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

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 dyst....

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

1. 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 food and water available ad libitum.
   NOTE: A complete description of how to generate severe 6-OHDA-lesioned rats is described elsewhere^4....

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,

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,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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