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>

作者没有利益冲突需要声明。

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 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

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

Research

JoVE Journal

Immunology and Infection

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

763 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 

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.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

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

科研

免疫与感染

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.

流感病毒在小鼠感染模型中的研究

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.

新生儿多药脓毒症的对照小鼠模型

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

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.

腹部主动脉瘤小鼠模型中脉冲传播速度、失散性和应变的测量

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.

细菌性心内膜炎小鼠模型中感染参数的内注射与定量

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.

在盲肠结扎和穿刺诱导的脓毒症小鼠模型中评估可靠的生物标志物

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

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

慢性肝纤维化小鼠模型用于研究胆道闭锁

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

小鼠自发性自身免疫性甲状腺炎模型的产生

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

建立 白色念珠菌 生物膜相关 PJI 的小鼠模型

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

作者聚焦：推进白色念珠菌生物膜相关人工关节感染的研究 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

小鼠假体周围关节 白色念珠菌 感染模型

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Methods Article

强直性脊柱炎小鼠模型中的杜艾灸