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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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&#39;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&#8217;s adjuvant (IFA)</td>
			<td>aladdin,Shanghai, China</td>
			<td>F393371</td>
			<td></td>
		</tr>
		<tr>
			<td>injection&#160;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&#160;TNF-&#945; ELISA Kit</td>
			<td>Wuhan&#160;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

818 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 

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 &#181;L. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 &#181;L of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 &#181;L sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 &#176;C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

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A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness