This work was supported by National Natural Science Foundation of China (No. 81922076, 81973951, 81873373), Construction project of collaborative innovation of shanghai (No. ZYJKFW201811010, ZYJKFW201701005).

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
<ol>
	<li>Set the length and width of the supporting device to 86 cm x 17 cm (Figure 1).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Establishment of a rat model of asthma
<ol>
	<li>Sensitizing the rats with OVA
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>With the right hand, perform an intraperitoneal injection in the rat with the prepared syringe with 1 mL of the sensitization solution.</li>
	</ol>
	</li>
	<li>Challenging rats with OVA
	<ol>
		<li>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 &#956;m, the nebulization rate is greater than 0.2 mL/min, and the ultimate pressure is 205 kPa.</li>
		<li>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.</li>
		<li>Prepare the challenge solution. Thoroughly mix 300 mg of OVA with 30 mL of normal saline.</li>
		<li>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.</li>
		<li>Repeat nebulization several times (once a day) or adjust the procedure according to the severity of asthma symptoms.</li>
	</ol>
	</li>
</ol>
3. Acupuncture treatment
<ol>
	<li>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).
	<ol>
		<li>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).</li>
		<li>Acupuncture the BL13 located below the third thoracic vertebra, on both sides of the intercostal spaces (lower edge of the scapula).</li>
		<li>Acupuncture the BL12 located below the second thoracic vertebra, on both sides of the intercostal spaces (below the scapula, position identified according to BL13).</li>
	</ol>
	</li>
	<li>Grasp the rats and place them on the high platform.</li>
	<li>Hold the disposable stainless-steel needle (0.3 mm x&#160;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.</li>
	<li>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.</li>
	<li>Quickly insert the needle after determining the specific location of the acupoint and rapidly remove the right hand.</li>
	<li>Evenly twisted the needles approximately 360&#176; 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).</li>
</ol>
4. The Penh measurement
<ol>
	<li>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.</li>
	<li>Instrument inspection
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>After checking the channels, ensure that the error value is less than 0.5%.</li>
	</ol>
	</li>
	<li>Setting parameters
	<ol>
		<li>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.</li>
		<li>Click Create Study Options and select Dose Response Study, select Rat of species and click Next.</li>
		<li>Select Simple Dose List, set the Number of Doses to 6 and click Next.</li>
		<li>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.</li>
		<li>Sequentially add the following solutions to the nebulizer (200 &#956;L 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).</li>
		<li>After all 6 solutions with different concentrations have been reacted, click File and End Session to complete the experiment.</li>
	</ol>
	</li>
</ol>

<ol>
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J., Li, J., Zeng, B. Y., Liang, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+acupuncture+research.">History of acupuncture research.</a> International Review of Neurobiology. 111, 1-23 (2013).</li><li>Yang, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+use+of+acupuncture+as+supplemental+therapy+for+patients+with+allergic+asthma.">Considerations for use of acupuncture as supplemental therapy for patients with allergic asthma.</a> Clinical Reviews in Allergy &amp; Immunology. 44 (3), 254-261 (2013).</li><li>Nurwati, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+interleukin-17+level+by+acupuncture+at+Feishu+(BL13)+is+strengthened+by+acupuncture+at+Zusanli+(ST36)+in+a+mouse+model+of+chronic+asthma:+An+experimental+study.">Reduction of interleukin-17 level by acupuncture at Feishu (BL13) is strengthened by acupuncture at Zusanli (ST36) in a mouse model of chronic asthma: An experimental study.</a> Medical Acupuncture. 27 (4), 278-282 (2015).</li><li>Nurwati, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+inflammation+and+airway+remodelling+after+acupuncture+at+BL13+and+ST36+in+a+mouse+model+of+chronic+asthma.">Improvement in inflammation and airway remodelling after acupuncture at BL13 and ST36 in a mouse model of chronic asthma.</a> Acupuncture in Medicine. 37 (4), 228-236 (2019).</li><li>Reddy, A. 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The authors have nothing to disclose.

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&#8217;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 stress19,20. 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&#160;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.

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 &#946;2-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 symptoms7,8. 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:728px;" width="728">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">0.3 mm x 13.0 mm acupuncture needle</td>
			<td style="width:192px;">China Suzhou Medical Device Factory</td>
			<td style="width:114px;"></td>
			<td style="width:154px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:24px;width:267px;">Al(OH)3</td>
			<td style="width:192px;">American Thermo</td>
			<td style="width:114px;">77161</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Compression nebulizer</td>
			<td style="width:192px;">Jiangsu Lude Medical Electronics Co., Ltd.</td>
			<td style="width:114px;">NB-212C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Fine Pointe V2.0</td>
			<td style="width:192px;">American Buxco</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Laboratory gas drying unit</td>
			<td style="width:192px;">American Drierite</td>
			<td style="width:114px;">26800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Methacholine</td>
			<td style="width:192px;">American Sigma</td>
			<td style="width:114px;">MKCF6054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Ovalbumin</td>
			<td style="width:192px;">American Sigma</td>
			<td style="width:114px;">A5503-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Phosphate Buffer solution (1x)</td>
			<td style="width:192px;">Gelifesciences</td>
			<td style="width:114px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Seal box</td>
			<td style="width:192px;">IRIS Corporation of Japan</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Whole-body plethysmography respiratory function measurement system</td>
			<td style="width:192px;">American Buxco</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

acupuncture in a rat model of asthma

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.

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% &#177; 10.70%, 27.55% &#177; 26.30%, 88.10% &#177; 45.63%, 427.60% &#177; 172.92% and 882.27% &#177; 121.97%, respectively, showing an effect that increases with increasing dose (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61762/61762fig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61762/61762fig2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61762/61762fig3.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61762/61762fig4.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61762/61762fig5.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61762/61762fig6.jpg" /> 
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 &#177; SEM (n = 3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. The nebulizer specifications. The nebulized particle size of the compressed nebulizer is 1 - 5 &#956;m, the nebulization rate is greater than 0.2 mL/min, and the ultimate pressure is 205 kPa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762supfig1large.jpg" target="_blank">Please click here to download this file.</a>
Supplementary Figure 2. The schematic positions of GV14 (Dazhui), BL12 (Fengmen), and BL13 (Feishu) in rat. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762supfig2large.jpg" target="_blank">Please click here to download this file.</a>
Supplementary Figure 3. Needle specifications. The length of the needle is 13 mm and the diameter is 0.3 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762supfig3large.jpg" target="_blank">Please click here to download this file.</a>
Supplementary Figure 4. The schematic of acupuncture manipulation. After inserting the needle, evenly twisted the needles approximately 360&#176; in different directions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61762/61762supfig4large.jpg" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Acupuncture in a Rat Model of Asthma at JoVE.com

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Nanyang Technological University

Research

JoVE Journal

Medicine

5.9K Views.  Shanghai University of Traditional Chinese Medicine. 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.

Video: Acupuncture in a Rat Model of Asthma

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of intraoperative arrest in the field of cardiac surgery. With recent advances in cardiac surgery, the demand for prolonged VF studies under perfusion has increased. However, the field lacks simple, reliable, and reproducible animal models of chronic ventricular fibrillation. This protocol induces long-term VF through alternating current (AC) electrical stimulation of the epicardium. Different conditions were used to induce VF, including continuous stimulation with a low or high voltage to induce long-term VF and stimulation for 5 min with a low or high voltage to induce spontaneous long-term VF. The success rates of the different conditions, as well as the rates of myocardial injury and recovery of cardiac function, were compared. The results showed that continuous low-voltage stimulation induced long-term VF and that 5 min of low-voltage stimulation induced spontaneous long-term VF with mild myocardial injury and a high rate of recovery of cardiac function. However, the low-voltage, continuously stimulated long-term VF model had a higher success rate. High-voltage stimulation provided a higher rate of VF induction but showed a low defibrillation success rate, poor recovery of cardiac function, and severe myocardial injury. On the basis of these results, continuous low-voltage epicardial AC stimulation is recommended for its high success rate, stability, reliability, reproducibility, low impact on cardiac function, and mild myocardial injury.

This protocol presents a model of long-term ventricular fibrillation in rat hearts induced by continuous stimulation with low-voltage alternating current. This model has a high success rate, is stable, reliable, and reproducible, has a low impact on cardiac function, and causes only mild myocardial injury.

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Tuina Therapy for Frozen Shoulders in a Rat Model

Tuina in a Frozen Shoulder Rat Model: An Efficient and Reproducible Protocol

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS patients in Chinese hospitals, has demonstrated excellent results, but its mechanisms are not fully understood. Building on a previous study, this work aimed to develop a Tuina protocol for an FS rat model. We randomly divided 20 SD rats into control (C; n = 5), FS model (M; n = 5), FS model Tuina treatment (MT; n = 5), and FS model oral treatment (MO; n = 5) groups. This study used the cast immobilization method to establish the FS rat model. The effect of Tuina and oral dexamethasone on the glenohumeral range of motion (ROM) was evaluated, and the histological findings were assessed. Our study showed that Tuina and oral dexamethasone were able to improve shoulder active ROM and preserve the structure of the capsule, with Tuina therapy proving to be more effective than oral dexamethasone. In conclusion, the Tuina protocol established in this study was highly effective for FS.

Author Spotlight: Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder 

This study develops an efficient and reproducible Tuina protocol for treating frozen shoulder established in a rat model. This approach will help to study the Tuina therapy treatment method for frozen shoulders.

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS ...

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.

Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...