Neuroprotection of Dopaminergic Neurons by Hominis Placenta Herbal Acupuncture in in vitro and in vivo Models of Parkinson’s Disease Induced by MPP+/MPTP Toxicity

Article information

Acupunct. 2015;32(1):23-36
Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Kyung Hee University
*Corresponding author: Department of Acupuncture & Moxibustion Medicine, Kangnam Korean Hospital, Kyunghee University, 225, Yeongdong-daero, Kangnam-gu, Seoul, 135-501, Republic of Korea, Tel: +82-2-3457-9000, E-mail: ackys@hanmail.net
Received 2015 February 24; Revised 2015 March 03; Accepted 2015 March 04.

Abstract

Objectives

This study was designed to investigate the neuroprotective effects of Hominis-Placenta (HP)on dopaminergic neurons.

Methods

We examined the effect of invitro administration of HP against 1-methyl-4-phenylpyridinium(MPP+)-induced dopaminergic cell loss in primary mesencephalic culture and also used behavioral tests and performed analysis in the striatum and the substantia nigra of mouse brain, to confirm the effect of HP on dopaminergic neurons in an invivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced PD mouse model. Animals were assigned to four groups: (1) Group 1(vehicle-treatedgroup), (2) Group 2(MPTPonlytreated group), (3) Group 3(MPTP+ saline-treated/ST36 group), and (4) Group 4(MPTP+HP-treated/ST36 group). HP at 20 μ L of 48 mg/kg dose was injected at ST36 for 4 weeks at 2-day intervals. MPTP in saline was injected intraperitoneally each day for 5 days from the 8th treatment of HP. We performed the pole test and rota-rod test on the first and seventh day after the last MPTP injection. To investigate the effect of HP on dopaminergic neurons, we performed analysis in the striatum and the substantia nigra of mouse brain after treatment with HP and/or MPTP.

Results

Treatment with HP had no influence on cell proliferation and caused no cell toxicity in PC12 and HT22 cells. Our study showed that HP significantly prevented cell loss and protected neurites against MPP+ toxicity. Although the invivo treatment of HP herbal acupuncture at ST36 showed a tendency to improve movement ability and protected dopaminergic cells and fibers in the substantia nigra and the striatum, it did not show significant changes compared with the MPTP treated group.

Conclusions

These data suggest that HP could be a potential treatment strategy in neurodegenerative diseases such as Parkinson’s disease.

Fig. 1.

Schedule of MPTP injections, HP pharmacoacupuncture treatment and behavioral tests

HP: Hominis Placenta. MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. NS: normal saline.

Fig. 2.

Effects of HP extract on cell viability in PC12 and HT22 cells. PC12 and HT22 neuronal cells were treated with HP extract(15.625–500 μg/mL) for a total 24 h(A and B, respectively)

Cell viabilities were determined using MTT assay and were expressed as a percentage of the normals. Values are indicated as the mean ± SEM of six replicates.

Fig. 3.

Effects of HP extract on toxin-induced neuronal damage in rat primary dopaminergic cells

The rat primary dopaminergic cells were treated with HP extract(1 or 10 μg/mL) for 6 h and with 10 μM 6-OHDA for an additional 18 h(A and B), or with HP extract for 1 h and with 12 μM MPP+ for an additional 23 h(C and D). Fixed cells were stained with anti-TH antibody and visualized with DAB. The numbers of TH-IR neurons were counted(A and C) and the neurite lengths of them were measured(B and D). Data were expressed as a percentage of the normals. Values are indicated as the mean ± SEM of four replicates.

***: p < 0.001 compared with the normal group. **: p < 0.01 compared with the normal group. #: p < 0.05 compared with the MPP+-only treated group.

Fig. 4.

The representative images of TH-IR cells for the effect of HP extract on 6-OHDA-induced toxicity in rat primary dopaminergic cells

The rat primary dopaminergic cells were treated with HP extract(1 or 10 μg/mL) for 6 h and with 10 μM 6-OHDA for an additional 18 h. Fixed cells were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 200 magnification. Normal group(A), 6-OHDA-only treated group(B), 6- OHDA + HP 1 μg/mL treated group(C), and 6-OHDA + HP 10 μg/mL treated group(D).

Scale bar = 100 μm.

Fig. 5.

The representative images of TH-IR cells for the effect of HP extract on MPP+- induced toxicity in rat primary dopaminergic cells

The rat primary dopaminergic cells were treated with HP extract(1 or 10 μg/mL) for 1 h and with 12 μM MPP+ for an additional 23 h. Fixed cells were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 200 magnification. Normal group(A), MPP+-only treated group(B), MPP+ + HP 1 μg/mL treated group(C), and MPP+ + HP 10 μg/mL treated group(D).

Scale bar = 100 μm.

Fig. 6.

Effect of HP pharmacoacupuncture on MPTP-induced hypokinesia in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervals and with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP pharmacoacupuncture. We conducted the rota-rod test after one day(A) and seven days(B) of the last MPTP injection. The time taken for the mice on the rod was recorded.

Values are indicated as the mean ± SEM of seven replicates.

***: p < 0.001 compared with the normal group.

Fig. 7.

Effect of HP pharmacoacupuncture on MPTP-induced bradykinesia in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervals and with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP phar-macoacupuncture. We conducted the pole test after one day(A and B) and seven days(C and D) of the last MPTP injection. The time to turn completely downward(T-turn A and C) and to arrive at the floor(T-LA B and D) were recorded.

Values are indicated as the mean ± SEM of seven replicates.

***: p < 0.001 compared with the normal group. *: p < 0.05 compared with the normal group.

Fig. 8.

Effect of HP pharmacoacupuncture on MPTP-induced dopaminergic neuronal damage in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervalsand with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP. Dopaminergic neurons and fibers in the substantia nigra and the striatum, respectively, were visualised with TH-staining. Quantification of the TH-IR cells was performed by counting the number of TH-IR cells in the substantia nigra(A) and by measuring the optical density in the striatum(B). Data were expressed as a percentage of the normals. Values are indicated as the mean ± SEM of four replicates.

***: p < 0.001 compared with the normal group.

#: p < 0.05 compared with the MPP+-only treated group. SN: substantia nigra. ST: Striatum.

Fig. 9.

The representative images of TH-IR cells in the substantia nigra for the effect of HP pharmacoacupuncture on MPTP-induced toxicity in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervalsand with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP pharmacoacupuncture. Free-floating tissues were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 100 magnification. Normal group(A), MPTP-only treated group(B), MPTP + saline-treated/ST36 group(C), and MPTP + HP pharmacoacupuncture-treated/ST36 group(D).

Scale bar = 200 μm.

Fig. 10.

The representative images of TH-IR fibers in the striatum for the effect of HP pharmacoacupuncture on MPTP-induced toxicity in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervals and with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP pharmacoacupuncture. Free-floating tissues were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 40 magnification. Normal group(A), MPTP-only treated group(B), MPTP + saline-treated/ST36 group(C), and MPTP + HP pharmacoacupuncture-treated/ST36 group(D).

Scale bar = 200 μm.

References

1. Oertel WH, Ellgring H. Parkinson’s disease-medical education and psychosocial aspects. Patient Educ Couns 1995;26:71–9.
2. Wang CC, Lin JD, Chen LL. Personal health maintenance: the perspective of traditional Chinese medicine. Hu Li Za Zhi 2010;57(2):10–5.
3. Kedar NP. Can we prevent Parkinson’s and Alzheimer’s disease? J Postgrad Med 2003;49(3):236–45.
4. Gupta M, Schwarz J, Chen XL, Roisen FJ. Gangliosides prevent MPTP toxicity in mice--an immunocytochemical study. Brain Res 1990;527(2):330–4.
5. Rajendran PR, Thompson RE, Reich SG. The use of alternative therapies by patients with Parkinson’s disease. Neurology 2001;57(5):790–4.
6. Doo AR, Kim ST, Kim SN, et al. Neuroprotective effects of bee venom pharmaceutical acupuncture in acute 1-methyl-4-phenyl-1,2,3,6-tetrahy- dropyridineinduced mouse model of Parkinson’s disease. Neurol Res 2010;32(Suppl 1):88–91.
7. Ferry P, Johnson M, Wallis P. Use of complementary therapies and non-prescribed medication in patients with Parkinson’s disease. Postgrad Med J 2002;78(924):612–4.
8. Kim SR, Lee TY, Kim MS, et al. 2009;Use of complementary and alternative medicine by Korean patients with Parkinson’s disease. Clinical Neurology and Neurosurgery 111:156–60.
9. Park HJ, Lim S, Joo WS, et al. Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol 2003;180(1):93–8.
10. Liu XY, Zhou HF, Pan YL, et al. Electro-acupuncture stimulation protects dopaminergic neurons from inflammation-mediated damage in medial forebrain bundle-transected rats. Exp Neurol 2004;189(1):189–96.
11. Kim YK, Lim HH, Song YK, et al. Effect of acupuncture on 6-hydroxydopamine-induced nigrostratal dopaminergic neuronal cell death in rats. Neurosci Lett 2005;384(1–2):133–8.
12. Jeon S, Kim YJ, Kim ST, et al. Proteomic analysis of the neuroprotective mechanisms of acupuncture treatment in a Parkinson’s disease mouse model. Proteomics 2008;8(22):4822–32.
13. Wang X, Liang XB, Li FQ, et al. Therapeutic strategies for Parkinson’s disease: the ancient meets the future-traditional Chinese herbal medicine, electroacupuncture, gene therapy and stem cells. Neurochem Res 2008;33(10):1956–63.
14. Lim SC, Seo JC, Lee KM, et al. Scavening effect of Hominis Placenta herbal acupuncture solution on nitric oxide. The Acupuncture & Moxibustion Society 2007;24(4):99–105.
15. Kim JI, Yang EJ, Lee MS, et al. Bee venom reduces neuroinflammation in the MPTP-induced model of Parkinson’s disease. Int J Neurosci 2011;121(4):209–17.
16. Lee SK, Lee JD, Koh HK, et al. The study on the Hominis Placenta aqua-acupuncture solution. The Acupuncture & Moxibustion Society 2000;17(1):67–74.
17. Hijikata Y, Kano T, Xi L. Treatment for intractable anemia with the traditional Chinese medicines Hominis Placenta and Cervi Cornus Colla (deer antler glue). Int J Gen Med 2009;30(2):83–90.
18. Shibasaki T, Odagiri E, Shizume K, et al. Corticotropin-releasing factor-like activity in human placental extracts. J Clin Endocrinol Metab 1982;55(2):384–6.
19. Goldfarb G, Tri Doan Ba, Duran A. Human placenta for chronic leg ulcers. Lancet 1980;2(8184):40.
20. Kim KW, Suh SJ, Lee TK, et al. Effect of safflower seeds supplementation on stimulation of the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells. J Ethnopharmacol 2008;115(1):42–9.
21. Jin UH, Kim DI, Lee TK, et al. Herbal formulation, Yukmi-jihang-tang-Jahage, regulates bone resorption by inhibition of phosphorylation mediated by tyrosine kinase Src and cyclooxygenase expression. J Ethnopharmacol 2006;106(3):333–43.
22. Chang GT, Min SY, Kim JH, et al. Anti-thrombic activity of Korean herbal medicine, Dae-Jo-Whan and its herbs. Vascul Pharmacol 2005;43(4):283–8.
23. Hong HT, Kim HJ, Lee TK, et al. Inhibitory effect of a Korean traditional medicine, Honghwain-Jahage(water extracts of Carthamus tinctorius L. seed and Hominis placenta) on interleukin- 1-mediated bone resorption. J Ethnopharmacol 2002;79(2):143–8.
24. Yeom MJ, Lee HC, Kim GH, et al. Therapeutic effects of Hominis placenta injection into an acupuncture point on the inflammatory responses in subchondral bone region of adjuvant-induced polyarthritic rat. Biol Pharm Bull 2003;26(10):1472–7.
25. Lee BH. The effects of Hominis Placenta herbal-acupuncture solution on the Alzheimer’s disease model induced by β A Department of Oriental medicine Graduate school of Daegu Hanny University; 2008.
26. Lee MS, Shin BC, Kong JC, et al. Effectiveness of acupuncture for Parkinson’s disease: a systematic review. Mov Disord 2008;23(11):1505–15.
27. Joh TH, Park HJ, Kim SN, et al. Recent development of acupuncture on Parkinson’s disease. Neurol Res 2010;32(Suppl 1):5–9.
28. Huang Y, Jiang X, Zhuo Y, et al. Complementary acupuncture treatment increases cerebral metabolism in patients with Parkinson’s disease. Int J Neurosci 2009;119(8):1190–7.
29. Rosen T, Krikun G, Ma Y, et al. Chronic antagonism of nuclear factor-kappaB activity in cytotrophoblasts by dexamethasone: a potential mechanism for antiinflammatory action of glucocorticoids in human placenta. J Clin Endocrinol Metab 1998;83(10):3647–52.
30. Matsumoto T, Sugiyama Y, Deguchi K, et al. An ADPase-like substance in placental extracts. Asia Oceania J Obstet Gynaecol 1990;16(3):267–74.
31. Hutton RA, Dandona P, Chow FP, et al. Inhibition of platelet aggregation by placental extracts. Thromb Res 1980;17(3–4):465–71.
32. Chang GT, Oh JY, Choi EY, et al. Purification and characterization of a new anticoagulant protein, PP27, from placenta. Thromb Res 2005;116(5):421–9.
33. Jang SY. Effect of Hominis Placenta on radiation-induced apoptosis and crypt regeneration in rat small intestine Department of Oriental medicine Graduate school of Kyunghee University; 2005.
34. Grünblatt E, Mandel S, Youdim MB. Neuroprotective strategies in Parkinson’s disease using the models of 6-hydroxydopamine and MPTP. Ann N Y Acad Sci 2000;899:262–73.
35. Ara J, Przedborski S, Naini AB, et al. Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine(MPTP). Proc Natl Acad Sci USA 1998;95(13):7659–63.
36. Rozas G, Lüpez-Martün E, Guerra MJ, et al. The overall rod performance test in the MPTP-treated-mouse model of Parkinsonism. J Neurosci Methods 1998;83(2):165–75.
37. Ju MS, Kim HG, Choi JG, et al. Cassiae semen, a seed of cassiaobtusifolia, has neuroprotective effects in Parkinson’s disease models. Food Chem Toxicol 2010;48(8–9):2037–44.
38. Fujikawa T, Miguchi S, Kanada N, et al. Acanthopanax senticosus Harms as a prophylactic for MPTP-induced Parkinson’s disease in rats. J Ethnopharmacol 2005;97(2):375–81.
39. Luchtman DW, Shao D, Song C. Behavior, neurotransmitters and inflammation in three regimens of the MPTP mouse model of Parkinson’s disease. Physiol Behav 2009;98(1–2):130–8.
40. Gorton LM, Vuckovic MG, Vertelkina N, et al. Exercise effects on motor and affective behavior and catecholamine neurochemistry in the MPTP-lesioned mouse. Behav Brain Res 2010;213(2):253–62.
41. L’Episcopo F, Tirolo C, Caniglia S, et al. Combining nitric oxide release with anti-inflammatory activity preserves nigrostriatal dopaminergic innervation and prevents motor impairment in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Neuroinflammation 2010;23(7):83.
42. Ferger B, Leng A, Mura A, et al. Genetic ablation of tumor necrosis factor-alpha(TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum. J Neurochem 2004;89(4):822.
43. Shim JS, Kim HG, Ju MS, et al. Effects of the hook of Uncaria rhynchophyllaon neurotoxicity in the 6-hydroxydopamine model of Parkinson’s disease. J Ethnopharmacol 2009;126(2):361–5.
44. Ju MS, Lee P, Kim HG, et al. Protective effects of standardized Thuja orientalis leaves against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells. Toxicol In Vitro 2010;24(3):759–65.
45. Lee CH, Hwang DS, Kim HG, et al. Protective effect of Cyperi rhizomaagainst 6-hydroxydopamine-induced neuronal damage. J Med Food 2010;13(3):564–71.
46. Choi JG, Kim HG, Kim MC, et al. Polygalae radix inhibits toxin-induced neuronal death in the Parkinson’s disease models. J Ethnopharmacol 2011;134(2):414–21.
47. Gao HM, Hong JS. Why neurodegenerative diseases are progressive: uncontrolled inflammation drives disease progression. Trends Immunol 2008;29(8):357–65.
48. Kang JM, Park HJ, Choi YG, et al. Acupuncture inhibits microglial activation and inflammatory events in the MPTP-induced mouse model. Brain Res 2007;1131(1):211–9.
49. Liberatore GT, Jackson-Lewis V, Vukosavic S, et al. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med 1999;5(12):1403–9.
50. McGeer PL, McGeer EG. Inflammation and neurodegeneration in Parkinson’s disease. Parkinsonism Relat Disord 2004;10(Suppl 1):S3–7.
51. McGeer PL, McGeer EG. Glial reactions in Parkinson’s disease. Mov Disord 2008;23(4):474–83.
52. Gao HM, Liu B, Zhang W, et al. Novel anti-inflammatory therapy for Parkinson’s disease. Trends Pharmacol Sci 2003;24(8):395–401.
53. Marchetti B, Abbracchio MP. To be or not to be (inflamed)-is that the question in anti-inflammatory drug therapy of neurodegenerative disorders? Trends Pharmacol Sci 2005;26(10):517–25.
54. Whitton PS. Inflammation as a causative factor in the aetiology of Parkinson’s disease. Br J Pharmacol 2007;150(8):963–76.
55. Tansey MG, Goldberg MS. Neuroinflammation in Parkinson’s disease: its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis 2010;37(3):510–8.
56. Chae HJ, Choi KH, Chae SW, et al. Placenta Hominis protects osteoporosis in ovariectomized rats. Immunopharmacol Immunotoxicol 2006;28(1):165–73.
57. Jhi YH. Effect of pre-treatment with the human placental extract on neuronal activity in the dentate gyrus of radiation-exposed mice Department of Oriental medicine Graduate school of Kyungwon University; 2007.
58. Chang GT, Oh JY, Choi EY, et al. Purification and characterization of a new anticoagulant protein, PP27, from placenta. Thromb Res 2005;116(5):421–9.
59. Jang SY, Park JW, Bu Y, et al. Protective effects of Hominis placenta hydrolysates on radiation enteropathy in mice. Nat Prod Res 2011;27

Article information Continued

Fig. 1.

Schedule of MPTP injections, HP pharmacoacupuncture treatment and behavioral tests

HP: Hominis Placenta. MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. NS: normal saline.

Fig. 2.

Effects of HP extract on cell viability in PC12 and HT22 cells. PC12 and HT22 neuronal cells were treated with HP extract(15.625–500 μg/mL) for a total 24 h(A and B, respectively)

Cell viabilities were determined using MTT assay and were expressed as a percentage of the normals. Values are indicated as the mean ± SEM of six replicates.

Fig. 3.

Effects of HP extract on toxin-induced neuronal damage in rat primary dopaminergic cells

The rat primary dopaminergic cells were treated with HP extract(1 or 10 μg/mL) for 6 h and with 10 μM 6-OHDA for an additional 18 h(A and B), or with HP extract for 1 h and with 12 μM MPP+ for an additional 23 h(C and D). Fixed cells were stained with anti-TH antibody and visualized with DAB. The numbers of TH-IR neurons were counted(A and C) and the neurite lengths of them were measured(B and D). Data were expressed as a percentage of the normals. Values are indicated as the mean ± SEM of four replicates.

***: p < 0.001 compared with the normal group. **: p < 0.01 compared with the normal group. #: p < 0.05 compared with the MPP+-only treated group.

Fig. 4.

The representative images of TH-IR cells for the effect of HP extract on 6-OHDA-induced toxicity in rat primary dopaminergic cells

The rat primary dopaminergic cells were treated with HP extract(1 or 10 μg/mL) for 6 h and with 10 μM 6-OHDA for an additional 18 h. Fixed cells were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 200 magnification. Normal group(A), 6-OHDA-only treated group(B), 6- OHDA + HP 1 μg/mL treated group(C), and 6-OHDA + HP 10 μg/mL treated group(D).

Scale bar = 100 μm.

Fig. 5.

The representative images of TH-IR cells for the effect of HP extract on MPP+- induced toxicity in rat primary dopaminergic cells

The rat primary dopaminergic cells were treated with HP extract(1 or 10 μg/mL) for 1 h and with 12 μM MPP+ for an additional 23 h. Fixed cells were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 200 magnification. Normal group(A), MPP+-only treated group(B), MPP+ + HP 1 μg/mL treated group(C), and MPP+ + HP 10 μg/mL treated group(D).

Scale bar = 100 μm.

Fig. 6.

Effect of HP pharmacoacupuncture on MPTP-induced hypokinesia in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervals and with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP pharmacoacupuncture. We conducted the rota-rod test after one day(A) and seven days(B) of the last MPTP injection. The time taken for the mice on the rod was recorded.

Values are indicated as the mean ± SEM of seven replicates.

***: p < 0.001 compared with the normal group.

Fig. 7.

Effect of HP pharmacoacupuncture on MPTP-induced bradykinesia in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervals and with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP phar-macoacupuncture. We conducted the pole test after one day(A and B) and seven days(C and D) of the last MPTP injection. The time to turn completely downward(T-turn A and C) and to arrive at the floor(T-LA B and D) were recorded.

Values are indicated as the mean ± SEM of seven replicates.

***: p < 0.001 compared with the normal group. *: p < 0.05 compared with the normal group.

Fig. 8.

Effect of HP pharmacoacupuncture on MPTP-induced dopaminergic neuronal damage in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervalsand with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP. Dopaminergic neurons and fibers in the substantia nigra and the striatum, respectively, were visualised with TH-staining. Quantification of the TH-IR cells was performed by counting the number of TH-IR cells in the substantia nigra(A) and by measuring the optical density in the striatum(B). Data were expressed as a percentage of the normals. Values are indicated as the mean ± SEM of four replicates.

***: p < 0.001 compared with the normal group.

#: p < 0.05 compared with the MPP+-only treated group. SN: substantia nigra. ST: Striatum.

Fig. 9.

The representative images of TH-IR cells in the substantia nigra for the effect of HP pharmacoacupuncture on MPTP-induced toxicity in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervalsand with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP pharmacoacupuncture. Free-floating tissues were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 100 magnification. Normal group(A), MPTP-only treated group(B), MPTP + saline-treated/ST36 group(C), and MPTP + HP pharmacoacupuncture-treated/ST36 group(D).

Scale bar = 200 μm.

Fig. 10.

The representative images of TH-IR fibers in the striatum for the effect of HP pharmacoacupuncture on MPTP-induced toxicity in a mouse PD model

Mice were treated with saline or HP pharmacoacupuncture at ST36 for 4 weeks at 2-day intervals and with MPTP(30 mg/kg, by intraperitoneal injection) for 5 consecutive days on the 8th treatment of HP pharmacoacupuncture. Free-floating tissues were stained with anti-TH antibody, visualized with DAB, and photographed with an optical light microscope at × 40 magnification. Normal group(A), MPTP-only treated group(B), MPTP + saline-treated/ST36 group(C), and MPTP + HP pharmacoacupuncture-treated/ST36 group(D).

Scale bar = 200 μm.