Prevotella bivia as a source of lipopolysaccharide in the vagina.

OBJECTIVES: To compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA). METHODS: Vaginal washes obtained from patients with (n=43) and without (n=59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts. RESULTS: The median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P<0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06+/-0.36 vs 5.4+/-2.2 log(10) CFU/ml, respectively, P<0.001). Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0+/-306.6 EU/ml) than either E. coli (4679.0+/-585.3 EU/ml) or G. vaginalis (0.07+/-0.01 EU/ml of LPS). The loss of DA neuron was 20-27% in cultures treated with vaginal washes from BV-negative patients and 58-97% in cultures treated with vaginal washes from patients with BV. CONCLUSION: P. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.

Altered glutathione homeostasis in animals prenatally exposed to lipopolysaccharide.

We previously reported that injection of bacterial lipopolysaccharide (LPS) into gravid female rats at embryonic day 10.5 resulted in a birth of offspring with fewer than normal dopamine (DA) neurons along with innate immunity dysfunction and many characteristics seen in Parkinson's disease (PD) patients. The LPS-exposed animals were also more susceptible to secondary toxin exposure as indicated by an accelerated DA neuron loss. Glutathione (GSH) is an important antioxidant in the brain. A disturbance in glutathione homeostasis has been proposed for the pathogenesis of PD. In this study, animals prenatally exposed to LPS were studied along with an acute intranigral LPS injection model for the status of glutathione homeostasis, lipid peroxidation, and related enzyme activities. Both prenatal LPS exposure and acute LPS injection produced a significant GSH reduction and increase in oxidized GSH (GSSG) and lipid peroxide (LPO) production. Activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, was up-regulated in acute supranigral LPS model but was reduced in the prenatal LPS model. The GCS light subunit protein expression was also down-regulated in prenatal LPS model. GSH redox recycling enzyme activities (glutathione peroxidase, GPx and glutathione reducdase, GR) and glutathione-S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GT) activities were all increased in prenatal LPS model. Prenatal LPS exposure and aging synergized in GSH level and GSH-related enzyme activities except for those (GR, GST, and gamma-GT) with significant regional variations. Additionally, prenatal LPS exposure produced a reduction of DA neuron count in the substantia nigra (SN). These results suggest that prenatal LPS exposure may cause glutathione homeostasis disturbance in offspring brain and render DA neurons susceptible to the secondary neurotoxin insult.

Age-related changes in glutathione and glutathione-related enzymes in rat brain.

The most reliable and robust risk factor for some neurodegenerative diseases is aging. It has been proposed that processes of aging are associated with the generation of reactive oxygen species and a disturbance of glutathione homeostasis in the brain. Yet, aged animals have rarely been used to model the diseases that are considered to be age-related such as Parkinson's or Alzheimer's disease. This suggests that the results from these studies would be more valuable if aged animals were used. The present study was designed to provide insight into the glutathione redox state in young and aged rat siblings of both genders by studying the enzyme activities related to glutathione synthesis, cycling, and usage. The results suggested a significant age-related reduction of reduced glutathione (GSH) level in all brain regions examined, associated with an increase of GSH oxidation to glutathione disulfide (GSSG) and decrease of the GSH/GSSG ratio. These changes were accompanied by diminished gamma-glutamylcysteine synthetase activity in de novo glutathione synthesis and increased lipid peroxidation. In addition, these changes were associated with increased enzyme activities related to the GSH usage (glutathione peroxidase, gamma-glutamyl transpeptidase, and glutathione S-transferase). The results indicate that aged animals are likely more vulnerable to oxidative stress and insinuate the roles of aged animals in modeling age-related neurodegeneration diseases.

Prenatal exposure to the bacteriotoxin lipopolysaccharide leads to long-term losses of dopamine neurons in offspring: a potential, new model of Parkinson's disease.

The cause of Parkinson's disease (PD) is currently unknown. Although a genetic cause has been implicated in familial PD, the vast majority of cases are considered idiopathic. Environmental toxins have been implicated as a cause for PD by many investigators. Unfortunately, the magnitude of this exposure would likely need to be very high and as a result, would likely have been identified by the many epidemiological studies performed to date. Recently, we inadvertently realized that exposure to neurotoxins while still in utero may also represent a risk factor. Thus, exposure to the bacteriotoxin, lipopolysaccharide (LPS) during a critical developmental window in rats, leads to the birth of animals with fewer than normal dopamine (DA) neurons. This DA neuron loss is apparently permanent as it is still present in 16 months old animals (the longest period studied to date). Moreover, the loss of DA neurons seen in these animals increases with age thereby mimicking the progressive pattern of cell loss seen in human PD. The DA neuron loss is accompanied by reductions in striatal DA, increases in DA activity, and increased production of the pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). These are also characteristics of the PD brain. This model therefore shares many of the same characteristics with PD, and most importantly exhibits a slow, protracted loss of DA neurons - a characteristics of this animal model not found in other models. Interestingly, a common complication of pregnancy is a condition known as bacterial vaginosis (BV), which is known to produce increased levels of LPS and pro-inflammatory cytokines in the chorioamniotic environment of the fetus. This raises the interesting possibility that BV may be a risk factor for PD. The possibility that prenatal toxin exposure may contribute to the development of a neurodegenerative disease of the aged raises interesting new pathogenic questions and draws attention to the possibility that in utero exposure to neurotoxins may represent a here to fore unrecognized cause of PD.

Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc). Although the exact mechanisms responsible for this cell loss are unclear, emerging evidence suggests the involvement of inflammatory events. In the present study, we characterized the effects of the proinflammatory bacteriotoxin lipopolysaccharide (LPS) on the number of tyrosine hydroxylase immunoreactive (THir) cells (used as an index for DA neurons) in primary mesencephalic cultures. LPS (10-80 microg/ml) selectively decreased THir cells and increased culture media levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well as nitrite (an index of nitric oxide (NO) production). Cultures exposed to both LPS and neutralizing antibodies to IL-1beta or TNF-alpha showed an attenuation of the LPS-induced THir cell loss by at least 50% in both cases. Inhibition of the inducible form of nitric oxide synthase (iNOS) by L-NIL did not affect LPS toxicity, but increased the LPS-induced levels of both TNF-alpha and IL-1beta. These findings suggest that neuroinflammatory stimuli which lead to elevations in cytokines may induce DA neuron cell loss in a NO-independent manner and contribute to PD pathogenesis.

Prenatal cocaine exposure induces an attenuation of uterine blood flow in the rat.

We have previously demonstrated that maternal cocaine injections result in a gradient of fetal brain cocaine levels that decrease as a function of the fetuses' proximity to the ovaries at embryonic (E) day 15. Our prior data suggest that cocaine-induced vasoconstriction may (1) limit cocaine's entry into the brain and (2) cause damage to DA neurons through injury associated with hypoxia or ischemia of the utero-placental junction. Therefore, using the microsphere technique (labeled with Ru(103)), the following study sought to determine whether the previously observed pattern of cocaine distribution among fetuses in the uterus were due to position-specific reductions in uterine or placental blood flow. On day 15, a single subcutaneous injection of 30 mg/kg cocaine HCl was administered to each rat. Thirty minutes after the cocaine injection, reference blood samples were drawn from the ventral tail artery. Uterine segments and placentae were removed and subjected to gamma counting. While results regarding placental blood flow were equivocal, cocaine significantly reduced average uterine blood flow by 54.6%. In addition, as one moves more proximal to the ovaries, cocaine progressively attenuates blood flow in uterine tissue segments. These data support the hypothesis that the pattern of drug distribution and subsequent brain alterations from prenatal cocaine exposure in our previous reports are likely due to differences in uterine blood flow.

Involvement of nigrostriatal pathway in Japanese encephalitis with movement disorders: evidence from 99mTc-TRODAT-1 and 123I-IBZM SPECT imagings.

PURPOSE: The purpose of this study was to evaluate molecular evidence of nigrostriatal pathway involvement in Japanese encephalitis (JE) survivors with movement complications. METHODS: Three JE patients were recruited. All had cranial magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT) studies with (99m)Tc-TRODAT-1 and (123)I-IBZM. RESULTS: Cranial MRI revealed involvement of bilateral thalami, substantia nigra, and medial temporal lobes in all three patients, but only case 1 had additional bilateral basal ganglia involvement. The (99m)Tc-TRODAT-1 SPECT for presynaptic dopamine transporter imaging disclosed asymmetrical decreases in bilateral striatal uptake in all three patients. However, the (123)I-IBZM SPECT imaging for postsynaptic D2 dopamine receptors (D2Rs) revealed inconsistent abnormalities including asymmetrical bilateral decreases (case 1), unilateral decrease (case 2), and bilateral increases (case 3) in striatal uptakes. CONCLUSION: Data have suggested that presynaptic dopaminergic neurons in JE patients are more susceptible to JE virus than postsynaptic striatal neurons. The degree of movement impairment was more closely correlated to the degree of D2Rs disruption seen in (123)I-IBZM SPECT imaging.

Prenatal lipopolysaccharide does not accelerate progressive dopamine neuron loss in the rat as a result of normal aging.

We previously demonstrated that in utero exposure to the bacteriotoxin lipopolysaccharide (LPS) led to the birth of rat pups with fewer than normal dopamine (DA) neurons. These animals exhibited significant neuroinflammation in the nigrostriatal pathway creating the possibility that they could exhibit further, progressive DA neuron loss over their lives. To study this possibility, we injected gravid female rats i.p. at 10,000 endotoxin units (EUs) of LPS per kg or saline at embryonic (E) day 10.5 and assigned pups to sacrifice groups at 4, 14 and 17 months such that littermates were sacrificed at each end point. The effects of prenatal LPS on DA cell counts and striatal DA were significantly reduced relative to controls whereas DA activity and numbers of activated microglia (OX-6ir cell) were statistically increased. However, the progressive DA neuron loss was parallel to that of the controls suggesting that prenatal LPS does not produce an accelerated rate of DA neuron loss. Interestingly, locomotor activity was increased after 3 months in animals exposed to LPS prenatally, but by 16 months, was significantly reduced relative to controls. Additionally, animals exposed to LPS prenatally exhibited Lewy body-like inclusions that were first seen in 14 month old animals. These data broadly support previous studies demonstrating that prenatal exposure to LPS, as frequently occurs in humans as part of Bacterial Vaginosis, leads to the birth of animals with fewer than normal DA neurons. The progressive DA neuron loss seen in these animals is, however, primarily a result of normal aging.

Dopaminergic and serotoninergic deficiencies in young adult rats prenatally exposed to the bacterial lipopolysaccharide.

We have reported previously that prenatal bacterial lipopolysaccharide (LPS) exposure at the gestation window of vulnerability could consistently lead to dopamine (DA) neuron loss in the substantia nigra (SN). Thus, we suggested that prenatal LPS exposure might represent as a risk factor for the development of Parkinson's disease (PD). Here, we report that the same exposure could lead to tryptophan hydroxylase (TPH, a serotonin neuron marker) immunoreactive cell loss in the dorsal raphe nucleus (DRN). Twenty two pups born to saline or LPS-injected gravid female rats at E10.5 were used in the current study. Twelve male pups at age of 4 months (6 from each of two prenatal groups) were used for the tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) immunochemistry studies. The other 10 (5 from each of two prenatal groups) males were used in the biochemistry studies. A 29% THir neuron loss in the substantia nigra (F(1,11)=17.573, P=0.002) and a 31% TPHir neuron loss (F(1,11)=44.005, P<0.001) in the DRN were seen. Significant DA and 5-hydroxytryptamine (5-HT) reductions (P<0.05) were found in the frontal cortex, nucleus accumbens, striatum, amygdala, hippocampus, and hypothalamus. The losses of DA and 5-HT were accompanied by the significant increases in homovanillic acid over DA and 5-hydroxyindoleacetic acid over 5-HT ratios in the most areas tested. These data further validate prenatal LPS exposure as a model of PD since DA and 5-HT changes similar to those seen in PD patients. They also suggest that prenatal LPS might be a risk factor for other diseases including mood disorders.

TNF-alpha knockout and minocycline treatment attenuates blood-brain barrier leakage in MPTP-treated mice.

Following intraparenchymal injection of the dopamine (DA) neurotoxin 6-hydroxydopamine, we previously demonstrated passage of fluoresceinisothiocyanate-labeled albumin (FITC-LA) from blood into the substantia nigra (SN) and striatum suggesting damage to the blood-brain barrier (BBB). The factors contributing to the BBB leakage could have included neuroinflammation, loss of DA neuron control of barrier function, or a combination of both. In order to determine which factor(s) was responsible, we assessed BBB integrity using the FITC-LA technique in wild-type (WT), tumor necrosis factor alpha (TNF-alpha) knockout (KO), and minocycline (an inhibitor of microglia activation) treated mice 72 h following treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared with WT mice, TNF-alpha KO mice treated with MPTP showed reduced FITC-LA leakage, decreased numbers of activated microglia, and reduced proinflammatory cytokines (TNF-alpha and interleukin 1beta) associated with significant MPTP-induced DA neuron loss. In contrast, minocycline treated animals did not exhibit significant MPTP-induced DA neuron loss although their FITC-LA leakage, numbers of activated microglia, and MPTP-induced cytokines were markedly attenuated. Since both TNF-alpha KO and minocycline treatment attenuated MPTP-induced BBB dysfunction, microglial activation, and cytokine increases, but had differential effects on DA neuron loss, it appears that neuroinflammation and not DA neuron loss was responsible for disrupting the blood-brain barrier integrity.

Progressive dopamine neuron loss following supra-nigral lipopolysaccharide (LPS) infusion into rats exposed to LPS prenatally.

Toxin-induced animal models of Parkinson's disease (PD) exhibit many of the same neuroinflammatory changes seen in patients suggesting a role for inflammation in DA neuron loss. Yet, despite this inflammation, the progressive loss of DA neurons that characterizes PD is rarely seen in animals. We infused lipopolysaccharide (LPS) or saline into 7-month-old rats that had been exposed to LPS or saline prenatally and assessed them for DA neuron loss and inflammatory measures (interleukin 1 beta, tumor necrosis factor-alpha, glutathione, and activated microglia) over a period of 84 days to examine the role of pre-existing inflammation in progressive DA neuron loss. LPS infusion into both prenatal treatment groups produced neuroinflammation during the 14 days of LPS infusion that subsequently reverted toward normal over the next 70 days. In animals with pre-existing inflammation (i.e., prenatal LPS), however, the acute changes seen were attenuated, but took much longer to return to normal suggesting a prolonged inflammatory response. These inflammatory changes were consistent with the greater acute DA neuron loss seen in the prenatal saline controls and the progressive DA neuron loss seen only in the animals exposed to LPS prenatally. Interestingly, both prenatal treatment groups exhibited increases in microglia over the entire 84-day course of the study. These data suggest that pre-existing neuroinflammation prolongs the inflammatory response that occurs with a second toxic exposure, which may be responsible for progressive DA neuron loss. This provides further support for the "multiple hit" hypothesis of PD.

Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice.

Mutations in the DJ-1 gene were recently identified in an autosomal recessive form of early-onset familial Parkinson disease. Structural biology, biochemistry, and cell biology studies have suggested potential functions of DJ-1 in oxidative stress, protein folding, and degradation pathways. However, animal models are needed to determine whether and how loss of DJ-1 function leads to Parkinson disease. We have generated DJ-1 null mice with a mutation that resembles the large deletion mutation reported in patients. Our behavioral analyses indicated that DJ-1 deficiency led to age-dependent and task-dependent motoric behavioral deficits that are detectable by 5 months of age. Unbiased stereological studies did not find obvious dopamine neuron loss in 6-month- and 11-month-old mice. Neurochemical examination revealed significant changes in striatal dopaminergic function consisting of increased dopamine reuptake rates and elevated tissue dopamine content. These data represent the in vivo evidence that loss of DJ-1 function alters nigrostriatal dopaminergic function and produces motor deficits.

Rotenone potentiates dopamine neuron loss in animals exposed to lipopolysaccharide prenatally.

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 (TNFalpha). 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 TNFalpha. 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 alpha-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.

Uterine Position Determines the Extent of Dopamine Reduction after Chronic Prenatal Cocaine Exposure.

Most studying the consequences of prenatal cocaine (COC) exposure employ rodents or other multiparous organisms in their models. We have previously shown that when pregnant Sprague-Dawley albino rats are administered a 30 mg/kg subcutaneous (s.c.) injection on embryonic day 15 (E15), fetal brain COC levels show a proximal-to-distal (in relation to the cervix) gradient that can vary by as much as 350%. The present study sought to determine whether this gradient translated into a similar gradient in brain dopamine (DA) levels. Pregnant rats were administered COC or saline (SAL) (30 mg/kg COC or 1 ml/kg SAL, b.i.d., E7-E19). On E20, dams were anesthetized with halothane, the fetuses immediately removed, their brains excised, frozen and subsequently processed for DA, dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA). High-performance liquid chromatography (HPLC) analysis revealed a proximal-to-distal gradient for DA in both COC- and SAL-exposed fetuses. Average fetal DA levels per litter were significantly lower in COC-exposed litters (57.39 ± 3.67 ng/hemibrain SAL; 48.29 ± 3.87 ng/hemibrain COC F7,1 = 11.66, p <0.05). The gradients for DA were in opposite directions such that COC litters showed the lowest levels of DA in the most distal uterine positions, whereas SAL-exposed litters showed the highest DA levels in the same location. These data suggest that a gradient in brain dopamine normally exists for fetuses based upon uterine position, and that cocaine can have selectively greater effects on this level as a function of fetal location.

In utero bacterial endotoxin exposure causes loss of tyrosine hydroxylase neurons in the postnatal rat midbrain.

We investigated whether in utero exposure to the Gram(-) bacteriotoxin lipopolysaccharide (LPS) induces dopamine (DA) neuron loss in rats. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) kills DA neurons and is elevated in the brains of patients with Parkinson's disease (PD). LPS is a potent inducer of TNF-alpha, and both are increased in the chorioamniotic environment of women who have bacterial vaginosis (BV) during pregnancy, suggesting that BV might interfere with the normal development of fetal DA neurons. Gravid female rats were injected intraperitoneally with either LPS or normal saline at embryonic day 10.5 and their pups were killed at postnatal day 21. The brains of the pups were assessed for DA and TNF-alpha levels and DA cell counts in the mesencephalon using tyrosine hydroxylase immunoreactive (THir) cells as a DA neuron marker. Prenatal LPS exposure significantly reduced striatal DA (29%) and increased DA activity (72%) as well as TNF-alpha (101%). Stereological cell counts in the mesencephalon were also significantly reduced (27%) by prenatal LPS exposure. Prenatal exposure to LPS, as might occur in humans with BV, produces a significant loss of THir cells in rats that is still present 33 days following a single injection of LPS. Since this cell loss is well past the normal phase of DA neuron apoptosis that occurs in early postnatal life, rats so exposed may have a permanent loss of DA neurons, suggesting that prenatal infections may represent risk factors for PD.