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。

<table><tbody><tr><td>4% Paraformaldehyde</td><td>Biosharp</td><td>BL539A</td><td></td></tr><tr><td>Adobe Photoshop</td><td>Adobe Inc.</td><td></td><td></td></tr><tr><td>Ammonia Solution</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2021070101</td><td></td></tr><tr><td>Anatomical Forceps</td><td>Jinzhong surgical instrument Co., Ltd</td><td>J3C030</td><td></td></tr><tr><td>Anhydrous Ethanol</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022070501</td><td></td></tr><tr><td>Automatic Dyeing Machine</td><td>Thermo scientific</td><td>Varistain&amp;trade; Gemini ES</td><td></td></tr><tr><td>Bone Microarchitecture Analysis Add-on</td><td>AnalyzeDirect, Inc</td><td></td><td></td></tr><tr><td>C57BL/6J mice</td><td>SPF (Beijing) Biotechnology Co., Ltd.</td><td></td><td></td></tr><tr><td>Carrier Slides</td><td>Nantong Mei Wei De Experimental Equipment Co., Ltd</td><td>220518001</td><td></td></tr><tr><td>Coverslips</td><td>Nantong Mei Wei De Experimental Equipment Co.</td><td>220518001</td><td></td></tr><tr><td>Decalcification Solution</td><td>Wuhan Xavier Biotechnology Co., Ltd</td><td>CR2203047</td><td></td></tr><tr><td>Delicate Scissors</td><td>Jinzhong surgical instrument Co., Ltd</td><td>ZJA010</td><td></td></tr><tr><td>Embedding box marking machine</td><td>Thermo scientific</td><td>&amp;nbsp;PrintMate AS</td><td></td></tr><tr><td>Embedding Machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-P5</td><td></td></tr><tr><td>Fiji: ImageJ</td><td>National Institutes of Health, USA</td><td></td><td></td></tr><tr><td>Film Sealer</td><td>Thermo scientific</td><td>Autostainer 360</td><td></td></tr><tr><td>Freezing Table</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-L5</td><td></td></tr><tr><td>H&amp;amp;E Staining Kit</td><td>Leagene</td><td>DH0020</td><td></td></tr><tr><td>Hydrochloric Acid Solution</td><td>Sichuan Xilong Science Co., Ltd</td><td>210608</td><td></td></tr><tr><td>ImageJ2 Plugin</td><td></td><td>BoneJ 7.0.16</td><td></td></tr><tr><td>Medical Gauze</td><td>Shandong Ang Yang Medical Technology Co.</td><td></td><td></td></tr><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></tr><tr><td>Neutral Balsam</td><td>Sinopharm Group Chemical Reagent Co., Ltd</td><td>10004160</td><td></td></tr><tr><td>Oven</td><td>Shanghai Yiheng Scientific Instruments Co., Ltd</td><td>DHG-9240A</td><td></td></tr><tr><td>PANNORAMIC Digital Slide Scanners</td><td>3DHISTECH Ltd.&amp;nbsp;</td><td>PANNORAMIC DESK/MIDI/250/1000</td><td></td></tr><tr><td>PBS buffer</td><td>Biosharp</td><td>G4202</td><td></td></tr><tr><td>Povidone-iodine solution 5%</td><td>Chengdu Yongan Pharmaceutical Co., Ltd</td><td></td><td></td></tr><tr><td>Quantum GX2 microCT Imaging System</td><td>PerkinElmer, Inc.</td><td></td><td></td></tr><tr><td>Rotary Microtome</td><td>Thermo scientific</td><td>HM325</td><td></td></tr><tr><td>Scalpel</td><td>Quanzhou Excellence Medical Co., Ltd</td><td>20170022</td><td></td></tr><tr><td>Scan &amp;amp; Browse Software</td><td>3DHISTECH Ltd.&amp;nbsp;</td><td>CaseViewer2.4</td><td></td></tr><tr><td>Single-Use Sterile Rubber Surgical Gloves</td><td>Guangdong Huitong Latex Products Group Co., Ltd</td><td>22B141EO</td><td></td></tr><tr><td>Sodium Chloride Solution 0.9%</td><td>Sichuan Kelun Pharmaceutical Co., Ltd</td><td></td><td></td></tr><tr><td>Sterile Hypodermic Syringes for Single Use</td><td>Shandong Weigao Group Medical Polymer Products&amp;nbsp; Co., Ltd</td><td></td><td></td></tr><tr><td>Sterile Medical Suture Needles</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.&amp;nbsp;</td><td>PW8068</td><td></td></tr><tr><td>Tissue Processor</td><td>Thermo scientific</td><td>STP420 ES</td><td></td></tr><tr><td>Tissue Spreading and Baking Machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JK-6</td><td></td></tr><tr><td>Tribromoethanol</td><td>Nanjing Aibei Biotechnology Co., Ltd</td><td>M2920</td><td></td></tr><tr><td>Wax Trimmer</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JXL-818</td><td></td></tr><tr><td>Xylene</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022051901</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

2.1K 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 

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

科研

生物工程

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

用 Imagej 插件对小梁骨的外边界辅助分割和量化

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

医学

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, ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17&#946;-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, &#946;-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

<meta charset="utf8"></head><body>八紫布参胶囊对去卵巢大鼠的雌激素样作用

Presented here is a protocol to show that Bazi Bushen capsule (BZBS) can regulate the RANKL/OPG signaling pathway in the ovariectomized rodent model through its estrogen-like effect.

八紫布参胶囊对去卵巢大鼠的雌激素样作用

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley ...

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

有条件 统计3 删除鼠标模型的骨骼表型分析

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

生物学

<meta charset="utf8"></head><body>类固醇诱导的股骨头骨坏死增强的 Elastic Tape 大鼠模型

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

<meta charset="utf8"></head><body>作者聚焦:开发用于负重干预的大鼠模型以研究股骨头骨坏死

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

类固醇诱导大鼠股骨头骨坏死的无张力负重模型

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure ...

骨质疏松症小鼠模型中的骨小梁微观结构评估

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半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

用 Imagej 插件对小梁骨的外边界辅助分割和量化

CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价

手术小鼠模型中骨关节炎的标准化形态学评价

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

八紫布参胶囊对去卵巢大鼠的雌激素样作用

八紫布参胶囊对去卵巢大鼠的雌激素样作用

有条件统计3 删除鼠标模型的骨骼表型分析

类固醇诱导大鼠股骨头骨坏死的无张力负重模型

骨质疏松症小鼠模型中的骨小梁微观结构评估

副本

摘要

探索更多视频

此视频中的章节

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

用 Imagej 插件对小梁骨的外边界辅助分割和量化

CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价

手术小鼠模型中骨关节炎的标准化形态学评价

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

八紫布参胶囊对去卵巢大鼠的雌激素样作用

八紫布参胶囊对去卵巢大鼠的雌激素样作用

有条件 统计3 删除鼠标模型的骨骼表型分析

类固醇诱导大鼠股骨头骨坏死的无张力负重模型

有条件统计3 删除鼠标模型的骨骼表型分析