This work was supported with funds from the National Natural Science Foundation of China (NSFC) Nos. 82374564 (to YJD) and Nos. 82074566 (to YJD) and from Sun Guojie Inheritance Base for TCM Acupuncture-Moxibustion of World Federation of Acupuncture-Moxibustion Societies in Wuhan, China (World Federation of Acupuncture-Moxibustion Societies [2019] No. 26) to YJD and the National Famous TCM Inheritance Studio construction project of the National Administration of Traditional Chinese Medicine, China (the National Administration of Traditional Chinese Medicine [2022] No. 5) to YJD.

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 &#177; 20 g, were used for the test. All rats were maintained on a 12 h light/dark cycle at 23 &#177; 1 &#176;C and 40%-50% humidity, with free access to food and water.
1. Pre-operative preparation
<ol>
	<li>Group design
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of rat osteoporosis model
	<ol>
		<li>For the rats in the model group and the fire-needle group, surgically remove their ovaries to establish an osteoporosis model as described in8,9,10.
		<ol>
			<li>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.</li>
			<li>After surgery, place the rats on a small animal heating pad and set the temperature to 32 &#176;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.</li>
			<li>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 healing9,10.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Selection of needle tools
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Selection and positioning of acupoints
	<ol>
		<li>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.</li>
		<li>Determine the acupoints selected for this experiment. Referring to the acupuncture points selected in previous studies14,15,16,17,18 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.</li>
		<li>Shave the acupuncture site with a pet electric clipper before positioning to expose the acupuncture point.</li>
		<li>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 12th 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.</li>
		<li>Mark them with an oil-based marker after locating the points.</li>
	</ol>
	</li>
	<li>Rat restraint method 
	NOTE: In order to reduce stress during immobilization and treatment of rats, the suspended rats bound with a secure coat20,21 was chosen as the immobilization method. The rat coat suspension method has the following advantages: Firstly, it makes full use of the rat&#39;s habit of preferring a dark environment and reduces the rat&#39;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.
	<ol>
		<li>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.</li>
		<li>Put on gloves, place the rat on a table that is not slippery, hold the rat&#39;s tail with the left hand and gently pull back. Open the port of the rat coat with the right hand. Using the rat&#39;s habit of darkness and burrowing, place it at the mouth of the rat coat, the rat would burrow into it on its own.</li>
		<li>When the rat burrows into the middle of the rat coat, use the left hand to hold both sides of the rat&#39;s shoulder blades, and pull out the rat&#39;s two forelimbs with the right hand.</li>
		<li>Tighten the ropes on the front of the coat, cross the ropes and wrap them around the rat&#39;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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Acupuncture treatment
<ol>
	<li>Disinfection
	<ol>
		<li>Sterilize the local area with 5% iodophor disinfecting solution 3x before acupuncturing. Add alcohol rub after each iodophor scrub.</li>
	</ol>
	</li>
	<li>Burning needles 
	NOTE: Safety precautions should be taken to prevent burns and injuries.
	<ol>
		<li>Take the hemostatic forceps to pick up the cotton ball, soaked with 95% alcohol, with the left hand and light it with a lighter.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Wait until the needle burns red, move the needle away from the flame and hold it above the acupoint.</li>
		<li>Point the needle tip vertically at the acupoint, quickly puncture and pull out. Ensure that the whole process is completed within 1 s.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Post-acupuncture management
<ol>
	<li>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.</li>
</ol>
4. Data collection and analysis
<ol>
	<li>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.</li>
	<li>Analyze the data statistically, using analysis software. Show measurements as mean and analyze using one-way ANOVA, with p &#60;0.05 being considered as statistically significant.</li>
</ol>

Osteoporosis Management in Rats Through Lingnan Fire-Needle Therapy

The authors declare that they have nothing to disclose.

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&#39;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.
The first step to using fire needles is selecting the appropriate type of needle. Fire needles can be categorized into three main categories: single-head, multi-head, and flat-head. The single-head fire needle is widely used in clinical practice and is commonly used for multi-system diseases. The multi-head fire needle combines three fine or three medium-coarse fire needles in a triangular shape, mainly used to treat skin diseases. The flat-head fire needle is commonly used to remove various kinds of moles, warts, and other excessive organisms on the skin25,26. The study selected single-head needles because they have a broader range of applications and more features, which makes it easier to obtain fine needles suitable for the size of rats.
Secondly, the proper use of fire-needle therapy requires critical criteria for cauterizing needles. Researchers27 have found that cauterizing needles to a specific temperature is necessary for their effectiveness. The stimulation may not alleviate the relevant pathological changes if the temperature is not high enough. Therefore, to achieve the desired therapeutic effect, the needles used in fire-needle therapy must be burned to a reddish or bright white color and then quickly inserted into the selected acupoints or parts. Furthermore, as the criteria for burning needles with fire needles vary depending on the fire-needling instruments, needling sites, and the diseases treated, we have compiled a comprehensive set of criteria for burning needles with fire needles that can be referenced in Table 2. In the present study, we used a single-head fire needle for multipoint stimulation instead of a multi-head fire needle. This was because multiple needles being too close together could not ensure that each needle would be cauterized uniformly.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Needle tools</td>
			<td>Degree of needle burning</td>
			<td>Indications</td>
		</tr>
		<tr>
			<td>Single-head Fire Needle</td>
			<td>Reddish-red.</td>
			<td>Bone and Joint Diseases and painful disease</td>
		</tr>
		<tr>
			<td>Multi-head Fire needle</td>
			<td>The tip of the needle is slightly hot.</td>
			<td>Skin diseases such as Acne</td>
		</tr>
		<tr>
			<td>Flat-head Fire Needle</td>
			<td>Bright white. The handle of the needle is slightly hot.</td>
			<td>Neoplasm such as filiform warts</td>
		</tr>
	</tbody>
</table>
Table 2: The criteria for burning needles.
Regarding the angle of fire needle insertion, it is typically vertical. As for how deep to insert the needle, there are no established standards yet. However, it is generally suggested that deeper insertion is better for areas with more muscle and thicker skin, whereas shallower insertion is more appropriate for areas with thinner skin and muscles. Additionally, many experts in the field believe that the depth of fire needling should be like that of filiform needle needling28. A study suggests the depth of fire needles should be shallower than filiform needles when needling acupoints and slightly deeper than filiform needles when needling pain points22. As a result, regarding the actual application of fire needles, referring to the standard for filiform needles first and then determining the depth of needling based on clinical experience is advisable.
Based on the collective opinion of 192 experts in Lingnan, the primary cause of OP in high-risk groups is kidney (Shen) deficiency, which leads to secondary blood stasis. Therefore, the treatment for OP involves addressing both the kidney (Shen) deficiency and blood stasis simultaneously29. Currently, acupuncture is a widely used treatment for OP. The different types of acupuncture treatments include filiform needle therapy, moxibustion, warm acupuncture, electro-acupuncture (EA), thread-embedding therapy, acupoint injection, a combination of acupuncture and medicine, and a combination of acupuncture and moxibustion. Among these, the most used treatments in clinical practice are thread-embedding therapy, moxibustion, and a combination of acupuncture and medicine30. Clinically, fire-needle therapy is widely used for 137 different diseases targeting several organ systems, such as the motor system, nervous system, endocrine system, digestive system, obstetrics, and gynecology, among which the most applied are motor diseases31. The Lingnan fire-needle therapy is a treatment that combines the benefits of acupuncture, three-edged needles, and scarring moxibustion. It can improve qi and blood circulation in the meridians and warm the kidney (Shen). This treatment can address both blood stasis and kidney (Shen) deficiency, which contribute to the pathogenesis of OP in TCM. In addition, it has the advantages of being low cost, having quick effects, a short treatment period, and being safe. Clinical trials also show that combining fire-needle therapy and acupuncture treatment is more effective in treating OP than simple acupuncture or mediciation5,6. Joint pain is one of the typical symptoms of OP, and it has been confirmed that the efficacy of fire-needle therapy is significantly better than filiform needle therapy in the treatment of joint pain32.
Bone strength depends on bone density, the amount of bone per unit volume. The number and distribution of bone trabeculae, a component of bone mass, significantly influences bone volume and, consequently, bone density. Bone volume fraction indicates the percentage of bone volume per unit of tissue volume and is an essential reference for bone mass23. As part of the skeletal system, the cancellous bone&#39;s primary function is supporting and facilitating movement. However, increasing its size can reduce bone density, decreasing bone strength. The skeletal structure of cancellous bone is spongy and composed of trabeculae. Therefore, the structure of the cancellous bone is determined by the trabeculae. To maintain the strength of cancellous bone, it is crucial to ensure sufficient trabeculae are present as a framework. Secondly, it is necessary to keep a relatively adequate number of connections between the trabeculae to ensure the structural stability of bone33. For the trabeculae to be able to carry a certain amount of load, their thickness must be maintained. The structure of cancellous bone is evaluated based on the number of trabeculae present, their connectivity, and their thickness34. After comparing the structural parameters of cancellous bone (Table 1), our findings revealed significant differences between the model group of rats and the normal group. The number of trabeculae, trabecular thickness, and bone volume fraction of the femur were reduced, while trabecular separation was elevated in the model group. These findings suggest that the cancellous bone structure changes the model group, possibly leading to osteoporosis or other bone diseases. Based on the three-dimensional structural image of the cancellous bone, it was observed that the middle part lacked trabeculae, and more than half of the trabeculae in the upper part of the periphery were also missing. This indicates that the bone structure integrity of the femur in rats after ovariectomy had been compromised. Compared to the model group, the femur in the fire-needle group showed increased trabeculae, trabecular thickness, bone volume fraction, and decreased trabecular separation. Additionally, when the three-dimensional structural diagram of the cancellous bone was considered, it was observed that the central cavity of the fire-needle group was reduced, and the peripheral bone trabeculae were complete (Figure 4). This study demonstrates that fire-needle therapy effectively prevents and controls damage to the cancellous bone structure of the femur bone in rats that have undergone ovariectomy.
The limitations of the ovariectomy model rats selected for this study are as follows. Estrogen reduction is one of the risk factors for primary OP35. For this reason, ovariectomy surgery is a dependable method for establishing OP rat models8,9,10. Additionally, SD rats are not only easily accessible but also docile, with relaxed feeding conditions. Consequently, we initially examined this model and recommended using fine fire-needles to stimulate acupoints. Our long-term plan is to expand our research scope by further validating the effectiveness of fire-needling therapy in alleviating OP in other models, such as aging rats. Finally, Table 3 summarizes the efficacy of acupuncture alone on osteoporosis model rats and its mechanism of action. We intend to conduct a more in-depth investigation into the possible mechanisms in the coming future36,37,38,39,40,41,42.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Outcome indicator</td>
			<td colspan="4">Mechanism of action</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td colspan="2">bone resorption</td>
			<td colspan="4">Acupuncture prevents bone loss and structural changes by suppressing bone resorption.</td>
			<td>36</td>
		</tr>
		<tr>
			<td colspan="2">bone strength</td>
			<td colspan="4">Acupuncture increased the trabecular bone volume and thickness, lowered the trabecular separation of tibiae and restricted the excretion of phosphorus and Dpd, while promoting the concentrations of serum osteocalcin.</td>
			<td>37</td>
		</tr>
		<tr>
			<td colspan="2">bone metabolism</td>
			<td colspan="4">Acupuncture can significantly downregulate serum ALP, BGP and TRAP levels in OVX rats.</td>
			<td>38</td>
		</tr>
		<tr>
			<td colspan="2">signaling pathway</td>
			<td colspan="4">Acupuncture improves bone mineral density, attenuates in vivo inflammatory response, and increases osteogenesis-influencing factor levels in rats by modulating the Wnt signaling pathway.</td>
			<td>39,40</td>
		</tr>
		<tr>
			<td colspan="2">endocrine-modulating effect</td>
			<td colspan="4">Acupuncture modulated the serum levels of estradiol, corticotrophin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone (CORT).</td>
			<td>41</td>
		</tr>
		<tr>
			<td colspan="2">bodyweight</td>
			<td colspan="4">Acupuncture modulates changes in leptin levels to abrogate the body weight gain.</td>
			<td>36,41</td>
		</tr>
		<tr>
			<td colspan="2">brain-derived neurotrophic factor</td>
			<td colspan="4">acupuncture can elevate BDNF and TrkB expression levels and accelerate the process of fracture healing after osteoporosis.</td>
			<td>42</td>
		</tr>
	</tbody>
</table>
Table 3: The effect of simple acupuncture on the efficacy of osteoporosis model rats.

Chinese medicine believes that factors such as climate and environment can affect a person&#39;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 medicine5,6,7. 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&#39;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.

<table><tbody><tr><td>75% Alcohol</td><td>Shandong Lircon medicaltechnology co., ltd</td><td></td><td>Disinfection</td></tr><tr><td>95% Alcohol</td><td>Shandong Lircon medicaltechnology co., ltd</td><td></td><td>Burn needles</td></tr><tr><td>Adult female Sprague-Dawley (SD) rats</td><td>Liaoning Changsheng biotechnology co., Ltd.</td><td>SCXK 2020-0001</td><td>Modeling of osteoporosis</td></tr><tr><td>Deluxe thermostat heat mat</td><td>Zhenhua Instruments</td><td></td><td>For maintaining the body temperature of rats.</td></tr><tr><td>Diclofenac sodium injection</td><td>Shandong Province Jianmu Bio-pharmaceutical Co.</td><td>150182913</td><td>Antipyretic and analgesic</td></tr><tr><td>Elevated fixation rack</td><td>Taizhou Shi Ai Scientific and Educational Instruments and Equipment Co.</td><td></td><td>Fix rats</td></tr><tr><td>Fire needles</td><td>Beijing Luoya Shanchuan Medical Equipment Co., Ltd.</td><td>20190240</td><td>A tool of needling</td></tr><tr><td>Hemostatic forceps</td><td>Qingdao Schultz Biotechnology Co.</td><td></td><td>Fix rats</td></tr><tr><td>Liguter</td><td>Shanghai Machi Trading Co.</td><td>H5</td><td>Ignite alcohol</td></tr><tr><td>Lodophor disinfecting solution</td><td>Qingdao Hai&#039;s Hainuo Inovation Disinfection Technology Co., Ltd.</td><td>2016-0604</td><td>Disinfection</td></tr><tr><td>Medical cotton ball</td><td>Jiangsu Misawa Medical Supplies Co., Ltd.</td><td>20172142357</td><td>Disinfection</td></tr><tr><td>pentobarbital sodium</td><td>Beijing Jiehui Biotechnology Co.</td><td>P3761</td><td>anesthesia</td></tr><tr><td>Pet electric clipper</td><td>Fenghua Xikou Lubao Knives &amp;amp; Scissors Electrical Appliance Factory</td><td></td><td>Shave the hair of rats</td></tr><tr><td>Self-made rat coat</td><td></td><td></td><td>Fix rats</td></tr><tr><td>Tweezers</td><td>Qingdao Schultz Biotechnology Co.</td><td></td><td>Pinch cotton balls</td></tr></tbody></table>

operation procedure and precautions of lingnan fire-needle therapy in osteoporosis model rats

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.

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 &#60;0.05), and the separation of trabecular bone (Tb.Sp) was elevated (p &#60;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 after de-osteoporosis, indicating obvious damage.
Compared with the model group, the trabecular thickness, trabecular number, and trabecular static parameter bone volume fraction of the femur in the fire-acupuncture group were significantly increased (p &#60;0.05), and trabecular separation was decreased (p &#60;0.05). This suggested that fire needle therapy can improve the damage to cancellous bone structure of femur in osteoporosis model rats induced by bone removal.
Figure 4 shows the three-dimensional structure images of rat bone cancellous matrix in each group. Figure 4A is the normal group, Figure 4B is the model group and Figure 4C is the fire-needle group. As can be seen from the figure, compared with the normal group, a large number of trabeculae were lost in the center and even the periphery of the femur cancellous matrix in the model group, and a large number of cavities appeared in the lower part. The overall continuity of the bone structure was damaged, with the appearance of part of the free bone trabeculae. Compared with the model group, there was a large increase in thickness of trabeculae in the center and periphery of the cancellous bone of the femur in the fire-needle group, and the continuity of the overall bone structure was improved. This suggests that fire-needles can protect the trabecular structure of the cancellous bone of the femur in the rat model of ovariectomy-induced osteoporosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65970/65970fig01v2.jpg" /> 
Figure 1: Rat fixation method: swing rats bound with coat.&#160;After dressing the rat in&#160; a rat&#160;coat, the rat is secured and suspended&#160;in the air by hooking the rat coat with&#160;two S-hooks.&#160;<a href="https://www.jove.com/files/ftp_upload/65970/65970fig01largev2.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/65970/65970fig02.jpg" /> 
Figure 2: Lingnan Fire-Needle. (A) Needle tools. Choose a fine fire needle with a size of 0.40 mm x 13 mm. (B) Burning needles. Put the needle tip and needle body on the alcohol-soaked cotton balls and heat it. <a href="https://www.jove.com/files/ftp_upload/65970/65970fig02large.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/65970/65970fig03.jpg" /> 
Figure 3: Fire needle acupuncture procedure at four acupoints. (A) Pishu (BL 20): Located between the ribs on the lower two sides of the 12th thoracic vertebra, 6 mm from the mid-dorsal line (B) Shenshu (BL 23): Located on the lower two sides of the second lumbar vertebra, 6 mm from the mid-dorsal line (C) Zusanli (ST 36): Located on the posterior and lateral side of the knee, about 3 mm below the small head of the fibula. (D) Sanyinjiao (SP 6): Located about 10 mm straight up from the tip of the inner ankle of the hind limb. <a href="https://www.jove.com/files/ftp_upload/65970/65970fig03large.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/65970/65970fig04.jpg" /> 
Figure 4: Comparison of the three-dimensional structure of the cancellous bone of the distal femur of rats in various groups. (A) The normal group. (B) The model group. (C) The fire-needle group. Scale bars: 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65970/65970fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>Tb.N(mm)</td>
			<td>Tb.Sp(mm)</td>
			<td>Tb.Th(mm)</td>
			<td>BV/TV</td>
		</tr>
		<tr>
			<td>normal group</td>
			<td>3.29&#177;0.47</td>
			<td>0.23&#177;0.06</td>
			<td>0.12&#177;0.03</td>
			<td>0.39&#177;0.14</td>
		</tr>
		<tr>
			<td>model group</td>
			<td>0.96&#177;0.78*</td>
			<td>0.66&#177;0.44*</td>
			<td>0.09&#177;0.01*</td>
			<td>0.17&#177;0.11*</td>
		</tr>
		<tr>
			<td>fire-needle group</td>
			<td>2.73&#177;0.76&#916;</td>
			<td>0.33&#177;0.20&#916;</td>
			<td>0.12&#177;0.02&#916;</td>
			<td>0.33&#177;0.16&#916;</td>
		</tr>
	</tbody>
</table>
Table 1: Fire needling effect on microstructural indices of femoral osteoporosis. Data shows mean &#177; SD, n= 10. For comparison performed with normal group, *p&#60;0.05; For comparison performed with model group, &#916;p&#60;0.05.

Read this Scientific Article Protocol about Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention at JoVE.com

Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention

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

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

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected ...

operation-procedure-precautions-lingnan-fire-needle-therapy

Nanyang Technological University

Research

JoVE Journal

Medicine

517 Views.  Hubei University of Chinese Medicine. Here, we present a protocol for the application of Lingnan Fire-Needle therapy in the intervention of osteoporosis model rats.

Video: Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Bioengineering

Improving Cognitive Functions in Rats via Thread-Embedding Acupuncture Therapy

Operation Procedure and Precautions of Thread-Embedding Acupuncture Therapy in Alzheimer

Thread-embedding therapy (TEAT) is a treatment that prevents and manages diseases by inserting a biodegradable suture into an acupoint, providing long-lasting stimulation. TEAT is a simple approach that avoids the discomfort of regular acupuncture and provides sustained therapeutic effects. This article discusses the potential impact of TEAT on the learning and memory abilities of rats with Alzheimer&#39;s disease-like symptoms. Since chemically induced neuronal degeneration and cognitive impairments in rats does not entirely reflect the true pathological changes observed in Alzheimer&#39;s disease. Consequently, our research group has designated these manifestations as Alzheimer&#39;s disease-like symptoms. A protocol has been established to outline the selection of acupoints, the operation process, and necessary precautions for the head and lower back. The experiment was conducted on three groups: a control group, a model group, and a TEAT group, each containing 6 rats. To induce Alzheimer&#39;s disease-like symptoms, rats were intraperitoneally injected with D-galactose for 7 weeks (49 days). The rats in the TEAT group received acupoint catgut embedding treatment. Following the intervention period, a Morris Water Maze (MWM) was conducted to evaluate the rats&#39; learning and memory. Subsequently, the rats were sacrificed, and their brain tissue was examined. A histological examination was performed to understand the effects of TEAT on the pathology of rats exhibiting symptoms of Alzheimer&#39;s disease. This study suggests that TEAT may improve learning and memory in rats with Alzheimer&#39;s disease-like symptoms, indicating a potentially promising new treatment approach for this neurodegenerative condition.

Author Spotlight: Exploring Acupuncture in Alzheimer's Research from Thread-Embedding Techniques to Clinical Trials

This study presents a protocol for thread-embedding acupuncture therapy in Alzheimer's disease-like rats.

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Neuroscience

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.

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Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Tuina Therapy for Frozen Shoulders in a Rat Model

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

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

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

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

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

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...

Elastic Tape Rat Model of Steroid-Induced Femoral Head Osteonecrosis Enhancement

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

Author Spotlight: Developing a Rat Model for Weight-Bearing Intervention to Investigate Osteonecrosis of the Femoral Head

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure ...

The Creation of a Rat Model for Osteosarcopenia via Ovariectomy

Osteosarcopenia (OS), a complex degenerative disorder, is characterized by the concurrent decline in skeletal muscle mass and bone mineral density (BMD), posing an enormous health hazard for the elderly population. Despite its clinical relevance, the pathophysiological mechanisms underlying OS are not fully understood, underscoring the necessity for a deeper comprehension of its etiology to facilitate effective treatment strategies. The development of a reliable animal model is pivotal in this endeavor. This study presents a refined protocol for the induction of postmenopausal osteosarcopenia in rats through bilateral ovariectomy, a method known to accelerate the onset of age-related muscle and bone loss. In this study, rats aged 12 weeks were stratified by body weight and randomly assigned to either a sham operation group or an ovariectomized (OVX) group. Tissue samples from the quadriceps and triceps muscles of the left hind limb, as well as the left femur, were systematically collected at 4, 8, and 12 weeks post-surgery. This methodical approach ensures a comprehensive evaluation of the effects of ovariectomy on muscle and bone health. Histological evaluation of muscle fiber atrophy and femoral morphology was conducted using hematoxylin and eosin (HE) staining, while bone mineral density was quantified using dual-energy X-ray absorptiometry (DXA). The temporal progression of OS was meticulously monitored at the aforementioned intervals, providing insights into the dynamic interplay between muscle and bone degeneration. This model not only accurately reflects the clinical manifestations of OS but also serves as a robust platform for investigating novel therapeutic approaches and their underlying mechanisms.

This current protocol outlines a procedure for creating a rat model of osteosarcopenia using ovariectomy.

Osteosarcopenia (OS), a complex degenerative disorder, is characterized by the concurrent decline in skeletal muscle mass and bone mineral density (BMD), ...