Long-term Levodopa Treatment Accelerates the Circadian Rhythm Dysfunction in a 6-hydroxydopamine Rat Model of Parkinson's Disease

Abstract

Background: Parkinson's disease (PD) patients with long-term levodopa (L-DOPA) treatment are suffering from severe circadian dysfunction. However, it is hard to distinguish that the circadian disturbance in patients is due to the disease progression itself, or is affected by L-DOPA replacement therapy. This study was to investigate the role of L-DOPA on the circadian dysfunction in a rat model of PD.

Methods: The rat model of PD was constructed by a bilateral striatal injection with 6-hydroxydopamine (6-OHDA), followed by administration of saline or 25 mg/kg L-DOPA for 21 consecutive days. Rotarod test, footprint test, and open-field test were carried out to evaluate the motor function. Striatum, suprachiasmatic nucleus (SCN), liver, and plasma were collected at 6:00, 12:00, 18:00, and 24:00. Quantitative real-time polymerase chain reaction was used to examine the expression of clock genes. Enzyme-linked immunosorbent assay was used to determine the secretion level of cortisol and melatonin. High-performance liquid chromatography was used to measure the neurotransmitters. Analysis of variance was used for data analysis.

Results: L-DOPA alleviated the motor deficits induced by 6-OHDA lesions in the footprint and open-field test ( P < 0.01, P < 0.001, respectively). After L-DOPA treatment, Bmal1 decreased in the SCN compared with 6-OHDA group at 12:00 ( P < 0.01) and 24:00 ( P < 0.001). In the striatum, the expression of Bmal1, Rorα was lower than that in the 6-OHDA group at 18:00 (P < 0.05) and L-DOPA seemed to delay the peak of Per2 to 24:00. In liver, L-DOPA did not affect the rhythmicity and expression of these clock genes (P > 0.05). In addition, the cortisol secretion was increased (P > 0.05), but melatonin was further inhibited after L-DOPA treatment at 6:00 (P < 0.01).

Conclusions: In the circadian system of advanced PD rat models, circadian dysfunction is not only contributed by the degeneration of the disease itself but also long-term L-DOPA therapy may further aggravate it.

Figure 1

Motor deficiency in 6-OHDA rats and L-DOPA improved it. (a) A simple illustration of the experimental procedure. (b) The body weight measurement (n = 14 for each group). (c) Rotarod test (n = 14 for each group, *P < 0.01). (d) Footprint test (n = 14 for each group, ^†P < 0.001) and (e) Open field test (n = 14 for each group, *P < 0.01, †P < 0.001). (f and g) Loss of TH neurons in the substantia nigra of 6-OHDA lesioned rats, as revealed by western blotting (n = 3 for each group, *P < 0.01) and immunofluorescence (n = 2 for each group). 6-OHDA: 6-hydroxydopamine.

Figure 2

QPCR analysis of the mRNA level of clock genes in the SCN (a-d), striatum (e-h) and liver (i-l). The left side of the brain was used for QPCR analysis, n = 3 for each time point, equals to n = 12 in each group. *P < 0.05, †P < 0.01, ‡P < 0.001 for 6-OHDA group versus sham group; §P < 0.05, ||P < 0.01 for 6-OHDA group versus L-DOPA group. 6-OHDA: 6-hydroxydopamine; SCN: Suprachiasmatic nucleus; QPCR: Quantitative polymerase chain reaction.

Figure 3

The concentrations of cortisol (a) and melatonin (b) in plasma were detected by ELISA method and expressed as ng/ml. n = 3 for each time point, equals to n = 12 in each group. *P < 0.01 for 6-OHDA group versus L-DOPA group. 6-OHDA: 6-hydroxydopamine; ELISA: Enzyme-linked immunosorbent assay.

Figure 4

Neurotransmitters content of DA, DOPAC, 5-HIAA were analyzed in the striatum (a-d) with HPLC, and the results were expressed as ng/mg wet tissue. The right side of the brain was used for HPLC analysis, n = 3 for each time point, equals to n = 12 in each group. *P < 0.05 for 6-OHDA group versus sham group. 6-OHDA: 6-hydroxydopamine; HPLC: High performance liquid chromatography.