Operation Procedure and Precautions of Lingnan Fire-Needle Therapy in Osteoporosis Model Rats

Summary

Here, we present a protocol for the application of Lingnan Fire-Needle therapy in the intervention of osteoporosis model rats.

Abstract

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected effects on some chronic diseases and incurable diseases. Osteoporosis (OP) has a high incidence in postmenopausal women and middle-aged and elderly men, and the treatment cycle is long. According to Traditional Chinese Medicine (TCM), Lingnan fire-needle therapy has shown potential in treating osteoporosis. However, there is still a long way to go before it can be widely used. This article focuses on the application of Lingnan fire-needle therapy in the intervention of OP in rats. It covers the selection of needle tools, acupuncture point selection, positioning of rats' bodies, and fixation methods. We also outline the steps and precautions to be taken during and after needling with fire needles. The experiment was done with three groups: a normal group, a model group, and a fire-needle group, each containing 10 rats. The rats in the fire-needle group were treated with fire-needle intervention for six sessions. After the intervention period, we collected femoral specimens and performed micro-CT scans. The results suggest that fire needling can enhance bone morphology and mineral density in OP rats. This information can serve as a methodological basis for conducting basic research on fire-needle therapy.

Introduction

Chinese medicine believes that factors such as climate and environment can affect a person's physique, which in turn affects the occurrence and development of diseases. The Lingnan region of China, mainly including Guangdong, Guangxi, and Hainan Island, is located in the tropical monsoon climate region. As a result, people living in this region for a long time are prone to a form of spleen deficiency. In some cases, it spreads to the kidney and liver, leading to the prevalence of osteoporosis¹. The spleen and stomach function in transporting and absorbing water and grains, which are the source of qi and blood biochemistry, while the liver helps maintain the marrows. So, bone nutrition relies on the supply of the spleen, stomach, and liver. In Chinese medicine, the kidney is capable of generating marrow and strengthening bones, and its vitality directly affects the strength and function of bones. Therefore, kidney deficiency is directly related to osteoporosis. According to TCM, liver (Gan)-kidney (Shen) deficiency and lack of essence (Jing) are the underlying causes. In some cases, malnutrition may occur due to a spleen (Pi) and stomach (Wei) deficiency or an imbalance of the two. That is, the deficiency of the liver (Gan), kidneys (Shen), spleen (Pi), and stomach (Wei) are the root causes, while external qi (Xie qi) blocks meridians and damages bones and muscles. According to the World Health Organization (WHO), OP is a medical condition characterized by low bone mass and tissue deterioration², which is a serious concern for the elderly. The selection of appropriate interventions is crucial for the prevention and treatment of osteoporosis.

Modern acupuncturists have developed a unique Lingnan characteristic of fire-needling therapy by combining the climate of Lingnan and the physical characteristics of Lingnan people³. Fire needle therapy is a kind of treatment method in which special needles are burned red and quickly pierced into specific parts or acupoints of the human body to treat various ailments. This method is widely used in skin diseases, musculoskeletal and joint diseases, neurological diseases, and other systemic diseases and shows good efficacy⁴. The treatment of osteoporosis with fire needles has been used in clinics for a long time, and it has a better curative effect than simple acupuncture or oral administration of traditional Chinese medicine^5,6,7. The Lingnan fire-needle therapy integrates acupuncture, three-edged needles, and scarring moxibustion. It can improve the qi and blood circulation in the meridians while warming the kidney. This treatment can address both blood stasis and kidney (Shen) deficiency, which contribute to the pathogenesis of OP in TCM. What's more, it has the advantages of being low cost, having quick effects, a short treatment period, and being safe. However, there is a lack of research on its mechanism. This paper introduces a specific procedure of Lingnan Fire-Needle in osteoporosis model rats and provides a methodological basis for conducting related basic research.

Protocol

All experiments were carried out following the National Institutes of Health - Guide for the Care and Use of Laboratory Animals and were approved by the Hubei University of TCM - Institutional Animal Care and Use Committee. Adult female Sprague-Dawley (SD) rats, weighing 220 ± 20 g, were used for the test. All rats were maintained on a 12 h light/dark cycle at 23 ± 1 °C and 40%-50% humidity, with free access to food and water.

1. Pre-operative preparation

1. Group design
   1. After genotyping 30, 3-month-old female rats, randomly divide them into three groups (n = 10 in each group): the normal group, the model group, and the fire-needle group. Treat the rats in the fire-needle group with fire-needle intervention 1x a day for 6 sessions, with 1 day of rest between sessions.
      NOTE: The normal group and the model group suspended the rat bound with a secure coat according to the same frequency and the same duration as the fire-needle group, but without the fire-needle treatment.
2. Preparation of rat osteoporosis model
   1. For the rats in the model group and the fire-needle group, surgically remove their ovaries to establish an osteoporosis model as described in^8,9,10.
      1. For ovariectomy, administer anesthesia by intraperitoneal injection of 2% pentobarbital sodium (0.2 mL/100 g) before the operation, and consider anesthetization successful when animals are unresponsive in a surgical plane of anesthesia based on lack of response from a toe pinch test. Use vet ointment on eyes to prevent dryness while under anesthesia. Inject buprenorphine (0.05 mg/kg) subcutaneously as an analgesic before the surgery. Maintain aseptic conditions during the operation.
         NOTE: If the surgery has not been completed and the rat shows signs of resuscitation, intraperitoneal injection of 2% pentobarbital sodium solution may be supplemented temporarily and appropriately, with each supplemental dose not exceeding 20% of the total amount injected.
      2. After surgery, place the rats on a small animal heating pad and set the temperature to 32 °C. Do not leave rats unattended until they regain consciousness and can crawl freely. Do not return them to the company of other animals until they have fully recovered.
      3. When the rats are revived, check the sutures for integrity and place the rats back in the cage. Inject all rats intramuscularly with penicillin sodium (50,000 U per day, for 3 days) and inject intraperitoneally with 0.1 mL 0.05% diclofenac sodium solution (per day, for 3 days) to prevent infection and accelerate healing^9,10.
   2. Observe the estrous cycle according to the vaginal exfoliated cell smear of female rats as described in¹¹. If the cycle is disordered, the model is established successfully¹¹.
      NOTE: It takes approximately 8-12 weeks for rats to develop osteoporosis after ovariectomy.
3. Selection of needle tools
   1. Purchase fire needles from medical device manufacturers. Ensure that needle tools are made of tungsten-manganese alloy, which is temperature resistant, is non-malleable, not easy to deform and gets uniformly heated. The fire needle is shaped like a filament needle. Ensure the needle is thick, the tip is sharp and straight, and the handle is wrapped with copper wire, which is heat-insulating and does not burn hands.
   2. Choose a single-head fire needle with a size of 0.40 mm x 13 mm because rats are small. Check whether the fire needle has barbs and cracks.
      NOTE: In clinical practice, most of the fire needles used are those developed by He Puren, a national medical master¹². Based on this, Lin Guohua created the Lingnan Long-Headed Fire Needle and Lingnan Three-Headed Fire Needle¹³. Choose different sizes of fire needles based on the need of the experiment.
4. Selection and positioning of acupoints
   1. According to the relevant data and literature, select acupoints for the treatment of postmenopausal osteoporosis.
      NOTE: The most frequently used acupoints are, in order of frequency, Shenshu (BL 23), Zusanli (ST 36), Pishu (BL 20), Xuanzhong (GB 39), and Dazhu (BL 11)^14,15. In basic research on acupuncture intervention for osteoporosis in de-ovulatory model rats, most used acupuncture points are Shenshu (BL 23), Sanyinjiao (SP 6), Guanyuan (CV 4) and Zusanli (ST 36)^16,17,18.
   2. Determine the acupoints selected for this experiment. Referring to the acupuncture points selected in previous studies^{14,15,16,17,18} and the pathogenesis of osteoporosis, select four acupuncture points, namely, Shenshu (BL 23), Pishu (BL 20), Zusanli (ST 36) and Sanyinjiao (SP 6), for this study. These correspond to the bladder meridian, the Du meridian, the spleen meridian, and the kidney meridian.
      NOTE: It is forbidden to use fire needles on parts with large vessels and nerve trunks.
   3. Shave the acupuncture site with a pet electric clipper before positioning to expose the acupuncture point.
   4. Location of acupoints refers to names and location of commonly used acupoints in¹⁹. Locate the acupoints as follows: the Shenshu (BL 23) is located on the lower two sides of the second lumbar vertebra, 6 mm from the mid-dorsal line; the Pishu (BL 20) is located between the ribs on the lower two sides of the 12^th thoracic vertebra, 6 mm from the mid-dorsal line; the Zusanli (ST 36) is located on the posterior and lateral side of the knee, about 3 mm below the small head of the fibula; and Sanyinjiao (SP 6) is located about 10 mm straight up from the tip of the inner ankle of the hind limb.
   5. Mark them with an oil-based marker after locating the points.
5. Rat restraint method
   NOTE: In order to reduce stress during immobilization and treatment of rats, the suspended rats bound with a secure coat^20,21 was chosen as the immobilization method. The rat coat suspension method has the following advantages: Firstly, it makes full use of the rat's habit of preferring a dark environment and reduces the rat's stress reaction during fixation. Secondly, the suspended rats are quieter and more cooperative during the whole process of fire-needling treatment due to the lack of force points on their limbs and toes. Thirdly, the acupoints on the back and limbs of the rats were sufficiently exposed to facilitate the treatment. A single treatment requires restraining the rat for 5-10 min. When secure in the coat, rats do not struggle in the restraint and are calm.
   1. Prepare the rat coat. Sew the cleaned fabric and make it into a 50 cm x 13 cm tube shaped rat coat. Cut holes for each of the four limbs and back of the rat coat and sew ties at the four corners.
   2. Put on gloves, place the rat on a table that is not slippery, hold the rat's tail with the left hand and gently pull back. Open the port of the rat coat with the right hand. Using the rat's habit of darkness and burrowing, place it at the mouth of the rat coat, the rat would burrow into it on its own.
   3. When the rat burrows into the middle of the rat coat, use the left hand to hold both sides of the rat's shoulder blades, and pull out the rat's two forelimbs with the right hand.
   4. Tighten the ropes on the front of the coat, cross the ropes and wrap them around the rat's back. Adjust the elasticity of the ropes and tie the knot on the back of the rat. Finally, fix the back end of the rat body in a similar way so that its bilateral hind limbs are located outside the rat coat.
   5. For the swing type suspension, take two S-hooks and hook them into the knot on the back of the rat coat and hook the hooks onto the iron frame. At this point, the rat is suspended from the strip of cloth, in a slightly swinging state (Figure 1).
      NOTE: The height of the strip of cloth from the ground is about 70 cm, for the experimenter sitting on a stool to operate comfortably and effortlessly. Place a thick padding to the flat surface below the rat as a precaution if animal slips.

2. Acupuncture treatment

1. Disinfection
   1. Sterilize the local area with 5% iodophor disinfecting solution 3x before acupuncturing. Add alcohol rub after each iodophor scrub.
2. Burning needles
   NOTE: Safety precautions should be taken to prevent burns and injuries.
   1. Take the hemostatic forceps to pick up the cotton ball, soaked with 95% alcohol, with the left hand and light it with a lighter.
   2. Hold the handle of the needle with the thumb, forefinger, and middle finger of the right hand in a slightly curved position, as if holding a pen.
   3. Put the needle tip and needle body on the alcohol cotton balls and heat it; first heat the needle body, and then heat the needle tip. Burn the needle until it is bright white or red, depending on the purpose of treatment (Figure 2).
      NOTE: The needle body should be fully burned bright white or red, otherwise it will affect the efficacy of acupuncture or cause postoperative infection and other issues.
3. Acupuncture
   NOTE: According to the speed, depth, range, needle retention time and treatment purpose, Lingnan Fire-Needle can be divided into various operating techniques such as point acupuncture, scatter acupuncture, dense acupuncture, fast acupuncture, and slow acupuncture²². The key to operating with Lingnan Fire-Needle is red, accurate, and fast.
   1. Wait until the needle burns red, move the needle away from the flame and hold it above the acupoint.
   2. Point the needle tip vertically at the acupoint, quickly puncture and pull out. Ensure that the whole process is completed within 1 s.
   3. The depth of acupoints penetration is about 1-2 mm, only pierce the red-hot needle tip.
      NOTE: The depth of acupuncture should be determined according to the condition, constitution, age, muscle thickness of the acupoint, the depth of blood vessels, and the distribution of nerves.
   4. Puncture each acupoint 3x-5x. Repeat this for all acupoints (Figure 3).
      NOTE: When performing the operation, be fully focused and agile. In the same procedure on a rat, the same fire needle should be used, with the needle being re-sterilized by flaming between every two punctures; the needle should not be placed down or become unsterile.

3. Post-acupuncture management

1. Lift the fire needle away from the skin and hold a sterile dry cotton ball to press the needle hole to prevent bleeding. Following the fire-needle therapy, the rats displayed unimpaired mobility and no detectable complications.
   NOTE: If bleeding after fire needling occurs, there is no need to stop the bleeding, just wipe with a dry cotton ball after it stops naturally.

4. Data collection and analysis

1. After 6 courses of fire-needling intervention, euthanize the rats by high dose intraperitoneal injection of pentobarbital sodium (150-200 mg/kg). Collect femur specimens and perform micro-CT scans²³.
2. Analyze the data statistically, using analysis software. Show measurements as mean and analyze using one-way ANOVA, with p <0.05 being considered as statistically significant.

Results

The data in Table 1 shows that compared with the normal group, the thickness of trabecular bone (Tb.Th), the number of trabecular bone (Tb.N) and the static parameter bone volume fraction of trabecular bone (BV/TV) in the model group were all significantly reduced (p <0.05), and the separation of trabecular bone (Tb.Sp) was elevated (p <0.05); indicating that the bone structure of trabecular bone in trabecular bone-enriched areas of the femoral epiphysis of the rat was significantly disrupted aft...

Discussion

Lingnan fire-needle therapy has a profound historical background. Records on fire needles and needling methods can be traced back to Huangdi Neijing. Modern acupuncturists Situ Bell and Zhang Jiawei combined Lingnan's climate and the physical characteristics of its people to create Lingnan fire-needle therapy³. Lin Guohua summarized Lingnan Fire-Needle therapy, as warming, dredging, tonifying, clearing, and eliminating, with warming as the essence²⁴.

Materials

Name	Company	Catalog Number	Comments
75% Alcohol	Shandong Lircon medicaltechnology co., ltd		Disinfection
95% Alcohol	Shandong Lircon medicaltechnology co., ltd		Burn needles
Adult female Sprague-Dawley (SD) rats	Liaoning Changsheng biotechnology co., Ltd.	SCXK 2020-0001	Modeling of osteoporosis
Deluxe thermostat heat mat	Zhenhua Instruments		For maintaining the body temperature of rats.
Diclofenac sodium injection	Shandong Province Jianmu Bio-pharmaceutical Co.	150182913	Antipyretic and analgesic
Elevated fixation rack	Taizhou Shi Ai Scientific and Educational Instruments and Equipment Co.		Fix rats
Fire needles	Beijing Luoya Shanchuan Medical Equipment Co., Ltd.	20190240	A tool of needling
Hemostatic forceps	Qingdao Schultz Biotechnology Co.		Fix rats
Liguter	Shanghai Machi Trading Co.	H5	Ignite alcohol
Lodophor disinfecting solution	Qingdao Hai's Hainuo Inovation Disinfection Technology Co., Ltd.	2016-0604	Disinfection
Medical cotton ball	Jiangsu Misawa Medical Supplies Co., Ltd.	20172142357	Disinfection
pentobarbital sodium	Beijing Jiehui Biotechnology Co.	P3761	anesthesia
Pet electric clipper	Fenghua Xikou Lubao Knives & Scissors Electrical Appliance Factory		Shave the hair of rats
Self-made rat coat			Fix rats
Tweezers	Qingdao Schultz Biotechnology Co.		Pinch cotton balls