Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model

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Experimental Neurology 180 (2003) 92–97

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Brief Communication

Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model Hi-Joon Park,a Sabina Lim,a Wan-Seok Joo,b Chang-Shik Yin,a Hyang-Sook Lee,a Hye-Jung Lee,a Jung-Chul Seo,c Kanghyun Leem,a Yang-Sun Son,a Youn-Jung Kim,a Chang-Ju Kim,a Yong-Sik Kim,b and Joo-Ho Chunga,* a Research Group of Pain and Neuroscience, Kyung Hee University, Seoul 130-701, Korea Department of Pharmacology, College of Medicine, Seoul National University, Seoul 110-799, Korea c Department of Acupuncture and Moxibustion, College of Oriental Medicine, Kyung San University, Kyungbuk 712-715, Korea b

Received 25 February 2002; revised 24 July 2002; accepted 17 October 2002

Abstract Parkinson’s disease (PD) is a chronic neurodegenerative disorder, and it has been suggested that treatments promoting survival and functional recovery of affected dopaminergic neurons could have a significant and long-term therapeutic value. In the present study, we investigated the neuroprotective effects of acupuncture on the nigrostriatal system in rat unilaterally lesioned with 6-hydroxydopamine (6-OHDA, 4 ␮g/␮l, intrastriatal injection) using tyrosine hydroxylase (TH) and receptor for brain-derived neurotrophic factor, trkB, immunohistochemistries. Two weeks after the lesions were made, rats presented with asymmetry in rotational behavior (118.3 ⫾ 17.5 turns/h) following injection with apomorphine, a dopamine receptor agonist (0.5 mg/kg, sc). In contrast, acupunctural treatment at acupoints GB34 and LI3 was shown to significantly reduce this motor deficit (14.6 ⫾ 13.4 turns/h). Analysis via TH immunohistochemistry revealed a substantial loss of cell bodies in the substantia nigra (SN) (45.7% loss) and their terminals in the dorsolateral striatum ipsilateral to the 6-OHDA-induced lesion. However, acupunctural treatment resulted in the enhanced survival of dopaminergic neurons in the SN (21.4% loss) and their terminals in the dorsolateral striatum. Acupuncture also increased the expression of trkB significantly (35.6% increase) in the ipsilateral SN. In conclusion, we observed that only acupuncturing without the use of any drug has the neuroprotective effects against neuronal death in the rat PD model and these protective properties of acupuncture could be mediated by trkB. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Acupuncture; 6-Hydroxydopamine; Substantia nigra; Striatum; Tyrosine hydroxylase; trkB

Introduction Parkinson’s disease (PD) is a chronic neurodegenerative disorder affecting about 1 in 200 people over the age of 55 (Cocker et al., 2001). PD is principally characterized by the selective loss of dopaminergic neurons of the substantia nigra (SN) pars compacta in the ventral midbrain (Blum et

* Corresponding author. Research Group of Pain and Neuroscience, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-gu, Seoul, 130-701, Korea. Fax: ⫹822-968-0560. E-mail address: [email protected] (J.-H. Chung).

al., 2001; Cocker et al., 2001). This loss of SN neurons is responsible for the reduction of dopamine release into the striatum and results in the neurological features of PD including resting tremor, rigidity of the limbs, poor balance, difficulty in initiating movements, and bradykinesia (Blum et al., 2001). Acupuncture has long been employed as a treatment for numerous disorders, and it has been traditionally used to relieve PD-like symptoms (Walton-Hadlock, 1999; Zhuang and Wang, 2000) and to delay the clinical progression of these symptoms (Walton-Hadlock, 1999). Recently, acupuncture has been demonstrated to possess the neurotrophic

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Brief Communication / Experimental Neurology 180 (2003) 92–97

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Table 1 The corresponding locations of human acupoints in rat Group

Acupoint

Location

Acu1

GB34 LR3 LI4 LI11

At point of intersection of lines from the anterior borders to the head of fibula Between first and second metatarsal bones, 0.5 mm proximal to margin of web Between first and second metacarpal bones, 0.3 mm proximal to margin of web At end of lateral transverse elbow crease at middle of connection between biceps tendon and lateral epicondylus of the humerus

Acu2

and neuroprotective effects in some experimental animal models of several pathologic conditions: acupuncture was shown to enhance brain-derived neurotrophic factor (BDNF) mRNA levels in the rat hippocampus after the induction of stress via immobilization (Yun et al., 2002), to elevate glial cell line-derived neurotrophic factor (GDNF) levels in peri-infarct cortex after focal ischemia (Wei et al., 2000), and to augment hippocampal neuronal cell proliferation following maternal separation in rats (Park et al., 2002). In the present study, the neuroprotective properties of acupunctural treatment against PD were investigated in an early stage of PD model produced by stereotaxic striatal injection of 6-hydroxydopamine (6-OHDA).

Materials and methods Animals Adult male Sprague–Dawley rats (200 –220 g) were used in the present study. They were housed at a room temperature (22 ⫾ 3°C) under standard 12-h light/dark cycles (lights on at 07:00 o’clock AM), with unlimited access to

food and water. The experimental procedures were carried out according to the animal care guidelines of the NIH and the Korean Academy of Medical Sciences. Surgery and grouping Rats were anesthetized with equithesin (3 ml/kg, ip) and given a unilateral stereotaxic injection of 20 ␮g 6-OHDA (4 ␮g/␮l) with 0.2 mg/ml L-ascorbic acid into the right striatum (AP 0.7, ML 2.6, DV 4.5; all coordinates represent millimeter adjustments from bregma) at a rate of 1 ␮l/min using a 26-gauge Hamilton syringe (Kim et al., 1998). After the operation, the rats were randomly assigned to one of three groups: (a) the control group (n ⫽ 5); (b) the Acu1 group, with acupuncture at the acupoints GB34 and LR3, known to relieve PD-like symptoms such as muscle and movement disorders in traditional Oriental medicine (Choi, 1991; Korean Association of Meridianology, 2002; Lin and Wang, 1999; Stux and Pomeranz, 1986) (n ⫽ 8, Table 1 and Fig. 1A); and (c) the Acu2 group, with acupuncture at the acupoints LI4 and LI11, known to regulate gastrointestinal functions (n ⫽ 6, Table 1 and Fig. 1A) (Choi, 1991), for comparison with the Acu1 group. The positions of these

Fig. 1. (A) The locations of the acupoints in rats. Acupoints GB34 and LR3 have been traditionally used to relieve PD-like symptoms in Oriental medicine, and acupoints LI4 and LI11 were used for comparison in this experiment. (B) Effects of acupunctural treatment on apomorphine (0.5 mg/kg, sc)-induced rotations in rats following unilateral striatal injections of 6-OHDA. Animals of the Acu1 group, which received acupunctural stimulation at the acupoints GB34 and LR3, showed significant decreases in the net number of rotations, while those of the Acu2 group, which received acupunctural stimulation at the acupoints LI4 and LI11 for comparison, displayed no significant differences in rotational behavior compared to the control group. The sham operation group and the SAcu1 group (sham operation ⫹ acupuncture at GB34 and LR3) did not show any increase in the number of rotations. Data are presented as means ⫾ SEM. *P ⬍ 0.01 compared to the control group.

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acupoints in rats correspond anatomically to their original locations in humans (Park et al., 2002; Koh, 1999; Korean Association of Meridianology, 2002). Clinically, combinations of acupuncture points are often used to relieve various kinds of disorders (Filshie and White, 1998; Stux and Pomeranz, 1986), and thus we utilized two acupoints in each of the acupuncture groups (Acu1 and Acu2) in accordance with the traditional theory of acupuncture (Choi, 1991; Koh, 1999). Animals of the sham operation groups were injected with the same amount of the vehicle (L-ascorbic acid) at the striatum, and they were also randomly assigned to one of two groups: (d) the sham operation group (n ⫽ 5) and (e) the SAcu1 (sham operation ⫹ Acu1) group, with acupuncture at the acupoints GB34 and LR3 (n ⫽ 5). Acupuncture treatment Starting on the day after the injections were given, daily acupunctural treatment was performed between 9:00 and 10:00 o’clock AM for 13 consecutive days. Rats of the acupuncture groups (Acu1, Acu2, and Sacu1) were lightly immobilized, and acupuncture needles were inserted to a depth of 3 mm at their respective acupoints bilaterally, turned at a rate of two spins per second for 15 s, and removed immediately afterward (treatment lasting 60 s). Animals of the control and sham operation groups were also immobilized in a similar fashion for 60 s (Choi, 1991).

for 1 h at room temperature. The sections were then incubated with ABC reagent (Vector Laboratories Inc., Burlingame, CA) for another 1 h at room temperature, washed in PBS, and then incubated for 5 min in 0.1 M Tris–HClbuffered saline (pH 7.5) containing 0.02% diaminobenzidine (DAB) and 0.003% hydrogen peroxide. After the DAB reaction, the tissues were rinsed with PBS, mounted on gelatin-coated slides, air-dried, dehydrated, and coverslipped using mounting fluid. To further observe the neuroprotective effect of acupuncture, immunohistochemistry specific for tyrosine kinase receptor trkB was performed. TrkB immunohistochemistry was performed using anti-rabbit trkB antibody (1:500, Santa Cruz, CA) and biotinylated anti-rabbit IgG (Vector Laboratories Inc, Burlingame, CA) with the same procedures of TH immunohistochemistry. Every fourth section was selected from each brain (level AP, ⫺5.2 ⬃ ⫺5.6 mm in the atlas of Paxinos and Watson), and the number of TH- or trkB-immunoreactive (ir) neurons in the SN was counted from each section using a bright-field microscope (Olympus, Japan) and analyzed using an image analyzer (Optimas Version 6.5, Media Cybernetics, MD). An independent investigator (Dr. Y. J. Kim) evaluated all sections in a blinded manner. All data were presented as mean ⫾ SEM and analyzed by ANOVA with Newman– Keuls post-hoc test.

Observation of rotational behavior

Results

Two weeks after 6-OHDA injection, changes in rotational behavior were assessed by monitoring body rotations induced by apomorphine (0.5 mg/kg, sc) in automated rotometer chambers (Kim et al., 1998). The net number of rotations (contralateral–ipsilateral) was recorded over a time span of 60 min. This behavioral test was performed blindly.

Fig. 1B shows the data from the assessment of apomorphine-induced changes in rotational behavior. Rotational asymmetry was not observed in sham-operated animals (4.0 ⫾ 8.9 turns/h for the sham operation group and ⫺2.8 ⫾ 16.5 turns/h for the SAcu1 group). However, rats of the control group exhibited rotational asymmetry in the direction contralateral to the lesion following apomorphine challenge (118.3 ⫾ 17.5 turns/h). A significant decrease in the net number of rotations was observed in the Acu1 group compared to the control group (14.6 ⫾ 13.4 turns/h, P ⬍ 0.01), but such a decrease was not observed in the Acu2 group (74.3 ⫾ 20.9 turns/h; Fig. 1B). TH immunohistochemistry performed in the present study revealed that unilateral injection of 6-OHDA produces a significant reduction of neuronal population in the SN, with loss of TH-ir terminals in the dorsolateral striatum ipsilateral to the lesion (Fig. 2). When expressed as a ratio of “lesioned” to “intact side,” the number of TH-ir neurons in the 6-OHDA-lesioned side showed a reduction of approximately 45.7% in the control group (Fig. 2). However, as was the case for rotational asymmetry, the number of TH-ir neurons in the ipsilateral SN was increased in the Acu1 group to 78.6% of the unlesioned side. The presence of TH-ir terminals in the dorsolateral striatum ipsilateral to the lesion was also observed in the Acu1 group (Fig. 2). Animals of the Acu2 group showed no significant increase in

Immunohistochemistry On the following day, the animals were anesthetized with pentobarbital sodium (50 mg/kg), and upon reaching a state of complete anesthesia, they were perfused and fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB). Brains were removed from the cranium, postfixed for 2 days, washed in 0.1 M PB, and immersed in a 30% sucrose solution for storage at 4°C prior to sectioning. Brains were frozen using a cryostat, sectioned into 40-␮mthick sections, and processed immunohistochemically for detection of tyrosine hydroxylase (TH) using a mouse monoclonal antibody as described previously (Cocker et al., 2001; Kim et al., 1998), with some modifications. In brief, the sections were incubated in mouse anti-TH antibody (Roche, IN) at a dilution of 1:3000 for 24 h at 4°C. After washing in 0.05 M phosphate-buffered saline (PBS), they were incubated with a secondary antibody (biotinylated anti-mouse IgG, Vector Laboratories Inc., Burlingame, CA)

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Fig. 2. (A) Quantification of TH-immunoreactive (ir) neurons in the SN 2 weeks after unilateral injection of 6-OHDA into striatum. Notice the significant increase in the number of TH-immunopositive neurons in the SN of the Acu1 group, with 6-OHDA-induced lesion and acupunctural treatment at the GB34 and LR3; while such an increase is absent in the Acu2 group, with 6-OHDA-induced lesion and acupunctural treatment at the points known to be ineffective in treating PD-like symptoms. The sham operation and the SAcu1 (Sham operation ⫹ Acu1) groups did not show any decrease in the number of TH-positive neurons in the SN. Data are presented as means ⫾ SEM. *P ⬍ 0.01 compared to the control group. The numbers of TH-ir neurons are as follows: control, 68.0 ⫾ 4.2 (i)/123.2 ⫾ 8.5 (c); Acu1 group, 87.7 ⫾ 5.0 (i)/111.6 ⫾ 3.6 (c); Acu2 group, 66.2 ⫾ 6.5/112.6 ⫾ 2.8 (c); Sham group, 117.5 ⫾ 7.2 (c)/120.4 ⫾ 10.3 (i); and SAcu1 group, 107.0 ⫾ 7.8 (i)/105.3 ⫾ 4.9 (c). “i” represents the ipsilateral side and “c” represents the contralateral side. (B) TH-specific immunohistochemical staining of the SN (a, b, c) and striatum (d, e, f) 2 weeks after intrastriatal infusion of 6-OHDA. (a, d) Control group, (b, e) Acu1 group, (c, f) Acu2 group. Notice the increased number of cell bodies in the SN and of their terminals in the dorsolateral striatum ipsilateral to the 6-OHDA-induced lesion in the Acu1 group compared to the control group. Arrows indicate syringe trajectories formed during striatal 6-OHDA injections. Bar ⫽ 200 ␮m.

TH-positive cells compared to the control group (58.8% of unlesioned side, Fig. 2). TrkB immonohistochemistry revealed that animals of the Acu1 group showed an increase of trkB-positive cells in the SN (35.6% increase compared to the unlesioned side, P ⬍ 0.01, Table 2). Animals of the control and Acu2 groups showed slight increases of trkB-positive cells in the lesioned side of the SN to the unlesioned side 2 weeks after 6-OHDA

injection, but the increases were not statistically significant (P ⫽ 0.14 and 0.09, respectively, Table 2). The sham operation and SAcu1 groups showed no changes in the SN. Discussion PD is a chronic neurodegenerative disorder mainly characterized by massive and progressive degeneration of the

Table 2 The number of trkB-positive cells in the SN per section Operation

Sham operation

6-OHDA injection

a b

Groups

Sham Acupuncture on acupoints GB34 and LR3 Control Acupuncture on acupoints GB34 and LR3 Acupuncture on acupoints LI4 and LI11

P ⬍ 0.01 vs contralateral part of same group. P ⬍ 0.05 vs ipsilateral part of control group.

TrkB-positive cells (mean ⫾ SEM) Unlesioned part

Lesioned part

32.3 ⫾ 4.5 36.5 ⫾ 7.1

34.0 ⫾ 4.5 38.0 ⫾ 8.1

32.9 ⫾ 5.2 33.2 ⫾ 4.3

41.9 ⫾ 3.8 56.8 ⫾ 4.4a,b

33.9 ⫾ 3.8

43.9 ⫾ 3.9

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nigrostriatal dopaminergic system, resulting in abnormal motor behavior (Blum et al., 2001). In animals, intrastriatal administration of 6-OHDA was shown to bring about a progressive degeneration of nigral dopamine neurons like that seen in PD over a course of several weeks (Blum et al., 2001; Cocker et al., 2001; Saver and Oertel, 1994). In the present study, immunohistochemical examination showed that unilateral injection of 6-OHDA into the striatum produces a significant neuronal reduction in the SN, with loss of TH-ir terminals in the dorsolateral striatum ipsilateral to the lesion, as was previously reported (Kim et al., 1998). It has been suggested that 6-OHDA produces free radicals that may initiate a death-cascade in affected neurons (Kim et al., 1998). The 6-OHDA terminal destruction model has been shown to induce apoptotic death in neurons of SN, resulting in a lasting decrease in the number of neurons (Blum et al., 2001). Considering the results of the behavioral and histological examinations undertaken in the present study, acupuncture appears to protect dopaminergic neurons against cell death induced by 6-OHDA. Together with recovery from aberration in rotational behavior, an increase in the number of TH-positive neurons in the SN and the presence of TH-ir terminals in the dorsolateral striatum ipsilateral to the lesion were observed in the Acu1 group. Animals of the Acu2 group, however, showed no significant increase in the number of TH-ir cells compared to the control group. This observation indicates that the enhanced survival of TH-ir neurons seen in the Acu1 group is not related to the stress induced by needling, and this discrepancy between the two groups implies that stimulation at different sets of acupoints will have different therapeutic effects, as one would have expected. TrkB is an essential component of the high-affinity receptor for BDNF (Numan and Seroogy, 1997). BDNF and its receptor are expressed within the mesostriatal system (Numan and Seroogy, 1997; Seroogy et al., 1994) and have been shown to enhance the survival, differentiation, and functional capacity of dopaminergic neurons in vitro and in vivo (Ho¨ glinger et al., 2001). Several previous studies have demonstrated that the degeneration of dopaminergic neurons seen in animal PD models can be attenuated by direct application of BDNF (Shults et al., 1995) or through BDNF gene therapy (Klein et al., 1999; Levivier et al., 1995). In the present study, animals of the Acu1 group showed an increase of trkB-positive cells in the SN compared to the unlesioned side. Those of the control and Acu2 groups showed slight increases of trkB-positive cells in the side of the SN lesioned to the unlesioned side, but the increases were not statistically significant. Lesions induced by 6-OHDA may activate a compensatory cascade of neurotrophic activity including BDNF and trkB within the nigrostriatal system as a physiological response to the loss of dopaminergic neurons in rats (Aliaga et al., 2000; Numan and Seroogy, 1997; Yurek and Fletcher-Turner, 2001) as shown in the control and Acu2 groups. However, the significant increase of trkB in the Acu1 group may involve

neuroprotective action, resulting in the protection of TH-ir neurons in the SN. Our recent reports also demonstrated increases of BDNF by acupunctural treatment in another animal model: electroacupuncture was shown to increase hippocampal BDNF mRNA levels after induction of stress via immobilization (Yun et al., 2002). We studied an acupuncture effect in quite an early stage of PD model (only within 2 weeks), and the long-term effect must be carried out for elucidating its therapeutic effect more clearly.

Conclusion In conclusion, the present study demonstrated the neuroprotective effects of acupuncture on the nigrostriatal dopaminergic system in rats with unilateral 6-OHDA-induced lesions in 2 weeks. As was observed in the Acu1 group, acupunctural treatment of 6-OHDA-treated rats resulted in significant recovery from lesion-induced aberration in rotational behavior. Along with this recovery, acupuncture also prevented the degeneration of dopaminergic cell bodies in the SN and their terminals in the striatum ipsilateral to the lesioned side, as shown from TH immunohistochemistry. These behavioral and histochemical observations are suggestive of neuroprotective effects of acupuncture and a possible use for acupuncture in the treatment of PD.

Acknowledgments We thank Mr. Junhoy Kim and Mr. Sebin Chung for editing the manuscript and Mee-Suk Hong for technical assistance. This work was supported by a research grant from the Ministry of Health and Welfare of Korea (02-PJ9PG1-CO03-0005).

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