Establishment Of Rat Model Of Levodopa Induced Dyskinesia And Study Of C-fos And ENK Expression In The Striatum Of The Model

0bjective To establish rat model of the levodopa-induced dyskinesia(LID)and examine the behavioral characteristics.also to study behavioral characteristics and the expression level of enkephalin (ENK) and c-fos in striatum,the functional alteration of the basal ganglia circuit in the pathogenesis of levodop-induced dyskinesias(LID),especially the function of indirect path in levodop-induced dyskinesias.Methods Hemi-parkinsomism rat models were made by 6-OHDA microinjection stereotaxically.The sucessful PD models were randomly applied into LID inducing group (n=22)and PD control group(n=22),20 normal rats were used as the normal group(n=20).The animals from the LID group and normal control group were treated with chronic intermittent l-dopa+benseride injection for 21 days,while the animals from the PD control group received equal volume saline treatment . The abnormal involuntary movement behavior was recorded by video and the abnormal involuntary movement(AIM) scores were estimated using the rat AIM rating scale.The limb movement function and the rotation behavior were also examined. Two weeks after the conclusion of l-dopa treatment,the animals in the LID inducing group received l-dopa injection again to check the reappearance of AIM,those showed AIM were considered sucessful LID model. Then six animals from each group were killed and the brain were colledted and prcessed for immunohistochemical staining to determine the expression level of c-fos and ENK in striatum .The behavior response of LID rats to D2 dopamine receptor agonist quinpirole and antagonist spiperone were checked to identify the pharmaceutical characteristics.Twelve LID rats were distributed into 3 groups: L-dopa(50mg/kg)+Benserazide (10mg/k)group(n=4),D2 dopamine receptor agonist quinpirol(e2mg/kg)group(n=4),L-dopa(50mg/kg)+Benserazide (10mg/kg)+ D2 dopamine receptor antagonist spiperone(2mg/kg)group(n=4),LID rats from each group received single acute drug injection as indicated above ,then AIM scores were estimated for 3 hours after drug injection. The limb movement function and the rotation behavior were also examined.The animals were killed and the brain specimen were collected immediately after behavior assesment .The paraffin sections including striatum area were made and studied by immunohistochemistry to determine the expression level of ENK and c-fos in striatum.Results 1.Establishment of LID model and its movement behavior characterics: Forty-four successful PD models were obtained from 80 rats. Treatment with levodopa in PD rats gradually induced abnormal involuntary movement(AIM), 18 of 22 PD rats which received L-dopa injection developed AIM, the ratio is 82%. the AIM score of the third day of L-dopa treatment was (31.13±0.32),and the scores of the 12th,15th,18th,21th day were (44.02±0.85),(47.47±0.65),(55.08±0.20),(59.10±1.01) respectively, the AIM scores of this group were significantly higer than that of the normal control group and the PD control group.Contralateral forepaw performance was significantly improved and the rotational behavior increased along with levodopa treatment.2.Effects of acute D2 receptor agonist and antagonist treatment on the movement behavior of LID rats : Two weeks after eatablishment of LID models, L-dopa treatment could still induce AIM (AIM score before treatment: 9.93±0.34,AIM score after treatment: 48.17±1.26,P<0.01);acute D2 receptor agonist stimulation could induce AIM in the LID rats, the AIM score increased from 10.23±0.52(before treatment)to 48.24±1.27(after treatment), the AIM scores were not significantly different from L-dopa treated group;while D2 receptor antagonist incombination with L-dopa stimulation could induce AIM(AIM score before treatment: 10.18±0.53,AIM score after treatment: 41.93±2.27,P<0.01),but the combined drug treatment induced AIM score was significantly lower than that of L-dopa treatment alone(P<0.05),suggesting D2 receptor antagonist can alleviate L-dopa-induced AIM.These results indicate this model is similar in response to pharmaceutical stimulation to human PD patients.3.Expression of c-fos and ENK in the striatum of LID rats : The expression level of c-fos in the striatum of LID group was significantly higher than that of PD control group as well as normal control group. The expression level of ENK in the striatum of LID group was also significantly higher than that of normal group, but there was no significant variability compared with PD group. In the LID group ,ENK and c-fos co-expression could be observed in some cells in striatum ,but in the PD group and the normal control group we hardly observe this co-expression phenomenon.4.Influence of acute D2 receptor agonist and antagonist treatment on the Expression of c-fos and ENK in the striatum of LID rats: The expression level of ENK and c-fos in the striatum of rats received acute(L-dopa+Benserazide) injection was significantly higher than that of LID group without pre-drug treatment.The expression levels of ENK and c-fos in the striatum of D2 receptor agonist group had no significant variability compared with LID group without pre-drug treatment and the L-dopa+Benserazide group ; the expression level of ENK in the striatum of D2 receptor antagonist group was significantly higher than that of LID group without pre-drug treatment and L-dopa+Benserazide group.Conclusion Chronic treatment with levodopa intermittently can induce AIM in hemi-parkinsomism rats. This rats showed similar behavioral and pharmaceutical characterization to PD patients with levedopa induced dyskinesia and can be used as a LID model.High levels of ENK and c-fos in striatum indicate that disturbed activities of striatum neurons was engaged in the production of LID. L-dopa induced c-fos expression in the striatum of LID animal was significantly enhanced by co-administration with D2 recetor antagonist suggested D2 receptor mediated tonic inhibition of indirect pathway of basal ganglia play a role in the LID mechanism.