Acupoint stimulation by microneedle alleviates motor dysfunction through modulating striatal dopamine and choline levels and reducing neuroinflammation in the Parkinson's disease mouse model

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the basal ganglia. Despite the development of numerous drugs to treat PD, the limitations of these drugs have led to the exploration of various therapies. Previous clinical trials and in vivo studies have demonstrated that stimulation of acupuncture points (acupoints) effectively improve PD phenotype. Recently, microneedles (MNs) have emerged as promising therapeutic tools and may offer a novel approach for easy acupoint stimulation. This study explored the effects of MN patches attached to acupoints on PD phenotypes. The MN patches were attached to the Fengchi (GB20) and Yanglingquan (GB34) acupoints in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced mice for 12 consecutive days. Following this treatment, tissue analysis was performed using immunohistochemistry and Western blot. Mice with MN patches showed significantly improved motor function in the pole and rotarod tests. In the SN of a mouse with MN patches attached, the expression of Heme Oxygenase 1 (HO-1)/Nuclear factor erythroid-2-related factor 2 (Nrf2) was increased, and dopaminergic neurons damaged by MPTP were protected. In the brain tissues of mice with MN patches, the balance of dopaminergic and cholinergic neurons was regulated, and the hyperactivation of microglia and astrocytes was inhibited. Furthermore, the levels of cytokines in the plasma of mice with MN patches were significantly decreased. Collectively, the stimulation of GB20 and GB34 acupoints by MN patches may be a novel therapy for delaying PD.

Graphical abstract

Abbreviations

PD

Parkinson's disease

DA

dopamine; acupoint, acupuncture points

MNs

Microneedles

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride

BSA

bovine serum albumin

PBS

phosphate buffered saline

PFA

paraformaldehyde

Iba-1

ionized calcium-binding adapter molecule-1

DAB

3,3-diaminobenzidine

TH

tyrosine hydroxylase

ChAT

choline acetyltransferase

HRP

horseradish peroxidase

GFAP

Glial fibrillary acidic protein

ABC

avidin–biotin complex

IL-6

Interleukin-6

TNF-α

tumor necrosis factor-α

ELISA

enzyme-linked immunosorbent assay

ACh

Acetylcholine

NOR

Normal

T-turn

times required for head down

T-LA

times required for landing

ST

striatum

SN

substantia nigra

H₂O₂

hydrogen peroxide

Ig G

immunoglobulin G

ANOVA

one-way analysis of variance

S.E.M

standard error of the mean

SNpc

SN pars compacta

HO-1

Heme Oxygenase

Nrf2

Nuclear factor erythroid-2-related factor 2

SNr

SN pars reticulata

GBM

gallbladder meridian

1. Introduction

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, causing motor dysfunction due to the loss of dopaminergic neurons.^1,2 Reduced brain dopamine (DA) levels lead to movement disorders, such as tremors and rigidity, typical PD symptoms.³ The currently identified causes of PD include various genetic and environmental factors that lead to oxidative stress, neuroinflammation, and misfolding of α-synuclein.⁴ Various drug treatments, including DA agonists, levodopa, and DA-degrading enzyme inhibitors, are currently used. However, these approaches are accompanied by side effects, prompting the exploration of various strategies, such as deep brain stimulation and physical therapy, for PD treatment.⁵

Recent studies have reported that stimulating specific acupuncture points (acupoints) has various effects, including suppressing inflammation, promoting nerve regeneration, and reducing pain.^6,7 In particular, acupoint stimulation has been reported to effectively improve PD symptoms and protect dopaminergic neurons through various mechanisms.8, 9, 10 Additionally, it is known that acupuncture to the Yanglingquan (GB34) acupoint in PD rodent models improves motor function by regulating striatal DA levels and suppresses gut microbial dysbiosis and neuroinflammation.^11,12 Clinical studies have shown that acupuncture applied to acupoints, such as Fengchi (GB20), GB34, Baihui (GV20), Dazhui (GV14), Sishencong (EX-HN1), Yintang (EX-HN3), and Zusanli (ST 36), significantly increases the Unified PD Rating Scale 2 and 3 scores in patients with PD.¹³ In addition to acupuncture, specific acupoint stimulation by moxibustion or laser has been found to protect dopaminergic neurons and improve movement disorders in rodent models.^14,15 Collectively, these previous studies suggest that stimulating specific acupoints can help improve PD.

Microneedles (MNs) refer to needles with a height of 25 to 2000 μm, and in most cases, MNs are used in the form of a patch with MNs lined up.^16,17 Owing to their advantage of not damaging blood vessels and nerves, MN is being developed to replace syringes for disease diagnosis or drug delivery.^18,19 Moreover, a previous study revealed that when MN patches containing immunostimulants were attached to the ST36 acupoint in immunosuppressed mice, the proliferative ability of the spleen and anxiety behavior improved, similar to acupuncture. In addition, significantly better effects were observed when the MN patches were attached to the acupoint rather than the non-acupoint.¹⁶ Therefore, acupoint stimulation using MN patches may be useful for treating or regulating various diseases, including PD.

This study explored the effects of MN patches attached to the representative acupoints, GB20 and GB34, on PD phenotypes in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-injected mouse model. Pole and rotarod tests were performed to assess their effects on motor function. The dopaminergic and cholinergic balance and inflammation-related factors, were assessed in the brains or plasma of mice to reveal how acupoint stimulation by MN patches improved motor function in mice with PD.

2. Materials and methods

2.1. Materials

MPTP, hydrogen peroxide, 3,3-diaminobenzidine, bovine serum albumin (BSA), tribromoethanol, phosphate buffered saline (PBS), paraformaldehyde (PFA) sucrose were purchased from Sigma Aldrich (St Louis, MO, United States). Rabbit anti-ionized calcium-binding adapter molecule-1 (Iba-1) was purchased from Fujifilm Wako (Chuo-Ku, Osaka, Japan). 3,3-diaminobenzidine (DAB), rabbit anti-tyrosine hydroxylase (TH), and anti-choline acetyltransferase (ChAT) were purchased from Merck Millipore (Burlington, MA, United States). Mouse horseradish peroxidase (HRP)-conjugated β-actin antibody and goat anti-Glial fibrillary acidic protein (GFAP) were purchased from Santa Cruz Biotechnology (Temecula, CA, USA). Biotinylated goat anti-rabbit antibody, avidin–biotin complex (ABC), normal goat serum, streptavidin-Alexa 594, and Alexa 488 were purchased from Vector Labs (Burlingame, CA, United States). Anti-rabbit HRP secondary antibodies were purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA). Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) mouse kits were purchased from BD Transduction Laboratories (Franklin Lakes, NJ, United States). DA ELISA kit was purchased from Abnova (Taipei, Taiwan). Choline/Acetylcholine (ACh) assay kit was purchased from Abcam (Cambridge, United Kingdom)

2.2. Animals

A total of 60 seven-week-old male C57BL/6J mice were purchased from Daehan Biolink (Eumseong, Republic of Korea). Mice were accommodated at a maintained condition (temperature: 23 ± 1 °C, humidity: 60 ± 10 % a 12 h light/dark cycle, and water and food ad libitum). All animal studies were performed in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th edition” (National Institutes of Health, 2011) and approved by the “Animal Care and Use Guidelines” of Kyung Hee University, Seoul, Republic of Korea (Approval number: KHSASP-23-357).

2.3. Experimental design

The mice were randomly divided into four groups:

• 1.Normal (NOR) group (vehicle injection plus non-patch group, n = 15).
• 2.MPTP group (MPTP injection plus non-patch group, n = 15).
• 3.Sham patch group (MPTP injection plus sham patch group, n = 15).
• 4.MN patch group (MPTP injection plus MN patch group, n = 15).

The acupoints were shaved 3 days before the start of the experiment to attach the MN patches. A new sham or MN patches were applied daily to the GB20 and GB34 acupoints for 2 h each day over a period of 12 days. Patches were attached to the mouse's acupoints by referring to previous papers.²⁰ The NOR and MPTP groups were not patched, and MPTP (30 mg/kg) was injected 1 h before applying the patch for the first 5 days.²¹ No mice died due to MPTP injection, and all mice were entered into the experiment. Out of 15 mice, eight were necropsied for immunohistochemistry after the behavioral experiments, and seven were necropsied for DA and ACh analysis and western blotting in the brain.

2.4. Preparation of MN patches for attaching mice

The MN patches were provided by Raphas (Seoul, Republic of Korea) and manufactured using a previously reported method.²² Biocompatible MNs (five arrays, 1 mm base width, and 350 μm height) were fabricated using the droplet extension method by drying a pharmaceutical-grade hyaluronic acid (HA) solution on top of a hydrocolloid patch. After dropping a viscous biocompatible polymer onto the bottom layer of the patch, causing the other substrates to came in contact with the dropped polymer. After that, the two substrates were then placed a certain distance apart to stretch the contacting viscous polymer materials. After the tensioning process, drying was performed to adjust the shape in the tensioned state. Finally, the middle part was cut so that the same MNs were shaped on two substrates (Fig. 1).

Fig. 1.

Schematic diagrams of making MN patches. HA, hyaluronic acid; MN, Microneedle.

2.5. Assessment of motor functions

2.5.1. Pole test

The pole test was performed six days after the last MPTP injection. The mice were placed head-up on a pole (diameter = 8 mm, height = 55 cm, rough surface). The times required for head down (T-turn) and landing (T-LA) were recorded.²³

2.5.2. Rotarod test

The rotarod test was performed 6 days after the last MPTP injection and the next day. The rotarod device contained a rotating spindle (7.3 cm in diameter) and five separate compartments for the simultaneous examination of five mice. The rotarod tests were performed at a constant speed between 10 and 12 rpm. The training was conducted by placing the mouse on the spindle whenever it fell tothe ground. During the test session, the test was performed at the same rotational speed as that of the training. The time that remained on the rotating spindle until the first drop (latency time) was recorded.²³

2.6. Tissue dissection and collection

Seven days after the last MPTP injection, the mice were anesthetized, and blood was collected from the heart. Eight mice per group were perfused transcardially with 0.05 M phosphate-buffered saline (PBS) and subsequently fixed with pre-chilled 4 % PFA in 0.1 M phosphate buffer. Whole brain tissues were post-fixed with 4 % PFA overnight, immersed in a solution containing 30 % sucrose in 0.05 M PBS, and stored at 4 °C until sectioning. The frozen brains were coronally sectioned on a cryostat at 25 μm and then stored in a storage solution at 4 °C. The remaining seven mice per group were decapitated, and the striatum (ST) and substantia nigra (SN) in their brains were isolated and stored at – 80 °C until used for western blotting and measuring DA, choline, and ACh levels. After collecting the blood, it was placed in tubes with K2-ethylenediaminetetraacetic acid and centrifuged at 3000 rpm for 10 min to obtain plasma.

2.7. Immunohistochemistry

Brain sections were rinsed in 0.05 M PBS and incubated with 1 % hydrogen peroxide (H₂O₂) in 0.05 M PBS for 15 min. Subsequently, sections were replaced with anti-TH antibody, anti-ChAT antibody, anti-Iba-1 antibody, or anti-GFAP antibody (1:1000 dilution) in 0.3 % Triton X-100, 1 % normal goat serum (Iba-1; TH), or 1 % normal horse serum (ChAT; GFAP) in 0.05 M PBS overnight at 4 °C. They were subsequently incubated in an ABC solution with biotinylated anti-rabbit immunoglobulin G (IgG) or anti-goat IgG antibodies (1:500 dilution). DAB was used to develop the color of each section, and images were photographed using an optical light microscope (K1-Fluo confocal microscope (Nanoscope Systems, Daejeon, Korea).²⁴ The areas or numbers of TH-, ChAT-, Iba-1-, and GFAP-positive cells in the ST or SN were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.8. Measurement of neurotransmitters and cytokines level

DA, choline, and ACh levels were measured in the right ST. Tissues were homogenized in 1 mM EDTA and 4 mM sodium metabisulfite to measure DA. Afterward, it was centrifuged at 12,000 rpm for 20 min, and the supernatant was used. Subsequent measurements of DA, choline, ACh, and cytokine levels were performed according to the manufacturer's instructions.

2.9. Western blot

The SN tissues were lysed in RIPA buffer containing a protease/phosphatase inhibitor cocktail. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5 % BSA for 30 min, then incubated at 4 °C with a primary antibody diluted in 1 % BSA overnight. After washing with Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.1 % Tween 20, the membrane was replaced with a secondary antibody at room temperature for 1 h. Protein was detected using ECL reagent, and visualization and quantitative assessment of bands were performed using Image Lab Software (Bio-Rad, CA, USA).²⁴

2.10. Statistical analysis

All datasets underwent the test for normal distribution using Shapiro-Wilk test, before analyzing the statistical significance. Differences among the groups were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). If significance was not found through one-way ANOVA, Students t-test was performed when a trend of change was found. All values are presented as mean ± standard error of the mean (S.E.M.). The differences were considered statistically significant at p < 0.05 and are expressed in each figure.

3. Results

3.1. MN patches attached to acupoints attenuate motor dysfunction in mice with MPTP-induced PD

By performing a pole test, we monitored motor behavior and observed that the T-turn and T-LA were significantly delayed in the MPTP group compared to the NOR group (p-value = 0.001 in T-turn, Student's t-test; p-value = 0.023 in T-LA, Student's t-test). However, the T-turn in mice with MN patches was significantly shorter than the MPTP group (p-value = 0.036, Student's t-test; Fig. 2A and B). Similarly, the MPTP group showed a shorter latency time in the rotarod test, which implied impaired motor function. In contrast, MN patch attachment restored motor function compared to the MPTP group (p-value = 0.01, Student's t-test; Fig. 2C). Additionally, in the rotarod test, latency time significantly increased in the MN patch group compared to the sham patch group.

Fig. 2.

Effects of MN patches attached to acupoints on motor behavior in MPTP injected mice (N = 8 per group). The T-turn (A) and T-LA (B) were measured in pole test. (C) Latency time on the rod were measured in rotarod test. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test or Student's t-test. #p < 0.05 and ##p < 0.01 compared to the NOR group and ∗p < 0.05 compared to the MPTP group using One-way ANOVA; $ p < 0.05 compared to the NOR group and & p < 0.05 Student's t-test. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal.

3.2. MN patches attached to acupoints regulate the balance of DA and ACh in the brains of mice with MPTP-induced PD

We aimed to determine how the MN patch ameliorates motor dysfunction in PD. TH expression levels in the ST and SN pars compacta (SNpc) regions were measured to determine whether the MN patch affected dopaminergic neurons. The TH expression level in the MPTP group was lower than in the NOR group for both the ST and SNpc regions. There was no significant difference between the sham patch and MN patch groups compared to the MPTP group in the ST region (Fig. 3B). In the SNpc, there was no difference in the sham patch group. However, the MN patch group showed significantly increased TH expression compared with the MPTP group (analyzed by Student's t-test; p-value = 0.2581 in One-way ANOVA). In addition, the MN patch group showed an increasing trend compared with the sham patch group (Fig. 3C). The sham patch attached to the acupoint did not affect the dopaminergic neurons. However, the MN patch attached to the acupoints protected the dopaminergic neurons in the SNpc (Fig. 3).

Fig. 3.

Effects of MN patches attached to acupoints on dopaminergic neuron in ST and SNpc of MPTP injected mouse (N = 8 per group). Representative images of TH staining in ST and SNpc (A). Graphs were represented as optical density of TH-immunoreactivity in the ST (B; Scale bar = 500 μm), and the number of TH-positive cells in the SNpc (C; Scale bar = 100 μm). TH-positive dopaminergic neurons in the ST and SNpc were microscopically visualized using immunofluorescence. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test or Student's t-test. ###p < 0.001 compared to the NOR group using One-way ANOVA; & p < 0.05 compared to the MPTP group using Student's t-test. The p-value (in One-way ANOVA followed by Dunnett's multiple comparisons test) is provided for cases where the result is not significant in the one-way ANOVA but significant in the Student's t-test. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; ST, Striatum; SNpc, Substantia nigra pars compacta; TH, Tyrosine hydroxylase.

In PD situation, DA depletion blocks the automatic inhibition of ACh release mediated by muscarinic autoreceptors. This results in excessive ACh release and ultimately prunes the spines of the indirect pathway projection neurons in the ST, disrupting motor commands from the cerebral cortex.^25,26 Therefore, we evaluated the expression of cholinergic neurons. We stained for ChAT, a representative cholinergic neuron marker, in the dorsal ST region (Fig. 4A). The ChAT levels were significantly higher in the MPTP group than in the NOR group. Moreover, compared with the MPTP group, there was no significant difference in the sham group. However, the ChAT expression in the MN patch was significantly reduced, similar to that in the NOR group (Fig. 4B). Therefore, the results showed that attaching an MN patch to the acupoints could regulate the striatal cholinergic pathway.

Fig. 4.

Effects of MN patches attached to acupoints on ChAT in dorsal ST of MPTP injected mouse (N = 8 per group). Representative images of ChAT staining (A). Graphs were represented as the number of ChAT-positive cells in the dorsal ST (B). ChAT-positive cells in the dorsal ST were microscopically visualized using DAB stain. Scale bar = 200 μm. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test. #p < 0.05 compared to the NOR group; ∗p < 0.05 compared to the MPTP group. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; ST, Striatum; ChAT, Choline acetyltransferase.

To investigate whether neurotransmitter balance was restored after MN patch attachment, DA, choline, and ACh levels were measured in the ST. Choline and DA levels were significantly reduced in the MPTP group than in the NOR group. Choline and DA levels were significantly higher in the MN patch group than in the MPTP group. However, they did not differ between the sham patch and MPTP groups, and DA levels were significantly increased in the MN patch group compared to the sham patch group. There was no significant difference in ACh levels among the groups (Fig. 5A–C). The ACh/choline ratio slightly increased in the MPTP group than in the NOR group, without statistical significance. Both DA/choline and DA/ACh were significantly reduced in the MPTP group than in the NOR group and were elevated in the MN patch group than in the MPTP group (Fig. 5D–F).

Fig. 5.

Effects of MN patches attached to acupoints on DA and ACh levels in ST of MPTP injected mouse (N = 7 per group). DA (A), choline (B), and ACh (C) levels were measured using quantitative assay kits. The ratios of DA/choline (D), DA/ACh (E), and ACh/choline (F) were calculated. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test or student's t-test. ##p < 0.01 and ###p < 0.001 compared to the NOR group and ∗ p < 0.05 compared to the MPTP group using One-way ANOVA; & p < 0.05 and && p < 0.01 using student's t-test. n.s.; not significant. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; ST, Striatum; DA, Dopamine; Ach, Acetylcholine.

3.3. MN patches attached to acupoints regulate HO-1/Nrf2 expression in the SN of mice with MPTP-induced PD

To understand how MN patch attachment to acupuncture points protects dopaminergic neurons in the SN, we evaluated the expression of Heme Oxygenase 1 (HO-1) and Nuclear factor erythroid-2-related factor 2 (Nrf2), signals associated with neuronal protection. As results, both HO-1 and Nrf2 levels were slightly increased in the MN patch group. (Fig. 6).

Fig. 6.

Effects of MN patches attached to acupoints on HO-1, Nrf2 and β-actin expression in SN of MPTP injected mouse (N = 7 per group). The protein levels of HO-1 (A), Nrf2 (B) and β-actin were measured by Western blot. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; SN, Substantia nigra; HO-1, Heme Oxygenase 1; Nrf2, Nuclear factor erythroid-2-related factor 2.

3.4. MN patches attached to acupoints reduce neuroinflammation in the brains of mice with MPTP-induced PD

Neuroinflammation is a major pathological mechanism in PD that aggravates disease progression through the loss of dopaminergic neurons. In addition, recent studies have shown that cytokines released by glial cells interact to regulate the release of neurotransmitters. We aimed to determine whether the MN patches attached to the acupoints affected neuroinflammation by measuring activation of glial cells. Microglial activation was evaluated by Iba-1 staining. Iba-1 positive cells were significantly increased in the ST by MPTP injection. In addition, compared to the MPTP group, both the sham patch group and MN patch group had significantly decreased expression levels of Iba-1. Similar results were observed for the SN pars reticulata (SNr); Sham and MN patches decreased Iba-1 (which MPTP increased). The reduction effect of the MN patch was significantly superior to that of the sham patch in both areas (Fig. 7).

Fig. 7.

Effects of MN patches attached to acupoints on activated microglia in ST and SNr of MPTP injected mouse (N = 8 per group). Representative images of Iba-1 staining in ST (A) and SNr (B, C). Scale bar = 200 μm. Graphs were represented as the number of Iba-1 positive cells in the ST (D), % Iba-1 positive area in the SNr (E). Iba-1-positive cells in the ST and SNr were microscopically visualized using DAB stain. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test. ###p < 0.001 compared to the NOR group; ∗p < 0.05, ∗∗∗p < 0.001 compared to the MPTP group; & p < 0.05 and &&& p < 0.001 using student's t-test. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; ST, Striatum; SNr, Substantia nigra pars reticulata; Iba-1, ionized calcium-binding adapter molecule 1.

We also investigated the activation of astrocytes and microglia using GFAP staining, which is closely related to neuroinflammation. The GFAP + area in the ST was significantly increased by MPTP injection and significantly decreased in the MN patch group. The GFAP + area was also increased by MPTP injection in the SNr (significant in the Student's t-test) and significantly decreased in the MN patch group than in the MPTP group. In both the SNr and ST, the GFAP + area decreased in the MN patch group than in the sham patch group (Fig. 8). The results suggest that stimulating acupoints could suppress neuroinflammation, significantly reducing it when the MN patch was attached.

Fig. 8.

Effects of MN patches attached to acupoints on activated astrocyte in ST and SNr of MPTP injected mouse (N = 8 per group). Representative images of GFAP staining in ST (A) and SNr (B, C). Scale bar = 100 μm (A); Scale bar = 200 μm (B, C). Graphs were represented as the number of % GFAP area in the ST (D), SNr (E). GFAP-positive cells in the ST were visualized using immunofluorescence and SNr were visualized using DAB stain. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test or student's t-test. ###p < 0.001 compared to the NOR group using One-way ANOVA; $ p < 0.05 compared to the NOR group using student's t-test; ∗p < 0.05, ∗∗∗p < 0.001 compared to the MPTP group using One-way ANOVA; & p < 0.05 using student's t-test. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; ST, Striatum; SNr, Substantia nigra pars reticulata; GFAP, Glial fibrillary acidic protein.

3.5. MN patches attached to acupoints reduce pro-inflammatory cytokines in the plasma of mice with MPTP-induced PD

Increased levels of pro-inflammatory cytokines are considered pathogenic mediators and biomarkers of PD. Based on the finding that the MN patch attachment reduced brain inflammation, we explored whether the effect of MNs was associated with regulating systemic inflammatory responses by measuring pro-inflammatory cytokine levels in the plasma. We observed that the levels of TNF-α and interleukin-6 were significantly increased in the MPTP group than in the NOR group. However, TNF-α levels tend to be significantly reduced in the sham group and the MN patch group. In contrast, MN patch attachment markedly lowered the pro-inflammatory cytokine levels in the MN patch group than in the MPTP and sham groups (Fig. 9).

Fig. 9.

Effects of MN patches attached to acupoints on pro-inflammatory cytokine levels in plasma of MPTP-injected mice (N = 8 per group). TNF-α (A) and IL-6 (B) in the plasma were measured using ELISA kits. Values are given as the mean ± S.E.M. Data were analyzed by One-way ANOVA followed by post hoc Dunnett's multiple comparisons test. #p < 0.05 compared to the NOR group and ∗ p < 0.05 and ∗∗p < 0.01 compared to the MPTP group. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MN, Microneedle; NOR, Normal; TNF-α, Tumor necrosis factor-α; IL-6, Interleukin-6.

4. Discussion

This study demonstrates the potential of MN patches stimulating acupoints GB20 and GB34 to improve symptoms and provide neuroprotection in PD, suggesting a promising novel therapeutic approach. The results revealed that the MN patches attached to the acupoints protected dopaminergic neurons in the SN by increasing HO-1/Nrf2 expressions and improved motor dysfunction by regulating the balance of striatal DA, choline, and ACh. In addition, MN patches attached to the acupoints significantly reduced the activation of microglia and astrocytes in the ST and SNr and cytokine levels induced by MPTP in plasma. Furthermore, to clarify the effect of the MNs, we verified the effect of sham patches attached to the same acupoints. The sham patches attached to the acupoints significantly inhibited microglia activation in the ST and SN regions. However, no significant effect was observed on the other factors. These results suggest that MNs effectively stimulate acupoints to alleviate PD symptoms and delay the disease.

Many studies have revealed that DA, choline, and ACh compete, cooperate, and maintain a balance in the ST.^26,27 In particular, overactivation of the cholinergic system and DA depletion in PD seriously deteriorate the function of the basal ganglia.²⁸ Therefore, regulating the balance between the two neurotransmitters in the ST maintains the basal ganglia's normal function and improves motor performance. The control of DA and ACh in patients with PD is a major way to improve motor function. Although MN patches attached to the acupoints did not significantly protect dopaminergic neurons in the ST, they have been shown to improve behavioral disorders by balancing choline, ACh, and DA.

Nrf2 and HO-1 are crucial regulators of cellular defense mechanisms against oxidative stress and inflammation, which are central to the pathophysiology of PD.²⁹ In PD, the degeneration of dopaminergic neurons is closely associated with increased oxidative stress and neuroinflammation, making the Nrf2/HO-1 pathway a significant focus of research for potential therapeutic interventions.³⁰ In the current study, Nrf2 and HO-1 levels did not change in the MPTP or sham groups compared to the NOR group, but were significantly increased by MN patch attachment.³¹ This increase is expected because MN attachment, similar to acupuncture, activates peripheral nerves and the peripheral immune system. Therefore, we propose that MNs exhibit acupuncture-like activity.

The results of this study show similar trends to those reported in previous studies. According to a previous study, electroacupuncture performed at the GV14 and GV20 acupoints improved behavioral disorders by regulating neurotransmitters, such as glutamate and ACh, rather than restoring DA in a PD rat model.³² In addition, electroacupuncture performed at the acupoints GB20 and GB34 reduced microglia-mediated neuroinflammation in the trigeminal nucleus caudalis in a migraine rat model.³³ These findings and the results of the present study provide evidence that various methods of stimulating specific acupoints control neurotransmitter balance and inhibit neuroinflammation effectively.

Studies have reported that stimulating both GB20 and GB34 is more effective than stimulating a single acupoint.^34,35 Neuroanatomically, GB34 is located at the junction of muscles and tendons and controls lower body movement, while GB20 is near the base of the skull, close to the cervical nerve that connects to the brainstem and upper spinal cord, controlling the upper nerves.^36,37 This creates a synergistic effect.⁸ Moreover, both acupoints belong to the Gallbladder Meridian (GBM) and are linked to the gallbladder, liver, and heart, targeting the treatment of pain, numbness, and spasms. The current study found results supporting this, with stimulation of GB20 and GB34 improving motor deficits and suppressing neuroinflammation.^13,38

Results related to inflammation in the current study showed a mild inhibitory effect even in the Sham patch group. Although there is no microneedle, it is assumed that some acupoint stimulation was applied during the patch attachment process. There are various ways to stimulate acupoints, including acupuncture, moxibustion or acupressure.^39,40 Because we attached and removed the sham patch to the acupoints every day for 12 days in the same way as the MN patch, it is expected that a weak stimulus was provided. However, the degree was so minor that it did not affect nerve cells or neurotransmitters, but it appears to have had some effect on inflammation.

The MNs used in this study were dissolved HA-based MNs. Dissolving MNs, which might use biodegradable polymers to avoid accumulation in the body, have the advantage of posing a lower risk of injury compared to other solid MNs.⁴¹ HA-based dissolving MNs are widely used because they are safe and offer several economic advantages.⁴² Through pilot studies, we devised a method to improve the skin irritation and adhesiveness of the patch. By comparing the length of the MN, we developed the safest and most effective form for mice. The length of the MNs used in this study was 350 μm, which was created under consideration of the depth of the mouse skin. The skin thickness of 4- to 6-week-old C57BL male mice is 540 ± 20 μm, and the dermal layer is approximately 250–300 μm.⁴³ In this study, the mice used for MN patch attachment were 8- to 9-week-old, and it is generally expected that the MNs penetrated the boundary between the dermal layer and adipose tissue. Therefore, the results of this study may be useful evidence for the production and application of MN patches in future clinical applications.

However, this study had some limitations. Firstly, no positive control group was included. Unlike MN patches, levodopa is mainly used for PD, which requires oral administration. Therefore, this study did not include a positive control group because it aimed to investigate MN patch attachment's effect on PD acupoints. However, in the future, we would like to evaluate the effect of MNs more clearly through comparison with a positive control group. Secondly, this study stimulated only two representative acupoints. Therefore, the effect on locations other than these acupoints was not considered. In future studies, it will be necessary to investigate whether the stimulation of acupoints has an effect by comparing the attachment of MNs to non-acupuncture points. Finally, there is controversy as to whether the acupoints of humans and mice are consistent. For this reason, verifying whether the current results can be applied to humans through future clinical studies is necessary.

In conclusion, the current study suggests that the stimulation of the GB20 and GB34 acupoints by MN patches could ameliorate MPTP-induced motor dysfunction. The results of this study are meaningful because they demonstrate the possibility that MN patches can effectively stimulate acupoints and reveal a mechanism for improving PD. Therefore, we propose that MN patch is a new method that can delay the progression of PD through acupoint stimulation.

Funding

This research was supported by grants from the National Research Foundation of Korea, funded by the Korean government (2022M3A9B6017813).

Author contributions

J.H.K.: Conceptualization, Investigation, Writing – original draft; I.G.J.: Investigation, Writing – original draft; Y.C.: Investigation; J.S.K.: Investigation; H.L.: Investigation; J.Y.O.: Methodology; K.H.L.: Resources; S.L.: Resources; D.H.J.: Resources; C.N.: Funding acquisition; H.J.P.: Funding acquisition, Project administration, Supervision; M.S.O.: Funding acquisition, Project administration, Supervision, Writing – review & editing.

Data availability statement

Data will be made available on request.

Ethical approval

All animal studies were performed in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th edition” (National Institutes of Health, 2011) and approved by the “Animal Care and Use Guidelines” of Kyung Hee University, Seoul, Republic of Korea (Approval number: KHSASP-20-137).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.