Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

Podsumowanie

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

Streszczenie

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.

Wprowadzenie

Acupuncture is a traditional therapy characterized by needling at specific acupoints or other positions to prevent and treat diseases. Electroacupuncture combines filiform needles with electrical stimulation¹, which allows for precise control over the amount of stimulation by adjusting relevant parameters, promoting the standardization of acupuncture treatment². In recent years, research on the mechanisms of electroacupuncture has continued to expand and deepen^3,4, which has led to an annual increase in studies focusing on the mechanisms of electroacupuncture⁵.

In basic research on electroacupuncture, rats are the most commonly used animals. During electroacupuncture treatment, the recipient must remain still to ensure a smooth procedure. Unlike in clinical trials with human subjects, small animals used in animal experiments cannot follow commands consciously during 15-30 min lengthy electroacupuncture procedures. This presents researchers with a primary challenge of achieving stable and reliable fixation. Improper fixation can lead to negative emotions such as anxiety, depression, and fear in animals⁶, which not only increases the risk of injury for rats and the chance of being bitten for the experimental personnel but also affects the stability and persuasiveness of research results.

In previous studies, researchers have often used anesthesia fixation⁷, high platform fixation⁸, binding fixation^6,9, or rat coat fixation¹⁰ before acupuncture or electroacupuncture operations. However, given the effects of anesthesia on the body¹¹ and brain function¹², and considering that acupuncture is performed on conscious patients in clinical cases, anesthesia may impact research results to a certain extent. Therefore, compared with anesthesia fixation, comfortable fixation of rats in an awake state is a better option.

However, in the non-anesthetized state, various fixation methods also possess their respective limitations, including restricted acupoint exposure due to coverage of the rat coat on rats¹⁰, the necessity of at least two individuals to perform the operation¹³, non-adjustable fixator size¹⁴, or the stressed state of rats in a forced position⁶. To address these technical issues, we designed a rat jacket and a fixation rack suitable for acupuncture manipulation in most parts of the rat body. This can help researchers efficiently carry out acupuncture procedures with rats.

The rationale for this protocol is as follows: firstly, the closed headgear on the rat's jacket takes advantage of the rats' natural inclination for darkness and burrowing, thus restricting their range of activities. Secondly, the binding ropes near the joints of the rats' limbs provide physical compression restraint, imposing certain limitations on their movements. However, rats are still able to jump and move horizontally even while wearing the jacket. Lastly, since rats cannot touch the ground without a fixed force point when suspended, we added a suspension fixation device to the rat jacket, further restricting their movements and ensuring needle retention. The composition of the fixation device is shown in Figure 1.

Figure 1: Schematic diagram of the rat jacket and suspension fixation. (A) The general appearance of the rat jacket in a flat view. (B) Three-dimensional view of the rat jacket. (C) Specially tailored rat jacket with an incision near the acupoints on the head and back waist. (D) Illustration of multiple rats simultaneously suspended on the elevated rack. 1: Fabric overall view; 2: Forelimb outlet; 3: Hind limb outlet; 4: Tying rope; 5: Cavity formed after suturing; 6: Incision for exposing head acupoints; 7: Incision for exposing back and waist acupoints. 8: Suture position. Please click here to view a larger version of this figure.

Protokół

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

1. Production of the rat jacket
   1. Weigh the rats and measure their body shape before making their jackets. To verify the suitability of this protocol for rats of varying weights, select rats weighing 220 g, 300 g, 400 g, and 520 g for independent measurements of their bodily dimensions, encompassing measurements from a to f, as depicted in Figure 2.
      NOTE: The correlation between the bodily proportions of rats and their corresponding jacket sizes is also presented in Figure 2. The pertinent body length parameters for rats of different weights are recorded in Table 1.
   2. Cut a rectangular piece of thick, dark fabric to make a rat jacket. Use the fabric with unfolded width equal to the circumference measured around the thickest part of the rat's abdomen. The length of the fabric is equal to the distance from the rat's nose tip to the base of its tail (see Figure 2).
   3. Fold the fabric in half and cut off the top left corner at an angle of about 45°-65° to form a right trapezoid (see Figure 3A).
   4. Sew the two sides on the left tightly with cotton thread. Refer to Figure 1B and Figure 3B for guidance.
   5. Cut two small holes, one on each side of the fabric, for the rat's upper and lower limbs to pass through (see Figure 3C). The distance between the two upper limbs determines the spacing between the upper holes of the rat jacket. The distance between the two lower limbs determines the spacing between the lower holes of the rat jacket.
   6. Use the longitudinal distance between the rat's armpit and the front of the ilium to serve as the spacing between the upper and lower holes of the rat jacket. Use the circumference of the shoulder joint to determine the circumference of the upper hole in the rat jacket. Use the circumference of the hip joint for the circumference of the lower hole in the rat jacket.
   7. Beside each hole, sew a long cloth strap that can be crossed and wrapped tightly around the body from the opening end of the jacket to fix the rat.
2. Rat jacket-wearing method
   1. Wear gloves before proceeding to dress the rat in the jacket. Begin by holding the middle and back part of the rat jacket with one hand on the table. With the other hand, hold the rat's tail from its feeding cage and gently place it into the jacket.
   2. As the rat moves forward, its head will reach the headgear of the jacket. At this point, use both hands to carefully pull its left and right upper limbs out of the holes in the jacket.
   3. Take the tying rope from the opening end of the jacket, cross it over from left to right, and wrap it securely around the rat's trunk. Tie it tightly on the opposite side of the trunk to ensure proper fixation. Following the restraint of the rat's upper limbs, repeat the same procedure to fix its lower limbs.
   4. Take precautions to avoid getting bitten or scratched by the rat's claws when pulling its limbs out of the jacket holes. Ensure that the tightness of the rope is appropriately adjusted, i.e., not too tight to cause local limb ischemia and hypoxia in rats, and not too loose, allowing the rat to escape easily (see Video 1).
3. Fixation rack and S-shaped iron hooks
   1. The fixation rack consists of two heavy iron trays (24 cm in length, 12 cm in width, and 2 cm in height) and an iron column (60 cm in length) that is firmly welded on each tray. Fix a solid iron stick (145 cm in length) at the same height on the two iron columns using a screw connection structure that can be adjusted and maintained at a fixed height (see Figure 4A).
   2. For connecting the rat wearing the jacket to the iron stick, use the S-shaped hooks (11 cm in diameter). Wrap one end of the hook around the strap on the jacket while the other end is hung on the iron stick. Use two S-shaped hooks to secure the rat onto the stick. Please refer to Figure 1D and Figure 4B for the overall structure.

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

1. Routine preparation
   1. Place the fixation rack on a horizontal desktop or ground.
   2. Prior to the needling procedure, remove the fur around the acupoints using an electric shaver to ensure complete exposure. When the acupoint to be punctured is located on the abdomen of the rat, such as Guanyuan (CV4, about 25 mm below the navel), shorten the width of rat clothing appropriately to expose abdominal acupoints.
   3. Locate the acupoint from the gap on the midline of the abdomen that is not covered by the rat jacket (see Figure 3D) and then inject the needle.
   4. When the acupoints to be punctured are on the head of the rat, such as Baihui (GV20, located at the middle of the parietal bone), make a small incision at the corresponding position on the head to fully expose the skin around GV20 (See Figure 1C).
   5. When acupuncturing acupoints on the limbs of rats, such as Neiguan (PC6, on the inner side of the forelimb, between the radial and ulnar sutures about 3 mm away from the wrist joint) in the upper limb or Zusanli (ST36, on the posterolateral side of the knee joint, about 2 mm below the fibula head) in the lower limb, due to the obvious exposure of the acupoints, use one hand to assist in fixing the limb, while the other can directly perform the acupuncture operation.
   6. When the acupoint is located on the back or waist of a rat, such as Shenshu (BL23, below the 2nd lumbar vertebrae, 6 mm apart from the midline of the back), cut an opening approximately 2 cm wide in the center of the rat's jacket to fully expose the skin of the corresponding site (See Figure 1C).
   7. Dress the rat in the jacket. After disinfecting the local skin of acupoints with alcohol swabs, begin the acupuncture procedure.
2. Needling technique: Locate the acupoint accurately. Insert a sterile disposable acupuncture needle (0.16 mm x 7 mm) into the rat's acupoint at the appropriate depth.
3. Electroacupuncture operation
   1. Wrap the electrode wire around the horizontal iron rod. This step reduces the risk of the needle falling out due to gravitational force acting on the electroacupuncture clip. Then, attach the end of the electrode wire securely onto the needle handle.
   2. Switch on the power and select the appropriate frequency, current, and time parameters. In this experiment, the parameters were set to 2 Hz, 1 mA, and 15 min. Once the parameters are adjusted, commence the electroacupuncture treatment process (See Video 2 and Video 3).
   3. Simultaneous electroacupuncture on rats: During treatment, suspend the rats in the same group together on the iron stick using the S-shaped hooks and let them undergo electroacupuncture intervention simultaneously.
   4. NOTE: The iron stick should not only be sturdy but also long enough to treat a group of rats at the same time. In this experiment, while ensuring appropriate spacing and no interference among the rats, 3-4 rats can be treated simultaneously (see Figure 4C).
4. Observation of rat responses during electroacupuncture treatment: Evaluate the suitability of this fixation method for electroacupuncture in rats based on the following factors, such as the number of personnel required to complete the operation, time taken to fix a rat, the rats' response during the electroacupuncture treatment, the number of needles dropped during the procedure, and any accidental deaths that might occur during the operation.
5. Removal of the needles
   1. Once the pre-set time is completed, the electroacupuncture apparatus will automatically shut off. Remove the small clip that is connected to the needle handle and carefully remove the acupuncture needle from beneath the rat's skin, thereby concluding the electroacupuncture treatment process.
   2. Loosen the straps, lift the head cover of the rat's jacket, and position the rat above the cage. The rat will then be free to release itself from the jacket and enter the cage independently (see Video 4).

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.

Wyniki

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 stabilit...

Dyskusje

Electroacupuncture has been shown to have good therapeutic effects on various diseases such as cognitive impairment^16,17, myocardial injury¹⁸, and inflammatory pain¹⁹. To understand its mechanism of action, animal experiments primarily using rats have been conducted. Fixation of the animals during the procedures is crucial for accurate results. While many studies have administered electroacupuncture subsequent to an...

Materiały

Name	Company	Catalog Number	Comments
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