The authors would like to acknowledge Danielle Jones (Clinical education specialist, Siemens Healthineers) and the entire CT technologist team at Houston Methodist DeBakey Heart and vascular center to support imaging protocols.

This protocol follows the ethical standards of the national research committee and with the 1964 Helsinki declaration. This protocol is approved by Houston Methodist Research Institute.
1. Patient selection and prior image review
NOTE: Dynamic CTA imaging should be considered as a follow-up imaging modality in patients with increasing aneurysm size and endoleak after stent-graft implantation, persistent endoleak after interventions, or in patients with increasing aneurysms sac size without demonstrable endoleak. Like conventional CT imaging, this technique involves iodinated contrast injection that may be relatively contraindicated in patients with severe renal failure.
<ol>
	<li>Before starting the actual scan, review the prior imaging studies for the presence of endoleak and stent-graft type. 
	&#8203;NOTE: This can provide information to decide the scan range and temporal distribution during the image acquisition. The most commonly available imaging is the conventional CTA scans with bi-(non-contrast scan and arterial scan) or triple-phase (non-contrast scan, arterial scan, and delayed scan).</li>
</ol>
2. d-CTA Image acquisition
<ol>
	<li>Position the patient in a supine position on the CT scanner table.</li>
	<li>Gain peripheral venous access. 
	NOTE: Ensure that access is gained by visualizing the venous back bleeding.</li>
	<li>Perform Topogram and Non-Contrast CT Image Acquisition using Sn-100 Tin filter (see Table of Materials) to reduce the radiation exposure and for the region of interest selection in the d-CTA scan. 
	NOTE: After the non-contrast scan, the location of the endograft will be visible. Place the region of interest just above the endograft.</li>
	<li>Perform timing bolus6 to check the contrast arrival time by placing a region of interest above the stent graft in the abdominal aorta.
	<ol>
		<li>Inject 10-20 mL of the contrast (see Table of Materials) through the peripheral venous access, followed by 50 mL of saline injection at a 3.5-4 mL/min flow rate. Acquire timing bolus scan. 
		NOTE: Contrast arrival is recorded by the CT scanner (see Table of Materials) based on Hounsfield unit change inside the aorta6.</li>
	</ol>
	</li>
	<li>By selecting the DynMulti4D menu point in the pop-up &#34;Cycle time window&#34; plan the distribution and the number of scans based on the contrast arrival time from timing bolus and the findings from prior imaging studies. 
	NOTE: If type I endoleak is suspected, perform more scans on the early phase of the contrast enhancement curve that is given by the timing bolus. If type II endoleak is suspected, perform more scans on the later phase.
	<ol>
		<li>For type I endoleak, include more scans during the earlier phase of the time-attenuation curve (scan at every 1.5 s at the beginning and then every 3-4 s).</li>
		<li>For type II endoleak that appear later, include more scans during the later phase of the time-attenuation curve.</li>
		<li>If no prior imaging studies are available, distribute the scans equally around the peak of the time-attenuation curve.</li>
	</ol>
	</li>
	<li>Optimize imaging parameters, including kV, scan range, etc., to reduce radiation exposure. Use settings shown in Table 1 for acquiring a dynamic scan with the CT scanner (see Table of Materials) used in this work.</li>
	<li>Inject the contrast for d-CTA acquisition: 70-80 mL of the contrast material, followed by 100 mL of saline injections at a 3.5-4 mL/min flow rate through the peripheral access.</li>
	<li>Start d-CTA image acquisition using the delay time based on the timing bolus describedin step 2.4. Breath-hold is not necessary during acquisition, given that the duration of d-CTA image acquisition ranges from 30-40 s.</li>
	<li>Send acquired, reconstructed images to Picture Archiving and Communication System (PACS) for qualitative and quantitative review of time-resolved angiographic images. To do this, select the data image and perform a mouse click on the bottom left side of the software.</li>
</ol>
3. Dynamic-CTA image analysis
<ol>
	<li>Open the software (see Table of Materials) for reading the image. Search for the patient&#39;s name or identification number to find the acquired images. Select the acquired d-CTA images and process them using the CT dynamic angio workflow. 
	NOTE: The layout is shown in Figure 1.</li>
	<li>Minimize respiratory motion artifacts between d-CTA images by selecting the dedicated software&#39;s Align Body motion correction menu item (Figure 1).</li>
	<li>Qualitative analysis: Check axial slices of CT images when maximum opacification of the aorta occurs to interpret any obvious endoleak.
	<ol>
		<li>Then analyze scans in multiplanar reconstruction mode; if endoleak is suspected, focus on the endoleak and use the timescale shown in Figure 1 to watch time-resolved images and infer the source of endoleak.</li>
	</ol>
	</li>
	<li>Quantitative analysis: Click on the Time Attenuation Curve (TAC) function shown in Figure 1. Select a region above the stent-graft (ROIaorta) and draw a circle using the TAC function, then select the endoleak (ROIendoleak) region and draw a circle there as well. 
	NOTE: Target vessels can be selected (ROItarget) to determine the role of the vessel to the endoleak (inflow or outflow).
	<ol>
		<li>Analyze the acquired TAC (Figure 2) to determine the endoleak characteristics. Subtract the time to the peak value of the endoleak from the aortic ROI curves to get the&#160;&#916; time to peak value. This value can be used for endoleak analysis6.</li>
	</ol>
	</li>
	<li>After qualitative and quantitative analysis, infer the type and source of endoleak. 
	&#8203;NOTE: Type I endoleaks appear as parallel contrast enhancement next to the graft, usually because of the inadequate sealing zone and have a shorter time difference between the aortic and endoleak enhancement curves (&#916; time to peak value) between aortic and endoleak ROI. Type II endoleaks are related to an inflow vessel with retrograde filling through collateral and have prolonged&#160;&#916; time to peak value between aortic and endoleak ROI. Based on experience, a &#916; time-to-peak value of higher than 4 s was not recorded for type I endoleaks.</li>
</ol>
4. Intra-operative image fusion guidance
<ol>
	<li>Position the patient supine on the hybrid operating room (OR) table.</li>
	<li>Load the selected dynamic CTA scan that has the best visibility of the endoleak in the hybrid OR workstation. Manually annotate critical landmarks on the scan: renal arteries ostia, internal iliac arteries ostia, endoleak cavity, lumbar artery(ies), or inferior mesenteric artery.</li>
	<li>Select 2D-3D image fusion in the workstation and acquire an anteroposterior and an oblique fluoroscopic image of the patient using the 2D-3D image fusion workflow. For this, move the C-arm to the required angle(s) with the joystick on the operating table and step on the CINE acquisition pedal.</li>
	<li>Electronically align the stent graft with markers from the 3D dynamic CTA scan with the fluoroscopic images using automated image registration, followed by manual refinement if necessary (Figure 3) in the 3D post-processing workstation (Drag one image for manual alignment). Check and accept the 2D-3D Image Fusion and Overlay the markers from d-CTA on the real-time 2D fluoroscopic image (Figure 4).</li>
	<li>Perform the endoleak embolization using the overlaid markers from d-CTA as guidance.</li>
</ol>

<ol>
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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=The+Society+for+Vascular+Surgery+practice+guidelines+on+the+care+of+patients+with+an+abdominal+aortic+aneurysm.">The Society for Vascular Surgery practice guidelines on the care of patients with an abdominal aortic aneurysm.</a> Journal of Vascular Surgery. 67 (1), 2-77 (2018).</li><li>Sommer, W. 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=Time-resolved+CT+angiography+for+the+detection+and+classification+of+endoleaks.">Time-resolved CT angiography for the detection and classification of endoleaks.</a> Radiology. 263 (3), 917-926 (2012).</li><li>Hou, 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=Dynamic+volumetric+computed+tomography+angiography+is+a+preferred+method+for+unclassified+endoleaks+by+conventional+computed+tomography+angiography+after+endovascular+aortic+repair.">Dynamic volumetric computed tomography angiography is a preferred method for unclassified endoleaks by conventional computed tomography angiography after endovascular aortic repair.</a> Journal of American Heart Association. 8 (8), 012011 (2019).</li><li>Berczeli, M., Lumsden, A. B., Chang, S. M., Bavare, C. 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J., Bockler, D., Krisam, J., Geisbusch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional-three-dimensional+registration+for+fusion+imaging+is+noninferior+to+three-dimensional-+three-dimensional+registration+in+infrarenal+endovascular+aneurysm+repair.">Two-dimensional-three-dimensional registration for fusion imaging is noninferior to three-dimensional- three-dimensional registration in infrarenal endovascular aneurysm repair.</a> Journal of Vascular Surgery. 70 (6), 2005-2013 (2019).</li><li>Madigan, M. C., Singh, M. J., Chaer, R. A., Al-Khoury, G. E., Makaroun, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occult+type+I+or+III+endoleaks+are+a+common+cause+of+failure+of+type+II+endoleak+treatment+after+endovascular+aortic+repair.">Occult type I or III endoleaks are a common cause of failure of type II endoleak treatment after endovascular aortic repair.</a> Journal of Vascular Surgery. 69 (2), 432-439 (2019).</li><li>Koike, 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=Dynamic+volumetric+CT+angiography+for+the+detection+and+classification+of+endoleaks:+application+of+cine+imaging+using+a+320-row+CT+scanner+with+16-cm+detectors.">Dynamic volumetric CT angiography for the detection and classification of endoleaks: application of cine imaging using a 320-row CT scanner with 16-cm detectors.</a> Journal of Vascular and Interventional Radiology. 25 (8), 1172-1180 (2014).</li><li>Macari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+aortic+aneurysm:+Can+the+arterial+phase+at+CT+evaluation+after+endovascular+repair+be+eliminated+to+reduce+radiation+dose.">Abdominal aortic aneurysm: Can the arterial phase at CT evaluation after endovascular repair be eliminated to reduce radiation dose.</a> Radiology. 241 (3), 908-914 (2006).</li><li>Brambilla, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cumulative+radiation+dose+and+radiation+risk+from+medical+imaging+in+patients+subjected+to+endovascular+aortic+aneurysm+repair.">Cumulative radiation dose and radiation risk from medical imaging in patients subjected to endovascular aortic aneurysm repair.</a> La Radiologica Medica. 120 (6), 563-570 (2015).</li><li>Buffa, V., 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=Dual-source+dual-energy+CT:+dose+reduction+after+endovascular+abdominal+aortic+aneurysm+repair.">Dual-source dual-energy CT: dose reduction after endovascular abdominal aortic aneurysm repair.</a> La Radiologica Medica. 119 (12), 934-941 (2014).</li><li>Apfaltrer, G., 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+analysis+of+dynamic+computed+tomography+angiography+for+the+detection+of+endoleaks+after+abdominal+aorta+aneurysm+endovascular+repair:+A+feasibility+study.">Quantitative analysis of dynamic computed tomography angiography for the detection of endoleaks after abdominal aorta aneurysm endovascular repair: A feasibility study.</a> PLoS One. 16 (1), 0245134 (2021).</li><li>Kinner, 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=Dynamic+MR+angiography+in+acute+aortic+dissection.">Dynamic MR angiography in acute aortic dissection.</a> Journal of Magnetic Resonance Imaging. 42 (2), 505-514 (2015).</li><li>Buls, N., 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=Improving+the+diagnosis+of+peripheral+arterial+disease+in+below-the-knee+arteries+by+adding+time-resolved+CT+scan+series+to+conventional+run-off+CT+angiography.+First+experience+with+a+256-slice+CT+scanner.">Improving the diagnosis of peripheral arterial disease in below-the-knee arteries by adding time-resolved CT scan series to conventional run-off CT angiography. First experience with a 256-slice CT scanner.</a> European Journal of Radiology. 110, 136-141 (2019).</li><li>Grossberg, J. A., Howard, B. M., Saindane, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+contrast-enhanced,+time-resolved+magnetic+resonance+angiography+in+cerebrovascular+pathology.">The use of contrast-enhanced, time-resolved magnetic resonance angiography in cerebrovascular pathology.</a> Neurosurgical Focus. 47 (6), 3 (2019).</li></ol>

ABL receives research support from Siemens Medical Solutions USA Inc., Malvern, PA. PC is a senior staff scientist at Siemens Medical Solutions USA Inc., Malvern, PA. Marton Berczeli is supported by Semmelweis University's scholarship: "Kieg&#233;sz&#237;t&#337; Kutat&#225;si Kiv&#225;l&#243;s&#225;gi &#214;szt&#246;nd&#237;j" EFOP-3.6.3- VEKOP-16-2017-00009.

Dynamic, time-resolved CTA is an additional tool in the aortic imaging armamentarium. This technique can accurately diagnose endoleaks after EVAR, including identification of inflow/target vessels4.
Third-generation CT scanners with bidirectional table movement capability can provide dynamic acquisition mode with better temporal sampling along the time-attenuation curve6. To achieve the highest accuracy in the protocol it is critical to personali...

Endovascular aortic aneurysm repair (EVAR) has shown superior early mortality results than open aortic repair1. The approach is less invasive but may result in higher mid to long-term re-intervention rates due to endoleaks, graft migration, fracture2. Hence better EVAR surveillance is critical to achieving good mid to long-term results.
Current guidelines suggest the routine use of duplex ultrasound and triphasic CTA3. Dynamic, time-resolved computed tomography angiography (d-CTA) is a relatively new modality used for EVAR surveillance4. During d-CTA, multiple scans are acquired in different time points along the time attenuation curve after contrast injection, hence the term time-resolved imaging. This approach has shown better accuracy in characterizing endoleaks after EVAR than conventional CTA5. An advantage of time-resolved acquisition is the ability to quantitatively analyze the Hounsfield unit changes in a selected region of interest (ROI)6.
The additional benefit of accurately characterizing endoleaks with d-CTA is that the scan can be used for image fusion during interventions, potentially minimizing the need for further diagnostic angiography. Image fusion is a method when previously acquired images are overlaid onto real-time fluoroscopy images to guide endovascular procedures and subsequently reduce contrast agent consumption and radiation exposure7,8. Image fusion in the hybrid operating room (OR) using a 3D dynamic CTA scan can be achieved by two approaches: (1) 3D-3D image fusion: where 3D d-CTA is fused with intraoperatively acquired non-contrast cone-beam CT images, (2) 2D-3D image fusion, where 3D d-CTA is fused with biplanar (anteroposterior and lateral) fluoroscopic images. 2D-3D image fusion approach has been shown to significantly lower the radiation compared with 3D-3D technique9.
This protocol describes the technical and practical aspects of dynamic CTA imaging for endoleak characterization and introduces a 2D-3D image fusion approach with d-CTA for intra-operative image guidance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Siemens Artis Pheno</td>
			<td>Siemens Healthcare</td>
			<td>https://www.siemens-healthineers.com/en-us/angio/artis-interventional-angiography-systems/artis-pheno</td>
			<td>Other commercially available C-arm systems can provide image fusion too</td>
		</tr>
		<tr>
			<td>SOMATOM Force CT-scanner</td>
			<td>Siemens Healthcare</td>
			<td>https://www.siemens-healthineers.com/computed-tomography/dual-source-ct/somatom-force</td>
			<td>Any commercially available third generation CT-scanner can perform such dynamic imaging</td>
		</tr>
		<tr>
			<td>Syngo.via</td>
			<td>Siemens Healthcare</td>
			<td>https://www.siemens-healthineers.com/en-us/medical-imaging-it/advanced-visualization-solutions/syngovia</td>
			<td>Any DICOM file viewer with 4D processing capabilities can review the acquired time-resolved images, TAC are software dependent.</td>
		</tr>
		<tr>
			<td>Visipaque (Iodixanol)</td>
			<td>GE Healthcare</td>
			<td>#00407222317</td>
			<td>Contrast material</td>
		</tr>
	</tbody>
</table>

time-resolved, dynamic computed tomography angiography for characterization of aortic endoleaks and treatment guidance via 2d-3d fusion-imaging

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach warrants good early results, but adequate follow-up imaging after EVAR is imperative to maintain long-term positive outcomes. Potential graft-related complications are graft migration, infection, fraction, and endoleaks, with the last one being the most common. The most frequently used imaging after EVAR is computed tomography angiography (CTA) and duplex ultrasound. Dynamic, time-resolved computed tomography angiography (d-CTA) is a reasonably new technique to characterize the endoleaks. Multiple scans are done sequentially around the endograft during acquisition that grants good visualization of the contrast passage and graft-related complications. This high diagnostic accuracy of d-CTA can be implemented into therapy via image fusion and reduce additional radiation and contrast material exposure.
This protocol describes the technical aspects of this modality: patient selection, preliminary image review, d-CTA scan acquisition, image processing, qualitative and quantitative endoleak characterization. The steps of integrating dynamic CTA into intra-operative fluoroscopy using 2D-3D fusion-imaging to facilitate targeted embolization are also demonstrated. In conclusion, time-resolved, dynamic CTA is an ideal modality for endoleak characterization with additional quantitative analysis. It can reduce radiation and iodinated contrast material exposure during endoleak treatment by guiding interventions.

The dynamic imaging workflow in two patients is illustrated here.
Patient I 
An 82-year-old male patient with chronic obstructive pulmonary disease and hypertension had a previous infrarenal EVAR (2016). In 2020 the patient was referred from an outside hospital for a possible type I or type II endoleak based on conventional CTA. and an adjunctive endoanchor placement in 2020 for type Ia endoleak. Dynamic CTA was performed that diagnosed a type Ia endoleak, and the patient...

Watch this Scientific Journal Video about Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging at JoVE.com

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

Dynamic computed tomography angiography (CTA) imaging provides additional diagnostic value in characterizing aortic endoleaks. This protocol describes a qualitative and quantitative approach using time-attenuation curve analysis to characterize endoleaks. The technique of integrating dynamic CTA imaging with fluoroscopy using 2D-3D image fusion is illustrated for better image guidance during treatment.

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach ...

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2.8K Views.  Houston Methodist Hospital. Dynamic computed tomography angiography (CTA) imaging provides additional diagnostic value in characterizing aortic endoleaks. This protocol describes a qualitative and quantitative approach using time-attenuation curve analysis to characterize endoleaks. The technique of integrating dynamic CTA imaging with fluoroscopy using 2D-3D image fusion is illustrated for better image guidance during treatment.

Video: Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

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

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

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

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

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 ...

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

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 ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

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

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

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

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Cone Beam Intraoperative Computed Tomography-based Image Guidance for Minimally Invasive Transforaminal Interbody Fusion

Transforaminal lumbar interbody fusion (TLIF) is commonly used for the treatment of spinal stenosis, degenerative disc disease, and spondylolisthesis. Minimally invasive surgery (MIS) approaches have been applied to this technique with an associated decrease in estimated blood loss (EBL), length of hospital stay, and infection rates, while preserving outcomes with traditional open surgery. Previous MIS TLIF techniques involve significant fluoroscopy that subjects the patient, surgeon, and operating room staff to non-trivial levels of radiation exposure, particularly for complex multi-level procedures. We present a technique that utilizes an intraoperative computed tomography (CT) scan to aid in placement of pedicle screws, followed by traditional fluoroscopy for confirmation of cage placement. Patients are positioned in the standard fashion and a reference arc is placed in the posterior superior iliac spine (PSIS) followed by intraoperative CT scan. This allows for image-guidance-based placement of pedicle screws through a one-inch skin incision on each side. Unlike traditional MIS-TLIF that requires significant fluoroscopic imaging during this stage, the operation can now be performed without any additional radiation exposure to the patient or operating room staff. After completion of the facetectomy and discectomy, final TLIF cage placement is confirmed with fluoroscopy. This technique has the potential to decrease operative time and minimize total radiation exposure.

The purpose of this article is to provide image-guidance for minimally invasive transforaminal interbody fusion.

Transforaminal lumbar interbody fusion (TLIF) is commonly used for the treatment of spinal stenosis, degenerative disc disease, and spondylolisthesis. ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...