This research was supported in part by the JPI-HDHL-INTIMIC &#34;GUTMOM&#34; Project: Maternal obesity and cognitive dysfunction in the offspring: Cause-effect role of the GUT MicrobiOMe and early dietary prevention (project no. INTIMIC-085, Italian Ministry of Education, University and Research Decree no. 946/2019).

Animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the International Guidelines on Handling of Laboratory Animals, demanded by the European Directive (Directive 86/609/EEC of 1986 and Directive 2010/63/UE) and Italian laws (D.Lgs. 26/2014).
1. Setup of the PET/CT imaging protocols and workflow
NOTE: The protocol presented here is specifically designed for cardiac imaging ...

The protocol presented in this paper focuses on a typical experimental procedure for translational cardiovascular research on small animal models of cardiac injury by using high-resolution PET/CT imaging. The presented results are indicative of the high quantitative and qualitative value of PET and Cine-CT images, providing both functional and structural information of the whole heart regarding its glucose metabolism, shape, and the dynamics of its contraction. Moreover, all the images obtained are 3D, time-resolved, and...

Small animal models are extremely important for the advancement of the understanding of cardiovascular diseases1,2. Non-invasive, diagnostic imaging tools have revolutionized the way we look at cardiac function in the last decades, both in clinical and preclinical settings. As far as small animal models of cardiac diseases are concerned, specific imaging tools have been developed with very high spatiotemporal resolution. Thus, such instruments can match the need for accurate quantification of the relevant metabolic and kinetic myocardial parameters on the very small and very fast-moving hearts of mice and rats in specific disease models, such as heart failure (HF)3 or myocardial infarction (MI)4. Several modalities are available for this purpose, each with their own strengths and weaknesses. Ultrasound (US) imaging is the most widely used modality due to its great flexibility, very high temporal resolution, and relatively low cost. The adoption of US cardiac imaging in small animals has increased considerably since the advent of systems using probes with ultra-high frequency5,6, featuring spatial resolutions below 50 &#181;m.
Among the main disadvantages of US for fully 3D cardiac imaging is the need for linear scans along the heart axis by mounting the probe on a motorized translation stage to create a full stack of dynamic B-mode images of the whole heart7. Eventually, this procedure gives rise (after accurate spatial and temporal registration of the images acquired in each probe position) to a 4D image with different spatial resolutions between the in-plane and out-of-plane directions. The same problem of non-uniform spatial resolution occurs in cardiac MR (CMR),8 which still represents the gold standard in the functional imaging of the heart. Real isotropic 3D imaging can be instead obtained using both computed tomography (CT) and positron emission tomography (PET)9. PET provides a very sensitive tool in terms of image signal per quantity of injected probe (in the nanomolar range), even though it suffers from a reduced spatial resolution compared to CT, MR, or US. The main advantage of PET is its ability to display the cellular and molecular mechanisms underlying the organ&#39;s pathophysiology. For instance, a PET scan following the injection of [18F]FDG allows the reconstruction of a 3D map of the glucose metabolism in the body. By combining this with dynamic (i.e., time-resolved) data acquisition, tracer kinetic modeling can be used to calculate parametric maps of the metabolic rates of glucose uptake (MRGlu), which will provide important information about myocardial viability10.
CT requires significant volumes of external contrast agents (CA) at high concentrations (up to 400 mg of iodine per mL) to provide a measurable enhancement of the relevant tissue components (e.g., blood vs. muscle), but it excels in spatial and temporal resolution, especially when using state-of-the-art micro-CT scanners designed for small animal imaging.11 A typical disease model in which the cardiac PET/CT can be applied is the experimental evaluation of myocardial infarction and heart failure and related response to therapy. A common way of inducing MI in small animals is by surgical ligation of the left anterior descending (LAD) coronary artery12,13 and then longitudinally evaluating the progression of the disease and the cardiac remodeling in the subsequent days4. Nevertheless, the quantitative morphofunctional evaluation of the heart in small animals is largely applicable also for other disease models, such as the evaluation of the effect of aging on cardiac function14 or altered receptor expression in models of obesity15. The presented imaging protocol is not restricted to any given disease model and, hence, could be of the widest interest in several contexts of preclinical research with small rodents.
In this paper, we present a start-to-end experimental protocol for cardiac imaging using small animal integrated PET/CT. Even though the presented protocol is designed for a specific bimodal integrated scanner, the PET and CT parts of the described procedure could be performed independently on separate scanners from different manufacturers. In the PET/CT scanner in use, the sequence of operations is organized in a preprogrammed workflow. The main branches of each workflow are one or more acquisition protocols; each acquisition protocol can have one or more branches for specific preprocessing protocols, and in turn, each preprocessing protocol can have one or more branches for specific reconstruction protocols. Both the preparation of the animal on the imaging bed and the preparation of the external agents to be injected during the imaging procedures are described. After the completion of the image acquisition procedure, example procedures for quantitative image analysis based on commonly available software tools are provided. The main protocol is specifically designed for mouse models; even though the mouse remains the most used species in this field, we also show an adaptation of the protocol for rat imaging at the end of the main protocol. Representative results are shown for both mice and rats, demonstrating the type of output that might be expected with the described procedures. A thorough discussion is made at the end of this paper to emphasize the pros and cons of the technique, critical points, as well as how different PET radiotracers could be used with almost no modification to the preparatory and acquisition/reconstruction steps.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sterile saline</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td>0.9% sodium chloride for injection</td>
		</tr>
		<tr>
			<td>1025L Physiological Monitoring</td>
			<td>Small Animal Instruments</td>
			<td></td>
			<td>Physiological monitoring system for small animal imaging</td>
		</tr>
		<tr>
			<td>5 mL syringes</td>
			<td>Artsana</td>
			<td></td>
			<td>Syringes with needle for injection of PET tracer</td>
		</tr>
		<tr>
			<td>Atomlab 500</td>
			<td>Else Nuclear</td>
			<td></td>
			<td>PET Dose calibrator</td>
		</tr>
		<tr>
			<td>Atrium software</td>
			<td>Inviscan</td>
			<td>Version 1.5.5</td>
			<td>PET/CT operating software</td>
		</tr>
		<tr>
			<td>Butterfly catheters</td>
			<td>Delta Med</td>
			<td></td>
			<td>27.5 G needle</td>
		</tr>
		<tr>
			<td>Carimas software</td>
			<td>Turku PET Center</td>
			<td>Version 2.10</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Fenestra VC</td>
			<td>Medilumine</td>
			<td></td>
			<td>Lipid emulsion iodinated contrast agent for small animals</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td></td>
			<td></td>
			<td>Heat lamp with clamp and switch</td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>Artsana</td>
			<td></td>
			<td>Syringes with needle for injection of CT CA</td>
		</tr>
		<tr>
			<td>Iomeron 400 mgI/mL</td>
			<td>Bracco</td>
			<td></td>
			<td>Iomeprol, vascular contrast agent</td>
		</tr>
		<tr>
			<td>IRIS PET/CT</td>
			<td>Inviscan</td>
			<td></td>
			<td>PET/CT scanner for small animals</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td></td>
			<td>Inhalation anesthetic, 250 mL</td>
		</tr>
		<tr>
			<td>OneTouch Glucometer</td>
			<td>Johnson&#38;Johnson Medical</td>
			<td></td>
			<td>Glucose meter kit</td>
		</tr>
		<tr>
			<td>Osirix MD software</td>
			<td>Pixmeo</td>
			<td>Version 11</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Air liquide</td>
			<td></td>
			<td>Compressed gas</td>
		</tr>
		<tr>
			<td>Rectal probe for 1025L</td>
			<td>Small Animal Instruments</td>
			<td></td>
			<td>Rectal probe with cable for SAII 1025L systems</td>
		</tr>
		<tr>
			<td>Respiratory sensor for 1025L</td>
			<td>Small Animal Instruments</td>
			<td></td>
			<td>Respiratory pillow with tubings for SAII 1025L systems</td>
		</tr>
		<tr>
			<td>TJ-3A syringe pump</td>
			<td>Longer</td>
			<td></td>
			<td>Motorized syringe pump for CT CA injection</td>
		</tr>
	</tbody>
</table>

high-resolution cardiac positron emission tomography/computed tomography for small animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

In this section, typical results are shown for both PET and CT analysis following the procedures described so far. Figure 6 shows the results of the automatic myocardial and LV cavity segmentation of the [18F]FDG PET scan of a control (healthy) CD-1 mouse. Even though the right ventricle is not always visible in the reconstructed images, the orientation axes based on the DICOM header can be used to correctly discriminate the interventricular septum from the other LV walls, as requ...

Watch this Scientific Journal Video about High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals at JoVE.com

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

The presented cardiac PET/CT protocol is useful for obtaining functional and morphological information in a variety of small animal models of cardiac disease. Advantages of PET/CT imaging as compared to other preclinical imaging modalities include, but are not limited to, high sensitivity and very high spatial temporal resolution, as well as robustness as it does not require manual positioning of probes by the operators. One of the main disease model that can be studied with this protocol is myocardial infarction.However, other cardiometabolic diseases and also response to therapy can be investigated. For instance, our group is interested in the role of obesity and diabetes on cardiac metabolism and function. The steps included in this protocol can be easily followed by users without experience.However, some steps such as caudal vein cannulations and blood sampling may require some training and experience to be done reproducibly. Demonstrating the procedure will be Federica La Rosa, a research fellow from our laboratory, Federico Granziera, a PhD student from Sant'Anna School of Advanced Studies, and Domiziana Terlizzi, veterinary doctor of Fondazione Toscana Gabriele Monasterio. To begin, place the mouse in the supine position head first on the scanner bed of the PET/CT tomograph putting its nose in the nose mask for anesthesia and gently blocking the head of the mask with adhesive tape.Fix the upper and lower limbs on the scanner bed to prevent involuntary movements during imaging procedures which may lead to motion artifacts. Monitor the body temperature using a rectal probe and respiration rate using a respiration pillow. For mice, draw 100 to 150 microliters of 10 megabecquerels fluorine-18 FDG using an insulin syringe.If the original concentration of the tracer in the vial is high, dilute the tracer with saline to a concentration of 50 to 100 megabecquerel per milliliter. Use the PET dose calibrator to measure the actual activity in the syringe. Annotate the pre-injection activity and time of measurement as these values will be used later using specific input modules of the PET scanner graphical user interface.If using iomeprol, set the injection rate to 10 milliliters per hour and volume to 0.5 milliliters. Connect the syringe to the syringe pump and set the pump for the actual syringe size and diameter, then connect the syringe to the CA tubing and needle and prefill the tubing with the CA.Set the injection rate to 10 milliliters per hour, limiting the injection volume to 0.5 milliliters. For iomeprol injection, use a syringe pump allowing slow injection at a constant rate, with an already set injection rate of 10 milliliters per hour.Limiting the injection volume to 0.5 milliliters, stop the injection after three minutes. After ensuring the tubing and needle are prefilled with CA, connect the needle attached to the CA tubing to the tail vein's cannula. Start the injection.Close the scanner's lid and prepare for the Cine-CT scan. Press the Continue button on the tomograph's user interface after 60 seconds from the beginning of the injection so that the Cine-CT acquisition is started. The injection of CA will stop roughly at the same time as the Cine-CT scan completion.Open the DICOM images of the dynamic PET scan. Select the heart plugin module. Zoom into the mouse or rat heart image and select the last timeframe or the sum of the last three to five timeframes for which most of the blood pool activity has already been washed out.Follow the onscreen instruction to reorient the image along the principal axis of the animal heart. Do this interactively by moving the displayed markers for the heart base and apex. Next, select the Segmentation tool.If the result of the automatic segmentation is not acceptable, refine the shape of the segmented myocardium or left ventricle cavity by enabling the manual mode ROI search disabled. In the modeling tool, select the appropriate kinetic model or dynamic PET analysis. In this case, select graphical and then Patlak to enable the Patlak plot analysis to compute the metabolic rate of glucose uptake for each cardiac sector.Next, in the polar map tool, select the correct number of displayed heart segments. In this case, select 17 segments. Now, press the Fit button to perform the fitting procedure of the Patlak analysis.At the end of the fitting procedure, observe the displayed polar map of the KI values. Load the DICOM images of the Cine-CT scan in the software. Then open the dynamic dataset with the built-in 4D viewer.And using the 3D multiplayer reformation or MPR tool, reorient the image data along the short axis. Export the reoriented data to DICOM, ensuring that the entire 4D data are exported with preserved slice thickness and image bit depth of 16 bits per voxel. Open the exported 4D MPR images using the 4D viewer, then select a timeframe corresponding to end-diastole and browse through all the timeframes with the time slider on the main toolbar to ensure that the correct cardiac phase is selected.On this timeframe, pick the closed polygon annotation tool and manually delineate the endocardial wall of the left ventricle. Do the same for 10 to 20 slices from the base to the apex ensuring that all the ROIs have the same name. Next on the ROI menu, select ROI volume, then generate missing ROIs to generate the ROIs on all the short axis slices by interpolation of the manually drawn ROIs.Then select ROI volume, followed by compute volume to calculate the volume of the ROI group with the same ROI name. Next, browse through the timeframes, select a phase corresponding to end-systole, and calculate the volume of the ROI group with the same ROI name as demonstrated. Finally, calculate the stroke volume and ejection fraction using the equation described in the manuscript.The results of the automatic myocardial and left ventricle cavity segmentation of a control CD1 mouse are shown here. Even in healthy subjects, lower reconstructed values around the apex are commonly observed in PET. The Patlak graphical analysis demonstrated an example of the regional KI, the scatter plot and linear regression analysis, and the values of the slope and intercept of the linear fit performed on each segment along with the corresponding coefficient of determination.In the healthy rat, different shapes and sizes of the left ventricle are shown for the end-diastolic and end-systolic phases. With the 3D reconstruction of the segmented left ventricle volume, the calculation of volumes resulted in end-diastolic volume of 0.361 milliliters and an end-systolic volume of 0.038 milliliters. A volume rendering of the same rat heart for the end-diastole and end-systole allows for depicting the iodine enhanced chambers and vessels so their value is more qualitative than quantitative.Maintain the level of anesthesia and often check the physiological parameters of the animal during the study. Moreover, an initial check of the patency of the cannulation must be done to avoid unsuccessful injections. Due to the non-invasive nature of this study, the animal can be recovered after the procedure and decay of the radioactive tracer so that virtually, any other investigation method can be applied.Otherwise, if the animal is going to be euthanized after PET/CT imaging, all standard ex vivo analysis on excited tissues can be performed.

high-resolution-cardiac-positron-emission-tomographycomputed

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

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