micro-computed tomography (ct) imaging of murine trabecular bone

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

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.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

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 Micro-Computed Tomography CT Imaging of Murine Trabecular Bone at JoVE.com

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.

micro-computed-tomography-ct-imaging-of-murine-trabecular-bone

Research

JoVE Journal

Medicine

157 Views.  Hospital of Chengdu University of Traditional Chinese Medicine. 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, Cu...

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

trabecular bone microarchitecture evaluation in an osteoporosis mouse model

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)...

https://cloudfront.jove.com/CDNSource/teasers/65880_b.jpg

analyzing micro-computed tomography (ct) images of murine trabecular bone

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

assessment of global ocular structure following spaceflight using a micro-computed tomography (micro-ct) imaging method

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

assessment of bone fracture healing using micro-computed tomography

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

proper positioning and restraint of a rat hind limb for focused high resolution imaging of bone micro-architecture using in vivo micro-computed tomography

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...

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

combined near-infrared fluorescent imaging and micro-computed tomography for directly visualizing cerebral thromboemboli

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using...

retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT)...

non-invasive skeletal muscle quantification in small animals using micro-computed tomography

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small...

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

micro-ct imaging and morphometric analysis of mouse neonatal brains

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &amp;#8211; A Micro-CT Protocol with Lugol&amp;#39;s Solution Contrast Agent

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

https://cloudfront.jove.com/CDNSource/teasers/65180_b.jpg

tissue preparation techniques for contrast-enhanced micro computed tomography imaging of large mammalian cardiac models with chronic disease

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast...

novel in vivo micro-computed tomography imaging techniques for assessing the progression of non-alcoholic fatty liver disease

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT)...

computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft...

induction and micro-ct imaging of cerebral cavernous malformations in mouse model

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular...

assessment of viability of human fat injection into nude mice with micro-computed tomography

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival.  Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and...

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

quantification of atherosclerotic plaque activity and vascular inflammation using [18-f] fluorodeoxyglucose positron emission tomography/computed tomography (fdg-pet/ct)

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography FDG-PET/CT

There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular...

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)

high resolution 3d imaging of ex-vivo biological samples by micro ct

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (...

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

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 ...

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.

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

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.