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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 6, 2004 as doi:10.1096/fj.03-0677fje. |
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,2
* Centre de Recherche en Neurosciences, CHUL, Sainte-Foy, Québec, Canada;
Département de Médecine, Université Laval, Sainte-Foy, Québec, Canada;
Département de Chimie-Biologie, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada; and
Service d’anatomopathologie et cytologie, Hôpital de l’Enfant-Jésus, Québec, Canada
2Correspondence: Centre de Recherche en Neurosciences CHUL, RC-9800, 2705 Boulevard Laurier Sainte-Foy, QC, G1V 4G2, Canada. E-mail: francesca.cicchetti{at}crchul.ulaval.ca
SPECIFIC AIMS
In recent years, there has been an enormous demand for further scientific development of animal models that can faithfully mimic the progressive motor impairments encountered in Parkinson’s disease (PD). The current weight of evidence strongly suggests that environmental neurotoxins may play an important role in the etiology of PD. However, the role of herbicides such as rotenone in PD development and its potential use as a model for toxin-induced nigrostriatal injuries, remains highly questionable and the relevance of this model deserves further examination. The present study was undertaken to examine the specificity of rotenone-induced degeneration from a behavioral, neuropathological and pathological perspective following systemic injection in rats.
PRINCIPAL FINDINGS
1. Systemic rotenone injection induces DA-related as well as non specific neuronal damage
We investigated the dopaminergic (DA) neuronal degeneration in animals subjected to systemic treatment of 2.5mg/kg/day of rotenone via subcutaneous delivery. Thirty rats were used and were assigned to 4 groups killed at 3, 5, 8, or 20 days post-surgery. Each group comprised 7 rats (experimental n=5, control n=2) except for the 8-day time point, which comprised 9 rats (experimental n=7, control n=2). Histological postmortem evaluation revealed that the average number of tyrosine hydroxylase (TH) immunoreactive cells in the substantia nigra pars compacta (SNpc) of rotenone-treated rats did not significantly differ from vehicle treated animals at any of the evaluated time points (3, 5, 8, and 20 days) and neurons of the SNpc depicted a normal and healthy appearance (Fig. 1
F, G). Degeneration of striatal TH immunoreactive fibers reached significance (P<0.01) only for animals that underwent an 8-day rotenone treatment. Severe loss of striatal TH immunoreactive fibers struck 5 animals out of 7 in the 8-day rotenone treatment group (Fig. 1D
), but less than 20% of the total number of animals. TH denervated areas had a very focal distribution in which several degenerating striatal neurons could be observed by Fluoro-Jade staining (Fig. 1E
). Absence of Fluoro-Jade staining in the SN of treated animals indicated that DA cells had not been targeted by rotenone toxicity. Striatal TH denervated areas were devoid of immunoreactivity for selective striatal markers of typical large and medium-sized interneurons including NADPH-diaphorase (P<0.005), choline acetyltransferase (P<0.005), calretinin (did not reach significance P<0.07), parvalbumin (P<0.005), and markers for projection neurons such as calbindin (P<0.005). Moreover, increase in density and intensity of 
-synuclein expression was clearly observable in TH denervated areas. Overall, the presence of this pathological hallmark matched the expression of degenerating neurons as indicated by Fluoro-Jade staining (Fig. 1E
).
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2. Motor impairment and peripheral toxicity correlates
Behavioral observations revealed prolonged hypokinetic conditions in rats killed at 3 and 5 days, and severe dystonic episodes in animals that had to be killed at 8 days. Animals that lived for 20 days showed no motor deficits. None of the animals comprised in this study showed signs of rigidity or tremors. At autopsy, macroscopic observations revealed severe damages to peripheral organs such as the stomach (Fig. 2
A, B) in animals treated with rotenone at earlier time points evaluated (3 and 5 days). These groups of rotenone treated animals suffered from severe health problems, weight loss and hypokinetic behaviors and had, with few exceptions, a stomach that was twice as large as vehicle treated animals (Fig. 2B
, inset). Further pathological analysis indicated severe muscle mass loss (Fig. 2B
) and confirmed a diagnosis of achlorhydria, a condition disabling the animals from producing gastric acid and digesting food. The liver was also noticeably affected in animals that were exposed to rotenone for a period of 20 days (Fig. 2D
) and which showed no signs of rotenone toxicity in the brain. Macroscopic examination of the liver revealed widespread softening and necrosis (Fig. 2D
).
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DISCUSSION
Throughout the experimental period, 80% of animals comprised in the study showed various signs of illness mainly characterized by a massive weight loss (>20% of body weight) necessitating immediate sacrifice. An attempt at force-feeding the animals was not successful and prompted general autopsy analysis, which revealed thinning of the stomach muscle lining and severe necrosis of the liver. Stomachs packed with undigested food prevented successful gavage.
In our study, less than 20% of all rotenone treated animals showed brain susceptibility to the toxin (8 days), as illustrated by a significant loss of DA striatal fibers in the center of the striatum. Although DA fibers were clearly affected in these animals, the average number of DA neurons of the SNpc did not differ in any of the treatment groups (3, 5, 8, and 20 days). The vast majority of striatal projection neurons, which can be observed by the presence of the calcium binding protein calbindin, was targeted by rotenone treatment, a pathological feature reminiscent of Huntington’s disease but clearly not of PD pathology. Fluoro-Jade staining, a marker of degenerating neurons, confirmed the presence or absence of degenerative process in the striatum and SNpc respectively. Loss of striatal projection neurons in the 8-day treated animals could feasibly account for our observation of dystonia in this group.
We also observed a marked increase of -synuclein protein expression in the central area of the striatum of 8-day treated animals. Similar to several of the striatal markers used in this study, -synuclein had a distribution that overlapped the TH-denervated area. Alpha-synuclein is normally observed at basal level everywhere in the brain parenchyma, but the protein accumulates in degenerated neurons in diseases such as PD. The significant -synuclein increase observed in our study is most likely due to the non-specific degeneration induced by rotenone in population of striatal neurons and interneurons. We did not observe any change in -synuclein expression in the SNpc, possibly reflecting absence of degeneration in this structure.
We also measured NGFI-B and Nurr1 mRNA expression in response to rotenone treatment. NGFI-B and Nurr1 are members of the "zinc finger" superfamily of nuclear receptors and they have been closely associated to the DA systems. Nurr1 colocalizes with TH and its expression is responsible for the development and maintenance of a DA phenotype in neurons of the midbrain (SN and the ventral tegmental area). We have repeatedly shown that alteration of the DA function modulates NGFI-B expression in the striatum. D2 receptor antagonists or that nigrostriatal 6-OHDA lesion increases NGFI-B expression in enkephalinergic projection neurons. In the present study, we observed a complete disappearance of NGFI-B mRNA signal in TH-negative territories of the striatum. This lack of compensatory adaptation in response to TH fiber loss further emphasizes a striatal post-synaptic defect in the rotenone model. At the level of the SN, Nurr1 expression was selectively decreased in animals showing the loss of TH-immunoreactive fibers in the striatum. Given the recently characterized regulatory role of Nurr1 in TH expression, the decrease in Nurr1 mRNA levels in vulnerable animals suggests that DA neurons have been targeted but have not reached identifiable degenerative stages.
A number of studies have also demonstrated that neurotoxins, which induce parkinsonism in animals along with DA cell loss in the SN, are associated with a potent immune response and microglial activation. In rotenone animals showing susceptibility to the toxin, the microglial cells may be overstimulated by intense signals coming from degeneration of several neuronal types and react by releasing several cytokines and reactive oxide species who could induce degeneration. A clear neuroinflammatory response was seen within the striatum of 8-day rotenone treated animals. On the contrary, no microglial response was observed in the SN of treated animals. This data further emphasizes that the initial insult takes place in the striatum, which may subsequently contribute to the reported toxicity in DA cells.
The idea that an inhibitor of complex 1 of the electron transport chain, which acts uniformly throughout the brain, could selectively target and induce degeneration of nigrostriatal neurons is very appealing. Rotenone, a lipophilic molecule, holds the capacity to penetrate all membrane types. In spite of this, what was initially considered an advantage for the use of this toxin could in fact be a shortcoming. Overall, this promising model suffers from much variability in the susceptibility of individual rats to the toxin as reported by others, and as documented for other toxins such as 3-nitroproprionic acid in Huntington’s disease models. Rotenone has thus far been administered orally, intravenously, stereotaxically, and subcutaneously without, prior to Betarbet et al.’s reports (2002), any specific effects on DA neurons. The recently described alternative subcutaneous administration route was also associated, in our hands, with a high degree of morbidity and mortality and failed to produce specific DA degeneration in the SN of rats exposed to the toxin. The non-specific effects actually mirror observations reported earlier and those of Sherer et al. (2003), who noted a 36% systemic toxicity in addition to describing nigrostriatal lesions in only 18 of the remaining 35 animals comprised in his study. Some cases of SN lesion were associated with highly circumscribed lesions in the striatum, and in "other instances" more diffuse lesions associated with loss of TH immunoreactivity in the SN.
Accumulating evidence has endorsed this importance of understating the contribution of environmental factors to PD prevalence. With the increasing use of pesticide in agricultural settings, it is extremely important to raise the awareness of potential dangers of rotenone poisoning. Although our study does not suggest the direct role of rotenone in PD induction, it provides evidence that this pesticide is toxic and damaging to the entire system and likely to cause peripheral and brain toxicity to a restricted percentage of susceptible individuals. The significance and reliability of the rotenone model recently questioned, concurrently with the data presented here as well as by others, introduces strong concerns regarding the scientific basis for the use of this model in the study of PD and thus data derived from this model should be interpreted with caution.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0677fje; doi: 10.1096/fj.03-0677fje 
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