Name: infrared thermography for the detection of changes in brown adipose tissue activity
Uploaded: 2022-09-28T14:30:00
Description: Watch this Scientific Journal Video about Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity at JoVE.com

This study was funded by the Croatian Science Foundation research grant (IP-2018-01- 7416).

All experimental procedures on laboratory animals were approved by the National Ethical Committee and the Ministry of Agriculture (EP 185/2018). The experiments were conducted in accordance with the Ethical Codex of The Croatian Society for Laboratory Animal Science and ARRIVE guidelines. All procedures performed in studies involving human participants were in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Zagreb, School of Medicine (UP/I-322-01/18-01/56). In this st...

<ol>
	<li>van Marken Lichtenbelt, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> New England Journal of Medicine. 360 (15), 1500-1508 (2009).</li><li>Morrison, S. F., Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+neural+pathways+for+thermoregulation.">Central neural pathways for thermoregulation.</a> Frontiers in Bioscience. 16 (1), 74-104 (2011).</li><li>Contreras, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+and+brown+fat.">The brain and brown fat.</a> Annals of Medicine. 47 (2), 150-168 (2015).</li><li>Chondronikola, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+improves+whole-body+glucose+homeostasis+and+insulin+sensitivity+in+humans.">Brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans.</a> Diabetes. 63 (12), 4089-4099 (2014).</li><li>Ouellet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outdoor+temperature,+age,+sex,+body+mass+index,+and+diabetic+status+determine+the+prevalence,+mass,+and+glucose-uptake+activity+of+18F-FDG-detected+BAT+in+humans.">Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans.</a> Journal of Clinical Endocrinology &amp; Metabolism. 96 (1), 192-199 (2011).</li><li>Pfannenberg, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+age+on+the+relationships+of+brown+adipose+tissue+with+sex+and+adiposity+in+humans.">Impact of age on the relationships of brown adipose tissue with sex and adiposity in humans.</a> Diabetes. 59 (7), 1789-1793 (2010).</li><li>Leitner, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+human+brown+adipose+tissue+in+lean+and+obese+young+men.">Mapping of human brown adipose tissue in lean and obese young men.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (32), 8649-8654 (2017).</li><li>Vosselman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+activity+after+a+high-calorie+meal+in+humans.">Brown adipose tissue activity after a high-calorie meal in humans.</a> American Journal of Clinical Nutrition. 98 (1), 57-64 (2013).</li><li>Hibi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+is+involved+in+diet-induced+thermogenesis+and+whole-body+fat+utilization+in+healthy+humans.">Brown adipose tissue is involved in diet-induced thermogenesis and whole-body fat utilization in healthy humans.</a> International Journal of Obesity. 40 (11), 1655-1661 (2016).</li><li>Fenzl, A., Kiefer, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+and+thermogenesis.">Brown adipose tissue and thermogenesis.</a> Hormone Molecular Biology and Clinical Investigation. 19 (1), 25-37 (2014).</li><li>Koksharova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+brown+adipose+tissue+content+in+supraclavicular+fat+depots+and+insulin+sensitivity+in+patients+with+type+2+diabetes+mellitus+and+prediabetes.">The relationship between brown adipose tissue content in supraclavicular fat depots and insulin sensitivity in patients with type 2 diabetes mellitus and prediabetes.</a> Diabetes Technology &amp; Therapeutics. 19 (2), 96-102 (2017).</li><li>Habek, N., Kordi&#263;, M., Jurenec, F., Dugand&#382;i&#263;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+thermography,+a+new+method+for+detection+brown+adipose+tissue+activity+after+a+meal+in+humans.">Infrared thermography, a new method for detection brown adipose tissue activity after a meal in humans.</a> Infrared Physics &amp; Technology. 89, 271-276 (2018).</li><li>Lee, P., Ho, K. K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+fat+in+a+cool+man:+Infrared+thermography+and+brown+adipose+tissue.">Hot fat in a cool man: Infrared thermography and brown adipose tissue.</a> Diabetes, Obesity and Metabolism. 13 (1), 92-93 (2011).</li><li>Ang, Q. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+infrared+thermography+for+quantification+of+brown+adipose+tissue+activation+in+healthy+adults+(TACTICAL):+A+randomized+trial.">A new method of infrared thermography for quantification of brown adipose tissue activation in healthy adults (TACTICAL): A randomized trial.</a> Journal of Physiological Sciences. 67 (3), 395-406 (2017).</li><li>Jang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+thermography+in+the+detection+of+brown+adipose+tissue+in+humans.">Infrared thermography in the detection of brown adipose tissue in humans.</a> Physiological Reports. 2 (11), 12167 (2014).</li><li>Dodd, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin+and+insulin+act+on+POMC+neurons+to+promote+the+browning+of+white+fat.">Leptin and insulin act on POMC neurons to promote the browning of white fat.</a> Cell. 160 (1-2), 88-104 (2015).</li><li>Habek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+brown+adipose+tissue+in+diet-induced+thermogenesis+is+GC-C+dependent.">Activation of brown adipose tissue in diet-induced thermogenesis is GC-C dependent.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 472 (3), 405-417 (2020).</li><li>Burke, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+difference+in+physical+activity,+energy+expenditure+and+obesity+driven+by+a+subpopulation+of+hypothalamic+POMC+neurons.">Sex difference in physical activity, energy expenditure and obesity driven by a subpopulation of hypothalamic POMC neurons.</a> Molecular Metabolism. 5 (3), 245-252 (2016).</li><li>Glumer, C., Jorgensen, T., Borch-Johnsen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalences+of+diabetes+and+impaired+glucose+regulation+in+a+Danish+population:+The+Inter99+study.">Prevalences of diabetes and impaired glucose regulation in a Danish population: The Inter99 study.</a> Diabetes Care. 26 (8), 2335-2340 (2003).</li><li>Sicree, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+height+explain+gender+differences+in+the+response+to+the+oral+glucose+tolerance+test-the+AusDiab+study.">Differences in height explain gender differences in the response to the oral glucose tolerance test-the AusDiab study.</a> Diabetic Medicine. 25 (3), 296-302 (2008).</li><li>van Genugten, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sex+and+hormone+replacement+therapy+use+on+the+prevalence+of+isolated+impaired+fasting+glucose+and+isolated+impaired+glucose+tolerance+in+subjects+with+a+family+history+of+type+2+diabetes.">Effects of sex and hormone replacement therapy use on the prevalence of isolated impaired fasting glucose and isolated impaired glucose tolerance in subjects with a family history of type 2 diabetes.</a> Diabetes. 55 (12), 3529-3535 (2006).</li><li>Williams, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+the+prevalence+of+impaired+fasting+glycaemia+and+impaired+glucose+tolerance+in+Mauritius.+Does+sex+matter.">Gender differences in the prevalence of impaired fasting glycaemia and impaired glucose tolerance in Mauritius. Does sex matter.</a> Diabetic Medicine. 20 (11), 915-920 (2003).</li><li>Stanford, K. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+regulates+glucose+homeostasis+and+insulin+sensitivity.">Brown adipose tissue regulates glucose homeostasis and insulin sensitivity.</a> Journal of Clinical Investigation. 123 (1), 215-223 (2013).</li><li>Brasil, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+on+the+role+of+infrared+thermography+in+the+brown+adipose+tissue+assessment.">A systematic review on the role of infrared thermography in the brown adipose tissue assessment.</a> Reviews in Endocrine and Metabolic Disorders. 21 (1), 37-44 (2020).</li><li>Byers, S. L., Wiles, M. V., Dunn, S. L., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+estrous+cycle+identification+tool+and+images.">Mouse estrous cycle identification tool and images.</a> PLoS One. 7 (4), 35538 (2012).</li><li>Crane, J. D., Mottillo, E. P., Farncombe, T. H., Morrison, K. M., Steinberg, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+infrared+imaging+technique+that+specifically+detects+UCP1-mediated+thermogenesis+in+vivo.">A standardized infrared imaging technique that specifically detects UCP1-mediated thermogenesis in vivo.</a> Molecular Metabolism. 3 (4), 490-494 (2014).</li><li>Hartwig, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+imaging+for+the+detection+of+brown+adipose+tissue+activation+in+women:+A+pilot+study+using+NIRS+and+infrared+thermography.">Multimodal imaging for the detection of brown adipose tissue activation in women: A pilot study using NIRS and infrared thermography.</a> Journal of Healthcare Engineering. 2017, 5986452 (2017).</li><li>James, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+infrared+thermography+in+the+measurement+and+characterization+of+brown+adipose+tissue+activation.">The use of infrared thermography in the measurement and characterization of brown adipose tissue activation.</a> Temperature. 5 (2), 147-161 (2018).</li><li>Folgueira, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+dopamine+signaling+regulates+brown+fat+thermogenesis.">Hypothalamic dopamine signaling regulates brown fat thermogenesis.</a> Nature Metabolism. 1 (8), 811-829 (2019).</li><li>Ratko, M., Habek, N., Kordi&#263;, M., Dugand&#382;i&#263;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+infrared+technology+as+a+novel+approach+for+studies+with+female+laboratory+animals.">The use of infrared technology as a novel approach for studies with female laboratory animals.</a> Croatian Medical Journal. 61 (4), 346-353 (2020).</li></ol>

Recent studies present growing evidence regarding the physiological regulation and importance of BAT activity in adult humans and animals in the development of obesity and diabetes mellitus. Furthermore, possible BAT activation by exogenous activators is becoming a target for pharmaceutical companies. To be able to estimate the physiological regulation and pathophysiological importance of BAT in very burdensome diseases, as well as discover a potential therapeutic approach, infrared thermography is becoming the method of...

Brown adipose tissue (BAT), in contrast to white adipose tissue (WAT), does not store but rather spends energy. Upon sympathetic stimulation, BAT utilizes fatty acids and glucose and produces heat by the activation of uncoupling protein 1 (UCP1). The function of UCP1 is to use an H+ gradient between two mitochondrial membranes to produce heat instead of ATP. The function of BAT is to increase heat production under cold conditions, which leads to an increase in energy expenditure1. After cold exposure, sensory inputs from the skin inhibit warm-sensitive neurons in the median preoptic (MnPO) nucleus of the hypothalamic preoptic area (POA), which diminishes the inhibitory effect of POA neurons on the rostral raphe pallidus (rRPa). The activation of rRPa neurons increases sympathetic activity, which is followed by an increase in BAT activity2,3. Cold-induced BAT activation improves insulin sensitivity in humans4, and this activity is decreased in humans with increased body mass index (BMI) and age1,5,6,7.
Apart from its role in cold-induced thermogenesis, after a meal, glucose uptake in the BAT increases in the lean male population, contributing to diet-induced thermogenesis (DIT), which is higher in BAT-positive male subjects8,9. The state-of-the-art technique used for measuring BAT activity is positron emission tomography computed tomography, known as PET-CT. This method determines BAT activity by measuring the accumulation of the radiotracer fluorodeoxyglucose (18F-FDG). However, PET-CT fails as the method of choice for detecting the activation of BAT after a meal. One of the reasons is that, after a meal, 18F-FDG competes with postprandial hyperglycemia for the same glucose transporter, which makes it unsuitable for determining BAT activation after a meal, especially when comparing BAT activity in healthy and diabetic participants with possible differences in blood glucose concentrations. Furthermore, BAT uses fatty acids as a source of energy for heat production which is not visible with PET-CT. 18F-FDG accumulation in BAT after a meal is barely visible10 and is, therefore, interpreted as a negative result in most cases. Unsurprisingly, recently, it was suggested that the activation of BAT is more pronounced in the human population than we had previously thought; therefore, a new approach to detect BAT activity and its involvement in metabolic disorders is necessary7. An attempt to solve this problem is to measure the volume of BAT with magnetic resonance imaging (MRI) in prediabetic patients and patients with diabetes mellitus type 2 (T2DM) with insulin resistance11. However, BAT volume measured by MRI is not a sufficient indicator for estimating the everyday function and usage of glucose and fatty acids by BAT. Therefore, to estimate real differences in BAT activity in healthy versus T2DM patients, a new approach is needed that offers a possibility to find out the pathological mechanism of BAT malfunction in T2DM patients.
To determine the activation of BAT, we performed measurements of BAT heat production before and after a meal using infrared (IR) thermography (Figure 1)12,13. Establishing IR thermography as a method of choice for measuring BAT activity after a meal in healthy and obese individuals or patients with diabetes mellitus will have a huge impact on the field. To this day, IR thermography is used for the determination of cold-induced activation of BAT13,14,15. In recent human history, cold-induced BAT activity is not very pronounced anymore (due to proper heating of habitats, proper clothing), while BAT activation after a meal occurs every day. Furthermore, the physiological regulation of these two BAT functions via the hypothalamus is completely different. After a meal, the activation of proopiomelanocortin (POMC)-expressing neurons in the hypothalamic arcuate nucleus (Arc) leads to an increase in sympathetic nerve activity via rRPa16. Cold-induced activation of BAT measured by IR thermography or PET-CT is improper when used as a measure for everyday BAT activity. Increased BAT activity after a meal is followed by glucose utilization, which is ultimately important for maintaining glucose homeostasis, insulin sensitivity, and the daily regulation of glucose concentration. Postprandial BAT activation leads to an increase in postprandial glucose consumption, followed by an increase in heat production and body temperature (DIT). This was shown to be gender, age, and BMI dependent12. Similar gender differences in BAT activation after a meal are observed in male and female laboratory mice17. These findings correspond to recently discovered gender differences in the regulation of BAT by Burke et al., who showed that the hypothalamic regulation of BAT browning via a subpopulation of POMC neurons differs in male and female mice18. The postprandial activation of BAT is smaller in women, older populations, and obese people. The lack of BAT activation after a meal (decreased glucose utilization) could lead to a higher prevalence of impaired glucose tolerance in women19,20,21,22. Unfortunately, the majority of studies on BAT activation were done only on men. By activating BAT after a meal, glucose uptake increases in the lean male population. It is not surprising that, after BAT activation, DIT is higher in BAT-positive male subjects8,9. Furthermore, BAT transplantation in male mice improves glucose tolerance, increases insulin sensitivity, and decreases body weight and fat mass23.
PET-CT fails as a method of choice for measuring BAT activity, especially after a meal. Therefore, a non-invasive and more sensitive method was developed. IR thermography enables the estimation of BAT activity in different laboratory animals (knock-out mice), as well as human participants, regardless of gender, age, or the effects of different pathological conditions on BAT activity. An additional benefit of this method is the simplicity for participants and laboratory animals, which allows us to estimate the potential benefits of BAT booster therapy. The recent studies using IR thermography for determining the physiological behavior of BAT after cold exposure or a meal are described in the recent publication of Brasil et al.24.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% cresyl violet acetate&#160;</td>
			<td>Commonly used chemical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Device for measuring air temperature and humidity</td>
			<td>Kesterl</td>
			<td>Kestrel 4200</td>
			<td>Certificat of conformity</td>
		</tr>
		<tr>
			<td>External data storage</td>
			<td>Hard Drive with at least 1 TB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscopic slides</td>
			<td>Commonly used</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small cotton tip swab&#160;</td>
			<td>Urethral swabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for analysis</td>
			<td>FLIR Systems, Wilsonville, OR, USA</td>
			<td>FLIR Tools</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for meassurements</td>
			<td>FLIR Systems, Wilsonville, OR, USA</td>
			<td>ResearchIR software</td>
			<td>FLIR ResearchIR Max, version 4.40.12.38 (64-bit)</td>
		</tr>
		<tr>
			<td>Thermac Camera</td>
			<td>FLIR Systems, Wilsonville, OR, USA</td>
			<td>FLIR T-1020</td>
			<td></td>
		</tr>
	</tbody>
</table>

infrared thermography for the detection of changes in brown adipose tissue activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

The easiest way to determine BAT activity is to subtract the maximal skin temperature above the BAT before and after a meal in human subjects. A better way to calculate BAT activity is to select two regions of interest: the skin area above the BAT, which is located in the supraclavicular area, and the interclavicular area of the skin where no BAT tissue is found in humans, designated as a reference area (according to PET-CT; Figure 1). BAT activity is then easily determined by the subtractio...

Watch this Scientific Journal Video about Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity at JoVE.com

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...

Infrared thermography enables the estimation of brown adipose tissue activity in laboratory animals and human participants, regardless of gender, age, or the effects of different pathological conditions. PET-CT fails as a method of choice for measuring brown adipose tissue activity, especially after a meal. Therefore, a noninvasive and more sensitive infrared thermography was developed.Using infrared thermography, we can determine that the pathophysiological changes in brown adipose tissue activity or bath activity in people with obesity and diabetes mellitus, and determine drug that can rejuvenate bad activity in these patients. This method could be used to determine the physiological effects of hormones or synthesize therapeutic substances on bad activity in humans and laboratory animals. Ask the participants if they are well-rested and fasted overnight.They will rest for at least 30 minutes before measurements to avoid possible brown adipose tissue activation. Perform the experiments in the morning hours. 15 minutes before the measurement, ask the participants to remove their upper clothing.While the participants are resting, mount the thermal camera on the tripod and position it one meter away from the spot where the participant will be seated. Connect the thermal camera to a computer and software as instructed by the manufacturer. Determine the reflected temperature of the room by recording wrinkled aluminum foil at a focal distance of one meter.To do so, in the camera software, input the distance of zero meters and emissivity of one. Then, enter values of atmospheric pressure and relative humidity. To determine reflected temperature, choose the square shape and select an ROI on the aluminum foil.Then, choose the first icon from the left in the main camera window and read the mean temperature, which is necessary for further analysis. Just before starting the measurements, determine the room air temperature and air humidity. In the software, above the main camera window, choose the third icon from the left.In the pop-up menu, choose Edit record settings, which opens a new window. In Record mode, select Record to disk and set the record for this duration at the desired time. In the Record Options of the same window, limit record rate to five hertz and choose the location where the recordings will be saved.Close the existing window, open Edit in the main menu, and select Preferences. Enter five in the target frame rate. Select Hotkey/Remote Start can stop record.And from the dropdown menu, select In Start/Stop Mode. At the time of measurement, ensure only the participant and the experimenter are present. Avoid air movement and ensure the participant is away from cold draft, sunlight, or any source of heat like light bulbs.Locate the supraclavicular area of the neck above the collarbone, where brown adipose tissue is located. Position the participant such that this area is at one meter focal distance. Record a short movie for 10 to 15 seconds at a frame rate of 5 FPS by pressing the F5 key.Ensure all the participants eat the same meal. After the meal, make a new recording at 30 minutes and then one, two, and three hours by pressing F5 using the same setting values. The day before the experiment, collect the animal's vaginal swab and spread the cells onto a glass slide.Allow it to air dry. Stain the cells with 500 microliters of 0.1%cresyl violet acetate for one minute, and then rinse them thrice with water. View the cells under a light microscope with 100 times magnification and brightfield illumination.Determine the phase of the estrous cycle based on the number of leukocytes and nucleated and quantified epithelial cells observed in the smear. On the morning of the experiment, prepare the thermal camera and recording settings. Carefully place the animal in a clean cage and place the cage under the thermal camera at a focal distance of one meter.Record a movie by pressing F5.Weigh the food pellet before giving it to each animal to calculate the food intake. Allow the animal to eat for 30 minutes in its cage and weigh the food pellet again after the meal. Repeat IR measurements 30 minutes, one hour, and two hours after starting a meal.After the experiment, test the phase of the estrous cycle in female animals, as shown earlier. For each movie, enter the variables into the camera software such as skin emissivity, reflected room temperature, air temperature, relative humidity, and distance to the object. Select the suitable frame from the movie by moving the playhead at the bottom of the screen or by pressing the Pause button.Select the region of interest by choosing the desired shape of the area on the left side of the main window. Choose the shape that best corresponds to the area of skin between the scapulas. Upon choosing the region of interest, the minimal, maximal, and average temperatures will be displayed on the right.In the image, the red triangle represents the point of maximum recorded temperature, and the blue triangle represents the minimum recorded temperature. Repeat this step for several frames to be sure that the measured temperature is stable during a few seconds of the recording. Subtract the maximal temperatures of the skin above the brown adipose tissue before a meal from the maximal temperatures after a meal to determine the increase in postprandial activity.Brown adipose tissue activity was determined by analyzing two skin areas:one above the brown adipose tissue and the second in the inter clavicular area as a reference point. The brown adipose tissue activity was calculated as 1.8 degrees Celsius from the differences between maximal temperatures in both skin areas. But the activity cannot be presented by maximal temperatures of the skin above the brown adipose tissue since there is no decrease in skin temperature even after three hours of the meal.In mice, the changes in activity after a meal were measured in diestrus females by measuring the average temperature of the skin above brown adipose tissue. Significant changes in activity were determined 30 minutes after a meal only when the maximal temperature was measured. Proper selection of matching control subjects is the hardest part of clinical studies since healthy controls and participants with pathological conditions should be as similar as possible and differ only in the disease investigated.In addition to this protocol, blood glucose concentration and body temperature could be measured, which allows the correlation analysis between bad activity and glucose utilization or increase in body temperature. This sensitive and non-invasive technique opens numerous possibilities in determining the physiological and pathophysiological changes of brown adipose tissue activity in other experimental settings and studies.

infrared-thermography-for-detection-changes-brown-adipose-tissue

Research

JoVE Journal

Medicine

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

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 ...

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

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

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

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

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. ...

Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several studies have focused their attention on mesenchymal stem cells derived from adipose tissue. Adipose-derived stem cells (ADSCs) exhibit morphological characteristics and properties similar to other mesenchymal cells, and are able to differentiate into several cellular lines. Moreover, these cells are also widely available in the subcutaneous tissue, representing 10 - 30% of the normal body weight, with a concentration of 5,000 cells per gram of tissue.
In the presented technique, the first step involves harvesting ADSCs from the abdomen and a process of microfracture and purification; next, the surgical procedure is performed entirely arthroscopically, with less soft tissue dissection, better joint visualization, and a faster recovery compared with standard open procedures. Arthroscopy is characterized by a first phase in which the lesion is identified, isolated, and prepared with microperforations; the second step, performed dry, involves injection of adipose tissue at the level of the lesion.
Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery with no reported complications. Functional scores at the latest follow-up are encouraging and confirm that the technique provides reliable pain relief and improvements in patients with osteochondral lesion of the talus.

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells.

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several ...

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 ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...