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Both epidemiological and pathological data suggest an inflammatory response including microglia activation and neuro-inflammation in the Parkinsonian brain. Treatments with lipopolysacchride (LPS), rotenone and paraquat have been used as models for Parkinson’s disease, as they cause dopaminergic neuron degeneration in culture and in animals. Recent studies have suggested that rotenone and paraquat induce neuro-inflammation, however, it is not known if they can directly activate microglia. Here, we use primary cultured microglia to address this question. Microglia activation was analyzed by morphological changes and release of nitric oxide and inflammatory cytokines. Treatment with LPS was used as a positive control. While LPS induced morphological changes characteristic of microglial activation and release of nitric oxide and inflammatory cytokines, rotenone and paraquat did not. Our results suggest that paraquat and rotenone do not act directly on microglia and that neuro-inflammation and microglial activation in animals treated with these agents is likely non-cell autonomous, and may occur as a result of dopaminergic neuron damage or factors released by neurons and other cells.
Brain inflammation has emerged as a possible risk factor in sporadic Parkinson’s disease. Severe head trauma has been associated with increased risk of developing Parkinson’s disease [5, 13]. While not directly proven, inflammation caused during such injury may be a contributing factor in the development of Parkinson’s disease later in life. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin use is negatively correlated with disease risk suggesting that suppression of the inflammatory response may be beneficial in the treatment of Parkinson’s disease [4, 7, 36].
From 1916–1927 a pandemic of Encephalitis lethargica swept through Europe and North America. Many of those who recovered from this disease developed post-encephalitic Parkinsonism [9, 25, 31]. The cause of this outbreak of encephalitis has not been positively identified. However, as encephalitis is inflammatory in nature it is possible that this increase in brain inflammation may have contributed to the later development of Parkinsonism. West Nile Virus (WNV) could prove to be a contemporary example of the connection between neuro-inflammation and Parkinsonism. Humans that have been infected with WNV exhibit encephalitis, neuro-inflammation and transient Parkinsonism [20, 33].
Lipopolysaccharide (LPS) is a bacterial coat protein and is used as an inflammatory model for Parkinson’s disease. LPS causes dopaminergic cell death and decreases locomotor activity in rodents [2, 19]. During an inflammatory response in the brain, microglia become activated, releasing interleukin (IL) -1β, IL-6, IL-8, tumor necrosis factor (TNF) -α, nitric oxide (NO) and superoxide. Postmortem studies examining the substantia nigra of Parkinson’s disease patients have revealed the presence of reactive microglia . These studies suggest that inflammation may be a key feature of Parkinson’s disease.
Epidemiological studies have suggested a potential link between general pesticide exposure and increased risk for Parkinson’s disease [3, 11, 14, 16]. Both rotenone and paraquat are pesticides used worldwide . Treatments with rotenone and paraquat induce selective dopaminergic neuron degeneration and have been used to model sporadic Parkinson’s disease [6, 17, 18, 28]. While LPS has been used as an inflammatory model for Parkinson’s disease, less is known about the inflammatory response resulting from rotenone or paraquat exposure. It has been reported that rotenone induces microglial activation in vitro and in vivo [12, 38]. Indeed, microglia were determined to be a key component in rotenone-induced dopaminergic cell death in vitro . Another study demonstrated that a single exposure to paraquat in mice induces activation of microglia even in the absence of observable dopaminergic neuron damage . However, these studies did not determine if rotenone or paraquat act directly on microglia to induce an inflammatory response.
Here, we use primary cultured microglia to determine the role rotenone and paraquat play in the inflammatory process. We analyzed microglia for morphological changes and nitric oxide and cytokine release and found that while LPS generates a significant inflammatory response, rotenone and paraquat did not produce similar results. These results suggest that rotenone and paraquat do not directly activate microglia or induce microglia-mediated inflammation.
All reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. Rotenone was dissolved in dimethyl sulfoxide (DMSO). LPS was dissolved in phosphate buffered saline (PBS). Paraquat was dissolved in water. Mouse β-III tubulin monoclonal antibody was purchased from Promega (Madison, WI). Rabbit ionized calcium-binding adaptor molecule 1 (Iba-1) polyclonal antibody was purchased from Wako (Richmond, VA). Rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody was purchased from Dako (Carpinteria, CA). Secondary Alexa Fluor® antibodies were purchased from Invitrogen (Carlsbad, CA).
All animal work was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Timed-pregnant C57BL/6 mice were purchased from Charles River. We cultured cortices from 3–4 day old mice under conditions selective for glia proliferation followed by isolation of microglia to be used for microglia-enriched experiments [22, 23]. Post-natal day 3–4 (P3-P4) pups were sacrificed and the cortex was isolated in Hank’s Balanced Salt Solution (Invitrogen). Cells were dissociated with 0.125% trypsin at 37°C for 25 minutes, followed by 50 μg/mL DNase (Worthington Biochemical Corporation, Lakewood, NJ) and mechanical triteration. Dissociated cells were then spun at 1000 rpm for 5 minutes and resuspended in D10C (Dulbecco’s modified eagle medium (DMEM), 10% F-12, 10% horse serum (Invitrogen), 2 mM glutamine (Invitrogen), 10 mM HEPES, 50 μg/mL penicillin/streptomycin) and plated in poly-D-lysine (PDL) coated T75 flasks overnight. The following day, media was changed to D10C plus 5% L929 conditioned media and incubated for 1 week.
Microglia were isolated by shaking the flask, collecting the media and spinning at 1000 rpm for 5 minutes. Cells were resuspended in DMEM-F12 (Invitrogen), 1% N2 supplement (Invitrogen), and 0.1% bovine serum albumen (BSA) and plated on PDL-coated 24-well plates. After overnight incubation, cells were treated with LPS, TNF-α, rotenone or paraquat for 24 or 72 hours. Microglia-enriched cultures were immunostained with Iba-1, β-III tubulin and GFAP to determine purity of microglia culture. Cultures were determined to be at least 95% pure microglia by determining the number of β-III tubulin-positive neurons and GFAP-positive astrocytes relative to Iba-1-positive microglia.
Culture medium from treated microglia was added to Griess reagent in a 1:1 ratio. After 15 minutes, optical density (OD) was measured at 540 nm using a colormetric plate reader. Nitric oxide concentration ([NO]) was determined by use of a nitrite standard reference curve. A 100 μM sodium nitrate solution and six 2-fold serial dilutions were used to generate the standard reference curve. Sample nitric oxide concentrations were then calculated from a 4-parameter equation based on this standard curve.
Cytokine levels of IL-1β, IL-2, IL-4, IL-10, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (INF) -γ, and TNF-α were measured in culture medium using the Bio-Plex mouse cytokine 8-plex Panel A (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Briefly, 50μL of each standard from a series of 4-fold dilutions and samples were incubated with target capturing beads on a 96-well plate for 30 minutes, followed by 30 minutes of incubation with specific biotinylated detection antibodies. Next, streptavidin R-phycoerythrin was added and incubated for 10 minutes. Filtration and washing were incorporated after each incubation step with wash buffer using vacuum manifold. Sample plate was incubated at room temperature with shaking. Samples were re-suspended in assay buffer prior to reading using the Luminex 100™ (Austin, TX) suspension array reader. All samples were analyzed in duplicates.
This was performed as previously described . Briefly, cells were fixed with 4% paraformaldehyde/4% sucrose at room temperature for 30 minutes and permeabilized with 0.5% Igepal® (Sigma) in PBS for 30 minutes. To block non-specific binding, cells were treated for two hours with 2.5% BSA, 5% horse serum, and 5% goat serum in PBS/0.1% Triton X-100. Cells were then incubated with mouse β-III tubulin (1:200) monoclonal antibody or rabbit Iba-1 (1:500), anti-GFAP (1:500) polyclonal antibodies overnight at 4°C. Secondary antibody (goat anti-mouse Alexa Fluor® 488 or 594, goat anti-rabbit Alexa Fluor® 488 or 594) was then added for one hour followed by nuclear staining with Hoechst 33258 (2.5 μg/ml) for 10 minutes.
For all experiments, data were results from at least three independent experiments, each with at least duplicate determinations. Statistical analysis of the data was performed using one-way analysis of variation (ANOVA) with statistically significant values representing an alpha level of 0.05 or below. Error bars represent standard error of means (SEM). *, p<0.05; **, p<0.01; ***, p<0.001; ns, not statistically significant (p>0.05). ND, data was below detectable levels and set at the lowest value of the dynamic range of detection.
Resting microglia are characterized by long bipolar or unipolar processes and elongated cell bodies, whereas activated microglia display rounded, amoeboid cell bodies . Morphology of microglia was analyzed immunocytochemically using ionized calcium-binding adaptor molecule 1 (Iba-1), a protein expressed selectively in microglia [1, 30]. Mouse primary cultured microglia were treated with vehicle control (DMSO or PBS), 1 ng/ml LPS, 10–50 nM rotenone or 5–50 μM paraquat. These concentrations of rotenone and paraquat were chosen because dopaminergic neuron degeneration in culture has been reported at rotenone and paraquat concentrations as low as 5 nM and 10 μM, respectively [17, 21].
LPS stimulated changes in microglial morphology, whereas no appreciable morphological changes were found in primary cultured microglia stimulated with rotenone or paraquat (Fig 1). Rotenone at levels as high as 50 nM did not have any effect on microglia morphologically. While paraquat-treated microglia underwent cell death at high levels (50 μM), there was no evidence of any morphological changes at lower doses. These data suggest that rotenone and paraquat do not directly activate primary cultured microglia.
Nitric oxide (NO) release from microglia is an indicator of an inflammatory response (as reviewed in [15, 35]). In addition, nitric oxide has been identified as toxic to dopaminergic neurons [12, 32, 37]. Here, LPS stimulated release of high levels of nitric oxide from microglia 24 and 72 hours post-treatment (Fig 2). Paraquat- or rotenone-stimulated microglia did not release significant amounts of nitric oxide, suggesting that these compounds do not activate one of the key inflammatory responses by microglial.
Upon activation, microglia release high levels of interleukins and other cytokines which are part of the inflammatory response (as reviewed in ). We measured the levels of IL-10, IL-1β, IL-2, IL-4, IL-5, TNF-α, GM-CSF and INF-γ released by primary cultured microglia after treatment with LPS, rotenone or paraquat. LPS treatment generated high levels of IL-10, IL-1β and TNF-α release at 24 and 72 hours post-treatment (Fig 3 A, B, and Fig 4 A). Increases in IL-2, GM-CSF and INF-γ release were also found following LPS stimulation, though to a lesser degree (Fig 3 C, Fig 4 B and C). Conversely, very little of any of these cytokines was detectable in the media of microglia cultures after rotenone or paraquat treatment. These data suggest that while LPS induces an inflammatory response in primary cultured microglia, rotenone and paraquat do not directly act on microglia to induce inflammation.
We analyzed microglia for morphological changes along with nitric oxide and cytokine release and discovered that while LPS generates a significant inflammatory response, rotenone and paraquat do not. LPS treatment of primary cultured microglia resulted in rounded, amoeboid-like cells that released high levels of NO, IL-10, IL-1β, and TNF-α. No significant changes in morphology or increase in inflammatory mediators were observed with rotenone or paraquat treatment. Our findings are important and suggest that rotenone and paraquat do not activate microglia directly.
Microglia activation occurs in vivo after rotenone treatment . Microglia play a key role in rotenone-induced dopaminergic cell death in mixed rat neuron/glia cultures . Rotenone-treated microglia generate high levels of superoxide, contributing to dopaminergic cell death. However, it is still unclear whether rotenone can directly cause microglial activation. Using primary cultured microglia from rats, Gao et al. failed to observe significant accumulation of NO, IL-1β or TNF-α after rotenone treatment . However, a later study, using the same type of culture preparation and treatment, found morphological changes characteristic of microglial activation and increased levels of TNF-α, cyclooxygenase 2 and prostaglandin E2, although no explanation was provided for this apparent discrepancy . Data presented in this study using highly enriched microglia preparation from mice showed that rotenone does not cause morphological changes of microglial or production and release of inflammatory cytokines, consistent with the findings of Gao et al. .
A single exposure of paraquat in mice induces activation of microglia even in the absence of observable dopaminergic neuron damage . This study was performed in vivo and did not identify if paraquat was specifically and directly affecting microglia or if paraquat was causing sub-lethal effects in other cells which resulted in subsequent microglial activation. Our results suggest that paraquat does not directly activate microglia.
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which specifically targets dopaminergic neurons via dopamine transporter uptake, is thought to also cause neuro-inflammation and microglia activation. Mice lacking inducible nitric oxide synthase, TNF receptors 1 and 2, or INF-γ receptor were protected from MPTP-induced reactive gliosis [8, 27, 34]. Since MPP+, the toxic metabolite of MPTP, acts specifically on dopaminergic neurons, perhaps MPP+-damaged neurons release TNF-α and INF-γ, which are known to activate inducible nitric oxide synthase in microglia. Thus, it seems likely that rotenone, paraquat, or MPTP, neurotoxicants used to induce models for sporadic Parkinson’s disease in vivo, induce neuro-inflammation and microglial activation indirectly through factors released from neurons or astrocytes.
We would like to thank Amanda Case, Nicole Nesser and Annette Zawalinski of the Department of Neurology at the University of Washington for their assistance with primary microglial cultures. This work was facilitated by the UW NIEHS sponsored Center for Ecogenetics and Environmental Health (NIEHS P30ES07033). This work was supported by NIH grants ES012215 and ES013696 (ZX) and the Environmental Pathology/Toxicology Training Grant (HK).
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