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Levodopa (L-DOPA) is the gold standard for symptomatic treatment of Parkinson's disease (PD); however, long-term therapy is associated with the emergence of L-DOPA-induced dyskinesia (LID). Nigral dopaminergic cell loss determines the degree of drug exposure and time required for the initial onset of LID. Accumulating evidence indicates that α-lipoic acid (ALA) decreases this nigral dopaminergic cell loss. However, until now, the precise mechanisms of ALA have only been partially understood in LID. Chronic L-DOPA treatment was demonstrated to develop intense AIM scores to assess dyskinetic symptoms. Rats in the LID group were administrated twice daily with L-DOPA + benserazide for 3 weeks to induce a rat model of dyskinesia. Moreover, other 6-OHDA-lesioned rats were treatment with ALA (31.5 mg/kg or 63 mg/kg) in combination with L-DOPA treatment. Furthermore, the authors investigated the level of malondialdehyde (MDA) and glutathione (GSH) activity, as well as IBa-1, caspase-3 and poly (ADP-ribose) polymerase (PARP) in substantia nigra by the way of western blotting and immunofluorescence. ALA reduced LID in a dose-dependent manner without compromising the anti-PD effect of L-DOPA. Moreover, ALA reduced the level of MDA and upregulated the GSH activity, as well as ameliorated IBa-1 positive neurons in the substantia nigra. Finally, it was identified that ALA could reduce L-DOPA-induced cleaved-caspase-3 and PARP overexpression in the substantia nigra. Based on the present findings, ALA could be recommended as a promising disease-modifying therapy when administered with L-DOPA early in the course of PD. The exact mechanism for this action, although incompletely understood, appears to relate to anti-oxidative stress and anti-apoptosis.
Levodopa (L-DOPA), the precursor of dopamine (DA), has provided obvious effective treatment for Parkinson's disease (PD) by replacing DA neurotransmission following the death of substantia nigra neurons (
Together with previous findings in LID models, this led to the overall impression that the development of LID is caused by a complex interaction of both pre- and postsynaptic changes, taking place not only in the DA system, but also involving a variety of other mechanisms (
α-lipoic acid (ALA), is an antioxidant naturally synthesized in human body with potential therapeutic value against a range of pathophysiological insults (
Experiments were conducted on 48 female Sprague-Dawley (SD) rats (age, 3–4 months; weight, 180–220 g), which were purchased from the Experimental Animal Center of China Medical University (Beijing, China). Upon their arrival, the animals were housed in clean cages with a maximum of four rats per cage under a 12 h light:12 h dark cycle, relative humidity of 55±10% and temperature 22.0±2.0°C. Animals had unrestricted access to standard chow and water, which were supplemented daily, and animal care was supervised by skilled veterinarians in the health care center (Medical School of Shanghai Jiaotong University, Shanghai, China). All experimental protocols involving the animals were reviewed and approved by the Ethical Committee of the Medical School of Shanghai Jiaotong University (Shanghai, China). Efforts were made to reduce to a minimum the number of animals required for statistically valid analyses and to minimize their suffering. The methods were carried out in accordance with the approved guidelines and regulations of the National Institutes of Health for the Care and Use of Laboratory Animals (Bethesda, MD, USA).
6-OHDA-lesioned PD rats were induced by the methods mentioned above (
Validated PD rats received vehicle or levodopa injection for 21 d. Apomorphine hydrochloride (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was administered (0.5 mg/kg). L-DOPA (Sigma-Aldrich; Merck KGaA, 25 mg/kg) with a fixed dose of the peripheral DOPA-decarboxylase inhibitor benserazide (Sigma-Aldrich; Merck KGaA, 6.25 mg/kg) were administered twice-daily (9:00 and 16:00). ALA was dissolved in normal saline and was administered i.p. (ALA-L group, 31.5 mg/kg; ALA-H group, 63 mg/kg, respectively) 30 min prior to L-DOPA intake for 3 weeks.
On testing days, rats were placed individually in transparent plastic cages 10 min prior to drug treatment. As described previously (
To assess the enzymatic activity of GSH and lipid peroxide in striatum, the tissues were homogenized in 0.1 mol/l PBS containing 0.05 mmol/l EDTA. The homogenate was centrifuged at 12,000 × g for 15 min at 4°C. The supernatants were kept for the measurement. Total GSH was assayed by 5,5-dithiobis (2-nitrobenzoic) acid (DTNB)-GSSG reductase recycling. GSSG was obtained by determining the absorbance of 5-thio-2-nitrobenzoic acid produced from the reaction of the reduced GSH with DTNB according to the manufacturer's protocols. The reduced GSH was obtained by subtracting GSSG from the total GSH. Absorbance was determined at 412 nm by using the microplate reader. GSH activity was assessed with GSH Assay kit (Beyotime Institute of Biotechnology, Haimen, China) by the mays of GSH/GSSG. The level of MDA, a product of lipid peroxidation, was measured with MDA Assay kit (Beyotime Institute of Biotechnology) based on modified thiobarbituric acid method.
IFC was carried out in free-floating sections using a standard avidin-biotin immunocytochemical protocol. Rats were rapidly anesthetized with 10% chloral hydrate (350 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde. Whole brains were post-fixed overnight in the 4% paraformaldehyde, stored at 4°C and then stored in a solution containing 30% sucrose. Sections (30 µm) were cut with a slicing machine and blocked for 10 min at room temperature in 5% normal donkey serum (Beyotime Institute of Biotechnology), and then incubated overnight at 4°C in the primary antibody solution (monoclonal rabbit anti-IBA; 1:200; cat. no. ab178680; Abcam, Cambridge, UK). Sections were rinsed in PBS and incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (cat. no. A0453; 1:200; Beyotime Institute of Biotechnology) for 1 h at room temperature. Subsequently, sections were again rinsed in PBS, mounted on slides, cover slipped, and examined with confocal microscopy. Digitized images were analyzed for distribution of immunoreactive cells in the lesioned hemisphere striatum and substantia nigra of rats.
Striatal tissues and substantia nigra were homogenized in 20 mM Tris-HCl (pH 7.4), containing 1 mM NaF, 150 mM NaCl, 1% Triton X-100 and freshly-added protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), and 100 µM phenylmethylsulfonyl fluoride. Cytosols were prepared by centrifugation at 12,000 × g for 10 min at 4°C. Proteins were separated by SDS-PAGE electrophoresis, using different percentages of gels based on the different protein weights (range, 6–12%) and transferred overnight to polyvinylidene difluoride membranes. Then, the membrane was incubated with polyclonal rabbit anti-caspase-3 (cat. no. 9661S; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and polyclonal rabbit anti-poly (ADP-ribose) polymerase (PARP; cat. no. 9542S; 1:1,000; Cell Signaling Technology) overnight at 4°C, respectively, and then incubated in horseradish peroxidase conjugated secondary anti-rabbit β-actin IgG (cat. no. A0208; 1:1,000; Beyotime Institute of Biotechnology). The signal was visualized by enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA) and quantified using Quantity One software (Image Lab).
The scores assigned for AIM and parkinsonian disability were non-parametric and were analyzed using a Kruskal Wallis followed by Dunn's test for multiple comparisons in the case of comparing data over multiple days. The western blot analysis and IHC conformed to normal distribution were performed using a one-way analysis of variance (ANOVA) followed by LSD post-hoc comparisons when appropriate as indicated in the figure legends. P<0.05 was considered to indicate a statistically significant difference. All analyses were conducted using SPSS software (version, 16.0; SPSS Inc., Chicago, IL, USA).
A total of 48 SD rats were unilaterally injected with 6-OHDA in the MFB (n=12 per group). The anti-dyskinetic potential of ALA against LID was evaluated at two different doses (31.5 and 63 mg/kg). As can be observed in
Following this, the authors sought to determine whether ALA improvement of LID without ablation of the therapeutic response to L-DOPA. The authors observed that PD rats treated with L-DOPA prefer to use the contralateral forelimb to touch the inner wall of the cylinder compared with the PD group (P<0.05;
In the current study, the MDA level as a measure of lipid peroxidation was remarkably increased in the LID group compared with the PD group (P<0.05;
IBa-1 is a protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells (
L-DOPA is the most successful approach to manage motor symptoms in PD patients. However, the emergence of LID with long-term use is a severe challenge for PD. The present study has demonstrated that chronic challenges of ALA (31.5 and 63 mg/kg) in combination with L-DOPA significantly alleviates dyskinesia. The major findings from this study were: (
Oxidative stress is a central event in a range of pathological conditions. Such a pathway appears to underlie the pathological processes of PD, in which the inhibition of the mitochondrial complex I elicited by the neuron toxicant increases the formation of ROS that cause the mitochondrial dysfunction finally result in PD occur (
Another related issue in the exploration of microglial activation phases is the reliance on Iba-1 immunoreactivity to report on their activation state (
Based on the present findings, ALA could be recommended as a promising disease-modifying therapy when administered with L-DOPA early in the course of PD. The exact mechanism for this action, although incompletely understood, appears to relate to anti-oxidative stress and anti-apoptosis.
The present study was supported by the Projects of National Science Foundation of China (grant nos. 81171203, 81471148, 81400925, 81171204 and 81200871), and the Projects of the Shanghai Committee of Science and Technology (grant nos. 11nm0503300 and 12XD1403800).
Time course of ALO AIMs development in 6-OHDA-lesioned rats during a 3 week treatment period with L-DOPA, ALA-L and ALA-H, respectively. For each day, a total AIM score was calculated as the sum of the basic score multiplied by the amplitude score for each AIM subtype. (A) global ALO AIM score (including ALO AIM score); (B) total AIM score (including ALO AIM score and locomotive score); (C) orolingual AIM score; (D) limb AIM score; (E) axial AIM score and (F) orolingual AIM score. Data are presented as mean ± standard deviation. *P<0.05 vs. LID group; #P<0.05 vs. ALA-L group (Kruskal Wallis followed by Dunn's test for multiple comparisons or a Mann-Whitney U test). ALO, axial, limb and orolingual; AIM, abnormal involuntary movements; L-DOPA, levodopa; ALA, α-lipoic acid; LID, L-DOPA-induced dyskinesia.
Unilaterally lesioned rats were tested for the percentage of impaired forelimb use compared with the total numbers. Animals were injected with either saline, L-DOPA + benserazide, ALA-L and ALA-H, respectively. The effective ALA dose did not interfere with the therapeutic motor effects of L-DOPA. Experiment consisted of three different sessions, namely 5, 13 and 20 days in the treatment period. Data are presented as mean ± standard deviation (n=8). *P<0.05 vs. LID group. L-DOPA, levodopa; ALA, α-lipoic acid; LID, L-DOPA-induced dyskinesia; PD, Parkinson's disease.
Effects of ALA on MDA and GSH levels. (A) MDA level as a measure of lipid peroxidation; (B) GSH level in striatum; (C) GSH/GSSG level in striatum. Data are presented as mean ± standard error of the mean. *P<0.05, **P<0.01 vs. LID group; #P<0.05 vs. ALA-L group, n=4 per group (one-way analysis of variance followed by least significant difference post-hoc analysis). ALA, α-lipoic acid; MDA, malondialdehyde; GSH, reduced glutathione; GSSG, oxidized glutathione; LID, levodopa-induced dyskinesia.
Representative examples of IBa-1 immunofluorescence studies in 6-OHDA-lesioned rats treated with L-DOPA, ALA-L or ALA-H, respectively. In 6-OHDA lesioned rats treated with L-DOPA induced approximately increase in IBa-1-positive neurons compared to other two group. These L-DOPA-induced increases in positive neurons decline in the ALA group. Shown is a coronal section of the striatum from the lesioned hemisphere. *P<0.05 vs. LID rats (one-way analysis of variance followed by least significant difference post-hoc analysis). Scale bar=200 µm. L-DOPA, levodopa; ALA, α-lipoic acid; LID, L-DOPA-induced dyskinesia.
Protein levels were evaluated by western blotting of proteins extracted from the ipsilateral striatum of the rat brain. They were assessed in extracts from 6-OHDA-lesioned rats treated with vehicle, levodopa, ALA-L and ALA-H. (A) Representative image and quantification of (B) pro-caspase-3, and (C) cleaved-caspase-3 level relative to actin level. (D) Representative image and quantification of (E) PARP, and (F) cleaved-PARP relative to actin level. (G) Representative image and (H) quantification of TH level relative to actin level. The data represent the mean of relative optical density ± standard deviation; *P<0.05, **P<0.01 vs. 6-OHDA group; #P<0.05, ##P<0.01 vs. LID group (one-way analysis of variance followed by least significant difference post-hoc analysis). LID, levodopa-induced dyskinesia; ALA, α-lipoic acid; TH, tyrosine hydroxylase; PARP, poly (ADP-ribose) polymerase.