Name: assessment of bone fracture healing using micro-computed tomography
Uploaded: 2022-12-09T13:30:00
Description: Watch this Scientific Journal Video about Assessment of Bone Fracture Healing Using Micro-Computed Tomography at JoVE.com

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

The following protocol was developed to characterize long-bone healing callus harvested from euthanized mice. However, most of the steps can be applied to rats and also used for in vivo scanning of fractured bones. The protocol describes a particular &#181;CT system and specific image processing, analysis, and visualization software (see Table of Materials), yet the methodology is generally applicable to other scanners and software. The protocol was approved by the Institutional Animal Care and ...

<ol>
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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 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 ...

Micro-CT is a very common approach for quantifying 3D morphology and quality of bone. Although analysis protocols are fairly established for characterizing intact cortical and trabecular bone, there is a less consensus on the protocol for analyzing fracture healing. This technique involves noninvasive imaging and accurate, calibrated 3D analysis using a good balance of manual and automated techniques.It also uses a sophisticated and flexible software environment. Demonstrating the procedure will be Hwabok Wee, a research associate from my laboratory. To begin, take a custom-developed 3D printed scanning fixture or similar that includes a miniature hydroxyapatite phantom for bone mineral density calibration.Place up to six long bone samples into the fixture for the simultaneous scanning of multiple samples. Place the prepared fixture in a syringe or a conical tube similar to the scanning field of view's diameter. Fill the syringe with a preservative, like saline, to prevent samples from drying out during the scanning process.After confirming the calibration of the micro-CT machine, align the sample fixture's center line with the approximate center line of the micro-CT to ensure that the samples are within the field of view and their long axes have orientation approximately coinciding with the axial direction of the resulting images. Next, set the scanning parameters of the micro-CT system by setting the energy or intensity to 55 kilovoltage peak, current to 145 microampere, isotropic voxel size to 10.5 micrometers, and integration time to 300 milliseconds. Then visually inspect the scout images in different views to cover the whole volume of all callus samples.Start the scan acquisition, and, once complete, convert the images to a DICOM stack to import them into the analysis software. To start with image cropping, pick one sample at a time, and crop each image stack, ensuring the whole sample is included in the cropped volume. Save the cropped image by clicking the File tab on the top left side of the screen, selecting Save Project As, and then selecting Minimize project size.To denoise the image, click the File tab and choose the image to be processed using Open Data, which opens the image in the Project View window in the top left corner of the screen. Right click to select Image Processing, followed by Filter Sandbox, and then click Create. In the Properties window on the left bottom corner of the screen, choose Data as the preview type.Select the filter type from the dropdown menu next to Filter and choose 3D for interpretation. Keeping Separable as the kernel type in the dropdown menu, fill in the values of standard deviation and kernel size factor in the available empty box. Then select Same as input from the dropdown menu next to the output.And finally, click Apply. For misaligned samples, the user may perform image realignment by creating a 3D-rendered image of the sample by selecting the filtered, cropped image from the Project View window. Right click to select Display and then Volume Rendering from the dropdown menu.Then click Create to visually check the 3D-rendered image in the sagittal and frontal planes. Then manually rotate the rendered volume to obtain a good alignment in the longitudinal axis. To apply the transformation in the rotated images, in the Properties window, click the Transform Editor.Then go to Transform Editor Manipulator and select Transformer from the dropdown menu. If needed, rotate, realign, and then click the Transform Editor again to lock the image. Next, to create new transverse plane image slices, resample the filtered image by selecting the image from the Project View window.Right click to select Geometry Transform, followed by Resample Transformed Image from the dropdown menu, and then click Create. In the Properties window, go to Data, and from the dropdown menu, select Standard for interpolation, choose Extended for mode and Voxel Size for preserve. Enter zero in the available blank box for padding value, and finally, click Apply.To define the volume of interest, go through the transverse image slices, identify the fracture callus's center plane, and define it based on the proximal and distal lens of the callus. If the callus ends are difficult to specify, define the volume based on a standardized distance away from the callus center plane. To segment the outer boundary of the callus following the reassembly of transformed images, click the Segmentation tab in the second tab row from the top of the screen.In the segmentation editor window, select transformed image from the dropdown menu next to the image. In the MATERIALS window, double click Add. By doing so, two tabs named Material3 and Material4 will appear.Right click to rename Material3 to callus and Material4 to cortical bone. Next, in the SELECTION window, click on the lasso icon. From the appearing options, select Freehand for the 2D mode, Inside for the 3D mode, and both Auto trace and Trace edges for options.Then use the lasso to mark the outer boundary of callus. Repeat the contouring steps with slices sampled across the volume of interest, and the slices can be spaced apart, for example, by 20 slices. To create a complete callus label, in the MATERIALS window, choose the callus file, click the Selection tab on the top of the screen, and select Interpolate from the dropdown menu.Then, in the Selection window, click on the plus sign. Next, segment the cortical bone, including the medullary cavity. Then interpolate the contoured periosteal cortex surface to create a cortical bone label as performed previously for the callus.To calculate the callus's contoured volume and mean gray value, click the Segmentation tab on the top row of the screen and select Material Statistics from the dropdown menu to generate a table of calculated values. Note that the values of the cortical bone and the callus are provided separately after subtracting the cortical bone. Export the generated table and save the data by clicking Export into Workspace.To convert the grayscale units to bone mineral density, crop the 3D image of the 4.5-millimeter HA phantom from the whole image and click Segmentation. To draw circles at the first and the last slices, in the MATERIALS window, click Add four times. Then right click to rename Material3, 4, 5, and 6 to Phantom1, 2, 3, and 4 respectively.Next, select Phantom1. Click the brush icon in the Selection window and use the slider to adjust the brush size such that the size of the circle is smaller than that of the phantom. To create a volume for each HA cylinder, apply interpolation by selecting Phantom1 in the MATERIALS window, clicking the Selection tab on the top row of the screen, and selecting Interpolate from the dropdown menu.Then, in the SELECTION window, click the plus sign. Repeat this process with the remaining HA cylinders. Use the generated 3D labels to calculate the mean gray values of the four analyzed HA cylinders.Plot the mean gray values and the corresponding bone mineral density, or BMD, values provided by the phantom manufacturer. Generate a correlation equation between BMD and the gray values using linear regression. To segment the mineralized callus, click Add in the MATERIALS window.Then right click to rename the new material to callus mineralized. Next, select Callus in the MATERIALS window, followed by clicking Select. Then click Threshold in the SELECTION window.Select the lower value and apply the calculated threshold from the HA phantom in threshold masking. Then click Select only current material in the SELECTION window, followed by clicking Select Masked Voxels. Then select callus mineralized in the MATERIALS window and click the plus sign in the SELECTION window.Finally, click 3D of callus mineralized in the MATERIALS window. The micro-CT images analyzed at three time points showed the substantial formation of mineralized callus on day 14. Incremental increases in bone fraction volume and bone mineral density were seen as healing proceeded from day 14 to days 21 and 28, consistent with bony bridging of the fracture gap.As expected, the callus underwent resorption or remodeling between days 21 and 28, as evidenced by a decline in total callus volume. Cortical bridging of the callus was more evident at day 28 than at any preceding time point. For complex fractures, we recommend carefully reviewing the resulting semi-automated segmentation by scrolling through all image slices, sometimes in different planes, and adjusting contours if needed.Once the fracture callus is accurately segmented, outcome parameters, in addition to the ones reported in this protocol, can be computed with additional scripts. These include, for example, moments of inertia and connectivity density.

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Biology

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 ...

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 ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

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 ...

Construction of a Realistic, Whole-Body, Three-Dimensional Equine Skeletal Model using Computed Tomography Data

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches have rarely been used to study equine athletic injury. Degenerative osteoarthritis caused by mechanical stress can originate from chronic postural dysfunction, which, because the primary dysfunction is often distant from the site of tissue injury, is best identified through modeling whole-body biomechanics. To characterize whole-body equine kinematics, a realistic skeletal model of a horse was created from equine computed tomography (CT) data that can be used for functional anatomical and biomechanical modeling. Equine CT data were reconstructed into individual three-dimensional (3D) data sets (i.e., bones) using 3D visualization software and assembled into a complete 3D skeletal model. The model was then rigged and animated using 3D animation and modeling software. The resulting 3D skeletal model can be used to characterize equine postures associated with degenerative tissue changes as well as to identify postures that reduce mechanical stress at the sites of tissue injury. In addition, when animated into 4D, the model can be used to demonstrate unhealthy and healthy skeletal movements and can be used to develop preventative and rehabilitative individualized therapies for horses with degenerative lamenesses. Although the model will soon be available for download, it is currently in a format that requires access to the 3D animation and modeling software, which has quite a learning curve for new users. This protocol will guide users in (1) developing such a model for any organism of interest and (2) using this specific equine model for their own research questions.

The purpose of this protocol is to describe the method of creation of a realistic, whole-body, skeletal model of a horse that can be used for functional anatomical and biomechanical modeling to characterize whole-body mechanics.

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches ...

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 ...