Name: induction and assessment of levodopa-induced dyskinesias in a rat model of parkinson's disease
Uploaded: 2021-10-14T14:30:00
Description: Watch this Scientific Journal Video about Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease at JoVE.com

We want to acknowledge the struggles of all those with Parkinson&#39;s disease and the strength and resilience they show every day, especially the beloved father of KSC, Mark Steece. The work represented here was supported by the National Institute of Neurological Disorders and Stroke (NS090107, NS110398) and the Parkinson Disease Foundation International Research Grant Program, now the Parkinson Foundation.&#160;We also would like to acknowledge Molly VanderWerp for her excellent editorial assistance.

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
<ol>
	<li>Postural Tail hang test11,12,13 
	NOTE: Asses...

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 research15,32,<sup class...

Although it has been more than 50 years since levodopa (L-DOPA) was first introduced as a treatment for individuals with PD1,2, 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 disease3,4. Inopportunely, with long-term treatment, most individuals with PD will develop&#160;L-DOPA induced dyskinesias (LID), including chorea, dystonia, and athetosis, which often significantly impact activities of daily living5,6,7.
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 lesioning9,10. 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.

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			<td height="21" style="height:21px;width:441px;">&#160;100 Minutes Digital Timer</td>
			<td style="width:193px;">Staples</td>
			<td style="width:182px;">1111764</td>
			<td style="width:559px;"></td>
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			<td height="21" style="height:21px;width:441px;">&#160;Compass CX Compact Scale</td>
			<td style="width:193px;">Ohaus</td>
			<td style="width:182px;">30428202</td>
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			<td height="42" style="height:42px;">5-(2-aminoethyl)-1,2,4-benzenetriol, monohydrobromide</td>
			<td style="width:193px;">Cayman Chemicals</td>
			<td style="width:182px;">25330</td>
			<td style="width:559px;">6-OHDA is a catecholaminergic neurotoxin that is used to induce dopaminergic lesions and parkinsonian symptoms in rodents.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Allentown cages</td>
			<td style="width:193px;">Allentown, LLC</td>
			<td style="width:182px;">Rat900</td>
			<td style="width:559px;">Allentown cages provide the ability to view the rats from all sides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">BD Allergist Trays with Permanently Attached Needle</td>
			<td style="width:193px;">BD</td>
			<td style="width:182px;">BD 305540</td>
			<td style="width:559px;">For subcutaneous L-DOPA injections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:441px;">Benserazide hydrochloride</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:182px;">B7283</td>
			<td style="width:559px;">Benserazide is a peripheral decarboxylase inhibitor used with L-DOPA to to induce dyskinesia in rodent models of PD.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Glass amber scintillation vials</td>
			<td style="width:193px;">Thermo Scientific</td>
			<td style="width:182px;">B7921</td>
			<td style="width:559px;">Used for storage of L-DOPA/benserazide at -20 &#176;C until mixed with sterile saline.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:182px;">D1507</td>
			<td style="width:559px;">L-3,4-Dihydroxyphenylalanine methyl ester is a precursor to&#160;L-DOPA that crosses the blood-brain barrierand use to treat parkinsonian symptoms in rodents.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Paper Mate Sharpwriter Mechanical Pencils</td>
			<td style="width:193px;">Staples</td>
			<td style="width:182px;">107250</td>
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			<td height="21" style="height:21px;width:441px;">Rodent nutritionally complete enrichment treats</td>
			<td style="width:193px;">Bio-Serv</td>
			<td style="width:182px;">F05478</td>
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			<td height="21" style="height:21px;width:441px;">Round Ice Bucket with Lid, 2.5 L</td>
			<td style="width:193px;">Corning</td>
			<td style="width:182px;">432129</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Standard Plastic Clipboard</td>
			<td style="width:193px;">Staples</td>
			<td style="width:182px;">1227770</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Steel wired 6&#39; long movable shelving units</td>
			<td style="width:193px;">Uline</td>
			<td style="width:182px;">H9488</td>
			<td style="width:559px;">Width/Height can be adjusted to need/number of rats per experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Sterile Saline 0.9%</td>
			<td style="width:193px;">Covidien/Argyle</td>
			<td style="width:182px;">1020</td>
			<td style="width:559px;">For mixing with L-DOPA/benserazide prior to subcutaneous injections.</td>
		</tr>
	</tbody>
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induction and assessment of levodopa-induced dyskinesias in a rat model of parkinson's disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;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.

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...

Watch this Scientific Journal Video about Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease at JoVE.com

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

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;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.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Our protocol provides a detailed method for a reliable induction and comprehensive rating of levodopa-induced dyskinesia in a rat model of Parkinson's disease. The main advantage of our dyskinesia rating scale is that it allows for quantification of both the intensity of a given abnormal involuntary movement and the amount of time spent exhibiting that behavior. Dependable animal models of levodopa-induced dyskinesia are critical in realizing the final goal of finding effective treatments to reduce or eliminate the side effect in individuals with Parkinson's disease.Start the preparation for the experiment by weighing rats weekly to calculate the appropriate drug quantity based on the weight changes. Provide the rats with nutritionally complete and highly palatable treats. On the first day of L-DOPA treatment, transfer rats to single housing supplied with institutional Animal Care and Use Committee approved enrichment.And maintain rats into the single housing throughout the study to avoid peer interference with the behavioral assessments. On the dyskinesia rating days, remove water bottles, food racks, and the enrichment in the cage to avoid interference with the behavioral assessments, and place the cages on a steel wire rack at an approximately 45 degree angle, then flip the identification tags upward. Before the L-DOPA injection, add the appropriate volume of sterile saline to the pre-weigh lyophilized L-DOPA and benserazide mix in the amber vial and shake well for 10 seconds.Immediately after the preparation, fill the required volume of the L-DOPA-Benserazide mix for each animal in individual syringes mounted with a 26 gauge needle and label each syringe with an individual animal identification. After bringing the first cage to the injection bench, remove the rat from its cage to place it on the injection surface. Use the nondominant hand to restrain the head and shoulders of the rat with the palm against the surface and scruff the skin on the back overlying the scapulae with the thumb and forefinger.Use the dominant hand to inject L-DOPA volume into the subcutaneous space between the fingers, keeping the needle parallel to the body to avoid intramuscular injection. Post injection, move the rat into the cage. Next, set the timer for one to two minutes and repeat the procedure injecting one rat every one to two minutes until all the rats are injected.On the rating day, position a timer next to the cage to observe levodopa-induced dyskinesia or LID behavior intensity while estimating the frequency of any given behavior during the rating period. Rate the intensity and frequency of the dystonic and hyperkinetic dyskinesia movements including the initial onset of LID behavior, peak behavior and the phase of decline at the desired number of time points. If using one minute rating intervals, set a timer for one minute to rate the first rat and continue the rating through all the rats at one minute intervals.The LID rating for abnormal involuntary behaviors in the Parkinsonian rats was categorized to include intensity and frequency of the behaviors. A range of abnormal involuntary movements, including dystonic, hyperkinetic, and stereotypic behaviors were observed in rats. The representative analysis provides an example of the data presented as peak dose LID over the entire experimental timeline and dosage schedule.The peak dose LID was defined as a time when the group average LID score was the greatest. Additionally, the LID rating data was examined over each daily rating time course at different L-DPOA concentrations with the groups combined and plotted as individual animals. Postmortem tissue from behaviorally evaluated subjects can be used to explore molecular mechanisms associated with levodopa-induced dyskinesias, and the impact of therapeutic interventions on such molecular mechanisms.Animal models of levodopa-induced dyskinesia have been useful in elucidating several mechanisms thought to underlie this behavioral malady. Such understanding has led to preclinical and clinical evaluation of several targets.

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