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

Coloration HE et micro-TDM pour évaluer la microarchitecture osseuse trabéculaire murine

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.

Pleins feux sur l’auteur : Une méthode économique et efficace pour l’évaluation quantitative de la microarchitecture osseuse dans un modèle d’ostéoporose murine 

Évaluation de la microarchitecture de l’os trabéculaire dans un modèle murin d’ostéoporose

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

Analyse des images d’hématoxyline-éosine (HE) du tissu osseux trabéculaire murin

Pour commencer l’analyse des images d’hématoxyline et d’éosine ou d’HE du tissu osseux de la souris, ouvrez les images HE avec le logiciel de visualisation de cas. Sélectionnez la région d’intérêt ou le retour sur investissement sur la diapositive et enregistrez-la en tant qu’image couleur. Ouvrez ensuite l’image dans l’image J.Sélectionnez l’outil baguette dans la barre d’outils, puis cliquez sur l’os trabéculaire.Pour ajuster les paramètres de tolérance et de contiguïté selon vos besoins, accédez à Image, puis à Réglages, puis à Noir et blanc. Et cliquez sur OK. Répétez les étapes pour d’autres zones de l’os.Inversez la sélection et remplissez-la de couleur blanche. Cliquez séquentiellement sur le processus binaire et convertissez-le en masque pour convertir l’image couleur en image binaire. Utilisez un plug-in dans le logiciel pour analyser les paramètres structurels.Exécutez la fraction volumique par zone pour calculer le volume osseux par rapport au volume total de l’os. L’analyse histopathologique réalisée par coloration HE du fémur de souris a montré que les os trabéculaires sous la plaque de croissance du fémur distal étaient réduits chez les souris modèles d’ostéoporose ovariectomisées par rapport au groupe opéré simulément. L’analyse quantitative des coupes de tissus a également indiqué que les souris OVX avaient moins de surface osseuse trabéculaire que celles du groupe simulé.

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

Research

JoVE Journal

Medicine

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

177 vues.  Hospital of Chengdu University of Traditional Chinese Medicine. Pour commencer l’analyse des images d’hématoxyline et d’éosine ou d’HE du tissu osseux de la souris, ouvrez les images HE avec le logiciel de visualisation de cas. Sélectionnez la région d’intérêt ou le retour sur investissement sur la diapositive et enregistrez-la en tant qu’image couleur. Ouvrez ensuite l’image dans l’image J.Sélectionnez l’outil baguette dans la barre d’outils, puis cliquez sur l’os trabéculaire.Pour ajuster les paramètres de toléranc...

Vidéo: Analyse des images d’hématoxyline-éosine (HE) du tissu osseux trabéculaire murin

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 ...

Recherche

Médecine

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.

Imagerie par micro-tomodensitométrie (TDM) de l’os trabéculaire murin

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.

Analyse d’images de microtomodensitométrie (TDM) de l’os trabéculaire murin

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.