We thank the support and assistance from the Zhuang Yao Medical Research Laboratory of Guangxi International Zhuang Medicine&#160;Hospital. This experiment was supported by the National Natural Science Foundation of China(82360982), the Doctoral Innovation Program (NO.YCBZ2023151) and Zhuang Medicine, Key Disciplines of Traditional Chinese Medicine in Guangxi (NO.GZXK-Z-20-60).

The animal experiments in this study were reviewed and approved by the Animal Experiment Ethics Committee of Guangxi University of Traditional Chinese Medicine (ethical license number DW20230411-45). The animal operation procedures strictly follow the content of the Guiding Opinions on Treating Experimental Animals (Guo Ke Fa Cai Zi [2006] No. 398) and the animal protection and use standards of the International Society for Pain Research (IASP).
NOTE: SPF-grade female rats, 6 weeks old, with a body weight of 200 g &#177; 20 g, were used in this study. Rats in the estrous phase were selected based on vaginal smears and divided into four groups using the random number method. They are divided into the vehicle group, the cold coagulation type dysmenorrhea model group (the model group), the cold coagulation type dysmenorrhea model group + Western Medicine Group (the medicine group), the cold coagulation type dysmenorrhea model group + Zhuang Medicine line moxibustion group (the moxibustion group), with 12 rats in each group. 
Vehicle group: The animals were treated with physiological saline (0.9% sodium chloride). 
Model group: During the modeling process of the cold coagulation syndrome dysmenorrhea model, no intervention was performed, and only the same restraint and fixation were performed as the moxibustion group. 
Medicine group: From the 6th day of sensitization, ibuprofen dissolved in 0.9% sodium chloride solution was given by gavage every day. Each rat was given 0.06 g/kg. 
Moxibustion group: From the 6th day of sensitization, daily moxibustion treatment with Zhuang medicine thread was performed. This group received treatment once daily at 8 a.m. for 7 consecutive days. 
All animals are kept at the Experimental Animal Science Center of Guangxi International Zhuang Medical Hospital (clean level). The rats were fed in plastic cages with 4 rats in one cage. They ate and drank normally, kept the circadian rhythm alternating, and should be dry indoors. The room temperature was controlled at 23 &#177; 1 &#176;C, and the humidity was 60% &#177; 5%.
1. Establishment of a rat model of dysmenorrhea with cold coagulation syndrome
<ol>
	<li>Freezing: On days 1-12, anesthetize the rats with an intraperitoneal injection of 0.3% pentobarbital (30 mg/kg), then place them on ice for 20 min daily for 12 consecutive days.</li>
	<li>Sensitization: On days 1-12, inject rats subcutaneously with estradiol benzoate to enhance the sensitivity of the uterus to pain-causing substances. On the 1st and 12th days, inject 0.8 mg/animal; on the 2nd to 11th days, inject 0.4 mg/animal.</li>
	<li>Pain induction: On the 12th day, 1 h after injection of estradiol benzoate, inject the rats intraperitoneally with 2 U of oxytocin, causing pain. 
	NOTE: The model was created using the whole-body freezing method combined with estradiol benzoate and oxytocin injection10.</li>
	<li>Treat each animal group as described in steps 1.4.1-1.4.4.
	<ol>
		<li>For the rats in the vehicle group, do not perform steps 1.1-1.3. Treat them with physiological saline (0.9% sodium chloride) alone.</li>
		<li>For the rats in the model group, perform steps 1.1-1.3. Restrain the animals in the group in a manner similar to the moxibustion group but without any drug/treatment intervention.</li>
		<li>For the rats in the medicine group, perform steps 1.1-1.3. From the 6th day of sensitization (step 1.2), dissolve ibuprofen in 0.9% sodium chloride solution and give it by gavage daily (0.06 g/kg).</li>
		<li>For the rats in the moxibustion group, perform steps 1.1-1.3. From the 6th day of sensitization (step 1.2), perform moxibustion treatment with Zhuang medicine thread (section 4) once daily at 8 a.m. for 7 consecutive days.</li>
	</ol>
	</li>
</ol>
2. Setting up the moxibustion simulator for Zhuang medicine threads
<ol>
	<li>Press and hold Button 1 for a long time to enter the setting state. Press Button 2 (Plus) or Button 3 (Minus) shortly, and a 132 code will appear. Set the button input to output 0.5 cm for each press.</li>
	<li>Repeat step 2.1 until code 130 appears, and the device can ignite automatically within 6 s after ignition.</li>
	<li>Repeat step 2.1 until code 131 appears, and the instrument automatically performs moxibustion twice.</li>
	<li>Rotate the speed control button to set the frequency to 2 times/s. 
	NOTE: The button number is detailed in Supplementary Figure 1.</li>
</ol>
3. Preparation of Zhuang medicine threads
<ol>
	<li>Preparation of medicinal liquor
	<ol>
		<li>Select medicinal liquor configurations according to the treatment of different diseases. In this experiment, 10 g of frankincense, 10 g of sandalwood, 10 g of woody incense, 10 g of peach kernel, 10 g of safflower, 10 g of Angelica sinensis tail, 10 g of Fangfeng, 10 g of red peony, 10 g of Sumu, 10 g of dry ginger, and 10 g of turmeric were used. Soak these ingredients in 1000 mL of 75% alcohol, and filter after 10 days to form a medicinal liquor.</li>
	</ol>
	</li>
	<li>Soaking the medicinal thread: Soak the ramie thread in the above-mentioned medicinal liquor for at least 10 days (Figure 2), take out the medicinal thread for moxibustion, and seal the medicinal thread in a bottle for later use.</li>
	<li>Take out a medicinal thread with a length of approximately 21 cm each time when using it (Figure 3).</li>
</ol>
4. Intervention methods of Zhuang medicine thread moxibustion
<ol>
	<li>Acupoint selection 
	NOTE: The rats are anesthetized by intraperitoneal injection of 0.3% pentobarbital (30 mg/kg).
	<ol>
		<li>Clean the hair in the abdominal area of rats using a small animal shaving machine.</li>
		<li>Based on the experience in treating dysmenorrhea with thread moxibustion, select acupoints11,12,13 such as Shenque, Guanyuan, Zhongji, and Uterine acupoints. Use a marker to mark the above acupoints on the lower abdominal area of the rat. 
		NOTE: Refer to Figure 4 for the acupoints. Shen que (CV8, the intersection of the upper 2/3 and lower 1/3 of the line connecting the xiphoid process and the upper edge of the pubic symphysis); Guan yuan (CV4, 25 mm below Shenque acupoint); Zhong ji (CV3, 8 mm below Guanyuan acupoint); Left uterine acupoint (left, EX-CA1, opening 25 mm to the left side of Zhongji acupoint); Right uterine acupoint (Right, EX-CA1, opening 25 mm to the right side of Zhongji acupoint).</li>
	</ol>
	</li>
	<li>Fixing the rats
	<ol>
		<li>Fix the rats on a transparent acrylic board using high elastic rubber bands (Acrylic board size 10 cm&#160;&#215; 15 cm&#160;&#215; 5 cm) to expose the abdomen.</li>
		<li>Place the fixed rat on the operating table of the Zhuang Medicine Line Moxibustion simulator, adjust the positioning ring to adhere to the rat&#39;s abdomen, and adjust the position of the rat to the left and right so that one marked acupoint is located at the center of the positioning ring (Figure 5).</li>
	</ol>
	</li>
	<li>Placing the medicated line
	<ol>
		<li>Take out one soaked medicine thread and insert it into the medicine thread control device.</li>
		<li>Rotate the screw knobs on the vertical and horizontal axes of the support frame. Adjust the shell of the drug line control device up and down so that the distance between the bottom of the shell and the bottom of the positioning ring is 2 cm (Figure 6), and adjust the ignition support shaft and positioning rod shaft accordingly.</li>
		<li>Click the wire entry button to expose the medicine wire 0.5 cm from the shell (set the wire end to exit 0.5 cm for each press) (Figure 7).</li>
	</ol>
	</li>
	<li>Point moxibustion
	<ol>
		<li>Press the ignition button, and the ignition device will automatically ignite. The default timing is 6 s. After that, the automatic moxibustion will be performed twice, with a frequency set to 2 times/s (Figure 8) 
		NOTE: It has been previously repeatedly tested that at a windless environment with a temperature of 23&#160;&#177; 1 &#176;C and humidity of 60%&#160;&#177; 5%, bead fire could form after 6 s.</li>
		<li>After the moxibustion of an acupoint, move the transparent acrylic frame to adjust its position so that the next acupoint requiring moxibustion is located at the center of the positioning circle. Repeat steps 4.3.3 and 4.4.1.</li>
	</ol>
	</li>
</ol>
5. Torsion scoring operation
<ol>
	<li>Inject rats with oxytocin (2 U) and place them in empty cages.</li>
	<li>Observe the twisting response of each rat within 20 min. 
	NOTE: Except for the vehicle group, 6 rats were selected for each group. If a twisting reaction occurs, the primary dysmenorrhea model was successfully established in rats14. The saline group was injected with equal doses of physiological saline. Behavioral scoring proposed by Schmauss15was used in this study: For normal exploration behavior, 0 point; The torso is tilted to one side, and the abdomen is inward concave due to contraction, 1 point; Extension of hind limbs, dorsiflexion of hind paws, trunk extension with pelvic rotation, 2 points; Abdominal muscle contraction, hind limb extension, 3 points (Figure 9) The latency, the number of twists, and the twist score of the twist response were all recorded within 20 min. The latency period of rat writhing is the time (min) from oxytocin injection (causing pain) to the first appearance of the writhing reaction in rats. 
	Twist score = [0 level (number of times)&#160;&#215; 0 points + 1 level (frequency)&#160;&#215; 1 point + 2 levels (frequency) &#215; 2 points + 3 levels (frequency) &#215; 3 points].</li>
</ol>
6. Infrared thermal imaging
<ol>
	<li>Anesthetize the remaining 6 rats in each group by intraperitoneal injection of 0.3% pentobarbital 30 mg/kg, exposing the uterine projection area.</li>
	<li>After the operation, place the rat in the field of view of the infrared thermal imager lens and record the temperature of the uterine projection area using the infrared thermal imager.</li>
	<li>Measure each acupoint 3 times and take the average value as the temperature of that acupoint.</li>
	<li>Export infrared thermal images using thermal imaging software (Figure 10). 
	NOTE: On the 12th day of modeling (i.e., the 7th day of intervention), the remaining 6 rats in each group were subjected to infrared thermal imaging observation. Primary dysmenorrhea rats with cold coagulation syndrome will experience a decrease in the surface projection temperature of the uterus. Observing the surface projection temperature of the uterus in rats can determine the severity of the cold coagulation syndrome. The lower the temperature, the more severe the cold coagulation syndrome.</li>
</ol>

Standardized Operation Process of Zhuang Medicine Line Moxibustion in Rats

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The authors have nothing to disclose.

As an experiential manual treatment method, Zhuang medicine thread moxibustion is divided into igniting Zhuang medicine thread, waiting for the formation of bead fire, and moxibustion contact with the skin. The key factor in the process of Zhuang medicine thread moxibustion operation is the amount of moxibustion stimulation, which is closely related to the intensity, frequency, and heat and has a direct impact on the biological effects of Zhuang medicine thread moxibustion. How to quantitatively apply the stimulation amount of moxibustion to the basic experimental research of Zhuang medicine line moxibustion is an urgent problem that needs to be solved, related to experimental results&#39; reliability, stability, and repeatability. Therefore, this article designs a Zhuang medicine line moxibustion simulator that maintains constant force, heat, and frequency. To compare the differences between manual moxibustion and Zhuang medicine thread moxibustion simulation device, we tested the uterine surface projection temperature of the model group, manual moxibustion group, and Zhuang medicine line moxibustion simulation device group. The results showed that the body surface projection temperature of the manual and device moxibustion groups was higher than that of the model group (p &#60; 0.05). There was no significant difference in body surface projection temperature between the manual and device moxibustion groups (p &#62; 0.05). However, from comparing the standard deviations between the two groups, it can be seen that the standard deviation of the device group is relatively small, indicating strong stability. The standard deviation of the manual moxibustion group is relatively large, indicating significant fluctuations in manual moxibustion operations (Supplementary Figure 2). At the same time, to compare whether the sudden increase in temperature during ignition had an impact on rats, we measured the abdominal skin before and at the ignition of the device group (using Shenque acupoint as the measurement point), and the results showed that the ignition moment did not have an impact on the abdominal skin temperature (Supplementary Figure 3).
Due to the susceptibility to wind interference while igniting the medicinal line, operating the Zhuang Medicine Line Moxibustion Simulator in a sheltered environment is advisable. After the medicinal thread is ignited, there are generally four types of heat conditions - open fire, strip fire, bead fire, and radial fire. The best effect is when the thread is in a &#34;bead fire&#34; shape, and it is important to avoid using open fire for moxibustion16. The time for the formation of bead fire varies after igniting different thicknesses of medicinal lines. Therefore, when replacing different thicknesses of medicinal lines, it is necessary to repeatedly test the formation time of bead fire, set a delay time on the instrument, and then perform moxibustion. The higher the number of times moxibustion is performed on the same acupoint simultaneously, the greater the stimulation. Therefore, it is necessary to choose the appropriate frequency of moxibustion according to experimental needs.
This experiment constructed a model of dysmenorrhea rats with cold coagulation syndrome. Through twisting experiments and the results of uterine surface projection temperature, it was found that Zhuang medicine thread moxibustion can effectively improve the pain response of dysmenorrhea rats with cold coagulation syndrome and increase their uterine surface projection temperature, suggesting that Zhuang medicine thread moxibustion positively improve the pain symptoms formed by dysmenorrhea with cold coagulation syndrome. Clinical studies have shown that pre-menstrual therapy at Shenque, Guanyuan, Zhongji, and uterine acupoints can effectively treat dysmenorrhea caused by coagulation and blood stasis. They have the effects of consolidating the foundation and nourishing the energy, tonifying the lower energizer, and dredging local qi and blood. Acupoints are stimulated by Zhuang medicine thread burning and moxibustion, combining the combined effects of warm heat conduction, acupoints, and medicinal liquor to achieve the regulation of qi and blood, promoting blood circulation and resolving blood stasis, and warming and dispersing cold, which can significantly improve the symptoms of dysmenorrhea.
This experiment still has certain limitations. Due to technological limitations, the formation nodes of bead fires are controlled through time delay in environments with temperature, humidity, and no wind. The next step is to optimize the instrument further and use the image recognition function to control the nodes where bead fires occur more accurately. In addition, due to the inability of the human-computer interaction panel to display detailed parameters, the instrument cannot visually display information such as frequency and temperature. In the future, the human-computer interaction panel of the instrument will be enriched to make its operation more intelligent.

Zhuang medicine thread moxibustion therapy is a unique external treatment method guided by Zhuang medicine theory, which uses ramie thread soaked in various Zhuang medicine preparation solutions. After being removed, one end of the ramie thread is ignited on a lamp to form a bead-shaped charcoal spark, which is then quickly burned directly onto a certain acupoint or part of the human body surface to prevent and treat diseases1. This therapy can treat various diseases and symptoms in clinical practice, especially chronic pain, geriatric diseases, insomnia, asthma, rheumatism, and other chronic diseases. In clinical practice, many researchers have conducted extensive theoretical discussions and conducted rigorous clinical controlled studies to optimize their operational techniques and explain their mechanism of action2,3,4. Due to the limitations of human experimental materials, the mechanism of action of Zhuang medicine thread moxibustion needs to be further validated through animal experiments. However, the therapeutic effect of Zhuang medicine line moxibustion is influenced by factors such as moxibustion intensity, heat intensity, and moxibustion frequency. Manual operation cannot precisely control each influencing factor. Therefore, it is necessary to design an instrument to maintain the influencing factors of Zhuang medicine line moxibustion, which is the direction of future experimental research.
This article designs a Zhuang medicine line moxibustion simulation instrument to maintain a constant force, heat, and frequency. The frequency and heat can be adjusted according to experimental needs, and it can be applied to the study of analgesic experiments to observe the analgesic effect of Zhuang medicine line moxibustion on pain animal models to solve the standardization problem of Zhuang medicine line moxibustion in the operation process of experimental animals.
Design Principle of Moxibustion Simulator for Zhuang Medicine Line 
To enable technical personnel in this field to understand better the technical solution of the Zhuang Medicine Line Moxibustion Simulator, the following is a detailed description of this instrument in conjunction with Figure 1. The Zhuang Medicine Line Moxibustion Simulator includes a base (1), support device (2), medicine line control device (3), ignition device (4), and locator (5). The base is a rectangular structure as a whole, with the top surface set as an operating table. The support device includes a vertical axis and a horizontal axis, with slots for vertical and horizontal movement, respectively on the vertical and horizontal axes. The medicine line control device includes a shell, a clamping pushing component, a medicine line, an incoming button, and a propulsion motor. The clamping-pushing component and the medicine line are set inside the shell, the incoming button is set on the shell&#39;s outer surface, and the incoming button is controlled and connected to the clamping-pushing component. The propulsion motor is arranged inside the base. The ignition device includes an ignition support shaft, an igniter, a wire, an ignition button, and a speed control button. The igniter is installed on the ignition support shaft, and the ignition support shaft is vertically arranged on the operating table. The igniter is connected to a speed control button set on the side of the base and an ignition button set on the surface of the igniter through a wire. The locator includes a positioning rod shaft and a positioning ring, wherein the positioning ring is set at the end of the positioning rod shaft, and the positioning rod shaft can also perform vertical movement. The ignition support shaft and positioning rod shaft are both &#34;L&#34; shaped telescopic rod structures, and the side of the base is also equipped with a power switch and power indicator light.
As one of the common gynecological diseases in adolescent women, primary dysmenorrhea (PD) has a high incidence rate and great harm, although it has no organic disease5. The survey results of relevant literature show that the prevalence of primary dysmenorrhea in different countries ranges from 45% to 90%6. Other studies have found that primary dysmenorrhea may be related to changes in prostaglandins and interleukins. Elevated levels of these factors can lead to uterine smooth muscle contraction, spasms, and reduced uterine blood flow7,8. In previous clinical studies, it has been found that cold coagulation syndrome accounts for more than half of the total number of primary dysmenorrhea patients. Currently, the modeling methods for dysmenorrhea related to cold coagulation syndrome include ice cold stimulation, ice water immersion with medication, low-temperature refrigerator, cold wind environment, and local limb low-temperature freezing method to simulate clinical cold coagulation syndrome. Patients with primary dysmenorrhea often experience systemic cold coagulation and blood stasis due to external cold, and pain is caused by obstruction. Therefore, we chose the ice-cold stimulation method. The dysmenorrhea model is established using the commonly recognized combination of estradiol benzoate and oxytocin9. As one of the most distinctive therapies of Zhuang medicine, Zhuang medicine thread moxibustion therapy is widely spread in the Guangxi region. Its therapeutic effect on primary dysmenorrhea, especially those with cold coagulation syndrome, is significant, but the mechanism of action is still unclear. This article observes the effects of Zhuang medicine thread moxibustion therapy on the twisting experiment and infrared thermography of the uterine region in rats with cold coagulation type dysmenorrhea and explores the possible mechanism of Zhuang medicine thread moxibustion treatment for cold coagulation type dysmenorrhea.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Estradiol Benzoate Injection</td>
			<td>Shanghai Quanyu Biotechnology Animal Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice maker</td>
			<td>Guoyi</td>
			<td>GYXH-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared thermal imager</td>
			<td>FLUKE</td>
			<td>Ti32</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxytocin Injection</td>
			<td>Sichuan Jinke Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ramie thread</td>
			<td>Guangxi Nanning Shuqin Trading Co., Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zhuang Pharmaceutical Line Moxibustion Simulator</td>
			<td>In-house</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

medicated thread moxibustion therapy of zhuang medicine and its application in a rat model of cold-congealing syndrome of primary dysmenorrhea

Zhuang medicine thread moxibustion therapy is one of the national intangible cultural heritages. It is a Zhuang medicine characteristic therapy that involves igniting ramie thread soaked in Zhuang medicine preparation solution and directly moxibustion on a certain acupoint or part of the body surface. This treatment method is characterized by combining drug, acupoints, and moxibustion, stimulating the skin's receptors at the acupoints through the warmth of moxibustion, affecting the biochemical metabolism of histiocyte and the function of the nervous system so that the drug ingredients can be absorbed through the skin. However, the therapeutic effect of Zhuang medicine line moxibustion is influenced by factors such as moxibustion intensity, heat intensity, and moxibustion frequency. Manual operation cannot precisely control each influencing factor. Here, we design a Zhuang medicine line moxibustion simulation instrument to maintain a constant force, heat, and frequency. The frequency and heat can be adjusted according to experimental needs, and it can be applied to the study of analgesic experiments to observe the analgesic effect of Zhuang medicine line moxibustion on pain animal models to solve the standardization problem of Zhuang medicine line moxibustion in the operation process of experimental animals.This study establishes a model of primary dysmenorrhea in rats with cold coagulation syndrome and provides a detailed introduction to the preparation, process, and precautions of Zhuang medicine thread moxibustion on the model of primary dysmenorrhea in rats with cold coagulation syndrome. The intervention of Zhuang medicine thread moxibustion on primary dysmenorrhea in rats with cold coagulation syndrome is evaluated by twisting score and infrared thermal imaging body surface temperature detection, intuitively demonstrating the therapeutic effect of Zhuang medicine thread moxibustion, and a preliminary analysis of its mechanism of action is conducted.

The Zhuang Medicine Line Moxibustion Simulator can simulate the clinical process of Zhuang Medicine Line Moxibustion, and the intensity, frequency, and heat are constant during the intervention of rats, avoiding human interference and minimizing experimental errors.
All three other groups experienced writhing reactions except for the vehicle group, indicating the successful preparation of the primary dysmenorrhea rat model. Compared with the model group, the writhing scores of the Western medicine and moxibustion groups were significantly reduced (p &#60; 0.05) (Figure 11).
Compared with the saline group, the model group showed a significant decrease in uterine surface projection temperature (p &#60; 0.05), while the moxibustion group showed an increase in uterine surface projection temperature (p &#60; 0.05) (Figure 12).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65669/65669fig01.jpg" /> 
Figure 1: Mechanical drawing. The figure shows 1-base, 2-housing, 3-incoming button, 4-propulsion motor, 5-vertical axis, 6-horizontal axis, 7-ignition support shaft, 8-igniter, 9-wire, 10-main switch, 11-speed control button, 12-positioning rod shaft, 13-positioning ring, 14-scale, 15-ignition button. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig01large.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/65669/65669fig02.jpg" /> 
Figure 2: Ramie thread (multiple bundles) soaked in medicinal liquor. The ramie thread in the medicinal liquor for at least 10 days. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig02large.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/65669/65669fig03.jpg" /> 
Figure 3: Single drug line diagram. The length of the medicinal thread is approximately 21 cm. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig03large.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/65669/65669fig04.jpg" /> 
Figure 4: Fixing the rats on a transparent acrylic board using high elastic rubber bands and marking the moxibustion acupoints. The acupoints used in this study are Shen que, Guan yuan, Zhong ji, the left uterine acupoint (left, EX-CA1), and the right uterine acupoint (Right, EX-CA1). <a href="https://www.jove.com/files/ftp_upload/65669/65669fig04large.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/65669/65669fig05.jpg" /> 
Figure 5: Positioning the rats. The rats are adjusted so that the acupoints that require moxibustion are located in the center of the positioning circle. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig05large.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/65669/65669fig06.jpg" /> 
Figure 6: Distance between the drug line device and the positioning ring. The distance from the drug line device shell&#39;s bottom to the positioning ring&#39;s bottom is 2 cm. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65669/65669fig07.jpg" /> 
Figure 7: Exposing the medicinal line. Press the feed button to expose the medication line 0.5 cm from the shell. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65669/65669fig08.jpg" /> 
Figure 8: Instrument moxibustion process. (A) Pressing the ignition button. (B) Waiting for the flame to turn into bead fire. (C) The instrument automatically performs moxibustion of the acupoints twice. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65669/65669fig09v2.jpg" /> 
Figure 9: Pain reactions in the rat after oxytocin injection. The pain reactions include abdominal muscle contraction and hind limb extension in rats after oxytocin injection. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig09largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65669/65669fig10.jpg" /> 
Figure 10: Infrared thermal imaging. The infrared thermal images are exported using the thermal imaging software. <a href="https://www.jove.com/files/ftp_upload/65669/65669fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65669/65669fig11.jpg" /> 
Figure 11: The writhing score of each group. Compared with the vehicle group (*p &#60; 0.05). Compared with the model group (#p &#60; 0.05). <a href="https://www.jove.com/files/ftp_upload/65669/65669fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65669/65669fig12.jpg" /> 
Figure 12: Infrared thermal imaging temperatures of each group. Compared with the vehicle group (*p &#60; 0.05). Compared with the model group (#p &#60; 0.05). <a href="https://www.jove.com/files/ftp_upload/65669/65669fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: The control buttons of the moxibustion simulator. <a href="https://www.jove.com/files/ftp_upload/65669/supplement Fig.1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Comparison between the device group and manual moxibustion operations group. <a href="https://www.jove.com/files/ftp_upload/65669/Supplement Fig 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Impact of ignition on the abdominal skin temperature. Abdominal skin temperature before and at ignition using Shenque acupoint as the measurement point. <a href="https://www.jove.com/files/ftp_upload/65669/Supplement Fig 3.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Author Spotlight: Demonstrating Therapeutic Effect of Automated Zhuang Medicine Line Moxibustion Simulator at JoVE.com

Author Spotlight: Demonstrating Therapeutic Effect of Automated Zhuang Medicine Line Moxibustion Simulator

This article demonstrates the standardized operation process of Zhuang medicine line moxibustion in experimental animals. To achieve repeatability and accuracy of Zhuang medicine line moxibustion, we developed a simulator to maintain strength, heat, and frequency. We constructed a dysmenorrhea model for experimental verification in rats with cold coagulation syndrome.

Medicated Thread Moxibustion Therapy of Zhuang Medicine and its Application in a Rat Model of Cold-Congealing Syndrome of Primary Dysmenorrhea

Zhuang medicine thread moxibustion therapy is one of the national intangible cultural heritages. It is a Zhuang medicine characteristic therapy that ...

medicated-thread-moxibustion-therapy-zhuang-medicine-its-application

Research

JoVE Journal

Behavior

749 Views.  Guangxi University of Chinese Medicine. This article demonstrates the standardized operation process of Zhuang medicine line moxibustion in experimental animals. To achieve repeatability and accuracy of Zhuang medicine line moxibustion, we developed a simulator to maintain strength, heat, and frequency. We constructed a dysmenorrhea model for experimental verification in rats with cold coagulation syndrome.

Video: Author Spotlight: Demonstrating Therapeutic Effect of Automated Zhuang Medicine Line Moxibustion Simulator

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

fMRI Validation of fNIRS Measurements During a Naturalistic Task

We present a method to compare brain activity recorded with near-infrared spectroscopy (fNIRS) in a dance video game task to that recorded in a reduced version of the task using fMRI (functional magnetic resonance imaging). Recently, it has been shown that fNIRS can accurately record functional brain activities equivalent to those concurrently recorded with functional magnetic resonance imaging for classic psychophysical tasks and simple finger tapping paradigms. However, an often quoted benefit of fNIRS is that the technique allows for studying neural mechanisms of complex, naturalistic behaviors that are not possible using the constrained environment of fMRI. Our goal was to extend the findings of previous studies that have shown high correlation between concurrently recorded fNIRS and fMRI signals to compare neural recordings obtained in fMRI procedures to those separately obtained in naturalistic fNIRS experiments. Specifically, we developed a modified version of the dance video game Dance Dance Revolution (DDR) to be compatible with both fMRI and fNIRS imaging procedures. In this methodology we explain the modifications to the software and hardware for compatibility with each technique as well as the scanning and calibration procedures used to obtain representative results. The results of the study show a task-related increase in oxyhemoglobin in both modalities and demonstrate that it is possible to replicate the findings of fMRI using fNIRS in a naturalistic task. This technique represents a methodology to compare fMRI imaging paradigms which utilize a reduced-world environment to fNIRS in closer approximation to naturalistic, full-body activities and behaviors. Further development of this technique may apply to neurodegenerative diseases, such as Parkinson&#8217;s disease, late states of dementia, or those with magnetic susceptibility which are contraindicated for fMRI scanning.

We present a method to compare functional brain activity recorded during a naturalistic task using fNIRS with activity recorded during fMRI.

We present a method to compare brain activity recorded with near-infrared spectroscopy (fNIRS) in a dance video game task to that recorded in a reduced ...

Signal Attenuation as a Rat Model of Obsessive Compulsive Disorder

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus which serves as a feedback cue. This feedback is later attenuated by repeated presentations of the stimulus without food (without the rat emitting the lever-press response). In the next stage, lever-pressing is assessed under extinction conditions (i.e., no food is delivered). At this stage rats display two types of lever-presses, those that are followed by an attempt to collect a reward, and those that are not. The latter are the measure of compulsive-like behavior in the model. A control procedure in which rats do not experience the attenuation of the feedback cue serves to distinguish between the effects of signal attenuation and of extinction. The signal attenuation model is a highly validated model of OCD and differentiates between compulsive-like behaviors and behaviors that are repetitive but not compulsive. In addition the measures collected during the procedure eliminate alternative explanations for differences between the groups being tested, and are quantitative, unbiased and unaffected by inter-experimenter variability. The major disadvantages of this model are the costly equipment, the fact that it requires some technical know-how and the fact that it is time-consuming compared to other models of OCD (11 days). The model may be used for detecting the anti- or pro-compulsive effects of pharmacological and non-pharmacological manipulations and for studying the neural substrate of compulsive behavior.

The goal of the protocol described in this paper is to induce compulsive-like behavior in rats for the study of obsessive-compulsive disorder (OCD). This behavior is precipitated by attenuating a signal indicating that a lever-press response was effective in producing food.

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus ...

Conscious and Non-conscious Representations of Emotional Faces in Asperger's Syndrome

Several neuroimaging studies have suggested that the low spatial frequency content in an emotional face mainly activates the amygdala, pulvinar, and superior colliculus especially with fearful faces1-3. These regions constitute the limbic structure in non-conscious perception of emotions and modulate cortical activity either directly or indirectly2. In contrast, the conscious representation of emotions is more pronounced in the anterior cingulate, prefrontal cortex, and somatosensory cortex for directing voluntary attention to details in faces3,4. Asperger&#39;s syndrome (AS)5,6 represents an atypical mental disturbance that affects sensory, affective and communicative abilities, without interfering with normal linguistic skills and intellectual ability. Several studies have found that functional deficits in the neural circuitry important for facial emotion recognition can partly explain social communication failure in patients with AS7-9. In order to clarify the interplay between conscious and non-conscious representations of emotional faces in AS, an EEG experimental protocol is designed with two tasks involving emotionality evaluation of either photograph or line-drawing faces. A pilot study is introduced for selecting face stimuli that minimize the differences in reaction times and scores assigned to facial emotions between the pretested patients with AS and IQ/gender-matched healthy controls. Information from the pretested patients was used to develop the scoring system used for the emotionality evaluation. Research into facial emotions and visual stimuli with different spatial frequency contents has reached discrepant findings depending on the demographic characteristics of participants and task demands2. The experimental protocol is intended to clarify deficits in patients with AS in processing emotional faces when compared with healthy controls by controlling for factors unrelated to recognition of facial emotions, such as task difficulty, IQ and gender.

Conscious and Non-conscious Representations of Emotional Faces in Asperger&#039;s Syndrome

An EEG experimental protocol is designed to clarify the interplay between conscious and non-conscious representations of emotional faces in patients with Asperger&#39;s syndrome. The technique suggests that patients with Asperger&#39;s syndrome have deficits in non-conscious representation of emotional faces, but have comparable performance in conscious representation with healthy controls.

Several neuroimaging studies have suggested that the low spatial frequency content in an emotional face mainly activates the amygdala, pulvinar, and ...

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

Establishment of a Valuable Mimic of Alzheimer&#039;s Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

Computerized Adaptive Testing System of Functional Assessment of Stroke

The computerized adaptive testing system of the functional assessment of stroke (CAT-FAS) can simultaneously assess four functions (motor functions of the upper and lower extremities, postural control, and basic activities of daily living) with sufficient reliability and administrative efficiency. CAT, a modern measurement method, aims to provide a reliable estimate of the examinee's level of function rapidly. CAT administers only a few items whose item difficulties match an examinee's level of function and, thus, the administered items of CAT can provide sufficient information to reliably estimate the examinee's level of function in a short time. The CAT-FAS was developed through four steps: (1) determining the item bank, (2) determining the stopping rules, (3) validating the CAT-FAS, and (4) establishing a platform of online administration. The results of this study indicate that the CAT-FAS has sufficient administrative efficiency (average number of items = 8.5) and reliability (group-level Rasch reliability: 0.88 - 0.93; individual-level Rasch reliability: &#8805;70% of patients had Rasch reliability score &#8805;0.90) to simultaneously assess four functions in patients with stroke. In addition, because the CAT-FAS is a computer-based test, the CAT-FAS has three additional advantages: the automatic calculation of scores, the immediate storage of data, and the easy exporting of data. These advantages of the CAT-FAS will be beneficial to data management for clinicians and researchers.

Here, we present a protocol to develop the computerized adaptive testing system of the functional assessment of stroke (CAT-FAS). The CAT-FAS can simultaneously assess four functions (two motor functions [upper and lower extremities], postural control, and basic activities of daily living) with sufficient reliability and administrative efficiency.

The computerized adaptive testing system of the functional assessment of stroke (CAT-FAS) can simultaneously assess four functions (motor functions of the ...

Pupillary Response as Assessment of Effective Seizure Induction by Electroconvulsive Therapy

Electroconvulsive therapy (ECT) is reported to be effective for severe neuropsychiatric disorders. In ECT, electrical stimulation is applied to the brain, inducing seizure activity. Adequate seizure induction with ECT is associated with seizure duration, symmetrical high amplitude waveforms during slow-wave activity, postictal suppression, and activation of the sympathetic nervous system. Sympathetic nervous system activation is influenced by anesthetic agents or cardiovascular drugs during ECT. Pupillary responses can reflect sympathetic nervous activity or the degree of brain damage. Pupillary response measurement can be conducted in a simple, precise, and objective way using an automated infrared pupillometer, enabling the measurement of pupil diameter (mm) to two decimal places. The white light used for measuring light reflexes is not overly bright, and patients do not typically report discomfort. Pupillary light reflexes were measured before anesthesia induction and immediately after electrical stimulation using this equipment. Pupil diameter is typically enlarged after brain damage or sympathetic nervous activation. Adequate seizure induction using ECT could induce pupillary enlargement immediately after electrical stimulation. In the current method, the constriction ratio of pupil size was calculated automatically and compared with seizure quality. Pupillary responses immediately after electrical stimulation may provide a useful assessment of the efficacy of seizure induction with ECT.

Pupillary responses (light reflex) were measured for assessment of adequate seizure induction by electroconvulsive therapy using an automated infrared pupillometer immediately after electrical stimulation. Constriction ratio was calculated and compared with seizure quality.

Electroconvulsive therapy (ECT) is reported to be effective for severe neuropsychiatric disorders. In ECT, electrical stimulation is applied to the brain, ...

Analysis of Learning and Memory Ability in an Alzheimer's Disease Mouse Model using the Morris Water Maze

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific research to assess the learning and memory of animals. Due to the extensive use of the MWM test, visual experimental protocols are essential for researchers. This manuscript uses the latest studies to introduce the protocol of the MWM test. Alzheimer&#8217; Disease (AD) is characterized by a progressive loss of memory and cognitive function. An alternative and complementary treatment used for AD is Manual Acupuncture (MA). To assess the learning and memory ability of AD model mice, the MWM test was conducted. The visible platform trial, hidden platform trial, probe trial, and reversal trial of MWM were used to evaluate spatial learning and memory ability. In the visible platform trial, the swimming speed and escape latency of mice in different groups was not significantly different. In the hidden platform and reversal trials, the AD group showed a long escape latency. The escape latency decreased significantly after the MA treatment. Low platform crossover number and the proportion of time in the SW quadrant in the probe trial increased after the MA treatment (p &#60; 0.05 or p &#60; 0.01). The results of the MWM tests suggest that MA can effectively improve the spatial learning and memory abilities of AD model mice. Rigorous experimental operations provided assurance of the reliability of the results.

Herein, a protocol to conduct the Morris water maze tests to evaluate the ability of learning and memory of Alzheimer&#8217;s Disease model mice and to assess the effect of manual acupuncture for treating them is described.

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific ...

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.