这项工作得到了四川省中医药管理局（2021YJ0175）和成都中医药大学临床医学院研究生科研创新项目（LCYJSKT2023-11）的支持。

该动物方案已获得成都中医药大学动物伦理委员会批准（备案号：2020-34）。雌性C57BL/6J小鼠（12周龄，n = 14）随机分为两组，假手术组（假手术组，n = 7）和模型组（OVX组，n = 7）。动物是从商业供应商处购买的（见 材料表）。将所有小鼠保持在22-26°C，湿度为45%-55%的单独笼子中，使其适应新环境1周，并免费获得水和饮食。所有动物实验研究均在成都中医药大学进行，并尽一切努力将动物的痛苦降到最低。
1.动物模型制备
<ol><li>通过腹膜内注射1.25%Avertin（叔戊醇中的三溴乙醇，参见 材料表）麻醉12周龄小鼠，剂量为0.02mL / g。将小鼠俯卧在无菌手术台上，并通过牢固地固定其四肢来固定其四肢。</li>
	<li>使用剪刀（见 材料表）修剪任何可能影响外科手术的头发。 注意：建议在准备手术部位时避免皮肤损伤。</li>
	<li>彻底洗手并戴上外科手套。用聚维酮碘对小鼠背部进行三次消毒（见材料表），并使用医用纱布（见材料表）将其擦干。 注意：谨慎;在消毒过程中让头发太湿可能会导致小鼠术后体温过低。</li>
	<li>使用手术刀在距鼠标背部中线三分之一 1 厘米处切开约 0.5-1.0 厘米长的切口（参见 材料表）。轻轻地分离筋膜并用组织剪刀切开肌肉（参见 材料表），直到卵巢可见。用不可吸收的缝合线结扎周围的血管（见 材料表）并切除卵巢。</li>
	<li>用0.9%生理盐水冲洗腔体，然后分别缝合皮肤、肌肉和筋膜（参见 材料表）。</li>
	<li>在另一侧重复相同的步骤序列。</li>
	<li>去除与卵巢大小相同的卵巢周围脂肪，并执行上述相同的手术程序，以建立假手术模型的其余步骤。</li>
	<li>手术后留出 1 周的恢复期。8周后，骨质疏松小鼠模型将成功建立9,10。</li>
</ol>2. 显微CT扫描
<ol><li>通过吸入过量的CO2 对小鼠实施安乐死。安乐死后尽可能多地从小鼠股骨中取出软组织，并获得新鲜样本进行扫描。 注意：虽然没有完美的样品保存解决方案，但可以采取某些步骤来最大限度地提高 Micro-CT 扫描结果的质量。固定前，注意尽可能多地去除周围组织。福尔马林或缓冲福尔马林是最优选的固定方法，储存在 PBS11,12 中。</li>
	<li>双击桌面上的显微CT成像系统软件图标启动系统（见 材料表）。选择与 18 mm x 18 mm 的样品视场 （FOV） 兼容的样品床。将适当的样品床装入机器并关闭舱口。</li>
	<li>单击“控制面板”中的“预热”按钮。 注意：需要很短的预热时间。请勿在产生 X 射线时尝试打开舱口。检查前面板和计算机显示器上的X 射线开启 指示灯，以确认是否正在生成X射线。</li>
	<li>单击 “菜单 ”按钮设置新数据库，然后创建新样品和研究。</li>
	<li>从菜单下拉列表中选择手动，然后在控制面板中输入自定义电压和电流值。将电压 （kV） 设置为 90，将电流 （μA） 设置为 80，将扫描模式设置为高分辨率 14 分钟，将 FOV （mm） 设置为 18 mm x 18 mm。 注意： 请参阅不同微型 CT 系统的手册以设置适当的参数。</li>
	<li>用塑料薄膜将骨头牢固地放置在样品床中。关闭舱口。按下机器上的调整按钮，确保拍摄对象在 X-capture 窗口中精确居中。 注意： 调整样品必须缓慢而轻柔，以免意外掉入机器中。</li>
	<li>单击 “开始 ”按钮开始 CT 扫描。</li>
</ol>3. CT数据分析
<ol><li>进入软件（见 材料表），选择要分析的数据。单击 “子 ”（Sub） 并将 “像素大小”（Pixel Size ） 设置为 10 μm。移动感兴趣区域 （ROI） 并调整其大小，以包括生长板上方的股骨远端。单击 “开始 ”（参见 图 1）。 注：亚体积重建像素大小选择在 10 μm，因为根据先前研究的组织学数据，成像小梁的厚度估计约为 20-30 μm13。如果信噪比不足以进行数据处理，则必须执行高分辨率 1 小时扫描。</li>
	<li>单击 “分析 3D ”按钮以获取生成的 3D 重建。</li>
	<li>将数据从显微CT系统导出到计算机进行分析。</li>
	<li>导入重建的 CT 数据。 单击“处理>图像计算器”，然后单击“ 区域填充”>“交互式”。将黄色复选框调整为适当的 ROI。 注意：感兴趣区域 （ROI） 从生长板近端约 540 μm 开始，向近端延伸 1600 μm，以评估实际的骨代谢和重塑。</li>
	<li>选择骨微结构分析 （BMA） 模块（参见 材料表）。单击 “分割皮层 ”，然后单击 “分割小梁”。 注意：小梁骨和皮质骨都是自动选择的。通常不需要手动调整。</li>
	<li>单击“ 保存最终对象映射 ”，然后单击 “ 测量骨骼”以计算骨骼形态测量指数。 注意：由于未添加对照，因此此处仅计算相对骨矿物质密度（BMD）。</li>
</ol>4.骨组织脱钙
<ol><li>将骨标本固定在4%多聚甲醛（见 材料表）中24小时。每次用PBS（参见 材料表）洗涤试样三次20分钟。</li>
	<li>每次用蒸馏水冲洗组织三次，每次20分钟。将组织转移到含有EDTA的脱钙溶液中（参见 材料表）并脱钙30天，每周更换溶液直至终点。 注意：当骨组织变软或针刺时没有阻力感时，使用针刺、手捏和夹紧来终止脱钙。物理检测方法会对组织结构造成一定的损伤，因此请尽量避免用力过大或重复检测。</li>
	<li>将组织从固定剂中取出，然后用手术刀在通风橱中修剪组织。将修剪后的组织和相应的标签放入脱水盒中。</li>
	<li>将脱水盒放入篮子中，用梯度酒精（75%乙醇4小时，85%乙醇2小时，90%乙醇2小时，95%乙醇1小时，无水乙醇30分钟，无水乙醇II30分钟，二甲苯I5-10分钟， 二甲苯II为5-10分钟，蜡I为1小时，蜡II为1小时，蜡III为1小时）。</li>
	<li>使用包埋机（见 材料表）嵌入蜡浸泡的组织。将熔化的蜡倒入包埋盒中，并在蜡变硬之前将脱水盒中的组织放入包埋盒中。</li>
	<li>根据包埋面定向组织并贴上相应的标签。在-20°C冷冻台上冷却（参见 材料表）。凝固后从包埋盒中取出蜡块，根据需要修剪蜡块（见 材料表）。</li>
	<li>在切片机上将修剪好的蜡块切成3μm厚的载玻片（参见 材料表）。将载玻片漂浮在组织铺展机上的40°C温水上（参见 材料表），使组织变平并用载玻片舀起。</li>
	<li>在60°C的烤箱中烘烤（见 材料表），直到水分蒸发，蜡融化。取出并存放在室温 （RT） 下以备后用。</li>
</ol>5. HE染色
<ol><li>将载玻片放入二甲苯I中20分钟，然后在二甲苯II中放置20分钟。此外，将载玻片分别置于无水乙醇I和无水乙醇II中5分钟，75%酒精中5分钟，然后用自来水洗涤载玻片（参见 材料表）。</li>
	<li>在苏木精（见 材料表）中孵育3-5分钟。用盐酸溶液区分试样（参见 材料表）。用氨溶液（见 材料表）处理载玻片发蓝，然后用水清洗载玻片。</li>
	<li>将载玻片放入85%，95%梯度酒精中脱水，并用伊红溶液（参见 材料表）染色5分钟。</li>
	<li>将载玻片放入无水乙醇I中5分钟，无水乙醇II中5分钟，无水乙醇III中5分钟。用二甲苯I处理载玻片5分钟，用二甲苯II处理5分钟，并用中性香脂安装（参见 材料表）。</li>
	<li>在显微镜下检查每个载玻片。然后，选择具有代表性的切片进行全景扫描（参见 材料表）。 注意：每个样品至少需要三个连续的切片。在全景扫描之前，必须在显微镜下检查切片，以确保它们完整和清晰。确保骨髓腔、皮质骨和松质骨完全显示，并且可以看到不同类型的骨细胞。</li>
</ol>6. HE图像分析
<ol><li>使用CaseViewer软件打开HE图像（参见 材料表）。在幻灯片上选择感兴趣区域 （ROI），并将其另存为彩色图像。 注意：ROI的选择与上述软件一致。</li>
	<li>在 ImageJ 中打开图像（参见 材料表）。从工具栏中选择 魔杖 工具。点击小梁骨。</li>
	<li>根据需要调整公差和连续设置。转到 “图像>调整”>“黑白”， 然后单击 “确定”。对骨骼的其他区域重复这些步骤。</li>
	<li>反转选区并用白色填充。将图像另存为掩码以供分析（参见图2）。 注：有关详细方法，请参阅先前发表的著作14。</li>
	<li>在分析软件15、16 中打开掩模（见 材料表）。运行 Process > Binary > Make Binary ，将彩色图像转换为二进制图像。</li>
	<li>使用软件中的插件17（参见材料表）分析结构参数：运行面积/体积分数以计算骨体积与骨总体积之比 （BV/TV [%]）18,19。 注：此方法适用于骨小梁和骨髓，填充HE图像中的整个ROI。</li>
</ol>

HE 染色和 micro-CT 评估小鼠骨小梁微结构

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+trabecular+architecture+differ+in+female+and+male+C57BL/6J+mice.">Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.</a> Journal of Bone and Mineral Research. 22 (8), 1197-1207 (2007).</li><li>Seeman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+and+biomechanical+basis+of+the+gain+and+loss+of+bone+strength+in+women+and+men.">The structural and biomechanical basis of the gain and loss of bone strength in women and men.</a> Endocrinology and Metabolism Clinics. 32 (1), 25-38 (2003).</li><li>Ticha, 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+novel+cryo-embedding+method+for+in-depth+analysis+of+craniofacial+mini+pig+bone+specimens.">A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens.</a> Scientific Reports. 10 (1), 19510 (2020).</li><li>Genant, H. K., Engelke, K., Prevrhal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CT+bone+imaging+in+osteoporosis.">Advanced CT bone imaging in osteoporosis.</a> Rheumatology. 47 (suppl_4), iv9-iv16 (2008).</li><li>Zaw Thin, M., Moore, C., Snoeks, T., Kalber, T., Downward, J., Behrens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+acquisition+and+image+processing+to+track+and+characterize+pulmonary+nodules+in+mice.">Micro-CT acquisition and image processing to track and characterize pulmonary nodules in mice.</a> Nature Protocols. 18 (3), 990-1015 (2023).</li></ol>

作者没有什么可透露的。

骨质疏松症可导致频繁骨折，代价高昂，可引起疼痛、残疾，甚至死亡，严重影响患者的生活质量20。多年来，卵巢切除术模型已被公认为研究骨质疏松症的标准方法之一21。骨质疏松症最常见的临床前动物模型是卵巢切除术（OVX）大鼠。尽管如此，对骨骼疾病（包括骨质疏松症）机制的大多数研究都是使用小鼠进行的 22.为了在成年雌性C57 / BL...

骨质疏松症是一种普遍存在的代谢性骨病，尤其是在老年人中，并且与脆性骨折的风险增加有关。随着骨质疏松症在中国变得越来越普遍1，对研究小动物骨骼结构的需求将越来越大2,3。以前的骨质流失测量方法依赖于二维双能X射线吸收测定法的结果。然而，这并不能捕捉到小梁结构微观结构的变化，而小梁骨是骨骼强度的关键因素4.骨骼的微观结构会影响其强度、刚度和抗断裂性。通过比较正常和病理状态下的骨微结构，可以识别骨质疏松症引起的骨组织形态、结构和功能的变化。这些信息有助于了解骨质疏松症的发展及其与其他疾病的关联。
显微计算机断层扫描 （Micro-CT） 成像最近已成为骨形态学评估的流行技术，它可以提供有关骨结构和密度参数（如骨体积分数、厚度和分离度）的准确和全面的数据 5,6。同时，Micro-CT结果会受到分析软件7的影响。各种商用 Micro-CT 系统使用不同的图像采集、评估和报告方法。这种不一致使得很难比较和解释不同研究报告的结果5.此外，它目前不能取代骨组织形态学，为研究人员提供有关骨骼系统中细胞水平参数的信息8。同时，组织学技术可以直接观察和测量骨骼的微观形态。苏木精和伊红 （HE） 染色是组织学中常用的染色技术，用于可视化细胞和组织的一般结构。它用于识别骨组织的存在及其微结构。
本文采用显微CT结合组织切片技术（苏木精-伊红[HE]染色）采集骨组织图像，对骨小梁骨进行定量分析，以评估骨质疏松症小鼠模型中骨微结构的变化。

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			<td>Jinzhong surgical instrument Co., Ltd</td>
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			<td>Fiji: ImageJ</td>
			<td>National Institutes of Health, USA</td>
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			<td>Film Sealer</td>
			<td>Thermo scientific</td>
			<td>Autostainer 360</td>
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			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-L5</td>
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			<td>Leagene</td>
			<td>DH0020</td>
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			<td>Hydrochloric Acid Solution</td>
			<td>Sichuan Xilong Science Co., Ltd</td>
			<td>210608</td>
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			<td>ImageJ2 Plugin</td>
			<td></td>
			<td>BoneJ 7.0.16</td>
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			<td>Medical Gauze</td>
			<td>Shandong Ang Yang Medical Technology Co.</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Mersilk 3-0 Silk Braided Non-Absorbable Sutures</td>
			<td>Ethicon, Inc.</td>
			<td>SA84G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Jinzhong surgical instrument Co., Ltd</td>
			<td>J32010</td>
			<td></td>
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			<td>Neutral Balsam</td>
			<td>Sinopharm Group Chemical Reagent Co., Ltd</td>
			<td>10004160</td>
			<td></td>
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			<td>Oven</td>
			<td>Shanghai Yiheng Scientific Instruments Co., Ltd</td>
			<td>DHG-9240A</td>
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			<td>PANNORAMIC Digital Slide Scanners</td>
			<td>3DHISTECH Ltd.&#160;</td>
			<td>PANNORAMIC DESK/MIDI/250/1000</td>
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			<td>G4202</td>
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			<td>Chengdu Yongan Pharmaceutical Co., Ltd</td>
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			<td>PerkinElmer, Inc.</td>
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			<td>Thermo scientific</td>
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			<td>Quanzhou Excellence Medical Co., Ltd</td>
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			<td>3DHISTECH Ltd.&#160;</td>
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trabecular bone microarchitecture evaluation in an osteoporosis mouse model

骨微观结构是指骨组织在微观水平上的排列和质量。了解骨骼的骨骼微观结构对于深入了解骨质疏松症的病理生理学并改善其治疗至关重要。然而，由于骨样本的坚硬和致密特性，处理骨样本可能很复杂。其次，专用软件使图像处理和分析变得困难。在该协议中，我们提出了一种具有成本效益且易于使用的骨小梁微观结构分析解决方案。提供了详细的步骤和注意事项。Micro-CT 是一种无损三维 （3D） 成像技术，可提供骨小梁结构的高分辨率图像。它可以对骨质量进行客观和定量的评估，这就是为什么它被广泛认为是骨质量评估的金标准方法。然而，组织形态测量仍然是必不可少的，因为它提供了关键的细胞水平参数，弥合了骨标本的二维 （2D） 和 3D 评估之间的差距。至于组织学技术，我们选择对骨组织进行脱钙，然后进行传统的石蜡包埋。综上所述，结合这两种方法可以提供更全面、更准确的骨微观结构信息。

显微CT分析 我们测量了两组小鼠的小梁微观结构参数，并在表1中报告了它们的平均值和SD。图3显示了每组内一些参数（即骨体积与总组织体积的比率、小梁厚度、小梁分离）的分布。
这些结果表明，OVX组和假手术组小鼠在Micro-CT估计的许多参数上存在显着差异。即OVX组骨体积与总组织体?...

Watch this Scientific Journal Video about An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model at JoVE.com

作者聚焦：一种经济有效的小鼠骨质疏松症模型中骨微结构定量评估的方法 

该协议提供了一种经济有效的方法，通过结合苏木精-伊红（HE）染色和显微计算机断层扫描（Micro-CT）技术，定量评估骨质疏松症小鼠模型中的骨微结构。

骨质疏松症小鼠模型中的骨小梁微观结构评估

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

我们的研究重点是中医或中医对骨质疏松症的影响。我们正在努力探索中医干预骨质疏松症的机制、疗效和安全性。显微计算机断层扫描成像最近已成为骨形态学评估的流行技术。它可以提供有关骨结构和密度参数的准确和全面的数据，例如骨体积、骨折厚度和分离。就身体的物理特性而言，骨骼系统是最独特的组织结构。它含有无机成分精氨酸磷酸钙，因此坚硬而坚硬。伤害骨骼症状的过程是乏味的。我们为研究人员提供了一个从建模到骨质疏松症的完整而详细的评估过程。我们相信，该技术的应用无疑将为骨质疏松症研究带来巨大的进步，在民族医学探索的背景下。

trabecular-bone-microarchitecture-evaluation-an-osteoporosis-mouse

Microsoft Bing

Research

JoVE Journal

Medicine

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

2.0K 次观看.  Hospital of Chengdu University of Traditional Chinese Medicine. 我们的研究重点是中医或中医对骨质疏松症的影响。我们正在努力探索中医干预骨质疏松症的机制、疗效和安全性。显微计算机断层扫描成像最近已成为骨形态学评估的流行技术。它可以提供有关骨结构和密度参数的准确和全面的数据，例如骨体积、骨折厚度和分离。就身体的物理特性而言，骨骼系统是最独特的组织结构。它含有无机成分精氨酸磷酸钙，因此坚硬而坚?...

视频: 作者聚焦：一种经济有效的小鼠骨质疏松症模型中骨微结构定量评估的方法 

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

科研

医学

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 ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

万古霉素滴入硫酸钙和自体骨治疗兔骨感染的改进模型

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

多囊卵巢综合征小鼠模型中肝葡萄糖产生的评价

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

评估培养骨细胞和分离骨干中的氨基酸消耗

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

<meta charset="utf8"></head><body>岭南火针疗法治疗大鼠骨质疏松症

Operation Procedure and Precautions of Lingnan Fire-Needle Therapy in Osteoporosis Model Rats

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected effects on some chronic diseases and incurable diseases. Osteoporosis (OP) has a high incidence in postmenopausal women and middle-aged and elderly men, and the treatment cycle is long. According to Traditional Chinese Medicine (TCM), Lingnan fire-needle therapy has shown potential in treating osteoporosis. However, there is still a long way to go before it can be widely used. This article focuses on the application of Lingnan fire-needle therapy in the intervention of OP in rats. It covers the selection of needle tools, acupuncture point selection, positioning of rats' bodies, and fixation methods. We also outline the steps and precautions to be taken during and after needling with fire needles. The experiment was done with three groups: a normal group, a model group, and a fire-needle group, each containing 10 rats. The rats in the fire-needle group were treated with fire-needle intervention for six sessions. After the intervention period, we collected femoral specimens and performed micro-CT scans. The results suggest that fire needling can enhance bone morphology and mineral density in OP rats. This information can serve as a methodological basis for conducting basic research on fire-needle therapy.

<meta charset="utf8"></head><body>作者聚焦：探索岭大火针疗法对骨质疏松症干预的影响

Here, we present a protocol for the application of Lingnan Fire-Needle therapy in the intervention of osteoporosis model rats.

岭南火针疗法治疗骨质疏松模式大鼠的操作流程及注意事项

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected ...