Static Strength Training Method for Type 2 Diabetic Mice

Özet

This protocol provides a simple method of making static training equipment for mice. The device maintains the muscle isometric contraction of the limbs of mice so as to verify the intervention effect of traditional exercise on type 2 diabetes (T2DM) and provides new exercise therapy for the clinical treatment of T2DM.

Abstract

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. Static strength training is one of the key forms of traditional sports in China. Research shows that static strength training is an effective clinical method for T2DM intervention, but there is no experimental device suitable for static training in mice. One of the difficulties in moving from clinical to basic research is to design appropriate experimental devices. In order to further study the mechanism of static training intervention in T2DM, a simple method for making a static training device for mice is introduced in this paper. This device has the advantages of simple operation, cheap material, and high feasibility. Previous studies conducted under this protocol have shown that static training can effectively reduce blood glucose levels and improve the mitochondrial function of skeletal muscle cells in T2DM mice. The purpose of introducing this device is to promote research on the mechanism of traditional exercise in the intervention of T2DM and to lay a foundation for the quantitative intervention of exercise.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by insulin resistance and β-cell dysfunction that is a significant threat to global health¹. Exercise is a crucial intervention in the management of type 2 diabetes. Numerous studies have shown that traditional Chinese exercise methods, such as Tai Chi and Ba Duan Jin, significantly improve blood glucose levels and quality of life for individuals with T2DM^2,3,4,5. To execute these movements, the trainer must maintain a stable body and joint position for a period of time. The static position is sustained by performing static muscle contractions, which is commonly referred to as static strength⁶.

However, the mechanism of static strength training intervention in T2DM has not been clarified. To answer this question, animal experiments are essential. During isometric exercises, muscles are activated, maintain a constant length, and safely achieve maximum tension⁷. In experiments with static strength training, the test animal is required to perform isometric muscle contractions and to maintain this state of muscular contraction. How to implement static strength training on mice, rats, and other laboratory animals has become a big problem in research. First, animals struggle to obey commands and contract their muscles as required. Secondly, it is difficult for the animal to maintain a stable position under resistance, and the purpose of isometric muscle contraction cannot be achieved. While letting animals train as required, it is important to address concerns related to animal welfare, such as relieving stress and anxiety, minimizing pain, and improving overall conditions. This protocol pertains to a static training model for rats^8,9, and here we introduce a simple device for static training of mice. When the hind legs of mice are lifted, their abdominal muscles contract due to the righting reflex, the forepaws grasp the cross-bar in front, and then the front and back limbs contract against gravity. The mice cannot move after grasping the short bar, resulting in their muscles being in a state of isometric contraction.

Protocol

All animal experiments were approved by the Animal Care and Use Committee of the Nanjing University of Chinese Medicine (permission no. 202209A033). Healthy male C57BL/6J mice with SPF grade, 8 weeks of age, and body weight of 20 ± 4 g were selected. The mice were housed in a 12 h light/dark cycle at a temperature of 20-22 °C, and a relative humidity of 45%-50% was maintained. The animals eat and drink freely.

1. Establishment of a mouse model of T2DM

1. Feed the mice for 1 week on a regular diet first and let the mice adapt to the new environment of the animal center. After 1 week, provide a high-fat diet (diet composition: 20.0% lard, 10.0% sucrose, 2.5% cholesterol, 1.0% cholate, 66.5% conventional diet) for a period of 4 weeks.
2. From the 1^st day of the 5^th week, intraperitoneally inject mice with streptozotocin solution at a dose of 35 mg/kg each day for 3 consecutive days. Prepare 1 mL of the streptozotocin solution with 10 mg of streptozotocin powder, with 0.1 M/L sodium citrate buffer as the solvent.
   NOTE: The injection should be completed within 30 min after the solution is prepared, and the solution should be kept away from light during use.
3. Randomly test blood glucose on day 5 and day 7 after the first injection of streptozotocin solution was administered. Animals with two random blood sugar levels higher than 16.7 mM/L are considered successful T2DM models.
4. Begin training intervention on day 8 after the first injection of streptozotocin solution.

2. Grouping and treatment in mice

1. Divide the T2DM mice into a model group, training group, and metformin group using the random number table method, with 6 mice in each group.
2. Perform no intervention for the model group.
3. Grind metformin tablets into powder and dissolve in pure water. Give metformin to the metformin group by gavage at a dose of 200 mg/kg, once per day, for 3 weeks.
4. Let the training group do 30 min of static strength training, once a day, 5 days a week, for 3 weeks. Training is held every Monday to Friday afternoon.
5. Randomly assign 6 non-T2DM mice as control group. These mice do not undergo modeling and intervention.

3. Manufacturing the static strength training device

1. Prepare a 5 mm thick transparent acrylic board with dimensions of 20 cm x 20 cm. Use a transparent board in order to observe the mice training.
2. Get wooden sticks with a diameter of 3 mm and a roll of tape with a width of 1 cm. Use a paper cutter to cut two 4 cm long sticks and four 1 cm long sticks.
3. Attach two sticks, each with a length of 1 cm, to the ends of a stick with a length of 4 cm using tape (Figure 1A). The short sticks should be fixed on the same side as the long stick.
4. Place the transparent board on a flat surface and attach two sets of 1 cm short sticks to the plate using a hot glue gun. Ensure the 4 cm wooden sticks do not come into contact with the board. Allow a 2 cm gap between the long stick and the board to hold the mice in place (Figure 1B). Let the two long sticks be aligned and parallel, with a distance of 6 cm between them (Figure 1C).

Figure 1: Assemble and secure the sticks to the transparent board. (A) Tape the 1 cm stick to both ends of the 4 cm stick. (B) Use hot melt adhesive to connect the 1 cm long stick and transparent board, and the gap length is 2 cm. (C) Two 4 cm sticks spaced 6 cm apart. Please click here to view a larger version of this figure.

4. Static strength training in mice

1. Cut two pieces of woolen yarn 15 cm long. Use a slipknot to tie a piece of woolen yarn on each of the mouse's upper ankle (Figure 2A).
   NOTE: Elastic rope cannot fix the posture of mice, and hemp rope will wear the ankles of mice, so inelastic but soft woolen yarn is chosen here.
2. Place the board horizontally on the table with sticks up. Press the mouse carefully between two long sticks, positioning its head and tail to align with the gaps below the sticks.
3. Pass the yarn through the gap at the end of the tail. Then, adjust the yarn until the ankles of the mouse fit snugly against the edge of the stick.
4. Secure the woolen yarn with tape (Figure 2B). The end of the woolen yarn should be secured otherwise, the mouse will climb the fallen wool. Pull the wool as tight as possible or the mouse will easily break free.
5. Invert the transparent board. Get 2 boxes of the same height (15-20 cm tall) on either end of the transparent board. Place the board horizontally at a certain height. The box is located at either end of the mouse's head and tail. If it is too high, the experimenter will not be able to handle it easily; if it is too low, the mouse may touch the tabletop.
6. Once the board is overturned, the mouse hangs upside down. Due to the righting reflex, mice curl their abdomen and extend their forelimbs to grasp their hindlimbs or sticks. At this stage, position a 20 cm stick in front of the mouse and carefully guide it to grasp the stick with its forelimbs. Repeat the process until the mouse becomes proficient at grasping the mobile stick.
7. Use the stick to move the forelimb of the mouse to another 4 cm stick on the transparent board. Constantly adjust the angle of the stick to enable the mice to actively grasp the stationary 4 cm stick on the board (Figure 2C).
8. Repeat the previous step as the mouse releases the front paw until the mouse is exhausted. After 30 minutes, most of the rats were unable to lift their upper body and grasp the stick with their forelimbs.
   NOTE: Before implementing the intervention, a week of habituation training on mice is necessary. The initial training duration is 10 minutes per day, which should then be increased by 5 minutes each day until reaching a total of 30 minutes per day.
9. After 30 min of training, immediately release the mouse and untangle the wool to prevent red and swollen feet caused by prolonged binding.
   1. If skin lesions appear on the ankle of the mice after training, stop training the mice and seek veterinary professional care until the ankle is healthy.
   2. Some mice may occasionally manage to free themselves through intense struggle during training. The mice struggle when their hind legs were not firmly fixed. To avoid injury due to struggle, observe the mice throughout the training. When it is found that the hind limbs are not firmly fixed or the mice begin to struggle, loosen the mice and re-fix.
      NOTE: Previous experiments have shown that hanging mice upside down for 30 min a day, 5 days a week, does not cause ankle wear, provided they are freed in due time¹⁰. Mice can learn and get used to the training in 1-3 sessions.

Figure 2: Fixation method in mouse. (A) Tie the top of the ankle with a slipknot. (B) The end of the rope is passed through the gap and pulled tight, then secured with tape. (C) Static strength training in mice. Please click here to view a larger version of this figure.

Representative Results

Following the above protocol, the hind limbs of the mouse are fixed, and the forelimbs autonomously grasp the front bar. The narrow range of motion keeps the mouse in a relatively fixed position. The muscles of mice can be confirmed to contract by touching the muscles of their abdomen and legs. This is consistent with the need for the state of isometric muscle contraction in static strength training. Training mice according to the protocol, with the increase in training times, will help the mice adapt to the training while reducing their desire to struggle. A struggle to escape can be avoided.

Effects on blood glucose and insulin level in T2DM mice
A total of 24 C57BL/6J mice were randomly divided into the control group (n=6) and the T2DM model group (n=18). The T2DM mice were then randomly divided into the model group, the training group, and the metformin group. No modeling or intervention was given to the control group, and no intervention was given to the mice in the model group. The mice in the training group received 30 min of static strength training once a day, 5 days a week for 3 weeks. Mice in the metformin group were given metformin 200 mg/kg once a day for 3 weeks. The results of fasting blood glucose (FBG) after 3 weeks of intervention are shown in Figure 3A. As can be seen from the figure, the FBG levels of the T2DM model mice were significantly higher than those of the control mice. Mice trained in static training showed significantly lower FBG levels compared to the model group, suggesting that static training is effective in reducing FBG in T2DM mice. Fasting serum insulin (FINS) levels in the model group were significantly lower than in the control group, as shown in Figure 3B. In the training and metformin groups, FINS levels increased compared to the model group. In Figure 3C, mice in the model group had a significantly higher insulin resistance index (HOMA-IR) than those in the control group, whereas the HOMA-IR of the static training and metformin groups was significantly lower than that of the model group, demonstrating their efficacy in alleviating the insulin resistance state of T2DM mice. These results show that the static strength training strategy provided by this regimen has similar effects to metformin in regulating the blood glucose level of T2DM mice.

Figure 3: Blood sugar and insulin levels. (A) Comparison of fasting blood glucose levels in T2DM mice after 3 weeks of intervention. * p <0.05 vs. Control group, # p <0.05 vs. Model group. (B) Comparison of fasting serum insulin levels in T2DM mice after 3 weeks of intervention. * p <0.05 vs. Control group, # p <0.05 vs. Model group. (C) Comparison of insulin resistance index in T2DM mice after 3 weeks of intervention. * p <0.05 vs. Control group, # p <0.05 vs. Model group. One-way analysis of variance (ANOVA) was performed for statistical analysis. Quantitative data are expressed as mean ± SEM (n=6). Please click here to view a larger version of this figure.

Effects on skeletal muscle in T2DM mice
The gastrocnemius of mice was observed by transmission electron microscopy. Compared with control mice, the skeletal muscle myocytes of T2DM mice were degenerated, the structure of myofibrils was loose, and the sarcomere arrangement was irregular (Figure 4A, B). After the static training described in the protocol, Figure 4C shows the tight structure of myogenic fibers in the muscle structure and the symmetrical arrangement of local muscle segments. This suggests that static strength training can regulate skeletal muscle morphology in T2DM mice. On the other hand, in the gastrocnemius of control mice, mitochondria were locally distributed, with intact membranes, and locally divided or fused mitochondria (indicated by red arrows). Similarly, in the skeletal muscle of mice after static strength training, the number of mitochondria is normal; some of them are obviously fused or divided (red arrow). In the T2DM model in mice, by contrast, the number of mitochondria is less, and there is low activity. (See Figure 4) This suggests that static strength training may affect mitochondrial function and activity in skeletal muscle cells.

Figure 4: Effect of static training on skeletal muscle of T2DM mice. (A) Control group; (B) T2DM model group; (C) Static training group. The M represent mitochondria. White scale bars represent 50µm, and red scale bars represent 10µm. The bottom image is a partial enlargement of the top image. Please click here to view a larger version of this figure.

Discussion

Static strength training can reduce fat accumulation, aid weight loss, and increase metabolism⁸. In addition, it enhances the expression of PGC-1α and mitochondrial biogenesis in skeletal muscle cells, leading to improved glucose metabolism in mice with type 2 diabetes mellitus and a consequent reduction in blood glucose levels¹¹. To confirm the impact and mechanism of static training on T2DM, appropriate devices need to be developed for performing static training on experimental animals.

This protocol introduces a device for static training in mice. The required equipment, such as acrylic plates, sticks, woolen yarn, and hot glue guns, are readily available, and the production method is simple and easy. This can effectively reduce the cost of the experiment and increase its feasibility and repeatability. Maintaining a fixed position for the mice during training can be challenging when designing a static training device. In this protocol, the mouse's feet can be secured to the front of the small stick by tightening the soft wool. The righting reflex causes the mouse to bend its upper body upwards, thereby enabling it to grasp the bar in front with its forepaws. The bar is short, limiting the range of motion for the forepaws. After two or three sessions of adaptive training, the mice were able to maintain a stable posture. Due to the limited strength of the forepaws, the mice frequently lose their grip on the crossbar. It is necessary for the experimenters to monitor mice the whole time in order to assist them in grabbing the crossbar with their forelimbs and preventing the mice from accidental injury. Through training, the mice can maintain a fixed position for 1-2 min at a time, repeated several times, for about 30 min after exhaustion. When exhausted, the mice no longer rolled their abdomens or raised their forelegs. When the mouse was guided by the stick to grasp with its forepaws, the mouse could grasp, but its upper body was unable to turn upward. Untie the knot as soon as possible after putting down mice, which can effectively avoid ankle edema and wear.

Preliminary experiments indicate that, following a period of static training, the blood glucose level of T2DM mice decreased significantly when compared with the control group of model mice. Skeletal muscle is a critical component in peripheral glucose metabolism and is essential for maintaining blood glucose homeostasis¹². Electron microscopy analyses indicated that severe degeneration occurred in gastrocnemius cells of T2DM mice, while static training was found to mitigate muscle tissue degeneration. These findings suggest that static training may be implicated in the regulation of skeletal muscle function. Research has shown that maintaining skeletal muscle function is significantly impacted by mitochondrial dynamics¹³. We observed a marked reduction in the number of mitochondria and a lack of mitochondrial activity in the gastrocnemius muscle cells of the T2DM mouse model group compared to wild mice and static exercise mice. These findings suggest that static training may promote skeletal muscle function by enhancing mitochondrial function in skeletal muscle cells. Based on the preceding experimental results, it can be determined that static training can considerably enhance blood glucose and insulin metabolism in T2DM mice. The intervention mechanism may be linked to the regulation of skeletal muscle mitochondrial function by static training.

This protocol has some limitations. Initially, there was difficulty in individually binding the hind limbs of mice by one person, as it required one individual to grip the hind legs while another performed the binding. With a few adjustments and increasing skill on the part of the experimenter, the mice became more amenable. This allowed for independent binding of the hind legs. Additionally, the soft wool may lessen the bruising in the ankles of the mice.

In conclusion, this protocol provides a simple method of making static training equipment for mice. Similarly, simple static training equipment for rats can be made by increasing the size of boards and sticks. The device can maintain the muscle isometric contraction of the limbs of mice so as to verify the intervention effect of traditional Chinese exercise on T2DM and provide a fresh perspective for the clinical treatment of T2DM.

Materials

Acrylic boards			Transparent acrylic boards with 5mm thickness. The size should be larger than 20cm×20cm
Boxes			Two boxes of the same height (15~20cm)
ELISA KIT		H203-1-2	Nanjing Jiancheng Bioengineering Institute
Hot melt glue gun			Avoid touching the gun head to cause burns
Knives			No special requirement
Metformin tablets		1396309	Sigma
scissors			No special requirement
Sticks			Several wooden sticks with a diameter of 3mm
Streptozotocin		S0130	Sigma
Tape			No special requirement
Transmission Electron Microscope (TEM)		HT7700	HITACHI