This study was financially supported by the Fonds de la recherche en sant&#233; du Qu&#233;bec, Canadian Institutes of Health Research, Canadian Foundation for Innovation and the Canada Research Chairs Secretariat (SCC). The Sherbrooke Molecular Imaging Center is part of the FRQS-funded &#201;tienne-Le Bel Clinical Research Center. The authors thank M&#233;lanie Fortier, Jennifer Tremblay-Mercier, Alexandre Courchesne-Loyer, Dr. Fabien Pifferi, Dr. M&#39;hamed Bentourkia, Dr. Otman Sarrhini, Dr. Jacques Rousseau, Caroline Mathieu, and M&#233;lanie Archambault for generous support and technical assistance. The authors would like to thank the image analysis and visualization platform (http://pavi.dinf.usherbrooke.ca) for their help.

All experiments were completed in accordance with the Animal Care and Use Committee at the Universit&#233; de Sherbrooke and with the Canadian Council on Animal Care. The experimental protocol&#160;was approved by the Institutional Animal Research Ethics Review Board (protocol #011-09).
1. Brain Anatomy with MRI
<ol>
	<li>Let rats acclimatize in the animal facility for a minimum of 7 days prior to the protocol. Perform brain MRI scans 1-2 weeks prior to the dual tracer PET protocol to allow ...

<ol>
	<li>Tremblay, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+synthesis+of+11C-acetoacetic+acid,+a+key+alternate+brain+fuel+to+glucose.">Automated synthesis of 11C-acetoacetic acid, a key alternate brain fuel to glucose.</a> Appl. Radiat. Isot. 65, 934-940 (2007).</li><li>Pifferi, 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=Mild+experimental+ketosis+increases+brain+uptake+of+11C-acetoacetate+and+18F-fluorodeoxyglucose:+a+dual-tracer+PET+imaging+study+in+rats.">Mild experimental ketosis increases brain uptake of 11C-acetoacetate and 18F-fluorodeoxyglucose: a dual-tracer PET imaging study in rats.</a> Nutr. Neurosci. 14, 51-58 (2011).</li><li>Lopez-Grueso, R., Borras, C., Gambiniy, J., Vina, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=El+envejecimiento+y+la+ovariectomia+causan+una+disminucion+del+consumo+cerebral+de+glucosa+in+vivo+en+ratas+Wistar.">El envejecimiento y la ovariectomia causan una disminucion del consumo cerebral de glucosa in vivo en ratas Wistar.</a> Revista Espanola de Geriatria y Gerontologia. 45, 136-140 (2010).</li><li>Fueger, B. J., 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+animal+handling+on+the+results+of+18F-FDG+PET+studies+in+mice.">Impact of animal handling on the results of 18F-FDG PET studies in mice.</a> J. Nucl. Med. 47, 999-1006 (2006).</li><li>Luo, 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=Characterization+of+7-+and+19-month-old+Tg2576+mice+using+multimodal+in+vivo+imaging:+limitations+as+a+translatable+model+of+Alzheimer's+disease.">Characterization of 7- and 19-month-old Tg2576 mice using multimodal in vivo imaging: limitations as a translatable model of Alzheimer's disease.</a> Neurobiol. Aging. 33, 933-944 (2012).</li><li>Poisnel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+regional+cerebral+glucose+uptake+in+an+APP/PS1+model+of+Alzheimer's+disease.">Increased regional cerebral glucose uptake in an APP/PS1 model of Alzheimer's disease.</a> Neurobiol. Aging. 33, 1995-2005 (2012).</li><li>Kenney, J. M., Marinelli, L. D., Woodard, H. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracer+studies+with+radioactive+phosphorus+in+malignant+neoplastic+disease.">Tracer studies with radioactive phosphorus in malignant neoplastic disease.</a> Radiology. 37, 683-690 (1941).</li><li>Cunnane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+fuel+metabolism,+aging,+and+Alzheimer's+disease.">Brain fuel metabolism, aging, and Alzheimer's disease.</a> Nutrition. 27, 3-20 (2011).</li><li>Shimoji, 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=Measurement+of+cerebral+glucose+metabolic+rates+in+the+anesthetized+rat+by+dynamic+scanning+with+18F-FDG,+the+ATLAS+small+animal+PET+scanner,+and+arterial+blood+sampling.">Measurement of cerebral glucose metabolic rates in the anesthetized rat by dynamic scanning with 18F-FDG, the ATLAS small animal PET scanner, and arterial blood sampling.</a> J. Nucl. Med. 45, 665-672 (2004).</li><li>Tantawy, M. N., Peterson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+[18F]FDG+image-derived+input+function+using+the+left+ventricle,+liver,+and+one+venous+blood+sample.">Simplified [18F]FDG image-derived input function using the left ventricle, liver, and one venous blood sample.</a> Mol. Imaging. 9, 76-86 (2010).</li><li>Roy, 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=The+ketogenic+diet+increases+brain+glucose+and+ketone+uptake+in+aged+rats:+A+dual+tracer+PET+and+volumetric+MRI+study.">The ketogenic diet increases brain glucose and ketone uptake in aged rats: A dual tracer PET and volumetric MRI study.</a> Brain Res. , (2012).</li><li>Yu, A. S., Lin, H. D., Huang, S. C., Phelps, M. E., Wu, H. M. <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+cerebral+glucose+metabolic+rate+in+mice+using+18F-FDG+and+small-animal+PET..">Quantification of cerebral glucose metabolic rate in mice using 18F-FDG and small-animal PET..</a> J. Nucl. Med.. 50, 966-973 (2009).</li><li>Bentourkia, 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=PET+study+of+11C-acetoacetate+kinetics+in+rat+brain+during+dietary+treatments+affecting+ketosis.">PET study of 11C-acetoacetate kinetics in rat brain during dietary treatments affecting ketosis.</a> Am. J. Physiol. Endocrinol. Metab. 296, 796-801 (2009).</li><li>Menard, S. 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=Abnormal+in+vivo+myocardial+energy+substrate+uptake+in+diet-induced+type+2+diabetic+cardiomyopathy+in+rats.">Abnormal in vivo myocardial energy substrate uptake in diet-induced type 2 diabetic cardiomyopathy in rats.</a> Am. J. Physiol. Endocrinol. Metab. 298, 1049-1057 (2010).</li><li>Liistro, 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=Brain+glucose+overexposure+and+lack+of+acute+metabolic+flexibility+in+obesity+and+type+2+diabetes:+a+PET-[18F]FDG+study+in+Zucker+and+ZDF+rats.">Brain glucose overexposure and lack of acute metabolic flexibility in obesity and type 2 diabetes: a PET-[18F]FDG study in Zucker and ZDF rats.</a> J. Cereb. Blood Flow Metab. 30, 895-899 (2010).</li><li>Croteau, 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=Image-derived+input+function+in+dynamic+human+PET/CT:+methodology+and+validation+with+11C-acetate+and+18F-fluorothioheptadecanoic+acid+in+muscle+and+18F-fluorodeoxyglucose+in+brain.">Image-derived input function in dynamic human PET/CT: methodology and validation with 11C-acetate and 18F-fluorothioheptadecanoic acid in muscle and 18F-fluorodeoxyglucose in brain.</a> Eur. J. Nucl. Med. Mol. Imaging. 37, 1539-1550 (2010).</li><li>Bergeron, M., Cadorette, J., Beaudoin, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+evaluation+of+the+LabPETTM+APD-based+digital+PET+scanner.">Performance evaluation of the LabPETTM APD-based digital PET scanner.</a> IEEE Trans. Nucl. Sci. 56, 10-16 (2009).</li><li>Freemantle, 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=Metabolic+response+to+a+ketogenic+breakfast+in+the+healthy+elderly.">Metabolic response to a ketogenic breakfast in the healthy elderly.</a> J. Nutr. Health Aging. 13, 293-298 (2009).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. sixth edn. , (2007).</li><li>Patlak, C. S., Blasberg, R. G., Fenstermacher, J. 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Critical steps
A critical step in this dual tracer PET protocol is to be able to simultaneously scan the heart&#39;s left ventricle and the brain at the same time. This requires a PET scanner with a sufficient axial length, i.e. a minimum of 7.5 cm. A few test scans are needed to determine the exact position of the scanner table (x, y, and z&#160;values), where the brain and the heart are scanned correctly.
Tracer injection is also a crucial point for a succes...

Context and Rationale
Positron emission tomography (PET) enables the minimally-invasive study of functional processes in the brain. Glucose is the brain&#39;s main energy substrate, but in conditions of glucose deficiency, ketones (acetoacetate [AcAc] and &#946;-hydroxybutyrate) are the main alternative energy substrates. Brain energy metabolism has been widely studied by PET using the most common PET tracer,&#160;18F-fluorodeoxyglucose (18F-FDG), a glucose analog. Our group recently developed a novel radiotracer -11C-AcAc - to measure brain ketone metabolism1. Magnetic resonance imaging (MRI) is a much higher resolution technique (0.1 mm &#215; 0.1 mm in-plane resolution) than PET, and is needed to clearly localize anatomical brain regions required for the regional PET analysis of brain energy metabolism.
PET data are commonly expressed as standardized uptake values (SUV)2-6. SUV are the tissue activity concentration normalized by the fraction of the injected dose/unit weight, as initially proposed over 70 years ago7. These units are still widely used because they require simpler PET acquisition and image analysis methodologies. However, an important limitation is that SUV are relative not absolute units, making it difficult to compare results across different studies. This difficulty of comparison may contribute to contradictory findings in the literature on brain glucose uptake in the elderly8. &#160;Therefore, the quantitative cerebral metabolic rate (CMR; &#956;mol/100 g/min) has particular advantages9-11. Generating CMR values requires a dynamic PET acquisition and the plasma radioactivity counts as a function of time, i.e. the plasma time-activity curve (TAC) or input function. The input function can be obtained by multiple blood samplings throughout the PET acquisition9,12&#160;or by the image-derived input function (IDIF) method, in which a region of interest is drawn on a blood pool (heart&#39;s left ventricle or major artery)11,13-16.
Goal
The aim of our method was to quantitatively compare for the first time the uptake of the brain&#39;s two key energy substrates, glucose and ketones, using PET and MRI in rodents. 11C-AcAc and 18F-FDG were used sequentially in the same animal. The protocol was designed to measure regional uptakes in different relevant brain structures (cortex, hippocampus, striatum, and cerebellum) clearly visible in the MR images. The protocol was also specifically intended to permit quantitative analysis of tracer brain uptake, i.e. CMR of both 18F-FDG and 11C-AcAc. Although this protocol was developed to study brain energy substrates, the radiotracers we used could be replaced by others, and the same methodology can be used to study different brain functional processes.
Advantages over existing methods
PET and MRI do not require the animal to be sacrificed after the acquisition. Therefore, follow-up studies of treatments are possible. Thus, baseline data followed by an experimental condition can be measured within the same animal, thereby reducing both biological variability and the number of animals required. A key advantage of our dual tracer PET protocol is to compare both tracers uptake in the same animal under the same physiological conditions within the same imaging session, thereby reducing even more biological variability and systematic discrepancies. This dual tracer PET protocol is feasible primarily because of the short physical half-life of 11C (20.4 min) and the fast biological washout of 11C-AcAc, which leave minimal residual 11C radioactivity during the second acquisition with 18F-FDG. The MRI scan is an important feature of this protocol as it enables the tracer uptake to be studied in specific brain areas. In addition, this method enables an absolute quantitative measure of brain tracer uptake in contrast to relative units obtained by the SUV method. Finally, 11C-AcAc images have a low signal-to-noise ratio because of relatively low brain AcAc uptake under physiological conditions, which makes automatic&#160;11C-AcAc and MR images registration challenging. Hence, because 11C-AcAc and 18F-FDG acquisitions are sequential (no motion of the animal), the 18F-FDG to MRI alignment can be applied to 11C-AcAc images.
Key papers where the protocol has been used
We have used the dual tracer PET protocol in a study involving the comparison of the fasted state and the ketogenic diet (KD) in young rats2. We showed that both fasting and the KD increase significantly both 11C-AcAc and 18F-FDG brain uptake. However, these were not quantitative results as we did not use the dynamic PET imaging and tracer kinetic modeling methodology at that time. Thereafter, we undertook a regional and quantitative study of brain metabolism in aged rats, where the effect of aging and a KD were evaluated on brain 11C-AcAc and 18F-FDG uptake11. We also showed that the percentage of distribution across brain regions was different between 11C-AcAc and 18F-FDG. Furthermore, not only the CMR of 11C-AcAc but also that of 18F-FDG was increased in the whole brain as well as in the striatum of aged rats on the KD.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MRI scanner</td>
			<td style="width:207px;">Varian</td>
			<td style="width:181px;"></td>
			<td style="width:224px;">7 Tesla</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small-animal PET scanner</td>
			<td>Gamma Medica</td>
			<td></td>
			<td>Lab-PET/-Triumph</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat mat</td>
			<td>Sunbeam</td>
			<td>PN 143937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heater system</td>
			<td>SA Instruments</td>
			<td>761 100 Rev B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Respiratory gating</td>
			<td>SA Instruments</td>
			<td>SAII&#39;s P-resp</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clinical chemistry analyzer</td>
			<td>Siemens Healthcare Diagnosis</td>
			<td>&#160;765000.931</td>
			<td>Dimension Xpand Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing 50&#160;</td>
			<td>Becton Dickinson</td>
			<td>427411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Injection pump</td>
			<td>KD Scientific</td>
			<td>Model 210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gamma-counter</td>
			<td>GMI</td>
			<td>Packard Cobra II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002416</td>
			<td>Heraeus Pico 21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMOD software</td>
			<td>PMOD Technologies</td>
			<td></td>
			<td>PMOD 3.2 version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Geiger counter</td>
			<td>Fluke Biomedical&#160;</td>
			<td>ASM-990</td>
			<td>Advanced Survey Meter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Name of the Reagent</td>
			<td style="width:207px;">Company</td>
			<td style="width:181px;">Catalogue Number</td>
			<td style="width:224px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Abbott Laboratories, Ltd</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% NaCl solution</td>
			<td>Hospira</td>
			<td>4888010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sandoz</td>
			<td>1004336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropenyl acetate</td>
			<td>Aldrich</td>
			<td>11778</td>
			<td>99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methyllithium</td>
			<td>Aldrich</td>
			<td>197343</td>
			<td>1.6 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">THF</td>
			<td>Aldrich</td>
			<td>87371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flex reagent cartridge glucose</td>
			<td>Siemens Healthcare Diagnosis</td>
			<td>DF40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizma base</td>
			<td>Sigma</td>
			<td>T6066-500G</td>
			<td>prepare tris buffer 100 mM pH 7.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium oxamate</td>
			<td>Sigma</td>
			<td>O2751</td>
			<td>20 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NADH</td>
			<td>Roche</td>
			<td>10128015001</td>
			<td>0.15 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Hydroxybutyrate dehydrogenase</td>
			<td>Toyobo</td>
			<td>HBD-301</td>
			<td>1 U/ml</td>
		</tr>
	</tbody>
</table>

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.

As seen in Figure 2,11C-AcAc uptake is low within the brain itself. As mentioned earlier, ketones consumption by the brain is very low on a short-term fasting.&#160;11C-AcAc uptake is higher in the tongue and cheek muscles. Indeed, ketones are rapidly taken up by rat skeletal muscles23. In contrast, 18F-FDG uptake is mostly in the brain and the cheek muscles. Figure 2 shows that during the coregistration process, MR images are fixed and PET ima...

Watch this Scientific Journal Video about A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

7.0K visualizações.  Université de Sherbrooke. Small-animal positron emission tomography enables the assessment of the brain's two main energy substrates: 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.