Name: rating l-dopa-induced dyskinesias in the unilaterally 6-ohda-lesioned rat model of parkinson's disease
Uploaded: 2021-10-04T14:30:00
Description: Watch this Scientific Journal Video about Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease at JoVE.com

This work was supported by S&#227;o Paulo Research Foundation (FAPESP, grant 2017/00003-0). We are grateful for the Coordination for the Improvement of Higher Education Personnel (CAPES). We thank Dr. Anthony R. West, Dr. Heinz Steiner, and Dr. Kuei Y. Tseng for support and mentoring.

All experiments were performed in accordance with The Ethics Committee of the Faculty of Philosophy, Sciences, and Letters of Ribeir&#227;o Preto (CEUA/FFCLRP 18.5.35.59.5).
1.&#160;6-OHDA lesion
<ol>
	<li>Use Sprague-Dawley male rats weighing 200-250 g at the beginning of the experiments (6 weeks). House the animals (2-3 per cage) under standard laboratory conditions (12:12 h light/dark cycle, lights on at 06:00 h, temperature-controlled facilities (22-24 &#176;C), with foo...

This protocol demonstrates how to induce and analyze AIMs in the rat model of PD induced by unilateral microinjection of 6-OHDA in the MFB. Chronic daily administration of low doses of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) produced the development of AIMs over the 3 weeks of treatment. Temporal analysis revealed a significant increase of AIMs, and the peak-dose dyskinesia is observed between 30 and 90 min after L-DOPA administration. AIMs are repetitive and purposeless movements affecting axial, limb,...

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) represents the most effective treatment for the motor symptoms of Parkinson&#39;s disease (PD)1. L-DOPA therapy may ameliorate motor symptoms associated with PD but loses effectiveness with time. Motor fluctuations such as &#34;wearing-off fluctuation&#34; or &#34;end-of-dose deterioration&#34; manifest clinically as a shortened duration of the effect of single L-DOPA doses2. In other cases, clinical manifestations consist of slow twisting movements and abnormal postures (dystonia)3 and occur when dopamine levels are low (off-period dystonia)4. On the other hand, L-DOPA-induced dyskinesias (LIDs) appear when dopamine levels in the plasma and the brain are high5.
LIDs produce debilitating side effects that include motor complications such as choreiform, dystonic, and ballistic6 movements. Once established, LIDs occur after every L-DOPA administration. Motor complications occur in 40%-50% of PD patients undergoing L-DOPA therapy for 5 years, and the incidence increases over the years7. Although the pathophysiological mechanisms involved in the development of LIDs in PD patients are not yet fully elucidated, the extent of dopaminergic denervation, pulsatile L-DOPA administration, downstream changes in striatal proteins and genes, and abnormalities in non-dopamine transmitter systems are factors that contribute to the development of these unwanted side effects6,8,9,10.
The neurotoxin 6-hydroxydopamine (6-OHDA) is a well-characterized tool to study PD in rodents11,12,13,14. Since 6-OHDA does not cross the blood-brain barrier, it must be injected directly into the nigrostriatal pathway. 6-OHDA-induced dopaminergic depletion is concentration- and site-dependent15. Unilateral administration of 6-OHDA at the medial forebrain bundle (MFB) can produce severe (&#62;90%) nigrostriatal damage in rodents16,17,18,19. Chronic administration of L-DOPA to severe unilaterally 6-OHDA-lesioned rodents causes the appearance of dyskinetic-like movements named abnormal involuntary movements (AIMs). Dyskinetic-like movements in rodents share similar molecular, functional, and pharmacological mechanisms related to LIDs in PD patients5. Therefore, 6-OHDA-lesioned rats20 and mice21 are valuable preclinical models to study LIDs. When treated chronically (7-21 days) with therapeutic doses of L-DOPA (5-20 mg/kg), unilaterally 6-OHDA-lesioned rats and mice show a gradual development of AIMs that affect the forelimb, trunk, and orofacial muscles contralateral to the lesion17,18,19,20,22,23,24. These movements are presented at a time course similar to L-DOPA-induced peak-dose dyskinesias in PD patients25 and are characterized by hyperkinetic movements and dystonia5. AIMs are usually scored based on their severity (e.g., when a specific AIM is present) and amplitude (e.g., characterized by the amplitude of each movement)5,23,25.
6-OHDA-lesioned rodent models of LIDs present face validity (i.e., the model has several characteristics that look like the human condition)5,11,26,27,28. Rodent AIMs, similar to what occurs in PD patients, are seen as hyperkinetic (forelimb and orolingual) and dystonic (axial) movements29 and mimics peak-dose dyskinesia. At the molecular and functional level, rodent models share many pathological characteristics with PD patients5, such as upregulation of FosB/&#916;FosB19,26,30,31,32,33 and serotonin transporter (SERT)34,35. Concerning predictive validity, drugs that reduce LIDs in PD patients (e.g., the N-methyl-D-aspartate (NMDA) receptor antagonist amantadine) present antidyskinetic efficacy in the rodent model22,36,37,38,39.
The rodent AIMs rating scale was created based on four AIMs subtypes that include AIMs affecting the head, neck, and trunk (axial AIMs), hyperkinetic forelimb movements (limb AIMs), and dyskinetic-like orolingual movements (orolingual AIMs). Although contralateral rotation (locomotive AIMs) is also present in unilaterally lesioned rodents20,22,23,25,40, it has not been scored as a dyskinetic-like movement since it may not represent a specific measure of LIDs22,37,41.
Here, we will describe how to induce and analyze dyskinetic-like movements (axial, limb, and orolingual AIMs) in the severe (&#62;90%) unilaterally 6-OHDA-lesioned rat model of PD. We organized our protocol based on the previous literature and our laboratory expertise.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-hydroxydopamine hydrobromide</td>
			<td>Sigma-Aldrich, USA</td>
			<td>H6507</td>
			<td>Neurotoxin that produces degeneration of catecholaminergic terminals</td>
		</tr>
		<tr>
			<td>Benzerazide hydrochloride</td>
			<td>Sigma</td>
			<td>B7283</td>
			<td>Peripheral dopa-decarboxylase inhibitor</td>
		</tr>
		<tr>
			<td>Camera Bullet IR Turbo HD (HD-TVI)&#160; 2.8mm B</td>
			<td>HIKVISION</td>
			<td>DS-2CE16C0T-IRP</td>
			<td>Camera used to record all behavior</td>
		</tr>
		<tr>
			<td>Imipramine hidrochloride</td>
			<td>Alfa Aesar</td>
			<td>J63723</td>
			<td>Norepinephrine transporter inhibitor (NET) used to protect noradrenergic neurons from 6-OHDA</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Ceva Animal Health</td>
			<td></td>
			<td>Anesthesia for surgical intervention</td>
		</tr>
		<tr>
			<td>L-3,4-dihydroxyphenylalanine (L-DOPA) methyl ester (hydrochloride)</td>
			<td>Cayman Chemical Company</td>
			<td>16149</td>
			<td>Dopamine precursor</td>
		</tr>
		<tr>
			<td>Mirrors</td>
			<td></td>
			<td></td>
			<td>Used to observe the behavior of animals during experiments in all directions</td>
		</tr>
		<tr>
			<td>Needles 0.30 x 13 mm</td>
			<td>PrecisionGlide</td>
			<td></td>
			<td>Needles used to inject drugs</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Samtec</td>
			<td></td>
			<td>Salt</td>
		</tr>
		<tr>
			<td>Syringes 1 ml Sterile</td>
			<td>BD Plastipak</td>
			<td></td>
			<td>Syringes used to inject drugs</td>
		</tr>
		<tr>
			<td>Transparent cylinders</td>
			<td></td>
			<td></td>
			<td>Used to record animal behavior during experiments</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td>Ceva Animal Health</td>
			<td></td>
			<td>Sedative, analgesic and muscle relaxant for surgical intervention</td>
		</tr>
	</tbody>
</table>

rating l-dopa-induced dyskinesias in the unilaterally 6-ohda-lesioned rat model of parkinson's disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Although the AIMs patterns observed in rats are simpler and limited compared to those observed in humans and nonhuman primates, this model reproduces both hyperkinetic and dystonic-like movements induced by chronic L-DOPA administration. Here we present data collected from a group (n = 10) of unilaterally 6-OHDA-lesioned rats chronically treated with L-DOPA (5 mg/kg combined with 12.5 mg/kg of benserazide) for 3 weeks (Monday to Friday). Note that the data presented in Figure 2, <strong clas...

Watch this Scientific Journal Video about Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease at JoVE.com

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

Rating L-DOPA-induced dyskinesias in the 6-hydroxydopamine rat model of the Parkinson's disease is an important preclinical tool to identify effective antidyskinetic interventions. This model is relatively simple, presents low cost, and has similarities to what happens in the clinic. The procedure will be demonstrated by Danilo Leandro Ribeiro, a PhD student in the laboratory.Begin the experiment with Sprague-Dawley male rats, weighing 200 to 250 grams. House two to three animals per cage under the standard laboratory conditions with food and water available ad libitum. Before the surgery, administer the norepinephrine transporter inhibitor, imipramine, to the rat intraperitoneally.After confirming the anesthesia by the lack of response to toe pinch, position the rat in a prone position in the stereotaxic apparatus on top of the heating pad. Once the rat is positioned, use a scalpel to make a one-centimeter-long incision at the region where the microinjection will occur. Then, clean the skull region with cotton swabs and ensure the bregma and lambda are exposed.Take the medial forebrain bundle, or MFB, stereotaxic coordinates from bregma at 4.3 millimeters anterior, 1.6 millimeters lateral to the right side, and 8.3 millimeters ventral from the durometer. When the coordinates are decided, administer the 6-hydroxydopamine, or 6-OHDA, unilaterally at a rate of 0.4 microliters per minute in the right MFB, with a 50-microliter Hamilton glass syringe. At the end of the surgery, suture the scalp incision and rehydrate the animal with warm, sterile, 0.9%saline solution subcutaneously, at the dose of 10 milliliters per kilogram.Remove the animal from the stereotaxic frame and place it in a warm recovery cage, while monitoring until consciousness is regained. At four weeks post-lesion, measure the effectiveness of the dopaminergic lesion using a stepping test, by assessing the akinesia of the forelimb contralateral to the lesion. Rats presenting three or fewer adjusting steps with the contralateral forelimb should be included in the study as putative, severely 6-OHDA-lesioned rats.Four weeks after the 6-OHDA lesions, start the chronic treatment on Monday and record abnormal involuntary movements on Wednesday. To do so, place the rat inside a transparent cylinder with 20 centimeters diameter and 40 centimeters height, and allow it to acclimatize for at least 15 minutes. Ensure that the floor is covered with the bedding material and mirrors are positioned behind the cylinder to observe the animal from all possible angles.Position a high-resolution video camera to allow the viewing of axial, limb, and orolingual abnormal involuntary movements. The mirrors behind the cylinder will allow tracking abnormal involuntary movements at a 360-degree angle. The camera can be positioned slightly below the plane at a 15-degree angle from animals to observe orolingual abnormal involuntary movements.When the position is decided, fix the camera directly to the bench. Once the animal is acclimatized for 15 minutes, remove the animal from the cylinder and administer five milligrams per kilogram of freshly prepared L-dopamine or L-DOPA combined with 12.5 milligrams per kilogram of benserazide, subcutaneously. Put the animal back in the cylinder and start a timer to track abnormal involuntary movements.Use a video camera to record the abnormal involuntary movements for 180 minutes post-injection, at 30-minute intervals, to carry out offline scoring. After the six observation periods, the scores must be given over one to two minute epochs and classified as axial, limb, or orolingual. Ensure not to include normal behaviors.The treatment with L-DOPA and benserazide should be continued for three weeks, once daily, from Monday to Friday. In the representative time course analysis, the scores applied to axial, limb, and orolingual abnormal involuntary movements over three weeks of chronic L-DOPA administration, are shown. It was noted that L-DOPA-induced, peak-dose dyskinesias occur between 30 to 90 minutes, with a gradual decrease after 120 minutes post-injection.Additionally, the sum of axial, limb, and orolingual abnormal involuntary movements was presented on individuals, scoring days and weekly over three weeks of chronic L-DOPA administration. The abnormal involuntary movements protocol is usually for the investigation of new therapeutic tools with antidyskinetic translational potential.

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