Parkinson’s disease (PD) is the most common neurological disorder that is characterized by debilitating motor abnormalities, including muscle rigidity, resting tumor, stiffer voluntary movements, and postural instability. Primary neuropathological condition of PD includes progressive degeneration of the dopaminergic neurons in the nigrostriatal area. Experimental models of PD are specifically designed to gain detailed insights into the pathological mechanisms of the disease. In addition to this function, animal models are essential in the development and evaluation of new therapeutic molecules and strategies. The introduction of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) has revolutionized the research in Parkinsonism.
The 6-hydroxydopamine molecule is transported to the dopaminergic and noradrenergic neurons to cause degeneration of nerve terminals. 6-OHDA neurotoxicity is developed as it inhibits the mitochondrial respiratory enzymes (chain complexes I and IV). Due to the blockade of these enzymes, the neurons could no longer exert their normal physiological duties and consequently die. Since in Parkinson’s disease, the dopaminergic nigrostriatal pathway is mainly subjected to degeneration, so the animal models have been developed in which 6-OHDA induced lesions of the dopaminergic system are generated. Regional selectivity for the nigrostriatal tract could be achieved by injecting the toxin into different parts of the ascending nigrostriatal pathway. In the preclinical PD research, rat models have been widely used in which 6-OHDA was injected into either one of three target sites: substantia nigra pars compacta (SNc), medial forebrain bundle (MFB), or the caudate-putamen unit (CPu). It remains unclear, however, which of these models is most suitable for PD modeling. To model PD, the animal model must mimic both the degeneration of dopaminergic neurons and the behavioral deficits associated with idiopathic PD. 6-OHDA model has contributed enormously to enhance the understanding of PD pathology (Blesa., Phani., Jackson-Lewis., & Przedborski., 2012).
Injection sites for 6-OHDA (Maciaczyk., Kahlert., Döbrössy., & Nikkhah., 2016)
6-OHDA injection into the CPu
6-OHDA injection into the CPu results in more selective neuron damage in the nigrostriatal dopaminergic system. Following the injection, because of its retrograde transport to SN pars compacta (SNc), the nigral dopaminergic neurons undergo degeneration and apoptosis. In this animal model, usually dorsomedial and ventrolateral striatum are targeted. In rodents, the ventrolateral portion of the CPu receives signals from motor and sensorimotor areas of the neocortex, whereas its dopaminergic innervations project from the SNc. Whereas, the dorsal part of the CPu has a mixed DA innervation from both SN and ventral tegmental area (VTA), and receives inputs from the limbic system as well as from the frontal cortical areas making it equivalent to the nucleus caudatus in humans. Also, it shows remarkable effects on locomotion and drug-induced rotation behavior by lesioning the dorsomedial part of the CPu, whereas the injection of the neurotoxin into the ventrolateral parts provokes difficulties in movement initiation, skilled motor behavior, and sensorimotor orientation. Therefore, the lesions in rodent ventrolateral CPu resemble more closely the depletion of dopaminergic innervation in the putamen of PD patients.
6-OHDA injection in the MFB
6-OHDA injection into the MFB leads to almost total destruction of the dopaminergic neurons of the SNc projecting to striatum as well as of the VTA heading to the nucleus accumbens, eventually causing a postsynaptic denervation sensitivity of DA receptors. In response to the lesion-induced imbalance between the nigrostriatal systems in both hemispheres, the test animals show unilateral sensorimotor deficits enabling the researchers to evaluate the lesions and intensity of the neurotoxic effect by behavioral analysis. The most robust manifestation of this is spontaneous postural motor asymmetry, which causes the animals to rotate toward their impaired hemisphere. This could be increased by stress and in particular due to drugs- such as D1/D2 receptor agonist apomorphine or DA reuptake inhibitor d-amphetamine induced rotations. Whereas, the bilateral MFB lesion of the nigrostriatal system in adult animals causes severe sensorimotor impairment with rapid aphagia and adipsia. Nevertheless, the standard 6-OHDA rat model, generated by unilateral injection of 6-OHDA into the MFB is more pragmatic and permits a direct comparison of lesion effects and therapeutic regimens within a single test subject by the comparison of both hemispheres.
6-OHDA injection into the SNc (Deumens., Blokland., & Prickaerts., 2002)
To generate more selective PD model, the neurotoxin is injected into SNc to cause less dramatic damage in the dopaminergic system. In this model, the animals receive unilateral injection either medial and/or a single lateral injection. With SNc injection, almost 90% dopaminergic neuron loss could be achieved. The single lateral 6-OHDA injection spares the dopaminergic cells in the medial SNc and efficiently manifests a neuropathological finding of PD patients with dopaminergic innervation damage mainly within the lateral SN. Consequently, the dopaminergic fibers at the lesion side within the lateral CPu diminish dramatically as compared to the fibers at the medial parts of the caudate-putamen unit. Moreover, the remaining DAnergic innervation in CPu corresponds clearly to the degree of DAnergic cell depletion in SNc. One of the major limitations of this model is the small size of the injection site which could not lesion the adjacent structures, i.e., VTA and makes it a very challenging task; therefore, restricting its application to rare experimental designs.
6-OHDA neonatal injection
For bilateral degeneration of the DAnergic nigrostriatal pathway in neonatal rats, the neurotoxin solution is injected transcutaneously into lateral ventricles on postnatal day 1. Bregma, the anatomical landmark to define the correct coordinates, is visible in this developmental stage. This bilateral lesion surgery could not result in severe akinesia and sensorimotor deficits.
6-OHDA administration (Mercanti., Bazzu., & Giusti., 2012)
- Prepare a tube containing the solution of sterile 0.9% saline with 0.02% (w/v) ascorbic acid and place it on ice.
- Aliquot small amounts of 1-2 mg of 6-OHDA-HCl into an Eppendorf tube and cover it with aluminum foil to avoid light exposure. Store these aliquots at -20°C.
- Set up the surgical area by putting all the surgical tools; and disinfect with 70% ethanol.
- Connect the needle to the Hamilton syringe. Test it by filling the syringe with the saline-ascorbic acid solution and press through the syringe and needle. This procedure needs extreme care to make sure that there is no occlusion as this could prevent the toxin from reaching the target structures at the required concentration.
- Fix the needle onto the stereotaxic frame and hold it vertically to avoid injection on the wrong site.
- Turn on the heating pad as the animal is placed on it after the stereotaxic procedure.
- Weigh and anesthetize the animal. Any one of the following anesthesia doses could be selected:
- Fentanyl citrate salt 50 μg/mL + medetomidine, HCl 50 μg/mL, 20:1 mixture, (intraperitoneal dose: 6.3 mL/kg). Use atipamezole hydrochloride 1 mg/kg as an antagonist.
- Ketamine 80 mg/kg + xylazine (intramuscular dose: 12 mg/kg).
- Chloral hydrate (i.p 400 mg/kg). Weigh 4 g of chloral hydrate and dissolve it in 100 mL of sterile 0.9% saline).
- Leave the animal in a cage with bedding until adequate anesthesia is achieved.
- Wipe the fur on the skull with cotton soaked in 70% ethanol and shave the head.
- Place the animal under the stereotaxic frame in flat-skull position; enter the ear bars, and fix the skull to avoid any head movement. After this, fix the teeth on the tooth bar to prevent the head from moving up and down. Correctly position the head so that the lambda and bregma are at the same horizontal level.
- Make a midline incision of about 2 cm on the skin starting between the eyes and remove the fascia above the bone to easily locate the bregma. Put two metal clips to keep the skin open.
- Injection coordinates are expressed in millimeters:
- For sensorimotor part of the striatum: AP = +0.5; L = −2.5; DV = −7.0 (4 μ L).
- To target the SNpc: AP = −5.7; L = −2.0; DV = −8.7 (4 μ L).
- For MFB: first injection AP = −4.4; L = −1.2; DV = −7.8, tooth bar = −2.3 (2 μ L); second injection AP = −4.0; L = −0.8; DV = −8, tooth bar = +3.4 (2 μ L).
- Find the bregma and position the needle exactly on it, read the coordinates anteroposterior (AP) and lateral (L) on the manipulator (x, y, and z), and retract the needle and calculate the injection location by subtracting the coordinates for different lesion sites.
- Move the manipulator on the target site and mark it.
- Pull out the needle and drill a small hole in the skull bone above the injection site. Make sure the meninges remain intact.
- Calculate the dorsal-ventral (DV) coordinates of the injection considering the dural surface as a reference plane, and make a small hole in the meninges with the help of a needle.
- Dissolve 4 mg of 6-OHDA-HCl in 1 mL of sterile saline with 0.02% ascorbic acid. The concentration of neurotoxin in this solution should be 3.6 mg/mL. Once dissolved, keep the solution on ice and in the dark as 6-OHDA is light and rapidly oxidizes. Prepare a fresh solution every 90 minutes.
- Wash the syringe with saline containing 0.02% ascorbic acid.
- Fill the injection with the 6-OHDA solution.
- Clamp the syringe on the pump, and then connect the needle with the PE tube to the Hamilton syringe.
- Fill the circuit with the 6-OHDA-HCl solution, and set the flow rate at 0.5 μ L/min.
- Slowly lower down the injection needle to the desired depth.
- Start the infusion pump and pressure-eject the 6-OHDA solution at a rate of 0.5 μ L/min.
- Let the needle in place for 5 minutes before slowly retracting it.
- Empty the syringe and wash it twice with ethanol and once with saline containing 0.02% ascorbic acid.
- Rehydrate the injection site with sterile water and suture it.
- Apply analgesic postoperatively.
- Inject 2 mL saline subcutaneously to prevent dehydration. The postoperative analgesia consists of:
- 5% Glucose solution: 3 mL subcutaneously
- Steroidal anti-inflammatory: Betamethasone disodium phosphate 1.5 mg/mL: 100 μ L intramuscular
- Nonsteroidal anti-inflammatory: Indomethacin, Liometacen, Chiesi: 100 μL intramuscular
- Antibiotics (i.e., ceftriaxone, Rocephin, Roche): 100 μ L subcutaneously.
- Place the animal in a clean cage until it recovers.
- Administer sterile physiological saline or glucose-saline solution subcutaneously (5 mL) to prevent dehydration.
- During surgery, the temperature of the animal body decreases. To cater this, place a wool pad under the animal’s body. After the surgery, return the animal to its cage and cover its body with a cloth and put a paper on the eyes.
- Monitor the animals for their normal activity, wound healing, and weight loss.
Assessing the neuroprotective effects of the drug (Sedaghat., Roghani., & Khalili, 2014)
- House the animals (three to four per cage) in a temperature-controlled room with water and food available ad libitum.
- Anesthetize the animals with an intraperitoneal injection of ketamine 80 mg/Kg and xylazine 10 mg/Kg.
- Inject the animals with unilateral intrastriatal 6-OHDA injection (left side) using a 5 μL Hamilton syringe at the stereotaxic coordinates: L –3 mm, AP +9.2 mm, V + 4.5 mm.
- Keep the needle in place for an additional 5 minutes and then withdraw it at a rate of 1 mm/min.
- Give a single injection of 5 μL of 0.9% sterile saline containing 2.5 μg/μL of 6-OHDA-HCL and 0.2% ascorbic acid (w/v) at a rate of 1 μL/min to the lesion group.
- Inject the drug receiving animals with the neurotoxin and thymoquinone (TQ) p.o. (using rodent gavage) dissolved in propylene glycol at doses of 5 and 10 mg/Kg respectively. Administer TQ daily from two days before surgery with an interval of 24 hours.
- Inject the third injection of thymoquinone 1 hour before surgery.
- Perform the behavioral tests as per experimental needs.
Modelling Parkinson’s disease (Blesa et al., 2012)
Parkinson’s disease could be efficiently modeled in laboratory animals using 6-OHDA neurotoxin that recreates specific pathological events and their behavioral outcomes. The development of PD animal models is valuable to test new neuroprotective agents and treatment strategies for PD. Parkinson’s disease is characterized by degeneration of dopaminergic neurons in the substantia nigra, loss of striatal DA fibers, and reduction of the striatal dopamine levels. Also, Lewy body formation is the most important neuropathological feature of PD. 6- hydroxydopamine (6-OHDA) is a powerful neurotoxin that selectively and rapidly destroys the catecholaminergic neurons. This model has been successfully applied to test preclinical therapies and new pharmacological and genetic therapeutic agents for Parkinson’s disease.
Assessment of the neuroprotective effect of Thymoquinone (Sedaghat., Roghani., & Khalili, 2014)
Parkinson disease (PD) is the most common neurological disorder with progressive degeneration of dopaminergic neurons for which only symptomatic treatments with no-prevention of disease progression are available. This study was conducted to assess the neuroprotective effects of the Nigella sativa bioactive compound thymoquinone (TQ). The effects of TQ on behavioral and cellular abnormalities and oxidative stress markers were also assessed in 6-OHDA experimental model of early PD in the rat. For this, the animals were treated with unilateral intrastriatal 6-hydroxydopamine (6-OHDA) injections with TQ. After 7 days, it was observed that apomorphine reduced the neurons on the left side of the substantia nigra pars compacta (SNC), and the malondialdehyde (MDA) and nitrite levels in midbrain homogenate were increased. While the activity of superoxide dismutase (SOD) was reduced in the 6-OHDA lesion group. It was also shown that the TQ pretreatment significantly improves turning behavior, prevents SNc neurons loss, and lowers MDA level. These results suggested that TQ possess excellent neuroprotection against 6-OHDA neurotoxicity and could provide a beneficial treatment regimen for neurodegenerative disorders, including PD.
Analyzing the expression of Tgfβ1 and inflammatory markers (Haas., Zhou., & Spittau., 2016)
Parkinson’s disease (PD) is characterized by complete loss of midbrain dopaminergic (mDA) neurons. Microglia-mediated neuroinflammation is a common hallmark of Parkinson’s disease and is believed to trigger the disease progression. 6-OHDA has been widely used to induce dopaminergic neuronal degeneration in rodents to mimic PD and to study neurodegeneration, neuroinflammation as well as the therapeutic approaches for PD. In this study, the temporal and spatial expression patterns of anti-inflammatory and pro-inflammatory markers was analyzed in 6-OHDA mouse model of PD. It was found that the activated microglia and neurons in the lesioned SNc and CPu express Transforming growth factor β1 (Tgfβ1). The data collected form this research presented an important role for Tgfβ1 as a lesion-associated factor that regulates microglial activation states in the 6-OHDA mouse model to prevent degeneration of uninjured neurons by releasing neurotoxic factors such as TNFα and nitric oxide (NO). This study validated the use of 6-OHDA rat model for analyzing the expression levels of inflammatory markers involved in Parkinsonism.
Gene therapy (Hernandes-Baltazar et al., 2017)
The 6-OHDA PD model has also been used to demonstrate the neurotrophic therapy (NT). Neurotrophic therapy consists of directed gene delivery of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), cerebral dopamine neurotrophic factor (CDNF), glial cell line-derived neurotrophic factor (GDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), and vascular endothelial growth factor (VEGF) through nanoparticles or viral gene vectors. The 6-OHDA model with neurotrophic therapeutic factors was used to prevent progressive neurodegeneration and to stimulate the functional regeneration of the nigrostriatal system. It was also studied that the recovery of dopaminergic neurons could improve motor function. It is therefore essential to identify further underlying mechanisms of oxidative stress, neurodegeneration, neuroinflammation, and neuronal death triggered by 6-OHDA. This knowledge is vital to discover novel therapies to treat Parkinson’s disease.
- Wear proper gloves and gown during the surgical procedure.
- Make sure the needle does not leak and is sealed.
- Carefully clean the needle to prevent infection and the blood clot formation, which may cause mechanical damage to brain tissue.
- Keep the head of the animal up in the stereotaxic frame to avoid suffocation.
- While drilling into the skull bone, hold the drill in an upright position, and avoid excessive pressure to avoid the drill penetrate the dura.
Strengths and limitations
- 6-OHDA experimental models of PD are widely used to gain detailed insights into the possible pathological mechanisms of Parkinson’s disease.
- Also, these models are essential in the assessment and development of new therapeutic strategies, whether pharmacological, neurotropic, or genetic.
- These neurotoxins efficiently kill the dopaminergic neurons through oxidative stress and mitochondrial failure.
- The 6-OHDA rat model of Parkinson’s disease efficiently mimics the behavioral and neurochemical outcomes of human disease.
- This model does not mimic all of the hallmarks of PD. However, dopamine depletion, nigral dopamine cell loss, and the associated neurobehavioral deficits have been successfully modeled using 6-OHDA.
- D. H. Baltazar., R. Nadella., M. R. Hernandez., L. M. Flores., & Jarquin., C. R. (2017). Animal Model of Parkinson Disease: Neuroinflammation and Apoptosis in the 6-Hydroxydopamine-Induced Model. In Experimental Animal Models of Human Diseases – An Effective Therapeutic Strategy.
- G. Mercanti., G. Bazzu., & Giusti., P. (2012). A 6-hydroxydopamine in vivo model of Parkinson’s disease. Methods Mol Biol, 864, 355-64.
- J. Blesa., S. Phani., V. Jackson-Lewis., & Przedborski., S. (2012). Classic and new animal models of Parkinson’s disease. J Biomed Biotechnol.
- J. Maciaczyk., U. D. Kahlert., M. Döbrössy., & Nikkhah., G. (2016). Stereotactic Surgery in Rats. In M. Janowski, Experimental neurosurgery in animal models. New York: Humana Press.
- R. Deumens., A. Blokland., & Prickaerts., J. (2002). Modeling Parkinson’s disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway. Exp Neurol, 175(2), 303-317.
- R. Sedaghat., M. Roghani., & Khalili, M. (2014). Neuroprotective Effect of Thymoquinone, the Nigella Sativa Bioactive Compound, in 6-Hydroxydopamine-Induced Hemi-Parkinsonian Rat Model. Iranian Journal of Pharmaceutical Research, 13(1), 227-234.
- S. J. Haas., X. Zhou., & Spittau., B. (2016). Expression of Tgfβ1 and Inflammatory Markers in the 6-hydroxydopamine Mouse Model of Parkinson’s Disease. Front Mol Neurosci, 9(7).