Name: visualization and quantification of brown and beige adipose tissues in mice using [18f]fdg micro-pet/mr imaging
Uploaded: 2021-07-01T14:00:00
Description: Watch this Scientific Journal Video about Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging at JoVE.com

We thank the support of National Natural Science Foundation of China (NSFC) - Excellent Young Scientists Fund (Hong Kong and Macau) (81922079), Hong Kong Research Grants Council General Research Fund (GRF 17121520 and 17123419), and Hong Kong Research Grants Council Collaborative Research Fund (CRF C7018-14E) for small animal imaging experiments.

The protocol described below follows the animal care guidelines of The University of Hong Kong. The animals used in the study were 8-week-old C57BL/6J mice.
1. Animal surgical procedures and cold challenge
<ol>
	<li>Perform interscapular BAT (iBAT) dissection.
	<ol>
		<li>Anesthetize the mice by intraperitoneal injection of ketamine/xylazine (100 mg/kg bodyweight ketamine and 10 mg/kg bodyweight xylazine). After anesthesia, shave the hair of the mouse from the neck to just below the scapulae...

<ol>
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In this study, a PET/MR -based imaging and quantification of functional brown and beige adipose tissue in small animal was described. This method uses the non-metabolizable glucose analog [18F]FDG as an imaging biomarker so as to identify the adipose tissues with high glucose-demand in a non-invasive manner. MR offers good soft tissue contrast and can better differentiate adipose fat tissues from the neighboring soft tissues and muscle. When combined with PET, this enables imaging and quantifying of the activa...

In response to changing nutritional needs, adipose tissue serves as an energy cache to adopt either lipid storage or mobilization mode to meet the needs of the body1. Moreover, adipose tissue also plays a key function in thermoregulation, via a process called non-shivering thermogenesis, also called facultative thermogenesis. This is typically achieved by the brown adipose tissue (BAT), which expresses abundant level of mitochondria membrane protein uncoupling protein 1 (UCP1). As a proton carrier, UCP1 generates heat by uncoupling the proton transport and ATP production2. Upon cold stimulation, thermogenesis in BAT is set in motion by activation of the sympathetic nervous system (SNS), followed by release of norepinephrine (NE). NE binds to the &#946;3 adrenergic receptors and leads to elevation of intracellular cyclic AMP (cAMP). As a consequence, cAMP/PKA-dependent engagement of CREB (cAMP response element-binding protein) stimulates Ucp1 transcription via direct binding on CREB-response elements (CRE)2. In addition to BAT, brown-like adipocytes are also found within white adipose tissue and are therefore named beige or brite (brown-in-white) cells1,3. In response to specific stimuli (such as cold), these otherwise quiescent beige cells are remodeled to exhibit multiple brown-like features, including multilocular lipid droplets, densely-packed mitochondria, and augmented UCP1 expression3,4,5.
Animal studies have demonstrated that brown and beige adipocytes possess multiple metabolic benefits beyond its fat-reducing effect, including insulin-sensitization, lipid-lowering, anti-inflammation, and anti-atherosclerosis6,7. In humans, the amount of beige/brown fat is inversely correlated with age, insulin resistance index, and cardiometabolic disorders8. Moreover, activation of beige/brown adipocytes in humans by either cold acclimation or &#946;3 adrenergic receptor agonist confers protection against a series of metabolic disorders4,9,10. These pieces of evidence collectively indicate that induction of brown and beige adipose tissue is a potential therapeutic strategy for management of obesity and its related medical complications8.
Interestingly, although they share similar function, beige and classical brown adipocytes are derived from different precursors and activated by overlapping but distinct mechanisms1. Therefore, in vivo imaging and quantification of brown and beige adipocytes are essential to achieve a better understanding of the molecular control of these adipose tissues. Currently 18F-fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) scan combined with computed tomography (CT) remains the gold standard for characterization of thermogenic brown and beige cells in clinical studies8. Magnetic resonance imaging (MRI) uses powerful magnetic fields and radio frequency pulses to produce detailed anatomical structures. Compared to CT scan, MRI generates images of organs and soft tissues with a higher resolution. Provided here is a protocol for visualization and quantification of functional brown and beige adiposes in mouse models after acclimation to cold exposure, a common and most reliable way to induce adipose browning. This method can be applied to characterize the thermogenic adipose depots in small animal models with high precision.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sterile saline</td>
			<td>BBraun</td>
			<td></td>
			<td>0.9% sodium chloride intravenous infusion, 500 mL</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Terumo</td>
			<td>SS05L</td>
			<td>5 mL syringe Luer Lock</td>
		</tr>
		<tr>
			<td>Dose Calibrator</td>
			<td>Biodex</td>
			<td></td>
			<td>Atomlab 500</td>
		</tr>
		<tr>
			<td>Eye lubricant</td>
			<td>Alcon Duratears</td>
			<td></td>
			<td>Sterile ocular lubricant ointment, 3.5 g</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Terumo</td>
			<td>10ME2913</td>
			<td>1 mL insulin syringe with needle</td>
		</tr>
		<tr>
			<td>InterView Fusion software</td>
			<td>Mediso</td>
			<td>Version 3.03</td>
			<td>Post-processing and image analysis software</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Chanelle Pharma</td>
			<td></td>
			<td>Iso-Vet, inhalation anesthetic, 250 mL</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Alfasan International B.V.</td>
			<td>HK-37715</td>
			<td>Ketamine 10% injection solution, 10 mL</td>
		</tr>
		<tr>
			<td>Medical oxygen</td>
			<td>Linde HKO</td>
			<td>101-HR</td>
			<td>compressed gas, 99.5% purity</td>
		</tr>
		<tr>
			<td>Metacam</td>
			<td>Boehringer Ingelheim</td>
			<td></td>
			<td>5 mg/mL Meloxicam solution for injection for dogs and cats, 10 mL</td>
		</tr>
		<tr>
			<td>nanoScan PET/MR Scanner</td>
			<td>Mediso</td>
			<td></td>
			<td>3 Tesla MR</td>
		</tr>
		<tr>
			<td>Nucline nanoScan software</td>
			<td>Mediso</td>
			<td>Version 3.0</td>
			<td>Scanner operating software</td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Reflex 7</td>
			<td>203-100</td>
			<td>7mm Stainless steel wound clips, 20 clips</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Alfasan International B.V.</td>
			<td>HK-56179</td>
			<td>Xylazine 2% injection solution, 30 mL</td>
		</tr>
	</tbody>
</table>

visualization and quantification of brown and beige adipose tissues in mice using [18f]fdg micro-pet/mr imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Three groups of mice (n = 3 per group) underwent micro-PET/MR imaging in this study, where they were housed at either thermoneutrality (30 &#176;C) or cold (6 &#176;C) for 7 days. One group of mice (n = 3) had their iBAT removed (iBATx) prior to cold treatment (Figure 1A). This method led to an alteration to the white adipose tissue activity in all three mice. In particular, a remarkable increase in [18F]FDG uptake was observed in iWAT using micro-PET/MR imaging (<strong class="xf...

Watch this Scientific Journal Video about Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging at JoVE.com

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

PET/MR imaging allows for both qualitative and quantitative measurements on these metabolically-active brown and beige adipocytes in live animals so that the activity of these so-called thermogenic adipocytes can be measured on a functional basis. The main advantage of this technique is that it allows acquisition and combination of PET and MRI scans on the same subject, thus enabling precise and simultaneous acquisition of thermogenic adipocytes in different depots of the small animal. This method can be easily transferred into any preclinical system or modified into a high throughput format by simultaneously imaging of multiple mice, thereby increasing the statistical power of the imaging data at a reduced cost and time.To harvest the interscapular brown adipose tissue, place an anesthetized 8-week-old C57 Black 6 mouse onto a heating pad and make a 2 centimeter incision along the dorsal midline. Remove the iBAT pads from the interscapular region. After the bleeding stops, use 7 millimeter stainless steel wound clips to close the incision.When all of the iBAT has been collected, house the mice in an intensive care unit for 14 days. Administer 5 milligram per kilogram Meloxicam to the mice for six days and remove the clips as soon as the wound has healed. To enable automated sequential PET/MR scans prior to imaging session, set the discrimination level to 400 to 600 kiloelectron-volts, the confidence mode to 1-3, the scanning time to 20 minutes, and the study isotope to F-18 to set the workflow for PET acquisition.To acquire T1-weighted magnetic resonance for attenuation correction, set the Gradient Echo-3D parameters. To acquire T2-weighted magnetic resonance as an anatomical reference, set the Fast-spin Echo 2D parameters. To allow reconstruction of the PET images, use the Tera-Tomo 3D algorithm with the coincidence mode set to 1-3 and with the decay, deadtime, random, attenuation, and scatter corrections set to create images with an overall voxel size of 0.3 cubic millimeters.One day before the start of the imaging study to check the accuracy of the PET quantitation, put on the appropriate PPE and fill a 5 milliliter syringe with F18-FDG as recommended by the manufacturer. Use a dose calibrator to record the activity of the syringe and note the time of the measurement. In the software, use the Interpolated Ellipse ROI to draw a volume-of-interest on the reconstructed image of the syringe and compare the recovered activity to the value measured using the dose calibrator.On the day of the imaging experiment, use forceps to carefully place the 18F-FDG stock vial behind an L-block tabletop shield and dilute an aliquot of radioisotope in 100 to 150 microliters of sterile saline to a total activity concentration of 200 to 250 microcuries. Draw the 18F-FDG solution into a 1 milliliter syringe equipped with a needle and measure the radioactivity with the dose calibrator as demonstrated. Record the weight of the mouse to be imaged before injecting the 18F-FDG solution into a tail vein.Take note of the injection time and the residual radioactivity of the syringe to enable decay correction. Then place the mouse back into its cage for 60 minutes. To calculate the injected 18F-FDG activity, subtract the activity in the syringe before the injection from the activity measured after the injection.At the end of the radioisotope uptake period, turn on the air heater for the mouse bed of the micro-PET/MR scanner. Place the anesthetized FDG-injected mouse onto a respiratory pad in the scanner bed and perform a scout view to determine the mouse position. Adjust the position of the mouse bed such that the entire body can be observed, ensuring that the center of the MRI field of view coincides with the center of the body.Under PET Acquisition, select Scan Range on Previous Acquisition to use the scout view position and click Prepare to move the animal bed from MR to PET. Select OK to initiate the PET scan. Record the injection dose and time measured before and after 18F-FDG administration in the Radiopharmaceutical Editor and enter the weight of the mouse under the Subject Information menu.Once the PET scan is complete, select Prepare to move to MR imaging and complete all MR acquisitions in the study list window. Select OK to start the MR scans. After the whole workflow has completed, briefly evaluate the quality of the acquired MR images in the post-processing software and click Home to move the mouse bed from MR to the original position.When all mice have been imaged, to reconstruct the data in the Raw Scan menu, select PET Acquisition to load the completed PET scan and select T1-weighted MR Acquisition to allow a material map to be created. Then use the Tera-Tomo 3D algorithm to reconstruct the data as demonstrated. The removal of iBAT prior to cold treatment alters the activity of the iWAT, as indicated by the remarkable increase in 18F-FDG uptake observed in iWAT by micro-PET/MR imaging.In addition, multilocular adipocytes, which are characteristically observed in beige adipose tissue, are more pronounced in iWAT from mice after iBAT removal compared to the adipocyte morphology observed in iWAT from sham-operated animals. Cold-treated, sham-operated mice demonstrate a markedly elevated 18F-FDG uptake in iBAT. Mice with their iBAT removed prior to cold treatment show the highest 18F-FDG uptake in the iWAT.Indeed, cold exposure causes a sevenfold increase in 18F-FDG uptake in the iBAT of sham-operated mice, while the removal of iBAT in the cold-induced mice results in an eightfold increase in 18F-FDG uptake compared to thermo-neutral mice in which only a modest twofold increase is observed. When attempting this procedure, placing each mouse in a same position for each PET/MRI acquisition is important to improve the visualization and quantification of brown and beige adipocytes in different regions. Using this method and together with other MRI methods, such as diffusion with imaging or thermometry, more insights into the spatial distribution and prevalence of brown and beige adipocytes in mice can begin, which can potentially be translated into humans.This method allows the researchers to study the function of these thermogenic brown and beige adipocytes. Therefore, they are especially powerful when studying the nonclinical thermogenic pathways wherein those classical markers are not altered or upregulated.

Research

JoVE Journal

Biology

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

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

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Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

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Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic ...