Elsevier

Experimental Neurology

Volume 190, Issue 2, December 2004, Pages 373-383
Experimental Neurology

Rotenone potentiates dopamine neuron loss in animals exposed to lipopolysaccharide prenatally

https://doi.org/10.1016/j.expneurol.2004.08.006Get rights and content

Abstract

We previously demonstrated that treating gravid female rats with the bacteriotoxin lipopolysaccharide (LPS) led to the birth of offspring with fewer than normal dopamine (DA) neurons. This DA neuron loss was long-lived and associated with permanent increases in the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Because of this pro-inflammatory state, we hypothesized that these animals would be more susceptible to subsequent exposure of DA neurotoxins. We tested this hypothesis by treating female Sprague–Dawley rats exposed to LPS or saline prenatally with a subtoxic dose of the DA neurotoxin rotenone (1.25 mg/kg per day) or vehicle for 14 days when they were 16 months old. After another 14 days, the animals were sacrificed. Tyrosine hydroxylase-immunoreactive (THir) cell counts were used as an index of DA neuron survival. Animals exposed to LPS prenatally or rotenone postnatally exhibited a 22% and 3%, respectively, decrease in THir cell counts relative to controls. The combined effects of prenatal LPS and postnatal rotenone exposure produced a synergistic 39% THir cell loss relative to controls. This loss was associated with decreased striatal DA and increased striatal DA activity ([HVA]/[DA]) and TNFα. Animals exposed to LPS prenatally exhibited a marked increase in the number of reactive microglia that was further increased by rotenone exposure. Prenatal LPS exposure also led to increased levels of oxidized proteins and the formation of α-Synuclein and eosin positive inclusions resembling Lewy bodies. These results suggest that exposure to low doses of an environmental neurotoxin like rotenone can produce synergistic DA neuron losses in animals with a preexisting pro-inflammatory state. This supports the notion that Parkinson's disease (PD) may be caused by multiple factors and the result of “multiple hits” from environmental toxins.

Introduction

Environmental factors are thought to contribute to the development of nonfamilial Parkinson's disease (PD). Von Economo's pan encephalitis (Casals et al., 1998) and manganese (Zheng et al., 1998) are considered long-standing members of a growing list of risk factors, while more recent additions include pesticides and fungicides such as dieldrin (Corrigan et al., 2000) and maneb (Thiruchelvam et al., 2000), respectively (see Adams and Odunze, 1991, Dawson et al., 1995, Di Monte, 2003; for reviews). That environmental factors serve as risk factors for PD was formalized by Calne and Langston (1983). They proposed that exposure to an environmental toxin such as 1-methyl, 4-phenyl, 1,2,3,6 tetrahydropyridine (MPTP) killed a portion of the DA neurons; but not enough cells were lost to produce PD. However, additional age-related DA neuron losses eventually produced sufficient cell losses to cause clinical symptoms. Unfortunately, the vast majority of PD patients cannot identify a specific exposure in their past that was sufficient enough to produce their disease. Although this might suggest that environmental factors are not responsible, it might also imply the involvement of low doses of toxins acting synergistically. This latter possibility is consistent with the emerging hypothesis that the cause of PD is multifactorial and a result of interacting “multiple hits” involving different toxins (Di Monte, 2003) or gene–toxin interactions (Greenamyre et al., 2003, Le Couteur et al., 2002).

The multiple hit hypothesis is attractive because it raises the possibility of synergistic interactions. This would imply that toxin exposure levels could be low or sporadic, and therefore difficult to detect epidemiologically. It has been widely assumed that these exposures occurred during adult life. However, we previously demonstrated that exposure to a low dose of the bacteriotoxin lipopolysaccharide (LPS) during a critical window of vulnerability during fetal development led to the birth of rat pups with fewer than normal DA neurons (Ling et al., 2002a, Ling et al., 2002b). This suggests toxin exposure relevant to PD can also occur in utero. Such exposure frequently occurs in humans as a result of bacterial vaginosis (Dammann and Leviton, 1998, Paige et al., 1998), which is known to complicate approximately 14% of normal births (Thorsen et al., 1998). Interestingly, animals born following prenatal exposure to LPS have long-lived elevations in the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). This is potentially significant because TNFα can kill DA neurons (McGuire et al., 2001), is increased by several DA neurotoxins (Hornykiewicz and Kish, 1987, Nagatsu et al., 2000), and is elevated in the brains of patients with PD (Boka et al., 1994, Mogi et al., 2000a). If animals exposed to LPS prenatally were exposed as adults to a neurotoxin that increased TNFα or other pro-inflammatory factors, synergistic DA neuron loss might occur.

We previously tested this hypothesis in vivo. Four-month-old male rats exposed to LPS prenatally received a moderate dose of the DA neurotoxin 6-hydroxydopamine (6OHDA; icv) (Ling et al., 2004). Both prenatal LPS exposure and postnatal 6OHDA produced significant DA neuron losses, although the combined effects of both factors were, at best, additive. Although the results supported the hypothesis that “multiple hits” to the DA system can combine to produce significant DA neuron loss, synergism was not seen. The young age of animals and the toxin used were discussed as possible reasons why synergistic cell losses were not seen. In the present study, we evaluated a different DA neurotoxin, the complex I inhibitor rotenone (Fiskum et al., 2003, Sherer et al., 2003), and evaluated it in aged female rats (17 months old) to explore the possibility of synergistic cell loss further. Rotenone is a pesticide that is found naturally in the environment and has been distributed even more widely by man (Gosalvez, 1983, Soloway, 1976). It selectively kills DA neurons in rats (Betarbet et al., 2000, Betarbet et al., 2002). Here we show that synergistic DA neuron losses do result from exposure to rotenone in animals treated with LPS prenatally.

Section snippets

Animals

Thirty-six timed-gravid, Sprague–Dawley (Zivic-Miller, Allison Park, PA) female rats were delivered to Rush's animal facility at gestational day nine ±12 h. All animals and their offspring were allowed access to food and water ad libitum and were maintained in an environmentally regulated animal facility for the duration of the study (lights on 0600–1800). The survival surgery was carried out under ketamine and xylazine anesthesia to minimize pain. The protocols and procedures used in these

Animals

Overall, the animals tolerated the LPS treatments without difficulty. At birth, the female pups were all full-term and no differences in birth weights were detected (data not shown). The animals developed normally and did not exhibit overt behavioral effects. We had performed a study similar to the current experiment except that 6-month-old males were used and 2.5 mg rotenone/kg per day was used. Unfortunately, at the dosage used, the animals became very sick, several died, and the experiment

Discussion

Results from the present study showed that exposure to rotenone, at a dose that failed to alter THir cell counts and had minimal effects on striatal DA in control animals, produced a synergistic loss of THir cells in animals exposed to LPS prenatally. This loss was accompanied by and was correlated with striatal DA losses. The combination of both treatments also increased DA activity and TNFα. This study extends those reported previously in that the DA cell losses and increases in TNFα seen in

Acknowledgments

This work was supported by ES10776, NS045316, and The Michael J. Fox Foundation.

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