Fixation and Operational Method for Abdominal Massage in T2DM Mice

Summary

This study introduces an abdominal massage device for mice that replicates manual massage while minimizing stress and tissue damage. The device significantly lowers blood glucose levels, improves lipid metabolism, and enhances insulin sensitivity in type 2 diabetes mice, offering a promising, non-invasive therapeutic approach with clinical potential.

Abstract

Type 2 diabetes mellitus (T2DM) is a rapidly growing global public health issue, affecting over 500 million people worldwide. Although abdominal massage has shown potential benefits in managing T2DM, its effectiveness remains unclear, particularly in animal studies, where challenges such as animal compliance and the need for precise pressure control complicate implementation. To address these challenges, this study introduces a novel abdominal massage simulation device specifically designed for mice. This device provides a practical solution for conducting abdominal massage interventions in a controlled manner while minimizing stress and tissue damage to the animals. It securely restrains the mice's limbs, allowing them to remain conscious during the massage, and offers precise control over both the pressure and frequency of the massage applied to the abdomen. The device's ability to simulate manual abdominal massage with accuracy opens new possibilities for experimental studies assessing its effects on T2DM. The primary goal of this protocol is to investigate the impact of abdominal massage on key T2DM markers such as blood glucose levels, lipid metabolism, and insulin sensitivity in mice. By providing a reliable and reproducible method for abdominal massage, this device can offer valuable insights into its potential as a non-invasive therapeutic intervention for T2DM. The findings from this research may contribute to advancing clinical strategies for diabetes prevention and treatment, particularly in enhancing the understanding of traditional therapies like abdominal massage in modern medical practice.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by insulin resistance and dysfunction of pancreatic β-cells. It is a nationwide public health issue, with a rapidly increasing incidence worldwide¹. According to the Global Metabolic Disease Burden Report, more than 500 million people globally have diabetes, with over 90% of them having T2DM². Abdominal massage is an important intervention for treating T2DM. A large body of research has shown that abdominal massage can significantly improve blood glucose levels, quality of life, gut microbiota composition, and its metabolic products in T2DM patients, thus enhancing insulin sensitivity^3,4,5. In addition, abdominal massage promotes gastrointestinal peristalsis, improves digestive function, reduces gastrointestinal stasis, and optimizes nutrient absorption and glucose metabolism^6,7,8. Despite the potential through which abdominal massage improves T2DM outcomes, have not been fully explored. Specifically, abdominal massage has been shown to improve insulin sensitivity and glucose metabolism by modulating metabolic pathways such as the insulin signaling pathway, but the precise physiological processes are not yet fully understood. This study aims to bridge this gap by providing more detailed mechanistic insights into how abdominal massage can improve T2DM in animal models.

The primary goal of this study is to design and develop a standardized, reproducible method to study the effects of abdominal massage on T2DM using an innovative device designed specifically for mice. This device aims to provide precise control over massage parameters, such as pressure and frequency, while minimizing animal stress and ensuring animal welfare compliance.

However, performing abdominal massage on animal models, especially mice, presents several challenges. Current research methods face challenges, including inconsistent massage application, difficulty maintaining uniform pressure and frequency, and ensuring animal compliance during the intervention. These limitations hinder reproducibility and introduce variability in results, emphasizing the need for a more effective and reliable technique. Animal experiments are essential to answer this question. For abdominal massage in animals, it is necessary to maintain a certain pressure and frequency. In practice, however, it is not easy to have animals comply with instructions and remain in the required position inside the device, as they are naturally active. It is also challenging to ensure consistent pressure and speed during the intervention, which makes it difficult to achieve the stability required for abdominal massage techniques. Therefore, performing abdominal massage interventions on mice, rats, and other animal models remains a significant challenge in research. Moreover, animal experiments must address issues related to animal welfare, such as alleviating stress and anxiety, reducing pain, and improving overall conditions. Existing methods, such as post-anesthesia massage or manual restraint, are often labor-intensive, introduce experimental bias, or fail to replicate natural physiological conditions in conscious animals. Previous solutions have involved rat abdominal massage models^9,10,11. To overcome these limitations, we introduce a novel abdominal massage device for mice. In this study, we introduce a novel abdominal massage device designed to overcome these limitations. Compared with traditional techniques, this device offers significant advantages, including stable operation, precise control over massage parameters, reduced animal stress, and improved reproducibility. Additionally, its cost-effective design enhances accessibility for broader applications in metabolic disease research. By integrating modern engineering with traditional therapeutic approaches, this device fills a critical gap in non-invasive experimental methodologies for T2DM.

This approach contributes to the wider body of literature on non-invasive therapeutic techniques for T2DM, particularly those derived from traditional Chinese medicine (TCM), which emphasizes the regulatory effects of abdominal massage on digestive and metabolic functions. Researchers may find this method particularly suitable for studies on non-pharmacological interventions for T2DM and other metabolic disorders. By addressing key experimental challenges, this method provides a foundation for advancing clinical and translational research in this field.

Protocol

All animal experiments were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine (Approval No: 202409A033). Here, 24 SPF-grade healthy male C57BL/6J mice, aged 8 weeks and weighing 22 g ± 2 g, were selected. The mice were housed under a 12 h light/dark cycle at a temperature of 20-22 °C and with a relative humidity of 45%- 50%. The animals had free access to food and water.

1. Establishment of the T2DM mouse model

1. Feed mice standard chow for 1 week to adapt to the new environment in the animal facility. After 1 week, randomly divide the 24 C57BL/6J mice divided into the control group (6 mice) and the T2DM model group (18 mice) using a random number table. Give the control group purified standard chow while the T2DM model group receives a high-fat diet. Continue the diet for 6 months.
2. Monitor the mice's drinking, eating, body weight, fasting blood glucose, and random blood glucose throughout this period. If two consecutive blood glucose readings exceed 7.8 mM/L, perform an insulin tolerance test (ITT). Consider the value of the area under the curve (AUC) greater than 35 as a successful T2DM model.
3. Start the Intervention once the T2DM model has stabilized.

2. Grouping and treatment of mice

1. After successful T2DM modeling, randomly assign the T2DM mice to one of the following groups using a random number table to ensure unbiased selection: model group, metformin group, and abdominal massage group, with six mice in each group.
2. Perform no intervention in the control group. Use the random number table to assign each mouse to one of the experimental groups, ensuring that the groups were comparable in terms of initial body weight and glucose levels to minimize bias in the assignment.
3. Feed the model group a high-fat diet throughout the experiment and administer 0.2 mL of physiological saline by gavage daily, 1x a day, 6 days a week, for 8 weeks.
4. Feed the metformin group a high-fat diet throughout the experiment. Ground metformin tablets and dissolve for gavage administration once daily at 200 mg/kg/day, diluted to 0.2 mL with physiological saline. Administer this 1x a day, 6 days a week, for 8 weeks.
5. For the abdominal massage group feed a high-fat diet throughout the experiment. Gavage the mice with 0.2 mL of physiological saline daily. Perform an abdominal massage once after 5 min of drug administration. Administer abdominal massage intervention 6 times a week at a fixed time, with one rest day, for a continuous period of 8 weeks.
   1. Fix mice using a homemade rodent restrainer and expose their abdomen while ensuring they remain calm for abdominal massage.
   2. Reference to the acupoint map for mice to locate the Shenque and Tianshu points. Use the distance from Shenque to Tianshu as the radius and perform a clockwise rotational massage for 30 min at 100-120 times per min. The pressure range applied during the intervention was 0.1-0.3 N/cm. Adjust the pressure to a level the mice can tolerate while remaining calm and monitoring using the FPS pressure testing system.

3. Preparing the abdominal massage training device

1. Prepare a 1 mm thick polypropylene board to create a fixation plate, shaping it into a semicircle with a radius of 4.5 cm on one side and 3 cm on the other. When unfolded, it forms a trapezoidal shape with an upper base of 8 cm, a lower base of 10 cm, and side lengths of 10 cm. Use a transparent plate to observe the mouse's fixation as shown in Figure 1A-C.
2. Depending on the size of the mice, punch four long holes in the fixation plate, as shown in Figure 1A.
3. Prepare a soft and durable nonwoven fabric, cutting it into an elliptical shape for the center with four elongated straight strips at the corners to serve as the fixation fabric. Ensure the long, straight strips can pass through the four elongated holes in the plate. Based on the size of the mouse, cut four round holes in the fabric strips to allow the mouse's limbs to pass through, as shown in Figure 1B.
4. Select a pressure-sensitive film sensor based on the mouse's abdominal size and attach the pressure film to the part of the fixation fabric corresponding to the mouse's abdomen. Connect the film to the pressure monitoring device, as shown in Figure 1D.

4. Abdominal massage procedure

1. Pass the four long strips of the fixation fabric through the holes in the fixation plate, creating a passage through which the mouse can easily pass.
2. Place the mouse on the side with the 4.5 cm radius, using its burrowing instinct to encourage it to move toward the side with the 3 cm radius, as shown in Figure 2A.
3. Observe the mouse's position from the back of the transparent fixation plate. When the mouse reaches the appropriate position, tighten the four fabric strips to bind the mouse to the central part of the fixation fabric. Tie the strips in knots to secure the mouse.
4. Flip the fixation plate over and use forceps to pull the mouse limbs out of the 4 round holes of the fixation cloth to fix the mouse further, as shown in Figure 2B.
5. Hold the plate with one hand, placing the fixed mouse device in the palm to position the mouse supine. Perform the abdominal massage on the mouse's abdomen with the thumb. During the procedure, closely monitor the values on the pressure monitoring device to ensure that the force applied to the pressure film sensor on the mouse's abdomen remains constant and uniform, as shown in Figure 2C - D.
   1. If the mouse's nose and lips appear purple during the abdominal massage, it may indicate that the fixation fabric is too tight, causing oxygen deprivation. In such cases, stop the procedure immediately, loosen the fabric strips, and release the mouse to recover from oxygen deficiency.
   2. During the abdominal massage, the mouse may defecate or urinate more frequently, which is a normal response to the abdominal massage. Perform prompt cleaning.
6. After 20 min of abdominal massage, loosen the knots on the fabric strips, allowing the mouse to free itself from the fixation device.
7. After the mouse adapts to the abdominal massage, the process should proceed quietly, with minimal struggle. If the mouse struggles excessively, check the device for issues, such as a broken fixation plate or overly tight fixation fabric. Constantly monitor the mouse's comfort and vital signs throughout the procedure.
8. Weigh the animals and perform blood glucose readings after the completion of the experiment.

Results

According to the above plan, the abdomen of the mice was kept fixed in place, and the relatively fixed position allowed the mice to maintain a stable posture. Massaging the abdomen at a fixed frequency, the pressure and duration of the massage were controlled within a manageable range. This approach is similar to traditional abdominal massage in Chinese medicine¹². By following this intervention plan, the intervention time can be appropriately extended, which helps the mice adapt to the training, ...

Discussion

Abdominal massage is a representative fundamental technique in traditional Chinese medicine (TCM) massage, widely applied in clinical prevention and treatment of various system diseases, especially in regulating and caring for the spleen and stomach digestive system^13,14,15. Abdominal massage directly affects the surface of the human body, covering most of the abdominal acupuncture points, including Zhongwan (CV12), Liangmen (ST...

Materials

Name	Company	Catalog Number	Comments
High-density lipoprotein cholesterol assay kit	A112-1-1	Nanjing Jiancheng Bioengineering Institute
Low-density lipoprotein cholesterol assay kit	A113-1-1	Nanjing Jiancheng Bioengineering Institute
Metformin tablets		Sino-US Shanghai Squibb Pharmaceutical Co
Pressure Thin Film Sensors	FSR16	Vickers Electronic Technology (Luoyang) Co.
Retainer sleeve		No special requirement，Preferably, the material should be PP.
Strips of cloth for fixing		1mm thick, unfolded to a trapezoidal shape with an upper base of 8cm, a lower base of 10cm and two waists of 10cm.
Total cholesterol assay kit	A111-1-1	Nanjing Jiancheng Bioengineering Institute
Triglyceride assay kit	A110-1-1	Nanjing Jiancheng Bioengineering Institute