Embryonic ventral mesencephalic grafts improve levodopa-induced dyskinesia in a rat model of Parkinson's disease

Brain (2000), 123, 1365–1379 Embryonic ventral mesencephalic grafts improve levodopa-induced dyskinesia in a rat model of Parkinson’s disease Chong S. Lee,1 M. Angela Cenci,2 Michael Schulzer1 and Anders Björklund2 1Neurodegenerative Disorders Centre, Vancouver Hospital and Health Sciences Centre, Vancouver, Canada and 2Wallenberg Neuroscience Center, University of Lund, Lund, Sweden Correspondence to: Dr Chong S. Lee, Neurodegenerative Disorders Centre, Purdy Pavilion, 2221 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 E-mail: Summary We investigated the role of dopamine neurons in the manifestation of levodopa-induced dyskinesia in a rat model of Parkinson’s disease. Daily treatment with a subthreshold dose of levodopa gradually induced abnormal involuntary movements (AIM) in 6-hydroxydopaminelesioned rats, which included stereotypy and contraversive rotation. After 4 weeks of levodopa treatment, rats with mild and severe AIM were assigned to two treatment subgroups. The graft subgroup received embryonic ventral mesencephalic tissue into the striatum, whilst the sham-graft subgroup received vehicle only. Rats continued to receive levodopa treatment for 3 months post-graft. Brain sections at the level of the basal ganglia were processed for autoradiography using a ligand for dopamine transporter, and in situ hybridization histochemistry for mRNAs encoding postsynaptic markers. Levodopa-induced AIM significantly improved in grafted rats. The severity of AIM correlated inversely with the density of dopamine nerve terminals in the striatum (P < 0.001), with almost no AIM when the density of dopamine nerve terminals was >10–20% of normal. Embryonic dopamine neuronal grafts normalized not only mRNA expression for preproenkephalin (PPE) in the indirect pathway, but also mRNA expression for prodynorphin (PDyn) in the direct pathway, which was upregulated by levodopa treatment. AIM scores correlated linearly with expression of PPE mRNA in the indirect pathway (P < 0.001) and also with PDyn mRNA in the direct pathway (P < 0.001). We conclude that embryonic dopamine neuronal grafts may improve levodopa-induced dyskinesia by restoring altered activities of postsynaptic neurons, resulting not only from dopamine denervation, but also from levodopa therapy, provided that the density of striatal dopaminergic nerve terminals is restored above a ‘threshold’ level. Keywords: Parkinson’s disease; 6-OHDA; levodopa-induced dyskinesia; embryonic dopamine neuronal graft Abbreviations: AIM ⫽ abnormal involuntary movements; GAD ⫽ glutamic acid decarboxylase; ISHH ⫽ in situ hybridization histochemistry; 6-OHDA ⫽ 6-hydroxydopamine; PDyn ⫽ prodynorphin; PPE ⫽ preproenkephalin; VM ⫽ ventral mesencephalic Introduction Various types of dyskinesias may manifest in patients with Parkinson’s disease. Certain dyskinesias are provoked by low or intermediate levels of dopaminergic stimulation (Marsden et al., 1982). However, the most common type of dyskinesia appears following administration of levodopa, in the form of chorea or dystonia, at peak dose or throughout the ‘on’ time. This levodopa-induced, peak dose dyskinesia presents in 30–80% of patients who are treated with levodopa, and increases in frequency and severity with progression of disease and duration of treatment (Marsden et al., 1982; Nutt, 1990). Since the early stage of the levodopa era, it has been recognized that levodopa-induced dyskinesia generally © Oxford University Press 2000 occurs in patients with a good therapeutic response to levodopa (Cotzias et al., 1967); it appears only after chronic treatment with levodopa, not with the first dose of levodopa; and it seems to appear earlier and be worse on the more severely affected side in asymmetrically affected patients (Mones et al., 1971; Kempster et al., 1989; Nutt, 1990). These clinical observations, in agreement with animal studies (Bedard et al., 1986; Clarke et al., 1989), indicate that both chronic levodopa therapy and nigrostriatal dopaminergic denervation play an important role in the pathogenesis of levodopa-induced dyskinesia. In an earlier experiment, we studied the effects of levodopa 1366 C. S. Lee et al. treatment in the rat with 6-hydroxydopamine (6-OHDA) lesions, in which the severity of dopaminergic denervation was static (Cenci et al., 1998). In this study, we explored the role of dopamine neurons in the pathogenesis of levodopainduced dyskinesia using a grafting technique to increase the density of dopaminergic nerve terminals in the striatum. We report here our observations that suggest therapeutic effects of intrastriatal embryonic dopamine neuronal grafts on levodopa-induced dyskinesia. Material and methods Female Sprague–Dawley rats, weighing 200–250 g at the time of lesion surgery, were given unilateral stereotaxic injections of 6-OHDA in the right ascending mesostriatal dopamine bundle. Two injections of 7.5 and 6 µg of freebase 6-OHDA (3 µg/µl in 0.02% ascorbate in saline) were given over 3–4 min under Equithesin anaesthesia [3 ml/kg body weight, intraperitoneally (i.p.)] at the following coordinates (in millimetres, relative to bregma and the dural surface): A ⫽ ⫺4.4, L ⫽ 1.2, V ⫽ 7.8, tooth bar ⫽ ⫺2.4; A ⫽ ⫺4.0, L ⫽ 0.75, V ⫽ 8.0, tooth bar ⫽ ⫹3.4. The toxin was infused at a rate of 1 µl/min, and the cannula was left in place for 3 min before being withdrawn. To determine lesion efficacy, rats were tested 1–2 weeks later with an automated rotometer following an injection of D-amphetamine (5 mg/kg, i.p.). Twenty-eight rats that displayed ⬎7 ipsilateral rotations/min for 90 min were selected for the study. Grafting Pre-graft induction period Six to eight weeks after 6-OHDA lesions, the rats started to receive 8 mg/kg of L-dopa methyl ester (Sigma, St Louis, Mo., USA), mixed with 15 mg/kg of benserazide (kindly provided by Hoffmann-La Roche, Basel, Switzland), i.p. twice daily for the first 3 weeks. In the fourth week, the dose of L-dopa methyl ester was increased to 12 mg/kg (plus 15 mg/kg of benserazide) to increase the number of rats with severe abnormal involuntary movements (AIM). Behavioural tests for AIM were carried out twice a week. At the end of the induction period, 23 rats that had developed mild to severe AIM were divided into two groups based on their final AIM scores. Group A included 13 rats with severe AIM (AIM score ⬎20), and group B included 10 rats with mild AIM (AIM score 艋20). The rats in each group were assigned to the graft and the sham-graft subgroups, at random, subject to approximately equal average AIM scores in the two subgroups. 14-day-old rat embryos (crown–rump length 11 mm) of the same strain in the form of a cell suspension (Bjorklund et al., 1983). Each recipient rat received two 2 µl deposits of the graft suspension into the dopamine-denervated striatum at the following coordinates (in millimetres, relative to bregma and the dural surface, and with the tooth bar set 2.3 mm below the interaural line): (i) A ⫽ (...truncated)