这项工作得到了济南市2020年科技发展计划（批准号202019059）、山东省中医药科技计划项目（批准号2021Q080）和齐鲁中医药学校传承项目（批准号[2022]93）的支持。

本研究经山东中医药大学附属医院伦理委员会批准（编号:AWE-2022-023）。
1.实验动物
<ol><li>将20只雄性Sprague-Dawley（SD）大鼠（7周龄，250-280g）饲养在标准条件下（室温[RT] 20-24°C，湿度40%-60%，12小时/12小时光/暗循环）。</li>
</ol>2.分组方式
<ol><li>将SD大鼠分为对照组（C）、FS模型对照组（M）、FS模型推拿治疗组（MT）和FS模型口服治疗组（MO），每组由5只大鼠组成。每个笼子养5只大鼠（同组）。</li>
	<li>驯化7天后，使用石膏固定固定3周以模拟FS，如下一节所述，固定M，MT和MO组中大鼠的一侧肩膀3周。</li>
	<li>如第4节（图1）所述，对MT组的大鼠进行推拿治疗2周。</li>
	<li>根据成人剂量（0.75mg /天）和大鼠与人体表面积的比例（0.018）计算每公斤大鼠（0.0675mg /天）所需的地塞米松剂量。</li>
	<li>每天上午7:00以0.067mg / kg /天对MO组大鼠施用每日胃内地塞米松溶液，持续2周。 注意:使用此分组方法确认推拿方案在本研究中的效果。根据不同研究中的实验目的进行分组方法。</li>
</ol>3. FS模型的开发
<ol><li>使用三溴乙醇（250mg / kg，通过腹膜内注射）麻醉大鼠11。 注:根据该机构伦理委员会的要求，将由摩擦乙醇（10g）和叔戊醇（10mL）组成的储备溶液储存在4°C下。 使用前，用蒸馏水稀释至2%。</li>
	<li>将石膏浸泡的绷带贴在大鼠的右肩和胸部，使右前肢保持肩关节内旋90°，持续3周（图2）12。 注意:监测大鼠以确保它们可以进行正常的生理活动，例如行走，进食和饮水。如果大鼠不能进行正常的生理活动，请重新固定石膏绷带。</li>
	<li>通过观察大鼠右肩关节僵硬、右上肢收缩、肌肉萎缩、跛行等症状的发展，确认FS模型的成功建立13。</li>
</ol>4.推拿法
注意:在整个过程中，研究者必须穿戴个人防护设备。只有一名专业的推拿医生必须执行所有操作（图 3、图 4 和图 5）。
<ol><li>通过智能按摩技术参数确定系统进行训练，该系统包括机械感受器和计算机（图3A）。<ol><li>在通过软件显示的三个方向上对机械感受器和力参数进行操作（图3B）。</li>
		<li>使用拇指手指以0.5kg的强度和100-120次/分钟的频率在旋转运动中执行旋转揉捏方法（图3C）。</li>
		<li>使用拇指指尖以0.5 kg的强度执行点压方法（图3D）。</li>
		<li>通过保持步骤4.1.2和4.1.3中提到的机械显示器1分钟，对大鼠进行推拿。</li>
	</ol></li>
	<li>握住大鼠直到它平静下来（~2分钟）。然后，执行操作。将大鼠置于侧卧位，但位置可能会根据不同的操作方法而改变。</li>
	<li>用右手食指和中指夹住大鼠的右前肢并弯曲并伸展数次，以确定大鼠肩关节、肘关节和肱骨的位置。</li>
	<li>用拇指髓顺时针旋转揉捏大鼠的右肩，前肢和背部，强度为0.5kg，频率为100-120次/分钟，持续3分钟（图4A-C）。<ol><li>在侧卧位操纵前肢肌肉。</li>
		<li>以俯卧姿势操纵肩部和背部肌肉。</li>
	</ol></li>
	<li>每个穴位用拇指指尖垂直按压 LI15（剑宇）、SI11（天宗）、HT01（吉泉）和 LI11（曲池）30 次，强度为 0.5 kg（图 4D-G）。<ol><li>使用大鼠穴位图谱定义每个穴位的位置（图5）14,15。</li>
		<li>按LI15，位于肩峰端前下凹陷处，俯卧位。</li>
		<li>按 SI11，位于肩胛棘中点冈下窝的凹陷处，俯卧位。</li>
		<li>按位于腋窝中心的 HT01，仰卧位。</li>
		<li>按LI11，位于侧卧位肘皱褶外侧端桡侧腕伸肌内侧的凹陷处。</li>
	</ol></li>
	<li>用左手拇指和中指握住肩关节，在内收、外展、前伸和后伸位置拉伸前肢10秒（图4H-K）。 注意:这种拉伸方法必须在大鼠中没有抵抗的情况下进行。</li>
	<li>如果大鼠变得激动，请暂停推拿程序。抚摸大鼠10秒使其平静下来，然后进行试验。</li>
	<li>每天执行该程序，持续 2 周。</li>
</ol>5. 盂肱ROM的测量
注意: 重要的是要尽快完成测量过程，以防止关节囊组织退化。
<ol><li>用过量剂量的三溴乙醇（3倍初始剂量，通过腹膜内注射）处死大鼠后，切除肩胛骨和近端三分之二的肱骨，暴露肩胛骨的下边缘。</li>
	<li>将注射针（1.2 cm x 0.45 mm）沿肱骨干插入肱骨头。</li>
	<li>将两根注射针垂直插入肩胛骨的上角和下角，用无菌手术片包裹塑料泡沫。</li>
	<li>将一根细线连接到肱骨干上的注射针上，然后用 5 g 的力将其拉到另一端，使其与肱骨干平行。测量肩胛骨下边缘与肱骨干之间的角度（图6）。 注意:为确保可靠的结果，请让单独的调查员进行测量。</li>
	<li>使用统计分析软件应用程序以均值±标准差 （SD） 的形式报告数据。 注:此处使用的是 SPSS 软件（SPSS 25.0 版）。</li>
	<li>使用单因素方差分析 （ANOVA） 分析组间差异。</li>
	<li>使用适当的软件获取条形图形。 注意:这里使用了GraphPad Prism 8。</li>
	<li>测量后使用 H&E 和 Masson 染色评估胶囊病理学。</li>
</ol>6. 章节准备
<ol><li>评估盂肱 ROM 后，将整个样品在 4% PFA 中固定 3 天，然后在 EDTA （pH 7.2） 溶液中再脱钙 2 个月。</li>
	<li>脱水后，将含有样品的包埋组织块切成5μm切片16。</li>
	<li>将切片在65°C干燥60分钟。</li>
	<li>对切片进行脱蜡。</li>
	<li>将切片浸泡在二甲苯I，二甲苯II和二甲苯III中7分钟，然后下降乙醇系列（无水乙醇，5分钟;95%乙醇，2分钟;80%乙醇，2分钟和70%乙醇，2分钟），最后在超纯水中浸泡2分钟。</li>
</ol>7. H&E染色
<ol><li>用苏木精染色切片5分钟，用1%盐酸乙醇冲洗3秒，用流水洗涤5分钟。</li>
	<li>用曙红染色切片3分钟，并用自来水洗涤。</li>
	<li>将切片浸泡在乙醇系列（95%乙醇I，3秒;95%乙醇II，3秒;无水乙醇I，3秒和无水乙醇II，1分钟）中，然后浸入二甲苯系列（二甲苯I，1分钟;二甲苯II，1分钟）中。</li>
	<li>在每个样品上滴一滴中性口香糖密封剂。用盖玻片密封每个样品。</li>
	<li>使用倒置荧光显微镜（比例尺= 100μm）收集图像。</li>
</ol>8. Masson染色
<ol><li>使用免疫组织化学笔，在切片周围画一个圆圈，然后将切片在37°C的Bouin溶液中孵育2小时以媒染。随后，用水清洗切片，直到黄色消失。</li>
	<li>用青金石蓝染料处理样品3分钟，然后用蒸馏水洗涤。</li>
	<li>用苏木精（Mayer）对切片染色2分钟后，在酸性乙醇分化溶液中处理切片3秒。然后，在流水中洗涤切片10分钟。</li>
	<li>用ponceau洋红色染料溶液染色切片10分钟，然后用水洗涤。</li>
	<li>将切片浸入磷钼酸溶液中10分钟。</li>
	<li>向切片中加入苯胺蓝染色液5分钟，然后用弱酸工作液洗涤2分钟。</li>
	<li>脱水并使部分透明，如步骤 7.3 中所述。</li>
	<li>在每个部分滴一滴中性口香糖密封剂，并用盖玻片盖住它。将部分放在通风橱中晾干。</li>
	<li>按照步骤 7.5 中的说明收集图像。</li>
</ol>

大鼠模型肩周炎的推拿疗法

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作者没有什么可透露的。

第一个关键步骤是模型选择。由于难以实现初级FS模型，通常使用石膏固定和手术内固定来建立FS大鼠模型9,12。在通过石膏固定 3 周12,20 建立的 FS 模型中观察到最严重的肩关节活动度限制和囊纤维化。在这项研究中，FS模型的成功率非常好，成功率为100%。
第二个关键步骤是该协议中使用的操作。本研究使用了三种操作（揉捏、按压和拉伸）。对肩部、肩胛骨和上臂进行软组织揉捏操作，以放松肌肉。通过对 LI15、SI11、HT01 和 LI11 等穴位施加压力来进行按压操作，这些穴位在 FS 5,21 的临床实践中最常用。LI15、SI11 和 HT01 位于肩囊周围的位置，可有效改善 ROM 和肩功能22。LI11 常用于治疗上肢运动障碍，与 LI15 位于同一经络中。这种穴位匹配方法有助于提高 LI1523 的功效。完全放松后，使用伸展技术来恢复功能性活动。
该协议中可能存在的问题是大鼠在推拿过程中表现出强烈的抵抗力，这可能是由恐惧引起的，而不是超过大鼠的耐受性。此时，应停止操作，直到大鼠平静下来（抚摸10秒使大鼠平静下来）。此外，应根据大鼠的症状调整拉伸程度。最初，肩关节的局限性明显，拉伸幅度较小。随着干预，大鼠肩关节功能逐渐恢复，伸展幅度逐渐增加。标准是大鼠可以接受无阻力的拉伸方法。最后，老鼠有一定的攻击性，推拿需要长时间与老鼠接触，所以穿戴个人防护装备很重要。
操作的定量控制是推拿实验中最困难的。虽然按摩操作模拟器可用于控制单次操作的强度和频率，但当涉及多个操作和治疗部位时，这种方法受到限制24,25。在临床实践中，推拿通常由从业者直接进行，在这项研究中，很难用医疗设备进行干预。为了控制刺激，可以采用智能按摩技术参数确定系统来规范推拿的训练。训练后，研究者可以在一定程度上对每只大鼠施加相同的力。该协议的主要局限性是无法完全控制操作。
中医推拿疗法在中国有着悠久的使用历史，医院的不同医生使用不同的操作和治疗部位组合。因此，为动物实验和临床研究建立可复制和有效的方案非常重要。在这项研究中，使用的操作和穴位是基于我们团队之前的一项研究，将我们的临床经验与FS动物模型21的特征相结合。本研究证明了所开发的推拿方案在改善FS大鼠肩关节功能和减少关节囊纤维化方面的有效性。这些发现为进一步研究推拿治疗机制奠定了基础。此外，该协议对于有兴趣探索FS替代医学治疗疗效的研究人员很有用。
既往研究发现，推拿干预纤维化的机制可能与TGF-β和CTGF的下调有关，同时调节MMP-1/TIMP-1的平衡，从而减轻细胞外基质（ECM）的产生26。推拿对肩囊纤维化的作用可以通过各种机制的调节来实现。然而，需要进一步的研究来充分了解这种改进所涉及的机制。

肩周炎（FS），也称为肩关节粘连性关节囊炎，是一种以肩痛和活动缺陷为特征的自限性疾病。它通常影响 30 至 70 岁的人群，平均年龄为 50 岁，在中国人群中的患病率约为 5%1。据报道，女性的 FS 发病率是男性的 1.6 倍2。FS 的患病率在糖尿病、葡萄糖和脂质代谢紊乱或其他相关疾病患者中较高，范围在 10% 至 36%2,3 之间。目前FS的临床治疗包括物理治疗、类固醇药物和手术治疗4。
推拿是一种传统中医 （TCM） 疗法，已被证明可有效缓解 FS 患者的肩部疼痛，改善他们的生活质量 5,6。然而，这种治疗的潜在机制尚不清楚。因此，使用动物模型研究推拿治疗FS的效果和机制至关重要。
大鼠肩关节具有与人肩关节相似的复杂结构，常用于FS7的机理研究。FS 大鼠模型的特征是盂肱 ROM 和包膜纤维化8 的下降。此外，该模型允许观察肩囊，并在修复损伤的同时进行病理学研究9。此外，口服皮质类固醇通常用作FS治疗研究的对照组10。本研究旨在为 FS 大鼠模型制定推拿方案，并通过比较推拿疗法和口服地塞米松的疗效来证明在推拿研究中进行动物实验的可行性。

<table><tbody><tr><td>4% paraformaldehyde</td><td>Solarbio</td><td>P1110</td><td></td></tr><tr><td>Embedding machine</td><td>Changzhou Paisijie Medical Equipment Co., Ltd</td><td>BM450A</td><td></td></tr><tr><td>Ethylene Diamine Tetraacetic Acid (EDTA)</td><td>Solarbio</td><td>E1171</td><td></td></tr><tr><td>Hematoxylin eosin (HE) staining kit</td><td>Sparkjade</td><td>EE0012</td><td></td></tr><tr><td>Intelligent-massage technique parameter determination system</td><td>Shanghai Dukang Intrument Equipment Co. Ltd</td><td>ZTC-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65440/65440eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>531CM-Y43</td><td></td></tr><tr><td>&lt;br/&gt; Modified Masson Trichrome Staining Solution</td><td>Shanghai yuanye Bio-Technology Co., Ltd</td><td>R20381-8</td><td>Bouin 50 mL;&lt;br/&gt; lapis lazuli blue dye 50 mL;&lt;br/&gt; Hematoxylin (Mayer) 50 mL;&lt;br/&gt; acidic ethanol differentiation solution 50 mL;&lt;br/&gt; ponceau magenta dye solution 50 mL;&lt;br/&gt; phosphomolybdic acid solution 50 mL;&lt;br/&gt; aniline blue staining solution 50 mL;&lt;br/&gt; &amp;nbsp;weak acid 50 mL</td></tr><tr><td>Tribromoethanol</td><td>Macklin</td><td>T903147-5</td><td></td></tr></tbody></table>

tuina in a frozen shoulder rat model: an efficient and reproducible protocol

肩周炎 （FS） 是一种常见疾病，没有明确的最佳治疗方法。推拿疗法是一种用于治疗中国医院FS患者的传统中医技术，已显示出良好的效果，但其机制尚不完全清楚。在先前研究的基础上，这项工作旨在为FS大鼠模型开发推拿方案。我们将20只SD大鼠随机分为对照组（C;n=5）、FS模型（M;n=5）、FS模型推拿治疗组（MT;n=5）和FS模型口服治疗组（MO;n=5）。本研究采用铸造固定化法建立FS大鼠模型。评价推拿和口服地塞米松对盂肱关节活动度（ROM）的影响，并评估组织学结果。我们的研究表明，推拿和口服地塞米松能够改善肩关节活动度并保留关节囊的结构，推拿疗法被证明比口服地塞米松更有效。总之，本研究中建立的推拿方案对 FS 非常有效。

观察大鼠的身体活动以评估FS模型的成功或失败。先前的一项研究表明，与正常大鼠相比，石膏固定显着减少了行进距离和步行速度17.另一项研究表明，FS 不影响行进距离，跛行是最常见的首发症状13。本研究显示，造模后大鼠出现右肩关节僵硬、右上肢收缩、肌肉萎缩和跛行。MT 组和 MO 组的这些病变在干预 2 周后完全消退。但M组没有明显变化。
评估推拿治疗 FS 有效性的主要标准是盂肱 ROM18 的测量。我们观察到C组盂肱ROM的平均值为149.3°±5.9°，M组为111.1°±3.9°，MT组为128.5°±2.8°，MO组为119.56°±2.9°。 如图7所示，M组大鼠盂肱ROM明显低于C组（P < 0.0001）。MT组和MO组的ROM显著高于M组（P < 0.05，P < 0.0001）。MO组ROM显著低于MT组（P < 0.0001）。这一发现表明，推拿可以显着改善FS大鼠的肩关节功能。
此外，H&E染色和Masson染色可以进一步证明推拿在保留结构和减少胶囊纤维化方面的作用。为了便于观察，盂肱关节的囊用于组织学检查。肩关节囊包括滑膜层和纤维层19。H&E染色显示M组滑膜细胞增殖、滑膜皱襞扁平、红细胞淤滞和血管增生，这是FS的典型特征（图8A，B）。在推拿和口服地塞米松治疗后，这些特征在一定程度上减弱（图8C，D）。与MT组相比，MO组也显示出更多的滑膜细胞。Masson染色显示每组纤维束的排列（黄色箭头）。胶囊由松散的网状纤维网络组成，纤维束以整齐的方向排列（图8E）。在M组中，纤维束排列无序，表明包膜纤维化（图8F）。MT组大鼠的荚膜显示纤维束分层整齐清晰，但MO组仍略有无序（图8G，H）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65440/65440fig01.jpg"> 图 1:建立 FS 模型和推拿干预的方案。 大鼠适应性喂养7 d，FS模型建立21 d，推拿治疗14 d。在第36天，所有大鼠都被处死。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65440/65440fig02.jpg"> 图 2:用于建立 FS 大鼠模型的铸件固定。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65440/65440fig03.jpg"> 图 3:操作的定量控制 。 （一）智能按摩手法参数确定系统。（B）三种力可以测量为沿X方向的平行力、沿Y方向的纵向力和沿Z方向的垂直力。（C）旋转捏合法的强度。红色曲线代表稳定的垂直力（0.5 kg）。橙色曲线表示规则的平行力。白色曲线表示规则的纵向力。（四）点压法的强度。红色曲线代表垂直力 （0.5 kg）。橙色和白色曲线表示非平行力和纵向力。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65440/65440fig04.jpg"> 图 4:推拿疗法中使用的操作。 （空调）揉捏右肩、前肢和背部的肌肉。（D-G）点压 LI15、SI11、HT01 和 LI11。（香港）在内收、外展、前伸和后伸位置拉伸前肢。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65440/65440fig05.jpg"> 图 5:LI15、SI11、HT01 和 LI11 在大鼠体内的解剖位置。 ● 外侧表面，○ 内侧表面。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65440/65440fig06.jpg"> 图 6:盂肱 ROM 的测量。 将一根细线连接到插入肱骨干的注射针上，并用 5 g 的力拉动另一端，使其与肱骨干平行。肩胛骨下缘与肱骨干之间的夹角测量为盂肱骨 ROM。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65440/65440fig07.jpg"> 图 7:三组大鼠的盂肱 ROM。值均值为 ± S.D.，n = 5。显著差异由单因素方差分析（a P < 0.001 和 bP < 0.0001）表示。 <a href="https://www.jove.com/files/ftp_upload/65440/65440fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65440/65440fig08.jpg"> 图 8:肩囊的组织学结果。 （A，E）对照组包含正常的胶囊结构（H&E和Masson染色）。（B，F）FS 模型组说明了胶囊结构的变化如下:滑膜皱襞扁平、胶囊纤维化和纤维束紊乱（H&E 和 Masson 染色）。（C，G）。FS模型结合推拿组说明，囊膜结构接近正常，纤维化不明显（H&E和Masson染色）。（D，H）FS模型联合口服地塞米松显示，胶囊结构接近正常，纤维化明显（H&E和Masson染色）。比例尺 = 100 μm。 HH:肱骨头;黑色箭头:滑膜皱襞;红色箭头:红细胞淤滞和血管增生;黄色箭头:纤维束。<a href="https://www.jove.com/files/ftp_upload/65440/65440fig08large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder at JoVE.com

作者聚焦:研究推拿疗法治疗肩周炎的有效性 

本研究开发了一种高效且可重复的推拿方案，用于治疗在大鼠模型中建立的肩周炎。这种方法将有助于研究肩周炎的推拿治疗方法。

冻肩大鼠模型中的推拿:一种高效且可重复的方案

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

本研究旨在了解推拿疗法的机制和临床疗效。基于之前的研究，我们正在开发专门针对肩周炎大鼠模型的推拿方案。本研究证明了开发的推拿方案在改善受影响大鼠肩关节功能和减少关节膜纤维化方面的有效性。推拿可以调节各种机制，以达到对肩囊纤维化的作用。本研究结果为进一步研究推拿治疗机制奠定了基础。将来，我们将继续使用该方案治疗大鼠肩周炎，并采用蛋白质组学技术研究与这些大鼠的包膜纤维化治疗相关的机制。

tuina-frozen-shoulder-rat-model-an-efficient-reproducible

Research

JoVE Journal

Medicine

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

1.8K 次观看.  Shandong University of Traditional Chinese Medicine. 本研究旨在了解推拿疗法的机制和临床疗效。基于之前的研究，我们正在开发专门针对肩周炎大鼠模型的推拿方案。本研究证明了开发的推拿方案在改善受影响大鼠肩关节功能和减少关节膜纤维化方面的有效性。推拿可以调节各种机制，以达到对肩囊纤维化的作用。本研究结果为进一步研究推拿治疗机制奠定了基础。将来，我们将继续使用该方案治疗大鼠肩周炎，并采?...

视频: 作者聚焦:研究推拿疗法治疗肩周炎的有效性 

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

科研

医学

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

肩上粘 Capsulitis 大鼠模型的建立

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

生物工程

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

主动脉阻塞后脊髓缺血大鼠模型中的可再生电机缺陷

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

大鼠膝骨关节炎的标准化推拿手法

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

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

作者聚焦:了解大鼠推拿操作的机制 

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

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

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

大鼠 DRG 压缩模型的标准化 Tuina 协议

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

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

作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用  

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

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

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

冻肩大鼠模型中的推拿:一种高效且可重复的方案

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采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

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肩上粘 Capsulitis 大鼠模型的建立

青光眼诱导在程序

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

主动脉阻塞后脊髓缺血大鼠模型中的可再生电机缺陷

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛