|Year : 2022 | Volume
| Issue : 1 | Page : 1-6
Motor function test protocol for parkinsonian triad in rodent model of Parkinson's disease
Mujittapha Umar Sirajo1, Kauthar Murtala2, John Chukwuma Oyem3, Azeez Olakunle Ishola4, Lukman Femi Owolabi5
1 Department of Anatomy, Bayero University, Kano, Nigeria
2 Department of Medical Laboratory, Ahmadu Bello University, Zaria, Nigeria
3 Department of Anatomy, University of Port Harcourt, Rivers State, Nigeria
4 Department of Anatomy, AfeBabalola University, Ado-Ekiti, Nigeria
5 Department of Anatomy, Bayero University, Kano, Nigeria; Department of Medicine, College of Medicine, University of Bisha, Bisha, Kingdom of Saudi Arabia
|Date of Submission||22-Jan-2022|
|Date of Acceptance||04-Mar-2022|
|Date of Web Publication||01-Apr-2022|
Mujittapha Umar Sirajo
Department of Anatomy, Faculty of Basic Health Sciences, Bayero University, PMB 3011, Kano
Source of Support: None, Conflict of Interest: None
Over the years, there has been an increase in research on parkinsonism in basic and translational neuroscience. Parkinson's disease (PD) is a progressive neurodegenerative disorder vehemently associated with motor function deficits and other unique features collectively called the Parkinsonian triad, which slightly differs from other movement disorders such as Wilson's disease, tardive dyskinesia, chorea, and athetosis. Parkinsonian triad combines three major motor phenotypes of PD including bradykinesia, rigidity, and resting tremors. Hence, there is a need to review motor deficits protocols to create a set of behavioral protocols that critically address the parkinsonian triad's quantification in PD models. Literature search on Medline and PubMed was conducted to access the articles on the motor function test in a rodent model of Parkinsonism. Search terms include parkinsonism, parkinsonian triad, bradykinesia, rigidity, resting tremors, stepping test, parallel bar test, pole test, and cylinder test. This review shows that bradykinesia characterized by difficulty in movement initiation could be assessed using a stepping test by measuring stepping length and time taken to initiate movement on a wooden ramp. It can also be assessed using a parallel bar test by measuring the time taken to make 90° turn. This turning hesitation is one of the critical features of akinesia. Rigidity is associated with an increase in muscle tone; it is assessed by using a pole test to measure the time taken for the rodent to slide down a smooth pole. Resting tremors is an involuntary, oscillatory movement of the distal part of the upper limb when not performing an action. It could be graded using a cylinder test when the rodent suspends its forelimb on the air in an attempt to climb the wall of the cylinder. In conclusion, the examinations and quantifications of the Parkinsonian triad are required to diagnose parkinsonism in rodent models.
Keywords: Cylinder test, bradykinesia, parallel bar test, parkinsonian triad, parkinsonism, pole test, resting tremors, rigidity, stepping test
|How to cite this article:|
Sirajo MU, Murtala K, Oyem JC, Ishola AO, Owolabi LF. Motor function test protocol for parkinsonian triad in rodent model of Parkinson's disease. J Neurobehav Sci 2022;9:1-6
|How to cite this URL:|
Sirajo MU, Murtala K, Oyem JC, Ishola AO, Owolabi LF. Motor function test protocol for parkinsonian triad in rodent model of Parkinson's disease. J Neurobehav Sci [serial online] 2022 [cited 2022 May 26];9:1-6. Available from: http://www.jnbsjournal.com/text.asp?2022/9/1/1/342497
| Introduction|| |
Parkinson's disease (PD) is a chronic and progressive brain disorder associated with many motor and nonmotor deficits., These deficits result from progressive degeneration of dopamine neurons in the substantia nigra pas compacta (SNc), leading to loss of dopamine in the basal ganglia.,, In most cases, PD occurs sporadically and is proposed to result from a complex interaction between environmental and genetic factors.,
For an excellent research to be carried out on parkinsonism using an animal model, it should fulfill the following criteria; first, face validity criterion; the animal model should express major and most common phenotypes of human PD, otherwise known as the parkinsonian triad. Second, constructive validity criterion; the model should be created with a sound scientific rationale, and finally, the predictive validity criterion, which indicates that the model should have the propensity to respond to therapy comparably to clinical therapy.
| Parkinsonian Triad|| |
Parkinsonian triad is the combination of bradykinesia, rigidity, and resting tremor, which are the three main phenotypes of PD., These three phenotypes are collectively called Parkinsonism or Parkinsonian syndrome. The diagnoses of Parkinsonism can be made with the clinical manisfestations of at least two of the triad.,
| Bradykinesia|| |
Bradykinesia means slow movement; it is the most common feature of Parkinsonism attributed to basal nuclei disorders. Bradykinesia encompasses difficulty planning, initiating [as seen in [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d], and executing movement, decreasing facial expression, including the rate of eye blink and sometimes the inability to perform sequential and simultaneous tasks effectively. It is hypothesized that bradykinesia results from the disruption in motor cortex activity mediated by reduced dopaminergic function and in some cases, bradykinesia presents itself in the form of akinesia, a sudden and transient inability to move for about 10 s., This is also called freezing or motor block; its features include start hesitation, turn hesitation, hesitation in narrow or tight quarters, destination hesitation, and open space hesitation.
|Figure 1: Diagrammatic and graphical interpretation of bradykinesia using the stepping test and parallel bar test. (a) Diagramatic illustration of stepping test set-up. (b) A pictorial representation of a wistar rat demonstrating the stepping test. (c) Stepping test result shows a significant (P < 0.001) increase in time taken to initiate steps by the four limbs of the dopamine-2 receptor knockout model parkinsonism. (d) Diagrammatic illustration of parallel bar test set-up|
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| Resting Tremors|| |
Resting tremor is an involuntary, rhythmic muscle movement involving oscillatory movement prominently in the distal part of the upper limb [as observed in [Figure 2]a and [Figure 2]b at a frequency of about 4−6 Hz when not performing an action., The tremors tend to stop when the hand is in action, such as in a flexed grip. Drug-induced Parkinsonism is less likely to be associated with tremor, although it can sometimes present with a tremor.,
|Figure 2: Diagrammatic and graphical interpretation of resting tremor using the cylinder test. (a) A pictorial representation of a parkinsonian rat exhibiting resting tremors in a cylinder test set up. (b) Cylinder test result shows a statistically significant (P < 0.05) increase in resting tremor in the dopamine-2 receptor knockout model of parkinsonism|
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| Rigidity|| |
Rigidity refers to increased resistance, stiffness, and inflexibility of the body's proximal part, such as the neck, shoulders, hips, and distal part of the body such as wrists and ankles [as seen in [Figure 3]b and [Figure 3]c]. Rigidity is accompanied by the “cogwheel” phenomenon, especially when associated with an underlying tremor present throughout the range of passive movement of a limb (flexion, extension, or rotation about a joint).,
|Figure 3: Diagrammatic and graphical interpretation of rigidity using the pole test. (a) Diagrammatic illustration of pole test set-up. (b) Pictorial representation of the pole test set-up. (c) Pole test result shows a significant increase in time taken to slide down the pole by the dopamine-2 receptor knockout model of parkinsonism|
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| Protocols for Motor Function Deficit Test|| |
Several different behavioral tests are imperative in assessing PD in rat models, each providing slightly different results. For the protocols reviewed in this paper, the rodents should be trained during the treatment phase of the designed experiment.
| Tests for Bradykinesia|| |
Stepping tests could be designed to detect motor activities' speed including hyperkinesia, normal motor activities, bradykinesia, or akinesia. The stepping test is conducted on a wooden ramp that is 1 m long connected to the rodent cage of 0.3 m height [as seen in [Figure 1]a and [Figure 1]b]. The rodent is gently placed on the wooden ramp with their heads pointing towards the cages. The time taken for each rat to initiate stepping by its four limbs is used to determine the difficulty of initiating movement. The distance covered by the rodent on the wooden ramp divided by the number of steps made by the rodent on the wooden ramp could be used to determine stepping length. Note that experimentally, the time the rodents use to reach their cage cannot be used to determine bradykinesia because the rodents tend to be distracted, exploring the edges of the wooden ramp.
Parallel bar test
A parallel bar test can be designed to assess bradykinesia, using the time taken to make 90° turns. As seen in the apparatus comprises two wooden frames of 60 cm height connected by two 1 m long plans and 1 mm thick parallel bars 3 cm apart [as seen in [Figure 1]d]. The test is conducted by placing the animal at the center of the two parallel bars (0.5 m), with its forelimb and hind limb on different bars, and allowed to roam freely on the bar. The time taken for the animal to make a 90° turns (latency of turn) is used to assess bradykinesia. In addition, the time taken to move from the center of the bars to either ends is used in assessing bradykinesia.,,
| Protocols for Resting Tremors|| |
Three scientists experienced in laboratory animal research are needed for accurate assessment of resting tremors using the cylinder test. This is to reduce misapprehension during the study. The volume and dimension of the cylinder to be used depends on the size of the rodent. Each animal should be placed in a transparent cylinder or beaker with an open top and allowed to explore the cylinder walls with its forelimbs while standing on the two hind limbs [as seen n [Figure 2]a]. As the animal raises it forelimb attempting to climb the container's wall, the three or more scientists conducting the test should grade the degree of resting tremors on a scale of 1–5 while the rodent suspends its forelimb on air during climbing attempts. Moreover, the number of times the animal places its forelimb against the container's wall can also be used to measure motor function deficit.,
| Protocol for Rigidity|| |
Pole test is designed to assess rigidity. Sirajo et al., who used the pole test to assess rigidity in a Wistar rat model of Parkinsonism, previously reported the use of pole test to assess rigidity. The Pole test is conducted on a smooth wooden pole of 1 m in length and 3 cm in diameter fixed in the middle of an empty cage [as seen in [Figure 3]a]. The rodent is placed facing downward, at the top of a wooden pole. The time taken for the Wistar rats to move down the Pole is used to assess rigidity.,
| Procedures|| |
For all the behavioural tests, the rodents should be familiarized with the behavioural test room 72 h before thecommencement of the tests.
- Clean the wooden ramp with methylated spirit and cotton wool before putting each rodent on the wooden ramp.
For movement initiation
- Hold the rodent by the tail and gently place it on the lowest height of the wooden ramp, with its face facing the direction of the cage
- Start a stopwatch immediately after placing the rodent on the wooden ramp
- Pause the stopwatch immediately the rodent makes steps with each of its four limbs
- Record time read by the stopwatch.
For stepping length
- Stain the tail of the rodent with a cleanable ink
- Place the rodent on the wooden ramp and carefully count the number of steps made by the rodent in 3 min
- Use a thread to trace the distance covered by the rodent
- Measure the length of the thread with a ruler
- Divide the length of the thread/distance covered by the rodent in 3 min over the number of steps made by the rodent in 3 min.
Parallel bar test
- Hold the rodent by the tail and gently place the rodent at the midpoint (0.5 m mark) on the parallel bars with it fore paws on the posterior pole and it hind paws on the anterior pole.
For turning hesitant
- Start a stopwatch immediately after placing the rodent on the parallel bars
- End the stopwatch immediately after the animal turn completely to the right or to the left
- 180 s is the maximum time allowed (cut-off time) for each rodent, terminate the experimentif the animal exceeds 180 s without making 90° turn
- Record the time read by the stopwatch.
- Start a stopwatch immediately after placing the rodent on the parallel bars
- End the stop watch when the rodent reaches the extreme end of the parallel bar
- Record the time read by the stopwatch.
- Clean the container with methylated spirit and cotton wool before putting each rodent inside it
- Set a stopwatch for 3 min (Duration of each experiment)
- Hold the rodent by the tail and gently put it vertically inside the container with the rodent facing downward
- The 3 laboratory scientist should grade the resting tremor of the rodent during attempt to climb the wall of the container.
- Hold the animal by the tail and place it at the peak of the wooden pole
- Immediately after placing the animal, start a stopwatch
- End the stop watch immediately the rodent reaches the ground/it cage
- Read and record the time taken for the rodent to reach it cage.
| Discussion|| |
In this article, we reviewed research protocols for quantifying and assessing parkinsonian triad in a Wistar rat model of PD. This review is relevant to basic and translational neuroscience researchers in developing countries who lacks funding for innovative research and whose research focus on designing therapeutic strategies for the management of PD.
Parkinsonian triad tests are designed to assess the degree of dopamine system damage in a rodent model of parkinsonism. It could also be used to assess a proposed therapeutic strategiy in rescuing damages in the dopamine system.
Parkinsonism leads to loss of motor coordination. Unfortunately, several other extrapyramidal syndromes manifest loss of motor coordination. However, loss of motor coordination in parkinsonism slightly differs from other motor functions diseases; in parkinsonism, the motor phenotypes are collectively called the Parkinsonian triad, a group of three major symptoms of PD (Bradykinesia, Rigidity and Resting tremor)., These symptoms lead to a loss of overall motor coordination.
Bradykinesia is the main clinical feature of parkinsonism., The diagnosis of PD is based on the manifestation of bradykinesia and at least one of the two other phenotypes of parkinsonian triad., Bradykinesia leads to an increase in time taken to initiate movement or increase in time taken to complete a voluntary movement. Bradykinesia can be presented in akinesia, a complete inability to initiate movements for a few seconds. The stepping test provides a window for detecting both akinesia and bradykinesia. Akinesia is assessed by measuring time taking to initiate movement, and bradykinesia by measuring stepping length. Significant (P < 0.001) increase in bradykinesia was seen in dopamine-2 receptor knockout model Parkinsonism when tested on a wooden ramp [as seen in [Figure 1]c].,
Although the brain circuitry that generates tremor is not well understood, tremor is regarded as an abnormal involuntary rhythmic and oscillatory movement. Tremors can be classified into resting tremors and action tremors. Resting tremors are associated with an involuntary activity. The examples of such tremors include PD tremor, physiological tremor, essential tremor, medication-induced tremor, lesional tremor, dystonic tremors, other neurodegenerative tremors, and psychogenic tremors. In PD, resting tremor occurs when the forelimb is not in contact with any object. Action tremors are associated with voluntary movements. Postural, kinetic, intention, task-specific, and isometric action tremors can be observed in mice when holding them by the tail, with their head facing downward. Tremor is often challenging to quantify and distinguish from other motor abnormalities in animals and this pose a major challenge in research. In this study, we showed that resting tremor can be quantified by observing and grading its frequency, amplitude, and phase. Our reviewed experimental result showed a less significant increase in resting tremors in the cylinder test [As seen in [Figure 2]b]. A less significant increase in resting tremor reaffirms the previous findings that drug-induced parkinsonism is less likely to be associated with resting tremors.,
Rigidity is associated with increased muscle tone, leading to stiffness, muscle's inflexibility, and a degree in the range of motion.,,, It is usually presented when the muscles are rapidly stretched. This phenotype has been evaluated using the pole test by measuring time taken to slide down a smooth wooden pole. Increased muscle tone tends to decrease the speed of sliding down [As shown in [Figure 3]c.
| Conclusion|| |
The characteristic motor function deficits observed in PD slightly differ from other movement disorders. The diagnosis of PD is based on the Parkinsonian triad, which can be effectively demonstrated using the stepping test, modified cylinder test, and pole test. Hence, these tests should be adopted to assess motor function deficits associated in PD, especially in developing laboratories that lacks advanced techniques for quantifying motor functions deficits.
It is indeed with a great sense of pleasure and privilege to express our profound gratitude to the management of Anatomy Department, Bayero University Kano, for allowing us to conduct the tests in Behavioural Neuroscience laboratory. Our sincere appreciation goes to Kabir Shehu and Onenson Kanu for their contributions in the experiments.
Patient informed consent
There is no need for patient informed consent.
Ethics committee approval
There is no need for ethics committee approval.
Financial support and sponsorship
No funding was received.
Conflicts of interest
There are no conflicts of interest to declare.
Author contribution subject and rate
- Sirajo Mujittapha Umar (35%): Design the research, data collection and analyses and wrote the whole manuscript.
- Kauthar Murtala (20%): Organized the research and supervised the article write-up.
- John C. Oyem (15%): Contributed with comments on manuscript organization and write-up.
- Ishola Olakunje Azeez (15%): Contributed with comments on research design and interpretations of the behavioural testing.
- Lukman Femi Owolabi (15%): Contributed with comments on research design and interpretations of the behavioural testing.
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[Figure 1], [Figure 2], [Figure 3]