Tunnel Handling and Environmental Enrichment for Repeated Whole-Body Plethysmography in Mice

Repeated whole-body plethysmography (WBP) is widely used for longitudinal assessment of respiratory function in mice. However, handling-induced stress can introduce physiological variability that confounds treatment effects and reduces experimental reproducibility. Traditional tail handling methods may produce sustained stress responses and poor habituation across repeated sessions, potentially affecting experimental outcomes. This protocol describes refined handling and housing methods integrating tunnel handling and environmental enrichment for repeated WBP sessions. Six wild-type 129S1/SvImJ mice (3 males, 3 females) received tunnel handling and continuous environmental enrichment throughout a 10-week study. Animals underwent repeated WBP sessions, and acclimation periods were analyzed at baseline, early-study, mid-study, and late-study timepoints. Respiratory frequency during initial chamber placement (finitial, 0-2 min) and stable baseline (fbaseline, 8-10 min) were quantified to assess longitudinal acclimation patterns. Progressive changes were observed across the study timeline under the implemented conditions. Mean initial respiratory frequency decreased from 345 ± 111 SD breaths/min at baseline to 267 ± 43 SD breaths/min during late-study sessions, accompanied by reduced coefficient of variation (32% to 16%). Baseline respiratory measurements also decreased from 255 ± 67 to 193 ± 43 breaths/min (p = 0.033), consistent with improved measurement stability during later sessions. Behavioral observations showed calm acclimation with minimal defecation, urination, or agitation. This protocol provides a practical approach for laboratories performing repeated respiratory assessments in mice. Tunnel handling and environmental enrichment were associated with progressive acclimation and reduced respiratory variability during longitudinal WBP studies.