本研究得到了国家自然科学基金项目（No.82174491）和齐鲁中医学院学术学校传承项目（No.陆维涵[2020]132）。感谢山东中医药大学实验动物中心为我们提供的实验条件。

所有动物实验均经山东中医药大学实验动物福利伦理审查委员会批准。SDUTCM20230831301）。
1.动物准备
<ol><li>提前确定实验组和每组动物数量。</li>
	<li>选择雄性BALb/c小鼠，将其饲养在22±1°C的恒温，60±5%的恒湿度和12小时的昼夜循环光照环境中。</li>
</ol>2. AS小鼠模型的建立
注:AS模型是用软骨蛋白多糖（PG）诱导的，这是一种经典的建模方法16,17,18,19,20,21。反复接种PG诱发肌腱炎和脊柱炎的BALB/c菌株小鼠。有关本协议中使用的材料、试剂和仪器的详细信息，请参阅材料表。
<ol><li>使用 200 μL 移液管将 200 μL PG 和 CFA（或 IFA）吸入 1.5 mL 离心管中。轻轻摇晃，使离心管中的液体混合。</li>
	<li>将离心管放入组织研磨机中匀浆，然后在冰上孵育4分钟，重复5次。在组织研磨机中设置以下参数: 60 Hz，研磨 30 秒，间隔 10 秒 ，共 6 个周期。</li>
	<li>用 1 mL 注射器从离心管中提取 0.2 mL 乳化 PG 蛋白。</li>
	<li>用左手抓住并固定鼠标。将鼠标置于仰卧位，保持头部低于尾部，以防止插入注射器时对腹部器官造成损害。</li>
	<li>用右手用酒精棉签对腹部进行消毒。</li>
	<li>右手拿着注射器，将针头皮下轻插入腹部下半部白线腹侧的左侧或右侧。</li>
	<li>将针头皮下平行于中腹线推下 3-5 mm，然后将针头以与皮肤成 45° 角推入腹腔。</li>
	<li>穿透腹膜，拔出针塞，缓慢注射乳化的PG蛋白。</li>
</ol>3. 杜艾灸
注意:提前准备Du-moxibation最重要的三种原材料（图1）。
<ol><li>准备艾灸锥。将天鹅绒拿在手中，两只手掌朝向相反的方向，施加扭转力，将天鹅绒揉成纺锤形（图2）</li>
	<li>将生姜切成小块，放入榨汁机中捣成泥（图3）。用棉纱布过滤姜泥。将姜汁和姜泥从姜泥中分离出来，为下一步做准备。</li>
	<li>准备 10 克姜泥和 20 毫升姜汁，放入纸杯中以备后用。</li>
	<li>称取 0.1 克中药粉（肉桂粉、丁香粉、黄芋粉、花蒽素粉和人造麝香的混合物）22 ，并在研钵中研磨。</li>
	<li>将小鼠置于动物麻醉机的麻醉诱导箱中，用2-3%异氟醚和氧气的混合物以2L / min的流速麻醉它们。</li>
	<li>首先通过观察是否存在惊吓反射来评估麻醉深度，并通过检查是否在轻轻捏脚趾时没有踏板反射来验证手术麻醉水平。</li>
	<li>鼠标达到手术平面麻醉后，将其从盒子中取出并以俯卧位固定在手术台上。将鼠标的嘴和鼻子与麻醉剂的出口对齐。</li>
	<li>通过连接到麻醉机的面罩进行麻醉，异氟醚和氧气通过非再呼吸回路输送。在整个操作过程中保持麻醉并将鼠标保持在37°C加热垫上。在眼睛上涂抹润滑剂以防止干燥。</li>
	<li>找到穴位并确定刺激区域（图4）。</li>
	<li>剃掉老鼠背部的毛发，直到看到老鼠背部的肉色皮肤。确保脱毛的宽度为~1.5厘米，长度为从第七颈椎到尾椎（图5A）。</li>
	<li>将准备好的姜汁用棉纱布涂抹在剃光区域（图3）。</li>
	<li>用毛笔将中药粉均匀地撒在鼠标背面的中线上，并用桑皮纸覆盖（图5B-D）。</li>
	<li>用姜泥制作一个长 6 厘米、宽 1.5 厘米、厚 3 毫米的梯形姜柱。将生姜柱固定在桑纸上（图5E）。</li>
	<li>在姜柱的中线上方做一个凹槽（图3），然后将艾灸锥放在姜柱上。</li>
	<li>点燃艾灸锥，在前一个艾灸锥烧尽后放置一个新的艾灸锥。检查生姜柱的硬度，以防止灼伤小鼠，共灼伤三个锥体（图5F）。</li>
	<li>艾灸锥燃烧后，擦去生姜和中药粉，清洁鼠标背面。此时停止麻醉。</li>
	<li>让老鼠在一个新的笼子里恢复意识，不受限制地获得食物和水。将笼子放在加热垫上以帮助恢复过程。监测动物，直到它恢复足够的意识。</li>
	<li>一旦鼠标完全恢复，在将其放回其常规外壳之前，确认是否存在矫正反射。 注意:在操作过程中，有必要监测小鼠的精神状态，以避免呼吸微弱。防止烫伤，但要保持老鼠温暖，避免爪子和尾巴瘀伤。</li>
</ol>4. 关节炎指数（AI）的评估
<ol><li>评估 AI 分数三次:建模前、建模后和治疗后。评定标准如下:0、无红肿;1、几个脚趾轻微发红或肿胀;2、大多数脚趾关节和脚趾发红肿胀;3、足部及踝关节以下严重发红肿胀;4、足部和踝关节发红肿胀23.</li>
	<li>计算所有四只爪子的 AI 分数，最大值为 16。</li>
</ol>5. Rotarod测试
注意:旋转测试用于通过记录小鼠在旋转鼓上花费的时间来评估运动协调和平衡。该实验由三项试验组成，间隔 20 分钟。官方测试数据记录在第三次试验中，前两次作为训练练习。
<ol><li>将鼠标放在旋转鼓上，该鼓在40秒24内从40到40rpm的连续加速度下旋转。</li>
	<li>测量延迟时间（鼠标从杆上掉下来之前的时间）。提供衬垫以防止脱落时受伤。</li>
</ol>6. 开放场测试（OFT）
注意:OFT用于评估自主神经运动的状态，旨在识别病理行为。四个方形的露天竞技场（50 cm x 50 cm x 40 cm）被放在一起形成设备。盒子的底部被分成九个相等的方块。
<ol><li>将实验室的背景噪音控制在65分贝以下。</li>
	<li>每次试验前用75%乙醇擦拭整个设备。</li>
	<li>在测试前1小时将所有小鼠置于测试室中，以使它们适应环境。</li>
	<li>将老鼠放在方形的空旷场地中，让它们探索 10 分钟。</li>
	<li>记录 10 分钟的总距离和移动速度。</li>
</ol>7. 安乐死和标本采集
<ol><li>将小鼠置于麻醉诱导箱中，并以2L / min的流速用2-3%异氟醚和氧气的混合物麻醉。一旦小鼠进入深度麻醉状态，确认缺乏踏板反射，将其转移到手术台上进行维持麻醉。</li>
	<li>用非惯用手抓住并固定鼠标。</li>
	<li>轻轻按压眼部区域，使眼球充血突出。</li>
	<li>用剪刀修剪老鼠的胡须，防止毛发引起的溶血。</li>
	<li>通过检查脚趾捏合时是否没有踏板反射来确认麻醉深度。用镊子夹住眼球并迅速取出，让血液从眼眶流入微量离心管。 注意:也可以使用其他采血方法，例如心脏穿刺。</li>
	<li>当滴血速度减慢时，轻轻按压鼠标的心脏区域以加快血液泵送并获得更多血液。然后，使用颈椎脱位对小鼠实施安乐死。</li>
	<li>使用非惯用手的镊子将腿保持在踝关节上方。然后，用剪刀剪掉踝关节上方的腿。</li>
</ol>8. 组织学分析
<ol><li>将踝关节标本浸入4%多聚甲醛中超过24小时。在 10% EDTA （pH = 7.4） 中脱钙 1 个月。</li>
	<li>将试样浸泡在4%多聚甲醛中2小时。</li>
	<li>然后，将样品浸泡在乙醇浓度增加的梯度中（70%，80%，90%，100%，100%，100%）。将每种乙醇浓度浸泡1小时。</li>
	<li>将试样浸泡在二甲苯中3小时。</li>
	<li>将踝关节标本浸入石蜡中7小时。将石蜡块放在4°C冰箱中。</li>
	<li>使用半自动石蜡切片机将石蜡块切片至6μm的厚度。</li>
	<li>将切片浸泡在二甲苯中4×5分钟以完成脱蜡。</li>
	<li>将切片浸泡在乙醇浓度降低的梯度（100%，100%，95%，75%）中，每次2分钟。在蒸馏水中浸泡2分钟。</li>
	<li>用苏木精染色切片10分钟，然后用蒸馏水冲洗5秒。</li>
	<li>将切片在分化液中浸泡30秒，然后用蒸馏水冲洗5秒。</li>
	<li>用曙红染色切片1分钟，然后用蒸馏水冲洗3分钟。</li>
	<li>将切片浸泡在乙醇浓度增加（80%，95%，100%，100%）的梯度中，每次1分钟。在二甲苯中浸泡 3 x 3 分钟，然后将切片密封 25。</li>
</ol>9. 酶联免疫吸附试验（ELISA）
<ol><li>根据制造商的说明，使用相应的试剂盒测量血浆中的IL-17和TNF-α浓度。</li>
</ol>10. 统计分析
<ol><li>将数据表示为均值±均值标准误差 （SEM）。</li>
	<li>通过单因素方差分析 （ANOVA） 或双向重复测量方差分析确定显着性，然后进行 Bonferroni 或 Tamhane T2 的事后检验。</li>
	<li>将 P < 0.05 视为具有统计学意义。</li>
</ol>

探索 Du-Moxibust 治疗强直性脊柱炎的治疗潜力

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J., Mikecz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+polyarthritis+induced+in+BALB/c+mice+by+aggrecan+from+human+osteoarthritis+cartilage.">Progressive polyarthritis induced in BALB/c mice by aggrecan from human osteoarthritis cartilage.</a> Arthritis Rheum. 41, 1007-1018 (1998).</li><li>Hu, B., Li, Y., Tan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+of+moxibustion+in+the+treatment+of+ankylosing+spondylitis.">Research progress of moxibustion in the treatment of ankylosing spondylitis.</a> Rheum &amp; Arthritis. 11 (05), 77-80 (2022).</li><li>Yang, Y., Dong, Q., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrine+induces+the+apoptosis+of+fibroblast-like+synoviocytes+derived+from+rats+with+collagen-induced+arthritis+by+suppressing+the+activation+of+the+JAK/STAT+signaling+pathway.">Matrine induces the apoptosis of fibroblast-like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway.</a> Int J Mol Med. 39 (2), 307-316 (2017).</li><li>Shiotsuki, H., 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=A+rotarod+test+for+evaluation+of+motor+skill+learning.">A rotarod test for evaluation of motor skill learning.</a> J Neurosci Methods. 189 (2), 180-185 (2010).</li><li>Liu, P., 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=A+mouse+model+of+ankle-subtalar+joint+complex+instability+induced+post-traumatic+osteoarthritis.">A mouse model of ankle-subtalar joint complex instability induced post-traumatic osteoarthritis.</a> J Orthop Surg Res. 16 (1), 541 (2021).</li><li>Taurog, J. D., Chhabra, A., Colbert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ankylosing+spondylitis+and+axial+spondyloarthritis.">Ankylosing spondylitis and axial spondyloarthritis.</a> N. Engl. J. Med. 374, 2563-2574 (2016).</li><li>Schett, G., 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=Enthesitis:+From+pathophysiology+to+treatment.">Enthesitis: From pathophysiology to treatment.</a> Nat. Rev. Rheumatol. 13, 731-741 (2017).</li><li>Duan, W. X., 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=Effect+of+different+concentrations+of+Moxa-smoke+on+lung+function+and+TNF-&#945;+and+IL-1+&#946;+levels+in+serum+and+lung+tissues+in+normal+rats.">Effect of different concentrations of Moxa-smoke on lung function and TNF-&#945; and IL-1 &#946; levels in serum and lung tissues in normal rats.</a> Zhen Ci Yan Jiu [Acupuncture Research]. 43 (2), 98-103 (2018).</li><li>Callhoff, J., Sieper, J., Wei&#223;, A., Zink, A., Listing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+TNF&#945;+blockers+in+patients+with+ankylosing+spondylitis+and+non-radiographic+axial+spondyloarthritis:+a+meta-analysis.">Efficacy of TNF&#945; blockers in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis: a meta-analysis.</a> Ann Rheum Dis. 74 (6), 1241-1248 (2015).</li><li>Schinocca, C., 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=Role+of+the+IL-23/IL-17+pathway+in+rheumatic+diseases:+an+overview.">Role of the IL-23/IL-17 pathway in rheumatic diseases: an overview.</a> Front Immunol. 12, 637829 (2021).</li><li>Gravallese, E. M., Schett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+IL-23-IL-17+pathway+on+bone+in+spondyloarthritis.">Effects of the IL-23-IL-17 pathway on bone in spondyloarthritis.</a> Nat Rev Rheumatol. 14 (11), 631-640 (2018).</li><li>Kenna, T. 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>

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AS临床表现为背部、腰椎和关节肿胀疼痛，严重者可出现脊柱畸形和关节僵硬26。早期诊断和治疗可以改善AS患者的生活质量，降低残疾率，并改善预后。根据中医（TCM），AS被认为是由肾脏和度经的阳气不足以及内部冷恶的积累引起的。中医认为，艾灸基于经络的概念，可以产生使身体表面温暖的气。然后，这股气沿着经络和穴位向动。在 《黄帝内经 》（一部非常经典和...

强直性脊柱炎 （AS） 是一种进行性恶化和致残的关节炎，主要影响中轴骨骼。根据最新的流行病学调查，世界上~0.01%-1.8%的人患有这种疾病1，其中大多数是年轻男性（男女比例为~2-3:1）2。AS在中国人群中的发病率为0.2%-0.4%。这种疾病主要累及脊柱和骶髂关节。脊柱与骶髂关节融合可能发生在疾病后期，导致脊柱僵硬和脊柱后凸，以及行走困难，严重影响工作和日常生活活动的质量，给患者、家庭和社会带来沉重的负担1。目前，AS尚无明确的解决方案，所提供的药物和非药物治疗主要集中在减轻疼痛、减缓疾病进展和提高生活质量上。近年来，由于药物治疗选择非常有限3，非药物治疗越来越受到关注，非药物治疗已成为AS的替代疗法。在中国，患者通常更喜欢由中医（TCM）组成的外部治疗，这种治疗副作用小，治疗过程非常方便。
艾灸是中医中使用的一种古老的治疗技术。度艾灸是一种外部治疗技术，由杜经脊柱段的"药泡艾灸"组成。它主要用于AS的治疗，并显示出安全性和有效性。与普通艾灸相比，杜艾灸具有艾灸面积广、艾灸锥大、火力强、耐高温4等特点。一项系统评价和荟萃分析表明，艾灸是治疗 AS5 的有效补充疗法。最近的一项研究证实，艾灸改善了AS患者的临床症状和一些炎症因素6。研究表明，度艾灸可增加脑内β内啡肽含量并发挥中枢镇痛作用7，下调HLA-B27基因8的表达，延缓AS的复发率。度艾灸还通过降低炎症指数 ESR （ESR） 和 C 反应蛋白 （CRP）、I 型胶原 C 末端肽 （CTX-I） 和 Dickkopf1 蛋白 （DKK1） 的水平来改善 AS 的进展9,10。
另一项临床研究表明，度艾灸可以进一步调节无序的T细胞亚群Ig和补体C3，以平衡免疫机制11。在骨代谢方面，艾灸可抑制血清碱性磷酸酶（ALP）和血清磷（S-P）的升高，增加血清钙（S-Ca）、骨密度（BMD）和骨强度12。此外，度艾灸可以在多个点修复脊柱功能，以缓解AS患者的疲劳症状13。一些动物研究揭示了艾灸治疗AS的潜在机制。研究表明，艾灸显著改善了整体健康状况，降低了脊柱韧带组织样本中爪子厚度的水平，并降低了IL-1β、PGE2、IL-6和TNF-α的水平。代谢途径分析将鉴定的代谢物与 TCA 循环以及脂质、氨基酸、肠道菌群和嘌呤代谢联系起来14。另一项研究表明，艾灸抑制促炎细胞因子IL-1β、TNF-α、IL-17和IL-6的表达; 降低 RANKL、RANK、ALP 和 OCN 的 mRNA 水平;并改善了AS小鼠的组织病理学特征15。
虽然这些研究表明艾灸可能有效治疗 AS，但需要更多的研究来证实这些发现并确定 AS 患者艾灸的最佳治疗方案。我们的团队基于中国传统艾灸的基本理论和实际操作，创建并改进了艾灸疗法，在中国临床实践中广泛开展和应用了35年以上。虽然关于独艾灸治疗AS的高质量研究很少，但其治疗AS的适应症已得到证据支持。然而，用度灸治疗AS的机制值得进一步研究。动物研究是探究杜艾灸治疗AS机制的重要方法。杜艾灸的临床操作比较成熟，但很少用于动物实验机理研究。本文详细介绍了杜艾灸治疗AS实验小鼠的操作及注意事项。

<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

强直性脊柱炎 （AS） 是一种进行性恶化和致残的关节炎，主要影响中轴骨骼。这种疾病主要累及脊柱和骶髂关节。脊柱与骶髂关节融合可能发生在疾病后期，导致脊柱僵硬和脊柱后凸，以及行走困难，严重影响工作质量和日常生活活动，给患者、家庭和社会带来沉重的负担。非药物治疗作为 AS 的替代疗法越来越受到关注。艾灸是中医（TCM）中使用的一种古老的治疗技术。度艾灸疗法是在普通艾灸的基础上发展起来的一种独特而创新的外用疗法，对AS有一定的治疗效果。度艾灸巧妙地结合了中医的相容技术，将经络、穴位、中草药、艾灸融为一体。本文详细介绍了实验小鼠度灸的操作规程和注意事项，为研究度艾灸治疗AS的机理提供了实验依据。

PG诱导的模型小鼠出现外周关节炎症状，以四肢和脚趾发红肿胀为特征，早在第14周就逐渐发展为中轴关节炎，与AS的表现非常相似。因此，如果最后一次注射后 14 周的关节炎指数 （AI） 超过 3 分，则 AS 小鼠模型被认为是成功的22。当小鼠的爪子出现肿胀时，每周进行一次度艾灸治疗，共计3次。治疗后，小鼠在异氟烷深度麻醉下通过颈椎脱位实施安乐死，采集其血液，切除脚踝?...

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

作者聚焦:评估传统中医疗法治疗小鼠强直性脊柱炎 

本文详细介绍了杜艾灸治疗实验小鼠强直性脊柱炎的操作规程和注意事项。

强直性脊柱炎小鼠模型中的杜艾灸

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

我们的研究范围集中在使用独艾灸（中国传统的外部疗法）治疗强直性脊柱炎。本文详细介绍了独艾灸治疗实验小鼠强直性脊柱炎的操作规程和注意事项。我们的研究发现，独艾灸可以改善AS小鼠的运动协调和自主运动，减少踝关节肿胀，并减轻炎症。首次详细介绍中医外疗杜艾灸，进行更多实验，为未来AS治疗机理研究奠定了基础。灸灸作为中医的一种外用治疗技术，巧妙地结合了中医的相容技术，将经络、穴位、中草药、艾灸融为一体，直接作用于脊柱区域，对相关部位进行整体调整。在未来的研究中，我们将关注不同参数的评估，如安慰剂艾灸剂量范围和治疗时间，以获得最有效的联合方案。在以后的研究中，成像方法可用于以更直观的方式评估治疗结果。

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

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JoVE Journal

Immunology and Infection

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

823 次观看.  Shandong University of Traditional Chinese Medicine. 我们的研究范围集中在使用独艾灸（中国传统的外部疗法）治疗强直性脊柱炎。本文详细介绍了独艾灸治疗实验小鼠强直性脊柱炎的操作规程和注意事项。我们的研究发现，独艾灸可以改善AS小鼠的运动协调和自主运动，减少踝关节肿胀，并减轻炎症。首次详细介绍中医外疗杜艾灸，进行更多实验，为未来AS治疗机理研究奠定了基础。灸灸作为中医的一种外用治疗技术，...

视频: 作者聚焦:评估传统中医疗法治疗小鼠强直性脊柱炎 

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 ...

科研

行为学

壮药系大鼠艾灸标准化操作流程

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.

作者聚焦：展示自动化壮药线艾灸模拟器的治疗效果

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.

壮药药用线灸疗法及其在原发性痛经冷凝证大鼠模型中的应用

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.

骨关节炎疼痛的鼠标Monoiodoacetate模式

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 ...

医学

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.

拉米内切手术和脊髓窗口植入小鼠

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

神经科学

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.

单关节膝关节疼痛小鼠模型疼痛的自发性和诱发性测量

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.

支气管哮喘的中医特色疗法:艾灸

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 ...

<meta charset="utf8"></head><body>双后爪注射增强镇痛测试

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.

<meta charset="utf8"></head><body>作者聚焦:评估镇痛效果的增强方法 — 小鼠双后爪角叉菜胶注射液

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

研究小鼠镇痛作用的改良炎症性疼痛模型

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 ...

强直性脊柱炎小鼠模型中的杜艾灸

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Moxibustion Strategies for Mice

壮药药用线灸疗法及其在原发性痛经冷凝证大鼠模型中的应用

壮药药用线灸疗法及其在原发性痛经冷凝证大鼠模型中的应用

壮药药用线灸疗法及其在原发性痛经冷凝证大鼠模型中的应用

骨关节炎疼痛的鼠标Monoiodoacetate模式

拉米内切手术和脊髓窗口植入小鼠

单关节膝关节疼痛小鼠模型疼痛的自发性和诱发性测量

支气管哮喘的中医特色疗法:艾灸

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

研究小鼠镇痛作用的改良炎症性疼痛模型