Name: segmentation and measurement of fat volumes in murine obesity models using x-ray computed tomography
Uploaded: 2012-04-04T13:00:00
Duration: 13 min 9 s
Description: Watch this Scientific Journal Video about Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography at JoVE.com

We warmly thank the Notre Dame Integrated Imaging Facility (NDIIF) and Carestream Health for financial support for this project.

1. Animals
<ol>
	<li>For the results reported below, three C57BL/6J mice and four B6.V-Lepob/J mice were obtained from Jackson Laboratories (Bar Harbor, Main, USA). B6.V-Lepob/J mice represent one of the earliest models of obesity and remain actively researched. B6.V-Lepob/J mice exhibit a phenotype characterized by increased adipocyte size and number and can weigh up three times more than wild type mice.7,8 Here, B6.V-Lepob/J mice were employed as a positive control for an obese phenotype to illustrate the feasibility of the Albira CT system for CT based fat content measurements.</li>
	<li>Imaging was performed when the animals reached approximately 12 weeks old. (Obesity in B6.V-Lepob/J mice is manifest after 4 weeks of age).</li>
	<li>Mice were anesthetized by Isofluorane (2.5% flow rate) and kept under at 2.5% via a nose-cone setup for imaging. Animals were positioned prone in the standard rat bed (M2M Imaging Inc. Cleveland, OH) in supplied with the Albira image station. Limbs were positioned lateral from the torso for a uniform CT acquisition. </li>
	<li>After image acquisition was completed, mice were removed from the nose cone and returned to a recovery cage until ambulatory.</li>
</ol>
2. Image Acquisition and Reconstruction
<ol>
	<li>Image acquisitions are performed using the Albira CT system (Carestream Molecular Imaging, Woodbridge, CT). Mice were anesthetized by Isofluorane (2.5% flow rate) and kept under at 2.5% via a nose-cone setup for imaging. Acquisitions were performed to scan a bed of 115 mm length, using 600 projections. The X-ray source was set to a current of 200 &mu;A and voltage of 45 kVp, and used a 0.5 mm Al filter to harden the beam.</li>
	<li>Approximate radiation deep dose equivalent for CT settings was 220 mSv, and shallow dose equivalent was 357.4 mSv. These doses are over 20-fold lower than reported LD50 values.9</li>
	<li>Images are reconstructed using the FBP (Filtered Back Projection) algorithm via the Albira Suite 5.0 Reconstructor using "Standard" parameters. These combined acquisition and reconstruction settings produce a final image with 125 &mu;m isotropic voxels, deemed sufficient for whole animal analysis. For detailed region specific analysis, a reconstruction with 35 &mu;m isotropic voxels may be selected for a final resolution of 90 &mu;m.</li>
</ol>
3. Image Analysis
Image analysis is performed using the PMOD (PMOD Technologies LTD, Zurich, Switzerland) analysis software. Images are segmented in PMOD according to tissue density-first for total volume and then for fat volume.
3.1 Images may be reduced for analysis to minimize computational demands.
<ol>
	<li>To reduce, navigate to the main View tab.</li>
	<li>Select Tools &gt; Reduce.</li>
	<li>Select X by 2, Y by 2, and Z by 2.</li>
	<li>Check Replace.</li>
	<li>Select Run.</li>
</ol>
The message: "Bounding box will change" displays once the reduction is complete.
3.2 Images can be masked to eliminate bed and nose-cone elements for subsequent volume-of-interest (VOI) analysis.
<ol>
	<li>To mask, navigate to Planes, Layouts, Rotations, Mirror, 3D Markers &gt; Planes &amp; Layouts.</li>
	<li>Select Show plane Z.</li>
	<li>Scroll to the nose cone in the Z plane.</li>
	<li>Select the main VOI tab &gt; Draw vertices.</li>
	<li>Draw a region of interest (ROI) around the animal nose-excluding the bed and nose cone.</li>
	<li>Select Copy Actual ROI.</li>
	<li>Move to the next slice, and paste ROI from buffer through the relevant planes of nose.</li>
	<li>Use Edit Group of Vertices to adjust the ROIs as necessary.</li>
	<li>Select Delete ROI at the first planes beyond the nose cones.</li>
	<li>Generate a new VOI to encompass the animal girth (excluding the animal bed) at the first planes beyond the nose cones.</li>
	<li>Navigate to VOI Tools &gt; Masking &amp; Algebra.</li>
	<li>Enter -1000 in the dialogue box provided.</li>
	<li>Select the Mask voxels outside the selected VOIs button.</li>
</ol>
The message: "Irreversible data operation. Do you want to continue?" displays.
<ol start="14">
	<li>Select Yes.</li>
	<li>Navigate to Planes, Layouts, Rotations, Mirror, 3D Markers &gt; Planes &amp; Layouts.</li>
	<li>Select Show all planes.</li>
	<li>Examine the VOI for integrity.</li>
	<li>Select Save.</li>
	<li>Save as Analyze.</li>
	<li>Change the file name prefix.</li>
</ol>
3.3 First, segment the image for total animal volume:
<ol>
	<li>Select Tools &gt; External.</li>
	<li>Select the Segmentation check box.</li>
	<li>Enter a range of -300 to +3500 (density range reference derived from abdominal fat-region density range).</li>
	<li>Select Run Segmentation.</li>
	<li>Examine the integrity of the segmentation.</li>
	<li>Select OK.</li>
	<li>Select Remove ROI.</li>
	<li>Select VOI statistics.</li>
</ol>
Reported statistics represent the total volume.
<ol start="9">
	<li>Record the reported volume.</li>
</ol>
3.4 Next, segment the image for fat volume:
<ol>
	<li>Return to the non-segmented masked image for fat-volume segmentation.</li>
	<li>To load the saved Masked data file, check the Analyze box in the Load window).</li>
	<li>Select Tools &gt; External.</li>
	<li>Select the Segmentation check box.</li>
	<li>Enter a range of -200 to -50.</li>
	<li>Select Run Segmentation.</li>
	<li>Examine the integrity of the segmentation.</li>
	<li>Select OK.</li>
	<li>Select VOI statistics.</li>
</ol>
The reported statistics represent the fat volume.
<ol start="10">
	<li>Record the reported volume.</li>
	<li>Select Save.</li>
	<li>Save as Analyze.</li>
	<li>Change the file name prefix.</li>
</ol>
Optional: If skin/peripheral density remains, the "Erosion and Dilation" protocol below may be performed to eliminate these regions for VOI analysis.
<ol>
	<li>Select Tools &gt; External.</li>
	<li>Select the Morphological check box. The Morphological view displays.</li>
	<li>Select Erosion.</li>
	<li>Select OK.</li>
	<li>Select Tools &gt; External.</li>
	<li>Select the Morphological check box. The Morphological view displays.</li>
	<li>Select Dilation.</li>
	<li>Select OK.</li>
</ol>
4. Visualization of CT Images
4.1 VolView v3.2 (Kitware, Clifton Park, NY, USA) was utilized to create rendered 3D visual displays of segmented images.
<ol>
	<li>Open the CT data set in Analyze format.</li>
	<li>Use the default settings in the pop-up window.</li>
	<li>Open the plug-ins menu.</li>
	<li>Under Utility, select Merge volumes.</li>
	<li>Uncheck Rescale Components.</li>
	<li>Click Assign Second Input.</li>
	<li>Choose the segmented fat data for the second input.</li>
	<li>Use the default settings in the pop-up window.</li>
	<li>Click Apply plug-in.</li>
	<li>Double-click Volume view window for a larger view of the subject mouse.</li>
</ol>
4.2 Return to the Color/Opacity tab. The component drop-down box refers to which data set is currently being edited. Two sliders are located at the bottom of the tab and determine the relative brightness of each component data set within the overlay, using values 0 to 1. For component 1, the CT, we prefer to use a grayscale color scheme. To change the color:
<ol>
	<li>In the Scalar Color Mapping section, double-click on one of the color sliders.</li>
	<li>To remove a slider, drag it out of the box.</li>
	<li>To add a new slider, click anywhere within the slider area.</li>
	<li>Remove one of the sliders.</li>
	<li>Make the left color slider black (scalar value (S)= -19000).</li>
	<li>Make right color slider white ((S) = 15000).</li>
	<li>From the Scalar Opacity mapping box, create a new point by clicking within the box. This will give a total of three points in the window.</li>
	<li>For the middle point, change the (S) to ~-3000, and the Opacity (O) value to 0.</li>
	<li>Select the third point at the right of the window.</li>
	<li>Change (S) to about 32000, and O to .25.</li>
	<li>The first point can be anywhere at left, just as long as the Opacity value is set to 0.</li>
	<li>Change to component two, which should edit the look of the fat.</li>
	<li>Change each of the color sliders to red by double-clicking and sliding the Hue (H) slider to the left to false-color map the fat to red. Very little else should be needed to adjust the look of the fat.</li>
</ol>
4.3 To create a three-panel rotation movie displaying the CT, fat, and overlay:
<ol>
	<li>Click and the drag the subject mouse into an upright position with the back facing you.</li>
	<li>Under Component Weights, set the value of Component two to 0 to display only the CT scan.</li>
	<li>Click Review &gt; Camera.</li>
	<li>Select a number of frames for the rotation movie (for the present case, we chose "36").</li>
	<li>Change the X rotation value to 360 degrees.</li>
	<li>Select Create.</li>
	<li>In the pop-up dialog box, create a new folder named CT, and save the file in tiff format, which will output a series of rotation images.</li>
	<li>Repeat this step for the fat image, as well as for the overlaid fat/CT image, saving them into individual folders each time.</li>
</ol>
4.4 ImageJ v 1.43u was used to generate a rotation movie file using the VolView output images.
<ol>
	<li>In ImageJ, select File &gt; Import &gt; Image Sequence.</li>
	<li>Select the first image in the CT folder. The software will automatically detect the other files and open them as a stack.</li>
	<li>Repeat to open the fat and overlay sequences.</li>
	<li>Open the ROI manager under Analyze &gt; Tools &gt; ROI Manager.</li>
	<li>Draw an ROI around the subject mouse, excluding needless background pixels.</li>
	<li>In the ROI manager, click Add to add the ROI.</li>
	<li>Select a different image sequence.</li>
	<li>In the ROI manager, click the ROI to apply it to the picture. In this fashion, each of the cropped stacks will match up perfectly.</li>
	<li>When the ROI is on all the stacks, right-click within the ROI.</li>
	<li>Select Duplicate.</li>
	<li>Select Check the Duplicate Stack Box to separate the ROI from the rest of the image.</li>
	<li>Close the larger image stacks.</li>
	<li>Repeat this procedure for all three image sequences.</li>
	<li>Go to Image &gt; Stacks &gt; Tools &gt; Combine to combine the stacks together.</li>
	<li>Select CT for Stack 1.</li>
	<li>Select Fat for Stack 2.</li>
	<li>Repeat, and select Combined Stacks for Stack 1 and Overlay for Stack 2.</li>
	<li>There is now a three-panel, image-rotation stack that can be previewed by selecting Play in the lower-left corner of the image window.</li>
	<li>To save the movie as an AVI, select File &gt; Save as&hellip; &gt; AVI&hellip;</li>
	<li>Click Save.</li>
</ol>
5. Representative Results
Results for three WT (C57BL/6J) mice and four obese (B6.V-Lepob/J) mice are reported here as a representative example of fat/total volume ratio measurements employing the Albira CT system. Figure 1 below provides a representative display created with VolView v3.2 for the segmentation (i.e. total volume and fat volume) of obese mice CT images.
<img alt="Figure 1" src="/files/ftp_upload/3680/3680fig1.jpg" /> 
Figure 1. Representative CT images segmented for fat. (A) Obese mouse (B6.V-Lepob/J) CT total volume in grayscale, (B) fat volume in red, and (C) image fusion. (D) WT mouse (C57BL/6J) CT total volume in grayscale, (E) fat volume in red, and (F) image fusion.
Total volumes, fat volumes, and calculated fat/total volume ratios are reported below in Table 1 for each WT mouse and each obese mouse. The averaged fat/total volume ratio for the WT group and the obese group was 0.09 and 0.42 respectively (Figure 2). The fat/total volume ratios for the WT mice versus the obese mice was found to differ significantly (p = 0.001).
<table border="1">
	<tbody>
		<tr>
			<td>WT (C57BL/6J)</td>
			<td>Total (cm3)</td>
			<td>Fat (cm3)</td>
			<td>Fat/Total Ratio</td>
			<td>Obese (B6.V-Lepob/) </td>
			<td>Total (cm3)</td>
			<td>Fat (cm3)</td>
			<td>Fat/Total Ratio</td>
		</tr>
		<tr>
			<td>Animal 1</td>
			<td>28.79</td>
			<td>3.00</td>
			<td>0.10</td>
			<td>Animal 1</td>
			<td>66.25</td>
			<td>26.75</td>
			<td>0.40</td>
		</tr>
		<tr>
			<td>Animal 2</td>
			<td>33.25</td>
			<td>3.05</td>
			<td>0.09</td>
			<td>Animal 2</td>
			<td>61.15</td>
			<td>26.31</td>
			<td>0.43</td>
		</tr>
		<tr>
			<td>Animal 3</td>
			<td>30.30</td>
			<td>2.63</td>
			<td>0.09</td>
			<td>Animal 3</td>
			<td>64.19</td>
			<td>25.7</td>
			<td>0.40</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Animal 4</td>
			<td>54.25</td>
			<td>23.78</td>
			<td>0.44</td>
		</tr>
	</tbody>
</table>
 
Table 1. Total volume, fat volume, and fat/total volume ratios for WT and obese mice. Total and fat volumes were derived from segmented images using PMOD VOI analysis.
<img alt="Figure 2" src="/files/ftp_upload/3680/3680fig2.jpg" /> 
Figure 2. Averaged fat/total volume ratios for WT mice versus obese mice. Averaged fat/total volume ratios for WT (C57BL/6J) and obese (B6.V-Lepob/J) found to be 0.09 and 0.42 respectively are displayed. (Error bars = single standard deviation). WT versus obese fat/total volume ratios were found to differ significantly (p-value = 0.001).

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	<li>Clarke, P. J., O'Malley, P. M., Schulenberg, J. E., Johnston, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midlife+and+Socioeconomic+Consequences+of+Persistent+Overweight+Across+Early+Adulthood:+Findings+From+a+National+Survey+of+American+Adults+(1986-2008).">Midlife and Socioeconomic Consequences of Persistent Overweight Across Early Adulthood: Findings From a National Survey of American Adults (1986-2008).</a> Am. J. Epidemiol. , (2008).</li><li>Brockman, G., Bevova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Mouse+Models+to+Dissect+the+Genetic+of+Obesity.">Using Mouse Models to Dissect the Genetic of Obesity.</a> TRENDS in Genetics. 18, 367-376 (2002).</li><li>Bray, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+Understanding+the+Genetics+of+Obesity.">Progress in Understanding the Genetics of Obesity.</a> Journal of Nutrition. 127, 940S-942S (1997).</li><li>Carroll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Obesity.">Mouse Models of Obesity.</a> Clinics in Dermatology. 22, 345-349 (2004).</li><li>Judex, S., Luu, Y. K., Ozcivici, E., Adler, B., Lublinsky, S., Rubin, C. T. <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+Adiposity+in+Small+Animal+Rodents+using+Micro-CT.">Quantification of Adiposity in Small Animal Rodents using Micro-CT.</a> Methods. 50, 14 (2010).</li><li>Luu, Y. K., Lublinsky, S., Ozcivici, E., Capilla, E., Pessin, J. E., Rubin, C. T., Judex, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Quantificaiton+of+Subcutaneous+and+Visceral+Adiposity+by+Micro+Computed+Tomography+in+a+Small+Animal+Model.">In Vivo Quantificaiton of Subcutaneous and Visceral Adiposity by Micro Computed Tomography in a Small Animal Model.</a> Med. Eng. Phys. 31, 34-41 (2009).</li><li>Medina-Gomez, G., Gray, S. L., Yetukuri, L., Shimomura, K., Virtue, S., Campbell, M., Curtis, R. K., Jimenez-Linan, M., Blount, M., Yeo, G. S., Lopez, M., Seppänen-Laakso, T., Ashcroft, F. M., Oresic, M., Vidal-Puig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PPAR+gamma+2+prevents+lipotoxicity+by+controlling+adipose+tissue+expandability+and+peripheral+lipid+metabolism.">PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism.</a> PLoS Genet. 3, e64 (2007).</li><li>Sakkou, M., Wiedmer, P., Anlag, K., Hamm, A., Seuntjens, E., Ettwiller, L., Tschöp, M. H., Treier, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.A+role+for+brain-specific+homeobox+factor+bsx+in+the+control+of+hyperphagia+and+locomotory+behavior.">M.A role for brain-specific homeobox factor bsx in the control of hyperphagia and locomotory behavior.</a> Cell Metab. 5, 450-463 (2007).</li><li>Patchen, M. L., MacVittie, T. J., Souza, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postirradiation+treatment+with+granulocyte+colony-stimulating+factor+and+preirradiation+WR-2721+administration+synergize+to+enhance+hemopoietic+reconstitution+and+increase+survival.">Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival.</a> International Journal of Radiation Oncology. 22, 773-779 (1992).</li></ol>

Todd A. Sasser, Shengting Li, Sean P. Orton, and Seth T. Gammon are employees of Carestream Molecular Imaging. Carlos Correcher is an employee of Oncovision, Gem-Imaging S.A. W. Matthew Leevy is a consultant for Carestream Molecular Imaging.

Here, utilizing B6.V-Lepob/J mice we have illustrated the feasibility of performing fat content measurements in a small animal model using the Albira CT system. These measurements are consistent with expectations for comparisons of intra-group and inter-group measurements. Firstly, representative results provided here highlight limited intra-group variability in measurements of fat/total volume ratios in both WT and obese mice groups using these procedures. Secondly, fat/total volume ratios for WT versus obese mice differ significantly. Finally, based on comparisons (not shown) with previous values reported for relative total fat mass and percent body fat for WT versus B6.V-Lepob/J mice, our measurements for fat/total volume ratios for WT versus B6.V-Lepob/J mice fall within an expected range,7, 8. 
 The methods detailed here could be applied or adapted to other models and/or study objectives. Modifications in reconstruction parameters may be required to achieve specific objectives. For example, Judex et al. (2010) reported that 50 &mu;m resolution images were required for some region specific analysis. One cm isotropic volumes of an image may be selected for 35 &mu;m reconstructions in the Albira 5.0 Suite Reconstructor using the "HR" reconstruction option. Once the Albira CT system has been utilized for region and organ specific fat content measurements the full benefits (i.e. simultaneous region and organ specific fat volume measurements and longitudinal measurements) of CT based fat content analysis may be realized for the Albira CT system. 
Conclusions:
 Here we provide a detailed, step by step method for the measurement of fat content in living mice using X-ray CT imaging. We acquired our CT data sets using an Albira image station, and performed subsequent segmentation and analysis using the PMOD software suite. Finally, we provide instructions to enable facile rendering and visualization of the fat tissue distribution within the whole animal.

segmentation and measurement of fat volumes in murine obesity models using x-ray computed tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Watch this Scientific Journal Video about Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography at JoVE.com

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

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Medicine

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

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 provide information about the burden of disease. However, despite multiple validation studies of CAC3-5, and C-IMT2,6, these modalities do not accurately assess plaque characteristics7,8, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13.
[18F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16, an important source of cellular inflammation in vessel walls. More recently, we17,18 and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22 and is also highly associated with overall burden of atherosclerosis23. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24 as well as longer term therapeutic lifestyle changes (16 months)25.
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28

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

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

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 dimensional&#160;(3D) diagnostic imaging in the interventional suite and can enhance minimally-invasive therapy beyond the limitations of 2D angiography alone. The role of CBCT has been recognized in transarterial chemo-embolization (TACE) treatment of hepatocellular carcinoma (HCC). The recent introduction of a CBCT technique: dual-phase CBCT (DP-CBCT) improves intra-arterial HCC treatment with drug-eluting beads (DEB-TACE). DP-CBCT can be used to localize liver tumors with the diagnostic accuracy of multi-phasic multidetector computed tomography (M-MDCT) and contrast enhanced magnetic resonance imaging (CE-MRI) (See the tumor), to guide intra-arterially guidewire and microcatheter to the desired location for selective therapy (Reach the tumor), and to evaluate treatment success during the procedure (Treat the tumor). The purpose of this manuscript is to illustrate how DP-CBCT is used in DEB-TACE to see, reach, and treat HCC.

Dual-phase cone-beam computed tomography (DP-CBCT) is a useful intraprocedural imaging technique for transarterial chemo-embolization treatment with drug-eluting beads of hepatocellular carcinoma. DP-CBCT has been used to perform three major steps in oncologic interventional radiology: tumor localization (see), navigation and intraprocedural catheter guidance (reach), and intraprocedural evaluation of treatment success (treat).

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 ...

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

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

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

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although several studies have investigated the role of VC as a direct hepatotoxicant, the concept that VC modifies sensitivity of the liver to other factors, such as nonalcoholic fatty liver disease (NAFLD) caused by high-fat diet (HFD) is novel. This protocol describes an exposure paradigm to evaluate the effects of chronic, low-level exposure to VC. Mice are acclimated to low-fat or high-fat diet one week prior to the beginning of the inhalation exposure and remain on these diets throughout the experiment. Mice are exposed to VC (sub-OSHA level: &#60;1 ppm) or room air in inhalation chambers for 6 hours/day, 5 days/week, for up to 12 weeks. Animals are monitored weekly for body weight gain and food consumption. This model of VC exposure causes no overt liver injury with VC inhalation alone. However, the combination of VC and HFD significantly enhances liver disease. A technical advantage of this co-exposure model is the whole-body exposure, without restraint. Moreover, the conditions more closely resemble a very common human situation of a combined exposure to VC with underlying nonalcoholic fatty liver disease and therefore support the novel hypothesis that VC is an environmental risk factor for the development of liver damage as a complication of obesity (i.e., NAFLD). This work challenges the paradigm that the current exposure limits of VC (occupational and environmental) are safe. The use of this model can shed new light and concern on the risks of VC exposure. This model of toxicant-induced liver injury can be used for other volatile organic compounds and to study other interactions that may impact the liver and other organ systems.

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of skeletal muscle mass and adipose tissues on Computed Tomography (CT) and/or Magnetic Resonance Imaging (MRI) may inform surgery risk evaluation and disease prognosis. This article describes two quantification methods originally described by Mourtzakis et al. and Avrutin et al.: tissue segmentation and linear measurement of skeletal muscle. Patients' cross-sectional image at the midpoint of the third lumbar vertebra was obtained for both measurements. For segmentation, the images were imported into Slice-O-Matic and colored for skeletal muscle, intramuscular adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue. Then, surface areas of each tissue type were calculated using the tag surface area function. For linear measurements, the height and width of bilateral psoas and paraspinal muscles at the level of the third lumbar vertebra are measured and the calculation using these four values yield the estimated skeletal muscle mass. Segmentation analysis provides quantitative, comprehensive information about the patients' body composition, which can then be correlated with disease progression. However, the process is more time-consuming and requires specialized training. Linear measurements are an efficient and clinic-friendly tool for quick preoperative evaluation. However, linear measurements do not provide information on adipose tissue composition. Nonetheless, these methods have wide applications in a variety of diseases to predict surgical outcomes, risk of disease progression and inform treatment options for patients.

Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification.

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of ...

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

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

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

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...