Bulk Electroacupuncture Operation for Mice or Young Rats

Summary

Here, we present a protocol for efficient and rapid electroacupuncture (EA) in mice or young rats using a three-dimensional (3D) printed holder. This technique allows simultaneous operation on multiple animals, saving time and increasing experimental efficiency.

Abstract

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering its promotion. The mechanistic study requires mouse or rat models to address this issue. However, these animals are not obedient to the experimental process, which is time-consuming. To solve these problems, we designed a 3D-printed small animal body bulk fixator to improve the efficiency of EA's animal experiments. This video shows in detail how to use the fixator to perform bulk EA on mice or young rats. For the selection of acupoints, the anterior oblique line of vertex temporal (MS6 head) and Tianshu point (ST25 belly) were chosen to verify the effect of the fixation device with prone positioning and supine positioning. Using the 3D-printed small animal holder allows multiple rodents to be immobilized and treated simultaneously, reducing the time and resources required for the experiment. This technique could be applied to other animal models by 3D printing different sizes and could potentially be used for various fixing conditions. The device is beneficial for the promotion of experimental scientific research in EA.

Introduction

Electroacupuncture (EA) therapy is a unique method in which acupuncture needles are inserted into the scalp and connected to an electro-machine to stimulate specific points¹. Unlike manual stimulation, EA allows better control of stimulation by stabilizing specific frequencies and waveforms to achieve optimal therapeutic effects². According to a survey, 81.2% of medical institutions in China use EA or manual acupuncture to treat cerebral palsy, neuralgia, facial palsy, and other conditions. Despite its popularity, the specific mechanism of efficacy of EA is still unknown, which has hindered its promotion and application in the rehabilitation treatment of neurological diseases^1,2,3. Further research is needed to fully understand the mechanism of EA's effectiveness and to promote its use in the rehabilitation treatment of neurological diseases.

As the influence of acupuncture expands worldwide, many studies have already investigated the mechanisms of EA performed on rodent models, such as rat or mouse models^1,2,3. Several issues are often encountered in EA's rodent experiments. The first is how to immobilize rodents without anesthesia, as acupuncture performed on awake subjects is more reflective of clinical practice. In addition, some experiments require animals to be awake to observe treatment effects^2,3. Another challenge is accurately locating the acupoints in mice or rats that correspond to those in humans. The precise localization of acupoints in experimental rodents is currently being studied by many scholars^4,5. In this protocol, MS6 and ST25 were selected, which have been clearly defined in rodents by the transformation of human anatomical localization. MS6 is commonly used for the treatment of some brain diseases, such as Parkinson's disease⁶. ST25 is usually used to treat gastrointestinal problems, such as diarrhea⁷. These two acupoints were chosen primarily to demonstrate how rodents can be effectively immobilized in both supine and prone positions. Moreover, these acupoints have been extensively studied and offer significant insights for research purposes^6,7.

The previous method of immobilizing a single rat for experiments is not only time-consuming but also difficult to handle by a single person⁸. Additionally, due to the non-cooperation of animals, the success rate is relatively low in practice. Therefore, there is a critical need to create an easily established animal model with stable characteristics to improve experiment efficiency. In this article, a 3D printed holder for small animals was introduced that could easily immobilize multiple rodents, leading to motor restriction. The aim of this paper is to administer EA treatments to a batch of young rats or mice, focusing on the strategies for bulk restriction of mice, identification, and stimulation of MS6 and ST25 acupoints.

Protocol

The experiments in this study comply with the "3Rs" principle of animal ethics and have been approved by the Laboratory Animal Welfare and Ethnic Committee of the Army Medical University (AMUWEC20234543).

1. Preparation of rodent body fixation device

1. Design a small animal holder based on the Chinese utility model patent⁹.
2. Produce the device through 3D printing technology.
3. Prepare a 2 m long rope and divide it into 6 ropes. Thread these 6 ropes through the holes in the fixator. Utilize the spring-loaded buckle to fasten the rope ends, facilitating loop size adjustment.
   ​NOTE: The fixator could be manufactured by any company equipped with 3D printing technology based on a design drawing. The design drawing of the device is shown in Figure 1. The fixator used in this study was produced by a 3D printer by a printing method called fused deposition modeling (FDM), also known as fused filament fabrication (FFF)¹⁰. This additive manufacturing method prints the parts of the design layer by layer by selectively depositing the material in the melted form in a predetermined path. The process uses thermoplastic polymers that come in filaments to create the final physical structure. The immobilization apparatus utilized in this study can secure three animals simultaneously, necessitating six ropes, with each animal requiring a pair of ropes: one for the neck and one for the trunk.

2. Preparation of rodents

1. Purchase 3 Sprague-Dawley rats (10-40 g, 7-45 days old) and 3 C57 BL/C mice (5-40 g).
2. Breed animals in a room with a 12 h day/night cycle, an ambient temperature of 21-23 °C, and a humidity of 45%-60%. Disinfect and ventilate the feeding environment regularly, and allow the animals to eat and drink freely.

3. Fixing rodents to the fixator (prone position)

1. Place the head of the rodent in the U-shaped hole in front of the fixator, with the rodent in a prone position. Pass the rodent's body through two rope loops.
2. Fasten the ropes around the neck and abdomen of the rodent, ensuring that it cannot escape, but not so tightly as to cause respiratory distress and death.
   1. The key step is to fix the neck in place. Leave a gap of 2-5 mm between the rodent's neck and the rope. Ensure that the rope is not too tight for the animals to strangle or too loose to escape.
3. Fix the other rodents in the same way in the device.
4. Repeat the above fixation steps (3.1-3.2) for 3 days to minimize the potential stress response caused by the fixation procedure. Repeat the fixation procedure for 3 consecutive days, with each session lasting 5 min per day.
   NOTE: To ensure that rodents were not over-stressed during the fixation procedure, we repeated the procedure for 3 consecutive days. Each session lasted 5 min per day, providing ample time for mice or rats to acclimate to the procedure and minimizing any potential discomfort.
5. Strat EA from Day 4.
6. To prevent the spread of disease, immerse a sterile cotton swab or ball in 75% alcohol and thoroughly wipe the holder to clean and disinfect it before and after each use. Use gloves and other protective equipment to avoid potential health risks when handling rodents.

4. Performing EA in mice or young rats (MS6)

1. Choose 0.25 mm x 13 mm disposable sterile acupuncture needles.
2. Find the acupoints by anatomical location and confirm the insertion depth at the acupoint.
   NOTE: MS6 is located on the lateral side of the head. This region in the head passes through the connection between Shencong (Ex-hn1) and Xuanli (GB6), also known as the anterior parietal (Du21). MS6 corresponds to the expression of the precentral gyrus of the cerebral cortex on the scalp. Ex-hn1 is located 0.5 cm behind the midpoint of the anterior-posterior midline of the head. GB6 is located at the midpoint of the front of the ear and the end angle of the eye. The superior supratrochlear nerve, supraorbital nerve, occipital nerve, and auriculotemporal nerve are distributed in it. MS6 is mainly used to treat stroke and encephalopathy, such as motor disorders.
3. Slowly twist and insert sterile needles bilateral into MS6 at a depth of 3/5 from the anterior Shenchong (Exhn1) to the Xuanli (GB6) along the subcutaneous in 3 rodents, respectively.
4. Connect one side of the connection cable to the EA instrument.
5. Connect another side of the connection cable containing the positive and negative EA clips to the acupuncture needle shanks on the left and right MS6.
6. Turn on the EA instrument.
   1. Rotate the button (Function/ Choose Switch) to set the frequency and function mode.
   2. Set the Frequency to 10 Hz and choose a Continuous Wave mode.
   3. Rotate the corresponding button (Adjust Intensity) to set the intensity. Each adjust intensity button controls the current strength of the rodents of the wire connected below it. Adjust the current intensity to the acceptable range of rodents, usually 1-2 mA, lasting 5 min.
      ​NOTE: It is important to monitor the breathing and behavior of the rodents during the procedure to ensure that they are not experiencing any distress or discomfort. If the rat appears to be struggling or in pain, the procedure should be stopped immediately.

5. Fixing rodents to the fixator (supine position)

1. Place the heads of the rodent in the U-shaped hole in front of the fixator, with the rodent in a prone position. Pass the rodent's body through two rope loops.
2. Fasten the rope around the neck and abdomen of the rodent, ensuring that it cannot escape, but not so tight as to cause respiratory distress and death.
   NOTE: The points to be noted during fixation are the same as in step 3.2.
3. Fix the other rodents in the same way.
4. As in step 3.4, repeat the above fixation steps for 3 days to reduce the stress response of rodents.
5. Strat EA from Day 4.
   ​NOTE: Disinfection is performed as mentioned in step 3.6.

6. Performing EA (ST25)

1. Choose 0.25 mm x 13 mm disposable sterile acupuncture needles.
2. Find the acupoints by anatomical location and confirm the insertion depth at the acupoint.
   NOTE: ST25 is located at the level of the umbilicus. Positioning this point in rodents requires first measuring the length of the rodent's paw. ST25 is located at 2/3 the length of the paw next to the navel. For example, if the measured paw of the rodent is 0.6 cm, then ST25 is located 0.4 cm to the right and left of the umbilicus.
3. Slowly twist and penetrate sterile needles into ST25 bilateral at a depth of 4-8 mm on 3 rodents, respectively.
4. Connect one side of the connection cable to the EA instrument.
5. Connect another side of the connection cable containing the positive and negative EA clips to the acupuncture needle shanks on the left and right ST25.
6. Turn on the EA instrument.
   1. Rotate the button (Function/ Choose Switch) to set the frequency and function mode.
   2. Set the Frequency to 10 Hz and choose Continuous Wave Mode.
   3. Rotate the corresponding button (Adjust Intensity) to set the intensity. Each adjust intensity button controls the current strength of the rat of the wire connected below it. Adjust the current intensity to the acceptable range of rodents, usually 1-2 mA, lasting 5 min.
      NOTE: The observations were the same as in step 4.6.

Representative Results

The floor plan of the animal body fixator we designed is shown in Figure 1. In addition, a 3D graphic model of this fixator was submitted to provide a comprehensive view of the design (Supplementary File 1). This is a device that allows 3 rodents to be immobilized and perform EA simultaneously. Rats and mice were restricted to the fixator in the awake state, and both prone and supine positions could be firmly fixed without causing injury to the rodents (Figure 2 and Figure 3). With the help of the fixator, the MS6 and ST25 acupoints were successfully located and needled in rodents (Figure 4 and Figure 5).

To control variables and validate the effectiveness of this fixation approach, one experimenter immobilized 3 rats (Video 1) or 3 mice for three consecutive trials in a comparative study (Table 1). The finding demonstrated that the average time for immobilization of 3 rats in prone and supine positions using this tool was 77.1 s ± 7.8 s and 74.9 s ± 8.6 s, respectively (mean ± SD). As for 3 mice, the average immobilization time in prone and supine positions using this fixator was 72.0 s ± 10.5 s and 62.3 ± 4.2 s, respectively (mean ± SD). Both groups of rats and mice were successfully immobilized on the device and did not escape during the 5-min EA treatment (Figure 2 and Figure 3). At 1-2 mA EA intensity, mice and rats felt comfortable and did not struggle violently. Respiration in mice and rats was stable, and no sudden deaths occurred in animals. After treatment, the mice and rats remained healthy and survived.

Figure 1. Batch rodent fixator floor plan. (1) Rodents holder device, divided into three parts. (2,3) Pillars: used to fix the limbs of mice or young rats. (4) Cave: used to fix the head. (5) Hole: used to fix the neck of the young rats. (6) Hole: used to fix the body. Please click here to view a larger version of this figure.

Figure 2: Fixed young rats in prone and supine positions. (A-C) EA treatment on MS6 acupoint.(D-F) EA treatment on ST25 acupoint. Please click here to view a larger version of this figure.

Figure 3: Fixed mice in prone and supine positions. (A-C) EA treatment on MS6 acupoint.(D-F) EA treatment on ST25 acupoint. Please click here to view a larger version of this figure.

Figure 4: MS6 in the cerebral region. MS6 is located on the lateral side of the head and is positioned at the connection point between Shencong (Ex-hn1) and Xuanli (GB6), which is also known as the anterior parietal (Du21) region. Please click here to view a larger version of this figure.

Figure 5: ST25 bilateral point in red. ST25 is located at the level of the umbilicus. Positioning this point in rodent requires first measuring the length of the rodent's paw. ST25 is located at 2/3 the length of the paw next to the navel. For example, if the measure paw of the rodent is 0.6cm, then the ST25 is 0.4 to the right and left of the umbilicus. Please click here to view a larger version of this figure.

Trials	Rats		Mice
	Prone position	Supine position	Prone position	Supine position
	(time in seconds)	(time in seconds)	(time in seconds)	(time in seconds)
Trial 1	75.5	65.1	60.2	62.3
Trial 2	70.2	80.3	80.1	70.6
Trial 3	85.5	79.5	75.7	65.9
Average time (Mean ± SD)	77.1 ± 7.8	74.9 ± 8.6	72.0 ± 10.5	62.3 ± 4.2

Table 1: Time spent on fixing rats and mice with prone and supine positions. The data (time in seconds) was obtained by one experimenter who immobilized 3 rats and 3 mice for 3 consecutive trials, respectively. The mean time required was determined by calculating the average and standard deviation of the time taken for the 3 trials.

Supplementary File 1: 3D printed bath rodent fixator design file. This 3D model can be dragged to achieve a full 360° view of the design. Please click here to download this File.

Video 1: Movie of prone and supine position fixation of young SD rats, as described in detail in the Figure 2 legend. This video demonstrates an experimenter restraining 3 rats in both supine and prone positions. Please click here to download this Video.

Discussion

Electroacupuncture (EA) is a form of acupuncture that involves the use of electrical stimulation on acupuncture needles¹¹. This technique involves the use of micro-pulse currents of specific intensity and frequency to stimulate acupuncture points and achieve enhanced therapeutic effects³. However, the mechanism of EA for healing is limited and requires extensive basic research to prove^1,2,3. Rodent models are commonly used in basic EA studies^8,12,13, and we aim to provide a fast and efficient method for conducting these experiments in rats and mice.

Currently, the EA experiment confronts many challenges. The primary challenge is to effectively immobilize rodents without anesthesia. Stable immobilization of animals is crucial and has a significant impact on the outcome of the animal experiment. Otherwise, the physiological functions of anesthetized animals are different from those of awake animals, and clinical acupuncture treatment is always performed on awake patients. Therefore, experimental studies with awake animals are more practical for clinical purposes. Previously, immobilization with a fixation plate was the preferred method, but it was time-consuming and only allowed for the operation of a single rodent¹⁴. Traditional immobilizers also required tying up the rodent's limbs, which was a time-consuming process and often resulted in pain due to the tight fastening. Several studies have implemented adhesive tape for mice, but their ability to easily escape poses significant challenges, particularly during acupuncture procedures⁸. When there are more experimental subjects, batch operation is often required to save time and effort². Therefore, we have developed a 3D printed small animal body batch fixator that offers several advantages over traditional fixation methods¹⁵: 1) it shortens the fixation time, greatly improving the experimental efficiency¹⁶; 2) it is simple and easy to use and is not affected by subjective factors^15,16; 3) It led less harmful to rodents; 4) Once fixed, the EA operation can be performed on several young rats at the same time, saving time and allowing simultaneous observation of treatment results.

The use of 3D printing technology for experimental studies of EA is a valuable expansion in the field of acupuncture research. The device can be adjusted to any size when 3D printed, which helps experimental acupuncture to be carried out more effectively. Fused deposition modeling (FDM), also known as fused filament fabrication (FFF), an additive manufacturing method^10, is used for 3D printing the fixator. This device is suitable not only for this experiment but also for performing experiments like CT or MRI on rodents^17,18. In addition, it can also be applied to any situation where bulk fixation is needed, except for EA studies. Due to the prevalence of 3D printing technology, this device is easily available. It can be made through a local 3D printing manufacturer. Furthermore, this fixing device can be modified and customized based on the design drawing. In this study, the fixing device is comprised of three sections, each capable of fixing a single rodent. If additional rodents or other animals need to be included, the device can be scaled proportionally to accommodate the larger number. For example, it can be designed as a device that can fix 5 mice or even as a fixing device for other animals such as rabbits.

Another challenge is to find acupoints in the rodents which correspond to humans. MS6 and ST25 acupoints were chosen to examine the effects of prone and supine fixation. Moreover, those two acupoints have been studied by many scholars^6,7,19. MS6 is most often used in brain disease research, such as Parkinson's disease, dysfunction after cerebral stroke, etc^6,13,19. MS6 could enhance the neurological function of rats suffering from ischemic stroke via modulation of the JAK/STAT signaling pathway that is mediated by IL-12 in a previous study¹³. ST25 is usually used for the treatment of the gastrointestinal tract, such as diarrhea and irritable bowel syndrome (IBS)^7,12. A previous study has reported that EA applied solely at the ST25 point showed the potential to alleviate visceral hypersensitivity while also restoring normal slow-wave frequency and colon rhythm in IBS rats¹². These two acupoints are also able to appropriately demonstrate the effects of supine and prone positioning in rodents. Also, these two acupoints have the significance of display^6,7,19. Since these two acupoints have been studied by many scholars, we provide a protocol on how to locate these two acupoints in rodents. It is helpful for animal experiments where MS6 and ST25 studies are being conducted. However, the current study has some limitations. For instance, the position and insert depth of the needle cannot be objectively measured and may vary based on the operator's level of experience.

Our experiments comply with the "3Rs" principle of animal ethics and have been approved by the Laboratory Animal Welfare and Ethnic Committee of the Army Medical University. The most challenging and crucial step in this experiment is to find a reliable and efficient method to securely immobilize rodents. This is because any attempt to escape or death of the rodents during the 5-min EA stimulation will result in the failure of the experiment. The design of the front cave takes advantage of the rodents' preference for digging holes. Placing the rodents' head inside the cave is intended to provide psychological comfort. Furthermore, the ropes used to fasten the head and body of the rodents should be moderately secure, remaining neither excessively slack nor overtight to avoid the escape of the rodent and prevent respiratory distress. Following electrical stimulation, the rodents' behaviors and responses were carefully monitored. Ideally, when applying a current of 1-2 mA, rodents would not exhibit aggressive struggling or vocalizations in response to electrical shock²⁰. The fixator can also be used to fix rodents in supine, prone positions and other types of fixations as needed. It is suitable for a variety of experimental needs, and the fixation operation is easy to perform²¹. However, this has led to a limitation that animals of varying sizes necessitate tailored immobilization equipment of varying sizes. Only animals with slight size discrepancies can be restrained using the same device. The batch immobilization fixator used in this protocol efficiently and reliably immobilizes rodents within the weight range of 10-40 g. For animals outside this weight range, customized batch immobilization fixators are needed to suit their specific requirements.

In addition, the 3D-printed batch rodent fixation used for bulk EA operations on rodents offers enhanced safety as it is resistant to rodent scratches and bites. Studies of EA on rodents have provided valuable insights into the potential benefits of this therapeutic modality in humans^{1,2,3,6,7,11,14}. The device is beneficial for the promotion of experimental scientific research in EA. This technique allows researchers to conduct animal experiments that require the immobilization of animals. Moreover, it presents the exact location of ST25 and MS6, which can serve as a reference for those studying these two acupoints in the future. Ultimately, this methodology is expected to lead to valuable animal models for EA research.

The 3D-printed batch mice fixator described in this paper offers a simple and efficient method for conducting EA experiments in rats or mice. Its design considers the comfort and safety of the animals, as well as the need for quick and efficient fixation. This device has the potential to save time and effort in conducting animal experiments, promoting the advancement of electroacupuncture research and ultimately leading to better treatments for various diseases.

Materials

Name	Company	Catalog Number	Comments
3D printing batch mice fixator	MESH INVENT	Custom made	The fixator produced by 3D printer. The printing method is called fused deposition modeling (FDM), also known as fused filament fabrication (FFF).
Electroacupuncture instrument	Hwato, Suzhou Medical Appliance Factory	SDZ-III	www.Hwato-med.com
Disposable sterile acupuncture needle	Suzhou Medical Appliance Factory	N/A	0.25 mm x 13 mm