analyzing the hematoxylin-eosin (he) images of murine trabecular bone tissue

HE 染色和 micro-CT 评估小鼠骨小梁微结构

Osteoporosis is a prevalent metabolic bone disease, especially among the elderly, and is associated with an increased risk of fragility fractures. As osteoporosis becomes more common in China1, there will be a growing demand for studying the bone structures of small animals2,3. The previous methods of measuring bone loss rely on the outcomes of two-dimensional dual-energy x-ray absorptiometry. However, this does not capture the changes in the architectural microstructure of the trabecular bone, which is a key factor for skeletal strength4. The microstructure of bone affects its strength, stiffness, and fracture resistance. By comparing bone microarchitecture in normal and pathological states, changes in bone tissue morphology, structure, and function caused by osteoporosis can be identified. This information contributes to the understanding of the development of osteoporosis and its association with other diseases. 
Micro-computed tomography (Micro-CT) imaging has recently become a popular technique for bone morphology assessment, where it can provide accurate and comprehensive data on bone structure and density parameters such as bone volume fraction, thickness, and separation5,6. At the same time, the Micro-CT results can be affected by the analysis software7. Different methods of image acquisition, evaluation, and reporting are used by various commercial Micro-CT systems. This inconsistency makes it hard to compare and interpret the results reported by different studies5. Also, it cannot currently replace bone histomorphometry in providing researchers with information on cellular-level parameters in the skeletal system8. Meanwhile, histologic techniques allow direct observation and measurement of the microscopic morphology of bone. Hematoxylin and eosin (HE) staining is a common staining technique used in histology to visualize the general structure of cells and tissues. It is used to identify the presence of bone tissue and its microarchitecture.
This article uses Micro-CT combined with tissue slicing technique (Hematoxylin-Eosin [HE] staining) to collect bone tissue images and perform quantitative analysis of trabecular bone to evaluate the changes of bone microstructure in an osteoporosis mouse model.

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 

骨质疏松症小鼠模型中的骨小梁微观结构评估

Our research focused on the effects of traditional Chinese medicine, or TCM, on osteoporosis. We are trying to explore the mechanism, efficacy, and safety of TCM intervention for osteoporosis. Micro-computed tomography imaging has recently become a popular technique for bone morphology assessment.It can provide accurate and comprehensive data on bone structure and density parameters, such as bone volume, fracture thickness, and separation. The skeletal system is the most unique tissue structure in terms of the physical properties of the body. It contains the inorganic component arginine calcium phosphate, and it is hard and stiff as a result.The process of hurting bone symptoms is tedious. We provided a complete and detailed process from modeling to osteoporosis in evaluation for researchers. We believe the application of the technology will undoubtedly bring great improvements to osteoporosis research in the context of ethnomedicine exploration.

Watch this Scientific Journal Video about Analyzing the Hematoxylin-Eosin HE Images of Murine Trabecular Bone Tissue at JoVE.com

分析小鼠小梁骨组织的苏木精-伊红 （HE） 图像

要开始分析小鼠骨组织的苏木精和伊红或HE图像，请使用病例查看器软件打开HE图像。在幻灯片上选择感兴趣区域或 ROI，并将其保存为彩色图像。然后打开图像J.从工具栏中选择魔杖工具，然后单击小梁骨。要根据需要调整公差和连续设置，请转到图像，然后转到调整，然后是黑白。然后单击确定。对骨骼的其他区域重复这些步骤。反转选区并用白色填充。依次单击处理二进制并转换为掩码，将彩色图像转换为二进制图像。使用软件中的插件来分析结构参数。按体积分数运行面积，以计算骨体积与骨总体积之比。对小鼠股骨HE染色进行的组织病理学分析显示，与假手术组相比，卵巢切除骨质疏松模型小鼠股骨远端生长板下的小梁骨减少。组织切片的定量分析也表明，OVX小鼠的小梁骨面积小于假手术组。

analyzing-hematoxylin-eosin-he-images-murine-trabecular-bone

Research

JoVE Journal

Medicine

Analyzing the Hematoxylin-Eosin (HE) Images of Murine Trabecular Bone Tissue

176 次观看.  Hospital of Chengdu University of Traditional Chinese Medicine. 要开始分析小鼠骨组织的苏木精和伊红或HE图像，请使用病例查看器软件打开HE图像。在幻灯片上选择感兴趣区域或 ROI，并将其保存为彩色图像。然后打开图像J.从工具栏中选择魔杖工具，然后单击小梁骨。要根据需要调整公差和连续设置，请转到图像，然后转到调整，然后是黑白。然后单击确定。对骨骼的其他区域重复这些步骤。反转选区并用白色填充。依次单?...

视频: 分析小鼠小梁骨组织的苏木精-伊红 （HE） 图像

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

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

科研

医学

Micro-Computed Tomography (CT) Imaging of Murine Trabecular Bone

To begin the micro-computed tomography, or micro CT imaging, remove as much soft tissue as possible from the bone. Open the micro CT imaging system software by double-clicking its icon on the computer. Select the sample bed compatible with the sample field of view, or FOV, of 18 millimeters by 18 millimeters.Load the sample bed into the machine and close the hatch. In the Control Panel, select Manual in the preset option in the Scan Acquisition Settings. Set Voltage kilovolts to 90, Current microamperes to 80, Scan Mode to high resolution 14 minutes, and FOV millimeters to 18 millimeters by 18 millimeters.Position the bone securely with plastic film in the sample bed and close the hatch. Click on the Live button to observe the sample. And using the adjustment buttons on the machine, center the subject precisely in the X-capture window.Click the Start button to start the CT scan.

小鼠骨小梁的显微计算机断层扫描 （CT） 成像

Analyzing Micro-Computed Tomography (CT) Images of Murine Trabecular Bone

To begin analyzing the micro-computed tomography, or micro CT images, in the micro CT imaging system software, select the data to be analyzed. Click Sub, and set pixel size to 10 microns. Move and resize the region of interest, or ROI, to include the distal femur above the growth plate.Then, click on Start. Next, click the Analysis 3D button to get the resulting 3D reconstruction. Export the data from the micro CT system to the computer for analysis.After importing the reconstructed CT data, click Process and Image Calculator. Then sequentially click Region Pad and Interactive. Adjust the yellow check box to the appropriate ROI.After exiting the image calculator, select the bone microarchitecture analysis, or BMA add-on, and click Segment Cortex and Segment Trabeculae. Finally, click on Measure Bone to calculate the bone morphometric indices. The trabecular micro-architectural parameters were measured in mice from two groups, the sham-operated group and the ovariectomized osteoporosis model group, or OVX group.The results indicated significant differences between mice of the two groups. The ratio of bone volume to total tissue volume in the OVX group was 3%lower than that in the sham group. The trabecular thickness in mice of the OVX group was lower than in the sham group, and the trabecular separation in the OVX mice was greater than that in the sham mice.3D displays of trabecular ROIs extracted from reconstructed bone volume revealed that compared to the sham group, in the OVX mice, the trabeculae was sparse and showed osteoporosis.

分析小鼠小梁骨的显微计算机断层扫描 （CT） 图像

To begin analysis of the Hematoxylin and Eosin or HE images of the mouse bone tissue, open the HE images with case viewer software. Select the region of interest or ROI on the slide and save it as a color image. Then open the image in image J.Select the wand tool from the toolbar, and click on the trabecular bone.To adjust the tolerance and contiguous settings as needed, go to image, then adjustments, followed by black and white. And click okay. Repeat the steps for other areas of the bone.Invert the selection and fill it with white color. Sequentially click on process binary and convert to mask to convert the color image to a binary image. Use a plugin in the software to analyze structural parameters.Run area by volume fraction to calculate bone volume to total volume of bone. The histopathological analysis performed by HE staining of mice femur showed the trabecular bones under the growth plate of the distal femur were reduced in the ovariectomized osteoporosis model mice when compared to the sham operated group. The quantitative analysis of tissue sections also indicated that the OVX mice had less trabecular bone area than those in the sham group.

分析小鼠小梁骨组织的苏木精-伊红 （HE） 图像

分析小鼠小梁骨组织的苏木精-伊红（HE）图像