The authors would like to express their gratitude to the Figdraw scientific research drawing platform of the Home for Researchers for providing the material used to create Figure 2 in this paper, with authorized license ID: URUUO5699e. This work has received support from grants provided by the National Natural Science Foundation of China (81873380, 82074566), the Natural Science Foundation of Hubei Province (2022CFCO43), and the National Famous Traditional Chinese Medicine Heritage Studio Construction Project of the National Administration of Traditional Chinese Medicine ([2022] No.5, [2022] No.75).

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&#39;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...

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+method+in+the+study+of+acupuncture+intervention+on+rats.">Fixation method in the study of acupuncture intervention on rats.</a> Journal of Shanxi College of Traditional Chinese Medicine. 20 (3), 169-171 (2019).</li><li>Wang, Y., Ning, Y., Wang, P. Y., Yang, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+Placing+a+Rat+on+an+Elevated+Platform+for+Acupuncture.">Method of Placing a Rat on an Elevated Platform for Acupuncture.</a> Shanghai Journal of Acupuncture and Moxibustion. 31 (5), 289-292 (2012).</li><li>Wang, F., Xiong, J., Zeng, F., He, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fixation+frame+for+acupuncture+and+moxibustion+of+Shu-Mu+acupoint+combination+in+rats.">A fixation frame for acupuncture and moxibustion of Shu-Mu acupoint combination in rats.</a> Acupuncture Research. 47 (10), 938-940 (2022).</li><li>Tu, S., P, C., Y, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+method+for+needle+warming+moxibustion+treatment+on+swinging+rats+bound+with+coat.">Fixation method for needle warming moxibustion treatment on swinging rats bound with coat.</a> Chinese Journal of Comparative Medicine. 31 (5), 128-132 (2021).</li><li>Redfors, B., Shao, Y., Omerovic, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+anesthetic+agent,+depth+of+anesthesia+and+body+temperature+on+cardiovascular+functional+parameters+in+the+rat.">Influence of anesthetic agent, depth of anesthesia and body temperature on cardiovascular functional parameters in the rat.</a> Laboratory Animals. 48 (1), 6-14 (2014).</li><li>Kiyatkin, E. A., Brown, P. 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W., Garber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. 85 (3), 963-965 (2011).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroacupuncture+ameliorates+beta-amyloid+pathology+and+cognitive+impairment+in+Alzheimer+disease+via+a+novel+mechanism+involving+activation+of+TFEB+(transcription+factor+EB).">Electroacupuncture ameliorates beta-amyloid pathology and cognitive impairment in Alzheimer disease via a novel mechanism involving activation of TFEB (transcription factor EB).</a> Autophagy. 17 (11), 3833-3847 (2021).</li><li>Su, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+and+Safety+of+Acupuncture+for+Vascular+Cognitive+Impairment:+A+Systematic+Review+and+Meta-Analysis.">Effectiveness and Safety of Acupuncture for Vascular Cognitive Impairment: A Systematic Review and Meta-Analysis.</a> Frontiers In Aging Neuroscience. 13, 692508 (2021).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+electroacupuncture+pretreatment+for+myocardial+injury+in+patients+undergoing+percutaneous+coronary+intervention:+A+randomized+clinical+trial+with+a+2-year+follow-up.">Efficacy of electroacupuncture pretreatment for myocardial injury in patients undergoing percutaneous coronary intervention: A randomized clinical trial with a 2-year follow-up.</a> International Journal of Cardiology. 194, 28-35 (2015).</li><li>Zhang, R., Lao, L., Ren, K., Berman, B. 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All authors have declared no potential conflicts of interest.

Electroacupuncture has been shown to have good therapeutic effects on various diseases such as cognitive impairment16,17, 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...

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 deepen3,4, 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 fixation6,9, 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&#39;s jacket takes advantage of the rats&#39; natural inclination for darkness and burrowing, thus restricting their range of activities. Secondly, the binding ropes near the joints of the rats&#39; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65652/65652fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/65652/65652fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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	<tbody>
		<tr>
			<td>75% alcohol</td>
			<td>Shandong Lircon medical technology co., ltd</td>
			<td>2002-0059</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>acupuncture needles</td>
			<td>Beijing Luo ya shan chuan medical equipment co., Ltd.</td>
			<td></td>
			<td>needling</td>
		</tr>
		<tr>
			<td>Adult Sprague-Dawley (SD) rats</td>
			<td>Liaoning Changsheng biotechnology co., Ltd.</td>
			<td>SCXK 2020-0001</td>
			<td>The recipient&#160; subjected to fixation</td>
		</tr>
		<tr>
			<td>an elevated fixation rack&#160;</td>
			<td>Taizhou shiai instruments and equipments for scientific and teaching research&#160; Co., ltd</td>
			<td>/</td>
			<td>Fix rats</td>
		</tr>
		<tr>
			<td>Hans electronic acupuncture apparatus</td>
			<td>Beijing Huayun Ante Science and Technology Co. Ltd.&#160;</td>
			<td>HANS-200A</td>
			<td>electroacupuncture</td>
		</tr>
		<tr>
			<td>Medical cotton ball</td>
			<td>Jiangsu Misawa Medical Supplies Co., Ltd.</td>
			<td>20172142357</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>Pet electric clipper</td>
			<td>Fenghua Xikou Lubao Knives &#38; Scissors Electrical Appliance Factory</td>
			<td>/</td>
			<td>Shave the hair of rats</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Qingdao Schultz Biotechnology Co., ltd</td>
			<td></td>
			<td>Pinch cotton balls</td>
		</tr>
	</tbody>
</table>

improved suspension fixation using rat jacket in rat acupuncture experiments

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.

Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

We developed an improved method for suspending and fixing rats with rat clothes in an acupuncture experiment. This protocol aims to provide detailed descriptions of its procedural steps, including important precautions, offering a reference for researchers conducting fundamental experiments in the field of acupuncture and moxibustion. In previously reported studies, researchers have often used anesthesia fixation, high platform fixation, binding fixation, or various other self-made fixators while performing acupuncture or electroacupuncture operations on rats.Given the side effects of anesthesia on the body and the real clinical conditions, fixation in an awakened stage offers the better alternative. However, in the non-anesthetized stage, the mentioned fixations also possess limitations such as restricted acupoint exposure, requirement of multiple labels, non-adjustable fixator size, or forced posture. This method utilizes the rat's preference for burrowing to create rat jackets that fit their body shape.It offers several advantages over other techniques. It is cost-effective, user-friendly, manpower-and space-saving, accommodates rats of varying weights, and can also be used for needle operation on most acupoints. In subsequent studies, we will focus on investigating the impact of the restraints and suspensions of rat clothing on physiological indicators and behavioral aspects related to emotional cognition in rats.This will enable us to quantitatively assess its potential influence on research and refine the fixation method.

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...

Watch this Scientific Journal Video about Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture at JoVE.com

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.

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...

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Securing the Rat in the Rat Jacket for Electroacupuncture

Wear a pair of gloves before proceeding to fit and secure the rat in the custom made rat jacket. Then using one hand hold the rat jacket's middle and back portion on the table. Using the other hand, gently hold the rat by its tail to pick it up from the feeding cage and place it into the jacket as the rat moves forward into the jacket and its head reaches the headgear point.Using both hands, carefully pull out its left and right upper limbs from the holes in the jacket. Securely wrap the tying ropes from the opening end of the jacket around the rat's trunk, and tie them tightly at the opposite end to adequately restrain its upper limbs. Tie the rope loose enough to prevent hypoxia or local limb ischemia, and tight enough to prevent the rat's escape.In a similar manner, secure its lower limbs.

Performing Electroacupuncture on Rat Immobilized Using Rat Jacket

Before beginning the procedure of electropuncture on the rat, dress the rat in the custom made rat jacket. Then disinfect the local skin of acupoints using alcohol swabs. After locating the acupoint accurately, insert the sterile disposable acupuncture needle into the spot at the appropriate depth.Gently suspend the animal from the horizontal iron rod with the help of the S-shaped hooks attached to the rat jacket. Wrap the electrode wire around the horizontal iron rod before securely attaching the small clip at the end of the electrode wire to the needle handle. Then after switching on the power, select the appropriate frequency, current, and time.Once the parameters are adjusted, commence the electroacupuncture treatment process. Using the S-shaped hooks, suspend the rats in the same group together on the iron rod for simultaneous electroacupuncture. When the treatment is over, remove the acupuncture needle along with the clamp from the animal.Then carefully take the needle out of the clamp and put it in the box. After removing the animal from the suspension hooks, 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.While performing a 15 minute electroacupuncture experiment on 12 rats only one rat exhibited intense struggle during the needle retention period resulting in needle drop. While other rats maintained proper needle fixation throughout the treatment. When compared to the other fixation methods reported in the literature, the demonstrated method exhibited the combined benefits of stability and secure fixation, efficiency in terms of time and number of personnel required, affordability, as well as relative comfort for the rats.Additionally, in terms of the exposed acupoint range, the specially designed rat jackets in this protocol met the requirements for exposing most acupoints on the body.

867 Views.  Hubei University of Chinese Medicine.  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. 

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

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