Aspirin attenuates L-DOPA-induced dyskinesia in hemi-parkinsonian rats

Background: At present, L-DOPA remained the gold standard therapy for motor symptoms of Parkinson’s disease (PD) patients. However, prolonged administration of L-DOPA led to the development of dyskinesia. Aspirin, a non-steroidal anti-inflammatory drug (NSAID), had been widely used to relieve pain and inflammation. Recent studies indicated aspirin produced neuroprotection against dopamine (DA) neuronal loss in animal model. This study aimed to explore the effects of aspirin pre-treatment and co-treatment with L-DOPA on DA neurotoxicity and L-DOPA-induced dyskinesia (LID) as well. Methods: Rats received a single 6-hydroxydopamine (6-OHDA) injection into substantia nigra to induce DA neuronal loss. For aspirin pre-treatment before L-DOPA studies: One day after 6-OHDA stimulation, rats were daily administrated with aspirin for 3 weeks followed by daily L-DOPA treatment along with aspirin for additional 3 weeks. For aspirin co-treatment with L-DOPA studies: Three weeks after 6-OHDA administration, rats were daily treated by L-DOPA together with aspirin for another 3 weeks. DA neurotoxicity was analyzed via rat PD-like behavior test and DA neuronal counting. The movement disorders of dyskinesia triggered by L-DOPA were determined by the abnormal involuntary movements (AIM) scores analysis. Results: we demonstrated both aspirin pre-treatment and co-treatment exerted anti-LID effects during L-DOPA treatment on 6-OHDA-lesioned rats. In addition, aspirin not only ameliorated DA neuronal damage, but also reduced the development of dyskinesia without affecting L-DOPA efficacy. Furtherly, inhibition of glial cells activation and the subsequent neuroinflammatory response might be involved in aspirin-attenuated dyskinesia. Conclusion: the present study suggested aspirin could have beneficial potential to attenuate LID in PD. the PD and additional studies are needed to prove the confirmed anti-dyskinetic effects of ASA in other animal models and even PD patients with LID. This study demonstrated ASA protected from DA neuronal damage and attenuated LID without affecting L-DOPA efficacy. These findings suggest ASA might be a potential alternative for attenuating LID in PD.

Treatment with the DA precursor, L-3, 4-dihydroxyphenylalanine (L-DOPA), is the most effective symptomatic therapeutic choice for PD patients [3]. However, prolonged administration of L-DOPA leads to the development of abnormal involuntary movements (AIM), known as L-DOPA-induced dyskinesia (LID) [4]. Dyskinesia is unfavorable for the quality of life, sometimes being more disabling than PD itself. Once dyskinesia appears, L-DOPA had little effects on PD symptoms, therefore remaining a significant challenge to mitigate several non-motor and motor impairment [5]. Therefore, strategies of promising synergistic effects in combination with L-DOPA might present a novel adjuvant therapy for PD.
Aspirin known as Acetylsalicylic Acid (ASA), a non-steroidal anti-inflammatory drug (NSAID), is widely used in 20th century to relieve pain and inflammation [6]. Recently, this oldest agent in medicine has been considered to be a potential new therapy for a range of neuropsychiatric disorders. A battery of studies confirmed that ASA had beneficial effects on mood disorders and schizophrenia. Also, ASA is associated with a reduced risk of Alzheimer's disease (AD) and PD [7,8]. In addition, ASA produced neuroprotection against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced DA neuronal loss [9]. This information might open the prospect of ASA for neurological disorders treatment.
The current study aimed to investigate the effects of ASA pre-treatment and co-treatment with L-DOPA on DA neurotoxicity and L-DOPA-induced dyskinesia as well. Rats lesioned with 6hydroxydopamine (6-OHDA) in SN were employed and treated with L-DOPA and ASA. DA neurotoxicity was analyzed via rat PD-like behavior test and DA neuronal counting. The movement disorders elicited by L-DOPA were determined by the AIM scores analysis. Especially, these findings might provide a more potential synergistic therapeutic strategy for PD.

Rotarod Test
Rotarod test was performed to study the muscular coordination. Rats were permitted to retain stationary for a while at 0 rpm. Then, the increasing speed from 10 rpm to 30 rpm over a 300-s period until animals fell off from rungs [11]. Rat behavior changes were detected and the mean duration time stayed on rod was recorded.

AIM Scores Analysis
To assess LID-like behavioral manifestations, AIM scores were employed to assess the properties of L-DOPA and ASA on rat behavior changes at 3 time points (7, 14 and 21 days after 6-OHDA treatment).
This scoring system was considered to be comparable to reflect human dyskinetic behavior [12,13].
Rat dyskinetic behaviors were quantified based on frequency during a 1 min-monitoring period within 4 different 30-min blocks for a total of 120 min. Next, the total AIM score corresponded to the sum of the individual scores for 3 AIM subtypes (axial, limb and orolingual) were summed [14]. For each subtype, the dyskinesia severity was scored by a 4-point scale (0: absent; 1: present less than 50% of the time; 2: present more than 50% of the time; 3: continuous but interrupted by strong sensory stimuli; and 4: continuous, with no interruption by strong sensory stimuli).

Forepaw Adjusting Steps (FAS) Test
The FAS test was applied to detect akinesia, a cardinal symptom of PD. Rats with unilateral DA depletion performed poorly stepping ability on the FAS test with the lesioned side of the body [15]. L-DOPA reversed this stepping deficit and thus FAS test could be used to determine whether any adjunctive treatment affected the efficacy of L-DOPA-mediated anti-parkinsonian [16]. For this test, experimenters were blinded to treatment condition, both hind legs and one forepaw were held, such that rat was bearing its weight on the forepaw to be tested. Rats were moved through the table at a speed of 90 cm/10 s and the number of adjusting steps taken on the weight-bearing forepaw was recorded. Rats were dragged for 6 trials per forelimb: 3 backhand and 3 forehand trials. Data were represented as the sum of the 3 trials per direction for each forepaw. The stepping results were shown as % intact stepping (lesioned steps/ intact steps). The lower % intact stepping score indicated the greater forelimb akinesia [17].
Immunohistochemistry Staining DA neurons were identified with anti-TH antibody, while microglia and astroglia were recognized with anti-IBA-1 and GFAP antibodies, respectively. Rats were sacrificed and perfused with PBS and 4% paraformaldehyde. Then, brains were fixed with 4% paraformaldehyde, and dehydrated with 30% sucrose solution until the brain sunk to the bottom at 4 °C. Rat brains were cut into 35 µm-transverse free-floating sections by freezing microtome. The section was stained with primary antibodies targeting TH antibody (1:800), IBA-1(1:1000) and GFAP (1:1000), respectively, followed by treatment with secondary antibodies. Images were acquired through an Olympus microscope with an attached Polaroid digital microscope camera (Polaroid®, Cambridge, MA, USA). Quantification of TH-positive neuronal cell bodies was performed blindly by two investigators. Then, the mean value for SN THpositive neuronal numbers was deduced by averaging the counts of 6 sections for each brain.

Western Blot Analysis
Brain tissues were homogenated in cold PBS and lysed in a radio immunoprecipitation (RIPA) lysis buffer. Subsequently, the lysate was centrifuged at 12,000 × g for 15 min at 4˚C. The protein concentrations were quantified using bicinchoninic acid (BCA) protein assay. The protein samples (10 µg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane [18]. Then, the membrane were blocked via 5% non-fat Milk However, pre-treatment of ASA attenuated L-DOPA-induced AIM scores aggravation. Similarly, cotreatment of ASA with L-DOPA reduced L-DOPA-increased these total and subtypes AIM scores (Fig. 2).
Collectively, these results suggested that both ASA pre-treatment and co-treatment with L-DOPA Consistent with DA neuronal counting analysis, similar results were indicated in TH protein detection (Fig. 3B).
For ASA co-treatment with L-DOPA, as shown in Fig. 4A

ASA Modulated Glial Cells Activation during L-DOPA-Mediated Anti-Parkinsonian Effects
The effects of ASA on glial cells activation in L-DOPA treatment against 6-OHDA-elicited DA neuronal damage were analyzed by immunofluorescence staining and western blot assay. As shown in Fig. 6, seven days after L-DOPA and ASA co-treatment (relative to 28 days after 6-OHDA administration), compared with the control group, 6-OHDA induced microglia and astroglia activation evidenced by the increased number of IBA-1-positive microglia and GFAP-positive astroglia and protein expressions of IBA-1 and GFAP. However, ASA alone, L-DOPA alone and ASA co-treatment with L-DOPA had no significant effects on glial cells activation. Twenty-one days after treatment (relative to 42 days after 6-OHDA application), ASA and ASA + L-DOPA attenuated 6-OHDA-induced astroglia activation, whereas no significant effects were shown after L-DOPA alone treatment. Moreover, ASA combined with L-DOPA inhibited IBA-1 protein expression although ASA or L-DOPA alone had no obvious inhibitory effects on microglial activation.
Next, pro-inflammatory mediators protein expressions in rat midbrain were detected. As shown in

Discussion
The present study demonstrated both aspirin pre-treatment and co-treatment exerted anti-LID effects during L-DOPA treatment on 6-OHDA-lesioned rats. In addition, aspirin not only ameliorated DA neuronal damage, but also reduced the development of dyskinesia without affecting L-DOPA efficacy.
Furtherly, inhibition of glial cells activation and the subsequent neuroinflammatory response might be involved in aspirin-attenuated dyskinesia.
L-DOPA has long been the standard therapy for treating symptoms of PD via the supplement of exogenous dopamine, which reverses the distinctive behavioral manifestations of PD. However, the eventual onset of motor fluctuations and LID complicates its utility in advanced PD [19][20][21]. In recent decades, various therapies have been developed for the treatment of LID. Nevertheless, PD patients remain disabled by the effects of LID. Amantadine is the only compound with anti-dyskinetic effects in PD patients, but several lines of evidence have demonstrated Amantadine produced short duration anti-dyskinetic effects [22]. Therefore, unveiling strategies to ameliorate LID still maintains a critical clinical hurdle [23]. ASA has been verified to exert neuroprotective effects in MPTP-and 6-OHDA-induced animal model against DA neuronal loss [24][25][26]. Therefore, ASA-mediated neuroprotection attracts an increasing attention. In this study, pre-treatment and co-treatment of ASA with L-DOPA attenuated L-DOPA-triggered dyskinesia in 6-OHDA-lesioned rats. Furthermore, the present study showed that L-DOPA attenuated motor symptoms of PD animal model rather than protecting DA neurons against 6-OHDA-indueced neurotoxicity. These findings confirmed the critical limitations of L-DOPA since its effects were merely symptomatic and not as fundamental. In addition, one such limitation concerned that L-DOPA might accelerate DA neuronal loss during the progression of PD [27]. Moreover, pre-treatment and co-treatment of ASA with L-DOPA still generated DA neuroprotection and ameliorated LID without affecting the therapeutic actions of L-DOPA, suggesting that ASA might be repurposed as an adjunct treatment to mitigate LID in PD therapy.
The mechanisms underlying ASA-mediated neuroprotection were controversial, but appeared to include the inhibition of neuroinflammation. Neuroinflammation has been considered to contribute not only to PD progression, but also to development of LID [28,29]. Substantia nigra is rich in microglia and astroglia, which are the main actors in neuroinflammatory responses, and their double role at the interface between immune and neurophysiological responses was widely studied [30]. Previous studies demonstrated that ASA down-regulated the inflammatory condition in activated microglia induced by lipopolysaccharide (LPS) [31]. In the present study, co-administration of ASA and L-DOPA

Consent for publication
Not applicable.

Availability of data and materials
All data mentioned in this article are available from the corresponding author on reasonable request. Figure 1 lesioned rats. Rats received a unilateral 6-OHDA (8 µg) injection to SN pars compacta on right side of rat brain. One day later, rats were daily administrated with ASA (10 mg/kg, p.o.) for 3 weeks followed by daily treatment of L-DOPA (25 mg/kg, i.p.) for additional 3 weeks.
Experimental design for 6-OHDA lesion, drug treatment and behavioral tests was shown (A).
single unilateral injection of 6-OHDA (8 µg) into rat SN pars compacta on right side of brain.

Figure 3
The effects of ASA pre-treatment before L-DOPA on 6-OHDA-induced DA neuronal loss.
were collected, respectively. Rat SN DA neurons in brain sections were immunostained and recognized with an anti-TH antibody and DA neuronal loss in the SN was analyzed via the quantification of TH-positive neurons (A). The "ellipse" presented the area of SN. Scale bar = 100 µm. The protein level of TH was determined by western blotting. The ratio of densitometry values of TH with β-actin was assessed and normalized to each respective control group (B). Data were mean ± SEM from 6 rats. *p<0.05 compared with the control group; #p< 0.05 compared with 6-OHDA group.

Figure 4
The effects of ASA co-treatment with L-DOPA on 6-OHDA-induced neurotoxicity. After L-DOPA and ASA co-treatment for 7 and 21 days (relative to 28 and 42 days after single 6-OHDA injection), respectively, DA neuronal lesion in SN was analyzed via the quantification of TH-positive neurons after immnunostaining with an anti-TH antibody (A and C). The "ellipse" presented the area of SN. Scale bar = 100 µm. TH protein level was determined by western blot assay. The ratio of densitometry values of TH with β-actin was assessed and normalized to each respective control group (B and D). Data were mean ± SEM from 6 rats. *p<0.05 compared with the control group; #p<0.05 compared with 6-OHDA group.  The number analysis of SN IBA-1-positive microglia and GFAP-positive astroglia from 6 evenly spaced brain sections of each rat was performed. In addition, rat midbrains were collected to detect the protein expressions of GFAP and IBA-1 28 and 42 days after 6-OHDA treatment via western blotting. The ratio of densitometry values of GFAP and IBA-1 with βactin was assessed and normalized to each respective control group. Data were mean ± SEM from 6 rats. *p<0.05 compared with the control group; #p< 0.05 compared with 6-OHDA group.