Research reportRotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats
Introduction
It is well known that in Parkinson's disease (PD) dopaminergic (DA-ergic) neurons degenerate, but the primary cause for this degeneration is still unknown. The disabling symptoms in PD are primarily due to a profound deficit in striatal dopamine (DA) content that results from the degeneration of DA-ergic neurons in substantia nigra pars compacta (SNpc) and the consequent loss of their projecting nerve fibres in the striatum.
Probably there is not one single factor responsible for neurodegeneration; it appears that several factors are acting in concert. One of these factors is mitochondrial complex I deficiency [9].
DA-ergic neurons are especially vulnerable to complex I inhibitors. MPTP (1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine), for example, which represents a widely used toxin to induce parkinsonian symptoms in animals, is a potent inhibitor of complex I. Also, rotenone is a complex I inhibitor and has been reported to have a rather selective toxicity on DA-ergic cells in vitro [7] and in vivo. However, there are differences between MPTP and rotenone. Rotenone facilitates the formation of alpha-synuclein fibrils [1] and crosses the blood–brain barrier as well as the cell membrane because of its lipophilic structure.
Tentatively, rotenone treatment may represent an animal model that shares basic processes with the disease. Non-familiar sporadic PD is characterised by 15–30% reduction of complex I activity [10]. Given that complex I deficiency persisting over the lifespan of a human being causally contributes to PD, rotenone treatment may mimic the processes that develop in humans and thus represents a unique model with construct validity (i.e. the highest degree of validating criteria for animal models [2]).
The selective toxicity of rotenone is especially relevant because it is widely used as a herbicide in private gardens and in several powders for delousing humans or animals, and thus a real threat that an environmental substance can cause PD does seems to exist.
In a previous study of Betarbet et al. [1], rotenone was administered continuously through minipumps, 2–3 mg/kg per day for 5 weeks, and it was found that the rats developed motor and postural deficits characteristic of PD. Immuno-assays also showed that ubiquitin and alpha-synuclein inclusions in brain shared the features of Lewy bodies.
The present study was designed to assess rotenone toxicity after intraperitoneal (i.p.) injection daily for 2 months at two different doses: 1.5 mg/kg (low dose) and 2.5 mg/kg (medium dose). Pulsatile administration was chosen because it may be similar to exposure in normal life, such as through inhalation, dermal contact and oral ingestion of pesticide residues and in foods such as vegetables, fruits and fish.
To assess quantitatively the transmitter concentrations, we measured both DA and its metabolites in the prefrontocortical and striatal tissue by means of high-performance liquid chromatography with electrochemical detection (HPLC–ECD) and tyrosine hydroxylase-immunoreactivity (TH-immunoreactivity) level in the striatum using Western blot analysis. Parkinsonian symptoms were assessed in behavioural tests (catalepsy and open field).
Section snippets
Animals
Thirty-six male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) aged 7 weeks were chosen for the experiment. At the beginning of the experiment, the rats weighed 220–240 g. They were divided into three groups, each containing 12 rats. Rats were housed in cages and kept in a room maintained at a constant temperature of 22 °C, 50–60% humidity and a 12/12 h light–dark cycle (7:00–19:00). They obtained dry food (Altromin, 1324) 12 g/day/rat, and water was available ad libitum. All experiments
Neurochemistry
The amount of DA and its metabolites was measured in the prefrontal cortex (PFC) and CPu for each group of animals. There was a decrease of DA in all these two regions of the brain in rotenone-treated animals.
In CPu, the amount of DA and its metabolites DOPAC and HVA was decreased in medium-dose-treated animals—DA: χ2=5.1635, df=2, P=0.0756; DOPAC: χ2=3.3147, df=2, P=0.1485; and HVA: χ2=5.5893, df=2, P=0.0611—as shown in Fig. 1a.
DA was decreased in low- and medium-dose-treated animals in PFC (χ2
Neurochemistry
HPLC analysis of homogenised brain tissue shows that rotenone depletes DA and reduces the concentration of DOPAC and HVA. NA and 5HT were not affected, showing the selectivity of rotenone toxicity (data not shown). The depletion of DA was sensitive to the action of rotenone in the posterior part of the striatum and PFC, suggesting the existence of a constitutive metabolic deficiency in the nigrostriatal DA-ergic neurons. This methodology also provides a good monitor of energy metabolism. In
Conclusion
The major goal of this study has been to test whether rotenone i.p. treatment could be used to establish a rat model of PD. Other studies have introduced rotenone either stereotactically or via intrajugular or intramuscular routes to produce PD symptoms. This gives rise to the controversial question of the relevance of these routes for PD patients in whom the disease may have been due to environmental exposure.
In this study a rotenone-based rat model of PD has been established: The chronic
Acknowledgements
Supported by the Deutsche Forschungsgemeinschaft SFB 430.
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