2.4. Breathing Rate Testing

Mouse respiratory rates were measured at baseline (prior to irradiation or sham) and every 4 weeks after irradiation or sham until death or euthanasia. A clear isoflurane sedation box connected to the Vetamec Compact II small animal anesthetic machine was utilized for sedation. In groups of 3–5 mice, mice were placed in the isoflurane sedation box with 3 L per minute (LPM) supplemental oxygen (no isoflurane). Isoflurane at a 2% concentration was then initiated, and mice were induced for 210 s. At 210 s, the isoflurane sedation box lid was opened for exactly 30 s to allow sedated mice to be oriented in the same direction and without overlapping body parts. The box lid was closed, and isoflurane 2% and 3 LPM supplemental oxygen were continued. A Nexigo N980P (T/N 920PJH; P/N USBL-625JH) wideview camera was placed on top of the clear isoflurane sedation box. A video recording of each mice group was started exactly 300 s after isoflurane initiation, and mice were recorded for 310 s (total of 610 s of isoflurane 2% sedation by the end of video recording). Between groups, the isoflurane box was allowed to “wash out” with 3 LPM oxygen (without isoflurane) with the lid open for 120 s plus with the lid closed for an additional 120 s. The process was repeated until all mice were sedated and video recorded. As previously described and validated, the open-source software RespiRate-x64 was utilized to track respiratory motion in video recordings and to calculate the respiratory rate for each mouse [23]. Video recordings were analyzed in 30 s segments at specific timepoints. In our validation measurements, the calculated respiratory rate matched the visually measured respiratory rate to within 5 breaths per minute (bpm) in all cases (and typically < 2 bpm) if the software’s respiratory motion waveform standard deviation (SD) was <0.1800. Thus, if the recorded respiratory motion waveform SD was not less than 0.1800, then video recordings were visually reviewed, and respiratory rate was visually measured instead.