Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by L-DOPA?

Abstract

Inflammatory mechanisms are proposed to play a role in L-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates L-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with L-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with L-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented L-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after L-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.

Keywords: COX2; extrasynaptic signalling; neuroinflammation; prostaglandin H-synthase; striatum interneurons; volume transmission.

Figure 1.

Striatal COX2 immunoreactivity in 6-OHDA-hemiparkinsonian rats with l-DOPA-induced dyskinesia. (a–b) Low-magnification photomicrograph showing NeuN (red) and COX2 immunoreactivity (green) in the striatum. There was COX2 immunoreactivity in the lesioned striatum (b, right) and occasional COX2 immunoreactivity in the contralateral one (a, left). (c–n) Photomicrographs of striatal sections showing immunofluorescence staining for COX2 antibody (green; c,f,i and l) in combination with antibodies (red) for neuronal protein (NeuN; d), dopamine-cAMP-regulated phosphoprotein-32 (DARPP-32; g); glial fibrillary acidic protein (GFAP; j), CD11b/c equivalent protein (OX-42; m). Co-localization with COX2 was observed for NeuN and DARPP-32. (e, h, k and n) correspond to merged images. Arrows indicate cells with co-localization. Arrowheads indicate cells with no co-localization. cc, corpus callosum; Cx, cortex; Str, striatum; V, ventricle. Fluorescent images were taken with the same setting. Scale bars: (a,b) = 100 µm; (c,f,i,l) = 50 µm.

Figure 2.

Expression of markers for striatal neurons co-localized with COX2-immunoreactive cells within the dopamine-denervated striatum following l-DOPA treatment. (a–l) Photomicrographs of striatal sections showing immunofluorescence staining with COX2 antibody (green; a, d, g and j) in combination with antibodies (red) for choline-acetyltransferase (ChAT; b), calbindin (CB; e), neuronal nitric oxide synthase (nNOS; h) and parvalbumin (PV; k). Co-localization with COX2 was observed for ChAT and CB. Arrows indicate cells with co-localization. (c,f,i,l) correspond to the merged images. Scale bars, 25 µm.

Figure 3.

Analysis of l-DOPA and 7NI effect on COX2 expression in the 6-OHDA-lesioned striatum of rats. (a) Representative Western blots of striatal COX2 (72 kDa) and α-tubulin (50 kDa, control). (b) Western blotting quantification of striatal protein extracts obtained from 6-OHDA-lesioned rats chronically treated with either VEH + SAL, 7NI + SAL, VEH + l-DOPA or 7NI + l-DOPA. Dashed line indicates the mean of COX2 expression in the contralateral side to the lesion. (c) Quantification of COX2-immunostaining cells in the 6-OHDA-lesioned striatum. VEH + l-DOPA treatment significantly increased COX2 protein expression in the dopamine-denervated striatum (p < 0.05 if compared with (*) all other groups, with (+) VEH + SAL and 7NI + SAL, with (#) VEH + SAL). (d) Graphics showing positive correlation between COX2 expression and abnormal involuntary movements induced by l-DOPA treatment in the hemi-Parkinsonian rats. (e,f and g) Photomicrographs showing COX2-immunopositive neurons in the dopamine-depleted striatum of VEH + l-DOPA (e,f and f’) and 7NI + l-DOPA (g and g’)-treated rats. (f’ and g’) show high magnifications of a COX2 neuron. Arrows indicate an example of a COX2-positive cell. AIMs, abnormal involuntary movements; LD, l-DOPA; 7NI, 7-nitroindazole; SAL, saline; VEH, vehicle; cc, corpus callosum; Cx, cortex; str, striatum; V, ventricle.