Auricular acupuncture plays a neuroprotective role in 6-hydroxydopamine-induced Parkinson's disease in rats

Abstract

Background: Parkinson's disease (PD) is the second-most common neurodegenerative disease. Currently, PD treatment is symptomatic and involves the use of dopamine-based therapies. This study investigated auricular acupuncture on motor and cognitive abilities in rats with 6-OHDA-induced PD.

Methods: A PD rat model was established by bilaterally injecting 6-OHDA into the lateral dorsal striatum. Then, 2- or 15-Hz auricular electroacupuncture (EA) was applied to the auricular CO15 and CO12 points bilaterally for 20 min three times a week for four consecutive weeks.

Results: Both the latency to fall and rest time of the open field test in the EA15 group were greater than in the 6-OHDA group (p < 0.001 and p < 0.05). The time spent on the two-object recognition task was greater in the EA15 group and EA2 group than in the 6-OHDA group (p < 0.01 and p < 0.05). More tyrosine hydroxylase (TH)-positive neurons and fibers were noted in the dorsolateral striatum and substantia nigra (SN) (all p < 0.05). TH expression in the SN was greater in the EA15 group than that in the 6-OHDA group (p < 0.05), while α-synuclein expression in the SN was stronger in the 6-OHDA group than in the EA15 group (p < 0.05). The l-DOPA level in the striatum was higher in the EA15 group than in the 6-OHDA group (p < 0.05).

Conclusion: According to the results, rats with 6-OHDA-induced PD may benefit from auricular EA in terms of motor and cognitive behavior as well as neuroprotection.

Keywords: 6-Hydroxydopamine; Auricular acupuncture; Parkinson's disease; Tyrosine hydroxylase; α-synuclein.

Graphical abstract

Fig. 1

Flowchart of experiments. First, we assessed the rats' behaviors by performing the rotarod test, open field test (OPF), and two-object recognition task (ORT) prior to 6-hydroxydopamine (6-OHDA) injection in the bilateral dorsal striatum. Then, 7 days after 6-OHDA injection (first week), 2- or 15-Hz auricular electroacupuncture (EA) was applied on Monday (1), Wednesday (3), and Friday (5) in the second (2nd), third (3rd), fourth (4th), and fifth (5th) weeks after 6-OHDA administration. The rats' behaviors were then assessed again by performing the rotarod test on the Saturday of the fifth week, the OPF on the Monday of the sixth (6th) week, the ORT on the Tuesday of the sixth week, and blood sampling from the heart and cerebrospinal fluid (CSF) collection from the cistern magna for the measurement of α-synuclein on the Wednesday of the sixth week after 6-OHDA administration. Subsequently, the rats were euthanized.

Fig. 2

Effect of auricular electroacupuncture on rotarod test time in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease rats. General motor performance was assessed by a rotarod test, with data displaying the latency to fall. The value presented is relative to the baseline value (before 6-OHDA administration). Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; n = 7/group; one-way ANOVA followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001.

Fig. 3

Effect of auricular electroacupuncture on the parameters of the open field test (OPF) in rats with 6-hydroxydopamine (OHDA)-induced Parkinson's disease. (A) The pictures represented 30-min trajectories of rats at 6 weeks post-surgery. (B) Locomotor activity is assessed by the total distance traveled in OPF. (C) Locomotor activity is assessed by total moving time in OPF. (D) Time of supported rearing in OPF. (E) Time of unsupported rearing in OPF. (F) Total resting time during OPF. The value presented is relative to the baseline value (before 6-OHDA administration). Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; n = 7/group; Kruskal-Wallis test followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001. This perseverative behavior is reflected in representative 10 min trajectories of a sham-treated (G), and a LAC-treated (H) mouse at 3 weeks post-surgery.

Fig. 4

Effect of auricular electroacupuncture on time spent on the two-object recognition task (ORT) in rats with 6-hydroxydopamine (OHDA)-induced Parkinson's disease. (A) The position of two objects and rats' trajectories in ORT. (B) The 20-min recording included exploration time with both familiar and unfamiliar objects. The ΔT value presented is the time calculated for pre- and post-treatment procedures. Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; n = 7/group; one-way ANOVA followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001.

Fig. 5

Effect of auricular electroacupuncture on tyrosine hydroxylase (TH) expression in rats with 6-hydroxydopamine (OHDA)-induced Parkinson's disease. (A–B) Representative photomicrographs of immunohistochemistry for TH-positive staining and quantitation of TH-positive signals in rats' striatum and substantia nigra. (C–D) Western blotting was performed to determine the expression of TH in substantia nigra and striatum of rats. DLS: dorsolateral striatum; DMS: dorsomedial striatum; SN: substantia nigra; Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; n = 7/group; one-way ANOVA followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001.

Fig. 6

Immuno-staining of tyrosine hydroxylase and α-synuclein in the substantia nigra and α-synuclein levels in the substantia nigra, serum and cerebrospinal fluid in 6-hydroxydopamine (OHDA)-induced Parkinson's disease rats. (A) Representative photomicrographs of immunofluorescence for TH and α-synuclein in substantia nigra. The yellow arrows in Fig. 6A showed the colocalization of tyrosine hydroxylase and alpha-synuclein in the substantia nigra. (B) The photomicrographs of immunohistochemistry for α-synuclein positive staining and number of positive cells in rats' substantia nigra (n = 7/group). (C) Western blotting was performed to determine the expression of α-synuclein and α-synuclein phospho-serine129 in substantia nigra of rats (n = 6/group). (D) The α-synuclein level in the serum of peripheral blood and (E) cerebrospinal fluid. CSF: cerebrospinal fluid; SN: substantia nigra; α-synuclein S129: α-synuclein phospho-serine129; Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; one-way ANOVA followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001.

Fig. 7

Effect of auricular electroacupuncture on glial fibrillary acidic protein (GFAP) expression in the striatum and caspase-3 and brain-derived neurotrophic factor expression in the substantia nigra in rats with 6-hydroxydopamine (OHDA)-induced Parkinson's disease. (A) Western blotting analysis of GFAP expression in the striatum. (B–C) Western blotting results of Caspase-3 and BDNF in substantia nigra. SN: substantia nigra; Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; n = 7/group; one-way ANOVA followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001.

Fig. 8

Effect of auricular electroacupuncture on neurotransmitters in the striatum and prefrontal cortex (PFC) in rats with 6-hydroxydopamine (OHDA)-induced Parkinson's disease. Levels of metabolites were determined by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS), including l-DOPA, Dopamine, Glutamate and Serotonine in striatum and PFC. Control: control group; 6-OHDA: 6-OHDA group; EA2: EA2 group; EA15: EA15 group; Data are presented as mean ± S.E.M; n = 7/group; Kruskal-Wallis test and one-way ANOVA followed by LSD post-hoc test. *p < 0.05, **p < 0.01; ***p < 0.001.