6-OHDA or 2,4,5-trihydroxyphenylethylamine is a specific catecholaminergic neu-rotoxin structurally analogous to both dopamine and noradrenalin. Acting as a "false-substrate," 6-OHDA is rapidly accumulated in catecholaminergic neurons. The mechanism of 6-OHDA toxicity is complex and involves alkylation; rapid auto-oxidization leading to the generation of hydrogen peroxide, superoxide, and hydroxyl radicals; and impairment of mitochondrial energy production (5,6). The 6-OHDA-induced rat model of PD was initially carried out by Ungerstedt in 1968, using stereotaxic bilateral intracerebral injections into the substantia nigra or lateral hypothalamus targeting the medial forebrain bundle (7). The bilateral administration of 6-OHDA resulted in catalepsy, generalized inactivity, aphagia, adipsia, and a high degree of animal morbidity and mortality. Consequently, the administration of 6-OHDA was modified to a unilateral intracerebral lesion targeting the substantia nigra and/or medial forebrain bundle. With unilateral lesioning, there was minimal postoperative morbidity, behavioral asymmetry, and a nonlesioned side to serve as a control (8,9). An additional modification of 6-OHDA administration is using chronic low dose striatal injections. This can lead to progressive dopaminergic cell death thought to more closely resemble the human condition (10). An important caveat of 6-OHDA lesioning is that the time course of cell death may be on the order of one to three weeks and must be taken into consideration when studies using intervention strategies are employed (10,11).
A distinctive behavioral feature of the unilateral lesioned model is rotation (12,13). This motor feature is due to asymmetry in dopaminergic neurotransmission between the lesioned and intact sides. Specifically, animals rotate away from the side of greater dopaminergic activity. Nomenclature describes the direction of rotation as either ipsilateral (toward) or contralateral (away) to the lesioned side. Initial reports of rotation examined both spontaneous and pharmacologically induced rotation. Spontaneous rotation consists of ipsilateral rotation, whereas pharmacologically induced rotation may be either contralateral or ipsilateral. For example, apomor-phine and other dopamine agonists induce contralateral rotation. This is due to their direct action on super-sensitized dopaminergic receptors on the lesioned side. Conversely, d-amphetamine phenylisopropylamine (AMPH) induces ipsilateral rotation by blocking dopamine re-uptake and increasing dopamine receptor activity on the nonlesioned side. In general, a greater than 80% depletion of dopamine is necessary to manifest rotation in this model (4,14). Circling behavior can be measured either by observation or by special devices called rotameters. The rate of rotation correlates with the severity of the lesion, and animals with more extensive striatal dopamine depletions are less likely to show behavioral recovery. This simple model of rotation away from the side with the most dopamine receptor occupancy has recently proven much more complex and less predictable than previously thought, especially in the context of various pharmacological treatments and neuronal transplantation (15,16). In addition to rotation, other behavioral assessments in the 6-OHDA model may include tests of forelimb use, bilateral tactile stimulation, single limb akinesia, and bracing [for review, see Ref. (17)]. A recent study on behavioral and electromyographic analysis in the 6-OHDA-lesioned rat showed gait impairment, differences in trajectory in lesioned versus nonlesioned rotation, and evidence of myoclonus (18).
The 6-OHDA-lesioned rat model has proven to be a valuable tool in evaluating the pharmacological action of new drugs on the dopaminergic system, the mechanisms of motor complications, the neuroplasticity of the basal ganglia in response to nigrostriatal injury, and the safety and efficacy of neuronal transplantation in PD. Extensive pharmacological studies have utilized the 6-OHDA-lesioned rat to investigate the role of various dopamine receptor (D1 through D5) agonists and antagonists, and other neurotransmitter systems (including glutamate, adenosine, nicotine, and opiods) on modulating dopamine neurotransmission. These studies elucidate the role of these compounds on electrophysiological, behavioral, and molecular (signal transduction) properties of the basal ganglia (13).
The 6-OHDA-lesioned rat model has also been an important tool in elucidating the mechanism(s) underlying motor complications (19,20). The chronic administration of levodopa (over a period of weeks) to the 6-OHDA rat has been demonstrated to lead to a shortening response similar to wearing off in idiopathic PD (21). This altered motor response occurs when greater than 95% of nigrostriatal dopaminergic neurons are lost. Studies using glutamate antagonists have demonstrated improvement in wearing off and have implicated the role of glutamate receptor subtypes in the development of motor complications (22-24). These findings have been supported by molecular studies that demonstrate alterations in the phosphorylation state of glutamate receptor subunits of the N-methyl-D-aspartate (NMDA) subtype (25). 6-OHDA-lesioned rats do not develop limb and truncal dyskinesia as seen in PD but a form more localized to the jaw (20,26).
In the context of neuroplasticity, the 6-OHDA-lesioned rat model demonstrates behavioral recovery and has been instrumental in characterizing the neuro-chemical, molecular, and morphological alterations within the basal ganglia in response to nigrostriatal dopamine depletion (27). These mechanisms of neuroplas-ticity in surviving dopaminergic neurons and their striatal terminals include increased turnover of dopamine and its metabolites; alterations in the expression of tyrosine hydroxylase, the rate-limiting step in dopamine biosynthesis; decreased dopamine uptake through altered dopamine transporter (DAT) expression; alterations in the electrophysiological phenotype (both pattern and rate of neuronal firing) of striatal and substantia nigra neurons; and sprouting of new striatal dopaminergic terminals. These molecular mechanisms may provide new targets for novel therapeutic interventions such as growth factors to enhance the function of surviving dopaminergic neurons.
The 6-OHDA-lesioned rat model has also been useful for determining important parameters for successful transplantation. These parameters include target site (striatum vs. substantia nigra); volume of innervation at the target site; number of cells transplanted; type and species of cells transplanted, including fetal mes-encephalon, engineered cell lines, and stem cells; age of host and donor tissues; pre-treatment of transplant tissue or host with neurotrophic factors, anti-oxidants, immunosuppressive therapy, or neuroprotective pharmacological agents; and surgical techniques, including needle design, cell suspension media, and transplant cell delivery methods (28,29). The near absence of dopaminergic neurons and terminals within the striatum due to 6-OHDA lesioning provides a template for the assessment of sprouting axons and terminals originating from the transplant. Measures of transplant success in this model include reduction in the rotational behavior and the survival, sprouting and innervation (synapse formation) of dopaminergic fibers within the denervated striatum. The reduction of rotational behavior suggests increased striatal dopamine production originating from the transplanted tissue. Interestingly, not all behavioral measures appear to respond to transplant. The advancements made in the 6-OHDA-lesioned rat provide a framework for the further testing of transplantation in nonhuman primates and human clinical trials.
Although the 6-OHDA-lesioned rat model has many advantages, it serves primarily as a model of dopamine dysfunction. Lesioning with 6-OHDA is highly specific for catecholaminergic neurons and does not replicate all of the behavioral, neuro-chemical, and pathological features of human PD. For example, the 6-OHDA-lesioned rat does not manifest alterations in the cholinergic and serotonergic neurotransmitter systems, which are commonly affected in PD. Stereotaxic injections of 6-OHDA to precise targets does not replicate the extensive pathology of PD where other anatomical regions of the brain (including the locus coeruleus, nucleus basalis of Meynert, and raphe nuclei) are affected. In addition, Lewy body formation, a pathological hallmark of PD, has not been reported in this model. Interestingly, a recent report using a regimen of chronic administration of 6-OHDA into the third ventricle did show a more extensive lesioning pattern reminiscent of human PD (30). In addition to the rat, other species, including the nonhuman primate (specifically the marmoset), have served as models for 6-OHDA lesioning (31,32). Lesioning in nonhuman primates provides for the analysis of behaviors not observed in the rat, such as targeting and retrieval tasks of the arm and hand. In addition, lesioning in the nematode Caenorhabditis elegans provides a potential genetic tool to investigate mechanisms involved in cell death with this toxin and to provide large-scale screenings (33,34).
Overall, lesioning with 6-OHDA has provided a rich source of information regarding the consequences of precise dopamine depletion and its effects on rotational behavior, dopamine biosynthesis, biochemical and morphological aspects of recovery, and serves as an excellent template to study both pharmacological and transplantation treatment modalities for PD.
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