Fixation and Operational Method for Abdominal Massage in T2DM Mice

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.

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.

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.

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.

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, alleviates their discomfort, and contributes to standardizing the experimental procedure, thus improving experimental efficiency.

Effect of abdominal massage on blood glucose and body weight in T2DM mice
Compared with the blank group, the Fasting Blood Glucose (FBG) and Random Blood Glucose (RBG) levels and body weight of mice in the model group were significantly increased, as shown in Figure 3, which indicated that the T2DM model was successfully established. Compared with the model group, the FBG and RBG levels of mice in the abdominal massage and metformin groups were reduced, as shown in Figure 3A-B. There was no significant difference between them, which indicated that both metformin and abdominal massage were effective in reducing the body weight and high level of blood glucose in the diabetes model; moreover, both interventions improved the body weight of mice, which might be related to their improvement in blood glucose control. These results suggest that the abdominal massage strategy provided by this protocol has similar effects to metformin in regulating blood glucose levels in T2DM mice.

Effect of abdominal massage on lipid metabolism in T2DM mice
In terms of lipid metabolism, compared with the blank group, the Total Cholesterol (TC), Low-Density Lipoprotein (LDL), and Triglyceride (TG) levels of the mice in the model group were elevated, as shown in Figure 4A,C,D, and the High-Density Lipoprotein(HDL) level was significantly reduced, as shown in Figure 4B. After the abdominal massage, the mice's TC, LDL, and TG levels significantly decreased, as shown in Figure 4A,C,D. In contrast, the HDL level was increased, as shown in Figure 4B, with no significant difference from the metformin group. This confirms that abdominal massage has a similar positive effect as metformin in optimizing lipid metabolism.

Effect of abdominal massage on insulin signaling pathway in T2DM mice
Compared with the blank group, the expression levels of IRS-1, IRS-2, PI3K, AKT, and GLUT4 mRNA in the liver tissues of mice in the model group were significantly reduced (p < 0.01), as shown in Figure 5. The liver tissue samples were collected at the end of the 8-week intervention period, following which RNA was extracted using TRIzol reagent and analyzed using quantitative PCR (qPCR). The expression levels of PI3K, AKT, and GLUT4 mRNA in the mice treated with abdominal massage and metformin were significantly increased (p < 0.01, p < 0.05). The difference between the abdominal massage and metformin groups was insignificant (p > 0.05), as shown in Figure 5C-E. These results suggest that both abdominal massage and metformin interventions effectively enhance insulin signaling by increasing the expression of key genes in the insulin signaling pathway, such as PI3K, AKT, and GLUT4, which play critical roles in glucose metabolism and insulin sensitivity. These findings indicate that abdominal massage has a therapeutic effect on T2DM comparable to metformin, likely by improving insulin sensitivity and promoting glucose homeostasis through similar molecular mechanisms. No significant differences between the abdominal massage and metformin groups suggest that abdominal massage could be a viable alternative or complementary approach to metformin in managing T2DM. The significance of these results lies in their potential to provide insight into non-pharmacological treatments for diabetes that modulate insulin signaling pathways.

Figure 1: Preparation of abdominal massage training devices. (A) A 1 mm thick polypropylene plate is shaped into a trapezoid with holes for fixation, and soft, nonwoven fabric is cut to fit, with four holes for the mouse's limbs. A pressure film sensor is placed on the fabric over the mouse's abdomen and connected to a pressure monitoring device. (B) The completed assembly of the device shows the front view of the fixed plate. (C) After the assembly is completed, the diagram shows the final result of the device with the reverse view of the fixed plate. (D) View of the effect after tightening the fixation cloth used to fixate the mice. Please click here to view a larger version of this figure.

Figure 2: Abdominal massage procedure in mice. (A) Place the mouse on the side with a radius of 4.5 cm and move the mouse to the side with a radius of 3 cm by utilizing the mouse's burrowing habit. (B) Further, manipulate the mouse's limbs to pull them out from the four holes of the fixing cloth to fix the mouse. (C) The operator holds the plate in one hand and places the mouse fixation device in the palm so the mouse is supine. (D) The thumb of the other hand can be used to massage the mouse's abdomen. Please click here to view a larger version of this figure.

Figure 3: Effects of abdominal massage on blood glucose and body weight levels in T2DM mice. (A) Comparison of fasting blood glucose (FBG) levels in T2DM mice after 8 weeks of intervention (n = 6 per group). (B) Comparison of insulin resistance index (IRI) levels in T2DM mice after 8 weeks of intervention (n = 6 per group). (C) Comparison of body weight changes in T2DM mice over the 8-week intervention period (n = 6 per group). Measurements were taken at baseline and after 8 weeks of intervention. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 denote significant differences between groups. Please click here to view a larger version of this figure.

Figure 4: Effect of abdominal massage on lipid metabolism in T2DM mice. (A) Comparison of LDL cholesterol levels among the intervention groups (n = 6 per group). (B) Comparison of high-density lipoprotein (HDL) levels in each group after intervention (n = 6 per group). (C) Comparison of total cholesterol (TC) levels in mice after intervention (n = 6 per group). (D) Comparison of triglyceride (TG) levels in T2DM mice after 3 weeks of intervention (n = 6 per group). Measurements were taken after 3 weeks of intervention. Error bars represent the standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistically significant differences between groups. Please click here to view a larger version of this figure.

Figure 5: Insulin signaling pathways in T2DM mice. (A) Comparison of IRS-1 mRNA expression levels across different groups following intervention (n = 6 per group). (B) Comparison of IRS-2 mRNA expression levels in each group after intervention (n = 6 per group). (C) Comparison of PI3K mRNA expression levels in mice after intervention (n = 6 per group). (D) Comparison of AKT mRNA expression levels in each group after intervention (n = 6 per group). (E) Comparison of GLUT4 mRNA expression levels in mice after intervention (n = 6 per group). Samples were collected at the end of the 8-week intervention. Error bars represent the standard error of the mean (SEM). Statistical significance was assessed using one-way ANOVA followed by Tukey's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 represent significant differences between groups. Please click here to view a larger version of this figure.

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 (ST21), Shenque (CV8), Tianshu (ST25), Guanyuan (CV4), and Qihai (CV6). The technique not only stimulates the surface abdominal acupuncture points but also deeply penetrates to stimulate internal organs, improving the body's metabolic function through uniform, gentle, firm, penetrating manipulation⁴.

In this study, a novel simulation device for abdominal massage in experimental mice was designed. This device consists of a transparent fixation plate, flexible fixation fabric, and pressure sensors capable of simulating the strength and rhythm of manual massage while avoiding unnecessary stress and tissue damage to the experimental mice. Regarding existing abdominal massage experimental protocols, such as traditional abdominal massage methods or post-anesthesia abdominal massage, while easy to operate, these methods cannot fully simulate the actual physiological responses in mice in a coma state¹⁵. Another method, involving lifting the mice for abdominal massage, may induce additional stimulation to the mouse spine, making it challenging to control single factors, leading to experimental bias. This observation was based on our experimental trials, where we encountered difficulties in maintaining consistent conditions during the intervention. In comparison, the device designed in this study allows for more controlled, repeatable, and stable conditions, offering clear advantages in terms of cost-effectiveness and reproducibility.

A critical step in this protocol involves fixing the mice on a stand, ensuring that the abdomen is accessible for massage while maintaining minimal movement. Precise control over the frequency and pressure applied is facilitated by the pressure sensors that provide real-time monitoring of the force. The adaptive training of the mice is another crucial aspect of the protocol, as it allows the mice to become acclimated to the process over several sessions, minimizing discomfort and reducing stress responses. After this training phase, the mice could tolerate 30-minute abdominal massages daily, a critical factor in ensuring the reliability and stability of the experiment. This process ensures that the animals adapt without undue stress, which is essential for the integrity of the results.

Recent studies have shown that abdominal massage has multiple regulatory effects on type 2 diabetes (T2DM), including lowering blood glucose levels and optimizing lipid metabolism^5,12,17. In this study, mice in the abdominal massage group showed significant reductions in fasting blood glucose (FBG) and random blood glucose (RBG) levels. Furthermore, the lipid metabolism indicators in the abdominal massage group significantly improved, with total cholesterol (TC), low-density lipoprotein (LDL), and triglyceride (TG) levels significantly decreased. In contrast, high-density lipoprotein (HDL) levels significantly increased. These results reveal the potential intervention value of abdominal massage for metabolic diseases and provide new directions for the comprehensive treatment of T2DM.

A deeper exploration of the molecular mechanisms underlying these improvements reveals that abdominal massage modulates the insulin signaling pathway, specifically by upregulating the expression of key genes involved in glucose metabolism, including PI3K, AKT, and GLUT4. These genes are integral to the insulin signaling pathway, which is responsible for regulating glucose uptake and insulin sensitivity. The upregulation of PI3K and AKT enhances the activation of downstream signaling molecules, which promotes glucose uptake and storage in tissues such as muscle and adipose tissue. Meanwhile, the increased expression of GLUT4 facilitates greater glucose transport into cells, improving overall glucose homeostasis. These molecular changes likely contribute to the observed improvements in glucose metabolism and insulin sensitivity, offering insight into the potential therapeutic effects of abdominal massage in T2DM management.

However, this protocol's experimental procedure still requires technical training for the researchers, particularly in familiarizing the mice with the device, which requires repeated practice to ensure that the mice experience minimal discomfort during the procedure. Another critical point for future studies is the optimization of massage strength and frequency to better understand their impact on experimental outcomes. The current findings are based on a mouse model, and further validation through more extensive animal experiments and clinical trials is essential to confirm the broader applicability and effectiveness of this method.

Looking forward, this protocol has numerous potential applications, particularly in studying metabolic diseases like diabetes, obesity, and even certain gastrointestinal conditions. The non-invasive nature of the technique makes it an attractive alternative to more invasive methods, offering a novel approach for clinical studies or potential therapeutic interventions. The device can also be adapted for use in other animal models, allowing for broader applicability in experimental settings. Furthermore, this device and protocol could play a significant role in modernizing traditional Chinese medicine therapies, providing a scientific platform to explore the mechanisms underlying their effects on metabolic diseases and other health conditions.

The authors have nothing to disclose.

This work has been supported by the second batch of special scientific research projects of the National Clinical Research Base of Traditional Chinese Medicine (JDZX2015127, based on Anhui Provincial Hospital of Chinese Medicine).