Use of Calm Breathing for Monitoring Responses to Hypoxia/Hypercapnia in Young and Middle‐Aged Mice in the Light and Dark Cycle
Authors: Eassa Brianna E , Receno Candace N , Cunningham Caitlin M , DeRuisseau Lara R 
Published: June 2021

Unrestrained barometric plethysmography (UBP) is a common technique used to quantify respiratory responses in mice; often air breathing values are compared to responses following respiratory stressors. Previous studies have reported a familiarization period (30–60 min), after which baseline measurements are quantified in mice, but these studies do not supply behavioral standards for what features determine a baseline. We had the overarching goal of quantifying a stable breathing pattern with air that could be compared to consistent breathing that is observed when mice are exposed to hypoxia and hypercapnia. The term calm breathing is used to define a segment of air breathing without sniffing and grooming, where >90% of breaths are accepted by the software. In order to compare a traditional method of baseline, we analyzed air breathing after habituation of 1 hour using a ten min average and compared it to a 30 s calm breathing segment collected during hours 1–3. Young (~4 months; n=7) and middle‐aged (~13 months; n=11) mice were tested during the light and dark cycle for breathing frequency (F; breaths/min), tidal volume (VT; ml/breath) and minute ventilation (VE; ml/min). Data were analyzed during exposure to air (20.93% O2, balanced N2), hypoxia (10 min; 10% O2, balanced N2), hypercapnia (10 min; 5% CO2, balanced air) and hypoxic hypercapnia (10 min; 10% O2, 5% CO2, balanced N2). Data are presented as mean±SD; 10 min air breathing vs. 30 s air breathing. The air breathing ten min segment was higher for F (light: 329±59, dark: 333±68 vs. light: 156±20, dark: 163±51; p=0.000) and VE (light: 114.0±37.1, dark: 138.5±50.4 vs. light: 65.7±16.4, dark: 89.7±44.2; p=0.004), but lower for VT (light: 0.38±0.14, dark: 0.44±0.13 vs. light: 0.41±0.08, dark: 0.57±0.31; p=0.004) vs. 30 s calm breathing. There was a main effect of circadian cycle for VE (p=0.020) and VT (p=0.021) as values during the dark cycle were higher than the light cycle. Percent change from the ten min air segment and the 30 s of calm breathing to the gas exposures were different for F (light: −17±21%, dark: −11±22% vs. light: 67±25%, dark: 84±37%; p=0.000), VT (light: 25±15%, dark: 38±39% vs. light: 10±13%, dark: 16±38%; p=0.006) and VE (light: 6±21%, dark: 24±34% vs. light: 90±37%, dark: 103±73%; p=0.000). Similar results were observed during hypercapnia and hypoxic hypercapnia for F, VT, and VE, although there was a significant main effect of circadian cycle only on frequency during hypoxia (p=0.048). Overall, the % change response to gases for the ten min air segment is lower for F and VE vs. calm breathing, but higher for VT. This response is due to the higher F and VE at baseline with the 10 min air segment. When air VE is normalized to VO2 (mL/min; 10 min: 171.69±42.0 ml/min, 30 s: 183.68±82.31 ml/min), no significant differences are observed (p=0.713) which suggests overall ventilation is not changing between the air segments, but the pattern of breathing is different. Therefore, documenting mouse behavior throughout UBP is essential. We recommend a shorter, calmer segment of air breathing for baseline.Support or Funding Information1 R15 HD076379‐01A1, 1 R15 HD076379‐01A1S1, American Physiological Society UGSRF (BE) and the Le Moyne College McDevitt Natural Science Fellowship (BE).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.