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.
