The overall goal of this diffuse optical spectroscopy technique is to develop a quantitative biomarker for describing acute radiation induced erythema. This method can be used in the radiation therapy field as a predictive biomarker for identifying patients at risk of severe radiation skin toxicity. So the main advantage of this technique is that it provides an objective and systematic metric to quantify radiation skin toxicities.
I will be demonstrating the diffuse optical spectroscopy technique, and Elina, a researcher in the Loo Lab, will be handling the mice. Turn on the electronics and allow the system to boot up. Then turn off all the fluorescent lights in the room and position incandescent lights at a distance from the measuring device to provide some working illumination.
Next, set the instrument for taking measurements from mouse skin. Set the signal parameters as follows. Set the collection time to 25 milliseconds, set the signal averages to 25, and set the boxcar filter width to one.
These parameters offer a reasonable balance between acquisition time and signal-to-noise. Next, using the custom programmed acquisition software, automatically acquire a background reading with the LED off. Then, acquire a reading with diffuse reflectance at two source detector separation distances.
Take one measurement at 260 microns, and the second at 520 microns. The acquisition time in total should be about two seconds. After anesthetizing the mouse, move it to the sterilized DOS probing area.
Position it on its side and secure its snout to a nose cone delivering 2%isoflurane gas to maintain the anesthesia. Now sterilize the probe with 70%ethanol, but do not attempt to sterilize the skin. Hold the sterilized probe gently to the flank skin.
Don't press too hard, as the local vasculature must not be dispersed by the probe's pressure. While holding the probe, acquire reflectance data over the two centimeter square area to be irradiated. Collect data in a five dot formation like on a die.
Keep this pattern and probing pressure consistent in all subsequent measurements. After making the measurements, place the mouse in a recovery cage. While the mouse recovers, repeat the procedure on the next mouse.
This procedure is tailored to the available irradiator. Tweak it as needed to irradiate a small section of skin. After anesthetizing a mouse, gently pinch some skin on its flank and tape the stretched skin to form a flap.
Then place the mouse onto a Plexiglass stage and cover its body with a customized lead jig. Use a lead box that is accessible on two sides and has a window for the skin to be irradiated. Then pull the flap of skin through the jig window, and tape the flap to the stage.
If the mouse isn't immobilized by the jig, give it an anesthetic injection. Then place the stage with the jig and the mouse into the irradiator. Calculate the required irradiation dose.
For example, a 160 peak kilovoltage x-ray source positioned 11 centimeters away would adequately irradiate the skin running at 6.3 milliamps for 2.5 minutes. Then, deliver the calculated dose. After dosing the animal, return it to a recovery cage.
Once recovered from the anesthesia, return to the mouse to its normal shared housing cage. Mice were irradiated and measured as described. Prior to irradiation, a baseline spectra was taken with a 260 micron source separation in an athymic mouse model of skin.
The thick green line shows a mathematical fit of the thin blue line. Compared to measurements made six days after a 40 gray irradiation, differences in the spectral shape between 550 and 600 nanometers were observed, likely due to an increase in oxygenated hemoglobin. A small rise in the absolute reflectance is also observed, and may be correlated to an increase in tissue scattering power.
The fitted data returns quantitative optical biomarkers that can tracked as a function of time post-irradiation. For example, tissue oxygen saturation measurements progressively increased following irradiation. This quantitative data correlated with a visual grade of the skin toxicity, which also progressively increased post-irradiation.
After watching this video, you should have a good understanding of how to employ diffuse optical spectroscopy for quantitative scoring of radiation skin toxicities. Once mastered, this technique can be performed in two or three minutes, if it is performed properly. While attempting this procedure, it's important to press the DOS probe down gently to avoid dispersing the vasculature.
This technique can be used in the field of radiation therapy as a way of linking physiological parameters in describing normal tissue response to radiation.
