This work was supported by&#160;the Zhejiang Provincial Natural Science&#160;Foundation, Exploring Program Q&#160;(LQ20H270012), Zhejiang Traditional Chinese&#160;Medicine Technology Plan (NO.2020ZB124), the&#160;Research Project of Zhejiang Chinese Medical&#160;University (NO.2021JKZKTS056B), and the&#160;Hospital-Level Project of the Third Affiliated&#160;Hospital of Zhejiang Chinese Medical&#160;University (NO.ZS22ZA01). We are grateful to&#160;the Experimental Animal Center of Zhejiang&#160;Chinese Medical University and the Key&#160;Laboratory of Acupuncture and Neurology of&#160;Zhejiang Province for providing us with the&#160;experimental conditions.

This experimental protocol was approved by the Experimental Animal Management and Ethics Review Committee of Zhejiang University of Traditional Chinese Medicine. All experimental operations on animals comply with the experimental animal welfare ethics and animal experimental safety regulations. The study used adult female Wistar rats aged 8-10 weeks from the Laboratory Animal Center of Zhejiang Chinese Medicine University. Prior to the experiment, rats were fed an adaptive diet for 1 week in a controlled environment (temperature: 23-25 &#176;C, humidity: 40%-60%). The rats were maintained in a 12-h light-dark cycle, with lights being turned on from 8:00 to 20:00, and had unrestricted access to food and water.The following protocol describes the PDM with cold coagulation and blood stasis model establishment and HPM on the navel therapy procedure. A representation of the procedure is shown in Figure 1.
1. Establishment of PDM with cold coagulation and blood stasis
<ol>
	<li>Screen the rats&#39; estrous cycles by vaginal lavage.
	<ol>
		<li>Insert the pipette gun with 60 &#181;L of 0.9% saline into the vaginal orifice of the rat, pipette up and down 3-4 times, and then suck out the liquid and drop it onto a glass slide. 
		NOTE: Wrapping the rat in a towel that could be laundered or disposed of would be appropriate.</li>
		<li>Fix the dried content by adding 60 &#181;L of 95% ethanol for 8 min, then rinse and dry the content. Add 0.1% methylene blue staining solution and incubate away from light for 10 min. Afterward, rinse and dry the content.</li>
		<li>Observe and obtain images under a microscope. 
		NOTE: If a large number of white blood cells and few nuclear epithelial cells are observed, the diestrus stage is confirmed (Figure 1B). These rats were randomly divided into the control group, model group, and moxibustion group (n = 6).</li>
	</ol>
	</li>
	<li>Establishment of PDM animal models with cold coagulation and blood stasis
	<ol>
		<li>Inject estradiol benzoate subcutaneously in the thigh for 10 days (0.5 mg per rat on the 1st and 10th days and 0.2 mg from the 2nd to the 9th days) to induce contraction of the uterus.</li>
		<li>Place the female rats in a refrigerator at -20 &#176; C for 4 h on the 1st to 5th day of modeling26. During the 4 h incubation period, open the refrigerator door midway to exchange air for 30 min and then close it.</li>
		<li>Inject oxytocin (2 U per rat) by intraperitoneal injection 24 h after the last administration to induce dysmenorrhea. 
		NOTE: This study is based on a previously published work that focused on cold coagulation and blood stasis dysmenorrhea rat model preparation27. The control group was injected with the same volume of saline, and the freezing procedure was the same as that of the model group.</li>
	</ol>
	</li>
</ol>
2. HPM navel therapy on PDM model female rats with cold coagulation and blood stasis
NOTE: Prepare the four most important materials for HPM navel therapy (see Table of Materials).
<ol>
	<li>Prepare moxa cones. Rub the moxa floss in the hand into a spindle shape (diameter 0.8 cm, height 0.8 cm) (Figure 2A).</li>
	<li>Make a dough bowl with a hole (diameter 1 cm, depth 0.8 cm) in the middle (Figure 2B).</li>
	<li>Weigh 0.3 g of TCM powder (Figure 2C). 
	NOTE: According to Du Dongqing28, TCM powder consists of Corydalis yanhusuo,Evodia fructus, Olibanum, Baishao (Paeoniae Radix Alba), Myrrh, Borneol, and Wulingzhi (Trogopterus Xanthipes).</li>
	<li>Prepare the rat pouch to fix the rat. This will keep the rat stable during the procedure.
	<ol>
		<li>Cut out cloth (length x width: 20 cm x 20 cm) and sew together diagonally. Cut out a small opening at the top for breathing and a hole (approximately 5 cm in diameter) on one side of the pouch for moxibustion.</li>
		<li>Sew a pair of cloth ropes of about 10 cm in length and 1 cm in width at the top and bottom ends of the bag, respectively, for tethering (Figure 2D). 
		NOTE: The experimenter can make a suitable size of the rat pouch according to the size of the tested animal.</li>
	</ol>
	</li>
	<li>Find the exact location of the Shenque (CV8) acupoint.
	<ol>
		<li>Referring to the literature29,30, locate the Shenque (CV8) on the midline of the rat&#39;s abdomen, at 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 bone (Figure 1D).</li>
	</ol>
	</li>
	<li>Remove hair around the Shenque acupoint.</li>
	<li>Place the rat in the rat pouch. Keep the rat&#39;s abdomen up and expose the lower limbs and tail. Fix the upper and lower parts of the rat&#39;s body and the tail on the operating table with tape. 
	NOTE: When tethering the rats, the operator must be gentle and not too hard, ensuring that it does not cause panic in the rats and affect the experimental results. Ensure that the fixing is loose and not applying pressure to the heart, which may lead to rat mortality.</li>
	<li>Disinfect the Shenque and its surrounding skin using 75 % ethanol.</li>
	<li>Place the bowl on the abdomen, aim its hole at the Shenque acupoint, and put the TCM powder on the Shenque acupoint.</li>
	<li>Put on the moxa cone on the TCM powder.</li>
	<li>Light the moxa cone and place a new one after the previous one burns out. Treat the rat for 30 min (Figure 1E). 
	NOTE: Do not use anesthesia during treatment. Be quiet. Observe the rat closely to prevent scald caused by excessive temperature during moxibustion.</li>
</ol>
3. Conducting the behavioral test
<ol>
	<li>Wipe the experimental box with 75% ethanol before each trial. 
	NOTE: The open field is a 40 cm x 40 cm x 40 cm cube without a wooden box cover.</li>
	<li>Place the rat in the cage after intraperitoneal injection of oxytocin (2 U) or 0.9% saline.</li>
	<li>Record the writhing behavior for 30 min with a video camera.</li>
	<li>Calculate writhing latencies (WLs) and writhing score (WS).
	<ol>
		<li>According to the method of Schmauss31, divide the twisting behavior into 4 levels from 0 to 3. Score the animals based on the method of Schmauss. 
		NOTE: Level 0: normal posture (hands and claws flat on the bottom of the cage or normal exploratory behavior); Level 1: body leaning to the left or right; Level 2: hind legs stretched out, back of the hind paws bent, and frequent rotation of the pelvis; Level 3: contraction of the abdominal muscles, hind legs stretched backward. The rats showed writhing responses (abdominal contraction, concavity, trunk and hind limbs extension, one limb rotation, uterine contraction), indicating successful preparation of cold-congealed-blood-stasis type PDM. The incubation period of writhing is the time from the injection of oxytocin to the occurrence of a writhing reaction. If the incubation period exceeds 30 min, it is calculated as 30 min. Twist score = (0 [times] &#215; 0) + (1 [times] &#215; 1) + (2 [times] &#215; 2) + (3 [times] &#215; 3).</li>
	</ol>
	</li>
</ol>
4. Taking the specimen after the behavioral test
<ol>
	<li>Hold the rat with the hand and inject 3% sodium pentobarbital (1 mL/kg) intraperitoneally for deep anesthesia.</li>
	<li>Weigh the rats to obtain the body weight.</li>
	<li>Incise the skin and muscle from the middle of the abdomen to expose the heart.</li>
	<li>Insert the heart from the apex position with a perfusion needle, fix the perfusion needle with hemostatic forceps, and make a small cut in the right auricle.</li>
	<li>Perfuse 0.9% saline through the left ventricle and ascending aorta until the liver and eyes of the rat become lighter and whiter in color.</li>
	<li>Use scissors to cut out the uterus and weigh it.</li>
	<li>Obtain the uterine index by calculating the ratio of uterus weight to body weight.</li>
</ol>

Herbal Moxibustion on Shenque Acupoint for Alleviating Primary Dysmenorrhea

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The authors have no conflicts of interest to declare.

At present, there are various modeling methods for PDM with cold coagulation and blood stasis type, but the main method is estradiol benzoate combined with oxytocin for dysmenorrhea modeling, which will be different on this basis. In this study, the whole-body freezing method combined with estradiol benzoate and oxytocin was used for modeling. The temperature of the whole body freezing method was -20 &#176;C. In the previous pre-experiment, it was found that when the temperature was &#8722;25 &#176;C, the rats would have...

Primary dysmenorrhea (PDM), also referred to as functional dysmenorrhea, is one of the most prevalent gynecological diseases affecting adolescent girls and menstruating young women1. It is characterized by side effects such as anguish and uterine compressions (spasms), together with inflammatory problems in the abdomen before and after menstruation without any evidence of pelvic pathology2. Lower abdominal cramps that start 8-72 h after the commencement of menstruation, peak in the first few days of increased menstrual flow3,4, and sometimes spread to the back and thighs are the characteristic symptoms of polycystic PDM. Due to differing definitions of the condition and a lack of standardized ways to evaluate the intensity of menstrual pain, the estimated prevalence of dysmenorrhea in women varies from 45%-95%3,4,5. About 10%-25% of women who are of reproductive age are thought to suffer from really severe PDM6. The prevalence of PDM prevalence is highest in the 20- to 24-year-old age group and decreases progressively thereafter2. PDM is characterized by periodic lower abdominal pain, which is frequently accompanied by other systemic symptoms such as low back pain, nausea, vomiting, headaches, anorexia, diarrhea7, and even mental symptoms like depression and anxiety8,9. During menstruation, women with dysmenorrhea experience a decline in overall quality of life4,8,10.
Furthermore, studies have found that repeated menstrual pain may affect the function of the central nervous system, leading to altered perception of pain in both painful or pain-free stages11. Due to the etiology of PDM being based on prostaglandins (PGs), the most common drug treatment for PDM is nonsteroidal anti-inflammatory drugs (NSAIDs)12,13, followed by oral contraceptives14. In addition to the side effects of drugs, 10%-20% of people do not respond to NSAIDs and contraceptives3. The majority of current research is concerned with the effects of dysmenorrhea on brain anatomy and physiology, with little attention paid to therapeutic approaches. Consequently, there is an urgent need to find a treatment strategy that can be widely applied in clinical practice, with established efficacy, and readily accepted by patients.
Herbs-partitioned moxibustion (HPM) is a characteristic external therapy in traditional Chinese medicine (TCM), which is widely used to treat diseases in China. Its benefits include minimal cost, no side effects, good long-term effects, easy operation, and easy acceptance by patients. It has been found that the effect of HPM on the navel on PDM is significant15, which may be closely related to regulating the cold and heat deficiency and excess of the uterus as well as mast cells and related receptors16. The research team has proved that HPM can immediately relieve menstrual pain17.
Zhang Zhong-jing initially recorded the concept of dysmenorrhea in the Women &#39;s Miscellaneous Diseases and Treatment of the Golden Chamber, writing that the menstrual flow is unfavorable under the belt, and the abdomen is full of pain18. According to its main symptoms, dysmenorrhea can be attributed to the category of menstrual pain and menstrual abdominal pain in traditional Chinese medicine. The syndromes of dysmenorrhea can be divided into five: qi and blood deficiency; cold coagulation and blood stasis; damp heat accumulation; qi stagnation and blood stasis; and liver and kidney deficiency16. According to relevant studies, the syndrome of cold coagulation and blood stasis is the most common syndrome of PDM19,20. TCM holds that PDM and cold pathogens are intimately connected21. During or around menstruation, there is an excessive flow of blood in Xuehai, leading to a quick depletion of qi-blood due to its energetic nature. Consequently, women are vulnerable to disease-causing causes due to a lack of sufficient healthy energy and the rapid fluctuations of qi and blood in the uterus and Chong and Ren meridians22. Women who have well-developed physiques should take precautions to avoid consuming raw or cold food and to prevent catching a cold during menstruation. Failure to do so may result in the intrusion of cold pathogens into the uterus, leading to the coagulation and stagnation of blood in the uterus and the Chong and Ren meridians. Consequently, individuals experience pain as a result of the stagnation of blood flow. Both the intrusion of cold pathogens from the outside and the presence of yin cold within the body can cause blockage and clotting of the meridians.
The location of dysmenorrhea is in the Chong and Ren meridians, and the onset is related to changes in qi and blood. In TCM theory, moxibustion works by stimulating Yang Qi, warming the meridians, dispersing cold, and increasing blood flow to relieve pain. According to Jinfu of Lingshu23, if the pulse and blood clot are in the middle, and there is congealed blood and cold blood, then it is appropriate for moxibustion; it is a typical external dysmenorrhea treatment. In a previously published work24 artemisia argyi leaves are described as bitter and pungent, capable of restoring the nearly exhausted original Yang, connecting the twelve meridians, running through the three Yin meridians, regulating Qi and blood, dispelling cold and dampness, and warming the uterus, moxibustion, with the help of corresponding acupoints, can expel pathogenic factors externally, regulate qi and blood internally, and thereby play a role in regulating the meridians, activating blood circulation, and stopping pain. The navel, also known as the Shenque (CV8), belongs to the Ren meridian, connecting the Du, Chong, and Dai meridians. According to TCM theory, Shenque is believed to be related to the five zang and six fu25. It is regarded as a congenital foundation and the pivot of the yin-yang balance. Anatomical, the PDM is in close proximity to the pelvis and is closely connected to the uterus. The PDM disease specifically affects the uterus, and its pathogenesis involves an imbalance of qi and blood as well as an imbalance of the viscera. By stimulating the Shenque point, it is possible to directly influence the qi and blood condition of the uterus and effectively treat menstrual cramps. Based on the aforementioned theory, we selected the stimulation of the Shenque acupoint located in the abdominal region for HPM therapy, with the aim of ameliorating dysmenorrhea symptoms and alleviating pain.
To mimic the intervention of TCM on PDM as closely as possible with cold coagulation and blood stasis, we replicated the HPM technique on the navel in female rats. It was discovered that HPM on the navel has a clear analgesic effect and can lessen the writhing reaction of dysmenorrhea rats. In order to investigate the mechanism of HPM on the navel to improve PDM and promote it, we have examined a technique of fixing rats and performing HPM at the Shenque acupoint in the belly.

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			<td>0.9% physiological saline</td>
			<td>Hangzhou Gaosheng Biotechnology Co., Ltd.</td>
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			<td>1 mL disposable sterile syringe</td>
			<td>Changzhou Yuekang Medical Equipment Co., Ltd.</td>
			<td>3000-0515-14</td>
			<td>For drug injection</td>
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			<td>3 mL (extended) Pasteur pipette</td>
			<td>Beijing Lanjieke Technology Co., Ltd.</td>
			<td>YCKJ-HC-007245</td>
			<td>For mounting</td>
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		<tr>
			<td>75% disinfection alcohol</td>
			<td>Hangzhou OUTOP Biotechnology Co., Ltd.</td>
			<td>N/A</td>
			<td>For sterilizing the treatment site</td>
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			<td>Adhesive-coated glass slide</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>188105W</td>
			<td>For HE staining</td>
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			<td>Biochemical incubator</td>
			<td>Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory</td>
			<td>N/A</td>
			<td>For tissue dewaxing in HE staining</td>
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			<td>Cloth material</td>
			<td>In-house preparation</td>
			<td>N/A</td>
			<td>Pouch to hold rat</td>
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		<tr>
			<td>Digital manager slice scanning system</td>
			<td>Hamamatsu</td>
			<td>C13210-01</td>
			<td>For HE slice scanning</td>
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			<td>Disposable latex gloves</td>
			<td>Guangzhou Lige Technology Co., Ltd.</td>
			<td>LG10-102-1I</td>
			<td>For use during operation</td>
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			<td>Estradiol benzoate</td>
			<td>Hefei Xinkexin Animal Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>For establishing dysmenorrhea model</td>
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			<td>Excelsior AS Fully automatic dehydrator</td>
			<td>Thermo Scientific</td>
			<td>Excelsior AS</td>
			<td>For HE staining tissue dehydration</td>
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			<td>Female Wistar rats</td>
			<td>Laboratory Animal Center of Zhejiang Chinese Medicine University</td>
			<td>N/A</td>
			<td>220&#8211;250 g</td>
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			<td>Flour</td>
			<td>In-house preparation</td>
			<td>N/A</td>
			<td>For herbs-partitioned moxibustion treatment</td>
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			<td>Forcep</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>lsnz</td>
			<td>For specimen collection</td>
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			<td>Formaldehyde&#160;</td>
			<td>Shanghai Lingfeng Chemical Reagent Co., Ltd.</td>
			<td>441244-1KG</td>
			<td>For tissue fixation</td>
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			<td>Gemini AS slide stainer</td>
			<td>Thermo Scientific</td>
			<td>Gemini AS&#160;</td>
			<td>Staining machine</td>
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			<td>Hemostat</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>lsnz</td>
			<td>For specimen collection</td>
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			<td>HistoCore</td>
			<td>Leica ( Arcadia H )</td>
			<td>14039357258</td>
			<td>For HE staining tissue embedding</td>
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			<td>Medical cotton ball sterile</td>
			<td>The Third Affiliated Hospital of Zhejiang Chinese Medical University</td>
			<td>N/A</td>
			<td>For sterilize the tratment site</td>
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		<tr>
			<td>Methylene blue staining solution (0.1%)</td>
			<td>Beijing Honghu United Chemical Products Co., Ltd.</td>
			<td>G1300</td>
			<td>For screening estrous cycle</td>
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		<tr>
			<td>Microdissection Shears</td>
			<td>Hangzhou Gaosheng Biotechnology Co., Ltd.</td>
			<td>XWJ12A</td>
			<td>For specimen collection</td>
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		<tr>
			<td>Microscope cover glass (24 mm x 50 mm)</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>10212450C</td>
			<td>For HE staining</td>
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			<td>Microtome</td>
			<td>Leica Microsystems</td>
			<td>RM2255</td>
			<td>For tissue sectioning</td>
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			<td>Midea freezer</td>
			<td>Hefei Midea Refrigerator Co., Ltd.</td>
			<td>N/A</td>
			<td>For establishing dysmenorrhea model with cold coagulation and blood stasis</td>
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		<tr>
			<td>Molar forceps</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>YGJD-11</td>
			<td>For specimen collection</td>
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			<td>Mounting medium</td>
			<td>Hangzhou Gaosheng Biotechnology Co., Ltd.</td>
			<td>CS9603-100mL</td>
			<td>For tissue mounting</td>
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			<td>Moxa stick</td>
			<td>The Third Affiliated Hospital of Zhejiang Chinese Medical University</td>
			<td>N/A</td>
			<td>For herbs-partitioned moxibustion treatment</td>
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		<tr>
			<td>Pentobarbital sodium</td>
			<td>Hangzhou Dawen Biological Co., Ltd.</td>
			<td>N/A</td>
			<td>For specimen collection</td>
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		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hangzhou Gaosheng Biotechnology Co., Ltd.</td>
			<td>P1010-2L</td>
			<td>For specimen collection</td>
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		<tr>
			<td>Scissor&#160;</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>lsnz</td>
			<td>For specimen collection</td>
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		<tr>
			<td>Sectioning blade</td>
			<td>Shanghai Saihan Technology Co., Ltd.</td>
			<td>LEICA-819-box</td>
			<td>For HE staining paraffin section preparation</td>
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			<td>Shaver</td>
			<td>Shenzhen Cordes Electrical Appliance Co., Ltd.</td>
			<td>N/A</td>
			<td>For shaving a rat&#39;s hair</td>
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			<td>Sodium Chloride</td>
			<td>Chemical Reagent Co., Ltd.</td>
			<td>A501218-0001</td>
			<td>For specimen collection</td>
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			<td>Sucrose</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>S11055-500g</td>
			<td>For gradient dehydration</td>
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			<td>Thermal insulation container</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>lsbwx8l</td>
			<td>For paraffin section dewaxing</td>
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		<tr>
			<td>Tissue embedding cassettes, rectangular in shape, with a breakable lid.</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>31050102W</td>
			<td>For HE staining tissue embedding</td>
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		<tr>
			<td>Wide adhesive tape</td>
			<td>Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.</td>
			<td>N/A</td>
			<td>For stabilizing rat during the treatment procedure</td>
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herbs-partitioned moxibustion on the navel in a rat model of primary dysmenorrhea with cold coagulation and blood stasis

Primary dysmenorrhea (PDM) refers to the occurrence of spasmodic colicky pain in the lower abdomen without any obvious pelvic pathology, often accompanied by other systemic symptoms, which significantly reduces the quality of life of women with PDM, 45%-95% of menstrual women are affected by it. Despite the high incidence of PDM, it is often not well treated and is ignored by relevant researchers and women themselves. Herbs-partitioned moxibustion (HPM) is a characteristic external therapy in traditional Chinese medicine (TCM), which is widely used to treat diseases in China; it treats diseases by applying a Chinese herbal formula on the patient's acupoints and then igniting moxa sticks on the herbal formula for moxibustion therapy. The primary effectiveness of the herbs utilized in this study is meridian activation and warmth, which encourages qi circulation to relieve pain. Moxibustion's heat stimulation could hasten the infusion of the herbs into the human body. Consequently, HPM, which combines acupoint, herbal, and heat stimulations, is appropriate for treating gynecological disorders, particularly functional diseases such as PDM. This paper provides a detailed description of the procedures and precautions for HPM at the Shenque (CV8) acupoint in female experimental rats, which 0provides an experimental basis for better promoting the application and development of HPM on the navel in PDM.

In this study, estradiol benzoate in combination with oxytocin was used to induce the PDM model of cold coagulation and blood stasis type to observe the effect of HPM navel therapy. First, by observing the rat&#39;s writhing behavior, determine whether the PDM rat with cold coagulation and blood stasis was successfully modeled, and evaluate the rat&#39;s pain behavior, modeling, and treatment on the uterus through WS, WLs, and uterine index. From the 6th to the 10th day of modeling, HPM treatment wa...

Herbs-Partitioned Moxibustion on the Navel in a Rat Model of Primary Dysmenorrhea with Cold Coagulation and Blood Stasis

Here, we describe in detail the operation procedures and precautions to be taken during herbs-partitioned moxibustion on the navel in the treatment of primary dysmenorrhea in rats with cold coagulation and blood stasis, which can significantly improve the writhing reaction of dysmenorrhea rats and alleviate pain.

Primary dysmenorrhea (PDM) refers to the occurrence of spasmodic colicky pain in the lower abdomen without any obvious pelvic pathology, often accompanied ...

herbs-partitioned-moxibustion-on-navel-rat-model-primary-dysmenorrhea

Research

JoVE Journal

Medicine

382 Views.  The Third Clinical Medical College of Zhejiang Chinese Medical University. Here, we describe in detail the operation procedures and precautions to be taken during herbs-partitioned moxibustion on the navel in the treatment of primary dysmenorrhea in rats with cold coagulation and blood stasis, which can significantly improve the writhing reaction of dysmenorrhea rats and alleviate pain.

Video: Herbs-Partitioned Moxibustion on the Navel in a Rat Model of Primary Dysmenorrhea with Cold Coagulation and Blood Stasis

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

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 ...

Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...