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 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
<ol>
	<li>Anesthesia with Ketamine/Xylazine 
	NOTE: The dose of ketamine used is 70 mg/kg, and the dose of xylazine is 10 mg/kg.
	<ol>
		<li>To prepare 1 mL of anesthetic using ketamine 100 mg...

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...

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 &#945;-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 movement), bradykinesia (difficulty in starting movements), muscle stiffness, and tremor at rest3,4,5.
As the molecular and physiological mechanisms involved in the onset of PD are still not fully understood, currently available principal treatments seek to alleviate the motor symptoms through pharmacotherapies, deep brain stimulation6,7, genetic therapies8, and cell transplantation9. Therefore, preclinical research is fundamental to elucidate the mechanisms involved in the onset of PD and discover new methodologies for the early diagnosis and new therapies to prevent or stop the degeneration of neurons affected by PD10.
Animal models have been widely used to simulate human pathology in the laboratory, contributing to the advancement of medicine and science11,12,13,14. However, it is essential to emphasize that the correct choice of the animal model is fundamental for the success of the study. Therefore, the animal model must be validated in three main aspects: i) face validity, in which the animal model must have the characteristics of human pathology; ii) constructive validity, in which the animal model must have a solid theoretical basis; and iii) predictive validity, in which animal models must respond to treatments in a similar way to clinical treatment.
Currently, several animals are used as animal models for PD. The main groups include mammals, such as rodents, primates, minipigs, dogs, and cats, and other groups such as drosophila and zebrafish. Rodents are the most classic animal model for PD and the most used due to their ease of handling and maintenance. In addition, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans15.
A review published by Kin and colleagues in 2019 analyzed the principal animal model methodologies used for PD in the 2000s and found that the most used animal model involved neurotoxins such as 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both neurotoxins cause mitochondrial dysregulation in dopaminergic neurons in the nigrostriatal pathway, leading to cell death16. Another widely used model involves genetic manipulation through mutation in specific genes involved in the onset of PD, causing mitochondrial dysregulation17. Neurotoxin models are commonly used to evaluate and compare therapeutics, whereas genetic models are used to study the development of preventive therapies and idiopathic PD15.
The neurotoxin MPTP was discovered to cause parkinsonism in the mid-1980s after seven patients used the substance and exhibited severe PD symptoms. In addition to the symptoms, the patients responded to treatment with L-DOPA, which made the researchers link the molecule directly to PD. After the case was published in 1986, several researchers began using MPTP in preclinical PD research18. Researchers have found that being a lipophilic molecule, MPTP can cross the blood-brain barrier (BBB) and be converted to MPP+19. This toxic substance accumulates inside neurons and causes damage to complex 1 of the mitochondrial respiratory chain, leading to the death of dopaminergic neurons20.
The 6-OHDA neurotoxin model was first used to induce the degeneration of monoamine neurons of the nigrostriatal pathway in 196821. The 6-OHDA model is commonly used to cause neurodegeneration in the nigrostriatal pathway as it is a dopamine analog and toxic for catecholamine-containing cells. After 6-OHDA enters the brain, it may be taken up by the dopamine transporter (DAT) in dopaminergic neurons, leading to degeneration of the nigrostriatal pathway22. Because 6-OHDA does not penetrate the BBB, it must be administered directly through intracerebral stereotaxic injection23. A noradrenaline reuptake inhibitor is often combined with 6-OHDA microinjection to preserve noradrenergic fibers and provide a more selective degeneration of dopaminergic neurons24.
After DAT takes up 6-OHDA, it will accumulate in the cytosol of neurons, producing reactive oxygen species (ROS) and leading to cell death15. Three different lesions models of 6-OHDA are frequently used: i) lesions to the SNc25,26; ii) lesions to the striatum27,28; iii) lesions to the MFB29,30. Lesions caused in the striatum result in a slow and retrograde degeneration of dopaminergic neurons in SNpc. In contrast, lesions caused in SNpc and MFB result in rapid and total degeneration of neurons, leading to more advanced parkinsonian symptoms31.
Unilateral or bilateral injection of 6-OHDA can cause neurodegeneration in dopaminergic neurons. 6-OHDA does not always cause severe damage to the neurons; sometimes, the injection results in partial damage, which is also used to simulate the early stages of PD32. The unilateral injection is more commonly used due to the model&#39;s ability to assess the animal&#39;s motor deficits and predict cell loss through tests such as amphetamine/apomorphine-induced rotation and the stepping test29. Bilateral injections are most used to evaluate spatial memory and recognition33.
The amphetamine/apomorphine-induced rotation test is a behavioral test commonly used to predict cell loss in the nigrostriatal pathway. It is defined as a process in which repeated administration of dopamine agonists leads to an intensification of rotational behavior in 6-OHDA-lesioned animals34. Rotational behavior consists of quantifying amphetamine-induced ipsilateral rotation or apomorphine-induced contralateral turns in unilaterally lesioned rodents. Drug-induced rotational behavior has been criticized because rotation does not correspond to PD symptoms in humans and can be affected by variables such as tolerance, sensitization, and &#34;priming&#34;35.
Priming is one of the most critical factors in these behavioral tests. Some cases have been reported wherein a single dose of L-DOPA led to a failure in rotational behaviors36. Additionally, another critical factor related to the combined application of the amphetamine-induced test and apomorphine-induced test for parallel use is that they measure different endpoints because of different mechanisms of action, reflecting the inactivation of different signaling mechanisms and pathways. Furthermore, the amphetamine-induced test is more accurate to measure nigrostriatal lesions above 50-60%, whereas the apomorphine-induced test is more accurate for lesions above 80%37.
The stepping test has emerged as a behavioral test that indicates deficits related to dopaminergic neuron degeneration and therapeutic effects. It enables the analysis of akinesia caused by a 6-OHDA lesion in dopaminergic neurons without a drug-induced procedure. Furthermore, the test has been well established and commonly used since 1995, when it was first described by Olsson et al.35. In 1999, Chang et al.38 also analyzed and compared the performance of rats in the stepping test with the level of degeneration caused by 6-OHDA and found that animals that performed worse in the stepping test also had a more significant degeneration of dopaminergic neurons.
The stepping test is an excellent method to predict severe dopaminergic nigrostriatal damage in 6-OHDA-lesioned rats. Evidence suggests that motor deficits appear in the contralateral forelimb of the 6-OHDA infusion during the stepping test when the degree of dopaminergic loss in SNc is &#62;90%39. This paper describes the protocols, methodologies, and materials used to perform stereotaxic surgery for the unilateral infusion of 6-OHDA into the MFB of rats and how to predict the dopaminergic lesions caused by the toxin through the stepping test.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-OHDA</td>
			<td>Sigma Aldrich</td>
			<td>H4381</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt&#38;region=BR&#38;cm_sp=Insite-_-caSrpResults_srpRecs_srpModel 
			_6-ohda-_-srpRecs3-1</td>
		</tr>
		<tr>
			<td>70% Alcohol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>795437</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sial/795437?lang=pt&#38;region=BR&#38;gclid= 
			Cj0KCQjw4cOEBhDMARIsAA3XD 
			RipyOnxOxkKAm3J1PxvIsvw09 
			_kfaS2jYcD9E5OyuHYr4n89kO 
			6yicaAot6EALw_wcB</td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drill or tap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 50 uL</td>
			<td>Hamilton</td>
			<td>80539</td>
			<td>https://www.hamiltoncompany.com/laboratory-products/syringes/80539</td>
		</tr>
		<tr>
			<td>Imipramine</td>
			<td>Alfa Aeser</td>
			<td>J63723</td>
			<td>https://www.alfa.com/pt/catalog/J63723/</td>
		</tr>
		<tr>
			<td>Infusion pump</td>
			<td>Insight</td>
			<td>EFF-311</td>
			<td>https://insightltda.com.br/produto/eff-311-bomba-de-infusao-2-seringas/</td>
		</tr>
		<tr>
			<td>Ketamine (Dopalen)</td>
			<td>Ceva</td>
			<td></td>
			<td>https://www.ceva.com.br/Produtos/Lista-de-Produtos/DOPALEN</td>
		</tr>
		<tr>
			<td>Machine for trichotomy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Maxicam 2%&#160; Ourofino)</td>
			<td>Ourofino</td>
			<td></td>
			<td>https://terrazoo.com.br/produto/maxicam-injetavel-2-50ml-ouro-fino/</td>
		</tr>
		<tr>
			<td>Metal Disposal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentabiotic</td>
			<td>Zoetis</td>
			<td></td>
			<td>https://www.zoetis.com.br/pentabiotico-veterinario.aspx</td>
		</tr>
		<tr>
			<td>Plastic waste garbage can</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-antibiotic</td>
			<td>Pentabiotic (Wealth)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel and blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scraper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>Insight</td>
			<td>EFF-331</td>
			<td>https://insightltda.com.br/produto/eff-331-estereotaxico-1-torre/</td>
		</tr>
		<tr>
			<td>Sterile saline solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Anasedan)</td>
			<td>Ceva</td>
			<td></td>
			<td>https://www.ceva.com.br/Produtos/Lista-de-Produtos/ANASEDAN</td>
		</tr>
	</tbody>
</table>

the 6-hydroxydopamine rat model of parkinson's disease

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.

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 (F2,74 = 93.63; p &#60; 0.0001; two-way repeated-measures...

Watch this Scientific Journal Video about The 6-hydroxydopamine Rat Model of Parkinson's Disease at JoVE.com

The 6-hydroxydopamine Rat Model of Parkinson's Disease

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.

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

This protocol demonstrates how to perform unilateral nigrostriatal lesions in the rat by injecting 6-hydroxydopamine in the medial forebrain bundle.This protocol also demonstrates how to assess motor deficits and predict lesions using the stepping test.This model reproduces the severe destruction of dopaminergic neurons and simulates Parkinsonian symptoms in rats.This rat model protocol makes it possible to study Parkinson's disease in the laboratory, covering several areas of interest, such as physiology, anatomy, and the pathological mechanism of the disease.The test is performed with both forelimbs, one at a time, as described in the pretest protocol.Perform the training sessions for three days.On the first day, in the first session, hold the rat for 1 to 2 minutes to allow the rat to familiarize itself with the handler or experimenter.In the second session, in order to familiarize the rat with the experimental setup for the stepping test, hold the rat for 20 seconds, and then place it on the table for 20 seconds.Repeat this training step for 3 minutes.On the second and third day, in the first training session, place both forepaws of the rat on the protocol table by holding its hind paws and back with one hand.Tilt the rat downwards, head first, at an angle of 45

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Abstract