Moxibustion Strategies for Mice

Summary

The present protocol describes a moxibustion application method for mice, with the benefits of reducing mouse fearfulness, guaranteeing their welfare, and improving efficiency for researchers.

Abstract

The field of moxibustion research is expanding, with a rapid increase in publications in recent years. Moxibustion is a therapy that ignites moxa on the skin of humans, with an increase in peripheral skin temperature and localized redness. During this treatment, the recipient must remain still to prevent scalding and expose intervention sites for easy manipulation; however, maintaining a fixed posture during moxibustion is a big challenge for animals. Thus, manipulating moxibustion in small animals, such as mice, can lead to several difficulties for researchers. In addition, an uncomfortable posture for animals can lead to fear and resistance to moxibustion, increased risk of injury, diminished animal welfare, and less valid research data. An efficient, comfortable moxibustion method is needed to protect animal welfare and minimize the adverse effects on experimental results. However, moxibustion methods are highly variable and often have limited efficacy. More importantly, an uncomfortable moxibustion posture might cause a stress response, such as those observed with anxiety, fear, and anger, which could influence the research data. Therefore, strategies for animal moxibustion that inflict the least harm possible during the intervention are required. This protocol introduces a mouse tethering method for moxibustion intervention, minimizing mouse discomfort and improving study efficiency. Essential strategies for tethering mice and application of moxibustion are highlighted, and the structure of the tethering instrument is described.

Introduction

Moxibustion is an external treatment used in traditional Chinese medicine¹. It has been widely applied for centuries as a means of keeping fit and preventing and curing diseases^2,3. However, the mechanism of moxibustion remains to be elucidated; thus, this field of research has expanded in recent years as researchers explore its effects and biological mechanisms.

During moxibustion, the recipient must remain still for the best results to be achieved⁴. Moxibustion treatment typically lasts for 15-30 min^5,6. Because of this, manipulation of moxibustion for animals, especially small animals such as mice and rats (favorable for use in animal experiments), has become a big challenge for researchers. Most moxibustion devices are suitable for clinical use and cannot be used for animals, especially for small animals such as mice^7,8,9. Besides this, unlike human beings, animals can barely follow orders and remain steady over a while. A fixed and uncomfortable posture for animals can lead to passive emotions, including anxiety, fear, anger, nervousness, and resistance to treatment, increasing the risk of injury, diminishing animal welfare, and resulting in less valid research data¹⁰. To solve this problem, in previous studies, anesthetics have been applied for extended periods during animal experiments involving external traditional Chinese medicine therapies, including acupuncture, moxibustion, and massage research^11,12,13. However, given that physical conditions change with anesthesia, and in most clinical cases, moxibustion is administered to people in conscious states, anesthesia might impact research data authenticity. Therefore, a method of tethering animals consciously and comfortably become a challenge. To solve these technical problems, specialized devices for mouse moxibustion have been introduced. This protocol provides a tethering method that will assist researchers in performing moxibustion treatment on animals, allowing the animals to remain steady, conscious, and comfortable.

Protocol

All experiments were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Chengdu University of TCM Institutional Animal Care and Use Committee. The care and maintenance of the animals in the laboratory were performed following References14,15. Adult male C57BL/6J mice, weighing 20-25 g, were used for the test. All mice were maintained on a 12 h light/dark cycle at 24 °C and 40%-50%…

Representative Results

Table 1 summarizes the different factors that affect the responses to moxibustion. Positive outcomes of mouse moxibustion have been indicated by multiple measures of mouse welfare and positive effects. Next, specific examples of results obtained when the moxibustion technique is paired with complete Freund's adjuvant (CFA)-induced injection procedures are outlined. The thermal withdrawal threshold (TWL) test was used to evaluate the efficacy of moxibustion in pain relief<s…

Discussion

The moxibustion technique is effective for many disorders, such as fatigue, insomnia, diarrhea, and pain syndrome^{20,21,22,23,24,25,26}. With the benefits of moxibustion treatment for many diseases, the mechanism of this technique has attracted the attention of researchers. The application of …

Materials

Complete Freund's Adjuvant (CFA)	Sigma-Aldrich	SF588102
Glue gun	Deli Group Co., Ltd.	DL5041
Laboratory glove	Boci  Co., Ltd	53625130323
Magnetic metal rectangle (thickness: 0.2 cm)	Sitoo Stationery Co., Ltd	1007908729
Moxa stick	Hanyi Airong factory	5613
Steel sheet	Rizhan metal materials Co., Ltd	1108
Syringe (1000 mL )	Xinmin Fuda Co., Ltd	796341
Timer	Bevoza Co., Ltd	KT003
Velcro belt	Minnesota Mining and Manufacturing Co., Ltd	3MSJ3550
Wooden cube	Chuang Hing Wood Chip Co., Ltd	2581457A
Wooden splint	Chuang Hing Wood Chip Co., Ltd	3410968M
Wooden stick	Chuang Hing Wood Chip Co., Ltd	2785476M
Wooden strip	Chuang Hing Wood Chip Co., Ltd	2374652S