Lipoic acid protects dopaminergic neurons in LPS-induced Parkinson’s disease model

Abstract

Parkinson’s disease (PD) is a chronic neurodegenerative disease of the central nervous system (CNS), characterized by a loss of dopaminergic neurons, which is thought to be caused by both genetic and environmental factors. Recent findings suggest that neuroinflammation may be a pathogenic factor in the onset and progression of sporadic PD. Here we explore the potential therapeutic effect of lipoic acid (LA) on a lipolysaccharide (LPS)-induced inflammatory PD model. Our results for the first time showed that LA administration improved motor dysfunction, protected dopaminergic neurons loss, and decreased α-synuclein accumulation in the substantia nigra (SN) area of brain. Further, LA inhibited the activation of nuclear factor-κB (NF-κB) and expression of pro-inflammatory molecules in M1 microglia. Taken together, these results suggest that LA may exert a profound neuroprotective effect and is thus a promising anti-neuroinflammatory and anti-oxidative agent for halting the progression of PD. Interventions aimed at either blocking microglia-derived inflammatory mediators or modulating the polarization of microglia may be potentially useful therapies that are worth further investigation.

Introduction

Parkinson’s disease (PD) is the second most prevalent age-related neurodegenerative disorder, affecting over 1 million people in the United States and 10 million population worldwide (Dorsey et al. 2007; Richardson and Hossain 2013). The pathological hallmarks of PD are the depletion of striatal dopamine caused by the loss of dopaminergic neurons in the substantia nigra (SN) of midbrain (He et al. 2013). Despite intensive investigation, the exact etiology of PD has not been fully elucidated. There is increasing evidence that the activation of microglia, with the attendant oxidative stress and neuroinflammation, may play an essential role in the pathogenesis of PD. (Richardson and Hossain 2013; Zhang et al. 2014) Activated microglia accelerate the expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokine interleukin- 1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) (Choi et al. 2012; Saijo et al. 2009; Glass et al. 2010), which are associated with degeneration of neurons in the SN of PD patients and in various PD models.

Lipolysaccharide (LPS), as a potent glial activator, has been used to activate glial cells in experimental models of PD. (Dutta et al. 2008) In the past few years, many LPS-induced PD models have been developed and explored via different routes, such as SN single injection, SN chronic infusion, and systemic, utero and intrapallidal LPS injection. Specific loss of SNpc (Substantia Nigra Pars Compacta) neurons and reduced striatal DA content were detected in these LPS models (Dutta et al. 2008). LPS administration has been shown to reduce motor activity, to progressively and selectively reduce dopaminergic neurons in the SN and to reduce striatal dopamine levels, while increasing α-synuclein accumulation and aggregation in the SN (Noworyta-Sokołowska et al. 2013; Tien et al. 2013; Qin et al. 2013). However, current LPS-induced PD models have two important limitations, i.e., a single intracranial injection of LPS was insufficient to induce degeneration and loss of DA neurons in the SN, while systemic LPS challenge did not provide a stable and uniform PD model, or even failure (Duty and Jenner 2011). In contrast, our previous study showed that nasal LPS-induced PD is purely inflammation-driven, displays the basic characteristics of PD pathology, and successfully mimics the chronic, progressive process of PD pathology (He et al. 2013). This model is, therefore, a unique addition to the current pool of PD models and provides a wide time frame for studying the inflammation-mediated chronic pathogenesis and searching for therapeutic intervention in the microglia-DA neuron pathway.

Lipoic acid (LA) is a natural thiol antioxidant that has been shown to have beneficial effects on oxidative stress parameters in various kinds of tissues (Biewenga et al. 1997). LA has been found to prevent hydrogen peroxide-induced neuron damage (Zhang et al. 2001), protect neurons from neurotoxicity (Li et al. 2013), and reduce oxidative damage following stroke through enhancing endogenous antioxidant level (Hall et al. 2010; Bilska and Wlodek 2005; Connell et al. 2011). In addition, LA has also capacity to inhibit LPS-induced inflammatory process (Zhang et al. 2007; Suh et al. 2014; Jiang et al. 2013).

Based on those properties of LA, we hypothesized that the administration of LA might be a potent neuroprotective agent in glial activation-induced PD by inhibiting oxidative stress and inflammatory response. We therefore explored the therapeutic potential of LA, and elucidated possible mechanisms in the intranasal LPS-induced PD model.

Materials and methods

Animal and treatment

Female C57BL/6 mice (8 weeks, 18–20 g) were obtained from Shanghai SLAC Laboratory Animal Company (Shanghai, China), housed under pathogen-free conditions and maintained at 12 h light/dark cycle (25 ± 2 °C). This study was approved by the Ethics Committee of Huashan Hospital of Fudan University, Shanghai, China and performed in accordance with the ethical standards of the International Council for Laboratory Animal Science. Mice were randomly divided into 3 groups: intranasal (i.n.) saline (normal control), i.n. LPS + intraperitoneally (i.p.) NS (NS-PD model), and i.n. LPS + i.p. LA (LA-PD). Each group contains 15 mice pooled from two independent experiments.

The chronic PD model was induced by a unilateral i.n. instillation with LPS, following a previously described protocol (He et al. 2013). Briefly, mice were slightly anesthetized with diethylether, 10 μl LPS (1 mg⁄ml in saline; Sigma-Aldrich, USA) was then slowly instilled with a micropipette (over 15 s) into the right nasal cavity and 10 μl saline solution into the left nasal cavity (contralateral control). Afterwards, the mice were immobilized in this position for ~10s, allowing the animal to sniff the solution into the upper nasal cavity, while preventing LPS infiltration into the left nasal cavity. This procedure was repeated every other day for 1 month. Mice that received saline nasally served as normal controls. At the same time, LA (Qidu Pharmacetical Co., Shandong, China) was dissolved in saline, with the PH adjusted to 7.4, and then sterilely filtered. LA was administered i.p. once a day at 100 mg/kg/d, as previously described (Zhang et al. 2007), and mice that received i.p. injection of saline served as control. On day 30, animals were sacrificed and brains were harvested for histological and immunohistochemistry examination.

Open-field test

To monitor general activity levels, mice were placed in the centre of an open behavioral chamber; before recording, mice were allowed to freely explore the chamber for 15 min to get used to the space in order to reduce novelty-induced stress (Rabbani et al. 2014); then their spontaneous activity was analyzed for 30 min by an automated tracking system and ambulatory episodes, counts and distance traveled were recorded. The apparatus was washed out after each test in order to avoid interfering with the activities of other mice.

Adhesive removal

To test forepaw sensitivity, including signs of neglect and motor impairment of the forepaw, the adhesive removal test was performed as previously described (Doeppner et al. 2014). Small adhesive stimuli (0.6 cm diameter) were placed on the plantar surface of both forelimbs with equal pressure, and the time to remove the stimulus was recorded. Prior to testing, two training trials were performed, which decreased the stress stimuli during the formal experiment. On the test day, each animal received two trials and the order of pasting the stimuli on right or left was alternated. The data on mice were removed if they were not able to remove the dot within 120 s.

Pole test

A pole test was performed to evaluate bradykinesia in the mice. The mouse was positioned head up at the top of a vertical rough-surfaced pole (10 mm diameter, 58 cm height). The time to turn and reach the floor was recorded. Two days before testing, each mouse was trained to descend the pole. On the test day, mice were allowed to practice five times and then tested three times, with each trial lasting for a maximum of 3 min.

Immunohistochemistry

Seven mice from each group were randomly perfused with saline, followed by 4 % paraformaldehyde. The brain was removed and immersed in different concentrations of sucrose for dehydration. Brain slices (10 μm) were cut from the SN area (−2.54 to −3.88 mm posterior to bregma based on the Paxinos atlas) for 90 sequential sections. For immunohistochemistry, slices were treated with 1 % bovine serum (Serotec, UK) to block non-specific binding sites, and permeabilized with 0.3 % Triton X-100/1 % BSA-PBS for 30 min. The section was then incubated at 4 °C overnight with anti-tyrosine hydroxylase (1:1000, anti-TH, Millipore, Billericay, MA), anti-α-synuclein (1:1000, Cayman Chemicals Company, USA), anti-CD11b (1:1000, eBioscience, San Diego, CA), anti-p-NF-кB/p65 (1:1000, Cell Signaling Technology, Danvers, MA), anti-TNF-α (1:500, Peprotech, Rocky Hill, NJ), anti-iNOS (1:500, Enzo Life Sciences, USA), anti-Arginase-1 (1:500, eBioscience, San Diego, CA), anti-IL-10 (1:500, eBioscience, San Diego, CA), or anti-IL-12 (1:500, Bioscience, San Diego, CA). After washing, the slices were incubated with anti-rat, anti-mouse or anti-rabbit secondary antibodies conjugated with Alexa 488 (1:1000, Thermo Scientific, Rockford, IL) or Alexa 555 (1: 1000, Thermo Scientific, Rockford, IL) at room temperature for 2 h. The nucleus was stained by Hoechst 33,342 (1 μg/ml, Sigma-Aldrich, USA). Control sections were run following identical protocols, but omitting the primary antibodies. Immunoflourescent double-labeled images were taken using an Olympus microscope. The horizontal sequential three brain sections in SN region were taken for Stereo count of immunohistochemistry staining positive cells.

Images were taken from the same location in all animals. Quantification of positive staining was determined using Image-Pro® Plus Software (Media Cybernetics®, Silver Springs, MD). Quantitative data (3 sections × 7 mice/group) were analyzed in a blinded fashion.

Western blot analysis

Eight mice from each group were used for Western blot analysis. The substantia nigra of brains were homogenized on ice with a microcontent motor-operated tissue homogenizer (KIMBLEKONTES, USA) in ice-cold lysis buffer (1 × PBS, 1 % Nonidet P-40, 0.5 % sodium deoxycholate, and 0.1 % SDS, RIPA) supplemented with protease inhibitors. Lysates were centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatants were collected. Protein concentrations were determined by a Bradford protein assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE, and transferred onto a polyvinylidene fluoride filter (PVDF) membrane (Millipore). Membranes were blocked with 5 % non-fat milk, and incubated at 4 °C overnight with anti-TH (1:1000, Millipore, Billericay, MA), anti-α-synuclein (1:1000, Cayman Chemicals Company, USA), anti-p-NF-кB/p65 (1:1000, Cell Signaling Technology, Danvers, MA) and anti-GAPDH (1:1000, Epitomics, USA). Bands were visualized by HRP-conjugated secondary antibodies (1:1000, Thermo Scientific, Rockford, IL) and chemiluminescence (ECL) kit under ECL system (GE Healthcare Life Sciences, USA).

Statistical analysis

All quantitative data are presented as mean ± SEM. Statistical analysis was assessed with one-way ANOVA using GraphPad Prism 4 (Cabit Information Technology Co., Ltd.). Individual differences between two groups were determined using post hoc test following one-way ANOVA and P < 0.05 was considered to be statistically significant.

Results

LA treatment partially improved motor dysfunction

To validate the effect of LA in the PD model, the model was induced in C57BL/6 mice by a unilateral (right side) intranasal instillation with 10 μl of LPS for 1 month. The neuroprotective effect of LA was visualized in behavioral analysis of motor functions, i.e., the general locomotor activities of mice in three groups were monitored by an open-field test. As shown in Fig. 1, LPS-induced PD mice showed a significant reduction in ambulatory episodes, ambulatory counts and distance travelled compared with saline-administered normal control mice (Fig. 1a–c; all P < 0.05). While there was no significant improvement in dyskinesia in LA-treated PD mice, significant improvement was observed in the adhesive removal test and the pole test. In the adhesive removal test, a sensitive method for determining focal sensorimotor deficits in all groups of mice, the reaction time was significantly increased in the side treated with LPS (Fig. 1d, e, middle bars) compared to contralateral saline control (Fig. 1d, e, left bars) and saline control mice (P < 0.05). The administration of LA significantly reduced the reaction time as compared to LPS instillation alone (P < 0.05) (Fig. 1d, e, right bars).

Fig. 1
figure1

LA partially improved behavioral symptoms of PD mice. General locomotor activities were analyzed in an open-field test the first month after PD induction and LA treatment, including ambulatory episodes, VM = voluntary movement (a), ambulatory counts (b) and distance travelled (c) for a period of 30 min. In an adhesive removal test, the average (d) and total (e) time to remove the stimulus was recorded. For the pole test, the total time to reach the floor (f) and time to turn (g) was recorded. Results are shown as mean ± SEM of 15 mice each group from two pooled independent experiments. *P < 0.05

In the pole test, which is another sensitive method for determining nigrostriatal dysfunction, the LPS-induced PD mice showed bradykinesia, with the time to climb down (T-LA) and time to turn at the top (T-turn) increased to 12.5 s and 6.8 s compared with the control (9.6 s and 2.5 s). However, after LA treatment, the mice took about 10.1 s and 2.8 s to turn downward and to turn at the top, respectively (Fig. 1 f, g; all p < 0.05). Taken together, the administration of LA improved motor dysfunction in LPS-induced PD mice.

LA prevented dopaminergic neuronal loss and α-synuclein accumulation

The protective effects of LA on dopaminergic neurons and nigrostriatal innervating fibers were also investigated after LPS-induced lesions had developed. The numbers of nigral TH-positive neurons and α-synuclein-positive cells were counted in the SN area. It was evident that LPS instillation induced a significant decrease in the TH-positive dopaminergic neurons and aggregation of α-synuclein. Next, we asked whether LPS, in addition to morphological damage to dopaminergic cell bodies, also blights nigrostriatal innervating fibers in the striatum (ST). Optical density of TH-positive fiber in ST reflects striatal TH levels. The increase in striatal TH staining shows protection from cell death for TH positive cells. Results showed that the optical density of TH-positive fibers in the ST was obviously decreased on the LPS-administered side, and LA treatment reduced the loss of nigrostriatal dopaminergic neurons and the aggregation of α-synuclein in the SN and/or ST, compared with mice that had LPS instillation alone (Fig. 2 a, b). We further explored the levels of TH and α-synuclein protein in the SN of brain by Western blot. The results showed that, compared with nasal saline control and contralateral brain (left side), the level of TH protein was decreased by 38.8 % and 45.4 %, and the aggregation of α-synuclein protein was increased by 223.9 % and 206.5 % in LPS-PD mice (Fig. 2c, P < 0.05). However, after LA treatment, the reduction of TH protein and up-regulation of α-synuclein were obviously attenuated (Fig. 2c, P < 0.05). These results indicated that LA treatment effectively protected dopaminergic neurons from LPS-induced cell death.

Fig. 2
figure2

LA prevented dopaminergic neuronal loss and α-synuclein accumulation. After behavioral studies, animals were sacrificed and brains were harvested for histological studies. (a) Representative photomicrographs illustrating that LA prevented the loss of dopaminergic neurons and α-synuclein accumulation in the SN of mice challenged with LPS inhalation and LA administration; (b) Representative photomicrographs illustrating that LA preserved the nigrostriatal innervating fibers in the ST of mice challenged with LPS inhalation and LA administration; (c) Western blot analyses of TH and α-synuclein in protein extracts from the left and right sides of the brain in mice challenged with LPS inhalation and LA administration. Results are expressed as the ratio of tyrosine hydroxylase (TH)/α-synuclein to GAPDH, which was used as a loading control, and are shown as mean ± SEM of 7 mice (a and b) and 8 mice (c), each group from two pooled independent experiments. *P < 0.05; **P < 0.01

LA inhibited M1 inflammatory microglia

Microglia, the resident macrophages of the brain, is known to be a double-edged sword, with both neurotoxic and neurotrophic effects in the CNS, and microglia/macrophage can adapt different phenotypes (M1 and M2) depending on the microenvironment (Trudler et al. 2014). We thus determined the effect of LA on M1/M2 regulation in the CNS. As shown in Fig. 3a, intranasal LPS stimulated the expression of both M1 molecules (iNOS, CD16/32, IL-12, TNF-α) and M2 molecules (Arg-1, CD206 and IL-10) on CD11b+ cells. LA treatment significantly inhibited LPS-induced expression of all M1 molecules examined (Fig. 3a), but did not influence M2 molecules (Fig. 3b). These results demonstrate that LA could inhibit M1 microglia, but not affect M2 microglia.

Fig. 3
figure3figure3

LA significantly inhibited M1 microglia. Immunostaining in the substantia nigra (SN) of brain was quantified by counting double-positive cells. The number of cells in seven brains was counted and averaged (cells/mm2). a Representative photomicrographs and quantitative analysis illustrating that LA down-regulated expression of M1 molecules CD16/32, nitric oxide synthase (iNOS), IL-12 and TNF-α on microglia; b Representative photomicrographs and quantitative for M2 microglia phenotype. Results are shown as mean ± SEM of 7 mice each group from two pooled independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001

LA attenuated the activation of NF-κB in microglia

Given the accumulating evidence that the nuclear factor-κB (NF-κB) signaling pathway contributes to inflammatory responses, we determined the activity of this pathway on microglia in SN by immunohistochemistry staining and Western blot. As expected, LPS-induced PD mice exhibited a high level of p-NF-kB/p65 expression on microglia, and this expression was overtly inhibited after LA treatment (Fig. 4a). When p-NF-κB level in the SN of brain tissues was further measured by Western blot, we found a 1.7 times increase in LPS-PD mice compared to saline-treated normal mice, and this increase was significantly reversed after LA treatment, down to a level similar to that in the contralateral brain and the brain of saline-treated normal mice (Fig. 4b).

Fig. 4
figure4

LA attenuated the activation of NF-κB in microglia. Immunostaining in the substantia nigra (SN) of brain was quantified by counting p-NF-kB/CD11b double-positive cells. The number of cells in seven brain sections was counted and averaged (cells/mm2). a Representative photomicrographs and quantitative analysis illustrating that LA inhibited the activation of NF-κB in microglia; b Phosphorylation of NF-κB was measured by Western blot. Results are shown as mean ± SEM of 7–8 mice each group from two pooled independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001

Discussion

The major finding of this study is that administration of LA attenuated motor dysfunction in LPS-induced PD mice, and prevented dopaminergic neuronal loss and α-synuclein accumulation. Further observation showed that LA remarkably inhibited inflammatory M1 microglia, and attenuated the inflammatory response by inhibiting NF-кB activation in these cells. These results demonstrate that administration of LA could protect dopaminergic neurons from loss, possibly through inhibiting inflammatory M1 microglia and subsequent neuroinflammation in the CNS.

In the later stages of human PD, there are clear signs of microglial activation and inflammation that may contribute to the progression of the disease (Taetzsch and Block 2013). Recent work has shown how extrinsic oxidative stress, such as that created by inflammation, could result in neuronal death in cells with high cytosolic calcium levels, suggesting that glial cell activation and its inflammatory response may contribute to the progressive degeneration of dopaminergic neurons in PD. (Song et al. 2013) It has been previously reported that intra-nigral and intra-striatal administration of LPS induced symptoms of PD, dopaminergic neuron death, and microglial activation (He et al. 2013; Dutta et al. 2008; Liu and Bing 2011; Tanaka et al. 2013). We have recently demonstrated that i.n. LPS instillation caused mice to exhibit the basic characteristics of PD, including reduced motor activity, progressive and selective loss of dopaminergic neurons in the SN, reduced striatal dopamine levels, α-synuclein aggregation, and microglial activation in the SN (He et al. 2013). This PD model is thus ideal for investigating the role of oxidative stress and neuroinflammation in dopaminergic neurons and the neuroprotective effect of a medication, e.g., LA, which inhibits microglial activation and subsequent oxidative stress and neuroinflammation.

The density of microglia in the healthy brain is remarkably higher in the SN compared with other midbrain areas and brain regions such as the hippocampus (Bilska and Wlodek 2005). Microglia have both neurotoxic and neurotrophic effects and serve as key components of brain inflammatory response, exerting a deleterious role on dopaminergic neurons in PD. (Trudler et al. 2014) Type 1 macrophages/microglia (M1) produce inflammatory cytokines and oxidative metabolites to exacerbate tissue injury, thus exerting a deleterious effect on dopaminergic neurons in PD. Conversely, M2 macrophages/microglia down-regulate inflammation to promote CNS repair (Trudler et al. 2014). Inflammatory M1 microglia release inflammatory cytokines, which are reported to be elevated in post-mortem PD brains, suggesting the possible involvement of inflammatory mechanisms in the progression of PD. (McGeer et al. 1988) Given that inflammatory responses induced with neurotoxin may also be derived from damaged neurons (Trudler et al. 2014), protecting neurons from inflammation-induced damage will be, in turn, an important mechanism underlying the anti-inflammatory effect of LA. In addition, activated microglia produce large amounts of superoxide radicals, which may be the major source of the oxidative stress responsible for dopaminergic cell death that occurs in PD patients (McGeer and McGeer 2008). Recent evidence suggests that glial cell activation and its inflammatory response may contribute to the progressive degeneration of dopaminergic neurons in PD. (Tanaka et al. 2013).

LA has been identified as a cofactor for mitochondrial α-ketoacid dehydrogenases (Smith et al. 2004), which easily crosses the blood–brain barrier (BBB) because of its small molecular size and high water and lipid solubility (Goraca and Asłanowicz-Antkowiak 2009). A number of studies support its use in the ancillary treatment of many diseases, such as diabetes, cardiovascular, neurodegenerative, autoimmune diseases, cancer and AIDS (Khalili et al. 2014; Shinto et al. 2014; Sancheti et al. 2013a; Gębka et al. 2014; Padmalayam 2012; Yamasaki et al. 2014; Jariwalla et al. 2008; Gorąca et al. 2011). In addition to its anti-oxidative role, LA supplementation in diet modulates the inflammatory response by inhibiting IL-6 and TNF-α expression (Li et al. 2014). LA significantly inhibited LPS-induced production of nitric oxide and TNF-α via an attenuated activation of NF-κB and activator protein-1 (Kiemer et al. 2002). In a LPS-stimulated BV-2 cell model, LA activated Akt, inactivated Glycogen synthase kinase 3 beta (GSK-3β) and inhibited LPS-stimulated inflammatory responses by inactivating Phosphatidylinositol 3-kinase (PI3K) and GSK-3β (Koriyama et al. 2013a). In addition, it has been found that LA stimulates nuclear factor–like 2 (Nrf2) signaling and promotes expression of heme oxygenase-1 (HO-1), an important anti-oxidative pathway that is also immunomodulatory (Koriyama et al. 2013b). LA treatment also protects PC12 neuronal cells from toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Li et al. 2013), reverses the impaired synaptic plasticity in 3xTg-AD mice (Sancheti et al. 2013b), improves spatial learning and memory in rat model of vascular dementia (Zhao et al. 2015), and promotes functional recovery after stroke in rats (Choi et al. 2015). Our findings thus propose a link between anti-inflammatory activity of LA in the LPS-induced PD model and the anti-oxidative pathway, indicating its preventive and therapeutic potential in PD, AD and stroke by anti-oxidative and anti-inflammatory effects..

In conclusion, our results demonstrate for the first time the therapeutic potential of LA in LPS-induced PD mice, accompanied by the preservation of dopaminergic neurons and reduction of α-synuclein accumulation in the SN. Based on these observations, it is postulated that neuroprotective effect of LA on the LPS-induced PD model is possibly through inhibition of inflammatory M1 microglia and the NF-κB inflammatory pathway, further indicating a therapeutic strategy for treating neurodegenerative diseases with an inflammatory component.

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Acknowledgment

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81,272,163, 81,070,957 and 81,371,414), the Natural Science Foundation of Shanxi (2,012,021,034–2), the Department of Science and Technology, Shanxi Province of China (No. 2,013,081,058) and the Shanxi University of Traditional Chinese Medicine (No. 2011PY-1). We thank Katherine Regan, Department of Neurology, Thomas Jefferson University, Philadelphia, USA, for editorial assistance.

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Correspondence to Bao-guo Xiao or Cun-gen Ma.

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Yan-Hua Li and Qing He contributed equally to this work.

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Li, YH., He, Q., Yu, Jz. et al. Lipoic acid protects dopaminergic neurons in LPS-induced Parkinson’s disease model. Metab Brain Dis 30, 1217–1226 (2015). https://doi.org/10.1007/s11011-015-9698-5

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Keywords

  • LPS-induced PD model
  • Lipoic acid
  • Microglia