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/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 solution. Administer the anesthetic solution at a final volume of 2 mL/kg. 
		NOTE: Ketamine along with xylazine can produce sedation for 60-80 min. If the animal still has reflexes (e.g., hind leg pitching and/or blinking reflex), administer an additional 10% of the individual dose.</li>
	</ol>
	</li>
	<li>Imipramine 
	NOTE: The individual dose of imipramine used is 20 mg/kg.
	<ol>
		<li>To prepare 1 mL of imipramine 20 mg/mL solution, combine 20 mg of imipramine and 1 mL of 0.9% sterile saline solution. Administer the imipramine solution at a final volume of 1 mL/kg.</li>
	</ol>
	</li>
	<li>Meloxicam 
	NOTE: The individual dose of meloxicam used is 1 mg/kg.
	<ol>
		<li>To prepare 1 mL of meloxicam 1 mg/mL solution, combine 0.05 mL of meloxicam 2% and 0.95 mL of 0.9% sterile saline solution. Administer the meloxicam solution at a final volume of 1 mL/kg once a day for two days.</li>
	</ol>
	</li>
	<li>Ascorbic acid 0.1%
	<ol>
		<li>To prepare 1 mL of 0.1% ascorbic acid, combine 1 mg of ascorbic acid and 1 mL of 0.9% sterile saline solution.</li>
	</ol>
	</li>
	<li>6-hydroxydopamine (6-OHDA) 
	NOTE: 6-OHDA is a neurotoxin used to selectively destroy dopaminergic and noradrenergic neurons in the brain. Avoid direct contact with skin and mucous membranes of the eyes, nose, and mouth. When handling 6-OHDA, wear double nitrile gloves, lab coat, disposable gown, eye protection, and surgical mask or face shield. The total infusion volume of the toxin is 4 &#956;L/animal, and the individual amount is 10 &#956;g of 6-OHDA/animal.
	<ol>
		<li>To prepare 1 mL of 6-OHDA at a final concentration of 2.5 mg/mL, mix 2.5 mg of 6-OHDA and 1 mL of 0.9% saline solution containing 0.1% ascorbic acid (described above). 
		NOTE: 6-OHDA is light-sensitive and degrades faster when exposed to bright light. It must be properly handled and stored in an environment protected from light. If the color of the solution is reddish, discard it.</li>
	</ol>
	</li>
	<li>Lidocaine hydrochloride (2%)
	<ol>
		<li>Prepare 2% lidocaine solution for local application to the animal. 
		NOTE: The maximum dose that can be applied is 7 mg/kg.</li>
	</ol>
	</li>
	<li>Poly-antibiotic suspension 
	NOTE: The poly-antibiotic suspension with streptomycins and penicillins (see the Table of Materials) must be prepared at the time of application with the entire volume of diluent, whose ampoule accompanies the vial with the powder.
	<ol>
		<li>Remove the metallic disc on the rubber stopper. Disinfect the rubber stopper with alcohol.</li>
		<li>Using a syringe with a needle of 23 G, inject the diluent into the vial. Remove the needle and shake the vial vigorously until the suspension is entirely homogenized. Inject a little air into the vial and withdraw the desired volume of suspension.</li>
		<li>Administer a deep intramuscular injection, pulling the plunger before injecting the drug to ensure that no blood vessel is reached. 
		&#8203;NOTE: The final volume of suspension to be applied is 0.5 mL/kg.</li>
	</ol>
	</li>
</ol>
2. Preparation of materials
NOTE: Always follow instructions provided with the material safety data sheet when handling chemicals.
<ol>
	<li>Stereotaxic apparatus
	<ol>
		<li>Place the stereotaxic device on a stable and clean bench with proper illumination to perform the surgery. Disinfect the apparatus with 70% ethanol.</li>
		<li>Check if the ear and incisor bars of the device are correctly aligned. Place a thermal blanket where the animal will be placed during surgery to stay warm during the procedure. Monitor the animal&#39;s temperature with an accurate rectal probe. 
		NOTE: The thermal blanket should be at 37.5 &#176;C so that the animal maintains a body temperature of 37 &#176;C body.</li>
	</ol>
	</li>
	<li>Microinfusion system
	<ol>
		<li>Fill (70-80%) a Hamilton syringe (50 &#956;L or as desired) attached to a medical-grade polyethylene microtubing and a needle with double distilled water (ddH2O) and check for leaks through the system.</li>
		<li>Pull air through the system so that a single air bubble separates the ddH2O in the syringe from the 6-OHDA solution in the microtube. 
		NOTE: This procedure avoids contaminating the Hamilton syringe with 6-OHDA and allows the use of several rats on the same experimental day.</li>
		<li>Position the Hamilton syringe on the infusion pump so that it is firmly attached and the plunger of the syringe is parallel to the frame that will move to push it. Set the infusion pump to a speed of 0.5 &#956;L/min so that the total application of 4 &#956;L of 6-OHDA lasts for 8 min. Test the infusion system by confirming that there are no leaks and that the infusion occurs according to the previously set time and volume.</li>
		<li>Attach the needle of infusion attached to the microtube to the apparatus at the end of the stereotaxic arm and check that the needle is positioned at a 180&#176; angle to the surface. Ensure that the needle is straight and not bent. 
		NOTE: Check all the described procedures carefully because if any of the items in the infusion system do not work correctly, it may jeopardize the success of the surgery.</li>
	</ol>
	</li>
	<li>Suture
	<ol>
		<li>Use a sterile nylon non-absorbable suture with a 3/8 circle needle to suture the incision after surgery.</li>
	</ol>
	</li>
	<li>Postsurgical recovery site
	<ol>
		<li>Place a clean and sterilized housing box where animals can be monitored until fully recovered (responsive to touch and manipulation). Put a thermal blanket in the box for thermoregulation. 
		&#8203;NOTE: As thermoregulation is important, include a supplemental heat source to maintain body temperature if necessary.</li>
	</ol>
	</li>
</ol>
3. Surgical procedure
NOTE: In this protocol, adult male Sprague-Dawley rats (200-250 g) were kept under controlled conditions of temperature (22 &#177; 2 &#176;C), air exchange (15-20 exchanges/hour), and light-dark cycles (12 h/12 h), grouped in boxes with 3 or 4 animals, with free access to food and water.
<ol>
	<li>Weigh the animals to monitor weight changes in the days following the surgery. Calculate the dose of drugs to be administered.</li>
	<li>Administer imipramine intraperitoneally 30 min before surgery (~10-15 min before administering anesthesia), using a 27 G needle and a 1 mL syringe. 
	NOTE: The imipramine will block the noradrenaline transporter (NAT) and prevent 6-OHDA uptake by noradrenergic neurons, making the lesion more selective to the dopaminergic neurons40.</li>
	<li>After 10-15 min of the administration of imipramine, administer the intraperitoneal ketamine/xylazine anesthesia using a 27 G needle and a 1 mL syringe. Wait until the animal is completely anesthetized. Verify that the animal is under deep anesthesia when the animal does not respond to hind leg pinching and does not show a blink reflex.</li>
	<li>Shave the rat&#39;s fur in the region of the head where the incision will occur.</li>
	<li>Position the rat in the stereotaxic apparatus.
	<ol>
		<li>Position the head over the incisor bar and fix the bar 3.3 mm below the interaural line.</li>
		<li>Position the ear bars, one side at a time. Position the incisor bar and the ear bars so that the top of the skull is straight and parallel to the surface.</li>
		<li>Adjust the nose clamp and test that the head is firm and does not move to either side.</li>
	</ol>
	</li>
	<li>Apply sterile ophthalmic ointment to the rat eyes to prevent corneas from drying out.</li>
	<li>Apply povidone-iodine to the area to be incised to disinfect the site.</li>
	<li>Apply local lidocaine for analgesia of the incision region; do not exceed 7 mg/kg.</li>
	<li>Administer the meloxicam subcutaneously using a 27 G needle and a 1 mL syringe. 
	NOTE: Meloxicam is a nonsteroidal anti-inflammatory analgesic that will help the animal recover post surgery.</li>
	<li>Administer the poly-antibiotic suspension intramuscularly using a 23 G needle and a 1 mL syringe. 
	NOTE: The poly-antibiotic suspension is administered as a prophylactic treatment to avoid possible bacterial infections in the postsurgery recovery.</li>
	<li>Check that the animal is in a state of deep anesthesia by checking for blink reflexes or hind limb reflexes by pinching the hind paw with tweezers.</li>
	<li>With a scalpel, make an incision of ~1.5 cm in the region where the microinjection will occur. 
	NOTE: Sterile techniques are applied from this point until the wound closure.</li>
	<li>Clean the skull region with cotton swabs and cotton buds until the Bregma and Lambda can be seen. Mark the Bregma and the Lambda with a sterilized fine&#160;pen.</li>
	<li>Check that the dorsal-ventral (DV) coordinates of Bregma and Lambda are similar. If they are different, readjust the rat in the stereotaxic apparatus as the rat&#39;s head is not correctly positioned.</li>
	<li>Note down the anteroposterior (AP) and mediolateral (ML) coordinates of the Bregma.</li>
	<li>Move to the AP and ML coordinates of the right MFB according to 41: AP: -4.3 mm, ML: 1.6 mm from Bregma.</li>
	<li>Mark the region of the trepanation with a sterilized fine&#160;pen.</li>
	<li>With a sterilized drill, slowly pierce the animal&#39;s skull, taking care not to injure the dura mater.</li>
	<li>Position the microinjection needle on the dura mater and note the DV coordinates. Take a thin needle and gently rupture the dura mater. Insert the needle to the DV coordinate (8.3 mm ventral) of the MFB, where the microinjection will take place.</li>
	<li>Operate the microinjection pump to release the 6-OHDA solution into the MFB. When the microinjection is finished, check the Hamilton syringe to see if 4 &#956;L of 6-OHDA has been injected. 
	NOTE: The microinjection should last 8 min.</li>
	<li>After administration of the 6-OHDA, wait for 10 min before removing the needle to avoid backflow of the drug. Remove the microinjection needle slowly from the animal&#39;s brain.</li>
	<li>Disinfect the incision region again with povidone-iodine.</li>
	<li>Suture the incision area with ~3-4 surgical knots. 
	NOTE: The knot should not be too strong or too loose.</li>
	<li>Remove the rat from the stereotaxic apparatus and place it in a clean box for recovery on the thermal blanket until the animal has fully recovered from anesthesia. Observe the animal every 15 min until it is fully awake from anesthesia.</li>
</ol>
4. Postoperative procedures
<ol>
	<li>Monitor the weight of the animals over the next four days after surgery. Treat them with meloxicam subcutaneously once a day for two days after surgery, adjusting the dose for each day&#39;s weight. 
	NOTE: All animals should be assessed for the need for analgesics on the third day after the surgery.</li>
	<li>Check the incisions daily for at least four days to ensure they are not infected. Look for heat, swelling, pain, discharge, and redness until the incisions heal.</li>
	<li>Check the appetite and water consumption by monitoring the animal&#39;s body weight. Give wet feed to encourage the animals to eat. Observe the general body condition, attitude, and mobility daily for at least four days after surgery. Remove the sutures 7-10 days after surgery. 
	&#8203;NOTE: Animals should be euthanized if the endpoints defined in the ethical procedures are reached.</li>
</ol>
5. Stepping test
<ol>
	<li>Training 
	NOTE: The animals should be trained for three days before the test. According to the protocol described below, training should occur twice a day, once in the morning and once in the afternoon, or with an interval of at least 2 h between sessions. Track the time using a timer.
	<ol>
		<li>Day 1
		<ol>
			<li>In the first session, handle the rat by holding it in gloves for ~1-2 min to allow the rat to familiarize itself with the handler/experimenter.</li>
			<li>In the second session, alternate between holding the rat for 20 s and placing it on the protocol table for 20 s. Repeat this training step for 3 min to familiarize the rat with the experimental setup for the stepping test.</li>
		</ol>
		</li>
		<li>Day 2
		<ol>
			<li>In the first 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 headfirst at an angle of 45&#176; to the flat surface of the protocol table. Move horizontally on the table from end to end, allowing the rat to step on the table with both paws (cover 90 cm in 4 s). Hold the rat in gloves for 10 s, allowing it to rest; repeat this pattern for 3 min.</li>
			<li>In the second session, place one forepaw of the rat on the protocol table by holding the other forepaw back with one hand and hold the rat&#39;s back and hind paws with the other hand (see step 5.1.2.1). Move horizontally on the table from end to end in 4 s, allowing the rat to step with its free paw. Hold the rat in gloves for 10 s, allowing it to rest, and repeat with another forepaw, followed by the rest period. Repeat this pattern, alternating between the two forepaws, and rest for 3 min.</li>
			<li>Repeat the training step 3 times for 1 min each.</li>
		</ol>
		</li>
		<li>Day 3
		<ol>
			<li>In the first session, follow the procedure described in step 5.1.2.2 for one forepaw. Repeat with another forepaw, followed by the rest period. Repeat this pattern, alternating between the two forepaws, and rest for 3 min.</li>
			<li>In the second session, follow the procedure described in step 5.1.2.2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Test 
	NOTE: The stepping test is performed before surgery, 2 and 4 weeks after stereotaxic surgery, to evaluate the akinesia of the contralateral forelimb and the possible injury caused by 6-OHDA.
	<ol>
		<li>Hold the rat at an angle of 45&#176; to the surface, immobilizing its hind limbs and allowing only one of the forelimbs to rest on the platform, as explained above, for day 3 of training.</li>
		<li>Drag the rat forward over a distance of 90 cm in 4 s, with the right or left paw resting on the surface.</li>
		<li>Take notes and quantify the number of forehand-adjusting steps taken with each paw in each direction.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to declare.

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><tbody><tr><td>6-OHDA</td><td>Sigma Aldrich</td><td>H4381</td><td>https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt&amp;amp;region=BR&amp;amp;cm_sp=Insite-_-caSrpResults_srpRecs_srpModel&lt;br/&gt; _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&amp;amp;region=BR&amp;amp;gclid=&lt;br/&gt; Cj0KCQjw4cOEBhDMARIsAA3XD&lt;br/&gt; RipyOnxOxkKAm3J1PxvIsvw09&lt;br/&gt; _kfaS2jYcD9E5OyuHYr4n89kO&lt;br/&gt; 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%&amp;nbsp; 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...

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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 ...

the-6-hydroxydopamine-rat-model-of-parkinson-s-disease

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10.5K Views.  University of São Paulo. Dit protocol laat zien hoe unilaterale nigrostriatale laesies bij de rat kunnen worden uitgevoerd door 6-hydroxydopamine in de mediale voorhersenenbundel te injecteren. Dit protocol laat ook zien hoe motorische tekorten kunnen worden beoordeeld en laesies kunnen worden voorspeld met behulp van de staptest. Dit model reproduceert de ernstige vernietiging van dopaminerge neuronen en simuleert Parkinson-symptomen bij ratten. Dit rattenmodelprotocol maakt het mogelijk om de ziekte van Parkinson i...

Video: The 6-hydroxydopamine Rat Model of Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Medicine

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Biology

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96&#160;mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p &#60; 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that ...

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.

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). ...

Generating the 6-hydroxydopamine Rat Model of Parkinson&#39;s Disease

Source: Guimar&#227;es, R. P., et al. <a href="https://app.jove.com/v/62923">The 6-hydroxydopamine Rat Model of Parkinson&#39;s Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a procedure to create a rat model of Parkinson&#39;s disease by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the brain. The neurotoxin infusion into the medial forebrain bundle (MFB) specifically targets dopaminergic neurons responsible for motor control, leading to their destruction and replicating Parkinson&#39;s disease symptoms.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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The 6-hydroxydopamine Rat Model of Parkinson's Disease

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