Name: a novel approach to assess motor outcome of deep brain stimulation effects in the hemiparkinsonian rat: staircase and cylinder test
Uploaded: 2016-05-31T13:00:00
Description: Watch this Scientific Journal Video about A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test at JoVE.com

This work was supported by Interdisziplin&#228;res Zentrum f&#252;r Klinische Forschung (IZKF), University Clinics W&#252;rzburg, Germany (project N-215).

Animal experiments were approved by the University of Wuerzburg and the legal state authorities of Lower Franconia in accordance with the animal protection guidelines and European Communities Council guidelines (approval number: 55.2-2531.01 76/11). All efforts were made to minimize pain or discomfort of the animals used.
Note: Electrode implantation was performed as described elsewhere18.
1. Cylinder Test (Figure 2)
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	<li>Prepare a clear plastic gl...

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This article describes a detailed training protocol for the cylinder and staircase test. The latter is designed to assess complex grasping behavior and fine motor movement due to skilled reaching in rats16,17. The outcome measurement is expressed as number of pellets eaten during the test, which is an objective measurement. The protocol can be used in rat models for Parkinson&#39;s disease and other motor disease models. The cylinder test involves a simple approach to evaluate paw use in rats. It requires no t...

Deep brain stimulation of the subthalamic nucleus (STN) is an effective treatment option for Parkinson&#39;s disease1 and other movement disorders. The underlying mechanisms are still poorly understood and multifactorial, but a key feature is the modulation of neuronal network activity by repetitive depolarization of axons in the vicinity of the stimulating electrode2-4. High-frequency (&#62;100 Hz) stimulation is required for a beneficial effect in most brain targets and for most indications of DBS. Side effects of deep brain stimulation result from inadvertent coactivation of other fibers, which are covered by the stimulation volume and which subserve different functions, such as the pyramidal tract. Hence, it would be desirable to develop stimulation parameters, which preferentially activate beneficial neural elements, while avoiding the coactivation of side effect elements5,6. Although neurophysiology may offer such fine tuning options of DBS, the scientific progress has been minimal during the past two decades, because programming strategies have primarily been assessed by &#34;trial and error&#34; in patients and restricted by the limited programming options of commercially available DBS devices, rather than using neurophysiological insight and defined experimental settings to systematically explore the full parameter space.
To overcome the translational roadblock in DBS research we are proposing a protocol to screen alternative stimulation parameters in rodent models of Parkinsonism prior to clinical exploration. Unilateral Parkinson&#39;s disease in rats is modeled using 6-hydroxydopamine injections into the right medial forebrain bundle7,8. The resulting lesion, described further as hemiparkinsonian, is assessed in the apomorphine test by evaluation of rotation score after low dose apomorphine injection and confirmed post mortem by tyrosine hydroxylase immunohistochemistry. The method is easy to apply and highly reproducible, while bearing a low mortality and morbidity. The resulting motor deficits are very discrete7,8; the animals exhibit a slight impairment of the contralateral left paw during both spontaneous exploration and complex grasping behavior9,10.
To assess the effectiveness of deep brain stimulation protocols tests are required which allow measuring a quick and reliable change in motor performance and can be repeated over time with different neurostimulation settings. Several groups have proposed different stimulation approaches and different tests to assess the motor functions in rats11 with highly variable and inconsistent outcomes11-14. This forced us to choose a set of tests with high predict validity and complementarity. Additionally, for assessment of motor outcome under deep brain stimulation conditions, tests were favored which could be performed by animals connected via cable to the stimulus generator. For these purposes we established our test battery consisting of one test for paw use asymmetry and one test for skilled reaching. The study design is illustrated in Figure 1.
For spontaneous paw use we performed the cylinder test described by Shallert15, which is a widely used test for paw use during vertical exploration. No training of the animal is required. For the assessment of more complex grasping behavior we established the staircase test according to Montoya16. Our protocol is modified according to Kloth17. The rats are trained for a period of twelve days in reaching pellets from the test box. After the training period the test can be applied to measure the complex grasping behavior by counting the success rate described as number of pellets eaten. The article presents the detailed training in the staircase box as well as the performance of both behavioral tests under na&#239;ve, hemiparkinsonian and deep brain stimulation conditions.

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			<td height="42" style="height:42px;width:216px;">Cylinder box</td>
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a novel approach to assess motor outcome of deep brain stimulation effects in the hemiparkinsonian rat: staircase and cylinder test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

All animals underwent a post mortem histological verification of both the dopaminergic lesion and the electrode location. Only animals with correct electrode placement inside the STN (Figure 6) and complete dopaminergic lesion (&#62;90% loss of dopaminergic neurons in substantia nigra) were included into the results section (Figure 7).
The cylinder test performed under lesioned condition showed ...

Watch this Scientific Journal Video about A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test at JoVE.com

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

The overall goal of this behavioral test battery, is to assess paw use assymetry in Hemiparkinsonian rats. This method can help answer key questions in the experimental deep brain stimulation field such as the motor outcome and the different stimulation patterns. The main advantage of this technique is that the cylinder test and the staircase test are complimentary and they can be both used easily in animal stimulated via cable.Demonstrating the procedure will be Dr.Milos Krstic, from my laboratory. Always perform behavioral tests at the same time of day to avoid circadian differences in activity. For the cylinder test, begin with cleaning a 40 centimeter tall, 19 centimeter diameter clear, plastic glass cylinder with a 0.1%acetic acid solution.Once well-cleaned, position two mirrors at a 90 degree angle behind the cylinder. Place the camera in front of the cylinder such are the distance between camera and the cylinder allows for a good view of the animal's paws when in the cylinder. Then, place the rat in the cylinder.If the animal is connected to the stimulus generator, make sure the cable is not twisted. Now, start the recording and flash a card to the camera with the actual date of the experiment and the rat's indentification number. After five minutes of recorded activity, return the rat to its home cage.Then, clean the cylinder again before running the next rat. In the video, count how often the front left and front right paws contact the wall. Also, count how often the rat stands on its hind paws with or without supporting itself with its front paws.These are called rearings. One day prior to the training, familiarize the rats with the staircase apparatus and pellet rewards. First, prepare a clear plastic glass staircase box by cleaning the box with 0.1%acetic acid.The staircase box is a two-compartment box with a raised platform and two stairs in a narrow compartment. The left steps on the stairs can be reached only with the left paw. The right steps can only be reached with the right paw.This is a standard commercially available box, but if the rats are to be stimulated via a cable, use a high box without a lid. Now, remove the staircase and fill the wells on each step with eight 45-milligram pellets. Then, replace the staircase and put eight additional pellets on the heightened platform.Now, place the rat in the box and after five minutes, return the rat to its home cage. Score how many pellets were eaten from the platform. Before the next test, wash the staircase and reload it with pellets.The acquisition phase should be performed once per day for three days. Over the next three days, run the free choice phase of the test. It is executed just like the acquisition phase, but now score how many pellets were eaten from the two staircases.If the animals have problems with grasping the pellet, add some more on the platform where they can be easily reached. Then, for six days, run the forced choice phase of the test. During this phase, only load pellets into the same side of the staircase as the occurred impairment.A lesion of the right hemisphere would require testing the left paw and thus loading the left staircase. Over the first three of these days, load one staircase side with eight pellets per step. But on the fourth through sixth days, only load that side with four pellets per step.For a successful staircase training, it is crucial to motivate a animal during the acquisition phase. It is important that the rat is able to grab some pellets at first grasping approach because the immediate reward resides in good training effect in the later phases. Another critical step of course, when the rat is not interested in the task, it can be then necessary to apply a mild foot restriction until the proper motivation level is reached.Tested animals were given dopaminergic lesions and STN-placed electrodes. Data was only included in the analysis when post-mortem analysis confirmed a correct electrode placement and lesion propagation. The cylinder test revealed that the lesioned paw use decreased to about 15%After 24 hours of deep-brain stimulation delivered to the STN for 60 microseconds, at 130 hertz frequency, the use of the lesioned paw increased to roughly 22%The result was encouraging, but not significant.In the staircase test, normal animals retrieved about half the pellets after 12 days of testing. Lesioned animals could only retrieve about 10%of the pellets. After deep-brain stimulation however, a significant improvement was made and they retrieved about 25%of the pellets.In neither test, did the stimulation cable effect the results. Animals reared as expected and ran the staircase as expected. After watching this video, you should have a good understanding how to train the rats in the staircase test and how to use the presented behavioral test battery to screen different stimulation patterns in rats.While attempting this procedure, it is important to remember that to handle the animals before starting the behavioral test. The test should be always performed by the same experimenter to avoid stress in the animals. Don't forget that while working with animals with implanted deep-brain stimulation electrodes, you should be extremely careful and the animals should be daily controlled for signs of pain or distress during the whole procedure.

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