ONO-2506 Can Delay Levodopa-induced Dyskinesia in the Early Stage

— Background : Levodopa-induced dyskinesia (LID) is a common motor complication of levodopa (L-DOPA) treatment for Parkinson’s disease (PD). In recent years, the role of astrocytes in LID has increasingly attracted attention. Objective : To explore the eﬀect of an astrocyte regulator (ONO-2506) on LID in a rat model and the potential underlying physiological mechanism. Methods : Unilateral LID rat models, established by admin-istering 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle through stereotactic injection, were injected with ONO-2506 or saline into the striatum through brain catheterization and were administered L-DOPA to induce LID. Through a series of behavioral experiments, LID performance was observed. Relevant indicators were assessed through biochemical experiments. Results : In the LID model of 6-OHDA rats, ONO-2506 signiﬁcantly delayed the development and reduced the degree of abnormal involuntary movement in the early stage of L-DOPA treatment and increased glial ﬁbrillary acidic protein and glutamate transporter 1 (GLT-1) expression in the striatum compared to saline. However, there was no signiﬁcant diﬀerence in the improvement in motor function between the ONO-2506 and saline groups. Conclusions : ONO-2506 delays the emergence of L-DOPA-induced abnormal involuntary movements in the early stage of L-DOPA administration, without aﬀecting the anti-PD eﬀect of L-DOPA. The delaying eﬀect of ONO-2506 on LID may be linked to the increased expression of GLT-1 in the rat striatum. Interventions targeting astrocytes and glutamate transporters are potential therapeutic strategies to delay the development of LID. (cid:1) 2023 The Author(s). Published by Elsevier Ltd on behalf of IBRO. This


INTRODUCTION
Parkinson's disease (PD) is a common neurodegenerative disease. Levodopa (L-DOPA) therapy is the gold standard for PD treatment, but longterm chronic treatment often leads to motor complications, and the therapeutic effect loses efficacy over time. Levodopa-induced dyskinesia (LID) is a common motor complication of L-DOPA treatment in patients with PD, which severely reduces the effectiveness of the treatment and adversely affects patients' daily activities (Iravani and Jenner, 2011). The exact mechanism underlying the occurrence and development of LID remains unclear, while the treatments for LID are limited. Thus, alleviating dyskinesia continues to be a major challenge in L-DOPA treatment for patients with PD. Therefore, exploring the pathogenesis of LID will significantly support the treatment and prevention of LID.
Astrocytes play a key role in the development of LID. In the striatum of LID model rats, astrocyte activity is significantly up-regulated, and the cell body of astrocytes is enlarged (Bortolanza et al., 2015a(Bortolanza et al., , 2015bMulas et al., 2016). Moreover, pro-inflammatory factors produced by activated astrocytes are closely associated with LID (Dos et al., 2020;Bortolanza et al., 2021). Activated astrocytes express more inducible nitric oxide synthase (iNOS) and produce high levels of nitric oxide, which may kill neurons under pathological conditions and may lead to excessive production of toxic substances in LID (Mander et al., 2005;Del-Bel et al., 2016). Additionally, LID is associated with the increase in vascular endothelial growth factor (VEGF) A, which is heavily expressed in astrocytes; histological evidence of angiogenesis in LID rat models and human patients have been reported (Ohlin et al., 2011). After L-DOPA treatment, regional cerebral blood flow in the motor cortex, striatum, and deeper basal ganglia nuclei increases temporarily and significantly. This increase is accompanied by a change in blood-brain barrier permeability (Ohlin et al., 2012). This maladaptive glial vascular response is con- ducive to the fluctuation of dopamine levels, which is related to the development of LID (Cenci, 2014a). In our previous study, voxel-based morphometry analysis showed that, compared with PD model rats, LID model rats had a significantly increased dorsal lateral volume of the striatum on the injured side . Astrocyte proliferation may be related to the increase in the striatal gray matter volume induced by L-DOPA in LID model rats. Therefore, astrocytes play a role in the occurrence of LID, but the specific underlying mechanism is unclear.
PD and LID may involve excessive glutamate release, while development of LID is associated with glutamatergic imbalance caused by L-DOPA treatment (Cenci, 2014b;Sebastianutto and Cenci, 2018;Pourmirbabaei et al., 2019). After chronic L-DOPA treatment in individuals with PD, transmission within the glutamate signaling pathway adaptively changes from the cortex to the striatum, resulting in excessive release of glutamate from the presynaptic end. This causes abnormal activity and distribution of N-methyl-D-aspartate (NMDA) receptors in medium spiny neurons (MSNs) and promotes the occurrence and development of LID (Mellone and Gardoni, 2018). Glutamate receptor antagonists, particularly NMDA receptor antagonists, have been shown to inhibit LID in PD animal models (Papa and Chase, 1996;Blanchet et al., 1998); however, they can also produce serious side effects (Tanaka, 2005). In addition to modulating glutamate receptor antagonists, clearing glutamate from synaptic spaces is an alternative method for reducing glutamatergic transmission (Anderson and Swanson, 2000;Rothstein et al., 2005). Reduction of glutamate efflux from the striatum or substantia nigra can block the occurrence of LID (Dupre et al., 2011;Paolone et al., 2015;Carta and Cenci, 2016;Garcia-Esparcia et al., 2018). Glutamate transporter 1 (GLT-1), one of the most abundant proteins ubiquitously expressed in the brain, is responsible for $90% of glutamate uptake (Takahashi et al., 2015). EAAT2 is the human homologue of GLT-1, and GLT-1 is mainly expressed in astrocytes (Rothstein et al., 1994;Maragakis et al., 2004). This transporter is crucial for maintaining extracellular glutamate concentrations below excitotoxic levels (Rothstein et al., 1995). In the 6-OHDA-induced PD rat model, increased GLT-1 expression contributes to glutamate uptake and clearance at stri-atal synapses and reduces the severity of LID (Kelsey and Neville, 2014;Chotibut et al., 2017).
Arundic acid ((2R)-2-propionoic acid, AA), also known as ONO-2506, is an astrocyte regulator that can specifically inhibit S100b protein in astrocytes (Asano et al., 2005), and has been widely used in the study of astrocyte function. ONO-2506 does not cause neuronal death, but in cultured astrocytes, it inhibits the changes caused by injury, such as increased S100b protein, secretion of nerve growth factor, disappearance of gammaaminobutyric acid (GABA) receptors, and decreased expression of GLT-1 and glutamateand aspartatetransporter (GLAST) (Pen˜a-Ortega et al., 2016). In an Alzheimer's disease mouse model, ONO-2506 treatment reduced S100b levels and significantly improved bamyloid plaque-associated reactive gliosis (Mori et al., 2006). In astrocyte cultures, ONO-2506 activated intracellular signaling proteins, such as those in the ERK, Akt, and NF-jB pathways, and upregulated astrocyte EAAT1/GLAST (Karki et al., 2018). In a murine glaucoma model, ONO-2506 increased the uptake of glutamate by Mu¨ller cells (retinal glial cells) through the up-regulation of EAAT1/GLAST and prevented the death of retinal ganglion cells (Yanagisawa et al., 2015). In a rat cerebral ischemia model, ONO-2506 increased glial EAAT2/GLT-1 activity after permanent middle cerebral artery occlusion (pMCAO) and inhibited the increase in glutamate concentration in the peri-infarct area after pMCAO (Mori et al., 2004). In an epileptic mouse model, ONO-2506 increased the extracellular levels of inhibitory transmitters and GABA in a dose-dependent manner and reduced K +induced L-glutamate release (Yamamura et al., 2013). The protective mechanism of ONO-2506, as an astrocyte regulator, in reducing glutamate levels has been widely confirmed in other fields of neurology, but there is no relevant research in the LID model. We speculated that ONO-2506 would reduce the severity of LID by regulating astrocyte function, increasing GLT-1 expression, and accelerating glutamate uptake.
Therefore, this study explores the effect of the astrocyte regulator ONO-2506 on LID in a rat model and investigate its potential underlying physiological mechanism to better understand the role of astrocytes in LID.

Animals
Forty 6-8-week-old male Sprague-Dawley (SD) rats, with a body weight of 210-230 g, were purchased from Beijing Spafford Biotechnology Co. ltd. (Beijing, China). All rats were raised in a specific pathogen-free (SPF) environment in the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (temperature: 21-23°C, humidity: 55-65%, 12-h day/night cycle, ad libitum food and water supply) and placed in the SPF environment for acclimation 1 week before the start of the study.
Drugs 6-OHDA was purchased from Sigma (St Louis, MO, USA). The solution was prepared in physiological saline containing ascorbic acid (mass/volume ratio: 0.02%) at a concentration of 2 lg/lL. We injected 2 lL of the solution each at two points into the medial forebrain bundle (MFB) of rats; a total of 4 lL of solution (8 lg of 6-OHDA) was administered to each rat.
Ascorbic acid was purchased from Sigma. We used physiological saline to prepare a solution with a mass volume ratio of 0.02%.
Apomorphine was purchased from Cayman Chemical (Ann Arbor, MI, USA). A solution was prepared with physiological saline containing ascorbic acid (mass volume ratio: 0.02%) at a concentration of 0.1 mg/mL. According to the body weight of the rats, 0.05 mg/kg apomorphine was injected subcutaneously into the back of the neck.
L-DOPA and benserazide were purchased from Sigma. A mixed powder of L-DOPA and benserazide was directly dissolved in physiological saline to concentrations of 12 and 6 mg/mL, respectively. Of this solution, 0.5 mL/kg was administered by intraperitoneal injection according to the body weight of the rats. In this study, the administration of ''L-DOPA" refers to the administration of this L-DOPA/benserazide mixture.
ONO-2506 was purchased from Bio-techne Corp. (Minneapolis, MN, USA). It was prepared in physiological saline at a concentration of 4 lg/lL. According to the body weight of the rats, 50 lg/kg ONO-2506 was administered to the right striatum of the brain through brain catheterization.
Establishment of a PD rat model with a complete unilateral lesion SD rats (body weight: 240-260 g) were ready for surgery after 1 week of adaptation to the environment. The rats were fasted for 12 h before surgery and were anesthetized with isoflurane (3% induction and 1.5-2% pure oxygen maintenance). When the rats reached deep anesthesia (no blink reflex), they were fixed onto a stereotactic device. After adjusting the stereo locator, the left and right ear rods and nose clips were fixed so that the head was stable and positioned in the middle of the locator. The hair on the top of the rat's head was removed to expose the skin. After iodophor disinfection, we incised the scalp along the sagittal suture, removed the fascia, the periosteum, and other tissues on the skull surface, rubbed the bone surface with 10% hydrogen peroxide to expose the front and rear fontanels clearly, and adjusted the dental bracket of the locator to keep the height of the front and rear fontanels consistent (no more than 0.1-mm difference). Taking the anterior fontanelle as the coordinate origin, we determined the coordinate position of the right MFB (anteroposterior [AV]: À4.4 mm, mediolateral [ML]: À1.5 mm, dura dorsoventral [DV]: À7.8 mm and dorsoventral: À7.9 mm) (Lindgren et al., 2009;Chen et al., 2017;Zhang et al., 2021). At this coordinate, we used a skull drill to drill a small hole vertically downward and slowed the speed when it was about to penetrate the skull to avoid damaging the brain tissue. After exposing the dura, we recorded its Z coordinate value, pricked the dura with sharp tweezers, and paid attention to hemostasis during the period. Taking the Z coordinate value as the origin, we used a 10-lL microinjector to insert the needle slowly into the target position and left the needle in place for 5 min. Then, we slowly injected 4 lL 6-OHDA, at an injection speed of 1 lL/min (2 lg/lL, 2 lL per injection site). We left the needle in place for 5 min after the injection and then slowly pulled out the microinjector. Subsequently, we evenly embedded three screws into the skull surface for catheter fixation. We adjusted and maintained the level of the anterior and posterior fontanelle. With the anterior fontanelle as the coordinate origin, we determined the dorsolateral position of the right striatum (AP À3.8 mm, ML + 0.2 mm, dura DV À3.5 mm), drilled a small hole vertically downward with a skull drill, punctured the dura, slowly placed the catheter into the dorsolateral side of the right striatum in approximately 2-3 min, and then filled the defect in the skull with bone wax. Finally, we fixed the catheter layer-by-layer with dental cement, applied erythromycin locally to prevent infection, and sutured the incision. Heated mats were used to keep the rats warm during surgery. After surgery, 400,000 units/kg/d penicillin was injected intramuscularly after surgery for 3 days to prevent infection.

Detection of PD rat model with complete unilateral lesion
After 2 weeks of postoperative recovery, the model of PD rats with unilateral complete damage was tested. Contralateral rotation of rats was induced by subcutaneous injection of apomorphine (0.05 mg/kg) into the back of the neck, and the number of rotations was recorded to evaluate the degree of dopaminergic neuron damage. Only rats with more than 200 cycles of contralateral rotation within 30 min were considered qualified PD models with complete destruction of dopaminergic neurons (Schwarting and Huston, 1996;Paille et al., 2010). These rats were used for subsequent investigations.

Treatment
Thirty successful unilateral PD model rats were randomly divided into the L-DOPA + ONO-2506 group (n = 16) and L-DOPA + saline group (n = 14). ONO-2506 (50 lg/kg) or the same volume of physiological saline was administered by placing a tube in the striatum of the brain for 7 days. The dosage of ONO-2506 was based on that used a recent study, in which ONO-2506 was administered to Wistar rats by intracerebroventricular injection at a dose of 1, 10, and 100 lg/kg, which was found to play a neuroprotective role (Vizuete et al., 2021). In our preliminary study, we observed that a 50 lg/kg dose provided more obvious effects than 25 lg/kg; thus 50 lg/kg was selected for all future analyses.
In previous studies, the administration period for ONO-2506 varied from a single dose to long-term daily doses lasting more than one month (Mori et al., 2006;Ishiguro et al., 2019;Mari et al., 2019). Hence, based on our LID model, we selected a 7-day administration period. In the control group, we administered the same amount of normal saline by placing a tube in the brain of the striatum to eliminate the effects caused by repeated brain administration between two groups. All rats were intraperitoneally injected with 6 mg/kg L-DOPA and 3 mg/kg benserazide 1 h after administration of ONO-2506 or saline. We used a smaller dose of L-DOPA to induce LID to observe the changes in the formation of LID better and avoided large doses of L-DOPA that could cause a rapid peak in LID. During the administration of L-DOPA, a blinded examiner regularly recorded the time and score of AIMs induced by L-DOPA as an evaluation index for LID.
For administration of ONO-2506 or saline, one experimenter used a fixator to fix and comfort the rat, while the other experimenter accurately extracted ONO-2506 or saline with a microinjector (50 lg/kg, according to the rat's body weight), unscrewed the catheter cover on the top of the rat's skull, discharged the bubbles at the front end, inserted the inner core of the syringe, and then injected the solution slowly (1 lL/min). After injection, the needle was left in place for 5 min, after which the inner core of the syringe was removed slowly, the catheter cover was again screwed, and the injection completion time was recorded. The catheter diameter was 1 mm, while the inner core of the casing was connected to the catheter cover. Typically, the inner core fills the casing to prevent dead cavity. During injection, we unscrewed the catheter cover and pulled the inner core out to ensure catheter patency. We observed the liquid level of the microinjector during the injection to ensure that the drug is injected into the striatum through the catheter.
Ten rats were excluded from the test. In the apomorphine test, the rats did not reach 200 cycles within 30 min, which is considered as failing to pass the detection of unilateral PD rat model, hence could not participate in the experiments that followed.

Behavioral testing
To minimize inter-examiner differences, all behavioral experiments were conducted and evaluated by the same experimenter, and the experimental data were recorded by another experimenter. Neither of the two experimenters knew the group allocation of the rats.

Adjusting-step test
The adjusting-step test was used to measure forelimb dyskinesia in rats and simulate the clinical symptoms of PD. Rats with striatal dopaminergic neuron loss exceeding 80% perform poorly in this behavioral assessment. L-DOPA treatment can partially reverse the behavioral defects induced by injury (Conti et al., 2016;Meadows et al., 2017).
The adjusting-step test was performed at 2 weeks after surgery and on days 16 and 22, 15 min before the administration of ONO-2506 or saline, 15 min post its administration, and 15 min post administration of L-DOPA (Chang et al., 1999). An experimenter gently lifted the hindquarters and contralateral forelimbs of rats from the table, and only the forelimb to be tested touched the table. Another experimenter counted the steps that the two forelimbs adjusted forward in a distance of 90 cm within 5 s (Pinna et al., 2007;Chen et al., 2017). The test was conducted three times, and the results were recorded and statistically analyzed.

Coat-hanger experiment
The coat-hanger test was performed at 2 weeks after surgery and on days 16 and 22, 15 min before the administration of ONO-2506 or saline, 15 min post its administration, and 15 min post administration of L-DOPA. An experimenter placed the rat naturally in the center of a clothes hanger (diameter 3 mm; horizontal length 35 cm; ground clearance 40 cm) by the front paw. Note, serious injury was prevented via the application of soft rat bedding under the hangers. Another experimenter observed the posture of the rat for 30 s and scored the performance of the rat. In addition, we also measured the time until the rat fell from the clothes hanger. The observation time was set to 30 s, and if the rats did not fall after 30 s, the time was recorded as 30 s. The test was conducted three times, and the results were recorded and statistically analyzed. The scoring criteria are presented in Appendix A (Moran et al., 1995;Voikar et al., 2002;Zhang et al., 2021).

Cylinder test
The cylinder test was performed 2 weeks post-surgery. The cylinder test is used to measure the asymmetric use of forelimbs when spontaneously exploring the wall of a cylindrical tube and is a frequently used motor function test after 6-OHDA injury (Schallert, 2006).
In the cylinder test, the animals were placed alone in a transparent plexiglass cylinder (diameter 21 cm; height 16 cm) in a dimly lit room and were observed for 5-10 min to evaluate their activity. The time limit for the cylinder test was 10 min or 20 times of front paw contact with the cylinder wall. In each test, the number of times the front paw on the damaged (contralateral), undamaged (ipsilateral) or both sides contacted the outer wall during exploration was recorded. The data are expressed as the percentage of injured forelimb use (Schallert, 2006).

AIM score
We used AIM scores to quantify the severity of LID. After intraperitoneal injection of L-DOPA on days 16, 17, 18, 20, and 22 of 6-OHDA injury (equivalent to days 1, 2, 3, 5, and 7 of L-DOPA administration, respectively), rats were assessed with the validated AIM scale, starting from the 20th min, observations were recorded every 20 min for a total of 140 min (Winkler et al., 2002;Chen et al., 2017). AIM subtyping was divided into three parts: axial (back arching, torsion), upper limb (scratching, tapping), and orofacial (licking, upper and lower occlusion, sniffing), divided into levels 0-4 according to severity (Lundblad et al., 2004). The sum of the axial, upper limb, and orofacial scores comprised the total dyskinesia score, with a maximum of 84 points. The scoring system has been used many times in previous studies by our research team (Chen et al., 2017;Tan et al., 2020;Zhang et al., 2021). The scoring criteria are listed in Appendix B.

Tissue preparation
On days 19 (n = 6) and 23 (n = 24), rats in each group were decapitated under isoflurane anesthesia for followup experiments at 1 h after L-DOPA administration. The striatum of the ONO-2506 group (day 19: n = 3, day 23: n = 10) and saline group (day 19: n = 3, day 23: n = 8) rats was quickly removed on ice and immediately stored in an À80°C refrigerator for Western blot.
Additionally, rats in the ONO-2506 group (n = 3) and saline group (n = 3) were perfused with 4°C 0.9% physiological saline through the aorta until bloodless clear liquid flowed out of the right atrial appendage, after which they were perfused with 4% paraformaldehyde until they were stiff. After careful and complete removal of the brain, it was fixed in pre-cooled 4% paraformaldehyde for 48 h. After dehydration, transparency, embedding, and sectioning, immunohistochemical staining was performed.

Immunohistochemistry
Referring to the rat brain atlas, the rat brain tissue was sectioned coronally, with a thickness of 4 lm. Paraffinembedded sections were prepared and stored at 20°C after baking. After dewaxing and hydration, antigen repair, blocking of endogenous peroxidase activity, the sections were incubated overnight with primary antibody at 4°C. After rewarming for 15 min the next day, sections were rinsed with Tris Buffered Saline (TBS) three times, after which the corresponding secondary antibody was added to the sections in drops and sections were incubated at room temperature for 1 h. Fresh DAB dye solution was used for staining, followed by hematoxylin staining for 3 min, after which the section was dehydrated and the slide was sealed. A slide scanner (NanoZoomer S360, Hamamatsu Photonics K.K, Hamamatsu, China) was used to scan slides under the same light intensity. ImageJ software (NIH, Bethesda, MD, USA) was used to analyze the cumulative optical density for statistical analysis. Image J process and antibodies used are listed in Appendix C.

Western blotting
Dorsolateral striatum tissues were dissected and homogenized to extract protein. The concentration of the extracted protein samples was measured using a bovine serum albumin (BSA) protein standard, and the protein concentration of each sample was adjusted to be consistent. Then, 40 mg of each protein sample was loaded onto a 10 or 12% polyacrylamide gel, and the target proteins were then electrophoretically separated. Proteins were subsequently transferred to PVDF membrane (Millipore, Burlington, MA, USA) and then blocked in 5% skim milk or 5% BSA at room temperature for 90 min. After washing, the membrane was incubated with the primary antibody overnight at 4°C. On the following day, the membrane was washed three times with Tris Buffered Saline Tween (TBST) and then incubated with the corresponding secondary antibody at room temperature for 1 h. After washing, bands were developed using a chemiluminescence kit (Biosharp, Hefei, China) and visualized using a fluorescent chemiluminescence gel imaging system (Syngene, Cambridge, UK). Each Western blotting was repeated three times. We used ImageJ software to analyze the gray values of bands for statistical analysis. The antibodies used are listed in Appendix D.

Statistical analysis
Data are expressed as mean ± standard error of mean. All data were analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA). Paired sample t-tests were used to compare the behavior changes in the same group before and after treatment and differences between the two sides within the same group. Independent sample t-tests were used to compare the differences between groups. Two-way analysis of variance (ANOVA) was used to compare the differences in AIMs between the two groups after L-DOPA administration at different time points (time Â treatment). Bonferroni multiple comparison post hoc tests was then used, p < 0.05 was considered statistically significant.

Verification of unilateral 6-OHDA injury rat model
After acute challenge with apomorphine (0.05 mg/kg), 30 of 40 rats injected with 6-OHDA demonstrated >200 contralateral rotations, which was considered to represent a completely damaged PD model. Subsequent behavioral test results confirmed that the injection of 6-OHDA into the right MFB also led to serious defects in the gait of the contralateral (left) forepaw, which was typically manifested by a significant reduction in the number of steps taken with the left limb ( Fig. 2(A)) and a low preference for using the left forelimb ( Fig. 2(B)). The behavioral performance of rats confirmed the success of unilateral 6-OHDA injury rat model.
After the experiment, we also stained the brain slices of rats in each group for tyrosine hydroxylase (TH). Immunohistochemical staining showed that TH was almost completely absent on the injured-side (Fig. 2(C-E)), which was consistent with a unilateral PD model.
There was no significant difference in TH loss between the ONO-2506 and saline groups (Fig. 2(F,G)).

ONO-2506 administration had no significant effect on motor deficits in 6-OHDA-injured rats
We evaluated the motor function of rats before and after ONO-2506 and saline administration based on the adjusting-step and coat-hanger tests. The behavioral test results of the ONO-2506 and saline groups on days 1 and 7 (total days 16 and 22, respectively) showed no significant change in the number of steps taken with the bilateral forepaws of rats after administration compared with the baseline test results (Fig. 3(A,B)), and no significant change in the scores of rats on the coat- Fig. 2. Validation of unilateral 6-hydroxydopamine injury rat model. (A) In the adjusting-step test, the number of steps on the left side of rats was significantly lower than that on the right side (left À right ± standard error of mean [SEM]: À6.944 ± 0.2186, ****p < 0.0001, t = 31.77, df = 23). (B) In the cylinder test, rats' preference for using the left forelimb was significantly reduced compared to that for using the right forelimb (left À right ± SEM: À67.82% ± 5.476%, ****p < 0.0001, t = 12.39, df = 23). (C, D) Representative images of tyrosine hydroxylase (TH) immunohistochemical staining in the saline (C) and ONO-2506 (D) groups. (E) Immunohistochemical staining showed that TH in the right striatum of the two groups was almost completely lost (injured side À healthy side ± SEM: À93.97% ± 3.713%, ****p < 0.0001, t = 25.31, df = 5). (F, G) Immunohistochemical staining showed that there was no significant difference in TH loss on both sides (F lesioned and G uninjured) between the ONO-2506 and saline groups (F: ONO-2506 group À saline group on the injured-side ± SEM: 17.67% ± 24.96%, NS p = 0.5182, t = 0.7077, df = 4; G: ONO-2506 group À saline group on the healthy side ± SEM: 0.2033% ± 9.496%, NS p = 0.9839, t = 0.02141, df = 4). hanger test (Fig. 3(C,D)) or the time of adherence to the coat hangers ( Fig. 3(E,F)).
L-DOPA administration improved the motor deficits of 6-OHDA-injured rats, and ONO-2506 did not affect the anti PD effects of levodopa We evaluated the motor function of rats before and after L-DOPA administration using the adjusting-step and coat-hanger tests. The behavioral test results on days 1 and 7 (total days 16 and 22, respectively) of L-DOPA administration showed that compared to the baseline test, L-DOPA treatment increased the number of steps taken with the injured-side forepaw of rats (Fig. 4(A,B)), score in the coat-hanger test (Fig. 4(C,D)), and duration of adhering to the coat hanger ( Fig. 4(E,F)). Moreover, the improvement in motor function in the L-DOPA + ONO-2506 group and L-DOPA + saline group was similar. The number of steps taken with the undamaged-side front paw did not significantly change.

ONO-2506 administration reduced the expression of L-DOPA-induced AIMs
ONO-2506 administration delayed the emergence of AIMs. We observed the time to emergence of the first AIMs in the ONO-2506 and saline groups after daily L-DOPA administration. Compared with that of the saline group, the AIM behavior of the ONO-2506 group took In the adjusting-step test, compared to that before ONO-2506 and saline administration, there was no significant change in the number of steps of the left and right front paws of rats (p > 0.05) on day 1 (A) or 7 (B). Similarly, there was no significant change in the number of steps taken with the bilateral forepaws between the ONO-2506 and saline groups on either day (p > 0.05). (C-F) In the coat-hanger test, compared with that before ONO-2506 and saline administration, the changes in the rats' scores (C, D) and duration of adhering to the coat hanger before falling off (E, F) were not significant on day 1 (C, E) or 7 (D, F) (p > 0.05). longer to emerge following L-DOPA administration, but the difference was not significant on the first day of L-DOPA administration (Fig. 5).

ONO-2506 administration reduced the total score of AIMs in rats
We observed the total AIM score of the ONO-2506 and saline groups during the observation period of 140 min post L-DOPA administration.
Two-factor ANOVA (rats in each group were matched with levodopa administration days) showed that levodopa administration days and intervention measures had a significant impact on the scores. Within 5 observation days, the AIM scores of the ONO-2506 group were lower than those of the saline group, and the difference between the two groups reached statistical significance on days 1, 2, 3, and 5. As the L-DOPA administration regimen continued, the difference between the two groups tended to decrease until the experiment ceased on day 7 (Fig. 6).

Time course of AIM severity post L-DOPA administration
On days 16, 17, 18, 20, and 22 (equivalent to days 1, 2, 3, 5, and 7 of L-DOPA administration), the AIM scores of rats from the 20th min, taken at 20-min intervals over a 140-min period, were evaluated with the AIM scale. Two-factor ANOVA (the rats in each group were matched with the time following levodopa administration) showed that time after levodopa administration had a significant effect, while the effect of different intervention measures was significant within 4 In the adjusting-step test, compared with that before L-DOPA administration, the number of steps taken with the left forepaw (injured-side) of the rats in the ONO-2506 and saline groups was significantly improved (****p < 0.0001) on days 1 (A) and 7 (B), but the number of steps taken with the undamaged-side forepaw did not significantly change (p > 0.05). There was no significant difference between the ONO-2506 and saline groups in terms of improvement in forepaw step on either day (p > 0.05). (C-F) In the coat-hanger test, compared with before L-DOPA administration, the rats in the ONO-2506 and saline groups obtained higher scores (C, D) and adhered to the coat hanger longer (E, F) on days 1 (C, E) and 7 (D, F) (**p < 0.01, ***p < 0.001, and ****p < 0.0001). There were no significant differences between the ONO-2506 and saline groups in these behavioral assessment (p > 0.05). observation days. During the 140-min observation period on five observation days, the LID of rats in the ONO-2506 group was significantly reduced compared with that of rats in the saline group, and AIMs were reduced, as shown by the scores, with an interval of at least four time points. With the repeated daily administration of L-DOPA, the difference in AIM scores between the ONO-2506 and saline groups gradually narrowed, while the score difference at only one scoring time point reached statistical significance (day 7). Fig. 7 presents the results for days 1, 2, 3, 5, and 7 post L-DOPA administration.
ONO-2506 administration significantly increased the expression of glial fibrillary acidic protein in rat brain striatum Astrocytes are heavily involved in the occurrence and development of LID (Del-Bel et al., 2016;Pisanu et al., 2018;Fletcher et al., 2020); glial fibrillary acidic protein (GFAP) is a marker commonly used for activated astrocytes. Western blotting results showed that the level of GFAP in the right striatum of the ONO-2506 group was significantly higher than that in the right striatum of the saline group on days 4 and 8 (Fig. 8(A-C)). The difference between the two groups on day 8 was greater than that on day 4. Consistent with the results of western blotting, immunohistochemical staining showed that the GFAP expression level in the right dorsolateral striatum in the ONO-2506 group was significantly higher than that in the saline group and was significantly higher than that in the left striatum (Fig. 8(D-H)).

ONO-2506 administration up-regulated the expression of GLT-1 in the striatum
We evaluated the expression of GLT-1 in our model using western blotting (Fig. 9(A-C)) and immunohistochemical staining ( Fig. 9(D-F)) to detect the expression of GLT-1 quantitatively in the striatum of each group. On days 4 and 8 following administration, the GLT-1 level in the right striatum of ONO-2506 group was higher than that of the saline group. The difference between the two groups on day 8 was smaller than that on day 4.

Effects of ONO-2506 administration on microglia, inflammatory factors, and vascular endothelial growth factor
We used western blotting and immunohistochemical staining to detect the expression of Iba-1, TNF-a and VEGF in the striatum of our model. The results showed no significant difference in the expression of Iba-1, TNFa, and VEGF in the striatum between the two groups ( Fig. 10).

DISCUSSION
This study explored the effect of ONO-2506 on LID in a rat model and the potential underlying physiological mechanism, and showed that ONO-2506 can delay the emergence of AIMs after L-DOPA administration, reduce the total AIM score in LID model rats, and delay LID development in the early stage of LID.
We showed that ONO-2506 significantly delayed the development and reduced the degree of AIMs during L-DOPA treatment and increased the expression of GFAP and GLT-1 in the striatum compared to saline, however did not affect motor function.
Furthermore, ONO-2506 could not improve the motor deficits of PD rats with unilateral damage caused by 6-OHDA and did not affect the anti-PD effect of L-DOPA, which was inconsistent with the early studies of Kato et al. (Kato, 2003;Kato et al., 2004;Himeda et al., 2006;Oki et al., 2008). In their series of studies, ONO-2506 administration had a protective effect on dopaminergic neurons in a 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP)-induced PD mouse model and could significantly reduce PD motor abnormalities. We speculate that the difference in models may be the reason for the different results. In the 6-OHDA-induced rat PD model, the degeneration of dopamine neurons  F (1, 22) = 13.71, p = 0.0012) had significant effects. There were significant differences in AIM scores between the two groups at the time points 60-120 min post administration. (E) On day 7, the time after L-DOPA administration (F (6, 132) = 64.34, p < 0.0001) had significant effects but ONO-2506 administration (F (1, 22) = 3.009, p = 0.0968) had no significant effects. The difference in AIM scores between the two groups was statistically significant only at the time point 120 min after administration. The results are expressed as mean ± standard error of mean. NS p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. occurred within 12 h of administration, peaked 1 week later, and remained on day 31 (Jeon et al., 1995). Compared with that in MPTP-induced mouse PD model, the pathological changes in the 6-OHDA-induced rat PD model appear earlier and are more severe, the degree of TH loss and dopaminergic neuron depletion is more extensive and intense and the resulting motion defects are more difficult to reverse (Hamadjida et al., 2019). The complete loss of dopaminergic neurons is the basis of LID formation (Espay et al., 2018). In our 6-OHDAinduced rat PD model, loss of dopaminergic neurons was irreversible; thus, ONO-2506 administration may not readily reverse bradykinesia associated with PD in long-term chronic PD models.
In this study, we injected ONO-2506 into the striatum of the damaged side of 6-OHDA through intracerebral striatum catheterization in the unilateral PD rat model, which can not only delay the emergence of AIMs after L-DOPA administration but also reduced the total AIM scores in rats in the early stage. These results suggest that astrocyte intervention may be a potential therapeutic target for LID and can be used to delay the occurrence and progression of LID. Appropriate intervention in astrocytes may be conducive to brain repair and functional recovery. However, with the repeated daily administration of L-DOPA, the gap between the AIM scores of ONO-2506 rats and saline rats gradually narrowed. Our study did not verify whether ONO-2506 plays a protective role over a longer time frame and whether ONO-2506 intervention is effective after formation of stable LID. However, our research shows that, at least in the early stage, ONO-2506 plays a role in delaying the emergence of LID. Therefore, we selected the early stage of the model to perform a series of pathological and biochemical tests to elucidate the mechanism by which ONO-2506 delays LID occurrence.
ONO-2506 administration significantly increased the expression of GFAP in the rat brain striatum, and a significant increase in GFAP+ astrocytes was observed in the dorsolateral striatum on the side of ONO-2506 administration. This result was consistent with that reported by Mari et al. (2019) who showed that, in the model of neonatal hypoxic-ischemic encephalopathy, at 2 days following cerebral hypoxia-induced tissue damage, ONO-2506 administration reduced the release of S100b into the cerebrospinal fluid and increased GFAP levels (Mari et al., 2019). However, Oki et al. reported that ONO-2506 administration reduced the increase of immunoreactive astrocytes in the substantia nigra and striatum 7 days post MPTP treatment (Oki et al., 2008). Vizuete et al. found a dual effect of ONO-2506 administra- Fig. 8. Effect of ONO-2506 administration on glial fibrillary acidic protein expression in rat brain striatum. (A-C) Western blotting results showed that compared with that in the saline group, the expression of glial fibrillary acidic protein (GFAP) in the right striatum of the ONO-2506 group was significantly increased (day 4: ONO-2506 group À saline group ± standard error of mean [SEM]: 319.7% ± 42.10%, p < 0.0001, t = 7.594, df = 16; day 8: ONO-2506 group À saline group ± SEM: 563.4% ± 143.5%, p = 0.0012, t = 3.926, df = 16). (D-G) Representative images of GFAP immunohistochemical staining in the ONO-2506 and saline groups. (H) Immunohistochemical staining showed that the level of GFAP in the right striatum of rats in the ONO-2506 group was higher than that in the right side of saline group (ONO-2506 group À saline group ± SEM: 137.4% ± 30.08%, p = 0.0103, t = 4.568, df = 4) and was higher than that in the left striatum (ONO-2506 group right À left ± SEM: 147.6% ± 34.13%, p = 0.0496, t = 4.324, df = 2). * p < 0.05; **p < 0.01. tion on astrocytes. They speculated that ONO-2506 may cause astrocyte proliferation at low doses, but this effect is absent or blocked at high doses (Vizuete et al., 2021). Thus, ONO-2506 administration may have different effects on astrocytes in different models and doses. In addition, in our study, the increase in GFAP expression was more obvious on day 8 than 4, inconsistent with the trend of diminishing the improvement of LID rats. The increase in GFAP may be attributed to ONO-2506 regulation of astrocytes activation, while GFAP might have had no role in the beneficial effects of ONO-2506. Our results showed that ONO-2506 delayed the occurrence of LID while significantly increasing GLT-1 expression, which indicates that the protective effect of ONO-2506 may be achieved by upregulating the expression of GLT-1. Excessive glutamate release may be involved in LID. Clearance of glutamate from the synaptic space is an alternative method to reduce glutamatergic transmission (Anderson and Swanson, 2000;Rothstein et al., 2005). The reduction of glutamate outflow from the striatum or substantia nigra can protect against LID (Dupre et al., 2011;Paolone et al., 2015;Carta and Cenci, 2016;Garcia-Esparcia et al., 2018). GLT-1 is a glutamate transporter, which is responsible for clearing synaptic glutamate (Danbolt, 2001), and is mainly expressed in astrocytes (Rothstein et al., 1994;Maragakis et al., 2004). In two studies on the use of ceftriaxone in the treatment of LID, intraperitoneal injection of ceftriaxone increased GLT-1 expression in astrocytes in a 6-OHDA rat model, contributed to glutamate uptake and clearance from the striatal synapses, and reduced the severity of LID (Kelsey and Neville, 2014;Chotibut et al., 2017). This further supports our findings that ONO-2506 increased the expression of GLT-1 and accel- showed that, compared with that in the right striatum of the saline group, the expression of glutamate transporter 1 (GLT-1) in the right striatum of the ONO-2506 group was increased (day 4: ONO-2506 group À saline group ± SEM: 86.62% ± 15.61%, p < 0.0001, t = 5.548, df = 16; day 8: ONO-2506 group À saline group ± SEM: 45.91% ± 11.66%, p = 0.0012, t = 3.938, df = 16). (D, E) Representative images of GLT-1 immunohistochemical staining in the ONO-2506 (D) and saline (E) groups (20 Â ). (F) Immunohistochemical staining showed that the level of GLT-1 in the right striatum of rats in the ONO-2506 group was slightly but significantly higher than that in the right striatum of rats in the saline group (ONO-2506 group À saline group ± SEM: 13.28% ± 2.640%, p = 0.0073, t = 5.032, df = 4). *p < 0.05; **p < 0.01. erated the uptake of glutamate between synapses. This may weaken the transmission via the glutamate pathway in cortical striatum, normalize the sensitization of postsynaptic dMSNs, and inhibit LID. In our study, the difference in GLT-1 expression between the ONO-2506 and saline group on day 8 was smaller than that on day 4, which may explain the decline in treatment effect of ONO-2506. However, we could not determine the reason behind the decline in the therapeutic effect of ONO-2506 or evidence for glutamatergic changes in synaptic transmission, hence further investigations remain warranted.
angiogenesis and development of new capillaries; thus, it may be related to LID (Carmeliet, 2000;Ohlin et al., 2011). However, in our model, there was no significant difference in the expression of VEGF between the two groups of the rat striatum on days 4 and 8 after administration. The significant increase in astrocytes induced by ONO-2506 administration was not accompanied by changes in angiogenesis-related indicators. The above results suggest that astrocytes have extensively varied and complex functions, some of which may even involve completely opposite effects. The phenotype of reactive astrocytes depends on the nature of the induced stimulation, while most of the gene expression changes associated with reactive gliosis are transient (Zamanian et al., 2012). Therefore, future studies should determine how to distinguish the respective functions and interactions occurring with the increase in different types of astrocytes and how to induce astrocytes to express a more favorable phenotype in a controllable form, while reducing the expression of adverse phenotypes.
ONO-2506 delays the emergence of L-DOPA-induced abnormal involuntary movements in the early stage of L-DOPA administration without interfering with the anti-PD effect of L-DOPA. The delaying effect of ONO-2506 on LID may be associated with increased expression of GLT-1 in the rat striatum. Interventions targeting astrocytes and glutamate transporters may be potential therapeutic targets for LID to delay the occurrence and development of LID. Our findings provide new insights into the prevention and management of LID in patients with PD.

AUTHOR CONTRIBUTIONS
Yuhao Yuan, Xuebing Cao, and Yan Xu conceived and designed the study. Yuhao Yuan, Xiaoqian Zhang, Yi Wu, and Piaopiao Lian performed the experiments. Yuhao Yuan and Xiaoqian Zhang analyzed the data. Yuhao Yuan wrote the manuscript. Xuebing Cao and Yan Xu revised the manuscript. All authors have read and approved the final manuscript.

ETHICS STATEMENT
Animal experiments were approved by the ethics committee of Huazhong University of Science and Technology. All feeding and experimental operations complied with the requirements of the ethical code for experimental animals and were performed according to the statutes on the administration of experimental animals issued by the Ministry of Science and Technology of China.

DATA STATEMENT
The original data presented in the study are included in the article/Supplementary Material.

FUNDING
This work was supported by a grant from the National Natural Science Foundation of China (grant number 81873734). The sponsor did not participate in the study design; collection, analysis and interpretation of data; writing of the report; and decision to submit the article for publication.

APPENDIX A. COAT HANGER EXPERIMENT
Score Behavior 0 Falling within 10 s 1 The rat does not fall for > 10 s; the two front claws remain on the hanger 2 Reach the standard of 1 point and try to climb the hanger at the same time 3 Two front claws plus at least one rear claw remain on the hanger 4 All four claws and tails are wrapped around the hanger 5 The rats try to escape to the edge of the hanger and may climb to the top of the hanger