Effects of levodopa on dopaminergic neurons and induced dyskinesia A radio-imaging study*☆
Xiuying Cai1, Yan Kong1, Hongru Zhao1, Bin Zhang2, Chunfeng Liu3

1Department of Neurology, First Hospital Affiliated to Soochow University, Suzhou  215006, Jiangsu Province, China
2Department of Nuclear Medicine, First Hospital Affiliated to Soochow University, Suzhou   215006, Jiangsu Province, China
3Department of Neurology, Second Hospital Affiliated to Soochow University, Suzhou  215004, Jiangsu Province, China

Xiuying Cai☆, Doctor, Associate chief physician, Department of Neurology, First Hospital Affiliated to Soochow University, Suzhou  215006, Jiangsu Province, China

Corresponding author: Chunfeng Liu, Doctor, Chief physician, Professor, Department of Neurology, Second Hospital Affiliated to Soochow University, Suzhou  215004, Jiangsu Province, China
liucf20@hotmail.com

Supported by: the Scientific Research Foundation Program of Ministry of Health, No. wkj.2005-2-030*

Abstract
BACKGROUND: Radio-imaging has been used in neurological diagnosis, in particular for ex-trapyramidal disease. Moreover, it has been extensively utilized for early diagnosis of Parkinson’s disease (PD) patients and in animal studies. However, it has rarely been utilized to assess drug-induced side effects in PD.
OBJECTIVE: To investigate changes in dopamine transporter expression in a rat model of PD through the use of radio-imaging taking 99mTc-TRODAT-1 as an imaging agent, and to explore the effect of levodopa (L-dopa) on dopaminergic neurons and the possible mechanisms of dyskinesia induction.
DESIGN, TIME AND SETTING: A randomized, controlled, animal study was performed at the Laboratory of Department of Nuclear Medicine, Soochow University from April 2006 to June 2007.
MATERIALS: 6-hydroxydopamine was purchased from Sigma, USA and L-dopa was purchased from Shanghai Fuda Pharmaceutical, China. 99mTcO4-fresh elutriant was provided by the Depart-ment of Nuclear Medicine, First Hospital Affiliated to Soochow University. TRODAT-1 image kit was provided by Jiangsu Atomic Energy Research Establishment, China. The SN-695B radioimmuno-assay gamma counter was purchased from Shanghai Hesuo Rihuan Photoelectric Instrument, China. The AZ-CA256eZ-Scope portable γ camera was purchased from Anzai Medical, Japan.
METHODS: A total of 34 healthy, male, Sprague Dawley rats were selected. Thirty were used to establish a PD model by injecting 6-hydroxydopamine into the right medial forebrain bundle, and four were injected with normal saline and served as the sham-surgery group. At the end of 4 weeks, 21 successful PD models were selected and randomly assigned to the L-dopa (n = 15, 20 mg/kg per day), model (n = 6, normal saline), and sham-surgery (n = 4, no treatment) groups. After 1 month of treatment, involuntary movement was evaluated twice weekly in each rat. A total of 0.2 mL 99mTc-TRODAT-1 was injected into the tail vein 2 days following drug termination, and images of dopamine transporters were acquired 2 hours later. The rats were sacrificed and the ratios of spe-cific radioactivity uptake were calculated.
MAIN OUTCOME MEASURES: Manifestations of abnormal involuntary movement (AIM) were observed and total AIM scores were calculated. Images of dopamine transporters were acquired using an eZ-Scope portable γ camera, and radioactive γ quantification of 99mTc-TRODAT-1 in the rat brains was assayed. The ratios of the left and right corpora striata were determined. The number and function of dopamine transporters was evaluated according to specific radioactivity uptake ratio (R) from the left and right corpora striata.
RESULTS: Of 15 PD rats, nine exhibited AIM following L-dopa treatment: five scored > 20, i.e., severe grade, four scored 8–16, mild grade, and the remaining exhibited normal behavior. There were no differences in specific radioactivity uptake of dopamine transporter between the left and right corpora striata in the sham-surgery rats, and the images were clear and symmetrically distrib-uted. Specific radioactivity uptake of the normal side (left) was significantly greater than the lesioned side (right) in the model group rats (P < 0.01), and the R value was significantly increased compared with the sham-surgery group (P < 0.01). The radio-ligand accumulation in the right corpus striatum was sparse. In the L-dopa group, specific radioactivity uptake was significantly decreased in the le-sioned (right) side of the AIM rats, and the R value was increased compared with the model group (P < 0.05). The amount of radio-ligand in the right corpus striatum was diminished. The R value was significantly reduced in the non-AIM rats compared with the AIM rats (P < 0.05), and specific radioactivity uptake was significantly increased in the lesioned (right) side compared with the normal side (P < 0.05). Moreover, radio-ligand accumulation was observed in the right corpus striatum, and differences in radio-ligand accumulation between the two sides were reduced.
CONCLUSION: Following L-dopa treatment, the number and function of dopamine transporter in some PD rats were reduced. L-dopa was shown to be toxic to dopaminergic neurons and induced dyskinesia.
Key Words: Parkinson’s disease; levodopa; dopamine transporter; 99mTc-TRODAT-1; neuroimaging; neural regeneration

INTRODUCTION
  
Treatment with levodopa (L-dopa) has been used exten-sively to treat Parkinson’s disease (PD), resulting in greatly improved PD symptoms. However, long-term application of L-dopa induces side effects, such as end-of-dose deterioration, on-off phenomenon, and server abnormal involuntary movement (AIM), also called L-dopa-induced dyskinesia[1]. Movement disorders pre-dominantly account for disability in PD patients. A better understanding of action pathways and curative effects, as well as motion complication mechanism[2-3], can help to prevent L-dopa adverse effects and minimize PD symptoms.
Results from studies addressing the effects of L-dopa on protection, regeneration, and toxicity to dopaminergic neurons[4-5] have varied. Factors, such as drug dose, frequency, course of treatment, disease severity, and progression, result in differences in sensitivity of surviv-ing dopaminergic neurons to extracellular L-dopa. The effect of disease severity on L-dopa action, under iden-tical drug dose and course of treatment, remains unclear. An increasing number of studies has demonstrated that dyskinesia may be related to dopamine receptor fluctu-ant stimulation of postsynaptic membrane and disease progression[6-7]. However, the precise mechanisms re-main uncertain. The present study further explored the effect of L-dopa on dopaminergic neurons and dyskine-sia induced by exogenous L-dopa.
The progressive loss of substantia nigra dopaminergic neurons in PD reduces the amount of dopamine trans-mitter, leading to required supplementation of exogenous L-dopa. The short half-life and instable levels of L-dopa in the blood induce presynaptic dopamine discontinuous release, forming non-physiological pulse stimulation to postsynaptic dopamine receptors, i.e., fluctuant stimula-tion[8]. Therefore, based on drug delivery approach and frequency[9-10], the present study established a dyskine-sia model via an intraperitoneal injection of L-dopa once a day.
In addition to the loss of dopaminergic neurons in PD, expression of the dopamine transporter (DAT) in the presynaptic membrane is also reduced. In addition, DAT levels may indirectly reflect quantity and function of dopaminergic neurons in the substantia nigra-striatum circuit. Therefore, DAT is considered to be a marker of dopaminergic neurons[11-12]. With the development of neuroimaging techniques, DAT functional imaging can be used for early clinical diagnosis and the study of PD[13-15], as well as the evaluation of drugs for the treatment of PD[16]. Moreover, the effects of dopaminergic drugs on brain DAT expression remain controversial. Imaging studies have shown that DAT density reduction following large-dose L-dopa treatment in PD patients is faster than in non-treated PD patients[17]. In addition, L-dopa and pramipexole (a DAT agonist) have been shown to downregulate DAT in a study using 11C-RTI-32 PET[18]. Nevertheless, these studies observed only the effect of drug on DAT, and the correlation between DAT expres-sion and drug therapeutic effects, as well as the side effects of drugs, remains unclear.
The present study established PD models through the use of 6-hydroxydopamine (6-OHDA) to explore the ef-fects of L-dopa on dopaminergic neurons in the brain through the use of radioactive γ-counting and the DAT imaging using 99mTc-TRODAT-1 as an imaging agent and to identify the therapeutic and side effects of L-dopa.

MATERIALS AND METHODS
 
Design
A randomized, controlled, animal study.
Time and setting
The experiment was performed at the Laboratory of Department of Nuclear Medicine, Soochow University from April 2006 to June 2007.
Materials
A total of 34 healthy, male, Sprague Dawley rats, aged 4–5 months and weighing 210–240 g, were provided by the Laboratory Animal Center of Soochow University Medical School (No. SYXK(su)2007-0035). The rats were housed at 18–25 °C with 60%–70% humidity and free access to water and food. The experimental proce-dure was in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the Peo-ple’s Republic of China[19].
Reagents and instruments are listed as follows:

Methods
Establishment of PD model
Rats were anesthetized by intraperitoneal injection of 4% chloral hydrate (10 mL/kg) and placed in a stereotaxic frame (upper incisor plane was 2.4 mm below the biau-ricular line). Following topical disinfection, a medial inci-sion was made at the scalp and through the subcutane-ous tissue to completely expose the anterior fontanel, which was rinsed with 3% H2O2 to expose bregma. Ac-cording to the stereotaxic atlas of the rat brain[20], the optimal three dimensional coordinates of the right medial forebrain bundle were identified: 0.2 mm anterior to the bregma, 2.5 mm lateral to the midline and 7.5 mm below the dura mater. The cranium was drilled (1–2 mm di-ameter) to the dura mater at the corresponding sites, and 4 μL 6-OHDA/0.2% ascorbic acid in a saline solution was injected. The holes were plugged with bone wax, and the skin was sutured and disinfected. Following model es-tablishment, the rats were housed and treated with anti-biotics for 3 consecutive days[21-22].
At 3–4 weeks after 6-OHDA injections, the animals were subcutaneously injected with 0.5 mg/kg apomorphine and tested for drug-induced rotational behaviors. The number of rotations (r) during a 30-minute period was recorded, and rats with rotations ≥ 7 r/min were consid-ered to be successful injury models[23].
Animal grouping
A total of 21 PD rats were screened at the end of 4 weeks following injury and randomly assigned to L-dopa (n = 15) and model (n = 6) groups. The L-dopa group rats were injected with 1.5–2.0 mL L-dopa, 20 mg/kg per day (i.p.), for 4 consecutive weeks. The model group rats were injected with 2 mL normal saline (i.p.) at the same time points for 4 consecutive weeks. The remaining four normal rats were injected with normal saline at the same coordinates as the sham-surgery group.
Abnormal involuntary movement scores 
AIM scores of PD rats were assessed twice weekly dur-ing L-dopa or normal saline injection[24]. Total AIM scores were assessed at 140 minutes after medication, as well as at four subsequent 30-minute periods, which included four parameters (upper limb, orofacial part, axiality, and motion). Moreover, each parameter was classified into five grades (0–4) according to symptom and severity: 0: no symptom; 1: occasional symptoms; 2: frequent symptoms; 3: continuous symptoms until stimulation; 4: continuous symptoms that were not terminated by sti-mulation. Theoretically, the highest rat score following one dose of medication should be 64.
99mTc-TRODAT-1 imaging in the rat brain
All rats were subjected to DAT functional imaging. 99mTc-TRODAT-1 was prepared[25] by adding 1 mL 99mTcO4 solution to the developing kit, followed by 100 °C for 30 minutes to label 99mTc-TRODAT-1 (label rate > 90%). After 1 month of L-dopa treatment, treatment was suspended for 2 days, and 0.2 mL 99mTc-TRODAT-1 was injected into the rats through the tail vein. Two hours later, using a portable γ-camera, the corpora striatum position in the rat brain was dynamically determined and an im-age was statically acquired for 1 minute.
DAT radioactive count assay
The rats were immediately sacrificed following image acquisition, and the corpora striata were resected and placed on ice. The wet weight of corpora striatum was recorded, and radioactive counts per gram in normal or lesioned brain tissues were quantified using a radioactive γ-measuring device. The ratio (R value) of specific ra-dioactive uptake in the corpora striata (left/right) was used to evaluate DAT quantity and function according to radioactive counting for each gram of brain tissue[26]. 
Main outcome measures
Total AIM scores and dyskinesia symptoms in PD rats; DAT image and radioactive γ quantification of 99mTc-TRODAT-1 in the brain; ratio of specific radioactive uptake in the corpora striata (left/right).
Statistical analysis  
Data were statistically processed using SPSS 10.0 software (SPSS, Chicago, IL, USA). All data were ex-pressed as Mean ± SD. The t-test was used to compare radioactive counts of each gram of brain tissue between the left and right corpus striata. Differences among groups were compared using one-way analysis of vari-ance and the LSD-t test was used to compare pairs. P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
Of 30 rats, successful model establishment failed in five. Therefore, 25 rats were included in the final analysis.
Performance of dyskinesia and AIM scores
In the L-dopa group, 9 out of 15 PD rats presented with dyskinesia 10–19 days after L-dopa treatment, with an incidence rate of 60%. According to AIM symptoms, the L-dopa group was assigned to AIM (n = 9) and non-AIM (n = 6) groups. AIM was characterized by rotary motion to the damaged side and mechanical movement. Rotary motion was similar to motion induced by apomorphine, and mechanical movements included rhythmical striking of the upper limb opposite to the damaged side, bending of the neck and body to the side opposite to the damage, unsteady posture of the seriously symptomatic rats, re-petitive chewing, involuntary licking on the opposite side, irritability, and increased movement.
The AIM scores of five rats were > 20 (serious), and four scored in the range 8 –16 (mild). AIM types and severity varied amongst the rats. However, AIM symptoms were similar in an individual rat following each dose of medi-cation, and symptoms commonly occurred after 1 to 2 hours.
Rats in the model and sham-surgery groups did not ex-hibit AIM manifestations. Results revealed that AIM was frequently observed in seriously lesioned rats from the L-dopa group (rotational behavior induced by apomor-phine, > 10 r/min).
99mTc-TRODAT-1 imaging in the rat brain 
DAT images revealed that 99mTc-TRODAT-1 specifically labeled the corpora striata and was symmetrically dis-tributed with a clear outline and low background, which was in accordance with normal DAT distribution. Moreover, distribution was sequential and predomi-nantly observed in corpora striata at 2–3 hours (Figure 1A).
In the model group rats, DAT images in the corpora stri-ata of PD rats revealed asymmetrical distribution. The radio-ligand outline in the left hemisphere (normal region) was normal, but specific radioactivity uptake was obvi-ously reduced in the right hemisphere (lesioned side). Moreover, the image outline was irregular (Figure 1B).
Following 1 month of L-dopa treatment, the radio-ligand accumulated in the normal corpora striata (left) area in the AIM rats, which was significantly denser than the lesioned (right) side (Figure 1C).
However, in non-AIM rats, radio-ligand accumulation in the corpora striata was similar following L-dopa treat-ment (Figure 1D).

Ratio of specific DAT radioactivity uptake in the rat brain
The binding rate (BR) of 99mTc-TRODAT-1 to DAT in the corpora striata was close to 1 in the sham-surgery group, and the difference between the two hemispheres was not significant (P > 0.05).
In the model group and AIM rats (following L-dopa treatment), specific radioactivity uptake in the normal hemisphere (left) was significantly greater than in the lesioned side (right, P < 0.01).
Specific radioactivity uptake in the lesioned hemisphere (right) was significantly reduced in the AIM group com-pared to the model group (P < 0.05; Table 1).
Differences of specific radioactivity uptake in the corpora striata in the non-AIM rats were reduced (Figure 1D, Table 1).
In the model, AIM, and non-AIM groups, specific radioac-tivity uptake was significantly greater in the normal hemisphere compared to the lesioned hemisphere (P < 0.01 or P < 0.05). R value was the greatest in the AIM group (P < 0.01, or P < 0.05; Table 1).

DISCUSSION

PD predominantly involves the substantia nigra and corpora striata. Animal models allow for the further study of PD, and the 6-OHDA-induced unilateral lesion has become a common method for establishing an animal model of PD[22-23]. 6-OHDA injection at different sites in the substantia nigra-corpus striatum system can be util-ized to establish a rat model of PD, and the use of the medial forebrain bundle as an injection point has been shown to have a success rate of 70%[27-28]. This process involves a pathological pathway with chronic progressive changes, which is similar to PD occurrence and devel-opment and partially simulates progressive PD[29]. Moreover, the state of the model rats was stable, and drug-induced rotary behavior was stable during the entire experiment.
DAT indirectly reflects the number and function of sub-stantia nigra-corpus striatum pathway dopaminergic neurons[11-12]. DAT expression is predominantly localized in the corpus striatum. After 1-3 hours, 99mTc-TRODAT-1 accumulation in the corpus striatum, rather than in other nuclei, indicated specific binding of 99mTc-TRODAT-1 to DAT. In addition, 99mTc-TRODAT-1 labeling objectively and sensitively reflects DAT regional distribution and function and assists in evaluation of DAT function and density in the corpus striatum[30]. Using a portable γ-camera, DAT imaging was performed 2 hours after 99mTc-TRODAT-1 injection. Radio-ligand accumulation was specifically observed in the corpus striatum. Be-cause of individual differences in γ quantification due to radioactive time limitations, the ratio of specific radioac-tivity uptake in the left and right corpora striata was used to further evaluate DAT alterations in the corpora striata.
 

Results from the present study demonstrated that DAT-specific uptake to the ligand was similar between left and right corpora striata in normal rats, and DAT images of bilateral brains displayed radio-ligand accu-mulation in the corpora striata, with a symmetrical dis-tribution and clear outline. The amount of DAT expres-sion in the lesioned hemisphere (right) decreased in the model group, and 99mTc-TRODAT-1-specific uptake was also significantly reduced. Moreover, radio-ligand accu-mulation was diminished in the right corpus striatum, which indicated that DAT radio-imaging with 99mTc-TRODAT-1 is a sensitive method to assess DAT and dopaminergic neuronal function, as well as diagnose PD. The use of a portable γ-camera to measure DAT levels in the rat brain is a novel method. It is simple, avoids animal sacrifice, and results in clear images.
As a dopamine precursor, L-dopa attenuates PD symp-toms by reconstructing normal dopamine neurotrans-mission. However, the incidence of dyskinesia is 30%–80%[31-32]. Studies have demonstrated that L-dopa-induced dyskinesia is associated with postsy-naptic membrane dopamine D1 receptor supersensitivity, and presynaptic membrane DAT alterations have been shown to be more obvious[33]. In the present study, PD rats undergoing L-dopa treatment were subdivided into AIM and non-AIM groups according to AIM occurrence. Results from the present study revealed different effects of L-dopa on brain DAT expression, even at the same dose. (1) There were no differences in DAT-specific ra-dioactivity uptake in the left hemisphere between AIM and non-AIM groups and the sham-surgery group; im-ages revealed radio-ligand accumulation in the left cor-pus striatum (normal hemisphere) in the model, AIM, and non-AIM groups. (2) Specific radioactivity uptake in AIM PD rats was significantly reduced compared with model rats, and radio-imaging displayed significantly reduced radio-ligand accumulation in the lesioned corpus striatum (right hemisphere). (3) Specific radioactivity uptake in the non-AIM rats was slightly increased, and differences in radio-ligand accumulation between left and right corpora striata was decreased. These results demonstrate that L-dopa has no effect on dopaminergic neurons in the normal substantia nigra-corpus striatum, but is toxic to dopaminergic neurons in PD rats with AIM, which leads to decreased DAT number and function. Moreover, DAT upregulation in non-AIM rats suggested the possibility that dopaminergic neurons are capable of regeneration and repair.
The effects of L-dopa on dopaminergic neurons remain controversial[4-5]. Results from the present study sug-gested that L-dopa exhibited toxicity on dopaminergic neurons in some PD rats, which was characterized by decreased DAT number and function. This further at-tenuated the capacity of dopaminergic neurons to alter synaptic space dopamine concentrations and to alter fluctuant stimulation accepted by postsynaptic dopamine receptors, which ultimately promoted dyskinesia. In the L-dopa group, DAT expression was significantly de-creased following L-dopa treatment in some PD rats, in particular the ratio of specific radioactivity uptake by the corpora striata was significantly greater than in the other groups. In addition, the incidence of dyskinesia was 60%, indicating that DAT variations contribute to L-dopa-induced dyskinesia. Moreover, results suggest that discontinuous exogenous L-dopa-induced fluctuant stimulations accepted by postsynaptic dopamine recep-tors plays an essential role in dyskinesia occurrence. Dyskinesia occurred in PD rats 10–19 days after L-dopa treatment, but did not occur in the model group. More-over, PD rats with severe lesion were more susceptible to dyskinesia following L-dopa treatment. Results demon-strate that dyskinesia correlated with PD severity and fluctuant medication, and that decreased DAT amount or function may be an important mechanism of dyskinesia.
In addition, following L-dopa treatment, differences in specific radioactivity uptake of DAT between the hemispheres were reduced, possibly because L-dopa promoted dopaminergic neuronal recovery and specific radioactivity uptake of DAT increased in the lesioned hemisphere, or because L-dopa is toxic to dopaminergic neurons and downregulates specific radioactivity uptake. Nevertheless, specific radioactivity uptake of DAT was increased in the lesioned hemisphere of non-AIM rats compared with the model group. Therefore, the former possibility is more plausible and requires further verification. Appropriate doses of L-dopa can promote repair in damaged dopaminergic neurons. Due to limited radioactivity time and the small number of experimental animals used in this study, further studies are needed to verify these results.

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 (Edited by Yan LR, Yang JP/Su LL/Song LP)