This research was funded by Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ; E-26/010.002643/2019 and E-26/201.335/2022), Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior -Brazil (CAPES)-001 Finance Code. Programa Institucional de bolsas de inicia&#231;&#227;o cient&#237;fica da Universidade Federal do Rio de Janeiro. National Council for Scientific and Technological Development (CNPq; FFB: 001. 306236/2022-2 TMO-C: 309339/2023-5). The authors acknowledge National Center for Structural Biology and Bioimaging (CENABIO)/ Universidade Federal do Rio de Janeiro for the use of its facilities, especially the microPET/SPECT/CT platform at the Small Animal Imaging Unit (UIPA). Supplemental&#160;Figure S1 was created with BioRender.com.

All methods were approved by the Institutional Animal Care and Use Committee of the Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro (IACUC - UFRJ; A16/23-025-20). Scans were performed on male C57BL/6 mice aged 6 and 22 months.
1. Animal preparation
<ol>
	<li>Anesthetize mice using 2% isoflurane in a mixture of oxygen delivered via vaporization. Use a precision vaporizer for both induction and maintenance with a mixture of 100% oxygen using a precision vaporizer.</li>
	<li>Position the anesthetized animal supine on the micro-CT scanning using a mouse bed. Secure the mouse with tape to minimize movement during the scan and then insert it into the gantry.</li>
	<li>Throughout the experiment, which lasts approximately 10 min, ensure that the animal wears a mask to keep it under the anesthetic plane while receiving the maintenance dose as needed.</li>
</ol>
2. Image acquisition and reconstruction
<ol>
	<li>Acquire body micro-CT scans using a high-resolution preclinical imaging system. Capture a total of 1,024 projections with an exposure time of 470 ms each, using a fly mode rotation at a voltage of 60 kV and a current of 480 &#181;A. Set the system to a 1.25 magnification, resulting in a 94.72 mm field of view (FOV) for a total acquisition time of 8.02 min. Capture images with a binning of 1 x 1, resulting in a resolution of 2,368 x 2,240 pixels. 
	NOTE: The equipment used in this manuscript for micro-CT images presents limitations in obtaining head images due to the overlap with the anesthetic mask. Additionally, the entire tail length is not captured. Thus, we excluded the head and tail analysis (see section 3).</li>
	<li>Perform a scan with the same parameters on an acrylic cylindrical phantom (diameter: 2.5 cm, length: 11.5 cm) half-filled with distilled water to determine the HU for air and water. Upon completion of the acquisitions, reconstruct the images using the acquisition software with settings optimized for noise reduction and the Filtered Back Projection (FBP) algorithm. Batch-reconstruct both animal and phantom images using a 147 &#181;m isotropic voxel size and a 640 matrix.</li>
	<li>Extract air and water HU from the phantom (raw data) using the referenced software. Convert the images into DICOM files and correct the HU values.</li>
</ol>
3. Image analysis
<ol>
	<li>Uploading a file 
	NOTE: Micro-CT is a technique that captures images temporally and spatially, generating an image sequence. Therefore, it is necessary to open the entire folder rather than a single image.
	<ol>
		<li>In the top left area of the interface, find the 3D Slicer menu, highlighted in a grayish-blue color. Click on Add Data (<img alt="Equation 1" src="/files/ftp_upload/67393/67393eq01.jpg" style="margin: auto;" />).</li>
		<li>When a window with two options appears, select the first option: Choose Directory to Add.</li>
		<li>Navigate to the folder containing the target animal DICOM images series and click on it. This action will open the folder within the software, allowing all the images to be viewed simultaneously.</li>
		<li>Observe the images displayed across three screens. Each screen represents a different anatomical plane: coronal, sagittal, and transverse, identified by the colors green, yellow, and red, respectively.</li>
	</ol>
	</li>
	<li>Segmentation
	<ol>
		<li>Locate the Segment Editor (<img alt="Equation 2" src="/files/ftp_upload/67393/67393eq02.jpg" style="margin: auto;" />) in the upper tab (Modules), and click on it.</li>
		<li>Click the green + Add button to create segments, defining the HU range.</li>
		<li>Create segments for bone, lean tissue, and adipose tissue by clicking 3x each.</li>
		<li>Double-click on each segment to name and color it according to the desired settings.</li>
		<li>Set the HU range for each segment using the Threshold function, located on the left side of the window. To do this, find the Threshold Range function <img alt="Equation 3" src="/files/ftp_upload/67393/67393eq03.jpg" style="margin: auto;" /> and enter the HU values for each tissue type: Lean tissue (-29 to 225 HU); adipose tissue (-190 to -30 HU); bone (500 to 5,000 HU). Click the Apply button. 
		NOTE: Refer to Supplemental Figure S1 for HU value comparisons.</li>
		<li>Repeat the segmentation process for each tissue type.</li>
		<li>Click Show 3D to generate a 3D rendering. 
		NOTE: The rendering will appear in the blue quadrant of the display.</li>
	</ol>
	</li>
	<li>Cleaning the image 
	NOTE: After segmentation and 3D rendering, extraneous items must be excluded to ensure they are not included in the volumetric measurements of lean tissue, adipose tissue, and bone. Removing these artifacts is essential to ensure accurate analysis.
	<ol>
		<li>Locate the &#34;Scissors&#34; tool (<img alt="Equation 4" src="/files/ftp_upload/67393/67393eq04.jpg" style="margin: auto;" />) in the Segmentation menu.</li>
		<li>Choose either the anatomical plane or the 3D rendering for a clearer view of the object to be removed.
		<ol>
			<li>If using an anatomical plane, click the maximize view button (<img alt="Equation 5" src="/files/ftp_upload/67393/67393eq05.jpg" style="margin: auto;" />) on the colored bar of the plane. Inside the anatomical plane window, use the mouse scroll button to navigate forward or backward through the CT scan. 
			NOTE: Each window can scroll independently.</li>
			<li>For 3D rendering, use the left mouse button to rotate along any axis and the right mouse button to zoom in. 
			NOTE: Arrow keys on the keyboard can also rotate the image.</li>
		</ol>
		</li>
		<li>With the Scissors tool selected, highlight the segmentation around the unwanted object and encircle it to remove it from the image. The software deletes the object automatically. 
		NOTE: Make sure the operation Erase Inside is selected. If the equipment used generates images for any nonbiological image, such as the bead, anesthetic, or surgical equipment, it is important to remove it. Additionally, if any anatomic area is not wholly obtained, it is fundamental to establish an anatomic reference to remove these areas that are not fully obtained to ensure repeatability between experiments. Unwanted objects must be removed in each plane.</li>
		<li>Click the restore view layout icon (<img alt="Equation 6" src="/files/ftp_upload/67393/67393eq06.jpg" style="margin: auto;" />) to return to the screen with the four windows, regardless of the window selected.</li>
		<li>Repeat the process for all windows where the object needs removal.</li>
	</ol>
	</li>
	<li>Segmentation volume
	<ol>
		<li>After segmentation, quantify the volumes of the segments using the quantification function found in the dropdown menu. 
		NOTE: All segmentations must be visible for this action.</li>
		<li>Navigate to Quantification | Segment Statistics. Click on Apply, and wait for the software to generate a table displaying values for each segmentation. The software provides two volume values for each segmentation: Labelmap (LM) and Closed Surface (CS). 
		NOTE: LM Value is generally suitable for calculating segment volumes, using the number of voxels multiplied by the volume of a single voxel. CS Value utilizes a smoothed surface model and integrates volume based on triangular surface elements. CS aligns more closely with the anatomical contours and is recommended for accurate measurements.</li>
		<li>To upload data from a new animal, save and close the scene at the file menu.</li>
	</ol>
	</li>
	<li>Corporal composition analysis
	<ol>
		<li>Use the volume measurements provided by the software in cm3 to convert these volumes to tissue mass and apply the appropriate density for each tissue type. Use the following tissue densities specified by the International Commission on Radiation Units and Measurements22: 0.95 g/cm&#179; (adipose tissue), 1.05 g/cm&#179; (lean tissue), and 1.92 g/cm&#179; (skeletal tissue)2.
		<ol>
			<li>To compute tissue mass, multiply the volume (cm&#179;) obtained from the software by the respective tissue density (g/cm&#179;): 
			Adipose Tissue Mass: Volumesegmentation X 0.95 = Massadipose 
			Lean Tissue Mass: Volumesegmentation X 1.05 = Masslean 
			Skeletal Tissue Mass: Volumesegmentation X 1.92 = Massskeletal 
			&#8203;Total Mass: Massadipose + Masslean + Massskeletal= MassTotal</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Bone length measurement 
	NOTE: To correct tissue mass by bone length for certain analyses, it is necessary to obtain specific bone lengths.
	<ol>
		<li>Return to the Segment Editor menu. Hide the adipose tissue and lean tissue segmentations by clicking on the eye icon (<img alt="Equation 7" src="/files/ftp_upload/67393/67393eq07.jpg" style="margin: auto;" />) next to each segment. 
		NOTE: Only the segmentation corresponding to skeletal tissue should remain visible.</li>
		<li>Select the Toggle Markups/Toolbar (<img alt="Equation 8" src="/files/ftp_upload/67393/67393eq08.jpg" style="margin: auto;" />) option in the upper corner of the main menu. This will open a secondary menu just below the main menu. Locate the create new line button (<img alt="Equation 9" src="/files/ftp_upload/67393/67393eq09.jpg" style="margin: auto;" />).</li>
		<li>After clicking on the create new line button, navigate to the 3D reconstruction and identify the bone to be measured. 
		NOTE: The recommended bones are the tibia or femur22.</li>
		<li>Once the bone is identified, left-click on one end of the bone and again on the other end of the tissue. This action allows the software to calculate the bone size. 
		NOTE: For consistent measurements, select the same anatomical region of the bone for each animal. For example, the femoral head is used as one end and the lateral condyle as the other end of the femur.</li>
	</ol>
	</li>
	<li>Data analysis and normalization 
	NOTE: The mass data can be obtained as explained in section 3.5 and used as it is. Alternatively, the lean, fat, and bone mass can be normalized in two ways.
	<ol>
		<li>Calculate the ratio between the tissue mass and the animal&#39;s weight on the day the images are obtained. 
		&#8203;Normalization to weight: Masslean &#247; Body weight</li>
		<li>Calculate the ratio between mass and bone size (for instance, femur or tibia). 
		Normalization to femur: Masslean &#247; Sizefemur 
		Normalization to tibia: Masslean &#247; Sizetibia 
		NOTE: The normalization parameters are chosen based on the experimental question. If changes in bone size are expected (for example: when comparing bone remodeling) the normalization by body weight is suggested (item 3.7.1). Additionally, if changes in body weight are expected in your experimental question, the long bone size is the suggested normalization parameter (item 3.7.2).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Engelke, K., Museyko, O., Wang, L., Laredo, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+skeletal+muscle+by+computed+tomography+imaging-State+of+the+art.">Quantitative analysis of skeletal muscle by computed tomography imaging-State of the art.</a> J Orthop Translat. 15, 91 (2018).</li><li>Peixoto da Silva, S., Santos, J. M. O., Costa e Silva, M. P., Gil da Costa, R. M., Medeiros, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cachexia+and+its+pathophysiology:+links+with+sarcopenia,+anorexia+and+asthenia.">Cancer cachexia and its pathophysiology: links with sarcopenia, anorexia and asthenia.</a> J Cachexia Sarcopenia Muscle. 11 (3), 619 (2020).</li><li>Lan, X. Q., 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=The+role+of+TGF-&#946;+signaling+in+muscle+atrophy,+sarcopenia+and+cancer+cachexia.">The role of TGF-&#946; signaling in muscle atrophy, sarcopenia and cancer cachexia.</a> Gen Comp Endocrinol. 353, 1-10 (2024).</li><li>Bloise, F. F., Oliveira, T. S., Cordeiro, A., Ortiga-Carvalho, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormones+play+role+in+sarcopenia+and+myopathies.">Thyroid hormones play role in sarcopenia and myopathies.</a> Front Physiol. 9, 560 (2018).</li><li>Yin, L., 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=Skeletal+muscle+atrophy:+From+mechanisms+to+treatments.">Skeletal muscle atrophy: From mechanisms to treatments.</a> Pharmacol Res. 172, 1-12 (2021).</li><li>Heymsfield, S. B., Adamek, M., Gonzalez, M. C., Jia, G., Thomas, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+skeletal+muscle+mass:+historical+overview+and+state+of+the+art.">Assessing skeletal muscle mass: historical overview and state of the art.</a> J Cachexia Sarcopenia Muscle. 5 (1), 9-18 (2014).</li><li>Cruz-Jentoft, A. J., 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=Sarcopenia:+revised+European+consensus+on+definition+and+diagnosis.">Sarcopenia: revised European consensus on definition and diagnosis.</a> Age Ageing. 48 (1), 16-31 (2019).</li><li>Faron, A., 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=Quantification+of+fat+and+skeletal+muscle+tissue+at+abdominal+computed+tomography:+associations+between+single-slice+measurements+and+total+compartment+volumes.">Quantification of fat and skeletal muscle tissue at abdominal computed tomography: associations between single-slice measurements and total compartment volumes.</a> Abdom Radiol (NY). 44 (5), 1907-1916 (2019).</li><li>Marra, M., 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=Assessment+of+body+composition+in+health+and+disease+using+bioelectrical+impedance+analysis+(BIA)+and+dual+energy+X-ray+absorptiometry+(DXA):+A+critical+overview.">Assessment of body composition in health and disease using bioelectrical impedance analysis (BIA) and dual energy X-ray absorptiometry (DXA): A critical overview.</a> Contrast Media Mol Imaging. 2019, 3548284 (2019).</li><li>Martin, L., 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=Cancer+cachexia+in+the+age+of+obesity:+skeletal+muscle+depletion+is+a+powerful+prognostic+factor,+independent+of+body+mass+index.">Cancer cachexia in the age of obesity: skeletal muscle depletion is a powerful prognostic factor, independent of body mass index.</a> J Clin Oncol. 31 (12), 1539-1547 (2013).</li><li>Schlaeger, S., 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=Association+of+paraspinal+muscle+water-fat+MRI-based+measurements+with+isometric+strength+measurements.">Association of paraspinal muscle water-fat MRI-based measurements with isometric strength measurements.</a> Eur Radiol. 29 (2), 599-608 (2019).</li><li>Kose, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+technical+aspects+of+human+magnetic+resonance+imaging:+present+status+and+50+years+historical+review.">Physical and technical aspects of human magnetic resonance imaging: present status and 50 years historical review.</a> Adv Phys: X. 6 (1), (2021).</li><li>Arnold, T. C., Freeman, C. W., Litt, B., Stein, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-field+MRI:+Clinical+promise+and+challenges.">Low-field MRI: Clinical promise and challenges.</a> J Magn Reson Imaging. 57 (1), 25-44 (2023).</li><li>Wang, H., 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=Preoperative+MRI-based+radiomic+machine-learning+nomogram+may+accurately+distinguish+between+benign+and+malignant+soft-tissue+lesions:+A+two-center+study.">Preoperative MRI-based radiomic machine-learning nomogram may accurately distinguish between benign and malignant soft-tissue lesions: A two-center study.</a> J Magn Reson Imaging. 52 (3), 873-882 (2020).</li><li>Brower, C., Rehani, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+risk+issues+in+recurrent+imaging.">Radiation risk issues in recurrent imaging.</a> Br J Radiol. 94 (1126), (2021).</li><li>Mainprize, J. G., Yaffe, M. J., Chawla, T., Glanc, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ionizing+radiation+exposure+during+pregnancy.">Effects of ionizing radiation exposure during pregnancy.</a> Abdom Radiol (NY). 48 (5), 1564 (2023).</li><li>Geethanath, S., Vaughan, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessible+magnetic+resonance+imaging:+A+review.">Accessible magnetic resonance imaging: A review.</a> J Magn Reson Imaging. 49 (7), e65-e77 (2019).</li><li>Ritman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+developments+and+applications+of+micro-CT.">Current status of developments and applications of micro-CT.</a> Annu Rev Biomed Eng. 13, 531-552 (2011).</li><li>Khoury, B. M., 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=The+use+of+nano-computed+tomography+to+enhance+musculoskeletal+research.">The use of nano-computed tomography to enhance musculoskeletal research.</a> Connect Tissue Res. 56 (2), 106 (2015).</li><li>DenOtter, T. D., Schubert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hounsfield+Unit.">Hounsfield Unit.</a> StatPearls [Internet]. , (2024).</li><li>Pinto, E. M., 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=Efficacy+of+Hounsfield+units+measured+by+lumbar+computer+tomography+on+bone+density+assessment:+A+systematic+review.">Efficacy of Hounsfield units measured by lumbar computer tomography on bone density assessment: A systematic review.</a> Spine (Phila Pa 1976). 47 (9), 702-710 (2022).</li><li>Beaucage, K. L., Pollmann, S. I., Sims, S. M., Dixon, S. J., Holdsworth, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+micro-computed+tomography+for+assessment+of+age-dependent+changes+in+murine+whole-body+composition.">Quantitative in vivo micro-computed tomography for assessment of age-dependent changes in murine whole-body composition.</a> Bone Rep. 5, 70-80 (2016).</li><li>Faot, F., Chatterjee, M., de Camargos, G. V., Duyck, J., Vandamme, K. <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+analysis+of+the+rodent+jaw+bone+micro-architecture:+A+systematic+review.">Micro-CT analysis of the rodent jaw bone micro-architecture: A systematic review.</a> Bone Rep. 2, 14-24 (2015).</li><li>Cooper, D., Turinsky, A., Sensen, C., Hallgrimsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+voxel+size+on+3D+micro-CT+analysis+of+cortical+bone+porosity.">Effect of voxel size on 3D micro-CT analysis of cortical bone porosity.</a> Calcif Tissue Int. 80 (3), 211-219 (2007).</li><li>Mrzilkova, J., 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=Morphology+of+the+vasculature+and+blood+supply+of+the+brown+adipose+tissue+examined+in+an+animal+model+by+micro-CT.">Morphology of the vasculature and blood supply of the brown adipose tissue examined in an animal model by micro-CT.</a> Biomed Res Int. 2020 (1), 7502578 (2020).</li><li>Li, F. W., 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=Optimal+use+ratio+of+the+stromal+vascular+fraction+(SVF):+An+animal+experiment+based+on+micro-CT+dynamic+detection+after+large-volume+fat+grafting.">Optimal use ratio of the stromal vascular fraction (SVF): An animal experiment based on micro-CT dynamic detection after large-volume fat grafting.</a> Aesthet Surg J. 39 (6), NP213-NP224 (2019).</li><li>Berndt, S., 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=Microcomputed+tomography+technique+for+in+vivo+three-dimensional+fat+tissue+volume+evaluation+after+polymer+injection.">Microcomputed tomography technique for in vivo three-dimensional fat tissue volume evaluation after polymer injection.</a> Tissue Eng Part C Methods. 23 (12), 964-970 (2017).</li><li>Rother, L., 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+micro-CT-based+standard+brain+atlas+of+the+bumblebee.">A micro-CT-based standard brain atlas of the bumblebee.</a> Cell Tissue Res. 386 (1), 29-45 (2021).</li><li>Ijiri, T., Todo, H., Hirabayashi, A., Kohiyama, K., Dobashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitization+of+natural+objects+with+micro+CT+and+photographs.">Digitization of natural objects with micro CT and photographs.</a> PLoS One. 13 (4), e0195852 (2018).</li><li>Fedorov, A., 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=3D+Slicer+as+an+image+computing+platform+for+the+Quantitative+Imaging+Network.">3D Slicer as an image computing platform for the Quantitative Imaging Network.</a> Magn Reson Imaging. 30 (9), 1323-1341 (2012).</li><li>Ponti, F., 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=Aging+and+imaging+assessment+of+body+composition:+From+fat+to+facts.">Aging and imaging assessment of body composition: From fat to facts.</a> Front Endocrinol (Lausanne). 10, 861 (2020).</li></ol>

The authors declare no competing interests.

Evaluation through tomography is an effective and non-invasive method for obtaining detailed body composition information. Micro-CT, in particular, offers valuable outcome measures for preclinical studies. In the bone field, micro-CT has different uses, as analyzing the micro-architecture23 and bone remodeling24 are particularly interesting. Assessing the morphology of internal biological structure is also relevant in biomedical research, as analyzing the density of vascula...

Size, mass, and composition are crucial skeletal muscle properties for studying muscle-related and metabolic disease mechanisms1. Sarcopenia, cachexia, atrophy, and myopathies share common phenotypes: reduction in mass, alteration in composition, and impaired muscle function2,3,4,5. However, quantifying the body composition in a living animal remains highly complex and technically challenging6.
The primary methodologies for in vivo imaging and body composition analysis are dual-energy X-ray absorptiometry (DXA), computed tomography (CT), and magnetic resonance imaging (MRI). These methods are primarily employed to screen and monitor diseases that lead to lean mass reduction7,8,9,10,11. DXA is the gold standard for body composition analysis due to its lower cost. However, DXA has a significant disadvantage compared to MRI and CT: its inability to spatially resolve muscle and adipose tissue1.
MRI uses strong magnetic fields and radio waves to generate detailed images of the body&#39;s internal structures12. One of its main advantages over CT is its superior contrast resolution, allowing excellent differentiation between distinct soft tissue types1,13,14. Unlike CT, MRI does not use ionizing radiation, making it safer for repeated use15,16. However, MRI is more expensive and less accessible, with longer scan times and higher maintenance costs13,17. Thus, MRI instruments adapted for small animal analysis are not usually available.
Micro-CT is similar to conventional CT but tailored for small structures and biomedical research18. Micro-CT is an advanced, non-invasive radiological assessment technique that enables detailed visualization of internal structures in small animal models. Micro-CT uses X-rays to create detailed images of the body&#39;s internal structures, relying on the differential attenuation of X-rays by various tissue types. During a micro-CT scan, the mouse lies on a table that moves slowly through a circular gantry. Inside the gantry, an X-ray tube rotates around the mouse, emitting X-rays from various angles. Detectors on the opposite side capture these X-rays after they pass through the body18.
The micro-CT scanner&#39;s software processes the data from these multiple angles to reconstruct two-dimensional cross-sectional images (slices) of the body. Through reconstruction, these slices can be combined to represent the internal anatomy comprehensively19. The images produced by micro-CT scans are based on the varying degrees of X-ray attenuation by different tissues within the body. This attenuation is quantified using Hounsfield Units (HU), a scale that standardizes radiodensity20,21. The HU scale is fundamental for segmentation, as each structure has a slightly different value.
In the present article, we used HU values to accurately differentiate between bone, lean tissue, and adipose tissue1. By referencing established HU ranges, we ensured precise analysis and comparison of body composition. Herein, we demonstrate how to acquire images using micro-CT and its application in visualizing and quantifying lean, fat, and skeletal mass.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3DSlicer, version 5.6.2</td>
			<td>3D Slicer platform</td>
			<td></td>
			<td>A free and open-source software for image analysis and scientific visualization.</td>
		</tr>
		<tr>
			<td>Amide, version 1.0.1</td>
			<td>Amide platform</td>
			<td></td>
			<td>A free and open-source software used for correcting the Hounsfield Unit values in the processed DICOM images.</td>
		</tr>
		<tr>
			<td>Amira, version 4.1</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Used to extract air and water Hounsfield Unit values from the phantom&#39;s raw data and to convert images into DICOM files.</td>
		</tr>
		<tr>
			<td>Isoforine</td>
			<td>Crist&#225;lia</td>
			<td></td>
			<td>Isoflurane is a non-flammable liquid anesthetic agent for use in general inhalation anesthesia by vaporization.</td>
		</tr>
		<tr>
			<td>Triumph XO subsystem</td>
			<td>Gamma Medica-Ideas Flex</td>
			<td></td>
			<td>Advanced imaging subsystem designed for preclinical small animal imaging, offering high-resolution CT and PET capabilities for quantitative and qualitative analysis.</td>
		</tr>
	</tbody>
</table>

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

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.

Proper anatomical positioning of the sedated animal on the imaging table ensures consistent and reproducible scan outcomes, highlighting the data acquisition effectiveness in achieving reliable results. Proper animal sedation throughout imaging, including specialized gas delivery systems and vaporizers, is fundamental for precise anatomical assessments (Figure 1).
Figure 2&#160;illustrates segmentation and rendering in different anato...

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 animals.

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

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

non-invasive-skeletal-muscle-quantification-small-animals-using-micro

Research

JoVE Journal

Biology

304 Views.  Universidade Federal do Rio de Janeiro. 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 animals.

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

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

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

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

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 damage.

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

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

Summary

Explore More Videos

Chapters in this video

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Mitochondrial Isolation from Skeletal Muscle

Purification of Progenitors from Skeletal Muscle

Procedures for Rat in situ Skeletal Muscle Contractile Properties

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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