Mouse Electroacupuncture Fixation Device Fabrication for Electroacupuncture Pretreatment in Diabetic Cardiomyopathy Mouse Model

Riepilogo

Here, we present a protocol to establish a mouse model of diabetic cardiomyopathy and a mouse electroacupuncture fixation device for the use of electroacupuncture in mice, which can fix the mice more gently during treatment.

Abstract

Diabetic cardiomyopathy (DCM) is a complication of diabetes that can progress to heart failure and is limited by the use of medications. Electroacupuncture is an effective way as a traditional Chinese medicine external treatment method to treat diabetic cardiomyopathy. A mouse electroacupuncture fixation device can easily and gently fix mice during electroacupuncture treatment and expose acupoints on the abdomen and limbs of mice. This article describes the methods used to fabricate the mouse electroacupuncture fixation device with all the steps and precautions, establishing a mouse model of diabetic cardiomyopathy with comprehensive measures to comprehensively record changes in blood biochemical indices such as blood glucose. The experiment was done with 3 groups: a control group, a model group, and a pretreatment group, with 10 mice in each group. The pretreatment group was treated by Biaoben acupoints electroacupuncture pretreatment once every alternate day for a total of 7 sessions. After electroacupuncture, the mice in the pretreatment group showed a decrease in blood glucose accompanied by a reduction in the symptoms of diabetes.

Introduzione

Diabetic cardiomyopathy (DCM) is a common complication of diabetes mellitus characterized by changes in cardiac structure and ventricular diastolic and contractile function of a specific lesion, further developing into heart failure. It is one of the leading causes of death in diabetes mellitus patients^1,2, and its increasing prevalence has attracted widespread attention in recent years³. It has been found that the pathogenesis of this disease mainly includes insulin resistance, inflammatory response, abnormal lipid metabolism, oxidative stress, mitochondrial damage^4,5.

At present, clinical treatment of diabetic cardiomyopathy is mainly based on oral medication for controlling blood glucose and alleviating the symptoms of heart disease, but it has certain limitations, such as drug reactions, kidney function effects, and dependence on long-term medication⁶. Acupuncture, as a traditional Chinese medicine external treatment method, has a better-regulating effect on blood glucose, blood lipids, and inflammatory response in patients with diabetic cardiomyopathy, and the clinical efficacy is remarkable⁷. Acupuncture has no negative effects on gastrointestinal and kidney functions, and long-term treatment does not produce dependence. The theory of Biaoben acupoints is an important method of selecting and using acupuncture points in the traditional theory of acupuncture⁸. It defines the acupoints that can improve the symptoms of diseases as Biao acupoints, and the acupoints that can regulate the basic functions of the body related to the diseases and treat the root causes of the diseases are called Ben acupoints. The stimulation of Biao acupoints and Ben acupoints at the same time with acupuncture can achieve the purpose of treating the diseases as well as increasing the body's resistance to the diseases and preventing the exacerbation and recurrence of the diseases⁹. It can prevent the aggravation and recurrence of the disease. Modern research has proved that acupuncture with Biaoben acupoints is effective for diabetes and its many complications, including diabetic heart disease, diabetic nephropathy, diabetic retinopathy, diabetic foot, and so on^10,11. An acupoint selection scheme in line with the theory of Biaoben acupoints is to select PC6 (Neiguan), which can regulate the functions of the heart and improve the inflammatory response as the Biao acupoint¹², and ST36 (Zusanli), which can improve blood glucose and blood sugar situation, improve insulin resistance, and reduce oxidative stress, as the Ben acupoint¹³.

Animal models are an important tool in the study of diabetic cardiomyopathy because it has been experimentally demonstrated that rat or mouse models well mimic the structural abnormalities and functional changes of the heart caused by diabetic cardiomyopathy and provide the samples required for biochemical detection indices of diabetic cardiomyopathy^14,15. The use of high-sugar and high-fat feed combined with Streptozotocin solution to induce mice can lead to the formation of diabetic models in 4-5 weeks, effectively shortening the modeling time and improving the success rate, and there are no special requirements for the rearing environment^16,17. Diabetic cardiomyopathy is a kind of cardiomyopathy caused by the damage to the cardiac structure and function caused by continuous hyperglycemia. With the prolongation of the course of the disease, the diabetic heart disease model can be naturally developed by the diabetic model under long-term hyperglycemia^18,19,20. Acupuncture pretreatment refers to the pretreatment of mice after the formation of diabetes and before the formation of obvious myocardial injury. However, after repeated experimental stimulation, mice often become mobile and difficult to immobilize, and violent experimental manipulation techniques may cause mice injury or make them manic or depressed²¹. Therefore, a suitable method to immobilize mice is needed. Commonly used methods of mouse immobilization include anesthesia and strapping²². Although the anesthesia method can better expose the acupoints, it may affect nerve conduction and ultimately affect the experimental results. The strapping method is a physical immobilization method and does not produce the same physiological effects as anesthesia, but the bound mice will experience significant tension, increased muscle tone, and enhanced defecation^22,23. In addition, the needle cannot easily reach the desired position. Here, we introduce a mouse electroacupuncture fixation device that can be used to ensure that mice do not move and also expose acupoints during electroacupuncture treatment so that electroacupuncture treatment can be performed conveniently, safely, nonviolently, and without sequelae.

This article describes in detail the fabrication of the mouse electroacupuncture fixation device and the establishment of a mouse model of diabetic cardiomyopathy. We treated diabetic cardiomyopathy by Biaoben acupoints electroacupuncture pretreatment, demonstrated how its operation was realized on the mice fixation device, and evaluated the therapeutic effect of Biaoben acupoints electroacupuncture pretreatment on diabetic cardiomyopathy by measuring indices related to glycolipid metabolism.

Protocollo

All animal experiments comply with the 3Rs principle of animal ethics and have been reviewed and approved by the Committee on the Ethics of Animal Experiments of Hubei University of Traditional Chinese Medicine (Ethical clearance number HUCMS00309300). Here, 30 SPF-grade mice were obtained from the Hubei Provincial Center for Disease Control and Prevention (Quality certificate NO.42010200009690).

1. Fabrication of the electroacupuncture fixation device

1. For the assembly of an electroacupuncture fixation device obtain a transparent acrylic hollow plastic tube and an electroacupuncture therapy instrument as the main body (Figure 1A). In the tube maintain a tube body and prepare closure tabs located in the openings at each end. Make grooves in the hollow tube in the center of each end and secure the closure tabs in the grooves at each end using manually rotatable screws.
2. In the middle of the hollow tube, make three oval holes about 2 cm long and 1 cm wide distributed adjacent to each other. The holes on both sides are used to pull out the forelimbs of the mouse for fixation for electroacupuncture treatment of the forelimbs, and the hole in the middle can be used to expose the acupuncture points of the thoracic and abdominal regions of the mouse for electroacupuncture treatment of the thoracic and abdominal regions.
3. At the end of the hollow tube, make an oval hole symmetrically distributed on both sides of the center groove, which can be used to pull out the hind limbs of the mouse for electroacupuncture treatment of the hind limbs (Figure 1B).
4. Check the integrity of the hollow plastic tubing and install the closure tabs. Move the closure tabs in the tube body as the screws slide in the grooves, ensuring that it can adjust the fixation length to suit the length of the mouse.
5. Turn on the electroacupuncture therapy instrument and make sure it is operational. Each button can be adjusted by pressing it once (Figure 1C).
6. Prepare the electroacupuncture therapy instrument to be placed next to the hollow plastic tubes for conducting electroacupuncture treatments on mice. The electroacupuncture therapy instrument consists of an electroacupuncture therapy machine and several pairs of energizing clips; each pair of energizing clips can provide two poles of energizing.
7. When using the clips to clip the needle handle to energize the needle, use a clip to fix the needle.
8. The electroacupuncture therapy machine has 11 buttons. Use the upper 5 to set the frequency, time, and waveform of electroacupuncture and are used from left to right for frequency, time, continuous wave, sparse and dense wave, and intermittent wave. Use the lower 6 to set the current strength of electroacupuncture. Each of these buttons has a notch underneath it for attaching energizing clips; the number of energizing clips can increase or decrease with the number of mice, up to a maximum of 6.

2. Establishing a mouse model of diabetic cardiomyopathy

1. Divide the 30 mice into 3 groups by the random number table method: a control group, a model group, and a pretreatment group, with 10 mice in each group. Subject a total of 20 mice in the model group and pretreatment group to establish the model of diabetic cardiomyopathy. Treat the pretreatment group with Biaoben acupoints electroacupuncture.
   NOTE: The model was built over a long period of time, and significant cardiomyopathy develops after 16 weeks. Our treatment time is from day 36 to day 70 (week 6 to week 10). This treatment was selected after the establishment of diabetes but before significant myocardial damage. At the end of 6 weeks, the mouse heart was removed and found to have significant diabetic cardiomyopathy. Therefore, the selection of treatment at this time point belongs to the category of pretreatment.
2. Feed mice in the control group with normal chow for 16 weeks. Feed mice in the model group and pretreatment group with a high-fat and high-sugar diet for 16 weeks to induce a metabolic state closely resembling the diabetic condition. The high-fat and high-sugar feed composition is 67.0% standard chow, 10.0% lard, 2.5% cholesterol, 20.0% sucrose, and 0.5% bile salts.
3. Inject intraperitoneally Streptozotocin solution to establish a mouse diabetic cardiomyopathy model.
   1. Prepare the 1% solution of Streptozotocin on day 29. Dissolve Streptozotocin in a buffer prepared by mixing citric acid with trisodium citrate (pH = 4.4).
   2. Provide just water and no food for 12 h on day 29. Then, give each mouse a single intraperitoneal injection of 150 mg/kg of Streptozotocin solution as described below.
   3. Grab the mouse by the tail with the right hand and move it out of the cage. Place the mouse on the cage's surface and gently pull its tail out. Immobilize the mouse with the left hand and flip it over to expose the abdomen.
   4. Inject the mouse intraperitoneally using the right hand with a 1 mL syringe prepared with the appropriate amount of the Streptozotocin solution already withdrawn.
4. Perform acupuncture treatment by Biaoben acupoints.
   1. Select the acupoints according to the theory of Biaoben acupoints¹³ including bilateral PC6 and ST36. For the pretreatment group, perform acupuncture on day 36, once every other day for a total of 18 treatments, with the treatment lasting a total of 5 weeks (from day 36 to day 70; Figure 2).
      1. Biao acupoint: Select bilateral PC6 as the Biao acupoint; the PC6 is located on the medial side of the forelimb, between the ulnar-radial sutures about 3 mm from the wrist joints. Ben acupoint: Select bilateral ST36 as Ben acupoints; the ST36 is located at the posterior-lateral aspect of the knee joint, approximately 2 mm below the fibular tuberosity.
   2. Grasp the mouse, pinch its tail, and place it on the cage's surface. Keep the mouth of the acrylic hollow plastic tube at an angle of 30° to the horizontal plane, with the small hole in the center facing the ground and the mouth of the tube aligned with the mouse's head.
   3. Accommodate the mouse's head to the mouth of the tube. Slowly rotate the mouse's head into the tube using voluntary movement and a slight push. Gently pat the mouse's buttocks so that the mouse is autonomously and completely pushed into the tube.
   4. Adjust the closure tabs, lift the mouse's tail out of the groove, tighten the nut, and use the closure tabs at the upper and lower ends to restrict the mouse's movement. At this point, the mouse can no longer move freely in the tube body.
   5. Use tweezers to gently pull out the four limbs of the mouse from each of the four small holes on both sides. After the four limbs are pulled out of the small holes, gently secure them to the outside of the tube body. At this point, the mouse's four limbs could no longer be displaced.
   6. Hold the disposable stainless-steel needle handle (0.3 mm x 13 mm) with the thumb and index finger of the right hand and hold the acrylic tube and the immobile mice in it with the left hand.
   7. Touch and locate where PC6 and ST36 are situated with the middle finger of the right hand, then gently prick the skin of the mouse with the needle tip. Quickly insert the needle injected obliquely around 3 mm and rapidly remove the right hand.
5. Perform electroacupuncture pretreatment
   1. Turn on the electroacupuncture therapy machine and adjust to intermittent wave, 1 Hz. For each mouse, carry out electroacupuncture treatment for 15 min.
   2. Use the matching energized clip of the electroacupuncture therapy machine to hold the needle handle, one clip for one needle handle.
   3. Adjust the current of the button with the clip attached to 1 mA (Figure 3).
   4. Wait for 15 min and observe the state of the mice during the electroacupuncture pretreatment.
   5. At the end of the treatment, release the clip and remove the acupuncture needle. Adjust the closure tabs and release the mice from the electroacupuncture fixation device.
   6. Examine the whole body of the mice and observe if the mice are injured or have any abnormalities.
6. Perform acupuncture treatments every other day after 7 days of injection of streptozotocin solution for a total of 18 treatments on a mouse electroacupuncture fixture from day 36 to day 70.
7. End experiment on day 112 (end of week 16).
   1. Anesthetize by intraperitoneal injection of 2% pentobarbital sodium and confirm anesthesia by toe pinch response. Measure the cardiac function of the mice using an echocardiography.
      1. Record left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS).
   2. Euthanasia the mice by cervical dislocation. Remove the heart, wash in physiological saline, and immerse in 4% paraformaldehyde.

3. Hematoxylin-eosin (HE) staining

1. Dehydrate, embed, slice, dewax the heart, and perform HE staining as per standard procedure.
2. Observe the pathological changes of mouse heart tissue under a light microscope.

4. Observations on mice

1. Measure blood sugar using a blood glucose meter on days 27, 36, and 49.
2. Observe how much the mice eat, quantify their water intake and urination volume every day, and record their weight changes.

5. Statistical analysis

1. Collect blood glucose data and use one-way ANOVA to identify statistical significance. Establish statistical significance as p < 0.05.
2. Quantify the data by assistants who are blind to the experimental conditions. Express the data as mean ± standard deviation.

Risultati

We followed the procedure described above and established a mouse model of diabetic cardiomyopathy. As can be observed in the HE-stained pictures (Figure 4), the mice in the control group had intact myocardial tissue structure, regular arrangement of cardiomyocytes, and no obvious lesions. In the model group, the myocardial cells were irregularly arranged, some myocardial cells were damaged and disintegrated (black arrow), and obvious inflammatory cell infiltration was observed in the inters...

Discussione

In this study, mice that had high blood glucose and showed signs of diabetes were obtained by intraperitoneal injection of the Streptozotocin solution, which is an effective and recognized method of modeling²⁷. PC6 was selected as the Biao point, and ST36 was selected as the Ben point according to the theory of Biaoben acupoints²⁸. All of these points are on the limbs of the mouse, so the acrylic tubes that can expose the abdomen and immobilize the limbs are well suited for...

Materiali

Name	Company	Catalog Number	Comments
acrylic hollow plastic	Henan Zhike	ZK-GDQ
acupuncture needle	Hwato	0.3 mm x 13 mm
C57BL/6  mice	Hubei Provincial Center for Disease Control and Prevention
citric acid monohydrate	Sigma	C1909
electroacupuncture therapy instrument	Hwato	SDZ-V
electronic balance	Henan Zhike	ZK-DST
glucose meters	LifeScan	OneTouch VerioVue
glucose test strip	LifeScan	OneTouch Verio
streptozotocin	Sigma	S0130
trisodium citrate dihydrate	Sigma	W302600
tweezers	Labshark	130302001