The Effect Of The Cortical Electrical Stimulation On The Expression Of The C-fos And ENK In Striatal Neurons Of Rats With Levodopa-induced Dyskinesia

Objective:To establish rat model of the levodopa-induced dyskinesia(LID) and to examine the effects of the cortical electrical stimulation on the expression of the c-fos,enkephalin(ENK) and extracellular regulated kinase1/2(ERK1/2) in straital neurons of rats with levodopa-induced dyskinesia(LID), in order to provide evidence of neural plasticity modulation of corticostriatal pathway in the pathogenesis of LID.Methods:Hemi-parkinsomism rat models were made by 6-OHDA microinjection stereotaxically.The successful PD models were randomly applied into LID inducing group (n=22)and PD control group(n=22),twenty normal rats were used as the normal control group (n=20).The animals from the LID group and normal control group were treated with chronic intermittent L-dopa 30mg/kg+Benseridel0mg/kg injection for 21 days,while the animals from the PD control group received equal volume saline treatment. The abnormal involuntary movement(AIM) behavior was recorded by video and the abnormal involuntary movement scores were estimated using the rat AIM rating scale. The cylinder tests of 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 successful LID model animals. Twenty successful LID models were obtained from twenty-two rats. Ten animals from each group recived corticostriatal electrical stimulations after behavior assessment. At the end of stimulation (about 20 minutes), the animal was anesthetized deeply and perfused slowly with a brief saline prewash followed by 150ml of 4% buffered paraformaldehyde. The brain was collected and processed for immunohistochemical staining. Serial paraffin sections(5-10μm thick) of striatum near the coronal suture(±1 mm) were cut in the frontal planes on a microtome. Immunohistochemical staining was performed to determine the expression level of c-fos, ENK, extracellular regulated kinasel/2(ERK1/2) and phosphorylated-ERK1/2 (p-ERK1/2) in striatum. Five brain sections from each animal (with the same coordinate for different animals) were examined under microscope in high multificatin field,the positive expression cells were counted and the staining intensities were analysed using auto-image analysis system in ten randomly selected field in the putamen for each slice. The coexpression of c-fos and ENK was also analysed by obversing the two successive sections which were immunochemically stained for c-fos and ENK expression respectively.Results1. Establishment of LID model and its movement behavior characterics:44 successful PD models were obtained from 80 rats,the ratio is 55%. Treatment with levodopa in PD rats gradually induced abnormal involuntary movement (AIM),20 of 22 PD rats which received L-dopa injection developed AIM, and the ratio is 91%. 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 cylinder tests showed the limb motor functions of animals was improved by levodopa treatment.The AIM never appeared and no significant changes of the limb motor functions were observed in both PD control group and the normal control group.2. The expression of the c-fos, ENK, ERK1/2, p-ERK1/2 in striatum:Compared with the contralateral side, the expression of the c-fos, ENK, ERK1/2 and p-ERKl/2 in ipsilateral striatum in each group increased significantly (P<0.01) after the electrical stimulation. The striatal expression of c-fos in LID rats (0.67±0.03) was significantly higher than that of PD rats (0.45±0.04) and normal rats(0.38±0.02) (P<0.05).ENK expressional level in LID group(0.21±0.07) was not significantly different from that of the PD group(0.18±0.08) (P>0.05).The expression of ERK1/2 was not significantly different among the three groups (P>0.05),but the p-ERK1/2 expressional level in LID rats (0.17±0.05) was significantly higher than that of the PD group (0.08±0.03) and the normal group (0.07±0.02) (P＜0.01)3.The co-expression of the c-fos and ENK:Co-expression of the c-fos and ENK in striatal neurons was observed in both LID group and PD group,however,the percentage of c-fos postive neurons co-expressing ENK in LID group(10.76%±2.34%) was higher than that of the PD group(7.43%±1.48%) (P＜0.05)Conclusion1.Chronic treatment with levodopa intermittently can induce AIM in hemi-parkinsomism rats. These rats showed behavioral and pharmaceutical characterization similar to levedopa induced peak-dose dyskinesia in PD patients and can be used as a rodent LID model.2. Electric stimulation on motor cortex can activate the corticostriatal pathway, which induced the expression of c-fos in striatum. Enhanced activity of the corticostriatal pathway was involved in the pathogenesis of LID which was suggested by significant higher level of c-fos expression in LID rats in relative to no-dyskinesia PD rats. The corticostriatal eletrical stimulation induced c-fos expression was correlated with phosphorylation modification of ERK1/2 but not with the total ERK1/2 expression, indicating that corticostriatal pathway stimulation induced c-fos expression may mediated by ERK1/2 phosphorylation but not by increasing ERK expression. 3. The increased co-expression of the c-fos and ENK in striatal neurons of LID rats induced by the electric stimulation of coticostriata pathway suggested enhanced activity of the indirect-pathway may be involved in the development of LID.