The 6-hydroxydopamine Rat Model of Parkinson's Disease

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 movement), bradykinesia (difficulty in starting movements), muscle stiffness, and tremor at rest^3,4,5.

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 stimulation^6,7, 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 science^11,12,13,14. 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 SNc^25,26; ii) lesions to the striatum^27,28; iii) lesions to the MFB^29,30. 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's ability to assess the animal'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 "priming"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 >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.

Protokół

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 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.
2. Imipramine
   NOTE: The individual dose of imipramine used is 20 mg/kg.
   1. 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.
3. Meloxicam
   NOTE: The individual dose of meloxicam used is 1 mg/kg.
   1. 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.
4. Ascorbic acid 0.1%
   1. To prepare 1 mL of 0.1% ascorbic acid, combine 1 mg of ascorbic acid and 1 mL of 0.9% sterile saline solution.
5. 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 μL/animal, and the individual amount is 10 μg of 6-OHDA/animal.
   1. 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.
6. Lidocaine hydrochloride (2%)
   1. Prepare 2% lidocaine solution for local application to the animal.
      NOTE: The maximum dose that can be applied is 7 mg/kg.
7. 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.
   1. Remove the metallic disc on the rubber stopper. Disinfect the rubber stopper with alcohol.
   2. 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.
   3. Administer a deep intramuscular injection, pulling the plunger before injecting the drug to ensure that no blood vessel is reached.
      ​NOTE: The final volume of suspension to be applied is 0.5 mL/kg.

2. Preparation of materials

NOTE: Always follow instructions provided with the material safety data sheet when handling chemicals.

1. Stereotaxic apparatus
   1. Place the stereotaxic device on a stable and clean bench with proper illumination to perform the surgery. Disinfect the apparatus with 70% ethanol.
   2. 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's temperature with an accurate rectal probe.
      NOTE: The thermal blanket should be at 37.5 °C so that the animal maintains a body temperature of 37 °C body.
2. Microinfusion system
   1. Fill (70-80%) a Hamilton syringe (50 μL 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.
   2. 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.
   3. 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 μL/min so that the total application of 4 μL 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.
   4. 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° 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.
3. Suture
   1. Use a sterile nylon non-absorbable suture with a 3/8 circle needle to suture the incision after surgery.
4. Postsurgical recovery site
   1. 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.
      ​NOTE: As thermoregulation is important, include a supplemental heat source to maintain body temperature if necessary.

3. Surgical procedure

NOTE: In this protocol, adult male Sprague-Dawley rats (200-250 g) were kept under controlled conditions of temperature (22 ± 2 °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.

1. Weigh the animals to monitor weight changes in the days following the surgery. Calculate the dose of drugs to be administered.
2. 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.
3. 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.
4. Shave the rat's fur in the region of the head where the incision will occur.
5. Position the rat in the stereotaxic apparatus.
   1. Position the head over the incisor bar and fix the bar 3.3 mm below the interaural line.
   2. 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.
   3. Adjust the nose clamp and test that the head is firm and does not move to either side.
6. Apply sterile ophthalmic ointment to the rat eyes to prevent corneas from drying out.
7. Apply povidone-iodine to the area to be incised to disinfect the site.
8. Apply local lidocaine for analgesia of the incision region; do not exceed 7 mg/kg.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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 pen.
14. 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's head is not correctly positioned.
15. Note down the anteroposterior (AP) and mediolateral (ML) coordinates of the Bregma.
16. Move to the AP and ML coordinates of the right MFB according to ⁴¹: AP: -4.3 mm, ML: 1.6 mm from Bregma.
17. Mark the region of the trepanation with a sterilized fine pen.
18. With a sterilized drill, slowly pierce the animal's skull, taking care not to injure the dura mater.
19. 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.
20. 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 μL of 6-OHDA has been injected.
    NOTE: The microinjection should last 8 min.
21. 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's brain.
22. Disinfect the incision region again with povidone-iodine.
23. Suture the incision area with ~3-4 surgical knots.
    NOTE: The knot should not be too strong or too loose.
24. 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.

4. Postoperative procedures

1. 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's weight.
   NOTE: All animals should be assessed for the need for analgesics on the third day after the surgery.
2. 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.
3. Check the appetite and water consumption by monitoring the animal'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.
   ​NOTE: Animals should be euthanized if the endpoints defined in the ethical procedures are reached.

5. Stepping test

1. 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.
   1. Day 1
      1. 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.
      2. 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.
   2. Day 2
      1. 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° 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.
      2. 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'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.
      3. Repeat the training step 3 times for 1 min each.
   3. Day 3
      1. 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.
      2. In the second session, follow the procedure described in step 5.1.2.2.
2. 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.
   1. Hold the rat at an angle of 45° 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.
   2. Drag the rat forward over a distance of 90 cm in 4 s, with the right or left paw resting on the surface.
   3. Take notes and quantify the number of forehand-adjusting steps taken with each paw in each direction.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
6-OHDA	Sigma Aldrich	H4381	https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt&region=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&region=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