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.

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% ± 10.7...

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 ...

Materials

List of materials used in this article

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