We would like to thank the Vanderbilt University Institute of Imaging Science MRI technologists David Pennell, Leslie McIntosh, and Kristen George-Durrett, and the team of Vanderbilt University Medical Center PET/CT technologists led by Martha D. Shone. This work was supported by the following grants from the NIH: NCATS/NIH UL1 RR024975, NIDDK/NIH R21DK096282, NCI/NIH R25CA136440, and NIBIB/NIH T32EB014841.

NOTE: The local ethics committee of this institute approved this study, and all subjects provided written informed consent prior to participation. To be eligible for the study, subjects must fulfill the following requirements: no known diabetes mellitus; no use of beta blockers or anxiety medications, currently or in the past; does not smoke or chew tobacco products, currently or in the past; no more than 4 cups of caffeine each day; no more than 2 glasses of alcohol each day; and if female, not pregnant or breastfeeding. 
NOTE: In this study, each participant undergoes four exams: two MRI and two PET-CT. Each exam is acquired on a different day, with each imaging modality performed under both thermoneutral 24.5 &#177; 0.7 &#176;C (76.2 &#177; 1.3 &#176;F), and cold 17.4 &#177; 0.5 &#176;C (63.4 &#177; 0.9 &#176;F) conditions. The scans are not scheduled in any particular sequence, helping to minimize any potential bias to the data due to heating or cooling the subject in a specific order. The total effective radiation dose for one PET-CT scan is 6.4 mSv (millisievert), and the radiologist on staff recommends a washout period of at least 24 hr between each scan.
1. General MRI Safety and Imaging Concerns
<ol>
	<li>Because the main magnetic field in MRI machines is always on, take care to ensure the safety of the patient and all personnel working in the MR area. Clear all magnetic objects from the subject and any persons working in the area.</li>
	<li>Ask subjects during the recruitment phase if they have any metal in their bodies43. In addition, have the subject complete a magnetic safety screening process44 to ensure that any metal in the body is approved for MRI. This initial check can help eliminate the possibility of consenting a subject who cannot complete the MRI scan.</li>
	<li>Additionally, if there is any metal in the subject&#8217;s body, which is compatible with MR, ensure that the metal is not near the tissue of interest. This is because metal can cause image distortion artifacts, which will make the analysis difficult if not impossible.</li>
</ol>
2. Obtaining Informed Consent
<ol>
	<li>Meet with the subject to obtain written informed consent. During this meeting, cover all details of the study, for example: the number of visits, the time commitment per visit, what the requirements are of the subject regarding limitations to exercise and/or food, what the subject can and cannot do during the visit (such as sleep), and any other specifics. Use this meeting to schedule the visits for the scanning, as it is usually easier to schedule these in person rather than using multiple emails.</li>
</ol>
3. Procedures Prior to Visit
<ol>
	<li>Instructions for the Subject
	<ol>
		<li>For 24 hr prior to arriving for the study, have the subject refrain from alcohol, caffeine, medication or any strenuous exercise or activity.</li>
		<li>Instruct the subject to fast and to avoid any caloric intake for 8 hr prior to arriving for the study. Subjects are allowed to drink water.</li>
	</ol>
	</li>
	<li>Contacting Volunteer
	<ol>
		<li>Remind the volunteer of the specific instructions the day before the start of their 24 hr preparation. This serves both as a reminder of the scan, as well as it helps to ensure that the subject remembers their restrictions, (i.e., no eating, no exercise, no alcohol, etc.).</li>
	</ol>
	</li>
</ol>
4. Procedure on Study Day &#8211; for MRI
<ol>
	<li>Temperature-Controlled Room Preparation
	<ol>
		<li>Use a small room as the temperature-controlled room where the subject is exposed to the desired temperature. 
		NOTE: By using a small room, it is possible to minimize temperature gradients in the room. For example, the room size used here is 7&#8217; x 6&#8217; 8&#8221; x 8&#8217; tall, (373.33 cubic feet).</li>
		<li>Prepare the room at least 60 min prior to the subject entering the room to allow sufficient time for the room to reach a stable temperature.</li>
		<li>Maintain the RT either with a portable air-conditioning unit and a rotating floor fan to keep the cool air circulating, or using a programmable portable heater, which oscillates to circulate the warm air around the room.</li>
		<li>Deactivate or minimize any existing thermostat controlling air-conditioning or heating of the room to avoid conflicting with the desired RT target of the portable devices.</li>
	</ol>
	</li>
	<li>Prior to Entering Temperature-Controlled Room
	<ol>
		<li>Have the subject change into standard medical shorts and shirt. Remove socks and shoes. If the subject is female, allow the wearing of a sports bra that does not contain any metal.</li>
		<li>Measure the subject&#8217;s height, weight, and waist circumference measurements after changing into the standard clothing.</li>
		<li>Measure the subject&#39;s body temperature using a sublingual thermometer.</li>
	</ol>
	</li>
	<li>In the Temperature-Controlled Room
	<ol>
		<li>Direct the subject to enter the temperature-controlled room. Ask the subject to sit quietly and not perform any activity that could change body temperature, e.g., exercising, typing, or falling asleep.</li>
		<li>After sitting in the room for 1 hr, measure the body temperature again using a sublingual thermometer.</li>
		<li>After the second hr of sitting in the temperature-controlled room, measure the body temperature again using a sublingual thermometer.</li>
		<li>On the MRI day when the subject is sitting in the cold room, use a cold vest to maintain a cold environment while the subject is transported to the MRI scanner. Place the cold vest on the subject prior to the subject leaving the temperature-controlled room.</li>
		<li>After 2 hr in the temperature-controlled room, transport the subject in a wheelchair to the MRI scanner. Use the wheelchair to keep the subject in a relaxed, sedentary state, and to minimize any &#8220;warming&#8221; that might occur from walking. Additionally, using the wheelchair helps to avoid any uptake of the PET tracer into skeletal muscles, though it would likely be minimal.</li>
	</ol>
	</li>
	<li>MRI Acquisition Protocol
	<ol>
		<li>Acquire MRI scans using a 3T MRI scanner equipped with two-channel parallel transmit capability, an extra-large 16-channel torso receive coil, and a modified tabletop.</li>
		<li>Hang the anterior portion of the torso receive coil from the top of the scanner bore in a fabric sling. Allow the sling to hang low enough to slide against the subject&#8217;s body in order to maximize signal-to-noise ratio (SNR).</li>
		<li>Place the posterior portion of the torso receive coil in a rolling &#8220;coil wagon&#8221; sandwiched between two layers of the tabletop. As the table moves through the scanner bore, hold the coil wagon at isocenter by straps attached to the scanner covers at the front and back of the scanner bore so that the posterior coil element remains stationary.</li>
		<li>Position the subject on the bed to enter the scanner feet first in a supine position.
		<ol>
			<li>If the subject is wearing the cold vest, remove the vest prior to the subject lying down.</li>
		</ol>
		</li>
		<li>Once lying down, have the subject place both arms inside a bag similar to a pillowcase, and lower the arms to either side of the body. This helps ensure the shoulders are positioned in a similar manner during both the MRI and PET/CT exams, which makes image co-registration easier. 
		NOTE: Allowing the subject to lie down on the scanner bed naturally, using the same amount of cushioning under the head during each scan, and using the pillowcase bag to support the arms, all helps to minimize differences between subject positioning between scans. Any support used for the subject during one scan, for example a pillow under the knees or lower back, should always be used in the same way for that subject, during both the MRI and PET/CT scans.</li>
		<li>Acquire fat-water MRI (FWMRI) using a multi-stack, multi-slice, multiple fast field echo (mFFE) acquisition with 7 stacks of 20 axial slices, covering from the crown of the head to upper thigh. Slices are contiguous with a 0 mm gap between slices.
		<ol>
			<li>Collect FWMRI scans using customized software to enable the acquisition of 8 echoes acquired as two interleaved sets of four echoes with a TR = 83 ms, TE1=1.024 ms and effective &#916;TE = 0.779 ms. Other acquisition protocol details include: flip angle = 20&#186;, water fat shift = 0.323 pixels, readout sampling bandwidth = 1346.1 Hz/pixel, axial in-plane field of view = 520 mm &#215; 408 mm, acquired voxel size = 2 mm x 2 mm x 7.5 mm, and sensitivity encoding (SENSE) parallel imaging factor = 3 (anterior posterior direction). Preparation phases for each station include center frequency (F0) optimization and first order linear shimming. Acquisition time is 27.8 sec for 20 slices.</li>
			<li>Perform breath holds for stations covering the pelvis to the shoulders with two breath holds per station, i.e., no breath hold is longer than 14 sec. At each table position, acquire a dual angle B1 calibration scan (acquisition time 15.1 sec) to enable optimized RF shimming (relative RF amplitude and phase adjustments) for the two-channel transmit capability of the scanner.</li>
			<li>Acquire a SENSE reference scan at each table position with an acquisition time of 12.1 sec. Recommended FWMRI parameters are listed in Table 1.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Procedure on Study Day &#8211; for PET-CT
<ol>
	<li>Temperature-Controlled Room Preparation
	<ol>
		<li>Use a small room as the temperature-controlled room where the subject is exposed to the desired temperature. 
		NOTE: By using a small room, it is possible to minimize temperature gradients in the room. For example, the room size used here is 7&#8217; x 6&#8217; 8&#8221; x 8&#8217; tall, (373.33 cubic feet).</li>
		<li>Prepare the room at least 60 min prior to the subject entering the room to allow sufficient time for the room to reach a stable temperature.</li>
		<li>Maintain the RT either with a portable air-conditioning unit and a rotating floor fan to keep the cool air circulating to achieve the cold stimulus temperature, or using an oscaillating portable heater to maintain the thermoneutral temperature.</li>
		<li>Deactivate or minimize any existing thermostat controlling air-conditioning or heating of the room to avoid conflicting with the desired RT target of the portable devices.</li>
	</ol>
	</li>
	<li>Subject Preparation
	<ol>
		<li>Direct the subject to the PET imaging suite to have an IV port placed in a hand or arm vein. This IV port allows the Radiology technician to inject the radiotracer later, when the subject is sitting in the temperature-controlled room.</li>
		<li>If the subject is female, perform a blood serum pregnancy test to ensure she is not pregnant. 
		NOTE: For this study, the internal review board requires a pregnancy test less than 24 hr prior to the PET/CT scan being acquired.</li>
	</ol>
	</li>
	<li>Prior to Entering Temperature-Controlled Room
	<ol>
		<li>Have the subject change into standard medical shorts and shirt. Remove socks and shoes. If the subject is female, allow the wearing of a sports bra that does not contain any metal.</li>
		<li>Measure the subject&#8217;s height, weight, and waist circumference measurements after changing into the standard clothing.</li>
		<li>Measure the subject&#39;s body temperature using a sublingual thermometer.</li>
	</ol>
	</li>
	<li>In the Temperature-Controlled Room
	<ol>
		<li>Direct the subject to enter the temperature-controlled room. Ask the subject to sit quietly and not perform any activity that could change body temperature, e.g., exercising, typing, or falling asleep.</li>
		<li>After sitting in the room for 1 hr, measure the body temperature again using a sublingual thermometer.</li>
		<li>On the PET-CT scan days after the first hour in the temperature-controlled room, have a radiology technician administer the injection of Fluorodeoxyglucose (18F-FDG) through the IV port. Inject 0.14 mCi/kg (approximately 10 mCi for a 70 kg subject) of 18F-FDG. Calculate exact dosage based on subject specific weight.</li>
		<li>After the second hr of sitting in the temperature-controlled room, measure the body temperature again using a sublingual thermometer. 
		NOTE: Unlike the cold MRI days, use of the cold vest is unnecessary on cold PET-CT days because the 18F-FDG tracer is taken up into the activated BAT during the hour post tracer injection. The tracer will not leave the tissue even if the subject becomes warm as he/she is being transported to the scanner. Therefore, because it is possible to detect the presence of activated BAT on the PET-CT images even if the BAT does not remain active during the PET-CT scan, the cold vest is not necessary.</li>
		<li>After 2 hr in the temperature-controlled room, transport the subject in a wheelchair to the PET-CT scanner. Use the wheelchair to keep the subject in a relaxed, sedentary state, and to minimize any &#8220;warming&#8221; that might occur from walking. Additionally, using the wheelchair helps to avoid any uptake of the PET tracer into skeletal muscles, though it would likely be minimal.</li>
	</ol>
	</li>
	<li>PET-CT Acquisition Protocol
	<ol>
		<li>Acquire PET-CT scans on a Discovery STE PET/CT scanner (STE stands for See and Treat Elite).</li>
		<li>Position the subject on the bed to enter the scanner head first in a supine position.</li>
		<li>Once lying down, have the subject place both arms inside a bag similar to a pillowcase, and lower the arms to either side of the body. This helps ensure the shoulders are positioned in a similar manner during both MRI and PET/CT exams, which makes image co-registration easier. 
		NOTE: The PET/CT imaging field of view covers from the crown of the head to mid-thigh in 7-9 bed positions, depending on subject height (2 min per bed position). Recommended PET-CT parameters are listed in Table 2.</li>
	</ol>
	</li>
</ol>
6. MRI Post Processing
<ol>
	<li>Save real and imaginary MR images for off-line processing.The signal measured by MRI is a vector quantity with both magnitude and direction that can be represented as a complex number with real and imaginary parts. In the clinical setting, the magnitude images are typically displayed. However, complex information is needed for processing into the fat and water images.</li>
	<li>Perform three-dimensional water/fat separation and R2* estimation based on a multi-scale whole-image optimization algorithm45 implemented in C++ for each individual slice stack. Fat is modeled using 9 peaks46.</li>
	<li>Discard the first echo of each 4-echo train to avoid potential contamination of eddy current in the complex water-fat signal model.</li>
</ol>
7. PET-CT Post Processing
<ol>
	<li>Load CT DICOM data into MATLAB and convert to Hounsfield units (HU) by applying the scanner-supplied rescale value to the data values.</li>
	<li>Load PET DICOM data into MATLAB and convert to Standardized Uptake Values (SUV) using the following formula: 
	<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/52415/52415eq1.jpg" /> 
	where &#8220;pixel value&#8221; is the stored value in the DICOM file for that pixel location. 
	<img alt="Equation 2" fo:content-width="3in" src="/files/ftp_upload/52415/52415eq2.jpg" /> 
	NOTE: The PET tracer activity is the radionuclide total dose, and can be read from the image meta-data (DICOM header file). 
	<img alt="Equation 3" fo:content-width="3in" src="/files/ftp_upload/52415/52415eq3.jpg" /></li>
	<li>Interpolate the PET data to have the same dimensions as the CT data.
	<ol>
		<li>Because PET and CT images are acquired with the same slice thickness, perform interpolation using a 2-dimensional spline function in the X-Y plane.</li>
	</ol>
	</li>
</ol>
8. Data Post Processing
<ol>
	<li>To analyze the images, co-register all 4 image-volumes for each subject using a rigid body registration algorithm47 via a semi-automated method with in-house developed 3-plane view software to verify registration in all three dimensions.</li>
	<li>Due to difficulties with registering the entire image volume across all four time-points, focus registration on the region covering the neck to the apex of the lungs. Use only the successfully registered region in further data processing.</li>
	<li>Following image registration, load FWMRI, CT HU and PET SUV data into MATLAB and use to define BAT regions of interest. 
	NOTE: Similar to previously published methods19,25,48 of distinguishing BAT using PET SUV and CT HU values, to be considered part of the BAT mask, each voxel in the image must satisfy the following: (1) HU value falls in the range of: -200 &#60; HU &#60; -1, on both cold and warm CT scans; (2) SUV &#62; 2.0 on the cold PET scan; (3) SUV signal fraction [(Cold SUV)/(Cold SUV + Warm SUV)] &#62; 0.55, i.e., the cold PET scan must generate more than 55% of the total observed SUV signal in that voxel; and (4) only contain foreground pixels from the MRI scan, where Otsu&#8217;s method49 is used to classify foreground pixels.</li>
	<li>If a voxel fulfills all these criteria, include the voxel in the binary mask of BAT identity.</li>
	<li>Apply the following binary morphology steps.
	<ol>
		<li>Create a matrix the same size as the images being processed. Each spatial location in the new matrix is the 3D sum of all its adjacent neighbors in the binary BAT mask, including diagonals. The maximum sum is 26.</li>
		<li>Threshold this new matrix to include only locations with 15 or more 3D neighbors. This matrix then forms the final binary BAT mask. 
		NOTE: These rules are sufficient to segment BAT tissue, and no further modification to the mask is necessary to eliminate non-BAT voxels. This forms a slice-by-slice mask of PET-CT confirmed BAT in the co-registered shoulder region.</li>
	</ol>
	</li>
	<li>Apply the mask to all the co-registered images to acquire the SUV, HU, fat signal fraction (FSF) and R2* values in the BAT regions, for both the cold and warm scans.</li>
</ol>

The authors declare that they have no competing financial interests or other conflicts of interests.

The described study protocol is designed to use both thermoneutral and cold-activated PET/CT to automatically segment BAT depots on a subject specific basis. These automatically generated regions of interest can then be applied to both thermoneutral and cold-activated MRI scans which have been co-registered to the PET/CT scans of the same subject. To the best of our knowledge, this is the first research to perform both MRI and PET/CT after thermoneutral and cold-activated conditions on the same healthy adult human volunt...

Due to the marked rise in obesity worldwide, there is an increased interest in research areas aimed at understanding energy balance. Obesity can result in costly and devastating medical conditions such as diabetes, liver disease, cardiovascular disease and cancer, making it a significant area of concern for public health1. One area of research aimed at understanding the balance of energy intake versus energy expenditure is the study of brown adipose tissue or BAT. Although termed an adipose tissue, BAT differs from the more common white adipose tissue (WAT) in many ways2. The function of white adipocytes is to store triglycerides in a single large lipid vacuole per cell, and to release these triglycerides as a source of energy into the blood stream when needed. In a very different manner, the function of brown adipocytes is to produce heat. One mechanism by which this occurs is through exposure to cold. This causes an increase in sympathetic nervous system activity, which in turn activates BAT. When activated, brown adipocytes generate heat. To do so, they use the triglycerides contained in the many small lipid vacuoles per cell, and through the presence of uncoupling protein 1 (UCP1) in the abundant mitochondria, convert the triglycerides to metabolic substrates without the production of ATP, resulting in entropic loss as heat generation. As the triglycerides stored in the small lipid vacuoles are depleted, the adipocyte takes up both glucose and triglycerides present in the blood stream3.
Interest in studying BAT has dramatically increased in recent years due to its contribution to non-shivering thermogenesis, its role in modulating the body&#8217;s energy expenditure, and the potential inverse relationship between BAT and obesity3&#8211;9. In addition, recent animal studies indicate BAT plays a critical role in clearing triglycerides and glucose from the blood stream, especially following ingestion of a high fat meal10,11. However, most of what we know about BAT is a result of research in small mammals, which contain many depots of BAT4,9,12&#8211;15. Notwithstanding a few early studies16&#8211;18, the presence of BAT in humans was widely thought to diminish with age until recently when interest in studying human BAT has been renewed. Recent research suggests that relatively small amounts of BAT persist into adulthood19&#8211;24. An additional limiting factor to studying BAT is that apart from biopsy and histological staining, the currently accepted unequivocal method for detecting BAT is 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). Modern PET scanners are typically combined with a computed tomography (CT) scanner. When activated by cold exposure, BAT takes up the 18F-FDG radiotracer, which is a metabolic analogue of glucose, and becomes visible on PET images, in comparison to the much lower level of 18F-FDG uptake when BAT is inactive20,21,23,25. CT images acquired during a PET exam on a PET-CT scanner help to differentiate between tissues with high 18F-FDG uptake by providing anatomical information. This use of PET-CT imaging exposes the subject to ionizing radiation (predominately from PET, though the dose from the CT scan is not negligible), and is therefore an undesirable method for BAT detection.
Although the number of studies on BAT in healthy adult humans is increasing, recent studies of human BAT have mainly been limited to retrospective PET-CT studies19,25, human infant cadavers26,27, human adolescents who have already been admitted to hospitals for other reasons27&#8211;30, and a few human studies of healthy adults31&#8211;35. One of the challenges with both studies of children and retrospective studies is the possibility of altered results when studying a patient population who is sick, which may affect BAT. Additionally, because glucose is not the preferred fuel source of BAT36, PET studies may not always detect activated BAT, and therefore may underrepresent the presence of BAT. Another difficulty in studying BAT with biomedical imaging is related to performing image segmentation to define the boundaries of tissue depots. Currently, segmentation of BAT in human studies often relies on some degree of manual image segmentation and is therefore vulnerable to misidentification of BAT depots, as well as inter-rater variability.
Because of these challenges, reliable spatial mapping techniques that can distinguish BAT from WAT distributions, along with automated segmentation methods, would provide investigators with a powerful new tool with which to study BAT. Magnetic resonance imaging (MRI) has the capability for identification, spatial mapping, and volumetric quantification of BAT, and unlike existing hybrid PET-CT imaging approaches that include a radioactive dose for the imaged subject, MRI involves no ionizing radiation and can be used safely and repeatedly. The ability to identify and quantify BAT using MRI can have a dramatic positive impact on clinical endocrinology and the pursuit of new avenues of obesity research. Previous fat-water MRI (FWMRI) studies of BAT in both mice and humans show that the fat-signal-fraction (FSF) of BAT is in the range of 40-80% fat, whereas WAT is above 90% fat15,26,27. We therefore hypothesize that this quantitative FWMRI metric, in conjunction with other quantitative MRI metrics, can be used in future work to visualize and quantify BAT depots in humans. This would provide the research community with a powerful tool with which to study BAT&#8217;s influence on metabolism and energy expenditure without the use of ionizing radiation.
Our research group has been studying BAT in adult humans for the past three years. Our first public presentation on the use of MRI to investigate suspected BAT in one adult human subject occurred in February 2012 at the International Society for Magnetic Resonance in Medicine (ISMRM) Fat-Water Separation Workshop in Long Beach, California37. Two months later, our group presented FSF values in suspected BAT in two adults at the 20th annual meeting of the ISMRM in April 2012 in Melbourne, Australia38. One year later at the 21st annual meeting of the ISMRM in April 2013 in Salt Lake City, Utah, the protocol described in this manuscript was used for the first (to the best of our knowledge) public presentation of MRI quantification of PET-confirmed BAT in adult human subjects39. Specifically, we presented evidence showing that the previously suspected BAT was confirmed to be activatable BAT using both cold-activated and thermoneutral 18F-FDG PET-CT imaging. Since 2013, our cohort of healthy adult human subjects imaged with both MRI and PET/CT under thermoneutral and cold-activated conditions has expanded to more than 20 subjects with results most recently presented in February 2014 at the workshop &#8220;Exploring the Role of Brown Fat in Humans&#8221; sponsored by the NIH NIDDK40. Specifically, we reported FWMRI FSF and R2* relaxation properties in regions of supraclavicular BAT confirmed by 18F-FDG PET-CT in adult humans, with the BAT ROIs delineated using automated segmentation algorithms based on the cold-activated and thermoneutral PET-CT scans. Most recently we presented results of temperature mapping in 18F-FDG PET-CT confirmed BAT in adult humans using advanced FWMRI thermometry41,42.
The procedure presented here acquires both MRI and 18F-FDG PET-CT scans on the same subject, each after exposure to both cold-activated and thermoneutral conditions. The cold-activated and thermoneutral 18F-FDG PET-CT scans are used to create automatically segmented BAT regions of interest (ROIs), on a subject specific basis. These BAT ROIs are then applied to the co-registered MRI scans to measure the MRI properties in the PET-CT confirmed BAT.
A limitation of this protocol is that the air temperature used when exposing subjects to either the warm or cold stimulus is consistent for every subject. This is a limitation because the temperature at which each subject experiences feeling warm or chilled can be different. Therefore, by running a trial session during which the air temperature is adjusted to fit the individual&#8217;s response, and then using these temperatures during the thermoneutral and cold-activation protocols, it could be possible to obtain better responses from the brown adipose tissue.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="100">
			<td height="100" style="height:100px;width:363px;">Name of Material/ Equipment</td>
			<td style="width:392px;">Company</td>
			<td style="width:375px;">Catalog Number</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">MRI</td>
			<td style="width:392px;">Philips</td>
			<td style="width:375px;">Achieva 3T</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:363px;">MRI Torso-XL coil</td>
			<td style="width:392px;">Philips</td>
			<td style="width:375px;">Philips SENSE XL Torso coil 16-elements</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:363px;">MRI X-tend Table</td>
			<td style="width:392px;">X-Tend</td>
			<td style="width:375px;">X-tend table, Acieva 3T compatible</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:363px;">X-tend armsupport</td>
			<td style="width:392px;">X-Tend</td>
			<td style="width:375px;">X-tend, accessories</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:363px;">X-tend fabricsling</td>
			<td style="width:392px;">X-Tend</td>
			<td style="width:375px;">X-tend, accessories</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">PET/CT</td>
			<td style="width:392px;">GE</td>
			<td style="width:375px;">Discovery STE</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Portable A/C Unit</td>
			<td style="width:392px;">Soleus Air</td>
			<td style="width:375px;">XL-140, 14000 BTU</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Floor fan</td>
			<td style="width:392px;">Lasko Pedestal Fan</td>
			<td style="width:375px;">2527</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Portable Heater</td>
			<td style="width:392px;">Lasko Ceramic Air</td>
			<td style="width:375px;">5536</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Chair</td>
			<td style="width:392px;">Winco Lifecare Recliner</td>
			<td style="width:375px;">585</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Sublingual Thermometer</td>
			<td style="width:392px;">WelchAllyn</td>
			<td style="width:375px;">SureTemp Plus 690</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Cold vest</td>
			<td style="width:392px;">Polar Products</td>
			<td style="width:375px;">Cool58 #PCVZ</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:363px;">Thermal IR Camera</td>
			<td style="width:392px;">FLUKE</td>
			<td style="width:375px;">TIR-125</td>
		</tr>
	</tbody>
</table>

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.

Acquiring both MRI and PET-CT scans on the same subject, and performing co-registration on all scans enables reliable measurement of quantitative MRI metrics of BAT. Figure 1 shows the unprocessed warm (TN) and cold (CA) PET-CT and MRI scans from one subject. By acquiring both TN and CA PET-CT data, it is possible to clearly distinguish the cold-activated BAT depots by the increased 18F-FDG uptake. After co-registering all four scans (Figure 2 and 3), it is po...

Watch this Scientific Journal Video about Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images at JoVE

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

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 ...

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Research

JoVE Journal

Medicine

12.1K Views.  Vanderbilt University.  Il metodo qui presentata utilizza 18 F-Fluorodeoxyglucose (18 F-FDG) tomografia ad emissione di positroni / tomografia computerizzata (PET-CT) e grasso-acqua separata risonanza magnetica (MRI), ciascuna scansione dopo 2 ore di esposizione a thermoneutral (24 ° C ) e freddo (17 ° C), al fine di mappare tessuto adiposo bruno (BAT) in soggetti umani adulti. 

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

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 ...

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 ...

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 ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

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

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...