This work was supported by the General Program of the National Natural Science Foundation of China (No.82174491) and the Qilu School of Traditional Chinese Medicine Academic School Inheritance Project (No. Lu Weihan [2020]132). Thanks to the Laboratory Animal Center of Shandong University of Traditional Chinese Medicine for providing us with experimental conditions.

All animal experiments were approved by the Experimental Animal Welfare Ethics Review Committee of Shandong University of Traditional Chinese Medicine (No. SDUTCM20230831301).
1. Animal preparation
<ol>
	<li>Determine the experimental groups and the number of animals per group in advance.</li>
	<li>Choose male BALb/c mice, house them at a constant temperature of 22 &#177; 1 &#176;C, constant humidity of 60 &#177; 5%, and 12 h diurnal cycle light environment.</li>
</ol>
2. Establishment of the AS mouse model
NOTE: The AS model was induced with cartilage proteoglycan (PG), which is a classic modeling method16,17,18,19,20,21. BALB/c strain mice were repeatedly immunized with PG to induce tendinitis and spondylitis. See the Table of Materials for details related to the materials, reagents, and instruments used in this protocol.
<ol>
	<li>Use a 200 &#181;L pipette to draw 200 &#181;L of PG and CFA (or IFA) into a 1.5 mL centrifuge tube. Shaking gently to mix the liquid in the centrifuge tube.</li>
	<li>Place the centrifuge tube in the tissue grinder for homogenization and then incubate on ice for 4 min. Repeat 5x. Set the following parameters in the tissue grinder: 60 Hz, grinding 30 s, interval 10 s for a total of 6 cycles.</li>
	<li>Extract 0.2 mL of emulsified PG protein from the centrifuge tube with a 1 mL syringe.</li>
	<li>Grab and fix the mouse with the left hand. Lay the mouse in a supine position, keeping the head lower than the tail to prevent damage to the abdominal organs when the syringe is inserted.</li>
	<li>Sterilize the abdomen with alcohol swabs using the right hand.</li>
	<li>With the syringe in the right hand, insert the needle subcutaneously slightly to the left or right of the linea alba ventralis in the lower half of the abdomen.</li>
	<li>Push the needle 3-5 mm subcutaneously parallel to the midabdominal line and then push the needle into the abdominal cavity at a 45&#176; angle to the skin.</li>
	<li>Penetrate the peritoneum, withdraw the needle plug, and slowly inject the emulsified PG protein.</li>
</ol>
3. Du-moxibustion
NOTE: Prepare in advance the three most important raw materials for Du-moxibustion (Figure 1).
<ol>
	<li>Prepare moxa cones. Take the velvet in the hand, with two palms facing opposite directions exerting a twisting force to rub the velvet into a spindle shape (Figure 2)</li>
	<li>Cut the ginger into small pieces and puree it in a juicer (Figure 3). Filter the ginger puree through cotton gauze. Separate the ginger juice and ginger mud from the ginger puree to prepare for the next step.</li>
	<li>Prepare 10 g of ginger mud and 20 mL of ginger juice and place them in paper cups for later use.</li>
	<li>Weigh 0.1 g of TCM powder (a mixture of cinnamon powder, clove powder, notopterygium powder, antharidin powder, and artificial musk)22 and grind it in a mortar.</li>
	<li>Place the mice in the anesthesia induction box of the animal anesthesia machine and anesthetize them&#160;with a mixture of 2-3% isoflurane and oxygen at a flow rate of 2 L/min.</li>
	<li>Assess the depth of anesthesia by observing the absence of a startle reflex initially and verify the level of surgical anesthesia by checking for the absence of a pedal reflex in response to a gentle toe pinch.</li>
	<li>After the mouse has reached surgical plane anesthesia, take it out of the box and fix it on the operating table in a prone position. Align the mouse&#39;s mouth and nose with the outlet of the anesthetic.</li>
	<li>Administer anesthesia via a mask attached to the anesthetic machine, through which isoflurane and oxygen are delivered via a non-rebreathing circuit. Maintain the anesthesia and keep the mouse on a 37 &#176;C heating pad during the entire operational procedure. Apply lubricant to the eyes to prevent desiccation.</li>
	<li>Locate the acupoints and identify the stimulation area (Figure 4).</li>
	<li>Shave the hair on the back of the mouse until the flesh-colored skin on the back of the mouse is seen. Ensure the width of hair removal is ~1.5 cm and the length is from the seventh cervical vertebra to the tail vertebra (Figure 5A).</li>
	<li>Apply the prepared ginger juice with a cotton gauze to the shaved area (Figure 3).</li>
	<li>Sprinkle the TCM powder evenly with a writing brush on the median line of the back of the mouse and cover it with mulberry paper (Figure 5B-D).</li>
	<li>Make a trapezoidal ginger column 6 cm long, 1.5 cm wide, and 3 mm thick with ginger mud. Fix the ginger column on the mulberry paper (Figure 5E).</li>
	<li>Make a groove above the midline of the ginger column (Figure 3), and place the moxa cone on the ginger column.</li>
	<li>Light the moxa cone, placing a new moxa cone after the previous one burns out. Check the firmness of the ginger column to prevent burning the mouse, burning a total of three cones&#160;(Figure 5F).</li>
	<li>After the burning of the moxa cones, wipe off the ginger and the TCM powder, and clean the back of the mouse. Discontinue the anesthesia at this point.</li>
	<li>Allow the mouse to regain consciousness in a new cage with unrestricted access to food and water. Place the cage on a heated pad to aid in the recovery process. Monitor the animal until it regains sufficient consciousness.</li>
	<li>Once the mouse has fully recovered, confirm the presence of the righting reflex before returning it to its regular housing. 
	&#8203;NOTE: During the operation, it is necessary to monitor the mental state of the mice to avoid weak breathing. Prevent scalding, but keep the mice warm, and avoid bruising of the claws and tail.</li>
</ol>
4. Evaluation of arthritis index (AI)
<ol>
	<li>Evaluate the AI score three times: before modeling, after modeling, and after treatment. The assessment standards are as follows: 0, no redness or swelling; 1, slight redness or swelling in a few toes; 2, redness and swelling in most toe joints and toes; 3, serious redness and swelling in feet and below ankle joint; and 4, redness and swelling in feet and ankle joint23.</li>
	<li>Calculate the AI scores of all four paws, with a maximum value of 16.</li>
</ol>
5. Rotarod test
NOTE: The rotarod test is used to assess motor coordination and balance by recording the time the mice spend on the rotating drum. This experiment consisted of three trials separated by 20 min intervals. The official test data were recorded from the third trial, with the first two serving as training exercises.
<ol>
	<li>Place the mice on rotating drums that rotate under continuous acceleration from 4 to 40 rpm over 300 s24.</li>
	<li>Measure the latency time (the time until the mouse falls off the rod). Provide padding to prevent injury when it falls off.</li>
</ol>
6. Open field test (OFT)
NOTE: The OFT is used to evaluate the state of autonomic movement, aiming to identify pathological behavior. Four square open field arenas (50 cm x 50 cm x 40 cm) were placed together to form the apparatus. The bottom of the box was divided into nine equal squares.
<ol>
	<li>Control the background noise of the laboratory to below 65 dB.</li>
	<li>Wipe the entire apparatus with 75% ethanol before each trial.</li>
	<li>Place all mice in the testing room 1 h before the test to allow them to adapt to their environment.</li>
	<li>Put the mice in the square open field arenas and allow them to explore for 10 min.</li>
	<li>Record the total distance and movement speed for 10 min.</li>
</ol>
7. Euthanasia and specimen collection
<ol>
	<li>Place the mouse in the anesthesia induction box and anesthetize it with a mixture of 2-3% isoflurane and oxygen at a flow rate of 2 L/min. Once the mouse enters a state of deep anesthesia confirmed by a lack of pedal reflex, transfer it to the operating table for maintenance anesthesia.</li>
	<li>Grab and fix the mouse with the non-dominant hand.</li>
	<li>Lightly press on the eye area to make the eyeballs congested and protrude.</li>
	<li>Use scissors to trim the whiskers of the mouse to prevent hemolysis caused by hairs.</li>
	<li>Confirm the depth of anesthesia by checking for the absence of a pedal reflex in response to a toe pinch. Use forceps to grasp the eyeball and quickly remove it, allowing the blood to flow into a microcentrifuge tube from the eye socket. 
	NOTE: Alternative blood collection methods, such as cardiac puncture, can also be used.</li>
	<li>When the blood dripping rate slows down, gently press on the mouse&#39;s heart area to speed up the blood pumping and obtain more blood. Then, euthanize the mouse using cervical dislocation.</li>
	<li>Hold the leg just above the ankle joint using forceps in the non-dominant hand. Then, cut the leg just above the ankle joint using scissors.</li>
</ol>
8. Histological analysis
<ol>
	<li>Immerse the ankle specimens in 4% paraformaldehyde for more than 24 h. Decalcify in 10% EDTA (pH = 7.4) for 1 month.</li>
	<li>Soak the specimens in 4% paraformaldehyde for 2 h.</li>
	<li>Then, soak the specimens in a gradient of increasing ethanol concentrations (70%, 80%, 90%, 100%, 100%, 100%). Soak each ethanol concentration for 1 h.</li>
	<li>Soak the specimens in xylene for 3 h.</li>
	<li>Immerse the ankle specimens in paraffin for 7 h. Set aside paraffin blocks in a 4 &#176;C refrigerator.</li>
	<li>Slice paraffin blocks to a thickness of 6 &#181;m using a semi-automatic paraffin sectioning machine.</li>
	<li>Soak the slices for 4 x 5 min in xylene to complete dewaxing.</li>
	<li>Soak the slices in a gradient of decreasing ethanol concentrations (100%, 100%, 95%, 75%) for 2 min each. Soak in distilled water for 2 min.</li>
	<li>Stain the slices with hematoxylin for 10 min and rinse with distilled water for 5 s.</li>
	<li>Soak the slices for 30 s in the differentiation fluid and rinse with distilled water for 5 s.</li>
	<li>Stain the slices with eosin for 1 min and rinse with distilled water for 3 min.</li>
	<li>Soak the slices in a gradient of increasing ethanol concentrations (80%, 95%, 100%, 100%) for 1 min each. Soak in xylene for 3 x 3 min and seal the slices25.</li>
</ol>
9. Enzyme-linked immunosorbent assay (ELISA)
<ol>
	<li>Measure the IL-17 and TNF-&#945; concentrations in plasma using the respective kits according to the manufacturers&#39; instructions.</li>
</ol>
10. Statistical analysis
<ol>
	<li>Express the data as mean &#177; standard error of the mean (SEM).</li>
	<li>Determine the significance through one-way analysis of variance (ANOVA) or two-way repeated measures ANOVA, followed by Bonferroni or Tamhane T2&#39;s post hoc tests.</li>
	<li>Consider P &#60; 0.05 to be statistically significant.</li>
</ol>

Exploring the Therapeutic Potential of Du-Moxibustion in Ankylosing Spondylitis

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+circulating+interleukin-17-secreting+interleukin-23+receptor-positive+&#947;/&#948;+T+cells+in+patients+with+active+ankylosing+spondylitis.">Enrichment of circulating interleukin-17-secreting interleukin-23 receptor-positive &#947;/&#948; T cells in patients with active ankylosing spondylitis.</a> Arthritis Rheum. 64 (5), 1420-1429 (2012).</li><li>Poddubnyy, D., Sieper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+axial+spondyloarthritis:+what+does+the+future+hold.">Treatment of axial spondyloarthritis: what does the future hold.</a> Curr Rheumatol Rep. 22 (9), 47 (2020).</li><li>van der, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ixekizumab,+an+interleukin-17A+antagonist+in+the+treatment+of+ankylosing+spondylitis+or+radiographic+axial+spondyloarthritis+in+patients+previously+untreated+with+biological+disease-modifying+anti-rheumatic+drugs+(COAST-V):+16+week+results+of+a+phase+3+randomised,+double-blind,+active-controlled+and+placebo-controlled.">Ixekizumab, an interleukin-17A antagonist in the treatment of ankylosing spondylitis or radiographic axial spondyloarthritis in patients previously untreated with biological disease-modifying anti-rheumatic drugs (COAST-V): 16 week results of a phase 3 randomised, double-blind, active-controlled and placebo-controlled.</a> Lancet. 392 (10163), 2441-2451 (2018).</li><li>Fu, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+properties+and+underlying+mechanisms+of+curcumin+and+prospects+in+medicinal+potential.">Pharmacological properties and underlying mechanisms of curcumin and prospects in medicinal potential.</a> Biomed Pharmacother. 141, 111888 (2021).</li><li>Hassan, A. S., Hofni, A., Abourehab, M. A. S., Abdel-Rahman, I. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginger+extract-loaded+transethosomes+for+effective+transdermal+permeation+and+anti-inflammation+in+rat+model.">Ginger extract-loaded transethosomes for effective transdermal permeation and anti-inflammation in rat model.</a> Int J Nanomedicine. 18, 1259-1280 (2023).</li></ol>

The authors have no conflicts of interest to declare.

AS is clinically manifested as back, lumbar, and joint swelling pain, and spinal deformity and joint rigidity may occur in severe cases26. Early diagnosis and treatment can improve the quality of life, reduce disability rates, and improve the prognoses of patients with AS. According to traditional Chinese medicine (TCM), AS is believed to be caused by a deficiency of Yang in the kidney and the Du meridian, as well as an accumulation of cold evil internally. TCM holds the view that moxibustion, bas...

Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. According to the latest epidemiological survey, ~0.01%-1.8% of people in the world suffer from this condition1, most of whom are young males (male to female ratio is ~2-3:1)2. The incidence of AS in the Chinese population is 0.2%-0.4%. This disease mainly involves the spine and the sacroiliac joint. Fusion of the spine and the sacroiliac joint may occur in the later stages of the disease, resulting in spinal stiffness and kyphosis, as well as difficulty in walking, which seriously affects the quality of work and daily living activities and imposes a heavy burden on the patient, family, and society1. Currently, there is no definitive solution for AS, and the provided treatments, both pharmacological and non-pharmacological, primarily focus on alleviating pain, slowing disease progression, and enhancing the quality of life. In recent years, because pharmacotherapeutic options have been very limited3, increasing attention has been paid to non-pharmacotherapy, which has become an alternative therapy for AS. In China, patients often prefer external treatment consisting of Traditional Chinese Medicine (TCM), which has minor side effects and great convenience during the treatment.
Moxibustion is an ancient therapeutic technique used in TCM. Du-moxibustion is an external treatment technique consisting of &#34;medicated moxibustion with foam&#34; on the spine segment of the Du meridian. It is mainly used in the treatment of AS and has shown safety and effectiveness. Compared with ordinary moxibustion, Du-moxibustion has the characteristics of a wide moxibustion area, a large moxa cone, strong firepower, and high temperature4. A systematic review and meta-analysis suggest that moxibustion is an effective complementary treatment for AS5. A recent study confirmed that the clinical symptoms and some inflammatory factors of AS patients were improved by moxibustion6. Studies have revealed that Du-moxibustion can increase the brain content of &#946;-endorphin and exert a central analgesic effect7, downregulate the expression of the HLA-B27 gene8, and delay the recurrence rate of AS. Du-moxibustion also has an ameliorating effect on the progression of AS by decreasing the inflammatory index ESR (ESR) and levels of C-reactive protein (CRP), C-terminal peptide of type I collagen (CTX-I), and Dickkopf1 protein (DKK1)9,10.
Another clinical study showed that Du-moxibustion can further adjust the disordered T cell subsets, Ig, and complement C3 to balance the immune mechanism11. In terms of bone metabolism, moxibustion can inhibit the rise of serum alkaline phosphatase (ALP) and serum phosphorus (S-P), increase serum calcium (S-Ca), bone mineral density (BMD), and bone strength12. Moreover, Du-moxibustion can repair spinal function at multiple points to relieve fatigue symptoms in AS patients13. Some animal studies have revealed the potential mechanism of moxibustion in treating AS. A study indicated that moxibustion significantly improved the overall health status, reduced the levels of paw thickness, and decreased the levels of IL-1&#946;, PGE2, IL-6, and TNF-&#945; in the ligament tissue samples of the spine. Metabolic pathway analysis linked the identified metabolites to the TCA cycle, as well as lipid, amino acid, intestinal flora, and purine metabolism14. Another study showed that moxibustion suppressed the expression of pro-inflammatory cytokines, IL-1&#946;, TNF-&#945;, IL-17, and IL-6; reduced the mRNA levels of RANKL, RANK, ALP, and OCN; and improved the histopathological features in AS mice15.
While these studies suggest that moxibustion may be effective in the treatment of AS, more research is needed to confirm these findings and determine the optimal treatment protocol for moxibustion in patients with AS. Our team created and modified the moxibustion therapy based on the basic theory and practical operation of traditional Chinese moxibustion, which has been widely carried out and applied in Chinese clinical practice for over 35 years. Although there are few high-quality studies on the treatment of AS by Du-moxibustion, its indication in treating AS has been supported by evidence. However, the mechanism of treating AS with Du-moxibustion is worthy of further study. Animal study is an important method to explore the mechanism of Du-moxibustion in the treatment of AS. The clinical operation of Du-moxibustion is relatively mature but rarely used in the study of the mechanism in animal experiments. This paper describes in detail the operation and precautions to be taken during Du-moxibustion in the treatment of AS experimental mice.

<table><tbody><tr><td>10% EDTA</td><td>Shanghai Macklin Biochemical Co., Ltd.,Shanghai, China</td><td></td><td></td></tr><tr><td>alcohol pads</td><td>HYNAUT, Qingdao Hainuo Biological Engineering Co., LTD, Qingdao, China</td><td></td><td></td></tr><tr><td>anesthesia machine</td><td>Medical Supplies &amp;amp; Services INT. LTD, Keighley, UK</td><td></td><td></td></tr><tr><td>centrifuge tubes</td><td>Axygen, Corning, NewYork, UAS</td><td></td><td></td></tr><tr><td>complete Frech&#039;s adjuvant (CFA)</td><td>aladdin,Shanghai, China</td><td>F393378</td><td></td></tr><tr><td>cotton ball</td><td>Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China</td><td></td><td></td></tr><tr><td>cotton gauze</td><td>Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China</td><td></td><td></td></tr><tr><td>cutting board</td><td>self-preparation</td><td></td><td></td></tr><tr><td>decorin from bovine articular cartilage</td><td>Sigma-Aldrich, MO, USA</td><td>D8428</td><td></td></tr><tr><td>depilatory cream</td><td>Veet, Reckitt Benckiser (China) Investment Co. LTD, Shanghai, China</td><td></td><td></td></tr><tr><td>electronic scale</td><td>Shanghai Yajin Electronic Technology Co., Ltd.,Shanghai, China</td><td></td><td></td></tr><tr><td>Eppendorf tube</td><td>Axygen, Corning, NewYork, UAS</td><td></td><td></td></tr><tr><td>eye lubricant</td><td>Beijing Shuangji Pharmaceutical Co., LTD., Beijing, China</td><td></td><td></td></tr><tr><td>ginger</td><td>self-preparation</td><td></td><td></td></tr><tr><td>GraphPad Prism 7 software</td><td>GraphPad Software,Boston, USA</td><td></td><td></td></tr><tr><td>hair clipper</td><td>Super human Group CO LTD, Jinhua, China</td><td></td><td></td></tr><tr><td>heating pads</td><td>Shenzhen Leshuo Tech Co., Ltd.,Shenzhen, China</td><td></td><td></td></tr><tr><td>incomplete Freund&amp;rsquo;s adjuvant (IFA)</td><td>aladdin,Shanghai, China</td><td>F393371</td><td></td></tr><tr><td>injection&amp;nbsp;syringe</td><td>Shandong Xinhua Ande Medical Supplies Co., LTD, Zibo, China</td><td></td><td></td></tr><tr><td>isoflurane</td><td>Shenzhen Rayward Life Technology Co., LTD, Shenzhen, China</td><td>R510-22-10</td><td></td></tr><tr><td>joss stick</td><td>Shijiazhuang Lidu Fragrant Industry Co., LTD.,Shijiazhuang, China</td><td></td><td></td></tr><tr><td>juicer</td><td>Braun (Shanghai) Co., Ltd.,Shanghai, China</td><td></td><td></td></tr><tr><td>knife</td><td>self-preparation</td><td></td><td></td></tr><tr><td>lighter</td><td>Zhejiang tiger-lighter Co. LTD</td><td></td><td></td></tr><tr><td>mortar</td><td>Luoyang Yinai Ceramic Technology Co., LTD.,Luoyang, China</td><td></td><td></td></tr><tr><td>Mouse IL-17 ELISA Kit</td><td>absin, Shanghai, China</td><td>abs520009-96T</td><td></td></tr><tr><td>Mouse&amp;nbsp;TNF-&amp;alpha; ELISA Kit</td><td>Wuhan&amp;nbsp;Sanying, Wuhan, China</td><td>KE10002</td><td></td></tr><tr><td>mulberry paper</td><td>Yishui County Mulinsang paper Co., LTD, Linyi, China</td><td></td><td></td></tr><tr><td>OFT</td><td>Xinruan,Shanghai, China</td><td>XR-XZ301</td><td></td></tr><tr><td>paper cup</td><td>self-preparation</td><td></td><td></td></tr><tr><td>pipettes</td><td>OXFORD BIO INSTRUMENTS INC.,Oxford, UK</td><td></td><td></td></tr><tr><td>refined moxa velvet</td><td>self-preparation</td><td></td><td></td></tr><tr><td>rotarod</td><td>Xinruan,Shanghai, China</td><td>XR-6C</td><td></td></tr><tr><td>scientz-48L cryogenic high throughput tissue grinder</td><td>Ningbo Xinzhi Biotechnology Co., LTD</td><td></td><td></td></tr><tr><td>scissors</td><td>Shandong Jiaren Medical Supplies Co., Ltd., Zibo, China</td><td></td><td></td></tr><tr><td>semi-automatic paraffin sectioning machine</td><td>Leica Camera AG, Watznach, Germany</td><td></td><td></td></tr><tr><td>SPSS 25.0 software</td><td>International Business Machines Corporation, NewYork, UAS</td><td></td><td></td></tr><tr><td>TCM powder</td><td>self-preparation</td><td></td><td></td></tr><tr><td>tips</td><td>Biosharp, Labgic, Beijing, China</td><td></td><td></td></tr><tr><td>writing brush</td><td>Yishui County Mulinsang paper Co., LTD, Linyi, China</td><td></td><td></td></tr></tbody></table>

du-moxibustion in a mouse model of ankylosing spondylitis

Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. This disease mainly involves the spine and the sacroiliac joint. Fusion of the spine and the sacroiliac joint may occur in the later stage of the disease, resulting in spinal stiffness and kyphosis, as well as difficulty in walking, which seriously affects the quality of work and daily living activities and imposes a heavy burden on the patient, the family, and society. Increasing attention has been paid to non-pharmacotherapy as an alternative therapy for AS. Moxibustion is an ancient therapeutic technique used in Traditional Chinese Medicine (TCM). Du-moxibustion therapy, a unique and innovative external treatment developed on the basis of ordinary moxibustion, has a definite therapeutic effect on AS. Du-moxibustion skillfully combines the compatible techniques of TCM to integrate meridians, acupoints, Chinese herbal medicine, and moxibustion. This paper describes the operation procedures and precautions to be taken during Du-moxibustion in experimental mice in detail to provide an experimental basis for the study of the mechanism of Du-moxibustion in the treatment of AS.

PG-induced model mice developed symptoms of peripheral arthritis, characterized by redness and swelling of the extremities and toes, and gradually developed axial arthritis as early as week 14, which is very similar to the manifestation of AS. Therefore, the AS mouse model was considered successful if the arthritis index (AI) was over 3 points at 14 weeks after the last injection22. When the paws of the mice appeared swollen, the Du-moxibustion treatment was carried out once a week for a total of ...

Watch this Scientific Journal Video about Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice at JoVE.com

Author Spotlight: Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice 

This paper describes in detail the operation procedures and precautions to be taken during Du-moxibustion in the treatment of ankylosing spondylitis in experimental mice.

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. This disease mainly ...

du-moxibustion-in-a-mouse-model-of-ankylosing-spondylitis

Research

JoVE Journal

Immunology and Infection

881 Views.  Shandong University of Traditional Chinese Medicine. This paper describes in detail the operation procedures and precautions to be taken during Du-moxibustion in the treatment of ankylosing spondylitis in experimental mice.

Video: Author Spotlight: Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice 

Moxibustion Strategies for Mice

The field of moxibustion research is expanding, with a rapid increase in publications in recent years. Moxibustion is a therapy that ignites moxa on the skin of humans, with an increase in peripheral skin temperature and localized redness. During this treatment, the recipient must remain still to prevent scalding and expose intervention sites for easy manipulation; however, maintaining a fixed posture during moxibustion is a big challenge for animals. Thus, manipulating moxibustion in small animals, such as mice, can lead to several difficulties for researchers. In addition, an uncomfortable posture for animals can lead to fear and resistance to moxibustion, increased risk of injury, diminished animal welfare, and less valid research data. An efficient, comfortable moxibustion method is needed to protect animal welfare and minimize the adverse effects on experimental results. However, moxibustion methods are highly variable and often have limited efficacy. More importantly, an uncomfortable moxibustion posture might cause a stress response, such as those observed with anxiety, fear, and anger, which could influence the research data. Therefore, strategies for animal moxibustion that inflict the least harm possible during the intervention are required. This protocol introduces a mouse tethering method for moxibustion intervention, minimizing mouse discomfort and improving study efficiency. Essential strategies for tethering mice and application of moxibustion are highlighted, and the structure of the tethering instrument is described.

The present protocol describes a moxibustion application method for mice, with the benefits of reducing mouse fearfulness, guaranteeing their welfare, and improving efficiency for researchers.

The field of moxibustion research is expanding, with a rapid increase in publications in recent years. Moxibustion is a therapy that ignites moxa on the ...

Behavior

Standardized Operation Process of Zhuang Medicine Line Moxibustion in Rats

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

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

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

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

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

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Medicine

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Neuroscience

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

A Traditional Chinese Medicine Characteristic Therapy for Bronchial Asthma: Moxibustion

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest tightness, or cough, as a result of increased airway reactivity. With high diurnal variation, these symptoms often occur or worsen at night or in the morning. By burning and roasting the Chinese medica above human acupoints, moxibustion is a type of treatment that stimulates the activity of the human meridians through drugs and heat stimulation to prevent and treat diseases. According to the principle of syndrome differentiation and treatment of traditional Chinese medicine, acupoints are selected on the corresponding parts, which has a definite effect. It is regarded as a characteristic traditional Chinese medicine therapy for bronchial asthma. This protocol elaborates the methods and steps of patient management, material preparation, selection of acupoints, operation, and postoperative nursing to ensure safe and effective moxibustion treatment to significantly improve the clinical symptoms and quality of life of patients with bronchial asthma.

This protocol presents a curative procedure of moxibustion in treating patients with bronchial asthma.

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest ...

Effects of Manual Therapy on a Chronic Non-Specific Low Back Pain Murine Model

Manual Therapy for a Chronic Non-Specific Low Back Pain Rat Model

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed with chronic non-specific low back pain (CNLBP) due to an unknown pathological cause. Manual therapy (MT) is an integral aspect of traditional Chinese medicine and is recognized as Tuina in China. It involves techniques like bone-setting and muscle relaxation manipulation. Despite its clinical efficacy in treating CNLBP, the underlying mechanisms of MT remain unclear. In animal experiments aimed at investigating these mechanisms, one of the main challenges is achieving normative MT on CNLBP model rats. Improving the stability of finger strength is a key issue in MT. To address this technical limitation, a standardized procedure for MT on CNLBP model rats is presented in this study. This procedure significantly enhances the stability of MT with the hands and alleviates common problems associated with immobilizing rats during MT. The findings of this study are of reference value for future experimental investigations of MT.

This study proposes a standardized procedure for manual therapy on chronic low back pain model rats, which will be of reference value for future experimental research on manual therapy.

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed ...

Dual Hind Paw Injection for Enhanced Analgesic Testing

A Modified Inflammatory Pain Model to Study the Analgesic Effect in Mice

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with an intraplantar injection of carrageenan in one hind paw. However, the findings from our laboratory showed that mice with a single-hind-paw injection of carrageenan lifted their paws to avoid thermal nociception during the hot plate test. Because of this response,&#160;previous injection method cannot accurately reflect the thermal pain threshold. Thus, we investigated a new method to avoid this issue. In the present study, we modified the previous method by injecting carrageenan into both hind paws to establish the model of inflammatory pain. The results demonstrated that both-hind-paw injection with carrageenan was sensitive and a better method to induce inflammatory pain when using the hot plate test than single-hind-paw injection. On the basis of these findings, we designed further experiments in which mice with either both-hind-paw or single-hind-paw injection of carrageenan were treated intragastrically with celecoxib (30 mg/kg). The results of the hot plate test showed that celecoxib augmented the thermal pain threshold in mice with both-hind-paw injection of carrageenan but not in mice with single-hind-paw injection of carrageenan. In summary, we developed a superior method to induce a model of inflammatory pain to evaluate analgesic effect.

Author Spotlight: Enhanced Method for Evaluating Analgesic Effects &#8212; Dual Hind Paw Carrageenan Injection in Mice

Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with ...

A Moxibustion Platform for Herbal Cake-Based Treatment of Rats with Renal Failure

Using Herbal-Cake-Separated Moxibustion for the Treatment of Rats with Chronic Renal Failure

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash falling, scalding skin, and poor curative effect. Such problems also exist in the rat moxibustion experiment. To simulate clinical practice, it is necessary to introduce an experimental instrument in animal experiments, that is, a moxibustion device with fixed rats and moxibustion treatment synchronization, which can make experimental rats receive moxibustion treatment quietly and comfortably under non-anesthesia.
Our research group designed a rat moxibustion experimental platform. The device was framed by a wooden board with a supporting base plate, multiple fixed components, and partitioned components. The device can achieve the operation mode of moxibustion in rats without binding, avoiding anesthesia and scalding and simultaneously exposing multiple acupoints on the back. This operation can avoid physical and mental injury to rats and operators, which improves the research efficiency and further promotes the development and research of moxibustion animal experiments.
The device has a simple structure, is easy to operate and popularize, is comprehensively and innovatively designed, reusable, and is suitable for rat experiments mainly based on moxibustion. This article mainly introduces the structure of the experimental platform device for rat moxibustion, the basic procedure of herbal-cake-separated-moxibustion in experimental rats using the device and describes the establishment of a rat model of chronic renal failure (CRF) and representative experimental results.

Author Spotlight: Understanding the Effect of Herbal-Cake-Separated Moxibustion in Rats with Renal Faliure

The moxibustion experimental platform can fix the rats without restriction, and the rats can undergo multi-acupoint moxibustion in the awake state, ensuring the welfare of the rats, protecting the safety of the operators, ensuring therapeutic efficacy, and improving the efficiency of the experiment.

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash ...

Herbal Moxibustion on Shenque Acupoint for Alleviating Primary Dysmenorrhea

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

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

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

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