COMBINED TOXICITY OF PRENATAL BACTERIAL ENDOTOXIN EXPOSURE AND POSTNATAL 6-HYDROXYDOPAMINE IN THE ADULT RAT MIDBRAIN Z. D. LING, a,b * Q. CHANG, a J. W. LIPTON, b C. W. TONG, a T. M. LANDERS a AND P. M. CARVEY a,b a Department of Pharmacology, 1735 West Harrison Street, Room 410, Rush University Medical Center, Chicago, IL 60612, USA b Department of Neurological Sciences, Rush University Medical Cen- ter, Chicago, IL 60612, USA Abstract—We previously reported that injection of the Gram () bacteriotoxin, lipopolysaccharide (LPS), into gravid fe- males at embryonic day 10.5 led to the birth of animals with fewer than normal dopamine (DA) neurons when assessed at postnatal days (P) 10 and 21. To determine if these changes continued into adulthood, we have now assessed animals at P120. As part of the previous studies, we also observed that the pro-inflammatory cytokine tumor necrosis factor  (TNF) was elevated in the striatum, suggesting that these animals would be more susceptible to subsequent DA neu- rotoxin exposure. In order to test this hypothesis, we injected (at P99) 6-hydroxydopamine (6OHDA) or saline into animals exposed to LPS or saline prenatally. The results showed that animals exposed to prenatal LPS or postnatal 6OHDA alone had 33% and 46%, respectively, fewer DA neurons than con- trols, while the two toxins combined produced a less than additive 62% loss. Alterations in striatal DA were similar to, and significantly correlated with (r 2 0.833) the DA cell losses. Prenatal LPS produced a 31% increase in striatal TNF, and combined exposure with 6OHDA led to an 82% increase. We conclude that prenatal exposure to LPS pro- duces a long-lived THir cell loss that is accompanied by an inflammatory state that leads to further DA neuron loss fol- lowing subsequent neurotoxin exposure. The results suggest that individuals exposed to LPS prenatally, as might occur had their mother had bacterial vaginosis, would be at in- creased risk for Parkinson’s disease. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Parkinson’s disease, TNF, IL-1, dopamine, prenatal, lipopolysaccharide. Although genetic factors account for some cases of Par- kinson’s disease (PD; Gasser, 1998; Polymeropoulos et al., 1997), the vast majority of PD is considered idiopathic. Several environmental factors, including chemical neuro- toxins, such as rotenone, dieldrin, and paraquat have been proposed as risk factors (Brooks et al., 1999; Thiruchelvam et al., 2000; Betarbet et al., 2000; Jenner, 2001). Although these known dopamine (DA) neurotoxins have been widely discussed, their potential roles as PD risk factors are sus- pect because of the large dosages that would be needed. However, if exposure were to occur in an individual that already had fewer than normal DA neurons, and especially if that hypo-dopaminergic condition was associated with a pro-inflammatory state, then lower dosages of neurotoxins would be needed. Recent studies in animals from our laboratory suggest that this is possible. We previously demonstrated that rat fetuses exposed to the Gram(-) bacteriotoxin, lipopolysaccharide (LPS) at embryonic day (E) 10.5 were born with fewer than normal DA neurons (Ling et al., 2002a,b). Assessment of tyrosine hydroxylase (TH) immunoreactive (ir) neuron counts (used as an index of DA neurons) in the substantia nigra (SN) of postnatal day (P) 10 and P21 rat pups revealed losses of 25 and 31%, respectively. Similar reductions in striatal DA and increases in DA activity ([homovanillic acid (HVA)]/ [DA]) were also seen. In addition, increased levels of the pro-inflammatory cytokine tumor necrosis factor  (TNF), were seen in the SN of these animals, indicating a pro- inflammatory state. These changes in DA and increases in TNF are consistent with the well-established effects of LPS. Thus, LPS, acting through the CD14 and toll-like receptor-4, is a well known inducer of pro-inflammatory cytokines and has been shown to kill DA neurons both in vitro and in vivo by several investigators (Castano et al., 1998, 2002; Gayle et al., 2002). Taken together, these studies suggested that LPS and infections that increase pro-inflammatory cytokines prenatally, may be involved in the etiology of PD. We hypothesized that individuals born to mothers with bacterial vaginosis (BV), a well known inducer of LPS and pro-inflammatory cytokines in the cho- rioamniotic environment (Fortunato et al., 1996; Menon et al., 1995), would be at increased risk for PD. Moreover, if prenatal LPS led to a continued pro-inflammatory state, then subsequent exposure to DA neurotoxins would pro- duce a more pronounced DA cell loss. This would then support the notion that PD is a consequence of the com- bined effects of multiple environmental factors with a pre- natal factor representing an entirely new exposure para- digm. We set out to assess this possibility in rats exposed to LPS prenatally followed by postnatal neurotoxin exposure. We chose the well-known DA neurotoxin, 6-hydroxydopa- *Correspondence to: Z. Ling, Department of Pharmacology, 1735 West Harrison Street, Room 410, Chicago, IL 60612, USA. Tel: +1- 312-563-2556; fax: +1-312-563-3552. E-mail address: zling@rush.edu (Z. D. Ling). Abbreviations: BV, bacterial vaginosis; DA, dopamine; E, embryonic day; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EU, endotoxin unit; HBSS, Hanks’ balanced salt solution; HPLC, high performance liquid chromatography; HVA, homovanillic acid; IL-1, interleukin 1; ir, immunoreactive; LPS, lipopolysaccharide; P, postnatal; PD, Parkinson’s disease; SN, sub- stantia nigra; TH, tyrosine hydroxylase; TNF, tumor necrosis factor ; VTA, ventral tegmental area; 6OHDA, 6-hydroxydopamine. Neuroscience 124 (2004) 619 – 628 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.12.017 619