Animals
This research was conducted on 49 wistar male rats weighting between 200 and 250 grams , following the ethical principles of working with laboratory animals . First , the animals were kept under conditions of 12 hours of darkness and 12 hours of light ( lighting time in 6 am ) and kept individually in transparent racks at a humidity of 45-55% and a temperature of 22 - 25 degrees centigrade in the laboratory animal keeping center of Semnan University of Medical Sciences .
Compressed food and city water were used to feed the animals . food and water were provided to them without any restrictions . the experimental protocols were approved by the Ethics Committee of Semnan University of Medical Sciences ( IR. SEMUMS.REC.1399.199 ) and animal care was according to the NIH Guide for the care and use of laboratory animals . 49 wistar male rats were randomly divided into the 7 groups ( n = 7 per group ) including the control , sham , healthy , Parkinsonism with levodopa and benserazide treatment , Parkinsonism with levodopa and benserazide and nortriptyline 5 mg / kg treatment , Parkinsonism with levodopa and benserazide and nortriptyline 10 mg / kg treatment , Parkinsonism with levodopa and benserazide and nortriptyline 20 mg / kg treatment .
Grouping
The rats were randomly divided into seven groups of seven each .
- Sham group : stereotaxic surgery was performed on this group , but neurotoxin injection was not done , so they did not develop parkinsonism , did not receive treatment , and were only kept during the 2 weeks of treatment of the other groups .
- Control group : this group did not receive any treatment two weeks after the injection of 6-ohda through stereotaxic and performing the rotational test and confirming the parkinsonian model , and only during the 2 weeks of treatment of the other groups , the vehicle ( drug solvent which is normal saline ) they receive daily by intraperitoneal injection .
- Treatment group ( P + N5 ) : 2 weeks after the injection of 6-OHDA and the elevated body swing test and confirmation of the parkinsonian model , this group was treated with nortriptyline at a dose of 5 mg/kg along with Levodopa at a dose of 10 mg/kg for 2 weeks and they received Benserazide at a dose of 2.5 mg/kg as a daily intraperitoneal injection .
- Treatment group ( P + N10 ) : 2 weeks after the injection of 6-OHDA and performing the elevated body swing test and confirming the parkinsonian model , this group was treated with Nortriptyline at a dose of 10 mg/kg along with Levodopa at a dose of 10 mg/kg for 2 weeks and they received Benserazide at a dose of 2.5 mg/kg as a daily intraperitoneal injection .
- Treatment group ( P + N20 ) : 2 weeks after the injection of 6-OHDA and performing the and elevated body swing test confirming the parkinsonian model , this group was treated with nortriptyline at a dose of 20 mg/kg along with Levodopa at a dose of 10 mg/kg for 2 weeks and they received Benserazide at a dose of 2.5 mg/kg as a daily intraperitoneal injection .
- Drug treatment group ( P + L ) : this group received only Levodopa treatment with a dose of 10 mg/kg and Benserazide with a dose of 2.5 mg/kg for 2 weeks , they received intraperitoneally daily .
- Completely healthy group without surgery .
Stereotaxic surgery
The rats were anesthetized with an intraperitoneal injection of mixed ketamine ( 100 mg/kg ) and xylazine ( 20 mg/kg ) and then , the rats were placed in a stereotax frame ( Stoelting, USA ) and fixed on the surgical table by the mouth piece and intra-ear rods , then the head of the rat was disinfected with alcohol cotton and after shaving the desired area with a scalpel , we made a longitudinal incision from between the two eyes to between the ears .
After removing the connective tissue from the skull and identifying the bregma area , we set the device pointer on the desired part and according to the coordinates extracted from the Paxinus and Watson atlas , the coordinates (Substantia Nigra Compacta , SNC) on the left side ( anterior , AP = - 5.0 4 mm , lateral , ML = - 2 mm , ventral , DV = - 8.1 mm) for destruction with neurotoxin 6-OHDA it was found [20, 21] .
Neurotoxin injection
After piercing the marked site with a hand drill , the animals were slowly injected with a 10 µl Hamilton syringe , 2 µl of 0.9% saline solution containing 4 µg/µl of neurotoxin 6-OHDA and 0.2% ascorbic acid by hand pump . five minutes after the injection , the syringe was slowly removed from the brain and after disinfection , the surgical site was sutured [20, 21] .
After stereotaxic , the injection site was checked and confirmed by fixing the brain tissue in 10% formalin and preparing a tissue section ( figure 1 ) .
Treatment phase
In all parkinson groups , seven days after the parkinsonization process by 6-OHDA , the models should be confirmed by elevated body swing test . then the treatment period with drugs and nortriptyline begins , after seven days of the treatment period , the elevated body swing test is taken from the rats again . fourteen days after the start of the treatment , the elevated body swing test is taken from the rats again , fourteen days after treatment , behavioral tests are taken from all animals . they are killed under anesthesia on the sixteenth day after the start of treatment .
Statistics methods
All behavioral tests were performed blindly . After confirming the statistical assumptions of normality and equality of variance, the groups were compared using one-way analysis of variance . Statistical assumptions were obtained using Shapiro-Wilk test and Levene's test , respectively . If the statistical hypotheses were not proven , we first tried to perform the hypotheses by changing a variable and the above tests were performed afterwards , otherwise the non-parametric Kruskal-Wallis test was used .
If the distribution is normal , the Tukey test is used for two-by-two comparisons , and if the distribution is not normal , the non-parametric Mann - Whitney U test with Bonferroni correction is used for two-by-two comparisons .
The confidence level in Leven's and Shapiro-Wilk tests is 99% and 95% for the rest of the tests .
In all the tests , the confidence level was equal to 95% and the significance level was considered less than 5% ; the software used for data analysis is SPSS statistical analysis software version 24 .
Behavioral tests
Elevated body swing test
This test is used to evaluate the severity of Parkinson's disease [22] .
The working method is that the tail of the rat is taken from the area of 2.5 cm where it connects with the body and raised , so that the animal's nose is 2.5 cm above the support surface . in this case , the animal turns its body to the right or left , and the number of these turns to each side indicates the severity of the disease . In this study , the number of turns to the right or left in a period of 1 minute (60 seconds) was counted .
Deviation in body swing as percentage ( L + R ) / L or percentage ( R + L ) / R , ( L : number of twists to the left , R : number of twists to the right ) , was calculated and displayed . When the rats were picked up by the tail , they showed twists to the left or right side of the body . In the parkinsonian rats with neurotoxin 6-OHDA , these twists are inclined to one side of the body or so called deviation . In order to confirm the parkinsonization of animals with neurotoxin 6-OHDA , the twisting of the animal should be towards the opposite side of the destruction of the dense area of the substantia nigra of the midbrain .
In our study , neurotoxin 6-OHDA was injected in the left side of the dense area of the substantia nigra of the rat midbrain , and the animal's twist should be to the right to confirm the parkinsonian model . a greater deviation indicates the severity of parkinson's disease , which should be above 70% , and the animals with a percentage lower than 70% are not parkinson's and are excluded from the study .
The steps of the elevated body swing test in the present study :
First elevated body swing test : before stereotaxic surgery and any invasive procedure .
Second elevated body swing test : seven days after stereotaxic surgery and parkinsonization of the animals , to confirm the parkinsonism model .
Third elevated body swing test : seven days after the treatment of different groups .
Fourth elevated body swing test : fourteen days after the treatment of different groups .
Open field test
This test is used to measure motor activities , anxiety and depression in rats .
The open field device consists of a black wooden box 50 x 72 x 72 cm , the floor of which is divided into 25 squares (18 cm x 18 cm) and defined by 2 areas , the center and the border . at the beginning of the experiment , each animal was placed in the central area, and the experiment began after 30 seconds of adaptation . the activity was recorded for 5 minutes ( 300 seconds ) . Etho Vision XT-7 software ( Noldus, Netherlands ) was used for data monitoring and analysis . Behavioral analysis for the field test included time spent in the central zone, number of line crossings between zones , and standing (standing on hind limbs) [23] .
In order to check the animal , we evaluated the following items :
- The number of times the animal crosses the grid lines of the floor of the test environment with all four paws is considered as a measure of movement activity , which is divided into two types of activity near the wall (periphery) and the center of the environment .
- On the floor of the test environment , a central environment is marked and the number of times the animal enters the center and periphery with all four paws is calculated .
- The time when the animal is in the center of the environment .
- The time period when the animal is on the edge of the environment .
- Animal movement speed .
- The distance traveled by the animal .
Elevated plus-maze test ( EPM )
This behavioral test is performed to measure the anxiety and movement ability of Parkinson's rats .
In addition , the maze is a device made of wood and has four arms in the shape of a positive (+) sign . the dimensions of the open and closed corridor are 50 x 10 cm , and the two sides and the end of the closed corridor have 40 cm high walls , and to prevent rats from falling , there is a 0.5 cm high edge of glass on both sides and the end of the open corridor . It has been installed . the four corridors lead to a central area with dimensions of 10 x 10 cm . the maze is placed by bases at a height of 50 cm from the ground . Rats were placed in the central area of the maze , facing an open corridor . Proper light is provided by a 100 watt lamp .
During the five minutes that the animal moves freely in different parts of the maze , for each animal , after counting the number of times it enters the open and closed arms and measuring the time of the animal's presence in the open and closed arms using a stopwatch , the number of times it enters the corridor (OAE) and the amount of time spent in the open corridor were calculated and then it was converted into a percentage [24] .
The amount of movement activities , which is the total number of times entering the open and closed arms of the maze , was measured by direct observation . the meaning of entering an open or closed corridor is when all four legs of the animal are placed in the corridor . the time spent in each corridor was calculated accordingly. all measurements were made in a very quiet environment with constant light (100 watt lamp) and at 10-15 in the morning [13-15, 25] .
Findings
Open field test results
In comparing the average time spent by the animal in the corner of the device , there was a significant difference between the group receiving nortriptyline with a dose of 10 mg/kg and the control group (P <0/01 ) , see ( figure 2 ).
It can be concluded that the anxiety-like behaviors in the group receiving nortriptyline with a dose of 10 mg / kg have significantly decreased compared to the control group .
In comparing the average time spent by the animal in the center of the device, there was a significant difference between the group receiving nortriptyline with a dose of 10 mg/kg and the control group . see ( figure 3 ) .
In the group receiving nortriptyline with a dose of 10 mg/kg , the amount of anxiety-like behaviors is significantly reduced compared to the control group .
In the comparison of the average number of times the animal entered the center of the device , which is considered as a measure of motor activity , there was no significant relationship between the different investigated groups ( P: 0.070 ) . it can be concluded that nortriptyline treatment in parkinsonian rats is not related to motor activity .
Post hoc statistical tests did not show a significant difference in the average movement speed of the animal between the different investigated groups .
According to this test that measures the animal's speed , it can be concluded that the animal's movement speed has decreased in the control group and the group treated with levodopa and benserazide compared to the healthy group . but in other treatment groups, the speed is not significantly different from the healthy group , and in the group receiving nortriptyline with a dose of 10 mg/kg , the distance covered is more than the control group .
EPM test results
In examining the number of times the animal entered the open and closed arms of the device , there was a significant difference between the group receiving nortriptyline with a dose of 10 mg/kg and the control group ( P < 0.001 ) and the group receiving nortriptyline with a dose of 5 mg/kg and the control group ( P : 0/002 ) , there was a significant difference . see ( figure 4 ) .
In examining the time spent by the animal in the open and closed arms of the device , there was a significant difference between the control group and the two groups receiving nortriptyline with doses of 5 and 10 mg/kg ( P < 0.001 ) .
Elevated body swing test results
The first elevated body swing test was performed before performing stereotaxic surgery and any invasive procedure . there was no significant difference between the average values of the first swing test between the healthy groups and the other 6 groups .
The second elevated body swing test was performed 7 days after stereotaxic surgery and parkinsonization of the animals to confirm the parkinsonism model .
There was a significant difference in the mean values of the elevated body swing test of the second round between the healthy group and the other 6 groups , as well as the sham group and the other groups . In other cases , no significant relationship was observed .
In the stage of the third elevated body swing test , which was performed 7 days after the treatment , post hoc statistical tests indicated a significant difference in the average values of the third time swaying test between the healthy group and the control group ( P = 0.010 ) .
In the fourth stage of the elevated body swing test , which was performed 14 days after the treatment , and we had the best response , there was a significant difference between the control group and the parkinsonian group treated with nortriptyline 10 mg/kg , levodopa and benserazide ( P : 0.002 ) . see ( figure 5 ) .