骨质疏松症小鼠模型中的骨小梁微观结构评估
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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…
Representative Results
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?…
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 4% Paraformaldehyde | Biosharp | BL539A | |
| Adobe Photoshop | Adobe Inc. | ||
| Ammonia Solution | Chengdu Kolon Chemical Co., Ltd | 2021070101 | |
| Analyze 12.0 | AnalyzeDirect, Inc | ||
| Anatomical Forceps | Jinzhong surgical instrument Co., Ltd | J3C030 | |
| Anhydrous Ethanol | Chengdu Kolon Chemical Co., Ltd | 2022070501 | |
| Automatic Dyeing Machine | Thermo scientific | Varistain™ Gemini ES | |
| Bone Microarchitecture Analysis Add-on | AnalyzeDirect, Inc | ||
| C57BL/6J mice | SPF (Beijing) Biotechnology Co., Ltd. | ||
| Carrier Slides | Nantong Mei Wei De Experimental Equipment Co., Ltd | 220518001 | |
| Coverslips | Nantong Mei Wei De Experimental Equipment Co. | 220518001 | |
| Decalcification Solution | Wuhan Xavier Biotechnology Co., Ltd | CR2203047 | |
| Delicate Scissors | Jinzhong surgical instrument Co., Ltd | ZJA010 | |
| Embedding box marking machine | Thermo scientific | PrintMate AS | |
| Embedding Machine | Wuhan Junjie Electronics Co., Ltd | JB-P5 | |
| Fiji: ImageJ | National Institutes of Health, USA | ||
| Film Sealer | Thermo scientific | Autostainer 360 | |
| Freezing Table | Wuhan Junjie Electronics Co., Ltd | JB-L5 | |
| H&E Staining Kit | Leagene | DH0020 | |
| Hydrochloric Acid Solution | Sichuan Xilong Science Co., Ltd | 210608 | |
| ImageJ2 Plugin | BoneJ 7.0.16 | ||
| Medical Gauze | Shandong Ang Yang Medical Technology Co. | ||
| Mersilk 3-0 Silk Braided Non-Absorbable Sutures | Ethicon, Inc. | SA84G | |
| Needle Holder | Jinzhong surgical instrument Co., Ltd | J32010 | |
| Neutral Balsam | Sinopharm Group Chemical Reagent Co., Ltd | 10004160 | |
| Oven | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | |
| PANNORAMIC Digital Slide Scanners | 3DHISTECH Ltd. | PANNORAMIC DESK/MIDI/250/1000 | |
| PBS buffer | Biosharp | G4202 | |
| Povidone-iodine solution 5% | Chengdu Yongan Pharmaceutical Co., Ltd | ||
| Quantum GX2 microCT Imaging System | PerkinElmer, Inc. | ||
| Rotary Microtome | Thermo scientific | HM325 | |
| Scalpel | Quanzhou Excellence Medical Co., Ltd | 20170022 | |
| Scan & Browse Software | 3DHISTECH Ltd. | CaseViewer2.4 | |
| Single-Use Sterile Rubber Surgical Gloves | Guangdong Huitong Latex Products Group Co., Ltd | 22B141EO | |
| Sodium Chloride Solution 0.9% | Sichuan Kelun Pharmaceutical Co., Ltd | ||
| Sterile Hypodermic Syringes for Single Use | Shandong Weigao Group Medical Polymer Products Co., Ltd | ||
| Sterile Medical Suture Needles | Shanghai Pudong Jinhuan Medical Products Co., Ltd. | PW8068 | |
| Tissue Processor | Thermo scientific | STP420 ES | |
| Tissue Spreading and Baking Machine | Wuhan Junjie Electronics Co., Ltd | JK-6 | |
| Tribromoethanol | Nanjing Aibei Biotechnology Co., Ltd | M2920 | |
| Wax Trimmer | Wuhan Junjie Electronics Co., Ltd | JXL-818 | |
| Xylene | Chengdu Kolon Chemical Co., Ltd | 2022051901 |
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Citer Cet Article
Li, J., Hu, Y., You, H., Li, R., Ran, Q., Ouyang, T., Huang, Y. Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model. J. Vis. Exp. (199), e65880, doi:10.3791/65880 (2023).