All procedures conducted on the rats in this study adhered to the National Standard for Laboratory Animal Welfare and Ethics Review Guidelines (2018) officially adopted by the People's Republic of China and Guide for the Care and Use of Laboratory Animals from the National Academies Press15. This protocol is composed of three main steps. Firstly, the rat jackets are tailored and fitted based on the individual body shapes of rats with different weights. Next, acupuncture manipulation is carried out following standard procedures. Lastly, the rats are suspended in their jackets while the needles remain in place to accommodate the need for simultaneous interventions among rats within the same group.
1. Pre-needling preparation
Figure 2: Corresponding diagram of rat body shape and matched rat jacket size. In the diagram, the parameter a represents the transverse distance at the back of the joint between the upper limbs and the rat's trunk. This value is utilized as the spacing measurement between the penetrating holes for the upper limbs of the rat jacket. Similarly, parameter b represents the transverse distance on the back where the bilateral lower limbs of the rat connect with the trunk. This measurement determines the spacing of the penetrating holes for the lower limbs of the rat jacket. The parameter c signifies the circumference of the thickest part of the rat's abdomen, which can be utilized as the width measurement for the fabric of the rat's jacket. The parameter d represents the distance from the rat's armpit to the front of its ilium, determining the longitudinal distance between the upper and lower holes of the jacket. The parameter e corresponds to the longitudinal distance from the rat's nose tip to the base of its tail, serving as the length measurement for the rat's jacket cloth. The parameter f denotes the circumference of the articulation connecting the upper limb and the rat's trunk, serving as the circumference of the hole through which the upper limbs pass. The parameter g represents the circumference of the joint connecting the lower limbs and the rat's trunk, determining the circumference of the hole through which the lower limbs pass. Please click here to view a larger version of this figure.
Body Weight (g) | Body Shape Parameters (cm) | ||||||
a | b | c | d | e | f | g | |
220 | 6.0 | 7.0 | 15.0 | 8.0 | 19.0 | 5.0 | 7.0 |
300 | 7.0 | 7.5 | 17.0 | 10.0 | 21.0 | 6.0 | 8.0 |
400 | 8.0 | 8.5 | 19.0 | 11.5 | 23.0 | 6.5 | 9.0 |
520 | 8.0 | 10.0 | 20.0 | 12.5 | 26.0 | 7.0 | 9.5 |
Table 1. The measurement results of body shape parameters of rats with different body weights.
Figure 3: Diagram depicting the physical appearance and usage of the rat jacket. (A) Plane exterior view of the rat jacket. The shape of the rat's jacket resembles a right-angled trapezoid, with separate openings on the left and right sides for the rat's upper and lower limbs to pass through. (B) Unfolded exterior view of the rat jacket. The closure of the headgear creates a semi-closed cavity, which serves as a crucial component to entice rats to enter. (C) Side view of a rat clothed with the jacket. Once the rat's limbs pass through the openings in the clothing, the straps are crossed over the ventral surface of the trunk and securely fastened at the back with appropriate tension. This ensures that the rat is relatively immobilized. (D) Abdominal view of a rat clothed with the rat jacket. The ventral surface of the jacket exposes the rat's abdominal skin, enabling convenient access for acupuncture at ventral points. Please click here to view a larger version of this figure.
Video 1: Putting on the rat jacket. This video shows a typical process of dressing the rat in the jacket. Please click here to download this Video.
Figure 4: Diagram depicting the physical appearance and usage of the elevated fixation rack. (A) Elevated fixation rack. This rack consists primarily of two vertical iron columns (60 cm in length) welded to each tray, along with a long horizontal iron stick (145 cm in length) placed parallel to the ground. Screws are used to secure the horizontal stick at the same height as the vertical columns. (B) Rat suspension on the fixation rack. Rats wearing the rat jackets are securely fastened to the fixation rack using two S-shaped hooks per rat. (C) Simultaneous electroacupuncture treatment in multiple rats. To prevent any potential interference resulting from tail movement, it was necessary to maintain proper spacing among the rats, equivalent to the length of a rat's tail. Please click here to view a larger version of this figure.
2. Electroacupuncture procedure
Video 2. Needling acupoints in the lumbar region. This video demonstrates the comprehensive and coherent procedure encompassing the attiring of the rat in the jacket, the insertion of acupuncture needles into acupoints at the waist, and the subsequent connection of an electroacupuncture apparatus. Please click here to download this Video.
Video 3. Needling acupoints in the abdomen and lower limb. This video demonstrates the comprehensive and coherent procedure encompassing the attiring of the rat in the jacket, the insertion of acupuncture needles into acupoints located in the abdomen and the lower limb, and the subsequent connection of an electroacupuncture apparatus. Please click here to download this Video.
Video 4. Removing the rat jacket. This video showcases the customary practice of removing the jacket from the rat. Please click here to download this Video.
In this experiment, a total of 12 rats were selected for electroacupuncture treatment, and their reactions under the fixation method were observed (as shown in Table 2). Among the 12 rats, only 1 rat exhibited intense struggle during the 15 min electroacupuncture needle retention period. As a result of its vigorous movements, the needle came off midway, accounting for 8.33% of the total number of rats. This outcome suggests that the fixation method used generally demonstrates a notable degree of stability and dependability. Despite the struggle exhibited by one rat, the majority of the rats in the group maintained proper needle fixation throughout the treatment. Moreover, during the 2-month experimental duration where this method was employed to immobilize rats for electroacupuncture treatment, no instances of rat mortality were recorded, suggesting the safety of this fixation method for rats.
Additionally, in Table 3, we compared this fixation method with four other methods reported in literature9,10,13,14. First, in terms of stability, apart from the slightly inferior stability of the fifth method using a newly made rat holder fixation, the other methods achieved relatively secure fixation. In terms of time, the average time required to fix one rat using this method was shorter than that of the rat coat swing fixation method. The remaining methods did not specify the time required for fixation in their respective papers; therefore, no discussion is provided. In terms of the number of personnel required to fix one rat, both this method and the rat coat swing suspension method only required one person to complete the operation, thus enhancing experimental efficiency. However, in terms of the exposed acupoint range, the specially designed rat jacket in this protocol can meet the requirements for exposing most acupoints on the body, while the rat coat in the swing suspension method mainly exposes the limbs. Regarding the materials used for fixation, the materials required for this method are relatively easy to obtain in both laboratory and everyday settings. Furthermore, in terms of the rat's position during fixation, the natural position is evidently more comfortable than the forced position. Overall, this method is similar to the rat coat swing suspension fixation method, but it has targeted improvements in the fabric and design of the rat jacket, allowing for more exposure of body acupoints. Moreover, compared to being suspended on a fabric strap, this protocol utilizes sturdier steel pipes, which reduces the adverse effects caused by individual rats' violent shaking on other rats when multiple rats are simultaneously undergoing electroacupuncture treatment on the fixation rack. This ensures the stability and reliability of experimental data.
Considering the aforementioned factors collectively, this approach exhibits the benefits of stability, efficiency, affordability, and relative comfort for rats, rendering it a valuable option for needle insertion fixation.
Fixation method | Number of struggling rats | Percent of struggling rats (%) | Number of rats with needle drop during treatment | Percent of rats with needle drop during treatment(%) | Number of rats died during operation | Percent of rats died during operation(%) |
Suspension fixation bound with jacket group | 1 | 8.33 | 1 | 8.33 | 0 | 0 |
Table 2. Response of fixed rats during electroacupuncture treatment (n=12).
Serial Number | Fixation method | Is the fixation of rats stable and secure | The time required to fix a rat | Number of personnel required to fix a rat | The location and range of acupoints that can be exposed | Accessibility of fixtures or materials | Comfort of fixed methods for rats: natural or forced position | Refer-ences | |||||||||||||||
1 | Suspension fixation bound with jacket in our experiment | √ | 50-63 s | 1 | Abdomen, head, back and waist, limbs | Readily available | Natural position | ||||||||||||||||
2 | A fixation frame for acupuncture and moxibustion for Shu-Mu acupoint combination (an acupoint selection that combines the alarm points with the back transport points) | √ | / | / | Head, limbs, back and waist | The fixation frame needs to be customized | Forced position | 9 | |||||||||||||||
3 | Fixation method of swinging rats bound with coat | √ | 85-93 s | 1 | Limb parts | Readily available | Natural position | 10 | |||||||||||||||
4 | A rat fixating device made from an old meshy cage cover | √ | / | ≥2 | The range of acupoints is not limited | Readily available | Forced position | 13 | |||||||||||||||
5 | A newly-made rat holder | After fixation, the front and rear limbs of the rats still have a certain degree of mobility. Furthermore, there is a certain risk that the intersection point of the neck fixing rod in rats may be too high, which can lead to suffocation in rats. If it is too low, the head fixing of rats may be unstable. | / | / | The range of acupoints is not limited | Rat holder requires special customization | Natural position | 14 |
Table 3. Comparison results of different fixation methods in rat acupuncture and moxibustion experiment.
75% alcohol | Shandong Lircon medical technology co., ltd | 2002-0059 | Disinfection |
acupuncture needles | Beijing Luo ya shan chuan medical equipment co., Ltd. | needling | |
Adult Sprague-Dawley (SD) rats | Liaoning Changsheng biotechnology co., Ltd. | SCXK 2020-0001 | The recipient subjected to fixation |
an elevated fixation rack | Taizhou shiai instruments and equipments for scientific and teaching research Co., ltd | / | Fix rats |
Hans electronic acupuncture apparatus | Beijing Huayun Ante Science and Technology Co. Ltd. | HANS-200A | electroacupuncture |
Medical cotton ball | Jiangsu Misawa Medical Supplies Co., Ltd. | 20172142357 | Disinfection |
Pet electric clipper | Fenghua Xikou Lubao Knives & Scissors Electrical Appliance Factory | / | Shave the hair of rats |
Tweezers | Qingdao Schultz Biotechnology Co., ltd | Pinch cotton balls |
In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device’s composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.
In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device’s composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.
In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device’s composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.