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

Summary

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.

Abstract

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson'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'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.

Introduction

Deep brain stimulation of the subthalamic nucleus (STN) is an effective treatment option for Parkinson'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 electrode^2-4. High-frequency (>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 elements^5,6. 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 "trial and error" 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's disease in rats is modeled using 6-hydroxydopamine injections into the right medial forebrain bundle^7,8. 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 discrete^7,8; the animals exhibit a slight impairment of the contralateral left paw during both spontaneous exploration and complex grasping behavior^9,10.

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 outcomes^11-14. 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 naïve, hemiparkinsonian and deep brain stimulation conditions.

Protocol

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)

1. Prepare a clear plastic glass cylinder (height: 40 cm, diameter: 19 cm) by cleaning the cylinder with a 0.1% acetic acid solution.
2. Prepare cards with date of experiment and each rat's identification number.
3. Place two mirrors in 90° angle behind the cylinder.
4. Place the camera in front of the cylinder such that the distance between the camera and the cylinder allows a good view of the paws.
5. Place the rat in the transport box.
   Note: The animals should be handled by the experimenter prior to the test to avoid stress.
6. Transport the rat from home cage to the cylinder using the transport box.
7. Place the rat in the cylinder (Figure 3).
   1. Always perform all behavioral tests at the same time of the day to avoid circadian differences in activity. If the animal is connected to the stimulus generator by cable make sure the cable is not twisted during the experiment.
8. Press the "record" button on camera. Show the card with the actual date of experiment and rat's identification number to the camera. Start recording.
9. After five minutes, remove the animal from the cylinder and put it back into the home cage using the transport box.
10. Clean the cylinder with a 0.1% acetic acid solution.
11. Evaluate the paw use from recorded video by counting the left and right paw wall contacts (paw use in percent) as well as rearings (standing on hind paws with or without supporting on the cylinder wall). The cylinder test can be also evaluated automatically by an appropriate software.
    Note: A healthy rat uses both paws equally. The hemiparkinsonian rat uses the paw affected due to lesion a lesser extent.

2. Staircase Test (Figure 4)

1. Acquisition Phase
   1. One day prior to the training familiarize the animals with the pellets used in the Staircase test.
      1. Optional: To increase the animal's motivation use a dietary restriction (10-15 g of standard laboratory chow to maintain body weight at 90% of the free feeding level¹⁶). However, this is not obligatory to achieve a positive training effect. This study was conducted without food restriction.
   2. Prepare a clear plastic glass staircase box (height: 34.5 cm, length: 35.5 cm, width: 12 cm and narrow compartment 6 cm) by cleaning the box with a 0.1% acetic acid solution. Note: The staircase box is a two-compartment-box with a raised platform and two stairs in the narrow compartment. The left steps on the stairs in the narrow compartment can be reached only with the left paw, the right steps only with the right paw.
      Note: The standard staircase boxes consist of two compartments with a lid, if it is to be used for experiments with rats stimulated via cable, use a high box without lid.
   3. Remove the staircase and fill the wells on each step with eight 45 mg pellets.
   4. Insert the staircase and put eight additional pellets on the heightened platform.
   5. Place the rat in the transport box.
   6. Transport the rat from home cage to the staircase box using the transport box.
   7. Place the rat in the staircase box (Figure 5).
   8. After five minutes, remove the animal from the staircase box and put it back into the home cage using the transport box.
   9. Note how many pellets were eaten from platform and (eventually) from right and left staircase.
   10. Refill the staircase by filling the wells on each step with eight 45 mg pellets.
   11. Clean the staircase box with a 0.1% acetic acid solution and place the additional pellets on the platform.
   12. Repeat this procedure (acquisition phase) three days in a row.
       Note: All experiments described were performed on male Sprague Dawley rats. The duration of the different training modules can differ in rats of different strain, sex and vender.
2. Free Choice Test
   1. Clean the staircase box with a 0.1% acetic acid solution.
   2. Remove the staircase and fill the wells on each step with eight 45 mg pellets.
   3. Place the rat in the transport box.
   4. Transport the rat from home cage to the staircase box using the transport box.
   5. Place the rat in the staircase box.
   6. After five minutes, remove the animal from the staircase box and put it back into the home cage using the transport box.
      Note how many pellets were eaten from right and left staircase.
   7. Note: If the animals still have problems with grasping pellet, add some more on the platform where they can be easily reached.
   8. Refill the staircase by filling the wells on each step with eight 45 mg pellets.
   9. Clean the staircase box with a 0.1% acetic acid solution for the next animal.
   10. Repeat this procedure (free choice phase) three days in a row.
       Note: The presented results were obtained by training conducted without a resting period between modules. Some groups prefer a resting day for consolidation, to support the training process.
3. Forced Choice Test
   1. Clean the staircase box with a 0.1% acetic acid solution.
   2. Remove the staircase and fill the wells on each step on the left staircase with eight (first three days of the module) or four (consecutive three days of the module) 45 mg pellets.
      1. Perform the forced choice test on the side, where the impairment will occur.
         Note: We perform the Parkinson lesion on the right hemisphere and therefore train selectively the left paw.
   3. Place the rat in the transport box.
   4. Transport the rat from home cage to the staircase box using the transport box.
   5. Place the rat in the staircase box.
   6. After five minutes, remove the animal from the staircase box and put it back into the home cage using the transport box.
   7. Note how many pellets were eaten from left staircase.
   8. Refill the staircase by filling the wells on each step with eight or four 45 mg pellets (number of pellets depends on training day).
   9. Clean the staircase box with a 0.1% acetic acid solution for the next animal.
   10. Repeat this procedure (forced choice phase) six days in a row.
4. Data Acquisition
   1. Perform the experiment as described for the forced choice module (four pellets in each well on the left staircase) on two consecutive days. Calculate the success rate (number of pellets eaten) as mean of the two days.

Results

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 (>90% loss of dopaminergic neurons in substantia nigra) were included into the results section (Figure 7).

The cylinder test performed under lesioned condition showed ...

Discussion

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 rats^16,17. 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's disease and other motor disease models. The cylinder test involves a simple approach to evaluate paw use in rats. It requires no t...

Materials

Name	Company	Catalog Number	Comments
Staircase box without lid	Glas Keil, Germany		custom made
Cylinder box	Glas Keil, Germany		custom made
Dustless precision pellets, 45 mg	Bio Serv	F0021