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).

Daniele Panetta received grants for the R&amp;D of micro-CT instrumentation from Inviscan Sas.

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 present isotropic pixel spacing; this is beneficial from the image processing point of view as it does not require pre-scan operator-dependent tasks to select specific slice orientations along the standard axes of the heart.
This paper includes a protocol based on the Patlak graphical analysis of dynamic PET data18. This type of analysis is useful to describe irreversible tracer uptake from the tissue, which is a good approximation in the case of [18F]FDG, where the effect of dephosphorylation or metabolites is generally negligible in the myocardium19. Within this approximation, the ratio between the decay-corrected tissue activity concentration CT(t) and the decay-corrected plasma activity concentration CP(t) can be approximated by the following equation (5):
<img alt="Equation 1" src="/files/ftp_upload/64066/64066eq01.jpg" style="margin: auto;" /> (5)
which holds for times&#160;t, after a certain start time, t*, that must be determined empirically. In the above equation, the constant Ki represents the net influx rate from blood to tissue, while IC is a constant involving the blood-volume fraction and the distribution volume of the tracer in the reversible compartment (i.e., the plasma). A more detailed mathematical derivation of this formula can be found elsewhere20. If the time activity curves (TACs) of both the plasma and tissue are available (e.g., from a dynamic PET scan and/or plasma sampling), a 2D scatter plot can be created by plotting <img alt="Equation 2" src="/files/ftp_upload/64066/64066eq02.jpg" style="margin: auto;" /> and <img alt="Equation 3" src="/files/ftp_upload/64066/64066eq03.jpg" style="margin: auto;" /> for each frame time, t, so that Ki and IC can be easily determined as the slope and intercept of the scatter plot by simple linear regression, restricted to those time points t &#62; t* after which linearity is observed. It must be underlined that the prolonged anesthesia can impact on the metabolic rate of the myocardium21. For this reason, it is very important to standardize the protocol so that inter-subject variations of all the relevant physiological parameters are kept to a minimum.&#160;The procedure described in the protocol, implemented in Carimas, allows regional Patlak graphical analysis of the myocardium; we have used the activity concentration of the whole blood in the LV cavity as an approximation of the plasma input function CP(t).
Some PET scanners might have lower spatial resolution and/or sensitivity, leading to the use of larger ROIs and consistent partial volume/spillover errors in the time activity curves (TACs) of the measurements, especially the plasmatic one used as the input function (IF). In this case, the analysis protocol can be modified by creating a hybrid IF based on the image values in the early phase after injection and on blood sample activity concentration (see protocol step 6.8) in the late phase (&#62;20 min). The corrected points of the hybrid IF can be calculated by interpolation, as shown by Shoghi et al.23. Within Carimas, it is possible to export the raw TACs of each myocardial segment, correct the arterial TAC, and load them again to perform Patlak analysis directly on the corrected curves. Due to the complexity of the operation required, we have not provided specific protocol operations to do that, as the results obtained in the case described in this protocol have a good level of reproducibility for most applications.
A possible application of the presented protocol is in small animal models of myocardial infarction. To avoid restrictions to such a specific field of imaging research, we did not add a specific protocol instruction for the induction of MI or other types of cardiovascular diseases. Detailed surgical procedures can be found elsewhere in the literature12,13, and they have been successfully applied in our group with the goal of depicting complementary information for both regional perfusion defects and ischemia-induced angiogenesis4. Nevertheless, the PET/CT imaging protocol presented in this paper can be useful in a variety of study designs when cardiac metabolism, function, and/or morphology are of concern, including, but not limited to, metabolic diseases27, response to therapy and/or to different diets28, and radiation-induced injuries29. Moreover, this type of investigation can be useful when validating new molecular probes for monitoring cardiac remodeling and neovascularization in correlation with global and regional cardiac function and morphology 4.
Here, we have discussed a typical PET image acquisition and analysis focused on the quantification of myocardial regional glucose uptake by means of [18F]FDG; for myocardial infarction imaging, for instance, this is useful and widely adopted for measuring the myocardial viability26 as complementary information about perfusion, which instead requires different tracers. Moreover, [18F]FDG is the most widely available tracer in PET imaging, and hence, we decided to tailor this protocol to this tracer to increase its applicability. With minor modifications to the analysis workflow, the same procedure can be used to quantify, for example, the regional myocardial blood flow (MBF), employing [13N]NH3 or [15O]H2O as blood flow tracers30.
In these cases, the PET acquisition protocol requires minor changes, considering the different radionuclide decay times of 13N (T1/2 = 9.97 min) and 15O (T1/2 = 2.04 min) with respect to 18F (T1/2 = 109.8 min). Further, appropriate kinetic models must be used instead of the one presented in this paper, which is commonly available in most quantitative image processing software for PET analysis; besides these points, the experimental procedure presented in this protocol is mostly suitable for other types of experimental investigation focused on the hearts of small animals. Although the protocol was specifically designed for cardiac imaging of mouse models, working with rats might imply some modifications to the actual protocol, mainly because of the bigger size of the animal (~10x heavier). However, additional information has been added to the protocol to indicate the required modifications for rat imaging for the sake of simplicity.
An advantage of the presented protocol is that it does not require the use of ECG probes on the animal, as the PET study can be reliably conducted without gating, and the CT study uses intrinsic (sensorless) retrospective gating. The algorithm at the base of the intrinsic gating software is based on the work of Dinkel et al.31. This method shows very high agreement with ECG-based (extrinsic) cardiac gating and may even potentially be better in the case of arrhythmias due to the dissociation of mechanical and electrical events31. Even though intrinsic gating could be implemented in fully automated workflows32, this protocol is based on an interactive method implemented in the IRIS CT scanner, giving more flexibility in the choice of the parameters. As discussed, minor adaptations of the procedures are required when using rats instead of mice, primarily regarding the injected doses, the need for attenuation correction (CTAC) scans when using larger animals, as well as some differences between the types of CT contrast agents. Regarding this last point, the usage of iodine-rich oil-in-water lipid emulsions on rats is also reported in the technical notes of small animal CA vendors. Due to the relatively large injection volumes involved, the relatively higher cost, and the less widespread availability of these specialized contrast agents, we have also presented a modification of the protocol based on commonly available vascular contrast agents, such as iomeprol, which is broadly applicable in clinical settings. Due to the very fast clearance of such standard vascular agents, a motorized injection pump allowing slow continuous injection is required in this case.
Limitations of the method 
The applicability of the presented PET/CT protocols relies on the availability of instrumentation that is generally less widespread and more expensive than other techniques (primarily US echocardiography), even though the contextual information about structure, function, and metabolism cannot be achieved by any other technique with the same sensitivity and flexibility on the choice of the molecular probe. However, the successful completion of the entire preparation/acquisition/analysis workflow with this methodology requires a strong collaboration between several professional figures, including biologists, veterinary doctors, chemists, physicists, and bioengineers. This is even more true when non-standard PET tracers are used, implying efforts in both radiosynthesis and mathematical modeling, as well as in the adaptation of the analysis software for correct and reliable quantification33,34,35.
In protocol section 9, we have described a very simple quantification procedure by using an image-derived input function (IDIF), pointing out that a mixed approach using IDIF and blood sample-derived IF for late frames can give better results. It must be noted that using activity measured from whole (venous) blood taken from the tail is considered a reliable approximation in [18F]FDG, but it requires further corrections for the activity of metabolites in the case of different tracers36,37. One of the most critical points of the entire protocol is the intravenous cannulation, giving venous access for injecting both the radioactive tracer for the PET scan and the iodinated contrast agent for the CT scan. Unsuccessfully carrying out this critical step results in useless images, as the effective quantity of circulating PET tracer or CT CA might be lower than required. Expert personnel with specific training for tail vein injection must be involved in this procedure to provide reliable results.
A drawback of CT for dynamic cardiac imaging is its relatively lower temporal resolution as compared to US and MRI, even though 3D cardiac imaging with ultrasound requires the use of a motorized translating stage for the probe and subsequent image registration to get correct results. The need for consistent volumes of CA to be injected for the correct discrimination of blood and myocardium in reconstructed images is one of the main concerns due to the intrinsic low sensitivity of the methodology. In this protocol, we have limited the volume of injection of CA for CT studies to 0.5 mL in mice and 2 mL in rats, using continuous infusion for 3 min at 10 mL/h in mice and for 5 min at 24 mL/h in rats. We have observed that these rates and volumes of injections are well tolerated by the animals. The quantities described here are in line with or are smaller than equivalent protocols found in the literature.
Nahrendorf et al. described a Cine-CT protocol for the depiction of murine myocardial infarction, involving a basal (pre-scan) bolus injection of 0.2 mL of oil-in-water lipid emulsion blood pool CA followed by a continuous injection of iomeprol at 1 mL/h for 1 h38. Badea et al. compared a similar cardiac Cine-CT protocol based on a 1 h infusion of Isovue 370 (iopamidol) with a bolus injection of 0.5 mL/25 g of body weight of Fenestra VC (oil-in-water lipid emulsion), finding better results in terms of image contrast in the second case39. The same manufacturer of the Fenestra VC contrast agent reported a recommended volume of injection of 0.4 mL/20 g of body weight for vascular imaging with micro-CT40. However, novel CAs with higher density such as eXIA 160 XL, MVivo Au, Aurovist 15 nm, or Exitron nano 12000 have recently entered the preclinical market and have the potential to reduce the injection volumes in cardiac micro-CT protocols. Nebuloni et al. conducted an extensive characterization of such CAs41. The radiation dose in gated CT is another common concern for longitudinal studies; in this case, the maximum dose for the described Cine-CT protocol is below 200 mGy for both mice and rats, as estimated on the basis of previous dosimetric characterization of our CT scanner42. This is roughly 5x lower than the reported dose in the literature for 4D cardiac CT scans38,39 and 30x lower than the average lethal dose for total body irradiation of small animals, estimated at 6 Gy43.
Applicability of the protocol to different instrumentation and software 
Even though the specific instructions presented in this protocol are unavoidably tailored for a specific PET/CT tomograph, the imaging tasks presented here can be adapted to different imaging systems. Regarding the PET section of this protocol, all state-of-art PET or PET/CT systems designed for small animal research have performance requirements (in terms of spatial and temporal resolution) suitable to carry out the protocol. As far as cardiac CT is concerned, the protocol might change depending on the specific cardio-respiratory gating system employed (e.g., extrinsic or intrinsic). The readers can refer to recent review articles and book chapters for a thorough discussion of current PET, CT, or PET/CT systems&#39; capabilities44,45,46. Of note, the CT and PET protocols presented in this paper can be performed independently, based on the capabilities and peculiarities of the tomographic instrumentation in use. We, therefore, believe that the presented procedures can provide a useful reference for any practitioner interested in conducting for the first time, a cardiac PET/CT study on small animals.
Any user with sufficient skills in the general protocol setup of their own PET/CT tomograph should be able to implement the required adaptations on the presented method to get equivalent results in their lab. The same arguments can be used for the section dedicated to image analysis. A thorough list of all the available software packages for cardiac PET and cardiac CT analysis is outside of the purpose of this paper. However, many other comparable software packages use a similar methodology for polar map generation and regional tracer kinetic analysis. The reader can refer to Wang et al.47 and references within for the task of PET quantification and to pertinent research articles48,49,50 for 4D CT quantification. In this case, we have decided to focus this protocol on Carimas51,52,53,54 and OsiriX55,56,57,58 for the quantitative analysis of cardiac PET and CT images, respectively. Due to the widespread use of these tools, we believe that this choice could be helpful in increasing the interest of the research community in the implementation and application of the presented methods, as compared to a discussion focused on closed, commercial, and scanner-specific analysis tools provided by some PET and CT scanner manufacturers.
Modifications to the quantitative image analysis protocol 
The example results shown here are just a simple outcome of a straightforward quantitative analysis task, which might be considered sufficient for most practical purposes in translational cardiovascular research experiments focused on small animal models of cardiac injury. However, many more analysis options are possible, starting from the DICOM images resulting from the acquisition/reconstruction protocol described in this paper. For instance, one might be interested in applying different compartmental models instead of the Patlak graphical analysis from dynamic [18F]FDG-PET data59,60,61. Moreover, the analysis of the cardiac function based on 4D Cine-CT images shown in this protocol was only global for the entire LV, but several different (mainly commercial) software allow users to perform strain analysis and regional wall motion, wall thickening, and regional EF analysis from the same images49. Nevertheless, we believe that the examples shown here represent a good starting point for more in-depth post-processing and quantitative tasks.

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><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&amp;amp;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 required for reliable identification of the standard sectors following the American Heart Association (AHA) recommendations25. In the case of myocardial ischemia, a regional lowering of tracer uptake appears as a typical sign of loss of myocardial vitality. This is not necessarily correlated with reduced perfusion, which would need a different tracer (e.g., [13N]NH3 or [15O]H2O) to be visualized in PET images. Even in healthy subjects, lower reconstructed values around the apex are commonly observed in PET (see Figure 6). This might arise from a more pronounced partial volume artifact due to a (generally) thinner myocardial thickness at the apex as compared to, for example, the left wall or the septum.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64066/64066fig06.jpg" /> 
Figure 6: Results of the automatic segmentation of the PET analysis software. The images were obtained with the Heart plugin of the Carimas software. Segmentation was done after standard reorientation according to AHA guidelines. The images shown refer to a healthy adult male CD-1 mouse (same as Figure 4) weighing 51 g and injected with 10 MBq of [18F]FDG, without cardiac gating, and sum the last 15 min of a 60 min PET scan. The images were reconstructed with an iterative 3D-OSEM algorithm with a voxel size of 0.85 mm3. Abbreviations: PET = positron emission tomography; AHA = American Heart Association; FDG = fluorodeoxyglucose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64066/64066fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In Figure 7, an example of the regional Ki obtained through Patlak graphical analysis18 is shown (top left). In the frame at the bottom, the Patlak scatter plot and the corresponding results of the linear regression analysis are shown. Each point in the scatter plot represents the ratio between tissue activity concentration and plasma activity concentration at a given time t (after correction for radioactive decay), CT(t)/CP(t), plotted against the time integral of the plasma activity concentration from the injection time t0 = 0 to time t. The table on the top-right frame of Figure 7 shows the values of the slope (Ki) and intercept (Ic) of the linear fit performed on each segment, along with the corresponding coefficient of determination (R2).
As far as cardiac PET is concerned, signs of bad execution of the protocol could include, but are not limited to, the following: (i) low or absent tracer uptake from the myocardium, which is typically a sign that a problem occurred during the tracer injection, such as an extravasated injection; (ii) similar problems as in the previous point if the animal temperature is too low during the PET scan (e.g., below 35 &#176;C) and, thus, altered tracer uptake occurs; (iii) evident image blurring, which could be due to a level of anesthesia that is too low or involuntary motion.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64066/64066fig07.jpg" /> 
Figure 7: Results of the Patlak graphical analysis. The images were obtained with the Heart plugin of the Carimas software. Top-left: parametric polar map of regional Ki of the LV resulting from the Patlak analysis. Top-right: average values of Ki and IC on each myocardial segment, along with the coefficients of determination of each linear fit (R2). Bottom: scatter plot of y(t) versus x(t) (see text for details) for the selected myocardial segment (segment 1 in this example). This result refers to the myocardial PET images shown in Figure 4 and Figure 6 (healthy adult male CD-1 mouse weighing 51 g and injected with 10 MBq of [18F]FDG). Abbreviations: PET = positron emission tomography; FDG = fluorodeoxyglucose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64066/64066fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64066/64066fig08.jpg" /> 
Figure 8: Example of manual segmentation of the LV of a rat. The image refers to the same animal as shown in Figure 5 and was obtained with Osirix MD. The resulting volumetric analysis of the LV at end-diastole and end-systole is shown at the bottom. From these results, EF and SV are calculated according to equations 3 and 4. Abbreviations: EF = ejection fraction; SV = stroke volume; ROIs = regions of interest; LV = left ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64066/64066fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64066/64066fig09.jpg" /> 
Figure 9: Volume rendering of the Cine-CT images. The images refer to the same rat shown in Figure 5 and Figure 8 (healthy adult male Wistar rat weighing 507 g and injected with 2 mL of iomeprol, 200 mg/mL, at the rate of 24 mL/h for 5 min, reconstructed with FBP with a voxel size of 0.24 mm3). Abbreviations: RA = right atrium; LA = left atrium; LV = left ventricle; RV = right ventricle; CT = computed tomography; FBP = Filtered BackProjection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64066/64066fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 8 and Figure 9 deal with the representative results of the Cine-CT cardiac analysis for a healthy rat. In particular, in Figure 8, the different shape and size of the LV are shown for the end-diastolic and end-systolic phases, along with the 3D reconstruction of the segmented LV volume in both phases. In this example, the calculation of the volumes according to equations 3 and 4 resulted in EDV = 0.361 mL and ESV = 0.038 mL, corresponding to a stroke volume of SV = 0.323 mL and an ejection fraction EF = 89.4%. This is in agreement with the results reported on similar protocols in the literature, showing normal EF of rats in the range of 70%-90%26. Infarcted hearts might lead to a reduced EF, in the range of 50%-70% or less, depending on the lesion severity and extension of the akinetic myocardium.
The following signs of bad execution of the experiment could occur for Cine-CT images: (i) reduced or absent image contrast between the heart chambers/vessels and the myocardium; in this case, it is likely that a problem in the contrast agent injection occurred; (ii) blurred contours of the myocardial walls; in this case, a problem in the reconstruction has occurred, probably due to wrong identification of the cardiac and respiratory peaks from the intrinsic gating signal, which, in turn, might depend on bad selection of the frequency bands (Figure 3) and/or bad selection of the gating signal ROI (Figure 2); (iii) evident motion artifacts, which might be due to a level of anesthesia that is too low or involuntary motion.
In Figure 9, a volume rendering of the same rat heart is shown for both the end-diastole and end-systole. This type of visualization only allows for depicting the iodine-enhanced chambers and vessels, so their value is more qualitative than quantitative. Nevertheless, reduced motility of the myocardial walls, such as that encountered in infarcted rats, will produce volumetric images with less evident differences between the end-diastolic and end-systolic phases.

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 ...

high-resolution-cardiac-positron-emission-tomographycomputed

Nanyang Technological University

Research

JoVE Journal

Medicine

3.2K Views.  CNR Institute of Clinical Physiology. Het gepresenteerde cardiale PET/CT-protocol is nuttig voor het verkrijgen van functionele en morfologische informatie in een verscheidenheid aan kleine diermodellen van hartaandoeningen. Voordelen van PET/CT-beeldvorming in vergelijking met andere preklinische beeldvormingsmodaliteiten omvatten, maar zijn niet beperkt tot, hoge gevoeligheid en zeer hoge ruimtelijke temporele resolutie, evenals robuustheid omdat het geen handmatige positionering van sondes door de operators vereist. Een van de...

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

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioengineering

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Cancer Research

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Biology

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 ...

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 ...

Four-Dimensional Computed Tomography-Guided Valve Sizing for Transcatheter Pulmonary Valve Replacement

The measurements of the right ventricle (RV) and pulmonary artery (PA), for selecting the optimal prosthesis size for transcatheter pulmonary valve replacement (TPVR), vary considerably. Three-dimensional (3D) computed tomography (CT) imaging for device size prediction is insufficient to assess the displacement of the right ventricular outflow tract (RVOT) and PA, which could increase the risk of stent misplacement and paravalvular leak. The aim of this study is to provide a dynamic model to visualize and quantify the anatomy of the RVOT to PA over the entire cardiac cycle by four-dimensional (4D) cardiac CT reconstruction to obtain an accurate quantitative evaluation of the required valve size. In this pilot study, cardiac CT from sheep J was chosen to illustrate the procedures. 3D cardiac CT was imported into 3D reconstruction software to build a 4D sequence which was divided into eleven frames over the cardiac cycle to visualize the deformation of the heart. Diameter, cross-sectional area, and circumference of five imaging planes at the main PA, sinotubular junction, sinus, basal plane of the pulmonary valve (BPV), and RVOT were measured at each frame in 4D straightened models prior to valve implantation to predict the valve size. Meanwhile, dynamic changes in the RV volume were also measured to evaluate right ventricular ejection fraction (RVEF). 3D measurements at the end of the diastole were obtained for comparison with the 4D measurements. In sheep J, 4D CT measurements from the straightened model resulted in the same choice of valve size for TPVR (30 mm) as 3D measurements. The RVEF of sheep J from pre-CT was 62.1 %. In contrast with 3D CT, the straightened 4D reconstruction model not only enabled accurate prediction for valve size selection for TPVR but also provided an ideal virtual reality, thus presenting a promising method for TPVR and the innovation of TPVR devices.

This study assessed a new methodology with a straightened model generated from the four-dimensional cardiac computed tomography sequence to obtain the desired measurements for valve sizing in the application of transcatheter pulmonary valve replacement.

The measurements of the right ventricle (RV) and pulmonary artery (PA), for selecting the optimal prosthesis size for transcatheter pulmonary valve ...

Retrospective Cardiac Gating with A Prototype Small-Animal X-ray Computed Tomograph

The CrumpCAT is a prototype small-animal X-ray computed tomography (CT) scanner developed at our research institution. The CMOS detector with a maximum frame rate of 29 Hz and similar Tungsten X-ray sources with energies ranging from 50 kVp to 80 kVp are widely used across commercially available preclinical X-ray CT instruments. This makes the described work highly relevant to other institutions, despite the generally perceived wisdom that these detectors are not suitable for gating the high heart rates of mice (~600 beats/min). The scanner features medium- (200 &#181;m) and high- (125 &#181;m) resolution imaging, fluoroscopy, retrospective respiratory gating, and retrospective cardiac gating, with iterative or filtered-back projection image reconstruction. Among these features, cardiac gating is the most useful feature for studying cardiac functions in vivo, as it effectively eliminates image blurring caused by respiratory and cardiac motion. 
Here, we describe our method for preclinical intrinsic retrospective cardiac-gated CT imaging, aimed at advancing research on in vivo cardiac function and structure analysis. The cardiac-gating method acquires a large number of projections at the shortest practical exposure time (~20 ms) and then retrospectively extracts respiratory and cardiac signals from temporal changes in raw projection sequences. These signals are used to reject projections belonging to the high motion rate inspiration phase of the respiratory cycle and to divide the remaining projections into 12 groups, each corresponding to one phase of the cardiac cycle. Each group is reconstructed independently using an iterative method to produce a volumetric image for each cardiac phase, resulting in a four-dimensional (4D) dataset. 
These phase images can be analyzed either collectively or individually, allowing for detailed assessment of cardiac function. We demonstrated the effectiveness of both approaches of the prototype scanner's cardiac-gating feature through representative in vivo imaging results.

We provide a comprehensive description of the intrinsic retrospective cardiac gating method of the CrumpCAT, a prototype small-animal X-ray computed tomography (CT) scanner designed and constructed at our research institution.

The CrumpCAT is a prototype small-animal X-ray computed tomography (CT) scanner developed at our research institution. The CMOS detector with a maximum ...

Cardiac Magnetic Resonance Imaging

Source: Frederick W. Damen and <a href="https://www-jove-com.remotexs.ntu.edu.sg/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
In this video, high field, small-bore magnetic resonance imaging (MRI) with physiological monitoring is demonstrated to acquire gated cine loops of the murine cardiovascular system. This procedure provides a basis for assessing left-ventricular function, visualizing vascular networks, and quantifying motion of organs due to respiration. Comparable small animal cardiovascular imaging modalities include high-frequency ultrasound and micro-computed tomography (CT); however, each modality is associated with trade-offs that should be considered. While ultrasound does provide high spatial and temporal resolution, imaging artifacts are common. For example, dense tissue (i.e., the sternum and ribs) can limit imaging penetration depth, and hyperechoic signal at the interface between gas and liquid (i.e., pleura surrounding the lungs) can blur contrast in nearby tissue. Micro-CT in contrast does not suffer from as many in-plane artifacts, but does have lower temporal resolution and limited soft-tissue contrast. Furthermore, micro-CT uses X-ray radiation and often requires the use of contrast agents to visualize vasculature, both of which are known to cause side effects at high doses including radiation damage&#160; and renal injury. Cardiovascular MRI provides a nice compromise between these techniques by negating the need for ionizing radiation and providing the user with the ability to image without contrast agents (although contrast agents are often used for MRI).
This data was acquired with a triggering Fast Low Angle SHot (FLASH) MRI sequence that was gated off of the R-peaks in the cardiac cycle and expiratory plateaus in respiration. These physiological events were monitored through subcutaneous electrodes and a pressure-sensitive pillow that was secured against the abdomen. To ensure the mouse was properly warmed, a rectal temperature probe was inserted and used to control the output of a MRI-safe heating fan. Once the animal was inserted into the bore of the MRI scanner and navigation sequences were run to confirm positioning, the gated FLASH imaging planes were prescribed and data acquired. Overall, high field MRI is a powerful research tool that can provide soft tissue contrast for the study of small animal disease models.

Cardiac MRI: Principle and Imaging

Source: Frederick W. Damen and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
In this video, high ...

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

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