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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 '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.

Protocol

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 Β°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

  1. Screen the rats' estrous cycles by vaginal lavage.
    1. Insert the pipette gun with 60 Β΅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.
    2. Fix the dried content by adding 60 Β΅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.
    3. 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).
  2. Establishment of PDM animal models with cold coagulation and blood stasis
    1. 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.
    2. Place the female rats in a refrigerator at -20 Β° C for 4 h on the 1st to 5th day of modeling. During the 4 h incubation period, open the refrigerator door midway to exchange air for 30 min and then close it.
    3. 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 preparation26. The control group was injected with the same volume of saline, and the freezing procedure was the same as that of the model group.

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).

  1. Prepare moxa cones. Rub the moxa floss in the hand into a spindle shape (diameter 0.8 cm, height 0.8 cm) (Figure 2A).
  2. Make a dough bowl with a hole (diameter 1 cm, depth 0.8 cm) in the middle (Figure 2B).
  3. Weigh 0.3 g of TCM powder (Figure 2C).
    NOTE: According to Du Dongqing27, TCM powder consists of Corydalis yanhusuo,Evodia fructus, Olibanum, Baishao (Paeoniae Radix Alba), Myrrh, Borneol, and Wulingzhi (Trogopterus Xanthipes).
  4. Prepare the rat pouch to fix the rat. This will keep the rat stable during the procedure.
    1. 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.
    2. 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.
  5. Find the exact location of the Shenque (CV8) acupoint.
    1. Referring to the literature28,29, locate the Shenque (CV8) on the midline of the rat'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).
  6. Remove hair around the Shenque acupoint.
  7. Place the rat in the rat pouch. Keep the rat's abdomen up and expose the lower limbs and tail. Fix the upper and lower parts of the rat'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.
  8. Disinfect the Shenque and its surrounding skin using 75 % ethanol.
  9. Place the bowl on the abdomen, aim its hole at the Shenque acupoint, and put the TCM powder on the Shenque acupoint.
  10. Put on the moxa cone on the TCM powder.
  11. 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.

3. Conducting the behavioral test

  1. 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.
  2. Place the rat in the cage after intraperitoneal injection of oxytocin (2 U) or 0.9% saline.
  3. Record the writhing behavior for 30 min with a video camera.
  4. Calculate writhing latencies (WLs) and writhing score (WS).
    1. According to the method of Schmauss30, 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] Γ— 0) + (1 [times] Γ— 1) + (2 [times] Γ— 2) + (3 [times] Γ— 3).

4. Taking the specimen after the behavioral test

  1. Hold the rat with the hand and inject 3% sodium pentobarbital (1 mL/kg) intraperitoneally for deep anesthesia.
  2. Weigh the rats to obtain the body weight.
  3. Incise the skin and muscle from the middle of the abdomen to expose the heart.
  4. 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.
  5. 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.
  6. Use scissors to cut out the uterus and weigh it.
  7. Obtain the uterine index by calculating the ratio of uterus weight to body weight.

Results

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's writhing behavior, determine whether the PDM rat with cold coagulation and blood stasis was successfully modeled, and evaluate the rat'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...

Discussion

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 Β°C. In the previous pre-experiment, it was found that when the temperature was βˆ’25 Β°C, the rats would have...

Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

This work was supported byΒ the Zhejiang Provincial Natural ScienceΒ Foundation, Exploring Program QΒ (LQ20H270012), Zhejiang Traditional ChineseΒ Medicine Technology Plan (NO.2020ZB124), theΒ Research Project of Zhejiang Chinese MedicalΒ University (NO.2021JKZKTS056B), and theΒ Hospital-Level Project of the Third AffiliatedΒ Hospital of Zhejiang Chinese MedicalΒ University (NO.ZS22ZA01). We are grateful toΒ the Experimental Animal Center of ZhejiangΒ Chinese Medical University and the KeyΒ Laboratory of Acupuncture and Neurology ofΒ Zhejiang Province for providing us with theΒ experimental conditions.

Materials

NameCompanyCatalog NumberComments
0.9% physiological salineHangzhou Gaosheng Biotechnology Co., Ltd.SLYSZ1AFor injecting the control groupΒ 
1 mL disposable sterile syringeChangzhou Yuekang Medical Equipment Co., Ltd.3000-0515-14For drug injection
3 mL (extended) Pasteur pipetteBeijing Lanjieke Technology Co., Ltd.YCKJ-HC-007245For mounting
75% disinfection alcoholHangzhou OUTOP Biotechnology Co., Ltd.N/AFor sterilizing the treatment site
Adhesive-coated glass slideZhejiang Tuzhi Pharmaceutical Technology Co., Ltd.188105WFor HE staining
Biochemical incubatorShanghai Boxun Industrial Co., Ltd. Medical Equipment FactoryN/AFor tissue dewaxing in HE staining
Cloth materialIn-house preparationN/APouch to hold rat
Digital manager slice scanning systemHamamatsuC13210-01For HE slice scanning
Disposable latex glovesGuangzhou Lige Technology Co., Ltd.LG10-102-1IFor use during operation
Estradiol benzoateHefei Xinkexin Animal Pharmaceutical Co., Ltd.N/AFor establishing dysmenorrhea model
Excelsior AS Fully automatic dehydratorThermo ScientificExcelsior ASFor HE staining tissue dehydration
Female Wistar ratsLaboratory Animal Center of Zhejiang Chinese Medicine UniversityN/A220–250 g
FlourIn-house preparationN/AFor herbs-partitioned moxibustion treatment
ForcepZhejiang Tuzhi Pharmaceutical Technology Co., Ltd.lsnzFor specimen collection
FormaldehydeΒ Shanghai Lingfeng Chemical Reagent Co., Ltd.441244-1KGFor tissue fixation
Gemini AS slide stainerThermo ScientificGemini ASΒ Staining machine
HemostatZhejiang Tuzhi Pharmaceutical Technology Co., Ltd.lsnzFor specimen collection
HistoCoreLeica ( Arcadia H )14039357258For HE staining tissue embedding
Medical cotton ball sterileThe Third Affiliated Hospital of Zhejiang Chinese Medical UniversityN/AFor sterilize the tratment site
Methylene blue staining solution (0.1%)Beijing Honghu United Chemical Products Co., Ltd.G1300For screening estrous cycle
Microdissection ShearsHangzhou Gaosheng Biotechnology Co., Ltd.XWJ12AFor specimen collection
Microscope cover glass (24 mm x 50 mm)Hangzhou Zhengbo Biotechnology Co., Ltd.10212450CFor HE staining
MicrotomeLeica MicrosystemsRM2255For tissue sectioning
Midea freezerHefei Midea Refrigerator Co., Ltd.N/AFor establishing dysmenorrhea model with cold coagulation and blood stasis
Molar forcepsHangzhou Zhengbo Biotechnology Co., Ltd.YGJD-11For specimen collection
Mounting mediumHangzhou Gaosheng Biotechnology Co., Ltd.CS9603-100mLFor tissue mounting
Moxa stickThe Third Affiliated Hospital of Zhejiang Chinese Medical UniversityN/AFor herbs-partitioned moxibustion treatment
Pentobarbital sodiumHangzhou Dawen Biological Co., Ltd.N/AFor specimen collection
Phosphate Buffered Saline (PBS)Hangzhou Gaosheng Biotechnology Co., Ltd.P1010-2LFor specimen collection
ScissorΒ Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.lsnzFor specimen collection
Sectioning bladeShanghai Saihan Technology Co., Ltd.LEICA-819-boxFor HE staining paraffin section preparation
ShaverShenzhen Cordes Electrical Appliance Co., Ltd.N/AFor shaving a rat's hair
Sodium ChlorideChemical Reagent Co., Ltd.A501218-0001For specimen collection
SucroseZhejiang Tuzhi Pharmaceutical Technology Co., Ltd.S11055-500gFor gradient dehydration
Thermal insulation containerZhejiang Tuzhi Pharmaceutical Technology Co., Ltd.lsbwx8lFor paraffin section dewaxing
Tissue embedding cassettes, rectangular in shape, with a breakable lid.Zhejiang Tuzhi Pharmaceutical Technology Co., Ltd.31050102WFor HE staining tissue embedding
Wide adhesive tapeZhejiang Tuzhi Pharmaceutical Technology Co., Ltd.N/AFor stabilizing rat during the treatment procedure

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Herbs partitioned MoxibustionPrimary DysmenorrheaCold CoagulationBlood StasisTraditional Chinese MedicineTCMAcupoint TherapyQi CirculationMoxibustion TherapyGynecological DisordersExperimental RatsShenque AcupointPain ReliefHerbal Formula

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