The authors gratefully acknowledge Susann Lehmann, Iloana Klamfu&#223; and Petra Wolff for animal housing and care and Matthias Wyss for support during the establishment of the online blood sampling system. The small animal PET/CT was funded by the Deutsche Forschungsgemeinschaft (INST 2268/6-1 FUGG).

<ol>
	<li>Schain, 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=Arterial+input+function+derived+from+pairwise+correlations+between+PET-image+voxels.">Arterial input function derived from pairwise correlations between PET-image voxels.</a> Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 33 (7), 1058-1065 (2013).</li><li>Schain, M., Zanderigo, F., Mann, J. J., Ogden, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+binding+potential+BPND+without+a+reference+region+or+blood+samples+for+brain+PET+studies.">Estimation of the binding potential BPND without a reference region or blood samples for brain PET studies.</a> NeuroImage. 146, 121-131 (2017).</li><li>Bentourkia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+Input+Function+at+the+Entry+of+the+Tissue+of+Interest+and+Its+Impact+on+PET+Kinetic+Modeling+Parameters.">Determination of the Input Function at the Entry of the Tissue of Interest and Its Impact on PET Kinetic Modeling Parameters.</a> Molecular Imaging and Biology. 17 (6), 748-756 (2015).</li><li>Phelps, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PET. , (2004).</li><li>Laforest, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+input+functions+in+rodents:+challenges+and+solutions.">Measurement of input functions in rodents: challenges and solutions.</a> Nuclear Medicine and Biology. 32 (7), 679-685 (2005).</li><li>Napieczynska, 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=Impact+of+the+Arterial+Input+Function+Recording+Method+on+Kinetic+Parameters+in+Small-Animal+PET.">Impact of the Arterial Input Function Recording Method on Kinetic Parameters in Small-Animal PET.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 59 (7), 1159-1164 (2018).</li><li>Sijbesma, J. W. A., 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=Novel+Approach+to+Repeated+Arterial+Blood+Sampling+in+Small+Animal+PET:+Application+in+a+Test-Retest+Study+with+the+Adenosine+A1+Receptor+Ligand+[(11)C]MPDX.">Novel Approach to Repeated Arterial Blood Sampling in Small Animal PET: Application in a Test-Retest Study with the Adenosine A1 Receptor Ligand [(11)C]MPDX.</a> Molecular Imaging and Biology: MIB: the Official Publication of the Academy of Molecular Imaging. 18 (5), 715-723 (2016).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> Journal of Visualized Experiments. (59), e3496 (2012).</li><li>Weber, B., Burger, C., Biro, P., Buck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+femoral+arteriovenous+shunt+facilitates+arterial+whole+blood+sampling+in+animals.">A femoral arteriovenous shunt facilitates arterial whole blood sampling in animals.</a> European Journal of Nuclear Medicine and Molecular Imaging. 29 (3), 319-323 (2002).</li><li>Liu, H. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anastomosis+of+submillimeter+vessels-a+training+model+in+rats.">Microvascular anastomosis of submillimeter vessels-a training model in rats.</a> Journal of Hand and Microsurgery. 5 (1), 14-17 (2013).</li><li>van der Weerdt, A. P., 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=Image-derived+input+functions+for+determination+of+MRGlu+in+cardiac+(18)F-FDG+PET+scans.">Image-derived input functions for determination of MRGlu in cardiac (18)F-FDG PET scans.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 42 (18), 1622-1629 (2001).</li><li>Alf, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+brain+glucose+metabolism+by+18F-FDG+PET+with+real-time+arterial+and+image-derived+input+function+in+mice.">Quantification of brain glucose metabolism by 18F-FDG PET with real-time arterial and image-derived input function in mice.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 54 (1), 132-138 (2013).</li><li>Eriksson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computerized+infusion+pump+for+control+of+tissue+tracer+concentration+during+positron+emission+tomography+in+vivo+pharmacokinetic/pharmacodynamic+measurements.">A computerized infusion pump for control of tissue tracer concentration during positron emission tomography in vivo pharmacokinetic/pharmacodynamic measurements.</a> BMC Medical Physics. 8, 2 (2008).</li><li>Burger, C., Buck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracer+kinetic+modelling+of+receptor+data+with+mathematical+metabolite+correction.">Tracer kinetic modelling of receptor data with mathematical metabolite correction.</a> European Journal of Nuclear Medicine. 23 (5), 539-545 (1996).</li><li>Iida, 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=Error+analysis+of+a+quantitative+cerebral+blood+flow+measurement+using+H2(15)O+autoradiography+and+positron+emission+tomography,+with+respect+to+the+dispersion+of+the+input+function.">Error analysis of a quantitative cerebral blood flow measurement using H2(15)O autoradiography and positron emission tomography, with respect to the dispersion of the input function.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 6 (5), 536-545 (1986).</li><li>Munk, O. L., Keiding, S., Bass, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+estimate+dispersion+in+sampling+catheters+and+to+calculate+dispersion-free+blood+time-activity+curves.">A method to estimate dispersion in sampling catheters and to calculate dispersion-free blood time-activity curves.</a> Medical Physics. 35 (8), 3471-3481 (2008).</li><li>Meyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+correction+for+tracer+arrival+delay+and+dispersion+in+CBF+measurements+by+the+H215O+autoradiographic+method+and+dynamic+PET.">Simultaneous correction for tracer arrival delay and dispersion in CBF measurements by the H215O autoradiographic method and dynamic PET.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 30 (6), 1069-1078 (1989).</li><li>Lanz, B., Poitry-Yamate, C., Gruetter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-derived+input+function+from+the+vena+cava+for+18F-FDG+PET+studies+in+rats+and+mice.">Image-derived input function from the vena cava for 18F-FDG PET studies in rats and mice.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 55 (8), 1380-1388 (2014).</li><li>Geworski, 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=Multicenter+comparison+of+calibration+and+cross+calibration+of+PET+scanners.">Multicenter comparison of calibration and cross calibration of PET scanners.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 43 (5), 635-639 (2002).</li><li>Boellaard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standards+for+PET+image+acquisition+and+quantitative+data+analysis.">Standards for PET image acquisition and quantitative data analysis.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 50, 11-20 (2009).</li></ol>

The authors have nothing to disclose.

The presented results are extracted from a larger-scale project on neuronal activity in a transgenic animal model of Huntington&#39;s disease compared to wildtype rats. Altogether 30 transgenic and wildtype rats were catheterized and manual and online blood sampling in parallel to [18F]FDG-PET/CT was performed. Three AIFs of wildtype rats are shown here to demonstrate the range of possible outcomes of the protocol. The results of the complete project on changes of neuronal activity in an animal model of Huntington&#39;s disease will be published elsewhere.
The here described method enables fast and accurate continuous blood sampling in a big cohort and provides a gapless AIF for kinetic modeling of dynamic PET/CT data in small animals. An external blood circulation is generated to detect actual time activity in the blood of the animals; consequently a loss of blood is avoided. The surgical procedure is based on Jespersen et al.8 and was modified to meet the needs for arterial blood sampling during the PET/CT measurements. The shunt system was validated by Weber et al.9. With the here used setup, an external blood volume of about 1.1 mL is running through the detector-pump system. A rat aged 4 month has a total blood volume of about 30 mL. The diameter of the femoral vein and artery is approximately 0.45-0.6 mm10 and needs to be a little starched to insert the catheter used.
The AIF can also be measured via sporadic manual blood collection or be reconstructed from early time points of the PET images itself (image-derived). Both approaches were performed with the here presented data and compared to the continuous blood sampling.
In comparison to manual blood sampling, with online blood sampling a noticeable higher temporal resolution (here: 1800 data points per 30 min) becomes possible. Manual blood draws (here: 5 data points per 30 min) are limited to the blood volume present in the small animal, as these samples are not pumped back into the circulation of the animal. Moreover, a maximum interval of 10-15 s is technically implementable and important information for kinetic modeling is missed. This can also be seen in the presented data, as a difference in the detected maximum of continuous and manual blood sampling is obvious (Figure 3A,C,E). With online blood sampling the detected peak was higher than with the image-derived input function of the ascending aorta11 (Figure 3B,D,F). The Imaged-derived input function is restricted to the spatial resolution of PET scanners which results in partial volume effects12 and is affected by the reconstructed time frames.
A general advantage of this continuous blood sampling procedure is that the tracer can be applied via the catheter, which is less prone to disturbance than injection via the lateral tail vein. Keep in mind that the tracer should be applied in a moderate volume to prevent the tracer from remaining in the beginning of the tube system. To ensure that no activity is remaining in the dead volume of the T-piece, it is flushed with heparinized saline solution afterwards. Moreover, the usage of an infusion pump is advised as it enables adjustment of the speed of the tracer injection and can contribute to more coordinated acquisition of the maximum radioactivity peak with manual blood sampling13.
There are a few possible difficulties that might occur during protocol processing and can be handled by the following troubleshooting. A sub-optimal position of the catheters might lead to an incomplete execution of the protocol, therefore ensure that they are accurately fixed with the proximal suture and that the catheter is pushed 2-3 cm proximal into the vessel. In addition, fibrin adhesive can be used. Also formation of thrombi can clog the catheters. This can be handled by increasing the heparin concentration and subsequent flushing of the catheters or the tube system. Such a sub-optimal outcome due to clogging of the catheters is shown in the results, the maximum peak is missed (Figure 3E). Another critical point concerning animal protection and well-being is the length of the extracorporeal blood flow. It is therefore suggested to reduce the length of the tube system to a minimum.
When blood sampling is performed, three corrections of the resulting AIF have to be taken into account. First, plasma correction. Tracers equilibrate between plasma and blood cells, mainly erythrocytes. Depending on how fast these diffusion processes are, the available tracer is mainly present in plasma. For some tracers, the ratio of plasma to whole blood needs to be considered, such as more lipophilic ones. In these cases, plasma activity has to be determined. If [18F]FDG is used, there is no need to centrifuge the blood to determine the plasma activity, as it equilibrates very fast between plasma and red blood cells and the availability of [18F]FDG in plasma is similar to that in the whole blood. Secondly, metabolite correction. Many tracers are metabolized in whole blood and some of these metabolites are still radioactively labeled14. This fraction is present in the AIF but is not available for tissue uptake. For some tracers metabolites need to be determined in whole blood or plasma and the AIF needs to be corrected. Thirdly, dispersion correction. Dispersion is caused by several factors, including (a) the systematic time difference between the tracer arrival times in the tissue relative to the peripheral sampling site (delay correction) and (b) and the smearing of the shape of the AIF, as the tracer transport within the tube system is influenced by its first order lag (PT1) kinetics. Several corrections based on deconvolution have been proposed, mainly based on the model by Iida et al.15, but most of them are susceptible to noise. A correction method which circumvents deconvolution and is therefore less prone to noise has been proposed by Munk et al.16. The necessary measurements to estimate the correction parameters have to be performed for every combination of tubing and tracer used. Dispersion correction should be done before time delay correction17. However, mainly fast tissue perfusion processes are affected by dispersion and it has also been shown, that for modeling of [18F]FDG studies a dispersion correction is not absolutely necessary18. Therefore, in the presented examples the dispersion correction of the AIF has not been applied.
A proper calibration of the on-site dose calibrator and its regular quality control is a prerequisite for the type of cross calibration procedures presented here. However, if the activity administered to the animal is measured with the same dose calibrator, any deviation in accuracy will be cancelled out, provided that the deviation is constant and the complete cross calibration procedure has been followed, including nuclide-specific corrections (e.g., for varying half-life or different branching ratio). Using such a calibration procedure for harmonizing PET/CT systems used in human health care and research, an accuracy of at least 5-10% could be achieved19,20.
The calibrated and corrected AIFs generated by successful implementation of this protocol enable quantification of PET/CT data for the characterization of animal disease models, testing of new therapy options, establishment of new tracers, and transferring of existing tracers into another species. Seemingly, continuous blood sampling in [18]FDG-PET/CT in rats delivers the most reliable information for the calculation of the input in bio-kinetic modeling. By taking into account the individual metabolism, especially liver clearance, a more precise assessment of the relevant pathological or therapeutical effects is possible. With this practicable protocol, a higher efficiency of preclinical PET/CT data analysis is easily implementable.

Positron emission tomography/computed tomography (PET/CT) is a nuclear imaging technology that enables visualization of metabolic processes in the body following injection of a radioactively labeled ligand, also called tracer. Whereas the ligand is a molecule that is involved in a metabolic pathway or targets cell surface proteins, the radioactive label is a positron-emitting radionuclide. Gamma rays are indirectly emitted by the positron decay and allow the detection of its distribution in the organism with extracorporeal PET detectors. In this way, different cellular molecules can be targeted: neurotransmitter receptors and transporters, metabolic processes like glycolysis or mitochondrial proteins like the translocator protein 18 kDa (TSPO) to detect activated glia cells.
In preclinical research, PET/CT is an attractive method to study biochemical processes in a non-invasive manner in vivo, thus allowing longitudinal studies. PET/CT data support the analyses of disease mechanisms, the assessment of the characteristics and pharmacokinetics of new drugs and the validation of both, current and novel radiotracers for translational research.
During PET/CT analyses three tracer states can be defined (example of the 2-tissue compartment model): First, the tracer flows within the blood after its application (state 1; conc.[blood]). Second, it enters the tissue via the capillary bed and can there either freely move within the extracellular space or is unspecifically bound to diverse cellular or extracellular structures (state 2; conc.[unspec]). Third, the tracer can be specifically bound (with or without metabolic trapping) to its target molecule (state 3, conc.[spec]). All these dynamic processes between the compartments are to some extent bidirectional and the diffusion processes are described by rate constants (K1, k2, k3, and k4). While the concentration of the tracer in the blood (i.e., state 1) is called &#34;Input&#34;, the concentration of unspecifically and specifically bound tracer (i.e., state 2 and state 3) is called &#34;Output&#34; and can be directly derived from the PET Image. This physiological relation can be displayed in the 2-tissue compartment model (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59701/59701fig1.jpg" /> 
Figure 1: The two-tissue compartmental model. The physiological conditions of the three different tracer states and the dynamic processes between them are displayed. <a href="https://www.jove.com/files/ftp_upload/59701/59701fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the ideal case, conc.[spec] is proportional to the concentration of its target molecule. However, the output of the PET/CT measurement is the sum of conc.[spec] and conc.[unspec]. To determine conc.[spec] in the region of interest, in parallel the conc.[unspec] of a reference region devoid of the target protein/pathway is determined. By using appropriate mathematical equations one can now calculate conc.[spec], most commonly using the compartment model (a bio-kinetic modeling approach). However, in many cases, such a reference region devoid of the target protein is not available1,2. In these cases, the conc.[blood] can be used to determine conc.[spec]. Since the conc.[blood] is varying due to different liver and kidney clearance, excretion, blood flow, different brain-blood barrier penetration and disease-related factors3, the current gold standard is to measure the conc.[blood] in parallel to the PET/CT scan by continuous blood sampling. This gives the arterial input function (AIF), which is defined as conc.[blood] over time4. Of note, performing continuous blood sampling is considered technically highly challenging, especially in small animals such as rats or mice5.
Here, we provide an easy and practical protocol to continuously sample blood from rats via an arteriovenous (a-v) shunt between the femoral vein and artery. Coupled to a commercially available detector-pump system, we are able to generate a real-time, continuous AIF during dynamic [18F]fluorodeoxyglucose ([18F]FDG)-PET/CT scans in rats and compared it to alternative approaches. PET/CT imaging was performed in male sprague dawley rats at an age of 4 months with an average weight of 462 g &#177; 33 g (mean &#177; standard deviation) using a multimodality PET/CT scanner.
Since a wide variety of devices is used during the series of measurements (dose calibrator, online blood sampler, PET/CT, and well counter), a quality control procedure referred as cross calibration is needed to check the quantitative accuracy of all systems and to compensate for differences. Cross calibration in the context of online blood sampling means that the count rate for a given activity concentration measured in corrected PET images can be converted into the concentration measured with the twilite system for the same concentration. Therefore, a cross calibration procedure between PET/CT, blood sampling system, and well counter has been established.
This highly standardized methodology provides a powerful approach to quantify metabolic and cellular processes in preclinical small animal research and is an elegant way to improve the reliability and reproducibility of the AIF. The AIF can then be used to quantify the specifically bound tracer in tissue in preclinical PET/CT data using bio-kinetic modeling.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Sugery for arteriovenous shunt&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>anesthesia station</td><td>Groppler</td><td></td><td></td></tr><tr><td>aneurysm clips</td><td>Aesculap</td><td>FT190T</td><td>5 mm, closing force 70 g</td></tr><tr><td>bulldog clamp</td><td>Aesculap</td><td></td><td>35 mm</td></tr><tr><td>dissectiong scissors BC165</td><td>Aesculap</td><td>490-866</td><td>dull, for skin preparation</td></tr><tr><td>heating mat</td><td></td><td></td><td></td></tr><tr><td>insulin syringe</td><td>Braun</td><td></td><td>30G</td></tr><tr><td>needle holder</td><td>medicon</td><td>11.62.18</td><td>micro surgical</td></tr><tr><td>pliers for aneurysm clips</td><td>Aesculap</td><td>FT 470T</td><td>Yasargil</td></tr><tr><td>portex fine bore polythene tubing</td><td>Smith Medical</td><td>800/100/200</td><td>ID 0.58 mm, OD 0.96 mm; PE50 equivalent tubing</td></tr><tr><td>surgical microscope with camera</td><td>Leica</td><td></td><td>M50 + MC120 HD</td></tr><tr><td>suture filaments 6.0</td><td></td><td></td><td>6.0, polypropylene</td></tr><tr><td>suture filaments 3.0</td><td></td><td></td><td>3.0, absorbable, braided</td></tr><tr><td>two anatomical forceps</td><td>Hammacher Soling</td><td>HSC601-11</td><td>micro surgery, 45&amp;deg;</td></tr><tr><td>vascular or corneal scissors</td><td>Geuder</td><td>G19605</td><td>micro surgery scissors</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;PET/CT imaging&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>dose calibrator ISOMED 2010</td><td>nivia instruments GmbH</td><td></td><td>for tracer portioning</td></tr><tr><td>Inveon PET/CT</td><td>Siemens</td><td></td><td></td></tr><tr><td>tracer (e.g. 18F-FDG)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;manuel bloodsampling&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>capillary blood collection EDTA tube</td><td>KABE Labortechnik GmbH</td><td></td><td>GK 150 EDTA 200 &amp;micro;l</td></tr><tr><td>test tubes</td><td>SARSTEDT</td><td></td><td>5 ml, 75 x 12 mm, PS</td></tr><tr><td>well counter CAPTUS 700t</td><td>Capintec</td><td></td><td>manuel measurement of blood activity</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;automatic blood sampling&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Venflon TM pro safety shielded IV catheter; 18 G (1.3 mm x 32 mm)</td><td>BD</td><td>3932269</td><td>luer connections (to fit in t-connections)</td></tr><tr><td>bloodsampler twilite two</td><td>swisstrace GmbH</td><td></td><td></td></tr><tr><td>combi stopper</td><td>Braun</td><td>4495101</td><td></td></tr><tr><td>heparin</td><td></td><td></td><td>50U/ml for tube flushing before the experiment and aspiration during catheter surgery</td></tr><tr><td>hypodermic needle</td><td></td><td></td><td>G23 x 1 1/4&quot; / 0.6 x 30 mm</td></tr><tr><td>microprocessor controlled tubing pump</td><td>Ismatec/Cole-Parmer</td><td>ISM596</td><td>12 rollers, 2 channels</td></tr><tr><td>PSAMPLE modul of PMOD</td><td>PMOD</td><td></td><td></td></tr><tr><td>reduction connectors</td><td>Ismatec/Cole-Parmer</td><td>ISM569A</td><td>from ID 2.5 mm to ID 1.5 mm</td></tr><tr><td>silicone pump tubes</td><td>Ismatec/Cole-Parmer</td><td>070535-17-ND /SC0065N</td><td>for roller pump (yellow/blue/yellow ID 1.52 mm, WT 0.84 mm, OD 3.2 mm)</td></tr><tr><td>silicone pump tubes - adapter tubing</td><td>Ismatec/Cole-Parmer</td><td>SC 0107</td><td>black/black/black ID 0.76 mm, WT 0.86 mm, OD: 2.48 mm</td></tr><tr><td>t-piece or t-connections</td><td>Ismatec/Cole-Parmer</td><td>ISM 693A</td><td>ID 2.5 mm</td></tr></tbody></table>

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.

The setup of the shunt system is displayed in Figure 2. Representative results of the continuous blood sampling data compared to manual blood sampling data in three wildtype rats over a time span of 30 min are presented in Figure 3A,C. At the beginning of the continuous blood sampling, an initial peak (maximum of radioactivity concentration) can be seen at 5 s after tracer injection. Afterwards, the activity in the blood declines rapidly and reaches a plateau at about 15 min. In the manual blood sampling data the detected Peak is smaller and the plateau is not easily to define (Figure 3A,C). The comparison of the continuous blood sampling to the image-derived data is displayed in Figure 3B,D. In the image-derived data, the peak and the starting point of the plateau are clearly visible, nevertheless the maximum of the peak is smaller compared to continuous blood sampling data for all animals (Figure 3B,D).
A sub-optimal outcome of continuous blood sampling with our setup is shown in Figure 3E,F. At the beginning of the continuous blood sampling, no data acquisition within the first 3.5 min was possible due to blood clotting. By disconnecting the tube system at connector orange and floating with heparinized saline solution, the flow in the tube system was restarted and the measurement continued. A peak can be seen at about 4 min, which does not record the maximum of radioactivity in blood (Figure 3E,F). Manual blood sampling (Figure 3E) and image-derived analyses (Figure 3F) were still possible and comparable to the correct outcomes.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59701/59701fig3.jpg" /> 
Figure 3: Representative results of continuous blood sampling compared to manual blood sampling. Typical arterial input functions derived from continuous blood sampling compared to manual blood sampling (left column) and continuous blood sampling compared to the image-derived approach (right column) are shown. Panels A-D demonstrate the results of correct implementation of the protocol in two different animals. Panels E and F illustrates a sub-optimal outcome of the measurement. All data shown were corrected for the cross-calibration factor and the background. <a href="https://www.jove.com/files/ftp_upload/59701/59701fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function at JoVE.com

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

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 ...

continuous-blood-sampling-small-animal-positron-emission

Research

JoVE Journal

Medicine

8.3K Views.  Rostock University Medical Center. In deze video is een protocol voor continue bloedafname voorzien. Bloedradioactiviteit wordt parallel aan een PET/CT gemeten met behulp van een extracorporale arterioveneuze shunt. Hierdoor wordt de arteriële inputfunctie voor latere kwantificering van PET/CT-gegevens gegenereerd en wordt later een nauwkeurige biokinetische modellering mogelijk gemaakt.De belangrijkste voordelen van dit protocol zijn dat het reproduceerbaar is, minimaal bloedverlies genereert en een hoge nauwkeurighei...

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

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 ...

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose analogue, 18F-fluordeoxyglucose (FDG-fPET), it is now possible to measure the dynamics of glucose metabolism with temporal resolutions approaching those of functional magnetic resonance imaging (fMRI). This direct measure of glucose uptake has enormous potential for understanding normal and abnormal brain function and probing the effects of metabolic and neurodegenerative diseases. Further, new advances in hybrid MR-PET hardware make it possible to capture fluctuations in glucose and blood oxygenation simultaneously using fMRI and FDG-fPET.
The temporal resolution and signal-to-noise of the FDG-fPET images is critically dependent upon the administration of the radiotracer. This work presents two alternative continuous infusion protocols and compares them to a traditional bolus approach. It presents a method for acquiring blood samples, time-locking PET, MRI, experimental stimulus, and administering the non-traditional tracer delivery. Using a visual stimulus, the protocol results show cortical maps of the glucose-response to external stimuli on an individual level with a temporal resolution of 16 s.

This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols.

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose ...

Behavior

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Neuroscience

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

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

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

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.

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 ...

PET-CT Scanning for Multi-Tracer Study of Cerebral Oxygen and Glucose Metabolism

Multi-Tracer Studies of Brain Oxygen and Glucose Metabolism Using a Time-of-Flight Positron Emission Tomography-Computed Tomography Scanner

The authors have developed a paradigm using positron emission tomography (PET) with multiple radiopharmaceutical tracers that combines measurements of cerebral metabolic rate of glucose (CMRGlc), cerebral metabolic rate of oxygen (CMRO2), cerebral blood flow (CBF), and cerebral blood volume (CBV), culminating in estimates of brain aerobic glycolysis (AG). These in vivo estimates of oxidative and non-oxidative glucose metabolism are pertinent to the study of the human brain in health and disease. The latest positron emission tomography-computed tomography (PET-CT) scanners provide time-of-flight (TOF) imaging and critical improvements in spatial resolution and reduction of artifacts. This has led to significantly improved imaging with lower radiotracer doses.
Optimized methods for the latest PET-CT scanners involve administering a sequence of inhaled 15O-labeled carbon monoxide (CO) and oxygen (O2), intravenous 15O-labeled water (H2O), and 18F-deoxyglucose (FDG)-all within 2-h or 3-h scan sessions that yield high-resolution, quantitative measurements of CMRGlc, CMRO2, CBF, CBV, and AG. This methods paper describes practical aspects of scanning designed for quantifying brain metabolism with tracer kinetic models and arterial blood samples and provides examples of imaging measurements of human brain metabolism.

Quantitative measurements of oxygen and glucose metabolism by PET are established technologies, but details of practical protocols are sparsely described in the literature. This paper presents a practical protocol successfully implemented on a state-of-the-art positron emission tomography-computed tomography scanner.

The authors have developed a paradigm using positron emission tomography (PET) with multiple radiopharmaceutical tracers that combines measurements of ...

Combined SPECT and CT Imaging to Visualize Cardiac Functionality

Source: Alycia G. Berman, James A. Schaber, and <a href="https://www.jove.com/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
Here we will demonstrate the fundamentals of single-photon emission computed tomography/computed tomography (SPECT/CT) imaging using mice. The technique involves injecting a radionuclide into a mouse, imaging the animal after it is distributed throughout the body, and then reconstructing the produced images to create a volumetric dataset. This can provide information about anatomy, physiology, and metabolism to improve disease diagnosis and monitor its progression.
In terms of collected data, SPECT/CT provides similar information as positron emission tomography (PET)/CT. However, the underlying principles of these two techniques are fundamentally different since PET requires the detection of two gamma photons, which are emitted in opposite directions. In contrast, SPECT imaging directly measures radiation via a gamma camera. As a result, SPECT imaging has lower spatial resolution than PET. However, it is also less expensive because the SPECT radioactive isotopes are more readily available. SPECT/CT imaging provides both noninvasive metabolic and anatomical information that can be useful for a wide variety of applications.

Combined SPECT and CT Imaging for Cardiac Functionality

Source: Alycia G. Berman, James A. Schaber, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
Here ...

Education

JoVE Science Education

Engineering

Biomedical Engineering

 Phase Contrast Magnetic Resonance Imaging to Assess Blood Flow in the Arteries of a Rat

<a href='https://app.jove.com/v/57304/phase-contrast-magnetic-resonance-imaging-rat-common-carotid'>Source: Chiu, S., et al. Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery. J. Vis. Exp. (2018).</a>This video demonstrates phase contrast magnetic resonance imaging (PC-MRI) to measure blood flow in the common carotid artery of a rat. After securing an anesthetized rat on the MRI bed and ensuring stable physiological parameters, the common carotid artery is scanned to capture images at defined intervals. Blood flow is then computed from these images to evaluate changes throughout the cardiac cycle.

Source: Chiu, S., et al. Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery. J. Vis. Exp. (2018).This video demonstrates phase ...

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

Samenvatting

Meer video's verkennen