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

Summary

This protocol describes tunnel handling and environmental enrichment procedures for repeated whole-body plethysmography to reduce stress-associated respiratory variability during longitudinal studies in mice.

Abstract

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.

Introduction

Whole-body plethysmography (WBP) enables non-invasive assessment of respiratory function in conscious, unrestrained rodents and has become an indispensable tool for longitudinal studies in respiratory physiology, inhalation toxicology, and disease modeling^1,2. The technique’s primary advantages, including its non-invasive nature and suitability for repeated measurements, make it particularly valuable for tracking respiratory changes across disease progression, therapeutic interventions, or exposure time courses. Glaab and Braun² highlighted that noninvasive head-out body plethysmo....

Protocol

Perform all procedures in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC Protocol #23-103) at the University of Miami. House mice in an AAALAC-accredited vivarium facility with environmental conditions maintained according to institutional standards.

1. Animal Selection and Housing Conditions

1. Select and prepare animals for the study
   1. Select animals from an established breeding colony or obtain them from a commercial vendor. For this study, use six wild-type 129S1/SvImJ mice (3 males, 3 females).
   2. Maintain animals under SPF (specific pathogen-free....

Results

Application of this tunnel handling and environmental enrichment protocol to six wild-type 129S1/SvImJ mice (3 males, 3 females) across a 10-week longitudinal WBP study yielded evidence of progressive habituation and improved physiological stability. Respiratory frequency measurements were obtained during repeated WBP sessions conducted at regular longitudinal intervals, with data analyzed from four study phases: Baseline (Week 0), Early-study (Weeks 2–3), Mid-study (Weeks 5–7), and Late-study (Weeks 8–10).

Discussion

This protocol describes an integrated approach combining tunnel handling, environmental enrichment, and repeated exposure in the context of longitudinal WBP. Under these conditions, respiratory frequency measurements showed progressive stabilization across repeated sessions, consistent with reduced arousal and progressive habituation. Several implementation factors appear important for successful protocol performance. First, tunnel handling should be introduced before experimental procedures begin. In this study, mice ha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aspen Chip Bedding	Lab Supply	Aspen 323	Aspen wood chip bedding. Autoclaved prior to use; placed to approximately 1 cm depth on cage floor.
Autoclaved Water	Facility-provided	Not applicable	Autoclaved municipal water provided ad libitum via standard water bottles.
Cardboard Tube	Facility-generated	Not applicable	Plain cardboard toilet paper roll. Used as rotated enrichment and shelter item.
Crink-I-Nest (Irradiated)	The Andersons / Lab Supply	CNKR	Shredded paper enrichment material. Gamma-irradiated. Provided as rotated enrichment on an alternating-week basis.
FinePointe Software	Data Sciences International	No catalog number	Respiratory data acquisition and analysis software, version 3.0. Used for WBP data collection and parameter calculation.
Individually Ventilated Cage (IVC) Rack	Allentown	EP5000E	Exhaust pak IVC rack, 140-cage capacity. Double-sided high-density system with automatic ventilation connections.
Irradiated Rodent Chow	Envigo	T.2918.15	Standard irradiated rodent diet. Provided ad libitum via wire-bar hopper.
Mouse Transfer Tunnel	Braintree Scientific	TRANS-TUBE 100X50MM	Clear polycarbonate tunnel, 100 mm length × 50 mm internal diameter. Autoclavable and reusable. Used for low-stress animal transfer procedures.
Nestlets (Irradiated)	Lab Supply	IRR	Compressed cotton nesting squares (~5 cm × 5 cm, ~2.5 g each). Gamma-irradiated. Provided as constant enrichment; one per cage.
Nitrile Gloves, Powder-Free	Kimberly-Clark	KC500	Purple nitrile powder-free exam gloves. Worn during all animal-contact procedures.
Non-Woven Gauze Sponge	Pivetal	21295040	Non-sterile, non-woven gauze sponge, 4-ply, 4 in × 4 in (10.16 cm × 10.16 cm). Used as enrichment material.
Paper Towel	Tork (Essity)	MB 540A	White multifold hand towel. Used as rotated enrichment material.
Smartphone Camera	Apple	No catalog number	iPhone smartphone camera. Used for weekly nest scoring documentation.
Whole-Body Plethysmography System	Data Sciences International	10222C (2-site); 770621 (4-site)	FinePointe FP Series unrestrained whole-body plethysmography system for mice. Available in 2-site and 4-site configurations.