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

Published: October 04, 2021

doi:

Keila Bariotto-dos-Santos*¹, Danilo Leandro Ribeiro*¹, Rayanne Poletti Guimarães, Fernando Eduardo Padovan-Neto

¹Department of Psychology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto,University of São Paulo

Summary

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.

Abstract

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.

Introduction

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) represents the most effective treatment for the motor symptoms of Parkinson's disease (PD)¹. L-DOPA therapy may ameliorate motor symptoms associated with PD but loses effectiveness with time. Motor fluctuations such as "wearing-off fluctuation" or "end-of-dose deterioration" 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 (>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/ΔFosB¹⁹^,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 (>90%) unilaterally 6-OHDA-lesioned rat model of PD. We organized our protocol based on the previous literature and our laboratory expertise.

Protocol

All experiments were performed in accordance with The Ethics Committee of the Faculty of Philosophy, Sciences, and Letters of Ribeirão Preto (CEUA/FFCLRP 18.5.35.59.5). 1. 6-OHDA lesion 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 °C), with foo…

Representative Results

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…

Discussion

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,…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by São 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.

Materials

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

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Bariotto-dos-Santos, K., Ribeiro, D. L., Guimarães, R. P., Padovan-Neto, F. E. Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson’s Disease. J. Vis. Exp. (176), e62924, doi:10.3791/62924 (2021).