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).

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 &#177; 4 g were selected. The mice were housed in a 12 h light/dark cycle at a temperature of 20-22 &#176;C, and a relative humidity of 45%-50% was maintained. The animals eat and drink freely.
1. Establishment of a mouse model of T2DM
<ol>
	<li>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.</li>
	<li>From the 1st day of the 5th 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.</li>
	<li>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.</li>
	<li>Begin training intervention on day 8 after the first injection of streptozotocin solution.</li>
</ol>
2. Grouping and treatment in mice
<ol>
	<li>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.</li>
	<li>Perform no intervention for the model group.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Randomly assign 6 non-T2DM mice as control group. These mice do not undergo modeling and intervention.</li>
</ol>
3. Manufacturing the static strength training device
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65556/65556fig01.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65556/65556fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Static strength training in mice
<ol>
	<li>Cut two pieces of woolen yarn 15 cm long. Use a slipknot to tie a piece of woolen yarn on each of the mouse&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>After 30 min of training, immediately release the mouse and untangle the wool to prevent red and swollen feet caused by prolonged binding.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65556/65556fig02.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65556/65556fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Exploring T2DM Exercise Interventions: A Static Training Device for Mice

<ol><li>Zheng, Y., Ley, S. H., Hu, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+aetiology+and+epidemiology+of+type+2+diabetes+mellitus+and+its+complications%5BTitle%5D)+AND+%22Nat+Rev+Endocrino%22%5BJournal%5D)">Global aetiology and epidemiology of type 2 diabetes mellitus and its complications.</a> Nat Rev Endocrino. 14 (2), 88-98 (2018).</li>
<li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+fitness+qigong+and+tai+chi+on+middle-aged+and+elderly+patients+with+type+2+diabetes+mellitus%5BTitle%5D)+AND+%22Public+Lib+Sci%22%5BJournal%5D)">Effects of fitness qigong and tai chi on middle-aged and elderly patients with type 2 diabetes mellitus.</a> Public Lib Sci. 15 (12), e0243989(2020).</li>
<li>Qin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+tai+chi+on+quality+of+life%2C+body+mass+index%2C+and+waist-hip+ratio+in+patients+with+type+2+diabetes+mellitus%3A+A+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Front+Endocrino%22%5BJournal%5D)">Effect of tai chi on quality of life, body mass index, and waist-hip ratio in patients with type 2 diabetes mellitus: A systematic review and meta-analysis.</a> Front Endocrino. 11, 543627(2020).</li>
<li>Yang, H., Wu, X., Wang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+conventional+medical+treatment+plus+Qigong+exercise+on+type+2+diabetes+mellitus+in+Chinese+patients%3A+A+Meta-analysis%5BTitle%5D)+AND+%22J+Trad+Chinese+Med%22%5BJournal%5D)">Effect of conventional medical treatment plus Qigong exercise on type 2 diabetes mellitus in Chinese patients: A Meta-analysis.</a> J Trad Chinese Med. 38 (2), 167-174 (2018).</li>
<li>Yu, X., Chau, J. P. C., Huo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effectiveness+of+traditional+Chinese+medicine-based+lifestyle+interventions+on+biomedical%2C+psychosocial%2C+and+behavioral+outcomes+in+individuals+with+type+2+diabetes%3A+A+systematic+review+with+meta-analysis%5BTitle%5D)+AND+%22Int+J+Nurs+Stud%22%5BJournal%5D)">The effectiveness of traditional Chinese medicine-based lifestyle interventions on biomedical, psychosocial, and behavioral outcomes in individuals with type 2 diabetes: A systematic review with meta-analysis.</a> Int J Nurs Stud. 80, 165-180 (2018).</li>
<li>Ağgön, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+dynamic+and+static+strength+training+on+hormonal+activity+in+elite+boxers%5BTitle%5D)+AND+%22Baltic+J+Health+Phys+Activity%22%5BJournal%5D)">Effect of dynamic and static strength training on hormonal activity in elite boxers.</a> Baltic J Health Phys Activity. 12 (3), 1-10 (2020).</li>
<li>Merico, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+combined+endurance+and+resistance+training+in+Amyotrophic+Lateral+Sclerosis%3A+A+pilot%2C+randomized%2C+controlled+study%5BTitle%5D)+AND+%22Eur+J+Translat+Myo%22%5BJournal%5D)">Effects of combined endurance and resistance training in Amyotrophic Lateral Sclerosis: A pilot, randomized, controlled study.</a> Eur J Translat Myo. 28 (1), 7278(2018).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eight+weeks+of+high-intensity+interval+static+strength+training+improves+skeletal+muscle+atrophy+and+motor+function+in+aged+rats+via+the+PGC-1%CE%B1%2FFNDC5%2FUCP1+pathway%5BTitle%5D)+AND+%22Clin+Interven+Aging%22%5BJournal%5D)">Eight weeks of high-intensity interval static strength training improves skeletal muscle atrophy and motor function in aged rats via the PGC-1α/FNDC5/UCP1 pathway.</a> Clin Interven Aging. 16, 811-821 (2021).</li>
<li>Jun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+model+for+static+massage+training%5BTitle%5D)+AND+%22Massage+Guid%22%5BJournal%5D)">Animal model for static massage training.</a> Massage Guid. (02), 5-6 (2000).</li>
<li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+by+which+static+exercise+improves+insulin+resistance+in+skeletal+muscle+of+type+2+diabetes%5BTitle%5D)+AND+%22Chinese+J+Tiss+Eng+Res%22%5BJournal%5D)">Mechanism by which static exercise improves insulin resistance in skeletal muscle of type 2 diabetes.</a> Chinese J Tiss Eng Res. 28 (08), 1271-1276 (2024).</li>
<li>Zhenrui, L., Fang, L., Wu, W., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+static+training+on+upper+limb+motor+ability+in+aged+rats+was+studied+based+on+PGC-1%CE%B1+signal+pathway+RNA+interference%5BTitle%5D)+AND+%22Lishizhen+Medicine+and+Materia+Medica+Research%22%5BJournal%5D)">The effect of static training on upper limb motor ability in aged rats was studied based on PGC-1α signal pathway RNA interference.</a> Lishizhen Medicine and Materia Medica Research. 30 (01), 247-249 (2019).</li>
<li>Lee, S. H., Park, S. Y., Choi, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+resistance%3A+From+mechanisms+to+therapeutic+strategies%5BTitle%5D)+AND+%22Diab+Metab+J%22%5BJournal%5D)">Insulin resistance: From mechanisms to therapeutic strategies.</a> Diab Metab J. 46 (1), 15-37 (2022).</li>
<li>Campos, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exercise+preserves+physical+fitness+during+aging+through+AMPK+and+mitochondrial+dynamics%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Exercise preserves physical fitness during aging through AMPK and mitochondrial dynamics.</a> Proc Natl Acad Sci U S A. 120 (2), e2204750120(2023).</li>
</ol>

The authors declare that they have nothing to disclose.

Static strength training can reduce fat accumulation, aid weight loss, and increase metabolism8. In addition, it enhances the expression of PGC-1&#945; 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&#39;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.&#160;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.

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by insulin resistance and &#946;-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 T2DM2,3,4,5. 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 rats8,9, 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.

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

static strength training method for type 2 diabetic mice

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.

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.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65556/65556fig03.jpg" /> 
Figure 3: Blood sugar and insulin levels. (A) Comparison of fasting blood glucose levels in T2DM mice after 3 weeks of intervention. * p &#60;0.05 vs. Control group, # p &#60;0.05 vs. Model group. (B) Comparison of fasting serum insulin levels in T2DM mice after 3 weeks of intervention. * p &#60;0.05 vs. Control group, # p &#60;0.05 vs. Model group. (C) Comparison of insulin resistance index in T2DM mice after 3 weeks of intervention. * p &#60;0.05 vs. Control group, # p &#60;0.05 vs. Model group. One-way analysis of variance (ANOVA) was performed for statistical analysis. Quantitative data are expressed as mean &#177; SEM (n=6). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65556/65556fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65556/65556fig04.jpg" /> 
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 50&#181;m, and red scale bars represent 10&#181;m. The bottom image is a partial enlargement of the top image. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65556/65556fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Author Spotlight: Advancing Diabetes Research with Static Exercise Training in Mice at JoVE.com

Author Spotlight: Advancing Diabetes Research with Static Exercise Training in Mice

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.

Static Strength Training Method for Type 2 Diabetic Mice

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-method-for-type-2-diabetic-mice

Universidad San Sebastian

Research

JoVE Journal

Medicine

435 Views.  Nanjing University of Chinese Medicine. 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.

Video: Author Spotlight: Advancing Diabetes Research with Static Exercise Training in Mice

Measuring the Strength of Mice

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score maximum on this task; it is a quick but insensitive gross screen, and the weights test described in this article will provide a finer measure of muscular strength.
There are also several strain gauge-based pieces of apparatus available commercially that will provide more graded data than the inverted screen test, but their cost may put them beyond the reach of many laboratories which do not specialize in strength testing. Hence in 2000 a cheap and simple apparatus was devised by the author. It consists of a series of chain links of increasing length, attached to a "fur collector" a ball of fine wire mesh sold for preventing limescale build up in hard water areas. An accidental observation revealed that mice could grip these very tightly, so they proved ideal as a grip point for a weight-lifting apparatus. A common fault with commercial strength meters is that the bar or other grip feature is not thin enough for mice to exert a maximum grip. As a general rule, the thinner the wire or bar, the better a mouse can grip with its small claws.
This is a pure test of strength, although as for any test motivational factors could potentially play a role. The use of scale collectors, however, seems to minimize motivational problems as the motivation appears to be very high for most normal young adult mice.

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Behavior

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Alleviation of Diabetic Tendon Injury via Activation of Tendon Fibroblasts Autophagy under Berberine Treatment

Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and possesses valuable pharmacological activities, including anti-inflammatory, anti-tumor, and alleviating several complications of type 2 diabetes mellitus (T2DM). However, the role of BBR in regulating diabetic tendon injury remains poorly understood. In this study, a rat model of T2DM was constructed, and cell apoptosis and autophagy were assessed in tendon tissues after BBR treatment through TdT-Mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemical analysis. Tendon fibroblasts were obtained from the rat Achilles tendon, and the role of BBR in regulating cell apoptosis, the production of inflammatory cytokines, and autophagy activation were assessed using flow cytometry, quantitative real-time PCR (qRT-PCR), and western blot analysis. We demonstrated that BBR treatment significantly increased autophagy activation and decreased cell apoptosis in tendon tissues of T2DM rats. In tendon fibroblasts, BBR repressed High glucose (HG)-induced cell apoptosis and production of proinflammatory cytokines. HG treatment resulted in a decrease of autophagy activation in tendon fibroblasts, whereas BBR restored autophagy activation. More important, pharmacological inhibition of autophagy by 3-MA weakened the protective effects of BBR against HG-induced tendon fibroblasts injury. Taken together, the current results demonstrate that BBR helps relieve diabetic tendon injury by activating autophagy of tendon fibroblasts.

Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and possesses valuable pharmacological activities, including anti-inflammatory, ...

Biochemistry

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

Neuroscience

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Biology

Versatility of Protocols for Resistance Training and Assessment Using Static and Dynamic Ladders in Animal Models

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way to gain insight into the underlying molecular mechanisms that orchestrate these adaptations. The aim of this article is to describe exercise models and training protocols designed for strength training and evaluation of resistance in animal models and provide examples. In this article, strength training and resistance evaluation are based on ladder climbing activity, using static and dynamic ladders. These devices allow a variety of training models as well as provide precise control of the main variables which determine resistance exercise: volume, load, velocity, and frequency. Furthermore, unlike resistance exercise in humans, this is a forced exercise. Thus, aversive stimuli must be avoided in this intervention to preserve animal welfare. Prior to implementation, a detailed design is necessary, along with an acclimatization and learning period. Acclimatization to training devices, such as ladders, weights, and clinical tape, as well as to the manipulations required, is necessary to avoid exercise rejection and to minimize stress. At the same time, the animals are taught to climb up the ladder, not down, to the resting area on the top of the ladder. Resistance evaluation can characterize physical strength and permit adjusting and quantifying the training load and the response to training. Furthermore, different types of strength can be evaluated. Regarding training programs, with appropriate design and device use, they can be sufficiently versatile to modulate different types of strength. Furthermore, they should be flexible enough to be modified depending on the adaptive and behavioral response of the animals or the presence of injuries. In conclusion, resistance training and assessment using ladders and weights are versatile methods in animal research.

The present protocol describes resistance training and testing using static and dynamic ladders in animal models.

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Murine Model for Diabetic Cardiomyopathy via High-Fat Diet and STZ Injection

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...