Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Summary

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson's disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Abstract

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson's disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor. It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10–15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Introduction

Although it has been more than 50 years since levodopa (L-DOPA) was first introduced as a treatment for individuals with PD^1,2, it remarkably remains the most effective therapy for parkinsonian motor symptoms. The clinical motor symptoms associated with PD stem from the loss of dopamine (DA) neurons in the substantia nigra (SN) pars compacta, resulting in the dramatic decrease in available dopamine in the striatum. L-DOPA effectively restores striatal DA levels, resulting in motor benefit early in the disease^3,4. Inopportunely, with long-term treatment, most individuals with PD will develop L-DOPA induced dyskinesias (LID), including chorea, dystonia, and athetosis, which often significantly impact activities of daily living^5,6,7.

While several behavioral models of LID in rodents exist, differences in modeling and behavioral assessment of LIDs have called into question the reproducibility of results between labs as well as the reliability of these experimental tools for pre-clinical PD research. Developed in association with a clinical movement disorder specialist⁸, the current protocol is a straightforward method for LID induction and rating and is appropriate for use in a rat model of PD utilizing 6-hydroxydopamine (6-OHDA)-induced unilateral nigral lesioning^9,10. The LID rating scale provided here includes scoring for both the intensity and frequency of dyskinetic behavior in various individual body parts. Pertinent information regarding workflow optimization of experiments and the appropriate care and handling of parkinsonian and dyskinetic animals is also provided.

Protocol

The animals presented here were maintained and handled in compliance with the institutional guidelines. All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC) in compliance with federal and state regulations.

1. Drug-free confirmation of 6-OHDA lesion status

1. Postural Tail hang test^11,12,13
   NOTE: Assess lesion status at least 1 week following 6-OHDA lesion induction (See^9,10,35 for details on lesioning) in experimental subjects (e.g., male or female, adult Sprague Dawley or Fisher 344 rats). 
   1. Suspend the rat approximately 6 cm above its cage, firmly holding at the base of the tail, for ~5 s.
   2. Record the direction of body contortion as + for a successfully lesioned animal twisting contralateral to the lesioned side and - for lack of twisting or twisting in both directions.
   3. NOTE: These tests are optional but recommended. See^14,15,16,17 for additional drug-free testing options/variations.
2. Step adjusting drag test (adapted from¹⁶)
   NOTE: Assess lesion status at least 1 week following unilateral 6-OHDA lesion induction in experimental subjects (e.g., male or female, adult Sprague Dawley or Fisher 344 rats).
   1. Hold the rat by the base of its tail, elevating the back feet off the surface by ~6 cm; drag backward across a flat, smooth but not slippery surface, ~75 cm, over 5–10 s.
   2. Observe and record the number of tapping/step adjusting movements of each forepaw over three repeated tests.
   3. Score the subject as + for successful unilateral lesioning when 0–2 forepaw taps are observed contralateral to the lesioned side, together with rapid tapping (~10 taps) from the forepaw ipsilateral to the lesioned side (e.g., animals unilaterally lesioned on the left side show tapping deficit (0–2 taps) with the contralateral right forepaw).
   4. Conversely, score moderate to rapid tapping (5–10 taps) from both forepaws as - to indicate incomplete or unknown lesion status.
      NOTE: An anxious animal can show rapid tapping/step adjusting even if successfully lesioned. If this is suspected, place the rat back in their home cage and re-test ≥30 mins later.

2. Preparation of reagents and supplies

1. Determine L-3,4-dihydroxyphenylalanine methyl ester hydrochloride (levodopa or L-DOPA) and benserazide hydrochloride, a peripheral decarboxylase inhibitor (see Table of Materials) dose, rating frequency, and experimental timeline that is appropriate for the investigational question^12,18,19,20 (Figure 1).
   NOTE: Investigational questions can be posed to seek any number of questions ranging from asking whether a specific therapy might reduce existing LID or prevent induction of LID. They can also explore whether therapeutic efficacy is dependent on the dose of levodopa or whether LID expression and/or therapeutic efficacy varies depending on the sex, species, and age of the subject.

Figure 1: Example of treatment timeline. Example L-DOPA dose-escalation timeline of 12 weeks in total length, with 8 weeks of L-DOPA injections beginning 3 weeks after 6-OHDA lesioning and 4 weeks following experimental treatment. In this example, L-DOPA is subcutaneously injected 5x per week (Monday–Friday) at approximately the same time each day, for 2 weeks at each prescribed L-DOPA dose (3, 6, 9, and 12 mg/kg). Behavioral LID ratings take place on days 1, 6, and 10 of each L-DOPA dosage level. Please click here to view a larger version of this figure.

2. Weigh rats weekly to calculate the appropriate drug quantity based on the ongoing weight changes during the study.
   NOTE: Due to increased activity in LID+ rats, there is potential for weight loss with long-term L-DOPA treatments. If weight loss occurs provide rats with nutritionally complete, highly palatable treats (see Table of Materials) following L-DOPA injections.
3. Calculate the amount of L-DOPA and benserazide required for each weekly concentration, weighing out lyophilized aliquots for each day of injection, and storing in combination for 1–2 weeks at -20 °C in glass amber vials until the day of treatment.
   NOTE: Target dose is 12 mg/kg or 12 mg L-DOPA/1000 g body weight. Example of calculations for determining the amount of L-DOPA and saline needed for each day of a week using 12 mg L-DOPA/kg body weight at an injection volume of 1 cc/kg of rat weight is given in Supplementary File 1.

3. Room and cage set up

1. On the first day of L-DOPA treatment 3-4 weeks following 6-OHDA lesion surgery, transfer rats to single housing, including IACUC-approved enrichment.
2. Maintain in single housing throughout the study to avoid peer interference with behavioral assessments.
3. On LID rating days, place the home cages on a steel wire rack, turned at an approximately 45^° angle for optimal viewing of the rat (Figure 2A). Flip the identification tags (Figure 2B) upward and remove water bottles, food racks, and all types of enrichment in the cage (Figure 2C) to avoid interference with behavioral assessments.

Figure 2: Example of the cage set up for LID ratings of large-scale rat experiments. (A) Multiple cages can be set up for LID rating using large metal racks that allow for optimal viewing of each animal. Cages should be spread apart at a 45° angle with ID cards flipped upward (B), food, water bottles, nesting materials, and other enrichment removed to limit visual obscureness of the rat and distractions to the rat while examining dyskinetic behaviors (C). The metal racks need to be a few feet away from any wall to allow the rater to examine the rat at the front or back of the cage as needed. It is essential to label enrichment apparatuses (e.g., C- red rat retreat houses) with individual animal IDs to replace them into the same cage from which they came. This is particularly important when using animals of different sexes not to increase stress to the experimental subjects. Please click here to view a larger version of this figure.

4. Levodopa injections and dyskinesia rating

1. Subcutaneous injections of L-DOPA^21,22
   1. Immediately preceding the daily injection of L-DOPA, add the appropriate volume of sterile saline to the pre-weighed lyophilized L-DOPA and benserazide mix in the amber vial and shake well for 10 s (step 2.3).
      NOTE: Target injection volume is 1 mL/1000 g body weight (with 12 mg L-DOPA per mL). The volume of the sterile saline will depend on the number of animals per study.
   2. Fill individual syringes (e.g., 1.0 or 0.5 mL with 26 G needle) with the required volume for each animal (1 mL/kg rat weight) and label each syringe with individual animal identification.
      NOTE: Keep the filled syringes protected from light in sterile pouches until the time of injection. L-DOPA rapidly oxidizes in the presence of oxygen and light in an aqueous environment^23,24,25,26.
   3. Bring the first cage to the injection bench.
   4. Remove the rat from its cage and place it on the injection surface.
   5. Gently restrain the head and shoulders against the surface on which the rat is resting with the palm of the non-dominant hand.
   6. Gently, scruff the skin on the back overlying the scapulae with the thumb and forefinger of the non-dominant hand, inject L-DOPA volume with the dominant hand into the subcutaneous space between/below the fingers, keeping the needle as parallel to the body as possible to avoid intramuscular injection.
      ​NOTE: The rats are not anesthetized before injection.
   7. Dispose of each used individual syringe in a sharp's container.
   8. Replace the rat into its individual cage and add nutritionally complete treats except on LID rating days to avoid interference with behavioral assessments until after ratings are complete.
   9. Set the timer for 1–2 min depending on the rating time desired and the number of rats in the study on rating days. Retrieve the next cage and inject the next rat when the timer indicates.
   10. Repeat this, injecting one rat every 1–2 min, until all the rats are injected.
2. Levodopa-induced dyskinesia rating post-injection
   1. Rate the intensity (Table 1) and frequency (Table 2) of dystonic and hyperkinetic dyskinesia movements at the desired number of timepoints, which should include the initial onset of LID behavior, peak behavior, and the phase of decline (see Supplementary File 2 for an example of LID rating log sheet).
   2. For male and female adult Sprague Dawley or Fisher 344 rats, and a sample size of N = 40 rats, begin the dyskinesia ratings 20 min after the first L-DOPA injection, and then at 50 min intervals until 220 or 270 min post-injection, depending on when LID behaviors have discontinued in 90%–100% of rats.
   3. If using 1 min rating intervals, set a timer for 1 min. Rate the first rat for one minute. Move to the next rat and rate it for 1 min. Continue through all the rats, rating for 1 min intervals.
   4. Have a timer positioned next to the cage visibly so that the LID behavior intensity (Table 1) can be observed while estimating the frequency of any given behavior (Table 2) during the rating period.
   5. After ratings for the first time point are completed start again with the first rat at the next time point (e.g; 70 min post-injection) and continue at the desired interval (e.g., every 50 min) until all time points are completed.
      NOTE: Due to the overlap of L-DOPA injection and LID rating tasks, two persons are needed on the rating days, one for injecting and one for behavioral ratings.

Results

LIDs in parkinsonian rats can manifest as a range of abnormal involuntary movements (AIMs), including dystonic, hyperkinetic, and stereotypic behaviors. LID rating criteria for such behaviors are presented here to include both intensity (Table 1) and frequency (Table 2). This provides an overall LID severity score for each rat that reflects both the quality (intensity) and quantity of time spent engaging (frequency) in these behaviors at each rating timepoint. The final LID severity scor...

Discussion

Presented here are details for the reproducible induction and rating of LIDs in a parkinsonian rat model following unilateral 6-OHDA lesioning of the nigrostriatal DA system. While it was once thought that rodents did not develop LID and that rotational asymmetry may be the analog of LID in rats³¹, rat and mouse models have been characterized over the past two decades and are a well-accepted tool for LID research^15,32,

Materials

Name	Company	Catalog Number	Comments
100 Minutes Digital Timer	Staples	1111764
Compass CX Compact Scale	Ohaus	30428202
5-(2-aminoethyl)-1,2,4-benzenetriol, monohydrobromide	Cayman Chemicals	25330	6-OHDA is a catecholaminergic neurotoxin that is used to induce dopaminergic lesions and parkinsonian symptoms in rodents.
Allentown cages	Allentown, LLC	Rat900	Allentown cages provide the ability to view the rats from all sides.
BD Allergist Trays with Permanently Attached Needle	BD	BD 305540	For subcutaneous L-DOPA injections
Benserazide hydrochloride	Sigma-Aldrich	B7283	Benserazide is a peripheral decarboxylase inhibitor used with L-DOPA to to induce dyskinesia in rodent models of PD.
Glass amber scintillation vials	Thermo Scientific	B7921	Used for storage of L-DOPA/benserazide at -20 °C until mixed with sterile saline.
L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride	Sigma-Aldrich	D1507	L-3,4-Dihydroxyphenylalanine methyl ester is a precursor to L-DOPA that crosses the blood-brain barrierand use to treat parkinsonian symptoms in rodents.
Paper Mate Sharpwriter Mechanical Pencils	Staples	107250
Rodent nutritionally complete enrichment treats	Bio-Serv	F05478
Round Ice Bucket with Lid, 2.5 L	Corning	432129
Standard Plastic Clipboard	Staples	1227770
Steel wired 6' long movable shelving units	Uline	H9488	Width/Height can be adjusted to need/number of rats per experiment
Sterile Saline 0.9%	Covidien/Argyle	1020	For mixing with L-DOPA/benserazide prior to subcutaneous injections.