The 6-hydroxydopamine Rat Model of Parkinson's Disease

Summary

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

Abstract

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

Introduction

The main neuropathological characteristics of PD are the chronic progressive neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies containing α-synuclein protein¹. As SNc dopaminergic neurons project their axons into the striatum through the nigrostriatal pathway, neurodegeneration of neurons in SNc results in a dopaminergic deficit in the striatum². The absence of dopamine in the striatum causes an imbalance in the activities of the direct and indirect motor control pathways, which is responsible for the main motor symptoms of PD: akinesia (slow moveme....

Protocol

All procedures involving animals followed the ethical principles of the National Council for the Control of Animal Experimentation (CONCEA) and the Arouca Law (Law 11.794/2008) and were approved by the local ethics committee (CEUA-FFCLRP/USP (18.5.35.59.5).

1. Preparation of drugs

1. Anesthesia with Ketamine/Xylazine
   NOTE: The dose of ketamine used is 70 mg/kg, and the dose of xylazine is 10 mg/kg.
   1. To prepare 1 mL of anesthetic using ketamine 100 mg/mL solution and xylazine 20 mg/mL solution, combine 0.35 mL of ketamine solution, 0.25 mL of xylazine solution, and 0.4 mL of 0.9% sterile saline sol....

Results

Dopaminergic lesion assessment
The stepping test enables the assessment of the akinesia of the anterior limb contralateral to the lesion and the selection of animals with a possible lesion of the nigrostriatal pathway induced by 6-OHDA infusion (Figure 1). The comparison of the performance of the contralateral forelimb stepping test presurgery and 2 weeks and 4 weeks after surgery revealed interaction (F_2,74 = 93.63; p < 0.0001; two-way repeated-measures AN.......

Discussion

This paper describes a protocol for performing surgery for unilateral microinfusion of 6-OHDA in the MFB, capable of causing robust lesions in the neurons of the nigrostriatal pathway and generating akinesia in the animal. Also described is the protocol for performing the stepping test, an easily applicable and noninvasive test that can be used to prove the success of the lesions and assess forelimb akinesia. As presented in the representative results, animals receiving 6-OHDA showed a reduction in the number of adjustin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-OHDA	Sigma Aldrich	H4381	https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt®ion=BR&cm_sp=Insite-_-caSrpResults_srpRecs_srpModel _6-ohda-_-srpRecs3-1
70% Alcohol
Ascorbic acid	Sigma Aldrich	795437	https://www.sigmaaldrich.com/catalog/product/sial/795437?lang=pt®ion=BR&gclid= Cj0KCQjw4cOEBhDMARIsAA3XD RipyOnxOxkKAm3J1PxvIsvw09 _kfaS2jYcD9E5OyuHYr4n89kO 6yicaAot6EALw_wcB
Cotton
Drill or tap
Gauze
Hamilton syringe 50 uL	Hamilton	80539	https://www.hamiltoncompany.com/laboratory-products/syringes/80539
Imipramine	Alfa Aeser	J63723	https://www.alfa.com/pt/catalog/J63723/
Infusion pump	Insight	EFF-311	https://insightltda.com.br/produto/eff-311-bomba-de-infusao-2-seringas/
Ketamine (Dopalen)	Ceva		https://www.ceva.com.br/Produtos/Lista-de-Produtos/DOPALEN
Machine for trichotomy
Meloxicam (Maxicam 2%  Ourofino)	Ourofino		https://terrazoo.com.br/produto/maxicam-injetavel-2-50ml-ouro-fino/
Metal Disposal
Paper towels
Pentabiotic	Zoetis		https://www.zoetis.com.br/pentabiotico-veterinario.aspx
Plastic waste garbage can
Poly-antibiotic	Pentabiotic (Wealth)
Povidone-iodine
Scalpel and blades
Scissors
Scraper
Stereotaxic apparatus	Insight	EFF-331	https://insightltda.com.br/produto/eff-331-estereotaxico-1-torre/
Sterile saline solution
Swabs
Temperature probe
Timer
Tweezers
Xylazine (Anasedan)	Ceva		https://www.ceva.com.br/Produtos/Lista-de-Produtos/ANASEDAN