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).