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.</li>
		<li>Place the mice on the heating pad after disinfection and make a 2 cm incision along the dorsal midline of the mice.</li>
		<li>Remove the iBAT pads (bilateral). In the sham-operated group, make the same incision but leave the iBAT pads intact.</li>
		<li>Close the incision using 7 mm stainless steel wound clips after the bleeding stops.</li>
		<li>After surgery, give meloxicam (5 mg/kg in drinking water) to the mice for 6 days and house them in an intensive care unit (ICU) for 14 days. Remove the clips as soon as the wound is healed (7-10 days).</li>
	</ol>
	</li>
	<li>Cold challenge of the mice: House the mice at thermoneutrality (30 &#176;C) for 14 days. On day 13, pre-chill the animal cages in cold (6 &#176;C) overnight. On day 14, put the mice at 6 &#176;C in the environmental chamber for 7 days. Place two mice in each cage.</li>
</ol>
2. Micro-PET/MR calibrations and workflow setup
NOTE: Micro-PET/MR imaging is performed using a sequential PET/MR system (see Table of Materials). Each mouse is placed on the imaging bed; first scan with the MR for an anatomical reference (scout view) before advancing to the center of the PET field-of-view (FOV) for a static [18F]FDG PET acquisition, followed by MR imaging for anatomic reference. An imaging workflow is created in the scanner-operating software (see Table of Materials) to enable automated, sequential PET/MR scans prior to the imaging session.
<ol>
	<li>Create an imaging workflow in the operating software that includes static PET acquisition, MRI acquisitions for attenuation correction, and anatomical reference using T1-weigthed 3D imaging and T2-weighted 2D imaging, respectively.</li>
	<li>To acquire PET, set 400-600 keV level discrimination, F-18 study isotope, 1-5 coincidence mode and 20 min scans.</li>
	<li>To acquire T1-weighted MR (for attenuation correction), set Gradient Echo-3D (TE = 4.3 ms, TR = 16 ms, FOV = 90 x 60 mm, number of excitations (NEX) = 3, 28 slices with 0.9 mm thickness, voxel size = 0.375 x 0.375 x 0.9 mm).</li>
	<li>To acquire T2-weighted MR (anatomical reference), set Fast-spin Echo 2D (TE = 71.8 ms, TR = 3000 ms, FOV = 90 x 60 mm, NEX = 5, 32 slices with 0.9 mm thickness, voxel size = 0.265 x 0.268 x 0.9 mm3).</li>
	<li>To reconstruct PET, use Tera-Tomo 3D (TT3D) algorithm (8 iterations, 6 subsets) with 1-3 coincidence mode, and with decay, dead-time, random, attenuation, and scatter corrections to create images with an overall of 0.3 mm3 voxel size.</li>
	<li>Perform a PET Activity Test of the micro-PET/MR scanner one day before the start of imaging study to check the accuracy of PET quantitation.
	<ol>
		<li>Prepare a 5 mL syringe filled with [18F]FDG as recommended by the manufacturer guidelines (140-220 &#956;Ci/5-8 MBq in water or saline).</li>
		<li>Record the activity of the syringe using a dose calibrator (see Table of Materials) and note the time of measurement.</li>
		<li>Select Interpolated Ellipse ROI to draw a volume-of-interest (VOI) on the reconstructed image to compare the recovered activity to the value measured as described above. The recovered activity for a well-calibrated scanner is accurate within &#177;5%.</li>
	</ol>
	</li>
</ol>
3. Injection of [18F]FDG
<ol>
	<li>Order a clinical dose of [18F]FDG (10 mCi/370 MBq) from the supplier for its arrival to the imaging lab approximately 30 min before the first scheduled injection. Make sure to wear appropriate personal protective equipment (PPE), such as a lab coat, gloves, personal radiation dosimeter e.g. Fingers, whole body when receiving the package containing radioactive materials. Change gloves regularly to prevent cross contamination of the radioactivity and increase distance from the radioactive source as much as possible.</li>
	<li>Use the forceps to carefully transfer the [18F]FDG stock vial behind an L-block table top shield.</li>
	<li>Dispense an aliquot of [18F]FDG and dilute with sterilized saline to give a total activity concentration at 200-250 &#956;Ci/7-9 MBq) in 100-150 &#956;L.</li>
	<li>Draw the [18F]FDG solution into a 1 mL syringe with needle (see Table of Materials), measure the radioactivity using a dose calibrator set to F-18, and record the time of measurement.</li>
	<li>Record the weight of the mouse prior to injection. Inject the prepared [18F]FDG solution via tail vein. Take note of the injection time and residue of the radioactivity of the syringe to enable decay correction.</li>
	<li>Put the mouse back in the cage and allow [18F]FDG uptake for 60 min before PET scans.</li>
	<li>Calculate the injected [18F]FDG activity using the following formula11: 
	Injected activity (&#956;Ci/MBq) 
	= Activity in the syringe before injection 
	- activity in the syringe after injection</li>
</ol>
4. Micro-PET/MR acquisition
<ol>
	<li>Turn on the air heater to the mouse bed to allow warm air to pass through it.</li>
	<li>Anesthetize the mouse using 5% isoflurane (1 L/min medical O2). Once induced, transfer the mouse to the warm mouse bed and maintain anesthesia at 2%-3 % isoflurane via a nose mask cone. Position the mouse head-prone onto the bite bar and make sure the mouse does not protrude outside of the diameter of the bed. Apply eye lubricant to avoid drying and formation of corneal ulcers.</li>
	<li>Monitor the body temperature and the respiratory rate by a thermal probe and a respiratory pad, respectively. Maintain the body temperature at 36-37 &#176;C, and the respiratory rate at 70-80 breaths per minute (bpm) by adjusting the isoflurane level.</li>
	<li>Perform a scout view to determine the mouse position. Adjust the mouse bed position to include the whole mouse body, and to ensure the center FOV of MR is in the center of mouse body.</li>
	<li>Under the PET Acquisition in the study list window, select Scan Range on Previous Acquisition to use the scout view position as described above. Click on 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. Enter the weight of the mouse under the Subject Information menu.</li>
	<li>Once the PET scan is completed, select Prepare to move to MR and complete all the MR acquisitions in the study list window. Select OK to start the MR scans.</li>
	<li>After the whole workflow is completed, briefly evaluate the quality of the acquired MR images using the post-processing software (see Table of Materials). Click on the Home button to move the mouse bed from MR to the original position.</li>
	<li>Carefully remove the mouse from the scanner and return it to a clean housing cage with a heated pad underneath to allow recovery in warm environment. Supply the mouse with food and water. The system is now ready for the next mouse in queue.</li>
	<li>To reconstruct data, select PET Acquisition under the Raw Scan menu to load the completed PET scan. Select T1-weighted MR Acquisition for material map creation. Reconstruct the data as described above (see step 2.5).</li>
	<li>Follow the local and institute regulations regarding the care and handling of post-PET imaging mouse. Consider all used syringes/needles, gloves, bedding, and fecal matter as radioactive waste that require special handling/disposal in accordance with the local regulations.</li>
</ol>
5. Post-imaging analysis
<ol>
	<li>Open the Image Analysis software (see Table of Materials) and click on Load DICOM Data to retrieve the corresponding MR and PET images.</li>
	<li>Perform co-registration of MR and PET image by dragging these images to the display window. Click on the Automatic Registration function.
	<ol>
		<li>Select Rigid transformation under the Registration Setup drop-down menu. Check Shift and Rotation under the Rigid/Affine menu.</li>
		<li>Select T1-weighted MR acquisition as the Reference and PET acquisition as the Reslice under the Global Role Selection menu.</li>
		<li>Inspect the registration in all three dimensions to make sure a perfect alignment between MR and PET images. To adjust it manually, click on Manual Registration.</li>
	</ol>
	</li>
	<li>Use Interpolated Ellipse ROI to draw VOI on the tissue of interest, i.e., iBAT and inguinal white adipose tissue (iWAT) using MR image for reference. Use the Brush Tool and Eraser Tool to define the VOI border; hence, the anatomy of tissues. Make sure there is no overlap uptake by using PET image to avoid spillover from the neighboring organs. Repeat the process slide-by-slide until the whole VOI is delineated. If necessary, edit the VOIs to maintain consistent VOI volumes between each mouse.</li>
	<li>Use Ellipsoid VOI to draw a 3 mm3 VOI on the lung as a reference organ. Avoid any spillover from the neighboring heart and muscle.</li>
	<li>Upon completion, click on Show ROI Table to rename each VOI. Record the mean radioactivity with the VOI and tissue volume into a spreadsheet. Archive the VOI drawings and the imaging data to a data storage device.</li>
	<li>Calculate the standardized uptake value (SUV) for all VOIs using the following equation11: 
	SUVmean&#160;= VOI radioactivity in kBq / (Decay - corrected injected dose in kBq / mouse body weight in kg), assuming a tissue density of 1 g/mL.&#160;</li>
</ol>

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The authors have no conflicts of interest to disclose.

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 activated adipocytes as a result of high glucose utilization in an accurate manner. The experimental conditions outlined here highlights the feasibility of using [18F]FDG PET to study up-regulation of iBAT and iWAT in vivo, and is potentially useful for evaluation of the thermogenic impact of new drug candidates. In addition, this protocol can be easily modified into high throughput format by simultaneously imaging multiple mice using a specially designed animal bed, thereby increasing the statistical power and confidence in the imaging data at a reduced cost and time12,13.
Currently, [18F]FDG PET/CT remains the most common approach to visualize BAT in humans and rodents and standard protocols have been well established8,11. In recent years, there are also several studies using [18F]FDG PET/MR imaging to assess BAT in humans14,15,16. In contrast, no detailed description on [18F]FDG PET/MRI for small animals is available. Described here is a detailed protocol that relies on the use of a combined PET and MR imaging system in mice. This method takes advantage of the higher resolution of MRI, especially on the detection of fat tissues, making them easy to identify and segment compared to the commonly used CT method. Therefore, the current approach enables an improved accuracy of PET quantification compared to the PET/CT method, which is of great value for studies in small animals with more delicate adipose depots. When analyzing the results of tissues of interest at their baseline uptake, MRI becomes an essential tool to accurately draw the VOIs to ensure consistency of their volumes between mice and avoid inclusion of neighboring organs. In addition, accurate image processing such as image registration and VOI delineation are important to allow reliable quantification. The anatomical location of the glucose-responsive BAT is distinct between humans and mouse. While the functional BAT locates at the interscapular region, [18F]FDG PET/MR imaging-based analysis mainly identify functional BAT in the supraclavicular region in humans14,15,16.
The fasting or fed status of the mice should also be taken into consideration when performing the [18F]FDG uptake experiment. In some studies, the mice are fasted for several hours or even overnight before the uptake experiment since it is supposed that endogenous glucose will compete with [18F]FDG. In the protocol, the [18F]FDG at fed status was measured and strong uptake signal was still observed in both iBAT and iWAT. This, thus, demonstrates that it is not necessary to put the mice at a fasted status for robust uptake signals, which is less physiologically relevant. Actually, caution should be taken when examining BAT and beige adipocytes in fasted animals since a previous finding has reported that the hypothalamic neuropeptide Y (NPY)-mediated hunger signal acts on the medullary motor systems to inhibit BAT thermogenesis by reducing the sympathetic innervation17. Consistently, in humans, it is suggested that upon high calorigenic diets, the thermogenic adipocytes burn out extra calories so as to maintain energy balance. In contrast, upon nutrient deprivation, counter-regulatory mechanisms are activated to suppress energy waste.
Another consideration for [18F]FDG PET imaging involves the routes for radiotracer administration into mice. Intraperitoneal and intravenous techniques are two common ways to inject [18F]FDG into mice, and both methods result in a relatively similar biodistribution of [18F]FDG in mice 60 min post injection18. While the intraperitoneal method is relatively easy to perform and the injection can be done quickly to avoid unwanted stress imposed onto the mice, direct injection accidentally into the bowel is common and is not immediately identified, leading to unreliable PET results19. Intravenous method is the preferred method and employed in this study. Successful tail vein injection can be determined when a visible blood flashback is observed prior to infusion, indicating that the needle is properly positioned inside the vein for infusion. One limitation to this technique is the difficulty to notice a visible blood flashback, potentially due to low blood pressure and the presence of dark hair on the tails. This can be overcome by warming the tail with a warm washcloth to increase the blood flow, hence improving the visibility of the vein for needle insertion.
An accurate scanner and a relevant equipment are other important factors for reliable PET image quantification. It is imperative to perform routine quality control examinations on PET and MR components of the scanner. MR quality control involves the signal-to-noise ratio assessment on different T1- and T2-weighted sequences, which should be performed on a weekly basis as recommended by the scanner manufacturer. For PET, accuracy of activity must be determined using a syringe containing a known concentration of radioactivity on a weekly basis or before the start of an important study. This quality control test also allows determination of co-registration of PET and MR images. Calibration must be performed if the recovered activity falls outside the recommended range or mis-registration between PET and MR images is found. In addition, the dose calibrator should be regularly calibrated according to the manufacturer guidelines since this is an important tool for quality control of the scanner as well as radioactivity measurement for PET imaging.
This study shows that the activation of adipose depots in both iBAT and iWAT in mice can be visualized and quantified using [18F]FDG PET/MR imaging upon exposure to cold temperature. However, the current study is limited by the fact that [18F]FDG uptake in iWAT was relatively low unless in the absence of iBAT. This indicates that compared to the iBAT that is readily activated by cold stimulus, beige adipocytes are relatively reluctant to be mobilized and act more like a backup thermogenic depot of iBAT in mice. More efficient methods to induce the [18F]FDG signal in the iWAT and/or other adipose depots in normal mice, such as the use of beige-specific activators or stronger cold challenge condition, are to be identified, which is beyond the scope of the current study.

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 (Figure 1B-C). This co-registered imaging data is demonstrated as maximal intensity projection (MIP), where iWAT was clearly delineated to allow quantification of the [18F]FDG uptake. Consistently, the multilocular adipocytes, which are characteristic morphology for beige adipocytes, were more pronounced in iWAT from iBATx mice, compared to the sham operated group (Figure 1D).
To verify whether changes on iBAT and iWAT activities upon this prolonged cold induction can be monitored by micro-PET/MR imaging, imaging studies were performed on the mice exposed to 30 &#176;C and 6 &#176;C and the results between groups were compared. PET/MR imaging also demonstrated that mice subjected to 6 &#176;C have markedly elevated [18F]FDG uptake on iBAT in sham operated mice (Figure 2A), which is consistent with the previous reported literature11. Mice with their iBAT removed (iBATx) prior to cold treatment showed the highest [18F]FDG uptake in the iWAT among the 30 &#176;C and 6 &#176;C group (Figure 2B). PET images were further quantified using an SUV-based approach. In iBAT, cold exposure caused a 7-fold increase in [18F]FDG uptake when compared to the 30 &#176;C group. In iWAT, [18F]FDG uptake was higher&#160;in cold-acclimated iBATx mice than the remaining groups (Figure 2C). Removal of iBAT in the cold-induced mice resulted in an 8-fold increase in the uptake of iWAT compared to the thermoneutrality mice, whereas only a modest increase (2-fold) was observed when iBAT was present in mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62460/62460fig1v3.jpg" /> 
Figure 1: Micro-PET/MR Imaging of inguinal white adipose tissue (iWAT) in mice. Interscapular brown adipose tissue was surgically removed (iBATx). After recovery, the mice were housed at 6 &#176;C for 7 days before analysis. (A) Flow chart for the surgical and the subsequent procedures. (B) Illustration of the position of the mouse and the PET/MR scanner. (C) Maximal intensity projection (MIP) of co-registered PET/MR images. White arrows: Location of iWAT. A: Anterior L: Left. (D) Hematoxylin and Eosin (HE) staining of iWAT in sham and iBATx mice after cold exposure. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62460/62460fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62460/62460fig02.jpg" /> 
Figure 2: Representative in vivo [18F]FDG uptake in brown adipose tissue at the interscapular region (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Mice housed at thermoneutrality (30 &#176;C), cold-acclimated (6 &#176;C) and cold-acclimated + iBATx were subjected to [18F]FDG PET/MR imaging. (A) Sagittal section of PET/MR images showing iBAT in mice. (B) Axial section of PET/MR images showing bilateral iWAT. (C) Quantitative analysis of [18F]FDG uptake in iBAT (left) and iWAT (right). Yellow arrows: Location of iBAT. White arrows: Location of iWAT. n = 3 for each group. Values of SUVratio are presented as mean &#177;SD. <a href="https://www.jove.com/files/ftp_upload/62460/62460fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

visualization-quantification-brown-beige-adipose-tissues-mice-using

Nanyang Technological University

Research

JoVE Journal

Biology

2.9K Views.  The University of Hong Kong. Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

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

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

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

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

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

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

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

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...