

Since calcium is one of the pro-aggregatory factor in catalytic break down of natural protein in to their fragments the possibility of calcium induced degeneration of transferrin might be reduced with Imatinib treatment.

CONCLUSION:

In the present study anti Parkinson’s disease effect of a tyrosine kinase inhibitor Imatinib is evaluated in 6 OHDA lesioned rat model. 6 OHDA induced lesion was confirmed by apomorphine induced rotational behavior of rats. The anti-parkinsonian effect of Imatinib was evaluated by carrying out various behavioral studies including rotational behavior test, rotarod test, test of catalepsy. Various biochemical parameters like estimation of total protein, estimation of reduced glutathione level, estimation of lipid per oxidation level and determination of calcium concentration in rat brain were also carried out.

The behavioral and biochemical estimations revealed that the tyrosinekinase inhibitor, Imatinib has showed siginificant anti parkinson’s activity in 6 OHDA lesioned rat models. The estimated parameters altered the normal behavior of the animals and the drug treatment protected the diseased brain of rat. Therefore further detailed molecular studies with this drugs in anti-parkinson’s pharmacology and proceeded.

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Received on 30.11.2018 Modified on 31.12.2018

Research J. Pharm. and Tech 2019; 12(8): 3745-3750.

DOI: 10.5958/0974-360X.2019.00641.3