Research reportBehavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson's disease in mice
Introduction
Parkinson's Disease (PD) is the second most common neurodegenerative disorder. It is characterized by a marked loss of dopaminergic neurons of mainly the substantia nigra (SN) pars compacta leading to a reduction of dopamine (DA) in the target structure, the striatum [22]. The dopaminergic deficit results in motor disabilities, such as rigidity, akinesia, tremor and postural abnormalities as well as cognitive and vegetative disturbances [16].
The most frequently used toxins in rodent models of PD is either the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), or 6-hydroxydopamine (6-OHDA) [13]. Although MPTP is a valuable model of PD in mice and non-human primates, it is limited for several reasons: MPTP injection causes a bilateral Parkinson syndrome, thereby ruling out all behavioral tests based on a side bias. Cell loss is strain-, age- and gender-dependent in mice [47]. More importantly, a spontaneous recovery of Parkinsonian symptoms has been described in both, monkeys [15], [50] and mice [45], [46] after MPTP administration, which causes concern to use this model for an assessment of long-term therapeutic effects of a compound or neural grafts.
6-OHDA is a neurotoxin that selectively destroys catecholaminergic neurons and it is typically injected unilaterally, since bilateral injections cause high mortality. The advantage using this model is that it lends itself to more easy assessment of motor impairments by utilizing tests that examine for a side bias, e.g. drug-induced rotation tests [53] and spontaneous motor tests. Furthermore, intracerebral injection of 6-OHDA into the rat nigrostriatal pathway has been shown to permanently degenerate virtually all dopaminergic neurons in the SN pars compacta [25], [26] leading to stable motor deficits over time. The toxin can be injected intrastriatally, into the median forebrain bundle (MFB) or directly into the SN. Only very few studies concerning mice with 6-OHDA-lesions have been published before. In these studies, 6-OHDA was injected mainly either intrastriatally [6], [9], [12], [31] or intraventricularly, and the mice were subjected to relatively little behavioral assessment [3], [4], [7]. More recently, new models of PD have been established that rely on genetic manipulations in mice. A number of laboratories have now generated different mouse strains that carry mutations in proteins or receptors that are critical for the function of the dopaminergic system. These include α-synuclein-overexpressing mice [32], α-synuclein-knockout mice [1], Parkin-knockout mice [20], [24], several knockout mice for dopamine receptors [5], [33], [49] and tyrosine hydroxylase (TH, the rate-limiting enzyme of dopamine synthesis), and a mouse with a UCH-L1 gene mutation [41]. With an increasing availability of genetically modified mice that model PD, we believe that characterizing toxin-induced lesions in mice is highly warranted. PD models are also used to demonstrate therapeutic effects of neural grafts as a promising approach to brain repair [14]. Thereby, in the future, new models could be created that combine genetic and toxin methodologies to investigate the pathogenesis of PD or the influence of a certain genetic background on graft properties, such as cell survival and fiber outgrowth.
In the present study, we injected the 6-OHDA unilaterally into the MFB to achieve retrograde dopaminergic cell loss in the SN.
We provide an extensive behavioral characterization of motor deficits by applying multiple behavioral tests, such as amphetamine- and apomorphine-induced rotation, the rotarod test, the cylinder test, the elevated body swing test (EBST) and the stride length test. By comparing behavioral outcome with the degree of nigral cell loss in individual mice, we have determined which of these tests best predict SN cell loss, and which tests should be used for selecting for adequately lesioned mice.
Section snippets
Animals
Hundred and ten adult female CBA mice (20 g, B&K, Stockholm, Sweden) were lesioned and all animals were tested for amphetamine-induced rotation at 3 and 6 weeks post-lesion. Out of these 110 mice, 53 mice were selected for the present study. This selection was based on their amphetamine rotation scores at 6 weeks after the 6-OHDA injections. We aimed to include mice with a large range of different amphetamine rotation scores for the study. The remaining mice were used in another study. Seven
Results
Fifty-three lesioned mice were included in the behavioral analysis based on their ipsilateral amphetamine-induced rotation scores ranging from −6.3 to 12.0 rotations/minute. Out of these mice, four were excluded due to a poor needle placement according to the analysis of the cresyl violet stains. Therefore, the statistical analysis is based on a total of 49 lesioned mice and seven non-lesioned control mice.
Discussion
We show that it is possible to achieve a permanent loss of midbrain dopaminergic neurons after injecting 6-OHDA unilaterally into the MFB. This study identified several behavioral motor tests sensitive to detecting this unilateral nigral cell loss in mice.
As mentioned above, we selected 53 mice out of 110 mice subjected to the lesion. The selected mice showed a wide variability regarding loss of dopaminergic SN neurons probably due to the small size of the MFB that makes it difficult to target
Acknowledgments
We would like to thank Birgit Haraldsson and Britt Lindberg for excellent technical assistance. The study was supported by the NIH Grant no 1 R21 NS043717-01A1, the Swedish Research Council and the Konung Gustaf V:s and Drottning Victorias Stiftelse.
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