Supported by a grant from The Memorial Foundation of Manufacturer Vilhelm Pedersen &#38; Wife. The foundation played no role in the planning or any other phase of the study.

A few clinical trials using this 64Cu PET/CT or PET/MRI protocol have been approved by the Regional Ethics Committee of&#160;Region Midt, Denmark [1-10-72-196-16 (EudraCT 2016-001975-59), 1-10-72-41-19 (EudraCT 2019-000905-57), 1-10-72-343-20 (EudraCT 2020-005832-31), 1-10-72-25-21 (EudraCT 2021-000102-25), and 1-10-72-15-22 (EudraCT 2021-005464-21)]. Written informed consent was obtained from the participants at enrollment. The inclusion criteria for all participants were age &#62;18, and for females the use of safe contraception. The exclusion criteria for Wilson disease patients were decompensated cirrhosis, a Model for End-stage Liver Disease (MELD) score &#62;11, or a modified Nazer score &#62;6. The exclusion criteria for all participants were a known hypersensitivity to 64Cu or other ingredients in the tracer formula, pregnancy, breastfeeding, or a desire to become pregnant before the end of the trial.
1. Preparation of&#160;64CuCl2
<ol>
	<li>Dissolve solid 64CuCl2 in hydrochloric acid (0.1 M) and add sodium acetate buffer (0.5 M) to increase the pH to ~5. Formulate with saline and filter sterilize the solution by passing it through a 0.22 &#181;m filter (see Table of Materials). 
	NOTE: Sodium acetate buffer (0.5 M) is produced from sodium acetate trihydrate and sterile water that is passed through a 0.22 &#181;m sterilizing filter.</li>
	<li>For quality control of the produced 64CuCl2 solution, perform pH measurement, bacterial endotoxin testing, radiochemical purity determination, and radio nuclidic identification7,8.</li>
	<li>Store the product in a lead container at room temperature and keep it in quarantine until all quality control specifications have been satisfactorily met. 
	NOTE: For the present study, 64CuCl2 was produced with a radio nuclidic purity &#8805;99% and a radiochemical purity &#8805;95%. Solid 64CuCl2, used as starting material, was obtained from a commercial source (see Table of Materials).</li>
</ol>
2. Preparation of PET scanner
<ol>
	<li>Perform a quality check (QC)9 on the scanner, following the manufacturer&#39;s protocol (see Table of Materials). 
	NOTE: QCs must be performed daily in the morning before patient scans.</li>
</ol>
3. Drawing of tracer for intravenous (IV) injection and per oral (PO) administration
<ol>
	<li>Wear plastic gloves and remove the lid from the lead container.</li>
	<li>Use long tweezers to disinfect the rubber membrane of the tracer-containing glass bottle inside the lead container with a disinfection swab.</li>
	<li>Use tweezers to insert a short cannula (~0.5 mm x 16 mm) into the membrane to avoid spill-over from the vacuum inside the bottle.</li>
	<li>Use tweezers to insert a longer cannula to draw from. This cannula should be long enough to reach the bottom of the bottle (usually 50 mm).</li>
	<li>Ensure that the dose calibrator (see Table of Materials) is calibrated for 64Cu. Calculate an approximate volume to draw for the first draw. 
	NOTE: From the chemical quality control reports, the activity amount and volume of the liquid will be available, allowing for the calculation of an approximate volume to draw.</li>
	<li>Wear plastic gloves, insert an appropriately sized plastic syringe into the long cannula, and draw the calculated volume. This volume will depend on the concentration of 64Cu in the product and how much 64Cu is decided for the protocol (see Dose calculations under the representative results).</li>
	<li>Use tweezers to hold the cannula while moving the syringe to the dose calibrator to measure the radioactivity.</li>
	<li>Keep drawing until the appropriate radioactivity amount is reached. Approximately 5% of the tracer will remain in the syringe and cannula after injection. 
	NOTE: The 64Cu should not be diluted in salt water, as the tracer may precipitate. Thus, the syringe cannot be rinsed with saline water after the injection (this is not relevant for PO administration).</li>
	<li>With the tweezers, apply a cannula with a cap (~16 mm cannula) to close the syringe and store it in a lead container until application.</li>
</ol>
4. Application of the tracer
<ol>
	<li>IV injection
	<ol>
		<li>Insert an intravenous cannula (~22 G, 25 mm), preferably in a cubital vein, and rinse with saline water to ensure correct placement. 
		NOTE: A worksheet with the participant&#39;s name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, injection, and leftover tracer should be available.</li>
		<li>Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.</li>
		<li>Transport the syringe in a lead container to the participant&#39;s bedside.</li>
		<li>If any spill-over occurs from the injection, place a napkin under the participant&#39;s elbow so the spilled radioactivity can be measured.</li>
		<li>With tweezers, remove the cap/cannula from the syringe, and with plastic gloves, connect the syringe to the IV access. Note the time on the worksheet and inject in one steady movement. 
		NOTE: As mentioned earlier, the syringe should not be rinsed with saline as the tracer may precipitate.</li>
		<li>Remove the syringe from the IV access, put on the cap/cannula, and place it in the lead container with the napkin if necessary.</li>
		<li>Rinse the IV access through with saline water.</li>
		<li>Note the time and leftover radioactivity in the syringe on the worksheet. 
		NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurements are entered into the PET scan protocol when the participant is scanned (see step 5).</li>
		<li>Dispose of the leftover material appropriately, according to institutional safety regulations.</li>
		<li>Remove the IV access. In case any allergic reactions appear, leave the IV access in for 30 min.</li>
	</ol>
	</li>
	<li>Oral administration 
	NOTE: A worksheet with the participant&#39;s name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, administration, and leftover tracer should be available.
	<ol>
		<li>In a disposable and soft plastic cup, pour around 100 mL of water or cordial; the 64Cu is tasteless. A disposable plastic straw and a small disposable plastic bag should be available.</li>
		<li>Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.</li>
		<li>Transport the syringe in a lead container to the participant&#39;s bedside. The participant should be seated in a bed or chair.</li>
		<li>Remove the cap/cannula from the syringe with tweezers and, wearing plastic gloves, inject the tracer into the cup, being careful not to spill any. Draw up a bit of the water/cordial and inject it into the cup again.</li>
		<li>Place a plastic straw in the cup (this is to minimize the risk of spill-over when the participant drinks).</li>
		<li>Note the time on the worksheet and let the participant drink. The cup should be as empty as possible.</li>
		<li>Put the empty cup and straw in the disposable plastic bag with the empty syringe and place them in the lead container.</li>
		<li>Note the time and measure the leftover radioactivity in the syringe. Note in the worksheet. 
		NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurement are entered into the PET scan protocol when the participant is scanned (see Scan).</li>
		<li>Dispose of the leftover material appropriately, according to institutional safety regulations. 
		NOTE: Observing the participant for acute allergic reactions for 30 min after the intake may be appropriate.</li>
	</ol>
	</li>
</ol>
5. PET scans
<ol>
	<li>Place the participant in a supine position in the scanner.</li>
	<li>Perform overview CT or MR scanning to plan the specific region to be examined during the PET scan.</li>
	<li>Note the time of the draw, injection, and leftover measurement, and the radioactivity at the draw and leftover measurement in the PET protocol.</li>
	<li>Perform PET scanning following the steps below. 
	NOTE: The PET scan protocol must be standardized with regard to scan duration and image reconstruction parameters for all participants in the same study; published reports should be followed10,11,12.
	<ol>
		<li>Perform static PET scans with a scan time of 4.5 min/bed position for up to 24 h after tracer administration, and 10 min/bed position for up to 68 h after tracer administration (for further elaboration, see Scan under the representative results). 
		NOTE: During dynamic PET scanning, decay is continuously recorded and subsequently segmented into a frame structure. This allows for selection of frames from short time intervals to emphasize the dynamics of 64Cu distribution, and frames from longer time intervals to prioritize sensitivity. Typically, shorter intervals are selected right after injection and gradually increased thereafter10.</li>
	</ol>
	</li>
</ol>
6. Image reconstruction
<ol>
	<li>Reconstruct the images using the best available corrections for attenuation, scatter, time-of-flight, and point-spread function. 
	NOTE: The image reconstruction parameters must be carefully selected to optimize the image properties, such as signal recovery and signal-to-noise. For multicenter studies, it is critical to standardize the image quality between centers.</li>
</ol>
7. Data analysis
NOTE: The present study describes a simple method to quantify 64Cu content in the liver. The PET signal is measured as standard uptake value (SUV), the tissue radioactivity concentration adjusted for participant weight injected activity and/or kilobecquerel (kBq) per mL of tissue.
<ol>
	<li>Download data to a suitable program, for example, Dicom files, to PMOD. 
	NOTE: There are likely many different programs to analyze PET images, such as Hermes or PMOD (see Table of Materials).</li>
	<li>Adjust the CT/MR scan tones to differentiate the anatomical structures.</li>
	<li>Ensure that the anatomical scan and PET scan are overlapping.</li>
	<li>Working in the horizontal plane with the best MRI or CT scan, localize the liver and the big structures.</li>
	<li>Place an appropriate volume of interest (VOI) or multiple VOIs in the liver. 
	NOTE: A VOI is a defined area of tissue where the SUV is measured. A VOI consists of multiple regions of interest (ROIs), which are tissue areas in one plane. Many programs have spherical VOIs as a pre-setting, meaning multiple ROIs (one in each plane) do not have to be drawn to constitute a VOI. The right liver lobe tends to be more homogenous, and thus a good position to place VOIs.</li>
	<li>Place multiple VOIs in the right liver lobe in different horizontal planes to achieve the most precise measure of activity, as the SUV may vary somewhat (~5%) in the right liver lobe. Calculate the mean SUV of these VOIs.</li>
	<li>To quantify the SUV, for example, in the entire liver, draw ROIs covering the whole liver volume in each plane for dosimetry studies. 
	NOTE: Avoid big structures such as arteries and veins when using this method.</li>
</ol>

<ol>
	<li>T&#252;mer, Z., M&#248;ller, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menkes+disease.">Menkes disease.</a> European Journal of Human Genetics. 18 (5), 511-518 (2010).</li><li>Ala, A., Walker, A. P., Ashkan, K., Dooley, J. S., Schilsky, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wilson's+disease.">Wilson's disease.</a> The Lancet. 369 (9559), 397-408 (2007).</li><li>Owen, 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=Absorption+and+excretion+of+Cu64-labeled+copper+by+the+rat.">Absorption and excretion of Cu64-labeled copper by the rat.</a> The American Journal of Physiology-Legacy Content. 207 (6), 1203-1206 (1964).</li><li>Osborn, S. B., Roberts, C. N., Walshe, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+radiocopper+by+the+liver.+A+study+of+patients+with+Wilson's+disease+and+various+control+groups.">Uptake of radiocopper by the liver. A study of patients with Wilson's disease and various control groups.</a> Clinical Science. 24, 13-22 (1963).</li><li>Vierling, J. 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=Incorporation+of+radiocopper+into+ceruloplasmin+in+normal+subjects+and+in+patients+with+primary+biliary+cirrhosis+and+Wilson's+disease.">Incorporation of radiocopper into ceruloplasmin in normal subjects and in patients with primary biliary cirrhosis and Wilson's disease.</a> Gastroenterology. 74 (4), 652-660 (1978).</li><li>Gibbs, K., Walshe, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+with+radioactive+copper+(64Cu+and+67Cu);+the+incorporation+of+radioactive+copper+into+caeruloplasmin+in+Wilson's+disease+and+in+primary+biliary+cirrhosis.">Studies with radioactive copper (64Cu and 67Cu); the incorporation of radioactive copper into caeruloplasmin in Wilson's disease and in primary biliary cirrhosis.</a> Clinical Science. 41 (3), 189-202 (1971).</li><li>Kume, 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=A+semi-automated+system+for+the+routine+production+of+copper-64.">A semi-automated system for the routine production of copper-64.</a> Applied Radiation and Isotopes: Including Data, Instrumentation and Methods for Use in Agriculture, Industry and Medicine. 70 (8), 1803-1806 (2012).</li><li>Ohya, T., 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=Efficient+preparation+of+high-quality+64Cu+for+routine+use.">Efficient preparation of high-quality 64Cu for routine use.</a> Nuclear Medicine and Biology. 43 (11), 685-691 (2016).</li><li>Koole, 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=EANM+guidelines+for+PET-CT+and+PET-MR+routine+quality+control.">EANM guidelines for PET-CT and PET-MR routine quality control.</a> Zeitschrift f&#252;r Medizinische Physik. , (2022).</li><li>Sandahl, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+Wilson's+disease+visualized:+A+human+64Cu+PET+study.">The pathophysiology of Wilson's disease visualized: A human 64Cu PET study.</a> Hepatology. 76 (6), 1461-1470 (2022).</li><li>Munk, D. E., 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=Effect+of+oral+zinc+regimens+on+human+hepatic+copper+content:+a+randomized+intervention+study.">Effect of oral zinc regimens on human hepatic copper content: a randomized intervention study.</a> Scientific Reports. 12 (1), 14714 (2022).</li><li>Kj&#230;rgaard, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+and+oral+copper+kinetics,+biodistribution+and+dosimetry+in+healthy+humans+studied+by+64Cu]copper+PET/CT.">Intravenous and oral copper kinetics, biodistribution and dosimetry in healthy humans studied by 64Cu]copper PET/CT.</a> EJNMMI Radiopharmacy and Chemistry. 5 (1), 15 (2020).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+acetate+for+the+treatment+of+Wilson's+disease.">Zinc acetate for the treatment of Wilson's disease.</a> Expert Opinion on Pharmacotherapy. 2 (9), 1473-1477 (2001).</li><li>Bush, J. 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=Studies+on+copper+metabolism.+XVI.+Radioactive+copper+studies+in+normal+subjects+and+in+patients+with+hepatolenticular+degeneration.">Studies on copper metabolism. XVI. Radioactive copper studies in normal subjects and in patients with hepatolenticular degeneration.</a> Journal of Clinical Investigation. 34 (12), 1766-1778 (1955).</li><li>Murillo, 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=High+value+of+64Cu+as+a+tool+to+evaluate+the+restoration+of+physiological+copper+excretion+after+gene+therapy+in+Wilson's+disease.">High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease.</a> Molecular Therapy - Methods &amp; Clinical Development. 26, 98-106 (2022).</li><li>Squitti, 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=Copper+dyshomeostasis+in+Wilson+disease+and+Alzheimer's+disease+as+shown+by+serum+and+urine+copper+indicators.">Copper dyshomeostasis in Wilson disease and Alzheimer's disease as shown by serum and urine copper indicators.</a> Journal of Trace Elements in Medicine and Biology. 45, 181-188 (2018).</li></ol>

The authors have no conflicts of interest.

The method is like any other PET method, but the long half-life of 12.7 h offers the opportunity to investigate long-term copper fluxes (we have good results from up to 68 h after IV tracer injection). All steps in the protocol must be handled by personnel familiar with PET, although they are no more critical than any other PET examination.
Troubleshooting 
Because we often use 64Cu for long-term investigations, the PET signal will be noisier than usual. This i...

Copper is a vital catalytic cofactor that drives multiple important biochemical processes essential for life, and defects in copper homeostasis are directly responsible for human diseases. Mutations in the ATP7A or ATP7B genes, encoding copper-transporting ATPases, cause Menke&#39;s and Wilson diseases, respectively. Menke&#39;s disease (ATP7A) is a rare lethal disorder of intestinal copper hyperaccumulation with severe copper deficiency in peripheral tissues and deficits in copper-dependent enzymes1. Wilson disease (WD) (ATP7B) is a rare disease characterized by the inability to excrete excess copper to bile, resulting in copper overload and subsequent organ damage, most severely affecting the liver and brain2.
Studies on copper metabolism have utilized radiolabeled copper (usually 64-copper [64Cu] or 67-copper) for decades, and these studies have proven invaluable for our understanding of mammalian copper metabolism, including absorption site and excretion pathways3,4,5,6. Previously, gamma counters were used to detect the radioactive signal with a limited anatomical resolution, but recently, 64Cu positron emission tomography (PET) combined with computed tomography (CT) or magnetic resonance imaging (MRI) has been introduced in both human and animal studies. Today, PET scanners have such a high sensitivity that it is possible to track 64Cu for up to 70 h after injection. The long half-life of 12.7 h for 64Cu allows for the long-term assessment of copper fluxes. This improvement in resolution has just recently entered the field of copper studies, and studies on normal and pathological copper metabolism, as well as studies evaluating the impact of specific treatments, are starting to emerge. Additionally, the introduction of whole-body PET scanners with an extended field-of-view will further enhance the sensitivity of these examinations.
This methodological paper aims to enable clinicians and scientists to add 64Cu PET CT/MRI to the existing repertoire of tools as a robust and easy-to-use method for assessing copper metabolism in a manner comparable between nuclear medicine departments. The production of 64Cu copper can be carried out using different methods and is usually performed at special facilities. Among the nuclear reactions, the 64Ni (p, n) 64Cu method is widely used, since a high production yield of 64Cu can be obtained with low energy protons in this route7,8. A detailed description of the production methods is out of the scope of this work, and availability will differ by country and region.
In this article, we first describe the preparation of the necessary radiochemistry and the tracer. Then, the principles for preparing the PET/CT or PET/MRI scanners are demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 micrometer sterilizing filter</td>
			<td>Merck Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula 21 G 50 mm</td>
			<td>BD Microlance</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula 25 G 16 mm</td>
			<td>BD Microlance</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>Dose calibrator</td>
			<td>Capintec CRC-PC calibrator</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PET/CT scanner</td>
			<td>Siemens: Biograph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PET/MR scanner</td>
			<td>GE Signa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMOD version 4.0</td>
			<td>PMOD Technologies LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution 0.9% NaCl</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate BioUltra</td>
			<td>Sigma Aldrich</td>
			<td>71188</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid 64CuCl2</td>
			<td>Danish Technical University Ris&#248;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Venflon 22 G 25 mm</td>
			<td>BD Venflon Pro Safety</td>
			<td>393280</td>
			<td></td>
		</tr>
	</tbody>
</table>

positron emission tomography using 64-copper as a tracer for the study of copper-related disorders

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in studying basic human and animal copper metabolism and copper-related disorders, such as Wilson disease (WD) and Menke&#39;s disease. A recent addition to this toolkit is 64-copper (64Cu) positron emission tomography (PET), combining the accurate anatomical imaging of modern computed tomography (CT) or magnetic resonance imaging (MRI) scanners with the biodistribution of the 64Cu PET tracer signal. This allows the in vivo tracking of copper fluxes and kinetics, thereby directly visualizing human and animal copper organ traffic and metabolism. Consequently, 64Cu PET is well-suited for evaluating clinical and preclinical treatment effects and has already demonstrated the ability to diagnose WD accurately. Furthermore, 64Cu PET/CT studies have proven valuable in other scientific areas like cancer and stroke research. The present article shows how to perform 64Cu PET/CT or PET/MR in humans. Procedures for 64Cu handling, patient preparation, and scanner setup are demonstrated here.

Dose calculation 
Based on dosimetry calculations, the effective radioactivity dose for IV administration is 62 &#177; 5 &#181;Sv/MBq tracer10. Thus, a 50 MBq dose is recommended depending on the time frame. Up to 75-80 MBq is applicable for longer examinations and provides good-quality images without exceeding an ethically approved dose. The effective dose for oral administration is 113 &#177; 1 &#181;Sv/MBq tracer, due to intestinal accumulation of the tracer. Thus, a lower...

Watch this Scientific Journal Video about Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders at JoVE.com

Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

The present protocol describes how to perform 64Cu PET/CT and PET/MRI imaging in humans to study copper-related disorders, such as Wilson disease, and the treatment effect on copper metabolism.

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in ...

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1.1K Views.  Aarhus University Hospital. The present protocol describes how to perform 64Cu PET/CT and PET/MRI imaging in humans to study copper-related disorders, such as Wilson disease, and the treatment effect on copper metabolism.

Video: Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&amp;#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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

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

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

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

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

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

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

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

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...