In developing neuroprotective or ‘disease-modifying’ treatments for PD, it has proven extremely difficult to translate positive results in animal studies to success in human clinical trials (Lang, 2006
). In this regard, there is a pressing need for new and improved animal models of the disease. The rotenone model of PD reproduces many features of PD, including systemic
mitochondrial impairment, oxidative damage, microglial activation, selective nigrostriatal dopaminergic degeneration, L-DOPA-responsive motor deficits, α-synuclein accumulation and aggregation with formation of Lewy body-like inclusions, impairment of ubiquitin-proteasome function, acidification and mitochondrial translocation of DJ-1, iron accumulation in substantia nigra and gastrointestinal dysfunction associated with α-synuclein accumulation and aggregation (Alam and Schmidt, 2004
; Betarbet et al., 2006
; Betarbet et al., 2000
; Sakai and Gash, 1994
). Thus, aside from preservation of dopaminergic neurons and related motor function – which are the most commonly employed endpoints in neuroprotection studies – there are numerous other relevant endpoints (e.g., α-synuclein accumulation and aggregation, ubiquitin-proteasome function or gastrointestinal dysfunction) that can be assessed in the rotenone model. Unfortunately, the use of this model for neuroprotective studies has been severely hampered by its variability. Here we report that daily intraperitoneal rotenone administration produces a highly reproducible lesion of the nigrostriatal dopamine system associated with α-synuclein pathology – a model that should be well-suited for the assessment of pathogenic pathways and experimental therapeutic interventions.
Previous studies using intraperitoneal injection of rotenone reported dopamine depletion and L-DOPA-responsive locomotor abnormalities (Alam and Schmidt, 2002
; Alam and Schmidt, 2004
); however, in those studies mortality was high (Biehlmaier et al., 2007
; Werner J. Schmidt, personal communication) and the pathological features of the model were not characterized. We now provide further evidence of progressive locomotor deficits after rotenone treatment. These behavioral deficits were responsive to the dopamine agonist, apomorphine, indicating that loss of dopamine contributes to the deficits. While rearing deficits have been reported previously in the rotenone model (Fleming et al., 2004
), variability was high, likely due to the low percentage of animals that exhibited histological evidence of a lesion to the nigrostriatal dopamine system. In our regimen, however, rearing proved to be a useful behavioral endpoint. Additionally, the postural instability test, which was recently developed in the unilateral 6-OHDA rat model of PD (Woodlee et al., 2008
), was also useful in the characterization of a bilateral PD model. Interestingly, rotenone animals exhibited significant improvement after apomorphine, which was not the case in 6-OHDA treated animals. In that report, dopamine depletion was likely high (~95%), given that 10 μg of 6-OHDA was administered into the medial forebrain bundle (Cannon et al., 2005
). Therefore, it may be that apomorphine was unable to produce improvement with a lesion of such magnitude. The use of this test to quantify postural instability is of particular interest, because postural instability is one of the cardinal manifestations of PD and unsteady gait is one of the first noticeable behavioral phenotypes in this model.
Intravenous or subcutaneous rotenone administration typically produces nigrostriatal dopamine system lesions in one-third of animals (Betarbet et al., 2000
). In contrast, here we report lesions in all animals treated with rotenone. Despite the fact that the current rotenone protocol is highly reproducible in terms of the ultimate behavioral phenotype and pathological features, there is still variability in the time from beginning rotenone treatment to the onset of a severe parkinsonian phenotype. This temporal variability in phenotype development was most apparent in the youngest animals. Indeed, older animals appeared to develop the phenotype more rapidly than younger animals. Rarely, animals exhibited behavioral resistance, or apparent decline followed by recovery, even while receiving daily rotenone injections (data not shown). A solution to this resistance was to increase the dose of rotenone by 10% per week after 30 days at 3.0 mg/kg/day. Under this regimen, the behavioral phenotype was observed in all animals. Alternatively, the use of older animals produces the phenotype with much less temporal variability. Interestingly, the dose-response curve for rotenone appears to be steep, as lower doses (2.0-2.5 mg/kg/day) for up to 60 days produce subtle behavioral features, without progression to a severe debilitating phenotype. A rotenone administration frequency of 24 hours was validated by a systemic complex I activity assay assessing rotenone–induced inhibition.
The debilitating phenotype observed includes bradykinesia, rigidity and postural instability. Postural instability in the form of an unsteady gait is one of the first signs of an ensuing severe parkinsonian phenotype. In this model, it should be noted that a trained observer should monitor the animals at least twice daily. While specialized behavioral tests are required to quantify behavioral deficits in unilaterally 6-OHDA lesioned animals (>95% dopamine depletion), even modest bilateral depletions can significantly impair motor function (Sakai and Gash, 1994
). It should also be noted that we have achieved a severe PD phenotype with <50% striatal dopamine depletion. This is a much smaller magnitude of depletion than required to achieve such a phenotype through 6-OHDA-elicted bilateral dopamine depletion (Deumens et al., 2002
; Sakai and Gash, 1994
). This observation may, in part, be explained by the focal nature of the striatal dopamine terminal loss, which was localized to the dorsolateral striatum. In this region, dopamine terminals appeared to be completely destroyed, and undoubtedly, the magnitude of dopamine loss was much greater here. However, neurochemical analysis was performed on a homogenate of the entire striatum, so the focal loss of dopamine was underestimated.
Loss of striatal dopamine terminals was readily apparent in animals treated at 3.0 mg/kg/day. However, animals treated at lower dose rotenone (2.75 mg/kg/day) achieved a debilitating PD phenotype, without an overt striatal lesion in all animals. Interestingly, there was little difference in the magnitude of nigral dopamine cell loss between the dosage groups. It may have been that in animals treated at lower doses, terminal sprouting occurred from surviving dopamine neurons as a response to rotenone.
Daily intraperitoneal rotenone caused striatal dopamine depletion, degeneration of nigrostriatal dopamine terminals, and loss of tyrosine hydroxylase-positive neurons of the substantia nigra. Remarkably, the magnitude and distribution of the lesion was very similar across animals. In contrast, rotenone administration via osmotic pumps was previously found to produce lesions with a wide range of magnitude, and the striatal distribution ranged from a diffuse, widespread decrease in tyrosine hydroxylase immunoreactivity to a focal loss of staining; the precise location of the terminal lesion within the striatum also varied (Betarbet et al., 2000
). With intraperitoneal administration, the loss of terminals is typically localized to the dorsolateral striatum and relatively focal in nature. Unbiased stereology showed that nigral dopamine cell loss was substantial, and it was particularly apparent in the lateral and ventral portions, the same regions that appear to be most vulnerable in human PD (Fearnley and Lees, 1991
; Gibb and Lees, 1991
As described with intravenous and subcutaneous administration of rotenone by osmotic pump, we have found that intraperitoneal rotenone also causes α-synuclein accumulation and aggregation in substantia nigra (Betarbet et al., 2006
; Betarbet et al., 2000
; Sherer et al., 2003
). Furthermore, in the regimen described here, we found intraneuronal, cytoplasmic inclusions containing α-synuclein and poly-ubiquitin which have a morphology very similar to human Lewy bodies. As noted above, this feature of the model, together with other relevant endpoints, such as inflammation, oxidative damage, ubiquitin-proteasome function and iron accumulation, make the rotenone model particularly attractive for studying disease-modifying therapies.
In summary we have presented a behavioral, neurochemical, and histological characterization of the neurological effects of chronic intraperitoneal rotenone. The ability of this environmental neurotoxicant to produce a consistent model of PD that recapitulates many key features of pathology and pathogenesis should be of great use in testing experimental therapeutics and examining gene-environment interactions.