Acupuncture in a Rat Model of Asthma

Summary

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

Abstract

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

Introduction

Asthma is a chronic disease characterized by bronchoconstriction and airway hyper-responsiveness¹. Approximately 339.4 million people worldwide are diagnosed with asthma². The prevalence of asthma is increasing globally, with 100 million new cases expected in the next decade³. Inhaled corticosteroids and long-acting β₂-agonists do not effectively control the symptoms of all patients⁴. Therefore, the use of alternative therapies such as acupuncture to treat asthma has been receiving increased attention⁵. An effective method for treating asthma is to needle GV14 (Dazhui), bilateral BL12 (Fengmen) and bilateral BL13 (Feishu)⁶. Acupuncture significantly reduces the levels of secretory immunoglobulin A (SIgA) in the saliva and nasal secretions of patients with allergic asthma and reduces peripheral blood eosinophil counts with a total effective rate of 85%⁶. Acupuncture at BL13 and ST36 (Zusanli) in mice with chronic asthma decreases the level of IL-17 by 40% and decreases the smooth muscle thickness by 33% compared with that of the ovalbumin (OVA) group, and thus relieving inflammation symptoms^7,8. However, the mechanism of acupuncture treatment for asthma is not completely clear.

Animal models are an important tool for asthma research⁹ because the animal model can be used for the continuous measurement of respiratory function to assess the effectiveness of the asthma treatment¹⁰. Meanwhile, studies of the pathophysiology of asthma require test samples, including samples from the trachea, lung and bronchoalveolar lavage to verify changes in the levels of key factors¹¹. Rat models of asthma are commonly used to assess asthma pathophysiology because they produce long-lasting airway responses and show immediate and late-phase airway responses¹². However, due to repeated experimental stimulation, the rats often become irritable. Therefore, a suitable method to fix rats is needed. Commonly used methods for rat fixation include anesthesia and binding¹³. Although the anesthesia method provides better exposure of the acupoints, it may affect nerve conduction and ultimately affect the experimental results¹⁴. The binding method does not induce the same physiological effects as anesthesia, but restrained rats will experience skeletal muscle tension. Also, the needle cannot easily reach the designated position¹⁵. Here, we introduce a high platform that can be used to fix rats without restricting them and fully expose the acupoints on the back. The rats can be placed on the high platform during the administration of acupuncture. The high platform is able to accommodate the rat only while it is standing, and as it is placed a certain distance from the ground, the rats will not move because of a fear of heights¹⁶.

This article describes in detail the fabrication of the high platform, the establishment of a rat model of asthma, the operation of needling a rat on the high platform and the measurement of respiratory function using a whole-body plethysmography (WBP) system.

Protocol

All animal experiments were reviewed and approved by the Committee on the Ethics of Animal Experiments of Shanghai University of Traditional Chinese Medicine (Ethical clearance number PZSHUTCM190308020).

1. Fabrication of the high platform

1. Set the length and width of the supporting device to 86 cm x 17 cm (Figure 1).
2. Fix the connecting device vertically to the supporting device. The connecting device should be approximately 30 cm long to prevent the rat from running to the top surface of the high platform and have a width of 14 cm.
3. Fix the horizontal pedal (5 cm x 5 cm) to the connecting device. The suspended height at the bottom of the horizontal pedal should be greater than or equal to 50 cm.
   NOTE: Depending on the number of experimental rats, multiple horizontal pedals can be used, with one rat placed on each pedal.

2. Establishment of a rat model of asthma

1. Sensitizing the rats with OVA
   1. Prepare the sensitization solution on day 0. Prepare each 1 mL of the sensitization solution with 1 mg of OVA and 10 mg of aluminum hydroxide, with normal saline as the solvent.
   2. Intraperitoneally inject each rat with 1 mL of the sensitization solution on day 0. Draw the sensitizing solution with a suitable syringe for later use.
   3. Grab the tail and lift up the rat, place it on a rough surface, and gently pull the tail back. Grasp the skin of both ears and neck with the left thumb and index finger while tightly holding the back skin with the remaining three fingers and palms (be careful to refrain from applying excess force that will suffocate the rat).
   4. With the right hand, perform an intraperitoneal injection in the rat with the prepared syringe with 1 mL of the sensitization solution.
2. Challenging rats with OVA
   1. Select a box with a lid (the length, width and height are 39 cm x 28 cm x 23 cm), and drill a small hole in the side. Connect the sealed box, nebulizing cup and the compression nebulizer with a rubber tube (Supplementary Figure 1).
      NOTE: The nebulized particle size of the compressed nebulizer is 1 - 5 μm, the nebulization rate is greater than 0.2 mL/min, and the ultimate pressure is 205 kPa.
   2. On the 14th day after sensitization, open the lid of the homemade sealed box and place the rat in the box (the box can accommodate 5 rats), and then put the sealed box in a biosafety cabinet.
   3. Prepare the challenge solution. Thoroughly mix 300 mg of OVA with 30 mL of normal saline.
   4. Turn on the ultrasonic nebulizer and add 30 mL of the challenge solution into the nebulizing cup. Adjust the dosage of challenge solution and the number of rats according to the volume of the homemade sealed box.
   5. Repeat nebulization several times (once a day) or adjust the procedure according to the severity of asthma symptoms.

3. Acupuncture treatment

1. Select acupoints, including GV14, bilateral BL12 and bilateral BL13, according to the theory of traditional Chinese medicine for treating asthma (Supplementary Figure 2). Administer manual acupuncture on day 2 and perform once every other day for a total of 7 session (Figure 2).
   1. Acupuncture the GV14 located between the seventh cervical vertebra and the first thoracic vertebra (the hollow of the scapula), on the midline of the back (Figure 3).
   2. Acupuncture the BL13 located below the third thoracic vertebra, on both sides of the intercostal spaces (lower edge of the scapula).
   3. Acupuncture the BL12 located below the second thoracic vertebra, on both sides of the intercostal spaces (below the scapula, position identified according to BL13).
2. Grasp the rats and place them on the high platform.
3. Hold the disposable stainless-steel needle (0.3 mm x 13.0 mm, Supplementary Figure 3) handle with the thumb and index finger of the right hand, and press the back of the rat with the palm of the right hand.
   NOTE: Disposable needles must be one-time-use only. Because repeated use will blunt the needle, which leads to increased pain in rats.
4. Touch the depression in the middle of the upper edge (GV14) or the lower edge of the scapula (BL12 and BL13), and then gently prick the skin of the rat with the needle tip to locate the acupoints.
5. Quickly insert the needle after determining the specific location of the acupoint and rapidly remove the right hand.
6. Evenly twisted the needles approximately 360° at a rate of 60 times/min for 10 - 20 s, manipulate them every 5 min, and then withdraw them after 20 - 30 min (Supplementary Figure 4).

4. The Penh measurement

1. Prepare methacholine: Place 100 mg of methacholine into a 5 mL centrifuge tube and add PBS to a volume of 2 mL (50 mg/mL methacholine). Then, dilute the 50 mg/mL methacholine solution to 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL and 3.125 mg/mL.
2. Instrument inspection
   1. Turn on the signal amplifier (the aerosol distribution system and the rodent bias flow supply system are all turned off, and the nebulization connector is closed).
   2. Check the channels: Click Calibrate, return the coarse adjustment baseline to 0 and switch to the direct current (DC) position. Fine-tune the baseline to 0 and inject 5 mL of gas with a syringe. Then, switch to the alternating current (AC) position.
   3. After checking the channels, ensure that the error value is less than 0.5%.
3. Setting parameters
   1. Turn on the aerosol distribution system and the rodent bias flow supply system. Adjust the flow rate to 4.5 mL according to the liquid level on the mercury ball.
   2. Click Create Study Options and select Dose Response Study, select Rat of species and click Next.
   3. Select Simple Dose List, set the Number of Doses to 6 and click Next.
   4. Select the Penh and Area parameters: Set the animal adaptation time to 5 min, the nebulization time to 2 min, the response time to 3 min, and the recovery time to 2 min.
   5. Sequentially add the following solutions to the nebulizer (200 μL per rat): methacholine (0 mg/mL, 3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL and 50 mg/mL). Real-time changes in Penh can be observed (Figure 4).
   6. After all 6 solutions with different concentrations have been reacted, click File and End Session to complete the experiment.

Representative Results

With acupuncture at the GV14, bilateral BL12 and bilateral BL13 points, the rat maintains a prone position. The rat can twist its head only to the outside and not the inside because one direction is blocked with a wooden board (Figure 5).

As the methacholine concentration increased, Penh shows an increasing trend and the growth rate gradually increases. At concentrations of 3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL and 50 mg/mL, Penh increased by 18.40% ± 10.70%, 27.55% ± 26.30%, 88.10% ± 45.63%, 427.60% ± 172.92% and 882.27% ± 121.97%, respectively, showing an effect that increases with increasing dose (Figure 6).

Figure 1. Fabrication of the high platform. The high platform consists of a supporting device, three connecting devices and three pedals. The length and width of the supporting device are 86 cm x 17 cm, the length and width of the connecting device are 30 cm x 14 cm, the length and width of the pedal are 5 cm x 5 cm. Each connecting device is separated by 20 cm. Please click here to view a larger version of this figure.

Figure 2. Protocol for establishing a rat model of asthma. Sensitization is performed on day 0, and acupuncture is performed every other day on the high platform from days 2 to 14. After challenge, the change in Penh is measured. Please click here to view a larger version of this figure.

Figure 3. Anatomical positions of GV14 (Dazhui), BL12 (Fengmen), and BL13 (Feishu) on the rats. (A) Anatomical dissection of the back of a rat showing the scapula, first rib, second rib, third rib, and second thoracic vertebra (T2). (B) The sagittal plane of the ribs and thoracic vertebra. (C) The cross section of the scapula. (D) GV14: Located in the hollow of the scapula, midline of the back; BL13: Located at the lower edge of the scapula; BL12: Located below the scapula, positioned according to BL13. Please click here to view a larger version of this figure.

Figure 4. Real-time changes in respiratory function. The blue curve shows real-time changes in Penh. The green curve shows the total change in the tidal volume (TVb) over a certain period. The purple curve shows the total change in Penh over a certain period. Please click here to view a larger version of this figure.

Figure 5. Acupuncture of GV14 (Dazhui), BL12 (Fengmen), and BL13 (Feishu) in rats on the high platform. (A) Top view of the positions of GV14, BL12 and BL13 in rats. (B) Lateral view of an acupunctured rat on a high platform. The rat is placed on the platform and maintained in the prone position during acupuncture. Please click here to view a larger version of this figure.

Figure 6. Measurement of Penh. WBP is used to measure the Penh of the normal rat. As the concentration of methacholine increases, the Penh also gradually increases. The data are presented as the mean ± SEM (n = 3). Please click here to view a larger version of this figure.

Supplementary Figure 1. The nebulizer specifications. The nebulized particle size of the compressed nebulizer is 1 - 5 μm, the nebulization rate is greater than 0.2 mL/min, and the ultimate pressure is 205 kPa. Please click here to download this file.

Supplementary Figure 2. The schematic positions of GV14 (Dazhui), BL12 (Fengmen), and BL13 (Feishu) in rat. Please click here to download this file.

Supplementary Figure 3. Needle specifications. The length of the needle is 13 mm and the diameter is 0.3 mm. Please click here to download this file.

Supplementary Figure 4. The schematic of acupuncture manipulation. After inserting the needle, evenly twisted the needles approximately 360° in different directions. Please click here to download this file.

Discussion

The GV14, bilateral BL12 and bilateral BL13 acupoints used in this study are located on the back, and thus the high platform is suitable for fixing rats. In other studies, an adequate fixation method should be selected according to the position of the acupoints¹⁵. For acupoints on the abdomen, the rat can be placed downward in a black cone (similar to a pastry bag used by bakers)¹⁷. A chain is attached to the restraint cone for fixation, and the hind leg of the rat is also fixed. In this way, the front of the animal’s body is firmly fixed in the cone, while the abdomen and the back are exposed. For acupoints on the leg, the rat can be placed vertically in a special transparent bucket, leaving the tail and hind legs naturally outside the bucket¹⁸. The transparent barrel covers the body of the rat, but it is convenient for acupuncture on the leg.

Several critical steps should be considered when performing these methods. Before the acupuncture treatment, the rats should be placed on the high platform for training for a few days in advance until the rats keep quiet and breathe smoothly on the high platform. For acupuncture manipulation, the harder the palm of the right hand presses the rat back, the easier it is to insert the needle. For respiratory function measurement, the WBP system measures changes in the Penh that reflect changes in pulmonary resistance of rats in a noninvasive manner and minimizes effects of psychological stress^19,20. The effect of the external environment on rats should be minimized. The rat should be placed in the testing room and then covered the testing room with a plastic bag (with 1cm x 1cm hole on the dorsal side for ventilation) to reduce the irritation caused by the external environment.

The limitation of this experiment is that the needle may fall off if the rat swings about during the acupuncture. Therefore, needling must be manipulated every 5 min to adjust the depth. In addition, the high platform is suitable for rats, but not for smaller animals, such as mice.

In summary, we provide a detailed description of method for the fabrication of a high platform for fixing rats without restriction during acupuncture. The high platform can completely expose the acupoints on the back of the rat. The use of the high platform and noninvasive respiratory function measurements may facilitate the analysis of acupuncture as a treatment for asthma. Studying the therapeutic mechanism of acupuncture on the back may bring new treatments to respiratory diseases such as asthma and chronic obstructive pulmonary disease.

Materials

Name	Company	Catalog Number	Comments
0.3 mm x 13.0 mm acupuncture needle	China Suzhou Medical Device Factory
Al(OH)3	American Thermo	77161
Compression nebulizer	Jiangsu Lude Medical Electronics Co., Ltd.	NB-212C
Fine Pointe V2.0	American Buxco
Laboratory gas drying unit	American Drierite	26800
Methacholine	American Sigma	MKCF6054
Ovalbumin	American Sigma	A5503-25G
Phosphate Buffer solution (1x)	Gelifesciences	SH30256.01
Seal box	IRIS Corporation of Japan
Whole-body plethysmography respiratory function measurement system	American Buxco