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Infrared Thermography for Estimating Supraclavicular Skin Temperature and BAT Activity in Humans: A Systematic Review

Objective Brown adipose tissue (BAT) is a thermogenic tissue with potential as a therapeutic target in the treatment of obesity and related metabolic disorders. The most used technique for quantifying human BAT activity is the measurement of 18F‐fluorodeoxyglucose uptake via a positron emission tomography/computed tomography scan following exposure to cold. However, several studies have indicated the measurement of the supraclavicular skin temperature (SST) by infrared thermography (IRT) to be a less invasive alternative. This work reviews the state of the art of this latter method as a means of determining BAT activity in humans. Methods The data sources for this review were PubMed, Web of Science, and EBSCOhost (SPORTdiscus), and eligible studies were those conducted in humans. Results In most studies in which participants were first cooled, an increase in IRT‐measured SST was noted. However, only 5 of 24 such studies also involved a nuclear technique that confirmed increased activity in BAT, and only 2 took into account the thickness of the fat layer when measuring SST by IRT. Conclusions More work is needed to understand the involvement of tissues other than BAT in determining IRT‐measured SST; at present, IRT cannot determine whether any increase in SST is due to increased BAT activity.


Introduction
Brown adipose tissue (BAT) is a thermogenic tissue with an important role in murine metabolism (1). Cold exposure leads to norepinephrine release by the sympathetic nervous system, which activates brown adipocytes to increase uncoupling protein 1 activity, thus generating heat (1). In 2009, several studies demonstrated the presence of metabolically active BAT in humans via the measurement of 18 F-fluorodeoxyglucose ( 18 F-FDG) uptake as determined by positron emission tomography/ computed tomography (PET/CT) scanning following exposure to cold (2)(3)(4). The high metabolic activity of BAT, and its ability to consume both glucose and fatty acids, suggest that it may have potential as a therapeutic target in the treatment of obesity (5).

OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
on the uptake of a glucose analogue, which is used to measure glucose uptake (quantitative measurement) or fractional FDG uptake in a reliable way (7). Alternative techniques for quantifying human BAT in vivo include PET/CT measurement of the uptake of 18 F-fluoro-6thia-heptadecanoic acid (8), 15 O-O 2 (9), 11 C-acetate (10), or adenosine (11), magnetic resonance imaging (12), and magnetic resonance spectroscopy (MRS) (13), but these are expensive, and some also expose individuals to radiation. A safe, inexpensive, reliable gold standard for human BAT quantification in vivo is thus lacking, as are alternative techniques (6).
Several studies have postulated that the supraclavicular skin temperature (SST) can be used as a surrogate marker of BAT volume (14) and activity (14)(15)(16). SST is commonly measured using iButtons (Maxim, Dallas, Texas) (14,15,17) (small devices attached to the skin) or infrared thermography (IRT) (18), although alternatives exist. IRT assesses superficial temperature using the emitted infrared radiation, and it is widely used in mice to quantify skin temperature at the site of the interscapular BAT (19). Because IRT is a low-cost, noninvasive, easy-to-use method (and more importantly, it poses no hazard to patients), its use in human studies of SST as a marker of BAT activity is becoming more popular. Recent narrative reviews (18,20) that addressed the use of IRT as a means of measuring BAT activity in humans assumed the method to be valid for that purpose, though this has not been established. The present work therefore systematically reviews the state of the art of using IRT for analyzing SST as an indirect marker of BAT activity in humans.

Methods
This systematic review was conducted adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (21) and was registered through the International Prospective Register of Systematic Reviews (PROSPERO registration number: CRD42018098837).

Search strategy
A literature search was conducted across the PubMed, Web of Science, and EBSCOhost (SPORTdiscus) databases from the time of their inception until April 20, 2019. The search terms used were "infrared thermography" OR "thermal imaging" OR "thermal camera" AND "brown adipose tissue" OR "BAT" OR "brown fat." The reference lists of the retrieved studies were reviewed to identify additional studies.

Selection criteria
The inclusion criteria were (1) type of study (randomized controlled trials and longitudinal and cross-sectional observational studies) and (2) the use of IRT to quantify SST. The exclusion criteria were as follows: (1) studies written in languages other than English or Spanish, (2) studies published in non-peer-reviewed journals, (3) reviews, and (4) studies in animal models. No restrictions were placed on participant age or body composition.

Study quality
The literature search, data extraction, and assessment of paper quality were independently performed by two observers, and inconsistencies were resolved by consensus. The PEDro Methodological Quality Scale (22) was used to assess the quality of the studies included. This scale, which consists of 11 items, assesses the external validity, internal validity, and interpretability of studies and can detect potential bias with good reliability (22,23). The score is calculated as the sum of the scores for items 2 to 11. Scores of ≤ 3 were deemed to describe studies of low methodological quality, 4 to 6 those of moderate quality, and ≥ 7 those of high methodological quality.

Comparisons of SST measured by IRT as a marker of human BAT activity
Eighteen of the twenty-four studies interpreted the changes in SST as changes in human BAT activity (16,25,26,29,30,(32)(33)(34)(35)(36)(37)39,40,(42)(43)(44), whereas six interpreted the results only as changes in SST (24,31,38,41,45,46). Ten studies took SST measurements by IRT and also quantified BAT activity via a nuclear medicine technique (n = 8 static 18 F-FDG-PET/CT scan (16,26,31,32,34,42,45,46), n = 1 magnetic resonance imaging (27), n = 1 MRS) (41) (Figure 2). Fourteen studies measured SST by IRT with no such quantification of BAT activity. Among the eight studies that measured SST by IRT and that quantified BAT activity via an 18 F-FDG-PET/CT scan, four aimed to compare the two techniques (16,42,45,46); the remaining four did not examine the agreement between them. Thuzar et al. (42) correlated the changes in SST by IRT following an intervention with the changes in 18 F-FDG uptake by BAT but reported no actual data regarding this finding. In another study by the same group (46), it was reported that SST measured by IRT after cold exposure positively correlated (r = 0.62; P = 0.03) with the BAT maximal standardized uptake of 18 F-FDG as measured by PET/CT. In another study comparing SST measured by IRT with 18 F-FDG-PET/CT, Law et al. (16) reported that SST relative to the sternal temperature correlated positively with BAT 18 F-FDG uptake in eight lean adults. When analyses were performed with absolute SST values, however, no correlation was detected. In other work, Martinez-Tellez et al. (45) reported that SST at the end of the cold exposure correlated inversely with 18 F-FDG-determined BAT volume (r = −0.764; P = 0.006) but did not correlate at all with BAT activity as measured by 18 F-FDG uptake after cold exposure. In addition, when they performed their analysis using values for SST relative to the sternal skin temperature, the former negative correlation they originally saw disappeared, and the latter lack of any significant correlation remained. One study compared SST as measured by IRT against SST as measured by MRS in lean adults (41) and found a positive correlation. Lastly, we observed that almost every study applied different cooling    protocols, which makes the comparison between studies even more difficult (Table 1).

Discussion
The present work reviews all human studies that have assessed IRTmeasured SST as a proxy of BAT activity. Only 5 out of 24 studies directly compared IRT-measured SST against BAT activity determined via a nuclear technique. Most of the studies in which some type of cold stimulus was applied reported an increase in SST although, in some studies, no change in SST was seen when reporting data as absolute values. All the studies found cold to have a great effect on SST in lean adults, independent of sex. Individuals with obesity seemed to have a 1: Eligibility criteria specified; 2: Individuals randomly allocated; 3: Allocation concealed; 4: Groups similar at baseline regarding most important prognostic indicators; 5: "Blinded" participants; 6: "Blinded" therapist; 7: "Blinded" assessors; 8: Measures of at least one key outcome obtained from more than 85% of participants; 9: Data analyzed by "intention to treat"; 10: Statistical comparisons between groups; 11: Point measures and measures of variation. a Item number 1 not used to calculate PEDro score because it influenced external validity but not internal or statistical validity of trial. N, criteria not satisfied; Y, criteria satisfied.

Figure 2
Type of cooling protocols (T.cooling) used in the studies included in this review. Supraclavicular skin temperature (SST) columns show the effect of cold exposure on SST. Red arrows mean an increase of SST after the cold exposure, whereas blue arrows mean a decrease of SST. Equal symbols ("=") mean that SST did not change upon cold exposure. Gray boxes mean those studies that compared SST measured by IRT with brown adipose tissue (BAT) measured by a nuclear medicine technique. Yellow boxes mean those studies that measured BAT with a nuclear medicine technique but that did not compare with SST measured by IRT.
White boxes mean those studies that used only SST measured by IRT as a proxy of BAT activity. CB, cooling blankets; CV, cooling vest; IRT, infrared thermography; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; 18 F-FDG-PET/CT, 18 F-fluorodeoxyglucose, positron emission tomography/computed tomography; ND, not described by the study.

Review
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY different response. Most studies assumed that the increase, or lack of change, in SST upon cold exposure involved BAT activation.
In 1987, Kellman et al. (47) performed a thorough study in which they analyzed the tissue composition of the supraclavicular fossa in humans and found it to be composed of the subclavian vessels, the brachial plexus, the omohyoid and scalene muscles, fat, and (to a lesser extent) lymph nodes and the posterior lung apex. This anatomical area is known to contain adipose tissue with a morphology and gene expression pattern indicative of the presence of brown or beige fat (48,49). However, the amount of active BAT present in humans is still questioned (50,51). Blondin et al. (52) observed that the deep and cervical muscles showed a greater 18 F-FDG uptake than other skeletal muscles after cold exposure, with the Longus colli and sternocleidomastoid muscles returning the highest values in the upper body and after applying a fixed cooling protocol. In addition, Hanssen et al. (53) found 18 F-FDG uptake by the scalene muscle to be higher than that seen in other skeletal muscles both before and after a 10-day cold-acclimation intervention and after applying an individualized cooling protocol. Using 15 O-O 2 -PET/CT scanning, U Din et al. (9) showed the deep and cervical muscles to be the main contributors toward the increase in energy expenditure upon cold exposure in humans. They also showed that the supraclavicular and cervical BAT contributed toward this increase in energy expenditure by only ~2%.
These results indicate that skeletal muscles contribute toward human cold-induced thermogenesis more than BAT does (54)(55)(56). Therefore, when skeletal muscles located in the supraclavicular fossa shiver and generate heat during cold exposure, they might be related to the increase in SST, although further studies are needed to confirm this hypothesis. Even after applying different cooling protocols, both Blondin et al. (52) and Hanssen et al. (53) observed that 18 F-FDG uptakes by skeletal muscles were different. Whether these different cooling protocols activate muscle shivering thermogenesis in different manners, and therefore could be contributing differently to SST, remains unknown.
The supraclavicular subcutaneous fat layer acts as an insulation tissue that, depending on its thickness, may influence the value of SST as measured by IRT (31). Indeed, IRT-measured SST following cold exposure may represent the sum of different tissues' features, including the thermogenic activity of skeletal muscles and of BAT, the temperature of vessels associated with the extent of blood flow, and insulation from the supraclavicular fat layer. Of note is that IRT is able to measure only skin temperature, not blood flow or heat production by the tissue (57). At best, IRT measures a reflection of the heat leakage, which is only a part of the heat transported from BAT. Therefore, from a theoretical point of view, probably at best, with IRT one can discriminate between no BAT activity and BAT activity. Thus, it remains unknown whether a direct link between changes in SST and BAT activation can be accurately detected. Because other thermogenic tissues are located in the same area, their possible contribution toward SST cannot be ignored.
Sarasniemi et al. (41) compared IRT-measured SST with the temperature of the supraclavicular depot as measured by MRS and observed a positive and significant correlation in lean adults but a negative correlation in adults with obesity. Moreover, Thuzar et al. performed two independent studies (42,46) in which they compared IRT-measured SST against BAT 18 F-FDG uptake as measured by a PET/CT scan on the same day, reporting a positive and significant association between them. In contrast, Law et al. (16) made the same kind of measurements on separate days and using different cold exposure times. Comparing the latter two studies is, therefore, not easy. It seems that IRT-measured SST might, however, be related to BAT 18 F-FDG uptake mostly in lean adults, although this positive association was seen only when SST results were presented relative to the sternal skin temperature. When absolute values were used, no association was seen at all. The present authors performed a similar study in which correlations between IRTmeasured SST (45) (absolute and relative to the sternal skin temperature) and BAT 18 F-FDG uptake were sought; an inverse correlation was seen. Sarasniemi et al. (41) reported the same thing. Recently, Leitner et al. (58) observed that 80 minutes of cold exposure (using a cooling vest) induced similar levels of BAT 18 F-FDG uptake as did 120 minutes of cold exposure. Whether shorter times induce similar BAT 18 F-FDG uptakes is unknown; the one study that did use a 10-minute cold exposure did not employ a nuclear technique to measure BAT activation. Furthermore, the cooling technique used (applying cold to the hands) could have activated BAT via a side pathway rather than through the classical cold pathway (59). More studies are needed to determine which protocol is the most appropriate for activating human BAT (5).

General limitations of studies discussed
Measurement of human skin temperature by IRT. Moreira et al. (57) published a consensus statement on the measurement of human skin temperature by thermography imaging in sports and exercise settings but did not focus on its use as a potential surrogate for BAT activity. They created a checklist of recommendations to be taken into account before starting any IRT measurement of skin temperature. The studies discussed in the present review did not follow these methodological recommendations, which may have affected the SST results obtained, especially in studies involving cold stimuli. For instance, almost no study reported how long the skin was exposed to the air temperature totally uncovered before performing the measurements to allow stabilization in skin temperature. Moreira et al. (57) also postulated relative humidity to be an important parameter that should be taken into account in line with thermography principles to properly correct the measures of temperature. Again, none of the studies discussed here recorded and corrected for this (Table 1). However, these recommendations themselves need to be revised and adapted for studies in which SST is measured to provide joined guidelines on both thermography and SST related to BAT.
Lack of gold standard. Currently, the method most used in the field of BAT quantification is 18 F-FDG-PET/CT scanning (60). It is important to understand that this measures the amount of 18 F-FDG (a glucose analogue) that a tissue consumes. It is well known, however, that BAT also consumes fatty acids; in fact, they are this tissue's preferred substrate for thermogenesis (61). The lack of a valid gold standard for measuring BAT activity therefore hampers the use of any PET/CT scan-based data as a means of validating IRT-based measurements. Radiolabeled fatty acids would more closely mimic the actual metabolic activity of BAT, but to date, this idea has been poorly developed. Thus, based on current knowledge, IRT-measured SST should be understood as complementary data that may represent the thermogenic activity of other tissues in addition to BAT. An accurate method for specifically measuring BAT activity in vivo in humans is, therefore, still needed.
Cooling protocols. There are almost as many cooling protocols as there are studies, which hinders the making of comparisons. The use of different cooling protocols could induce different BAT activities, and the SST response may therefore differ. Certainly, the lack of any changes in IRT-measured SST when employing 5-minute cooling times may reflect that such short cold stimuli are insufficient to induce BAT activation (it should be noted that the majority of studies did not report the temperature of other zones as controls). However, extremely long cooling protocols might activate BAT via pain rather than cold pathways (59). Basically, different cooling protocols could induce different responses, and the contribution of the tissues in nonshivering and shivering thermogenesis could be dissimilar. It is important that future studies use longer cooling protocols and report the temperatures of different areas.

IRT-measured SST in different human populations
The aforementioned limitations in IRT use for assessing SST may manifest themselves differently in different human cohorts, and certainly the thickness of the subcutaneous fat layer might lead to different results being recorded in individuals with obesity and lean individuals. The relative value of IRT-based measurements, and their validation in pediatric populations, deserve further research.

Conclusion
IRT-measured SST is still to be unequivocally validated as a tool for indirectly quantifying human BAT activity. It remains to be seen whether there is a direct link between changes in IRT-measured SST and BAT activation. Because other thermogenic tissues are located in the same area, their contribution to SST cannot be ignored. Future human studies must take into account the thickness of the subcutaneous fat layer if SST in individuals with different body compositions is to be compared. Finally, further studies are needed to identify the biological factors that determine the temperature of the supraclavicular area. O