This work was supported by National Institutes of Health (NIH) R01 DK121327 to R.A.E and R01 AR071968 to F.K.

<ol>
	<li>R&#252;egsegger, P., Koller, B., M&#252;ller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtomographic+system+for+the+nondestructive+evaluation+of+bone+architecture.">A microtomographic system for the nondestructive evaluation of bone architecture.</a> Calcified Tissue International. 58 (1), 24-29 (1996).</li><li>M&#252;ller, R., 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=Morphometric+analysis+of+human+bone+biopsies:+a+quantitative+structural+comparison+of+histological+sections+and+micro-computed+tomography.">Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography.</a> Bone. 23 (1), 59-66 (1998).</li><li>Waarsing, J. 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=Detecting+and+tracking+local+changes+in+the+tibiae+of+individual+rats:+a+novel+method+to+analyse+longitudinal+in+vivo+micro-CT+data.">Detecting and tracking local changes in the tibiae of individual rats: a novel method to analyse longitudinal in vivo micro-CT data.</a> Bone. 34 (1), 163-169 (2004).</li><li>Boyd, S. K., Davison, P., M&#252;ller, R., Gasser, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+individual+morphological+changes+over+time+in+ovariectomized+rats+by+in+vivo+micro-computed+tomography.">Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography.</a> Bone. 39 (4), 854-862 (2006).</li><li>Christiansen, B. A. <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+micro-computed+tomography+voxel+size+and+segmentation+method+on+trabecular+bone+microstructure+measures+in+mice.">Effect of micro-computed tomography voxel size and segmentation method on trabecular bone microstructure measures in mice.</a> Bone Reports. 5, 136-140 (2016).</li><li>Holdsworth, D. W., Thornton, 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=Micro-CT+in+small+animal+and+specimen+imaging.">Micro-CT in small animal and specimen imaging.</a> Trends in Biotechnology. 20 (8), 34-39 (2002).</li><li>Schambach, S. J., Bag, S., Schilling, L., Groden, C., Brockmann, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+micro-CT+in+small+animal+imaging.">Application of micro-CT in small animal imaging.</a> Methods. 50 (1), 2-13 (2010).</li><li>Bouxsein, M. 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=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> Journal of Bone and Mineral Research. 25 (7), 1468-1486 (2010).</li><li>Morgan, E. 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=Micro-computed+tomography+assessment+of+fracture+healing:+Relationships+among+callus+structure,+composition,+and+mechanical+function.">Micro-computed tomography assessment of fracture healing: Relationships among callus structure, composition, and mechanical function.</a> Bone. 44 (2), 335-344 (2009).</li><li>O'Neill, K. R., 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=Micro-computed+tomography+assessment+of+the+progression+of+fracture+healing+in+mice.">Micro-computed tomography assessment of the progression of fracture healing in mice.</a> Bone. 50 (6), 1357-1367 (2012).</li><li>Bissinger, O., 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=Fully+automated+segmentation+of+callus+by+micro-CT+compared+to+biomechanics.">Fully automated segmentation of callus by micro-CT compared to biomechanics.</a> Journal of Orthopaedic Surgery and Research. 12 (1), 108 (2017).</li><li>Brown, M. 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=Delayed+fracture+healing+and+increased+callus+adiposity+in+a+C57BL/6J+murine+model+of+obesity-associated+type+2+diabetes+mellitus.">Delayed fracture healing and increased callus adiposity in a C57BL/6J murine model of obesity-associated type 2 diabetes mellitus.</a> PLOS One. 9 (6), 99656 (2014).</li><li>Khajuria, D. K., 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=Aberrant+structure+of+fibrillar+collagen+and+elevated+levels+of+advanced+glycation+end+products+typify+delayed+fracture+healing+in+the+diet-induced+obesity+mouse+model.">Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model.</a> Bone. 137, 115436 (2020).</li><li>Sigurdsen, U., Reikeras, O., Hoiseth, A., Utvag, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+between+strength+and+quantitative+computed+tomography+measurement+of+callus+mineralization+in+experimental+tibial+fractures.">Correlations between strength and quantitative computed tomography measurement of callus mineralization in experimental tibial fractures.</a> Clinical Biomechanics. 26 (1), 95-100 (2011).</li><li>Duvall, C. L., Taylor, W. R., Weiss, D., Wojtowicz, A. M., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+angiogenesis,+early+callus+formation,+and+late+stage+remodeling+in+fracture+healing+of+osteopontin-deficient+mice.">Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice.</a> Journal of Bone and Mineral Research. 22 (2), 286-297 (2007).</li><li>Gerstenfeld, L. C., 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=Comparison+of+effects+of+the+bisphosphonate+alendronate+versus+the+RANKL+inhibitor+denosumab+on+murine+fracture+healing.">Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing.</a> Journal of Bone and Mineral Research. 24 (2), 196-208 (2009).</li><li>Alentado, V. 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=Validation+of+the+modified+radiographic+union+score+for+tibia+fractures+(mRUST)+in+murine+femoral+fractures.">Validation of the modified radiographic union score for tibia fractures (mRUST) in murine femoral fractures.</a> Frontiers in Endocrinology. 13, 911058 (2022).</li><li>Yu, K. E., 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=Enhancement+of+impaired+MRSA-infected+fracture+healing+by+combinatorial+antibiotics+and+modulation+of+sustained+inflammation.">Enhancement of impaired MRSA-infected fracture healing by combinatorial antibiotics and modulation of sustained inflammation.</a> Journal of Bone and Mineral Research. 37 (1), 1352-1365 (2022).</li><li>Nyman, J. 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=Quantitative+measures+of+femoral+fracture+repair+in+rats+derived+by+micro-computed+tomography.">Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography.</a> Journal of Biomechanics. 42 (7), 891-897 (2009).</li><li>Fiset, 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=Experimental+validation+of+the+radiographic+union+score+for+tibial+fractures+(RUST)+using+micro-computed+tomography+scanning+and+biomechanical+testing+in+an+in-vivo+rat+model.">Experimental validation of the radiographic union score for tibial fractures (RUST) using micro-computed tomography scanning and biomechanical testing in an in-vivo rat model.</a> The Journal of Bone and Joint Surgery. 100 (21), 1871-1878 (2018).</li><li>Shefelbine, S. 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=Prediction+of+fracture+callus+mechanical+properties+using+micro-CT+images+and+voxel-based+finite+element+analysis.">Prediction of fracture callus mechanical properties using micro-CT images and voxel-based finite element analysis.</a> Bone. 36 (3), 480-488 (2005).</li><li>Liu, Y., 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=Glucocorticoid-induced+delayed+fracture+healing+and+impaired+bone+biomechanical+properties+in+mice.">Glucocorticoid-induced delayed fracture healing and impaired bone biomechanical properties in mice.</a> Clinical Interventions in Aging. 13, 1465-1474 (2018).</li><li>Watson, P. J., Fitton, L. C., Meloro, C., Fagan, M. J., Gr&#246;ning, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+adaptation+of+trabecular+bone+morphology+in+the+mammalian+mandible.">Mechanical adaptation of trabecular bone morphology in the mammalian mandible.</a> Scientific Reports. 8 (1), 7277 (2018).</li><li>Nie, C., Wang, Z., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+depression+on+fracture+healing+and+osteoblast+differentiation+in+rats.">The effect of depression on fracture healing and osteoblast differentiation in rats.</a> Neuropsychiatric Disease and Treatment. 14, 1705-1713 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The purpose of this study is to describe a detailed protocol for &#181;CT analysis with the goal of accurate quantification of the 3D mineralized callus structure, which is often fundamental in bone and fracture healing studies. The protocol utilizes a general-purpose state-of-the-art 3D image analysis software platform which facilitates image visualization, segmentation/labelling, and measurements ranging from simple to complex.
The most time-consuming task in the protocol is semi-automated s...

Micro-computed tomography (&#181;CT) imaging has been widely used in preclinical bone research, providing noninvasive, high-resolution images to evaluate the microstructure of bones1,2,3,4,5. &#181;CT involves a large number of X-ray images, obtained from a rotating sample or by using a rotating X-ray source and detector. Algorithms are used to reconstruct 3D volumetric data in the form of a stack of image slices. Clinical CT is the gold standard for 3D imaging of human bones, and &#181;CT is a commonly used technique for evaluating bone healing efficiency in experimental animals1,2,3,4,6,7. Mineralized bone has excellent contrast to X-ray, while soft tissues have relatively poor contrast unless a contrast agent is used. In the assessment of fracture healing, &#181;CT generates images that provide detailed information about the 3D structure and density of the mineralized callus. In vivo &#181;CT scanning can also be used for longitudinal, time-course assessment of fracture healing.
The quantification of intact cortical and trabecular bone using &#181;CT is generally well-established and standardized8. Although preclinical studies use a variety of quantification methodologies to analyze fracture healing9,10,11, a detailed protocol of &#181;CT image analysis for callus quantification has not been published yet.Therefore, the aim of this study is to provide a detailed step-by-step protocol for &#181;CT imaging and analysis of bone healing callus.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% neutral buffered formalin&#160;</td>
			<td>Fisher chemical</td>
			<td>SF100-20</td>
			<td>Used for bone tissue fixation</td>
		</tr>
		<tr>
			<td>Avizo</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Image processing and analysis software</td>
		</tr>
		<tr>
			<td>Hydroxyapatite phantom&#160;</td>
			<td>Micro-CT HA D4.5, QRM</td>
			<td>QRM-70128</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Processing Language</td>
			<td>Scanco</td>
			<td></td>
			<td>Used to convert raw images to DICOM images</td>
		</tr>
		<tr>
			<td>Micro-Mosquito Straight Hemostatic Forceps</td>
			<td>Medline</td>
			<td></td>
			<td>Used to remove the intramedullary pin&#160;</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>Scanco mCT system (vivaCT 40)</td>
			<td>Scanco</td>
			<td></td>
			<td>Used for &#181;CT imaging&#160;</td>
		</tr>
	</tbody>
</table>

assessment of bone fracture healing using micro-computed tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

To monitor bone formation during fracture healing, a mid-diaphyseal open tibial fracture was induced in adult, male C75BL/6J mice. The fracture was stabilized using an intramedullary nail, an established model of secondary healing13. Callus tissues were harvested at days 14, 21, and 28 post-fracture12. These timepoints represent different phases of healing. Endochondral bone formation during secondary bone healing proceeds via initial formation of a fibro-cartilagi...

Watch this Scientific Journal Video about Assessment of Bone Fracture Healing Using Micro-Computed Tomography at JoVE.com

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.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

assessment-of-bone-fracture-healing-using-micro-computed-tomography

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1.7K Views.  The Pennsylvania State University College of Medicine. Micro-computed tomography (µCT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on µCT procedures for analyzing the bone healing callus. This study provides a step-by-step µCT protocol that allows the monitoring of fracture healing. 

Video: Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

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 ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.