A Mechanical Acupuncture Instrument for Reproducible Vibratory Stimulation of Acupuncture Points in Rats

Summary

This protocol describes a standardized mechanical acupuncture method using a vibratory stimulation device that delivers reproducible needle stimulation at the HT7 acupuncture point in rats. Validated in a cocaine-induced locomotor activity model, this method provides a quantitative and reproducible platform for investigating the peripheral and central neurophysiological mechanisms underlying acupuncture-mediated effects.

Abstract

Although manual acupuncture is widely used in experimental and clinical research, its application is limited by poor reproducibility resulting from practitioner-dependent variability in needle manipulation. To overcome this limitation, a mechanical acupuncture instrument (MAI) was developed to deliver controlled vibratory stimulation through an inserted acupuncture needle without the use of electrical current. This article presents a standardized protocol for applying MAI stimulation to the HT7 acupuncture point in rats, including animal preparation, localization of the acupuncture point, needle insertion, configuration of stimulation parameters, and behavioral assessment. Representative validation studies showed that MAI stimulation at HT7 reduced cocaine-induced locomotor activity and preferentially activated mechanoreceptor-associated A-fiber pathways. Further studies confirmed the involvement of peripheral ulnar afferents, the dorsal column pathway, and mesolimbic reward circuitry in mediating these effects. The representative results presented in this article are based on previously published validation studies using the MAI platform. This protocol provides a reproducible and quantitative method for investigating the neurophysiological mechanisms of acupuncture stimulation in experimental animal models.

Introduction

Manual acupuncture is a traditional needle-based stimulation method that delivers dynamic mechanical input to peripheral tissues through direct needle manipulation and has been widely used in both clinical and experimental settings^1,2. Unlike electroacupuncture (EA), manual acupuncture generates complex mechanical stimulation, including tissue deformation and activation of peripheral mechanoreceptors, through physical manipulation of the inserted needle². These mechanical components are considered important contributors to the characteristic physiological effects of acupuncture and are thought to engage somatosensory afferent pathways differently from electrical stimulation^3,4.

Despite its physiological relevance and broad use, the experimental application of manual acupuncture remains limited by poor reproducibility². The therapeutic effects of manual acupuncture largely depend on operator-dependent factors, including needle manipulation techniques, stimulation intensity, frequency, and duration, which can vary substantially among practitioners and experimental conditions¹. This variability presents a major obstacle for mechanistic studies attempting to establish causal relationships between acupuncture stimulation and neurophysiological outcomes.

To overcome these limitations, EA has frequently been adopted as an alternative method because it enables precise control of stimulation parameters such as frequency, amplitude, and duration. However, EA introduces electrical input that may recruit neural pathways distinct from those activated by manual needling⁵. Consequently, it remains difficult to isolate and evaluate the specific contribution of mechanical stimulation, which represents a fundamental component of traditional manual acupuncture. Therefore, a reproducible method capable of delivering controlled mechanical stimulation without electrical confounds is needed for mechanistic acupuncture research.

To address this limitation, a mechanical acupuncture instrument (MAI) was developed in 2013 to reproduce vibration-based mechanical stimulation while enabling quantitative control of stimulation parameters^3,5. This device delivers controlled vibratory stimulation directly to an inserted acupuncture needle, thereby standardizing the mechanical input applied to tissue. Using this approach, previous studies demonstrated that mechanical acupuncture modulates somatosensory, reward-related, and autonomic neural circuits across multiple experimental models^4,6,7,8. For example, mechanical stimulation at HT7 acupoints attenuated cocaine-induced behavioral responses and alcohol dependence through modulation of mesolimbic dopamine circuitry, endogenous opioid pathways, and somatosensory relay circuits^6,7. In addition, combined mechanical and electrical stimulation applied to neurogenic spots suppressed hypertension through opioid-mediated mechanisms in central autonomic regions⁵. Collectively, approximately 30 studies using this platform have contributed to the mechanistic understanding of acupuncture in addiction, autonomic regulation, and other experimental conditions.

Unlike general vibration-only stimulation systems, this protocol provides an integrated workflow for needle-coupled, non-electrical, vibration-based mechanical acupuncture in awake rats. It combines device assembly, needle-motor coupling, insertion-depth standardization, accelerometer-based calibration, acupuncture point localization, and representative behavioral validation using HT7 stimulation in a rat cocaine-induced locomotor activity model. Thus, this article provides a reproducible experimental protocol for controlled mechanical acupuncture stimulation rather than presenting a new therapeutic device.

The standardized and quantitatively controlled output of the MAI may also provide a useful platform for future integration with data-driven analytical approaches. Because stimulation parameters such as vibration frequency, acceleration, insertion depth, and stimulation duration can be precisely defined and reproduced, these variables may serve as structured inputs for computational modeling. Recent studies have shown that machine learning, deep learning, and transfer learning approaches can improve feature extraction, signal classification, pattern recognition, and optimization in complex biomedical images and vibration-based signal datasets^9,10,11,12. Similar approaches could potentially be applied to MAI-based acupuncture research by integrating stimulation parameters with behavioral, neurophysiological, and vibration-output data to support signal analysis, behavioral classification, stimulation-parameter optimization, and prediction of acupuncture-related responses. Thus, future studies combining MAI-based stimulation with machine learning methodologies may further enhance the precision, reproducibility, and mechanistic interpretation of acupuncture research.

Importantly, the representative validation data presented in this article are reproduced, adapted, or summarized from previously published studies using the MAI platform. The primary objective of this article is not to report new experimental findings, but rather to provide a detailed, step-by-step protocol for the reproducible application of standardized mechanical acupuncture stimulation in experimental animal models.

Protocol

All animal protocols described in this article were approved by the Institutional Animal Care and Use Committees (IACUCs) of Daegu Haany University (DHU2012-008, DHU2022-12) and Yonsei University College of Medicine (# 2023-0006) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Preparation of the mechanical acupuncture instrument

1. Assemble the mechanical acupuncture instrument (MAI) (Figure 1).
   1. Prepare a mechanical acupuncture instrument (MAI) consisting of a custom-made control unit and a mechanical vibrator connected to an acupuncture needle (Figure 1D). Use the control unit to adjust stimulation intensity, vibration frequency, and stimulation duration^3,4,13.
   2. Use a sterile acupuncture needle with a diameter of 0.10 mm, a 10-mm needle shaft, and a 10-mm handle. Fix a rubber stopper to the needle shaft at a distance of 3 mm from the needle tip to standardize insertion depth.
   3. Attach the vibration motor to the acupuncture needle using a stable clip-type connector. Confirm that the needle does not slip during vibration and that the stopper remains fixed during stimulation.
2. Configure stimulation parameters.
   1. Set the stimulation condition to continuous vibration at 85 Hz and 1.3 m/s² for 20 s, which generates stable acceleration waveforms during stimulation (Figure 1E).
      ​NOTE: Previous validation experiments¹³ showed that manual acupuncture generated vibration frequencies of approximately 70–90 Hz and acceleration values averaging approximately 1.3 m/s² (Figure 1A,B). Based on these measurements, the MAI was configured to deliver vibratory stimulation at 85 Hz and 1.3 m/s², which closely approximates the mechanical characteristics of manual acupuncture while providing stable and reproducible output. The 20 s stimulation duration was adopted from previously published parameter-optimization studies¹³ in a rat cocaine-induced locomotor activity model, in which different stimulation durations and acceleration intensities were compared to identify an effective and well-tolerated stimulation condition. In this protocol, “frequency” refers to the dominant frequency component of needle-tip vibration measured from the accelerometer signal. This value reflects the vibration transmitted to the needle tip during manual handle-flicking manipulation rather than the slower rhythm of repeated hand movements by the operator.
   2. Apply the 85 Hz, 1.3 m/s² condition to generate a stable and reproducible vibration pattern comparable to manual needle manipulation (Figure 1E).
3. Validate vibration output.
   1. To validate stable vibration output, firmly attach the needle tip to the accelerometer so that mechanical vibration from the motor is directly transmitted from the needle to the sensor.
   2. Record the acceleration signal during the stimulation period using a data acquisition system and analyze the signal with LabVIEW or equivalent software.
   3. Define the acceleration value as the stable needle-tip acceleration measured during the plateau phase of stimulation after the initial transient response, and calculate the dominant vibration frequency from the acceleration signal.
   4. Perform calibration before each experimental session and whenever the motor, connector, needle, or needle-motor coupling is replaced or adjusted.
   5. Proceed with animal experiments only when the measured dominant frequency and acceleration remain stable during the plateau phase and are within an acceptable range of the target stimulation parameters, such as approximately ±10% of 85 Hz and 1.3 m/s².
      NOTE: Representative acceleration traces generated during manual acupuncture and MAI stimulation are shown in Figure 1B,E.

2. Animal preparation and cocaine-induced locomotor activity model

1. Prepare experimental animals.
   1. Use male Sprague-Dawley rats weighing 270–320 g for cocaine-induced locomotor activity experiments^3,4,5. House animals under a 12-h light/dark cycle with free access to food and water.
   2. Assign 6–8 animals to each experimental group unless otherwise specified.
2. Habituate animals.
   1. Habituate each rat to the experimental procedures 1 day before testing. Perform gentle handling and sham acupuncture manipulation without needle insertion for 2–3 min.
   2. Place the animals in the locomotor activity chamber for 90 min to reduce stress-related behavioral responses during awake stimulation procedures.
   3. Before the acupuncture experiment, habituate rats to grab-handling and light manual restraint daily for the same duration as the planned acupuncture procedure.
   4. During stimulation, gently hold the animal by hand to stabilize the body and forelimb without excessive pressure. Lightly restrain non-acupuncture control animals in the same manner and for the same duration, but without needle insertion, to control for generalized effects of immobilization stress.
      NOTE: Operators should be trained to perform restraint, needle insertion, and stimulation consistently.
   5. If persistent struggling, vocalization, escape attempts, or other excessive stress responses are observed, stop the procedure and provide additional habituation before testing.
      NOTE: Perform all acupuncture stimulation procedures in awake animals under light manual restraint.

3. Acupuncture point localization

1. Locate HT7 and LI5 acupuncture points.
   1. Locate acupoints using the transpositional method, in which the human acupoint location is mapped onto the corresponding anatomical site in the animal.
   2. Identify HT7 (Shenmen) at the transverse crease of the forepaw wrist, radial to the tendon of the flexor carpi ulnaris muscle^3,4,5,8.
      ​NOTE: This location corresponds anatomically to the ulnar tunnel/Guyon’s canal region near the ulnar nerve. In contrast, LI5 is located at the radial end of the dorsal side of the wrist, between the tendons of the extensor pollicis muscle and the extensor pollicis brevis muscle. LI5 is positioned on the opposite side of HT7 and is approximately 5 mm apart (Figure 1F). These anatomical landmarks were used to ensure reproducible localization of the acupuncture points¹⁴.
2. Locate control acupuncture points.
   1. Use LI5 as a nearby wrist control point. Locate LI5 approximately 5 mm from HT7 near the distal end of the radius between the tendons of the palmaris longus and flexor carpi radialis muscles.

4. Mechanical stimulation procedure

1. Perform locomotor activity experiments.
   1. Place the rat in the open-field chamber and allow 60 min of habituation. Record baseline locomotor activity for 30 min.
   2. Inject cocaine intraperitoneally at 15 mg/kg to induce locomotor activation^3,4,5. Lightly restrain the animal 1 min after cocaine injection.
   3. To standardize insertion depth, mount a rubber stopper on the needle shaft at a point 3 mm from the needle tip (Figure 1D). Ensure the stopper is securely fastened so it remains stable during insertion and vibration.
   4. During needle insertion, advance the needle until the stopper gently contacts the skin surface, while avoiding excessive skin compression that could alter the effective insertion depth.
2. Apply mechanical stimulation.
   1. Apply continuous vibratory stimulation at 85 Hz and 1.3 m/s² for 20 s, the optimized stimulation condition identified in parameter validation experiments (Figure 2A–C). Maintain the needle in place for 1 min after insertion, then withdraw it.
   2. Continue recording locomotor activity for up to 60 min after cocaine injection. Analyze locomotor activity in 10-min intervals or as total distance traveled over 60 min (Figure 1C).
   3. Apply 20-s stimulation as the standard condition because this duration produced robust inhibition of cocaine-induced locomotor activity in previous validation experiments (Figure 2A–C).
      NOTE: Continuous 40-s stimulation induced stress-like behaviors, including vocalization and avoidance responses, and was therefore excluded from standard stimulation conditions.
3. Perform stimulus-parameter validation experiments.
   1. Compare stimulation durations of 0 s, 10 s, 20 s, and 40 s or compare different acceleration settings, such as 0.7 and 1.3 m/s², to validate stimulation parameters (Figure 2).
   2. Apply 50-Hz stimulation as a Meissner-corpuscle-biased condition and 200-Hz stimulation as a Pacinian-corpuscle-biased condition for mechanoreceptor-related experiments, as previously described^4,8.

5. Data acquisition and statistical analysis

1. Measure locomotor activity using an overhead video-tracking system. Place animals in a square open-field chamber (40 cm × 40 cm × 45 cm) and quantify total distance traveled using video-tracking software.
2. Record acceleration-time traces from accelerometer output during vibration calibration experiments. Report dominant frequency, acceleration amplitude, and stable vibration duration (Figure 1D,E).
3. Assess data distribution for normality before parametric statistical analysis. When data satisfied the assumptions of normality, analyze group differences using one-way or two-way analysis of variance, followed by appropriate post hoc comparisons.
4. Perform behavioral tracking and analysis by investigators blinded to experimental group allocation whenever possible.
5. Express behavioral and physiological data as mean ± SEM. Analyze locomotor activity using one-way or two-way repeated-measures analysis of variance (ANOVA) followed by Tukey post hoc testing.
   NOTE: Sample sizes were selected based on previous studies using the same rat cocaine-induced locomotor activity model and mechanical acupuncture stimulation protocol. To ensure sufficient statistical power, 6–8 rats were used per group. Animals were excluded from analysis only when predefined technical or procedural issues occurred, including incorrect acupuncture point localization, device malfunction, unstable vibration output during calibration, or failure of video tracking.

Results

Validation of reproducible mechanical stimulation

The representative device configuration and vibration-output validation shown in Figure 1 were generated for the present manuscript, whereas the quantitative reference ranges for manual acupuncture-derived vibration parameters were summarized from prior validation studies.

The MAI was validated by comparing the mechanical output generated during manual acupuncture manipu...

Discussion

This study presents a standardized protocol for applying mechanical acupuncture using a vibratory stimulation device that enables reproducible delivery of vibration-based mechanical stimulation. By providing quantitative control of stimulation frequency, acceleration intensity, and duration, this approach addresses a major limitation of traditional manual acupuncture, which is inherently dependent on practitioner technique and difficult to standardize.

One of the major strengths of this protoc...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accelerometer	PCB Piezotronics	PO-AXA-12-01	Used for vibration measurement
Acupuncture needle (0.10 mm × 10 mm)	Dongbang Acupuncture Inc.	DB102	Sterile disposable acupuncture needle
Alligator clip connector	N/A	N/A	Used to connect vibrator to acupuncture needle
Bupivacaine hydrochloride (0.5%)	Huons Pharm, Korea	HIRA Code (199502333)	Used for local anesthesia experiments
Cocaine hydrochloride	Macfarlan Smith Ltd	53-21-4	Used for cocaine-induced locomotor experiments
Control unit	Custom-made	N/A	Used to adjust stimulation frequency, acceleration intensity, and duration
Data acquisition software	LabVIEW	LabVIEW 7 Express (7.0)	Used for vibration signal analysis
Data acquisition system	National Instruments	DAQ-NI USB-6200	Used for signal digitization
Mechanical acupuncture instrument (MAI)	Custom-made	N/A	Custom-built device consisting of a control unit, vibration motor, clip-type connector, acupuncture needle, and rubber stopper
Open-field locomotor chamber	Custom-made	square open field box (40 cm × 40 cm× 45cm) made of black acrylic.	Used for locomotor activity analysis
Rubber stopper	N/A	N/A	Mounted 3 mm from the needle tip to standardize insertion depth
Sprague-Dawley rats	Orient Bio	Crl:CD(SD)IGS	Male, 270–320 g
Vibration motor	Motor Bank	MB-0412V	Used for mechanical vibratory stimulation
Video tracking software	EthoVision XT	Ethovision 3.1	Used for locomotor tracking